mice after UVB irradiation (Figure 3). A previous study also demonstrated similar protective effects in the corneas of MIF Tg mice in response to acute UVB light (Kitaichi et al., 2008). MIF is upregulated by UVB irradiation in the mouse cornea, and MIF Tg mice showed fewer apoptotic corneal cells. Similarly, other studies have reported that MIF-deficient mice showed a significant increase in p53 activity and reduced tumor incidence compared to WT mice after acute and chronic exposure to UVB radiation, respectively (Martin et al., 2009). MIF has also been shown to have an inhibitory effect on UVB-induced photo-damage by blocking the expression of apoptosis-regulatory genes (Honda et al., 2009).

Recent evidence suggests that the pathways responsible for the removal of UV-mediated DNA damage can be modulated by several inflammatory cytokines. It has been observed that IL-12, which is an immunomodulatory cytokine, reduces UV-induced apoptosis *in vitro*. This was confirmed *in vivo,* because an injection of IL-12 into the skin of UV exposed mice significantly reduced the number of apoptotic keratinocytes (Schwarz et al., 2002). IL-18 seems to exert similar effects as IL-12. The injection of IL-18 into the UV exposed skin of mice reduced the amount of DNA damage (Schwarz et al., 2006).

#### **4.2 MIF overexpression accelerates photocarcinogenesis**

In addition to decreasing protective DNA damage repair, MIF also exhibits direct proneoplastic activity. In many tumor cells and pre-tumor states, for example, in prostate (Meyer-Siegler et al., 1998), colon (Legendre et al., 2003), and hepatocellular cancers (Ren et al., 2003), adenocarcinomas of the lung (Kamimura et al., 2000), glioblastomas (Rogge, 2002), and melanomas (Shimizu, 1999b), increased MIF mRNA levels can be detected. The proneoplastic role of MIF has been studied by several groups. Fingerle *et al*. reported that embryonic fibroblasts from MIF-deficient mice exhibit p53-dependent growth alterations, increased p53 transcriptional activity, and resistance to ras-mediated transformation (Fingerle-Rowson et al., 2003). The concurrent deletion of the p53 gene *in vivo* reversed the observed phenotype of cells deficient in MIF. *In vivo* studies showed that fibrosarcomas are smaller in size and have a lower mitotic index in MIF-deficient mice relative to their WT counterparts. The authors concluded that there is direct evidence for a functional link between MIF and the p53 tumor suppressor (Fingerle-Rowson et al., 2003).

Using an anti-MIF antibody has also been reported to be effective for reducing tumor growth and neovascularization in lymphoma cells and vascular endothelial cells *in vivo* (Nishihira, 2000). Consistent with this finding, anti-MIF antibodies were found to be effective for reducing tumor angiogenesis in melanoma cells (Shimizu, 1999b) This was demonstrated *in vitro* by using recombinant MIF in fibroblasts, where growth-factorinduced stimulation of these cells resulted in increased MIF concentrations, activation of the extracellular regulated kinase/mitogen-activated protein (ERK-MAP) kinase pathway, and a subsequent increase in cell proliferation (Takahashi et al., 1998). In addition, it has been shown that exposure to TGF-β results in increased MIF expression in a colon cancer cell line (Meyer-Siegler et al., 2006). Furthermore, other investigators observed an increase in the level of cytotoxic T lymphocytes following MIF inhibition achieved by using specific antibodies, and the number of apoptotic tumor cells increased following MIF inhibition (Abe et al., 2001). Tumors arising in the MIF-knockdown cells grew less rapidly and also showed an increased degree of apoptosis (Hagemann et al., 2007). These findings, therefore, suggest that once keratinocytes acquire mutations by UVB-induced DNA damage, they may become malignant, and MIF may thus play a dual role in promoting the growth of these tumor cells and inhibiting their apoptosis.

#### **4.3 MIF and UV-Induced skin inflammatory responses**

UVB stimulates the production of several proinflammatory cytokines, including TNF-α, IL-1 and IL-6 in the skin, and these proinflammatory cytokines are considered to be closely related to the progression of skin carcinogenesis (Moore et al., 1999, Murphy et al., 2003, Kim et al., 2010). UVB-induced inflammatory responses, such as the production of cytokines and the infiltration of inflammatory cells, are clearly associated with the development of skin tumors. The inhibition of this inflammatory response via topical application of an antiinflammatory drug inhibits the acute inflammatory responses after UVB exposure, and decreases tumor formation after chronic exposure (Wilgus et al., 2003).

MIF plays a direct role in both inflammation and tumorigenesis (Bach et al., 2008). UVB exposure in MIF Tg mice resulted in greater leukocyte infiltration than in WT mouse skin (Honda et al., 2009). Moreover, UVB irradiation enhances the expression of MIF in the epidermis, and MIF Tg mice showed higher levels of MIF mRNA expression after UVB exposure (Shimizu, 1999c). Once released, MIF acts as a proinflammatory cytokine to induce the expression of other inflammatory cytokines, including IL-1, IL-6 and TNF-α (Toh et al., 2006, Sanchez-Zamora et al., 2010, Donnelly, 1997). Since the intense inflammation in the skin in response to UVB irradiation was found to correlate with the early onset of carcinogenesis and a higher incidence of tumors after chronic UVB exposure, this finding confirms the likely role of MIF in this process.

#### **5. Conclusions**

148 Skin Cancers – Risk Factors, Prevention and Therapy

mice after UVB irradiation (Figure 3). A previous study also demonstrated similar protective effects in the corneas of MIF Tg mice in response to acute UVB light (Kitaichi et al., 2008). MIF is upregulated by UVB irradiation in the mouse cornea, and MIF Tg mice showed fewer apoptotic corneal cells. Similarly, other studies have reported that MIF-deficient mice showed a significant increase in p53 activity and reduced tumor incidence compared to WT mice after acute and chronic exposure to UVB radiation, respectively (Martin et al., 2009). MIF has also been shown to have an inhibitory effect on UVB-induced photo-damage by

Recent evidence suggests that the pathways responsible for the removal of UV-mediated DNA damage can be modulated by several inflammatory cytokines. It has been observed that IL-12, which is an immunomodulatory cytokine, reduces UV-induced apoptosis *in vitro*. This was confirmed *in vivo,* because an injection of IL-12 into the skin of UV exposed mice significantly reduced the number of apoptotic keratinocytes (Schwarz et al., 2002). IL-18 seems to exert similar effects as IL-12. The injection of IL-18 into the UV exposed skin of

In addition to decreasing protective DNA damage repair, MIF also exhibits direct proneoplastic activity. In many tumor cells and pre-tumor states, for example, in prostate (Meyer-Siegler et al., 1998), colon (Legendre et al., 2003), and hepatocellular cancers (Ren et al., 2003), adenocarcinomas of the lung (Kamimura et al., 2000), glioblastomas (Rogge, 2002), and melanomas (Shimizu, 1999b), increased MIF mRNA levels can be detected. The proneoplastic role of MIF has been studied by several groups. Fingerle *et al*. reported that embryonic fibroblasts from MIF-deficient mice exhibit p53-dependent growth alterations, increased p53 transcriptional activity, and resistance to ras-mediated transformation (Fingerle-Rowson et al., 2003). The concurrent deletion of the p53 gene *in vivo* reversed the observed phenotype of cells deficient in MIF. *In vivo* studies showed that fibrosarcomas are smaller in size and have a lower mitotic index in MIF-deficient mice relative to their WT counterparts. The authors concluded that there is direct evidence for a functional link

Using an anti-MIF antibody has also been reported to be effective for reducing tumor growth and neovascularization in lymphoma cells and vascular endothelial cells *in vivo* (Nishihira, 2000). Consistent with this finding, anti-MIF antibodies were found to be effective for reducing tumor angiogenesis in melanoma cells (Shimizu, 1999b) This was demonstrated *in vitro* by using recombinant MIF in fibroblasts, where growth-factorinduced stimulation of these cells resulted in increased MIF concentrations, activation of the extracellular regulated kinase/mitogen-activated protein (ERK-MAP) kinase pathway, and a subsequent increase in cell proliferation (Takahashi et al., 1998). In addition, it has been shown that exposure to TGF-β results in increased MIF expression in a colon cancer cell line (Meyer-Siegler et al., 2006). Furthermore, other investigators observed an increase in the level of cytotoxic T lymphocytes following MIF inhibition achieved by using specific antibodies, and the number of apoptotic tumor cells increased following MIF inhibition (Abe et al., 2001). Tumors arising in the MIF-knockdown cells grew less rapidly and also showed an increased degree of apoptosis (Hagemann et al., 2007). These findings, therefore, suggest that once keratinocytes acquire mutations by UVB-induced DNA damage, they may become malignant, and MIF may thus play a dual role in promoting the growth of

blocking the expression of apoptosis-regulatory genes (Honda et al., 2009).

between MIF and the p53 tumor suppressor (Fingerle-Rowson et al., 2003).

these tumor cells and inhibiting their apoptosis.

mice reduced the amount of DNA damage (Schwarz et al., 2006).

**4.2 MIF overexpression accelerates photocarcinogenesis**

Chronic exposure to UV irradiation induces early-onset skin carcinogenesis, and many inflammatory cytokines are related to the induction and progression of UV-induced skin cancer (Table 1). Chronic UVB exposure enhances MIF production, which may inhibit the p53-dependent apoptotic processes, thereby inducing photocarcinogenesis in the skin (Figure 4). This newly identified mechanism may positively contribute to our overall understanding of photo-induced skin damage, which ultimately results in carcinogenesis.

Table 1. Skin cancer and related inflammatory cytokines

Macrophage Migration Inhibitory Factor (MIF)

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Fig. 4. MIF inhibits p53-dependent apoptotic processes.

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**Part 2**

**Skin Cancer Diagnosis**

**Part 2**

**Skin Cancer Diagnosis**

156 Skin Cancers – Risk Factors, Prevention and Therapy

Wilgus, T.A., Koki, A.T., Zweifel, B.S., Kusewitt, D.F., Rubal, P.A. and Oberyszyn, T.M.

Young, A.R. (1990). "Cumulative effects of ultraviolet radiation on the skin : cancer and

Zhao, Y., Shimizu, T., Nishihira, J., Koyama, Y., Kushibiki, T., Honda, A., Watanabe, H.,

the eye lens." Proc Natl Acad Sci USA 90(4): 1272-5.

photoaging." Semin Dermatol 9(1): 25-31.

wounds." Am J Pathol 167(6): 1519-29.

(2003). "Inhibition of cutaneous ultraviolet light B-mediated inflammation and tumor formation with topical celecoxib treatment." Mol Carcinog. 38(2): 49-58. Wistow, G.J., Shaughnessy, M.P., Lee, D.C., Hodin, J. and Zelenka, P.S. (1993). "A

macrophage migration inhibitory factor is expressed in the differentiating cells of

Abe, R., Tabata, Y. and Shimizu, H. (2005). "Tissue regeneration using macrophage migration inhibitory factor (MIF)-impregnated gelatin microbeads in cutaneous

**7**

*Brasil*

**Optical Imaging as Auxiliary**

**Tool in Skin Cancer Diagnosis**

S. Pratavieira, C. T. Andrade, A. G. Salvio, V.S. Bagnato and C. Kurachi

Clinical detection of early malignant and premalignant skin lesions is not simple due to their similar features with the more common benign lesions. The traditional skin cancer screening is based on careful whole body visual inspection, and if a suspected lesion is noticed, further examination with biopsy is required. Visual examination relies heavily on the experience and skills of the physician to identify and delineate the initial changes related to carcinogenesis and cancer progression. White light dermatoscope is the most used conventional tool for skin cancer screening, used by dermatologists for a magnified visualization of the skin. Optical imaging at widefield and microscopic levels has been presented as clinical tools for aiding lesion detection and discrimination. Optical techniques are non-invasive, with fast response through an *in situ* interrogation, and are potentially sensitive to biochemical and structural changes presented in skin cancer development. For skin cancer diagnostics the main used techniques are: widefield imaging, optical spectroscopy and microscopy imaging. Widefield imaging allows the examination of large areas and has the potential of improving detection of occult lesions, margin delimitation, and of guiding biopsy site determination. Optical spectroscopy presents more detailed information on tissue composition, through the light intensity for each collected emission wavelength, which might be later correlated to specific biomolecules. Microscopy imaging has a main advantage of the evaluation of the tissue characteristics at cellular level, but only a small fraction of the lesion volume is interrogated. Distinct optical techniques offer different tissue/cellular information that is complimentary, even though for cancer screening, widefield optical imaging may represent the first best option, when compared to point spectroscopy or optical microscopy. The combination of information from different imaging modalities has the potential of revealing tissue features resulting in an improved diagnostic performance, due to their distinct provided tissue information. In this chapter an overview of the optical clinical methods and the instruments developed for skin cancer

The electromagnetic radiation extends, in order of increasing energy, from radio waves with low energy (long wavelengths), to high-energy radiation (X-ray and gamma radiation). Include regions of radiation with intermediate energies between the ultraviolet and

**1. Introduction**

detection of published studies will be discussed.

**2. Interaction visible light – skin**

 *Instituto de Física de São Carlos - Universidade de São Paulo, São Carlos, SP,*

### **Optical Imaging as Auxiliary Tool in Skin Cancer Diagnosis**

S. Pratavieira, C. T. Andrade, A. G. Salvio, V.S. Bagnato and C. Kurachi  *Instituto de Física de São Carlos - Universidade de São Paulo, São Carlos, SP, Brasil*

#### **1. Introduction**

Clinical detection of early malignant and premalignant skin lesions is not simple due to their similar features with the more common benign lesions. The traditional skin cancer screening is based on careful whole body visual inspection, and if a suspected lesion is noticed, further examination with biopsy is required. Visual examination relies heavily on the experience and skills of the physician to identify and delineate the initial changes related to carcinogenesis and cancer progression. White light dermatoscope is the most used conventional tool for skin cancer screening, used by dermatologists for a magnified visualization of the skin. Optical imaging at widefield and microscopic levels has been presented as clinical tools for aiding lesion detection and discrimination. Optical techniques are non-invasive, with fast response through an *in situ* interrogation, and are potentially sensitive to biochemical and structural changes presented in skin cancer development. For skin cancer diagnostics the main used techniques are: widefield imaging, optical spectroscopy and microscopy imaging. Widefield imaging allows the examination of large areas and has the potential of improving detection of occult lesions, margin delimitation, and of guiding biopsy site determination. Optical spectroscopy presents more detailed information on tissue composition, through the light intensity for each collected emission wavelength, which might be later correlated to specific biomolecules. Microscopy imaging has a main advantage of the evaluation of the tissue characteristics at cellular level, but only a small fraction of the lesion volume is interrogated. Distinct optical techniques offer different tissue/cellular information that is complimentary, even though for cancer screening, widefield optical imaging may represent the first best option, when compared to point spectroscopy or optical microscopy. The combination of information from different imaging modalities has the potential of revealing tissue features resulting in an improved diagnostic performance, due to their distinct provided tissue information. In this chapter an overview of the optical clinical methods and the instruments developed for skin cancer detection of published studies will be discussed.

#### **2. Interaction visible light – skin**

The electromagnetic radiation extends, in order of increasing energy, from radio waves with low energy (long wavelengths), to high-energy radiation (X-ray and gamma radiation). Include regions of radiation with intermediate energies between the ultraviolet and

Optical Imaging as Auxiliary Tool in Skin Cancer Diagnosis 161

Fig. 1. Main optical events that occur when a light beam is incident to the skin surface. These

During neoplastic progression, the optical properties of both superficial epithelium and underlying stroma are altered. Optical effects like reflectance and fluorescence are considerably modified during the time evolution of the lesion. The perceived loss of fluorescence in dysplatic and malignant tissues compared to normal tissues has been mainly attributed to decreasing collagen signal from the stroma, due to fiber crosslink breakdown. Reflectance analysis, that is the analysis of the diffuse scattering, can detect local tissue changes in scattering and absorption properties. Absorption is affected by changes in some endogenous cromophores, blood content and tissue oxygenation, which are related to altered tissue metabolism and neovascularization presented at carcinomas (Gillenwater, et

Optical imaging can be performed at cellular level (microscopy) or tissue level (widefield). The advantage of the microscopy imaging is the evaluation of cellular characteristics, but in a small fraction of the lesion volume. Widefield imaging allows the examination of large areas and has the potential of improving detection of occult lesions, margin delimitation, and of guiding biopsy site determination. Distinct optical techniques offer different tissue/cellular information that is complimentary, but widefield optical imaging may represent the best option for cancer screening due to the possibility of analysis of large areas

Nowadays, conventional examination for skin cancer screening is based on the visualization provided by a dermatoscope, a well-established modality in Dermatology.

schematic diagrams are illustrative for single photo pathways.

with fast response and less inter-operator variance (Laine, et al. 2008).

al. 2008, Kwasniak and Garcia-Zuazaga 2011).

**3. Clinical procedures**

microwave, passing by the visible light. Each of these regions has distinct ways to be produced and detected, and each interacts with matter differently, which may reveal certain characteristics of the system investigated. Due to the distinct energy characteristics, different light/biological tissue interaction occurs. This is the reason, for instance, why the x-rays may induce DNA damage (Karagas, et al. 1996, McLaren, et al. 1975), and the violet light is used for jaundice treatment of new-borns (Bauer, et al. 2004). Optical diagnostic systems are the system that uses the visible light for diagnostics. The visible light or white light is composing by all colors, with the range of wavelength of 400 – 700 nanometers (when the light is understood as a wave). All optical diagnostic systems are based on the one or more types of light-tissue interactions to provide biochemical and/or structural tissue information. This idea relies on the fact that tissue changes alters light-tissue interaction, and if the re-emitted light exiting the tissue is collected and analyzed, a correlation may be obtained. Each specific light wavelength (or color) has a particular interaction with the tissue.

When the light is incident in a biological tissue, the main following events occur:

These are the most important events of the light-tissue interaction (figure 1), but there are many others phenomena that can be explored by an optical technique. Light polarization, Raman scattering, lifetime fluorescence, phase coherence, non-linear effects, are some examples. All these effects can be used to form an optical image. Biological tissues, especially the skin, are considered turbid media, where the diffuse scattering is the most important optical event in the visible range, i.e., the incident photons have multiple scattering before they are absorbed. Biological fluorophores will emit fluorescence in several directions, and these fluorescent photons will scattered and can also be absorbed by other chromophores. Tissue native fluorescence will result from the contribution of all fluorophores present in the interrogated tissue. This detected tissue fluorescence is also influenced by the presence and concentration of absorbers and scatterers. The influence of absorbance and structural tissue properties is even more relevant whereas the collected fluorescence is obtained at the tissue surface result from the exiting photons.

The main endogenous fluorophores are tryptophan, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), collagen, elastin, and keratin. Main absorbers non-fluorescent are hemoglobin and melanin; and the scatterers are the cell membrane, organelles, and collagen fibers, that indirectly modifies the intensity and shape of the fluorescence collected spectrum. For the imaging widefield devices, the most important light-tissue interactions are the fluorescence and diffuse reflectance (de Veld, et al. 2005).

microwave, passing by the visible light. Each of these regions has distinct ways to be produced and detected, and each interacts with matter differently, which may reveal certain characteristics of the system investigated. Due to the distinct energy characteristics, different light/biological tissue interaction occurs. This is the reason, for instance, why the x-rays may induce DNA damage (Karagas, et al. 1996, McLaren, et al. 1975), and the violet light is used for jaundice treatment of new-borns (Bauer, et al. 2004). Optical diagnostic systems are the system that uses the visible light for diagnostics. The visible light or white light is composing by all colors, with the range of wavelength of 400 – 700 nanometers (when the light is understood as a wave). All optical diagnostic systems are based on the one or more types of light-tissue interactions to provide biochemical and/or structural tissue information. This idea relies on the fact that tissue changes alters light-tissue interaction, and if the re-emitted light exiting the tissue is collected and analyzed, a correlation may be obtained. Each specific light wavelength

When the light is incident in a biological tissue, the main following events occur:

a. Specular scattering: the light is reflected at the tissue surface with the same incident

b. Diffuse scattering: the light is diffusely scattered inside the tissue. Due to the different refractive indexes of biological components, the photon (when the light can be understood as constitute by photons) travelling within the tissue suffers multiple

c. Transmission: the light is transmitted through the tissue and may exit without changing

d. Absorption: the light is absorbed by biological chromophores and this energy may result in molecular vibration (heat), and luminescent emission. The most important

e. Luminescence: after light absorption, some excited biological chromophores may lose this excess of energy through light emission (fluorescence and phosphorescence). The

These are the most important events of the light-tissue interaction (figure 1), but there are many others phenomena that can be explored by an optical technique. Light polarization, Raman scattering, lifetime fluorescence, phase coherence, non-linear effects, are some examples. All these effects can be used to form an optical image. Biological tissues, especially the skin, are considered turbid media, where the diffuse scattering is the most important optical event in the visible range, i.e., the incident photons have multiple scattering before they are absorbed. Biological fluorophores will emit fluorescence in several directions, and these fluorescent photons will scattered and can also be absorbed by other chromophores. Tissue native fluorescence will result from the contribution of all fluorophores present in the interrogated tissue. This detected tissue fluorescence is also influenced by the presence and concentration of absorbers and scatterers. The influence of absorbance and structural tissue properties is even more relevant whereas the collected

The main endogenous fluorophores are tryptophan, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), collagen, elastin, and keratin. Main absorbers non-fluorescent are hemoglobin and melanin; and the scatterers are the cell membrane, organelles, and collagen fibers, that indirectly modifies the intensity and shape of the fluorescence collected spectrum. For the imaging widefield devices, the most important light-tissue interactions are the fluorescence and diffuse reflectance (de Veld, et al. 2005).

prevalent optical luminescent event in biological tissues is the fluorescence.

fluorescence is obtained at the tissue surface result from the exiting photons.

(or color) has a particular interaction with the tissue.

angle relative to the normal orientation.

biological absorbers are hemoglobin and melanin.

direction changes.

its incident direction.

Fig. 1. Main optical events that occur when a light beam is incident to the skin surface. These schematic diagrams are illustrative for single photo pathways.

During neoplastic progression, the optical properties of both superficial epithelium and underlying stroma are altered. Optical effects like reflectance and fluorescence are considerably modified during the time evolution of the lesion. The perceived loss of fluorescence in dysplatic and malignant tissues compared to normal tissues has been mainly attributed to decreasing collagen signal from the stroma, due to fiber crosslink breakdown. Reflectance analysis, that is the analysis of the diffuse scattering, can detect local tissue changes in scattering and absorption properties. Absorption is affected by changes in some endogenous cromophores, blood content and tissue oxygenation, which are related to altered tissue metabolism and neovascularization presented at carcinomas (Gillenwater, et al. 2008, Kwasniak and Garcia-Zuazaga 2011).

Optical imaging can be performed at cellular level (microscopy) or tissue level (widefield). The advantage of the microscopy imaging is the evaluation of cellular characteristics, but in a small fraction of the lesion volume. Widefield imaging allows the examination of large areas and has the potential of improving detection of occult lesions, margin delimitation, and of guiding biopsy site determination. Distinct optical techniques offer different tissue/cellular information that is complimentary, but widefield optical imaging may represent the best option for cancer screening due to the possibility of analysis of large areas with fast response and less inter-operator variance (Laine, et al. 2008).

#### **3. Clinical procedures**

Nowadays, conventional examination for skin cancer screening is based on the visualization provided by a dermatoscope, a well-established modality in Dermatology.

Optical Imaging as Auxiliary Tool in Skin Cancer Diagnosis 163

for this purpose (Marcon, et al. 2007, Ramanujam 2000, Marghoob, et al. 2003). Main advantages are a non-invasive and objective in situ procedure with real time response and a potential technique to the identification of occult lesions under the conventional with light examination (Carli, et al. 2001, Bono, et al. 2002, Braun, et al. 2002, MacKie, et al. 2002,

There are several optical imaging systems with direct visualization (naked eye) or visualization through electronic devices. Both methodologies allow the visualization of fluorescence and/or reflectance. When a set of images of the same skin location is taken at different spectral bands (reflectance and/or fluorescence); the result can be a multispectral or hiperspectral image. The difference between a multispectral or hiperspectral image is the number of images acquired. A multispectral imaging is a technique with a promising potential for *in vivo* skin diagnostics. Fluorescence usually has as tissue targets the NADH/FAD from the epithelial cells, and the collagen at the stroma. Reflectance using different excitation wavelength will focused on the absorption by specific chromospheres

There are some multispectral systems, acquiring reflectance images (RL) with several excitation wavelengths, or fluorescence images (FL) with several excitation/emission wavelengths (Dhawan, et al. 2009, Carrara, et al. 2005, Kapsokalyvas, et al. 2009). More recently, a few systems with combination of both modalities have been used and proposed for the diagnostics of skin cancer (Pratavieira, et al. 2009) and other cancer types as oral (Roblyer, et al. 2009), and cervical (Benavides, et al. 2003, Agrawal, et al. 2001). Individual analysis of fluorescence, narrowband reflectance or white light image may not be sufficient for the final discrimination, characterization, and classification of the lesion. Combining information from different imaging modalities has the potential of revealing tissue features resulting in an improved diagnostic performance, due to their distinct provided information. In table 1 we present the most important tissue information provided by each

Several commercial systems are available: Dermlite (3Gen, LLC, Dana Point, CA, U.S.A.), Dyaderm (Biocam GmbH, Regensburg, Germany), Dermascope (AMD Telemedicine, Elizabeth, NJ, U.S.A.), MelaFind (Electro-Optical Sciences, Irvington, NY, U.S.A.), Molemax (Derma Medical Systems, Vienna, Austria), Microderm (Visiomed Comp.,Bochum, Germany), Multiderm (Light4tech, Scandicci, Fi, Italy), Nevoscope (Translite LLC, Sugar Land, TX, U.S.A.), Siascopy (Astron Clinica, Cambridge, UK), SolarScan (Polartechnics Ltd, Sydney, Australia), and many others. All have been specifically designed for the detection/interpretation of skin lesions. Some of those commercial imaging systems are

RL ME=530nm Absorption of hemoglobin, morphology and identification of blood vessels.

Table 1. Most relevant tissue information for cancer diagnostics provided by some optical

McGovern 2003).

imaging modality.

multispectral.

modalities.

*Image Modality: Tissue information by optical detection:*

RL ME=470nm Absorption of keratin and melanin.

RL White Light Global analysis.

FL, ME=400nm Fluorescence of Collagen, Elastin, NADH, and Flavins. RL ME=400nm Absorption of Collagen, Elastin, NADH, and Flavins.

RL ME=630nm Observation of scatter structures and surface texture.

mainly hemoglobin and melanin.

Dermatoscopy (or dermoscopy or epiluminescence microscopy) uses a magnifying lens through an oil/gel interface, the conventional immersion contact dermatoscopy, or crosspolarizing optical filters, the non-contact dermatoscopy. This last one method reduces the amount of reflected light, resulting in the visualization of deeper skin structures. The clinical features that are important for analysis using a dermatoscope are color, dimensions (symmetry), and the presence or absence of certain structures in the lesion. The main idea of this diagnostic tool is to improve the visualization of the skin lesions, based on magnification, so the physician can detect better the clinical features to discriminate and distinguish the lesion types. In this method, the white light is used for this visualization (Ferris and Divito 2010, Tanaka 2006).

Total body investigation and time monitoring of suspicious lesions and benign nevi would be the ideal skin screening, but this is unpractical because of the required clinical time. Some studies have showed improved early detection of melanoma when the dermatoscopy is associated with time series photography. The disadvantage of this method, concerning health policies, is the high cost that is around thousands US\$ per person. Incidence of new and changed nevi and melanomas detected using baseline images and dermoscopy in patients at high risk for melanoma (Banky, et al. 2003).

When the clinician detects a lesion, its visual characteristics as color, architecture, texture, and its palpation are carefully evaluated under white light illumination, the ABCD (Asymmetry, Border, Color, Diameter) rule. If malignant features are suspected, a biopsy is indicated to provide tissue sample for histological analysis, the golden standard for skin cancer diagnostics. In the cases of punch biopsies, the biopsy site is chosen based on the individual clinician criteria to determine the more representative area of the lesion. This procedure is highly subjective and variable comparing dermatologists with distinct experience and training (Kittler, et al. 2011, Bono, et al. 1999). The biopsy is indicated when the clinician recognizes clinical features that are potential indicatives of a malignant condition. At non-homogenous and extent lesions, the biopsy site determination may not be simple. It is not unusual that a lesion shows distinct histological patterns, as different dysplasia degrees, carcinoma *in situ*, and even invasive carcinoma. As a result, the chosen lesion sample may not be representative, and initial carcinoma can be missed. On the other hand, multiple biopsies at once are not well accepted by the patient, since it is an invasive procedure. After tissue removal, the material is sent to a laboratory for processing and analysis by a pathologist. This method takes time and cannot be performed *in situ*, and besides the fresh frozen tissue protocol for resected specimens, the pathology result is usually obtained only after a few days (Patel, et al. 2008).

#### **4. Optical systems for aid in skin cancer diagnostics**

Visual examination and the methods of the last section relies heavily on the experience and skills of the physician to identify and delineate premalignant and early cancer changes, which is not simple due to the similar clinical characteristics compared to the more common benign lesions. Gold standard diagnostics is obtained only after a tissue sample removal and processing for microscopic evaluation and its classification based on morphological characteristics at cellular level.

Enhancing the visual contrast between normal and altered skin has the potential to contribute for biopsy site determination, as well as for lesion margin delimitation before surgical resection. Optical widefield imaging has been presented as an attractive technique

Dermatoscopy (or dermoscopy or epiluminescence microscopy) uses a magnifying lens through an oil/gel interface, the conventional immersion contact dermatoscopy, or crosspolarizing optical filters, the non-contact dermatoscopy. This last one method reduces the amount of reflected light, resulting in the visualization of deeper skin structures. The clinical features that are important for analysis using a dermatoscope are color, dimensions (symmetry), and the presence or absence of certain structures in the lesion. The main idea of this diagnostic tool is to improve the visualization of the skin lesions, based on magnification, so the physician can detect better the clinical features to discriminate and distinguish the lesion types. In this method, the white light is used for

Total body investigation and time monitoring of suspicious lesions and benign nevi would be the ideal skin screening, but this is unpractical because of the required clinical time. Some studies have showed improved early detection of melanoma when the dermatoscopy is associated with time series photography. The disadvantage of this method, concerning health policies, is the high cost that is around thousands US\$ per person. Incidence of new and changed nevi and melanomas detected using baseline images and dermoscopy in

When the clinician detects a lesion, its visual characteristics as color, architecture, texture, and its palpation are carefully evaluated under white light illumination, the ABCD (Asymmetry, Border, Color, Diameter) rule. If malignant features are suspected, a biopsy is indicated to provide tissue sample for histological analysis, the golden standard for skin cancer diagnostics. In the cases of punch biopsies, the biopsy site is chosen based on the individual clinician criteria to determine the more representative area of the lesion. This procedure is highly subjective and variable comparing dermatologists with distinct experience and training (Kittler, et al. 2011, Bono, et al. 1999). The biopsy is indicated when the clinician recognizes clinical features that are potential indicatives of a malignant condition. At non-homogenous and extent lesions, the biopsy site determination may not be simple. It is not unusual that a lesion shows distinct histological patterns, as different dysplasia degrees, carcinoma *in situ*, and even invasive carcinoma. As a result, the chosen lesion sample may not be representative, and initial carcinoma can be missed. On the other hand, multiple biopsies at once are not well accepted by the patient, since it is an invasive procedure. After tissue removal, the material is sent to a laboratory for processing and analysis by a pathologist. This method takes time and cannot be performed *in situ*, and besides the fresh frozen tissue protocol for resected specimens, the pathology result is

Visual examination and the methods of the last section relies heavily on the experience and skills of the physician to identify and delineate premalignant and early cancer changes, which is not simple due to the similar clinical characteristics compared to the more common benign lesions. Gold standard diagnostics is obtained only after a tissue sample removal and processing for microscopic evaluation and its classification based on morphological

Enhancing the visual contrast between normal and altered skin has the potential to contribute for biopsy site determination, as well as for lesion margin delimitation before surgical resection. Optical widefield imaging has been presented as an attractive technique

this visualization (Ferris and Divito 2010, Tanaka 2006).

patients at high risk for melanoma (Banky, et al. 2003).

usually obtained only after a few days (Patel, et al. 2008).

characteristics at cellular level.

**4. Optical systems for aid in skin cancer diagnostics**

for this purpose (Marcon, et al. 2007, Ramanujam 2000, Marghoob, et al. 2003). Main advantages are a non-invasive and objective in situ procedure with real time response and a potential technique to the identification of occult lesions under the conventional with light examination (Carli, et al. 2001, Bono, et al. 2002, Braun, et al. 2002, MacKie, et al. 2002, McGovern 2003).

There are several optical imaging systems with direct visualization (naked eye) or visualization through electronic devices. Both methodologies allow the visualization of fluorescence and/or reflectance. When a set of images of the same skin location is taken at different spectral bands (reflectance and/or fluorescence); the result can be a multispectral or hiperspectral image. The difference between a multispectral or hiperspectral image is the number of images acquired. A multispectral imaging is a technique with a promising potential for *in vivo* skin diagnostics. Fluorescence usually has as tissue targets the NADH/FAD from the epithelial cells, and the collagen at the stroma. Reflectance using different excitation wavelength will focused on the absorption by specific chromospheres mainly hemoglobin and melanin.

There are some multispectral systems, acquiring reflectance images (RL) with several excitation wavelengths, or fluorescence images (FL) with several excitation/emission wavelengths (Dhawan, et al. 2009, Carrara, et al. 2005, Kapsokalyvas, et al. 2009). More recently, a few systems with combination of both modalities have been used and proposed for the diagnostics of skin cancer (Pratavieira, et al. 2009) and other cancer types as oral (Roblyer, et al. 2009), and cervical (Benavides, et al. 2003, Agrawal, et al. 2001). Individual analysis of fluorescence, narrowband reflectance or white light image may not be sufficient for the final discrimination, characterization, and classification of the lesion. Combining information from different imaging modalities has the potential of revealing tissue features resulting in an improved diagnostic performance, due to their distinct provided information. In table 1 we present the most important tissue information provided by each imaging modality.

Several commercial systems are available: Dermlite (3Gen, LLC, Dana Point, CA, U.S.A.), Dyaderm (Biocam GmbH, Regensburg, Germany), Dermascope (AMD Telemedicine, Elizabeth, NJ, U.S.A.), MelaFind (Electro-Optical Sciences, Irvington, NY, U.S.A.), Molemax (Derma Medical Systems, Vienna, Austria), Microderm (Visiomed Comp.,Bochum, Germany), Multiderm (Light4tech, Scandicci, Fi, Italy), Nevoscope (Translite LLC, Sugar Land, TX, U.S.A.), Siascopy (Astron Clinica, Cambridge, UK), SolarScan (Polartechnics Ltd, Sydney, Australia), and many others. All have been specifically designed for the detection/interpretation of skin lesions. Some of those commercial imaging systems are multispectral.

Table 1. Most relevant tissue information for cancer diagnostics provided by some optical modalities.

Optical Imaging as Auxiliary Tool in Skin Cancer Diagnosis 165

most common technique is the previous topical application of a 15-20% aminolevulinic acid (ALA) or metil-ALA cream, a pro-drug for photodynamic therapy. ALA or Me-ALA enhances the cellular production of protoporphyrin IX (PpIX), a fluorescent biomolecule. When the pretreated tissue with ALA or Me-ALA is visualized with widefield fluorescence imaging, a strong red fluorescence, originated from the produced PpIX, lesions discrimination is improved. This technique is known as photodynamic detection (Hewett, et

Fig. 3. Typical configuration of a spectroscopy system, the light is emitted by a lamp, laser or a LED. The light is guided by the optical fiber to the skin. The re-emmited light is analyzed

at a spectrometer, which shows the intensity light emitted at each wavelength.

al. 2000, Orenstein, et al. 1998, Hewett, et al. 2001).

The image, in general, can be obtained placing the instrument in contact with the skin, where the surface reflection of light is reduced by either an oil-glass interface at the skin or through cross-polarization setup to remove the specular reflectance before forming the image. Other systems do not have any contact with the interrogated skin. Some limitations of the widefield fluorescence image are very limited interrogation depth, photobleaching of biomolecules, and high signal background. The main advantages are lower instrumentation cost, when compared to others optical techniques, and with potential portable and friendlyuse systems.

In figure 2 an example of this instrumentation is presented. This system was assembled by our group, this is based on light emitting diodes (LED) illumination for reflectance modality at red, green and blue (RGB) spectrum and white light, and for fluorescence modality at 400 nm excitation (Pratavieira, et al. 2009). The main optical components a widefield fluorescence system are presented at figure 2, the light source, some lens and filters to remove the backscattering light at the excitation wavelength and the detection mode. For the detection, the visualization can be performed directly with the naked eye, or it can be digitalized and saved as an image by a conventional digital camera or a CCD. Besides the LED illumination other sources can be also chose as a lamp or laser.

When the image is digitalized, a mathematical processing, using computational algorithm, can be performed for image segmentation. The segmentation performed may be helpful for enhancing lesion discrimination, in choosing the biopsy site and in the delimitation of lesion margins, aiding in surgical resection. Also this technique provides a quantitative and less subjective analysis of the image, compared to only its visualization. The computational algorithm may vary from simple image addition or/and subtraction, the widely accepted ABCD (Asymmetry, Border, Color, Diameter) rule when comparing skin lesions against the database, until advanced texture analysis using fractal geometry (Schaefer, et al. 2011, Tasoulis, et al. 2010, Ogorzalek, et al. 2009).

Fig. 2. Possible configuration of an image system. The light interacts with the skin and is reemitted. The emission can be magnified by lens and the colors selected by optical filters. The visualization can be performed by a camera or naked eye.

Fluorescence imaging can also be performed using an exogenous component for increasing contrast between lesion and normal skin. In this case, the fluorescence analysis is not based on the contribution of the endogenous biomolecules present in the lesion, but on the presence of a fluorescent marker with enhanced concentration at the malignant cells. The

The image, in general, can be obtained placing the instrument in contact with the skin, where the surface reflection of light is reduced by either an oil-glass interface at the skin or through cross-polarization setup to remove the specular reflectance before forming the image. Other systems do not have any contact with the interrogated skin. Some limitations of the widefield fluorescence image are very limited interrogation depth, photobleaching of biomolecules, and high signal background. The main advantages are lower instrumentation cost, when compared to others optical techniques, and with potential portable and friendly-

In figure 2 an example of this instrumentation is presented. This system was assembled by our group, this is based on light emitting diodes (LED) illumination for reflectance modality at red, green and blue (RGB) spectrum and white light, and for fluorescence modality at 400 nm excitation (Pratavieira, et al. 2009). The main optical components a widefield fluorescence system are presented at figure 2, the light source, some lens and filters to remove the backscattering light at the excitation wavelength and the detection mode. For the detection, the visualization can be performed directly with the naked eye, or it can be digitalized and saved as an image by a conventional digital camera or a CCD. Besides the

When the image is digitalized, a mathematical processing, using computational algorithm, can be performed for image segmentation. The segmentation performed may be helpful for enhancing lesion discrimination, in choosing the biopsy site and in the delimitation of lesion margins, aiding in surgical resection. Also this technique provides a quantitative and less subjective analysis of the image, compared to only its visualization. The computational algorithm may vary from simple image addition or/and subtraction, the widely accepted ABCD (Asymmetry, Border, Color, Diameter) rule when comparing skin lesions against the database, until advanced texture analysis using fractal geometry (Schaefer, et al. 2011,

Fig. 2. Possible configuration of an image system. The light interacts with the skin and is reemitted. The emission can be magnified by lens and the colors selected by optical filters. The

Fluorescence imaging can also be performed using an exogenous component for increasing contrast between lesion and normal skin. In this case, the fluorescence analysis is not based on the contribution of the endogenous biomolecules present in the lesion, but on the presence of a fluorescent marker with enhanced concentration at the malignant cells. The

LED illumination other sources can be also chose as a lamp or laser.

Tasoulis, et al. 2010, Ogorzalek, et al. 2009).

visualization can be performed by a camera or naked eye.

use systems.

most common technique is the previous topical application of a 15-20% aminolevulinic acid (ALA) or metil-ALA cream, a pro-drug for photodynamic therapy. ALA or Me-ALA enhances the cellular production of protoporphyrin IX (PpIX), a fluorescent biomolecule. When the pretreated tissue with ALA or Me-ALA is visualized with widefield fluorescence imaging, a strong red fluorescence, originated from the produced PpIX, lesions discrimination is improved. This technique is known as photodynamic detection (Hewett, et al. 2000, Orenstein, et al. 1998, Hewett, et al. 2001).

Fig. 3. Typical configuration of a spectroscopy system, the light is emitted by a lamp, laser or a LED. The light is guided by the optical fiber to the skin. The re-emmited light is analyzed at a spectrometer, which shows the intensity light emitted at each wavelength.

Optical Imaging as Auxiliary Tool in Skin Cancer Diagnosis 167

Fig. 4. Configuration of the fluorescence imaging device used.

 Fig. 5. Basocellular carcinoma under white light illumination (left hand picture) and

Another important issue in skin diagnostics is the lesion discrimination between melanoma and seborrheic keratosis. At conventional white light illumination, without magnification, both lesions may appear similar. The fluorescence widefield imaging has the potential to overcome this problem. In figure 6 and 7, we present the examination of two patients, one with a seborrheic keratosis (fig.6) and another with melanoma. At white light illumination

visualized using the widefield fluorescence modality (right hand picture).

Another commonly investigated technique is the point spectroscopy by fluorescence (FL) and/or reflectance (RL), with single or multiple wavelengths. In this case, the result is not an image, but a graph showing the spectral information present at the re-emitted light. The spectroscopy methods show good specificity and sensitivity on cancer discrimination however, the obtained information is punctual, which makes the analysis difficult for large areas and the diagnostics is dependent on and specific for the interrogated site. Point spectroscopy uses optical fibers that deliver the excitation light to the tissue surface and collect the re-emitted light. Depending on the spectrometer and optical design, fluorescence or reflectance is investigated. The result showed after this optical measurement is a graph with the emitted light intensity as a function of the wavelength (Sterenborg, et al. 1994, Utz, et al. 1994, Kollias, et al. 2000, Ramanujam 2000, Panjehpour, et al. 2002, Panjehpour, et al. 2002). In figure 3 a typical configuration of this setup is presented.

Microscopy imaging is another punctual technique. For skin investigation, common systems are confocal scanning microscopy, and multiphoton tomography, where a laser is used to excite the skin fluorophores, or scatterers components. For one or two photon excitation some commercial system are available such as Lucid's VivaScope (Rochester, NY, U.S.A.), and DermaInspect (JenLab GmbH, Jena, Germany). This technique has a great advantage in comparison with the others (widefield, spectroscopy) because in this method, the image resolution is at cellular level. For some instrumentation, the cellular and tissue features may be similar to what is visualized by histological slides. Characteristics used for conventional diagnostics as cellular shape, nuclei structure and tissue organization may be also obtained. The microscopy images has the advantage of very high resolution, very low out-of-focus signal, but the main limitation is still the high cost (Roberts, et al. 2011, Koehler, et al. 2011, Garbe, et al. 2011, Breunig, et al. 2010).

#### **5. Widefield fluorescence image in skin cancer diagnostics**

In this section, we are going to present a widefield fluorescence device assembled by our group and some of the images taken at patients with skin lesions. The illumination device is based on an array of two LEDs composing an UV-blue range light. This spectral region was chosen for its already well-known use to excite endogenous fluorophores at skin. Light intensity is around 50 mW/cm2. A handheld device enclose the illumination part and also the optical components to provide fluorescence imaging: a dichroic mirror, a longpass filter to remove the light at excitation wavelengths, and another filter to remove the yellow spectral range. This set of filters allows the fluorescence viewing in the green and red range (Costa, et al. 2010). A schematic diagram of the configuration of the widefield fluorescence image is presented at figure 4.

An example of the obtained fluorescence image is showed at figure 5. A basocellular carcinoma lesion at a Caucasian patient was visualized using the system. The comparison between the discrimination provided at a white light illumination and the fluorescence imaging can be observed. The cancer lesion is usually visualized at the fluorescence image as a darker region, correlated with the less tissue fluorescence, mainly due to the changes on collagen structure induced by the carcinoma progression. The tumor margins can be better discriminate using the fluorescence, the contrast between lesion and normal tissue is enhanced.

Another commonly investigated technique is the point spectroscopy by fluorescence (FL) and/or reflectance (RL), with single or multiple wavelengths. In this case, the result is not an image, but a graph showing the spectral information present at the re-emitted light. The spectroscopy methods show good specificity and sensitivity on cancer discrimination however, the obtained information is punctual, which makes the analysis difficult for large areas and the diagnostics is dependent on and specific for the interrogated site. Point spectroscopy uses optical fibers that deliver the excitation light to the tissue surface and collect the re-emitted light. Depending on the spectrometer and optical design, fluorescence or reflectance is investigated. The result showed after this optical measurement is a graph with the emitted light intensity as a function of the wavelength (Sterenborg, et al. 1994, Utz, et al. 1994, Kollias, et al. 2000, Ramanujam 2000, Panjehpour, et al. 2002, Panjehpour, et al. 2002). In figure 3 a typical configuration of this setup is

Microscopy imaging is another punctual technique. For skin investigation, common systems are confocal scanning microscopy, and multiphoton tomography, where a laser is used to excite the skin fluorophores, or scatterers components. For one or two photon excitation some commercial system are available such as Lucid's VivaScope (Rochester, NY, U.S.A.), and DermaInspect (JenLab GmbH, Jena, Germany). This technique has a great advantage in comparison with the others (widefield, spectroscopy) because in this method, the image resolution is at cellular level. For some instrumentation, the cellular and tissue features may be similar to what is visualized by histological slides. Characteristics used for conventional diagnostics as cellular shape, nuclei structure and tissue organization may be also obtained. The microscopy images has the advantage of very high resolution, very low out-of-focus signal, but the main limitation is still the high cost (Roberts, et al. 2011, Koehler, et al. 2011,

In this section, we are going to present a widefield fluorescence device assembled by our group and some of the images taken at patients with skin lesions. The illumination device is based on an array of two LEDs composing an UV-blue range light. This spectral region was chosen for its already well-known use to excite endogenous fluorophores at skin. Light intensity is around 50 mW/cm2. A handheld device enclose the illumination part and also the optical components to provide fluorescence imaging: a dichroic mirror, a longpass filter to remove the light at excitation wavelengths, and another filter to remove the yellow spectral range. This set of filters allows the fluorescence viewing in the green and red range (Costa, et al. 2010). A schematic diagram of the configuration of the widefield fluorescence

An example of the obtained fluorescence image is showed at figure 5. A basocellular carcinoma lesion at a Caucasian patient was visualized using the system. The comparison between the discrimination provided at a white light illumination and the fluorescence imaging can be observed. The cancer lesion is usually visualized at the fluorescence image as a darker region, correlated with the less tissue fluorescence, mainly due to the changes on collagen structure induced by the carcinoma progression. The tumor margins can be better discriminate using the fluorescence, the contrast between lesion and normal tissue is

presented.

Garbe, et al. 2011, Breunig, et al. 2010).

image is presented at figure 4.

enhanced.

**5. Widefield fluorescence image in skin cancer diagnostics**

Fig. 4. Configuration of the fluorescence imaging device used.

Fig. 5. Basocellular carcinoma under white light illumination (left hand picture) and visualized using the widefield fluorescence modality (right hand picture).

Another important issue in skin diagnostics is the lesion discrimination between melanoma and seborrheic keratosis. At conventional white light illumination, without magnification, both lesions may appear similar. The fluorescence widefield imaging has the potential to overcome this problem. In figure 6 and 7, we present the examination of two patients, one with a seborrheic keratosis (fig.6) and another with melanoma. At white light illumination

Optical Imaging as Auxiliary Tool in Skin Cancer Diagnosis 169

Fig. 8. Basocellular carcinoma under white light illumination (upper left hand picture), visualized using the widefield fluorescence modality (upper right hand picture), and under

 Fig. 9. Nevi under white light (right hand picture) and the photodynamic diagnostic image

Photodynamic diagnostics does not show satisfactory results for hyperkeratinized lesions, since the keratin layer is a physical barrier for ALA penetration, and so the induction of PpIX production does occur properly. For these lesions, it is recommended a superficial

photodynamic diagnostic image using ALA (lower picture).

using ALA (left hand picture).

lesion curettage before ALA solution application.

the clinical features may appear similar for a not well trained physician, but at the fluorescence image the discrimination is evident due to the presence of the corneal pseudocists. All pigmented lesions show low fluorescence compared to the normal skin, and at seborrheic keratosis, the corneal pseudocists are evident due to its high fluorescence emission.

Fig. 6. Seborrheic keratosis under white light illumination (left hand picture) and at fluorescence imaging visualization (right hand picture).

Fig. 7. Melanoma under white light illumination (left hand picture) and at fluorescence imaging visualization (right hand picture).

To improve the contrast visualization of tumor imaging using fluorescence, a biomarker can be used, such as protoporphyrin IX (PpIX), which is a photosensitizer that occurs in cells, but at low levels. In the example showed at figure 8, the basocellular carcinoma lesion was pre-treated with a topical application of a 10% ALA solution. Sixty minutes after, a high concentration of PpIX production is observed at the lesion and not at the surrounding normal cells. This photodynamic optical diagnostics may be useful for the detection of occult lesions, but the efficacy of this indication needs still to be defined.

To show the potential use of the photodynamic diagnostics to aid in the differentiation of benign and malignant lesions, the same protocol was used for the investigation of a nevi. As it is showed at figure 9, no PpIX production is observed.

the clinical features may appear similar for a not well trained physician, but at the fluorescence image the discrimination is evident due to the presence of the corneal pseudocists. All pigmented lesions show low fluorescence compared to the normal skin, and at seborrheic keratosis, the corneal pseudocists are evident due to its high fluorescence

 Fig. 7. Melanoma under white light illumination (left hand picture) and at fluorescence

To improve the contrast visualization of tumor imaging using fluorescence, a biomarker can be used, such as protoporphyrin IX (PpIX), which is a photosensitizer that occurs in cells, but at low levels. In the example showed at figure 8, the basocellular carcinoma lesion was pre-treated with a topical application of a 10% ALA solution. Sixty minutes after, a high concentration of PpIX production is observed at the lesion and not at the surrounding normal cells. This photodynamic optical diagnostics may be useful for the detection of

To show the potential use of the photodynamic diagnostics to aid in the differentiation of benign and malignant lesions, the same protocol was used for the investigation of a nevi. As

occult lesions, but the efficacy of this indication needs still to be defined.

it is showed at figure 9, no PpIX production is observed.

Fig. 6. Seborrheic keratosis under white light illumination (left hand picture) and at

fluorescence imaging visualization (right hand picture).

imaging visualization (right hand picture).

emission.

Fig. 8. Basocellular carcinoma under white light illumination (upper left hand picture), visualized using the widefield fluorescence modality (upper right hand picture), and under photodynamic diagnostic image using ALA (lower picture).

Fig. 9. Nevi under white light (right hand picture) and the photodynamic diagnostic image using ALA (left hand picture).

Photodynamic diagnostics does not show satisfactory results for hyperkeratinized lesions, since the keratin layer is a physical barrier for ALA penetration, and so the induction of PpIX production does occur properly. For these lesions, it is recommended a superficial lesion curettage before ALA solution application.

Optical Imaging as Auxiliary Tool in Skin Cancer Diagnosis 171

Costa, M. M. ;, C. ; Kurachi, V. S. ; Bagnato, and L. Ventura. "Development of an Optical

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Hewett, J., V. Nadeau, J. Ferguson, H. Moseley, S. Ibbotson, L. Brancaleon, D. Birkin, W.

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#### **6. Conclusion**

Several optical modalities, including spectroscopy and imaging techniques, have been presented as potential tools to improve cancer diagnostics. Widefield fluorescence imaging is the most attractive technique for skin cancer screening. Tissue fluorescence has correlated information of biochemical and structural features, which can be used for discrimination of normal and tumor tissues. Some examples of fluorescence images obtained by a homemade device were presented, showing a better discrimination of basocellular carcinoma and melanoma lesions based on tissue autofluorescence. Clinical studies must be performed to determine the efficacy of the optical techniques for the skin cancer diagnostics, based on specificity and sensitivity rates. We believe that the optical technologies have great potential to become important auxiliary tools for clinical examination of skin.

#### **7. Acknowledgments**

The authors acknowledge the support provided by CNPq (INOF – INCT Program), FAPESP (CEPOF – CEPID Program and S.P. scholarship) and CAPES scholarship.

#### **8. References**

Several optical modalities, including spectroscopy and imaging techniques, have been presented as potential tools to improve cancer diagnostics. Widefield fluorescence imaging is the most attractive technique for skin cancer screening. Tissue fluorescence has correlated information of biochemical and structural features, which can be used for discrimination of normal and tumor tissues. Some examples of fluorescence images obtained by a homemade device were presented, showing a better discrimination of basocellular carcinoma and melanoma lesions based on tissue autofluorescence. Clinical studies must be performed to determine the efficacy of the optical techniques for the skin cancer diagnostics, based on specificity and sensitivity rates. We believe that the optical technologies have great potential

The authors acknowledge the support provided by CNPq (INOF – INCT Program), FAPESP

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Phototherapy of Neonatal Jaundice Does Not Increase the Risk for Melanocytic

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**6. Conclusion**

**7. Acknowledgments**

**8. References**

**Part 3**

**Prevention and Therapy for Skin Cancer**

## **Part 3**

### **Prevention and Therapy for Skin Cancer**

172 Skin Cancers – Risk Factors, Prevention and Therapy

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**8**

*Argentina*

**Cyclooxygenase-2**

**Overexpression in Non-Melanoma Skin**

**Targets for Prevention and Treatment**

*University of Buenos Aires, Pharmacy and Biochemistry School*

Daniel H. González Maglio, Mariela L. Paz,

Eliana M. Cela and Juliana Leoni

**Cancer: Molecular Pathways Involved as**

Non melanoma skin cancers (NMSCs) are the most frequent malignancies in humans (Geller & Annas, 2003). It comprises many different types of tumors, such as Squamous Cell Carcinoma (SCC) and Basal Cell Carcinoma (BCC), which are the main ones among the NMSCs. There are 100.000 new cases of these neoplasms per year in the UK (Aitken *et al.*, 2007) and about a millon in the US (Kuchide, 2003). NMSC incidence is estimated to increase in a 10% by 2060 -taking into account the Copenhagen Amendments scenario for protection

The development of both SCC and BCC implies a malignant transformation of keratinocytes, the most abundant cells in the epidermis. In addition to genetic factors, there are a variety of stimulus that can induce this transformation, like viral or bacterial infections and chemical agents (such as 7,12-dimethyl benzanthracene -DMBA- or Benzo[a]pyrene-7,8 diol-9,10-epoxide -B[a]PDE-), (Aubin *et al.*, 2003; Bouwes Bavinck *et al.*, 2001; Madan *et al.*, 2006). However, the most important carcinogen of the skin is the solar ultraviolet radiation (UVr), mainly the UVB component (280-310 nm), which is able to reach the earth surface

All of those carcinogens promote molecular and cellular changes, modifying different functions like cell cycle control or apoptotic cell death. In this way, the affected cells lose their capacity to regulate their proliferation properly and have the potential to generate an uncontrolled linage of cells, which may ultimately lead to tumor formation

There are many gene products that have been found to be involved in skin cancer development. Some of them are related to cell cycle -like cyclin D1- (Liang *et al.*, 2000), apoptosis -like Bax, Bcl-2, and others- or in both processes -like p53 and p21- (Coultas & Strasser, 2003; Murphy *et al.*, 2002). Additionally, skin UVB irradiation has been proven to induce an increment in the levels of a significant number of immune system mediators, such as tumor necrosis factor-α (TNF-α), interleukins 6 (IL-6), 10 (IL-10), 8 (IL-8), 4 (IL-4), prostaglandins (PGs), nitric oxide (NO) and vascular endothelial growth factor (VEGF),

**1. Introduction**

of ozone layer- (Slaper *et al.*, 1996).

(Armstrong & Kricker, 2001).

and growth.

### **Cyclooxygenase-2 Overexpression in Non-Melanoma Skin Cancer: Molecular Pathways Involved as Targets for Prevention and Treatment**

Daniel H. González Maglio, Mariela L. Paz, Eliana M. Cela and Juliana Leoni *University of Buenos Aires, Pharmacy and Biochemistry School Argentina*

#### **1. Introduction**

Non melanoma skin cancers (NMSCs) are the most frequent malignancies in humans (Geller & Annas, 2003). It comprises many different types of tumors, such as Squamous Cell Carcinoma (SCC) and Basal Cell Carcinoma (BCC), which are the main ones among the NMSCs. There are 100.000 new cases of these neoplasms per year in the UK (Aitken *et al.*, 2007) and about a millon in the US (Kuchide, 2003). NMSC incidence is estimated to increase in a 10% by 2060 -taking into account the Copenhagen Amendments scenario for protection of ozone layer- (Slaper *et al.*, 1996).

The development of both SCC and BCC implies a malignant transformation of keratinocytes, the most abundant cells in the epidermis. In addition to genetic factors, there are a variety of stimulus that can induce this transformation, like viral or bacterial infections and chemical agents (such as 7,12-dimethyl benzanthracene -DMBA- or Benzo[a]pyrene-7,8 diol-9,10-epoxide -B[a]PDE-), (Aubin *et al.*, 2003; Bouwes Bavinck *et al.*, 2001; Madan *et al.*, 2006). However, the most important carcinogen of the skin is the solar ultraviolet radiation (UVr), mainly the UVB component (280-310 nm), which is able to reach the earth surface (Armstrong & Kricker, 2001).

All of those carcinogens promote molecular and cellular changes, modifying different functions like cell cycle control or apoptotic cell death. In this way, the affected cells lose their capacity to regulate their proliferation properly and have the potential to generate an uncontrolled linage of cells, which may ultimately lead to tumor formation and growth.

There are many gene products that have been found to be involved in skin cancer development. Some of them are related to cell cycle -like cyclin D1- (Liang *et al.*, 2000), apoptosis -like Bax, Bcl-2, and others- or in both processes -like p53 and p21- (Coultas & Strasser, 2003; Murphy *et al.*, 2002). Additionally, skin UVB irradiation has been proven to induce an increment in the levels of a significant number of immune system mediators, such as tumor necrosis factor-α (TNF-α), interleukins 6 (IL-6), 10 (IL-10), 8 (IL-8), 4 (IL-4), prostaglandins (PGs), nitric oxide (NO) and vascular endothelial growth factor (VEGF),

Cyclooxygenase-2 Overexpression in Non-Melanoma Skin Cancer:

exhibiting similar structures in mouse, horse, cow and rat.

**2.2 Stimulus and mechanisms involved in COX-2 expression**

Rundhaug *et al.*, 2007), which are able to induce the expression of COX-2.

three subgroups: p38 MAPK, ERKs and JNK-.

the transcription of target genes, like COX-2.

section.

types.

Molecular Pathways Involved as Targets for Prevention and Treatment 177

In 1995 Inoue et al. analyzed the sequence of the human COX-2 5'-flanking region, describing several transcription regulatory elements (Figure 1), which include a TATA box, a CRE motif, an E-box, a NF-IL6 motif, three Sp1 sites, two AP-2 sites and two NF-κB sites (Inoue *et al.*, 1995). Many animal COX-2 5'-flanking sequences have been characterized,

All of these sequence elements have a potential role in the induction of the expression of COX-2. Some inducers and the molecular pathways involved in the activation of transcription factors, which finally lead to COX-2 expression, are described in the next

Prostaglandins and thromboxanes production are intimately related to inflammation, besides the fact that they produce a regulation of the overall inflammatory response (Ricciotti & FitzGerald, 2011). In this way, it is reasonable that many cytokines and molecules involved in the inflammatory process may play some role in the production of PGs and TXs. Certainly, TNF-α, IL-1 and Toll-like receptors agonists (like bacterial lipopolysaccharide -LPS-) can highly induce the expression of COX-2 gene in many cell

Besides the three mentioned molecules, there are other stimuli which are capable of inducing COX-2 expression. Other cytokines and different growth factors –like IL-6, EGF, FGF and PDGF- may lead to its expression (Tanabe & Tohnai, 2002). Keratinocytes, the most abundant cells in the skin, are a cell type that is particularly exposed to natural UVr. This stimulus is sufficient to induce the production of many inflammation-related molecules by the keratinocytes -such as TNF-α, IL-6 and COX-2 itself- among others (Paz *et al.*, 2008). Finally, there are many chemical agents used as tumor promoters (Lim *et al.*, 2011;

There are two main molecular pathways involved in COX-2 expression (Figures 2 and 3). The first one includes the activation of a transcription factor (NF-κB, nuclear factor kappa B) and the second one involves the mitogen-activated protein kinases (MAPKs) cascade -with

NF-κB is normally inhibited by IκB binding; this inhibitor can be phosphorylated and degraded in the proteasome, releasing the nuclear factor to translocate to the nucleus and to start its transcriptional effects. In this pathway the trigger stimuli is related to the interaction of membrane receptors with their corresponding agonists, like TNF-α receptor (TNFR), IL-1 receptor (IL-1R) and Toll-like receptor 4 (TLR-4). As it is shown in Figure 2 there are several molecules involved in the pathway, relating the membrane receptors with the phosphorylation of IκB. TNFR recruits TRADD (TNFR-associated death domain protein), which recruits TRAF-2 (TNFR-associated factor 2), leading to the final common path: phosphorylation of NIK (NF-κB-inducing kinase), IKK (IκB kinase complex) and the final phosphorylation (and consequent inactivation) of IκB. The other side of this pathway comprises IL-1R and TLR-4. Both molecules use MyD88 (Myeloid differentiation factor 88) as a linker for the recruitment of IRAK (IL-1R-activated kinase) and the consequent phosphorylation of TRAF-6 (TNFR-associated factor 6), which leads to the same final common path above mentioned. Once released, NF-κB translocates to the nucleus activating

among others (Paz *et al.*, 2008; Trompezinski *et al.*, 2001). Some of these molecules are known to mediate an important outcome of UVB irradiation: immunosuppression. It has been vastly proved since Margaret Kripke's first description (Fisher & Kripke, 1977) that UVr-induced DNA damage is not sufficient to lead to skin cancer development; there must be a concomitant decrease in normal immunosurvillance (Aubin, 2003; Ullrich & Kripke, 1984). This immunosuppression is mainly mediated by IL-4, IL-10 and prostaglandin-E2 (PGE2) (Shreedhar *et al.*, 1998).

PGs -including PGE2- and thromboxanes (TXs), are subproducts of key enzymes in the inflammatory response: cyclooxygenases (COXs). There are two isoforms described for COX: COX-1, which is constitutively expressed in many tissues and the inducible isoform COX-2, which is up-regulated under physiological and pathophysiological conditions.

The relationship between COX-2 over-expression and non-melanoma skin cancer development and progression has been very well documented and described. Along this chapter we will discuss different aspects concerning this relationship, including molecular pathways involved in COX-2 expression, cellular effects of COX-2 products and experimental treatments for non-melanoma skin cancer, which comprises the modulation of the COX-2 pathway.

#### **2. Molecular pathways involved in COX-2 expression**

COX-2 is a single copy gene, whose location was identified in chromosome 1 by a fluorescence in situ hybridization technique (locus 1q25.2-25.3) (Tay *et al.*, 1994). Its gene size is about 8 kb and it includes 10 exons with 9 introns, resulting in a primary mRNA of 4.5 kb. The expression of this gene, as it was mentioned before, is inducible. There are many factors which can contribute to the induction of COX-2 expression, including cytokines, growth factors, hormones and bacterial components.

#### **2.1 COX-2 gene structure**

COX-2 was first described in 1991 by Kujubu et al. (Kujubu *et al.*, 1991) as a primary response gene, which encodes an amino acid sequence sharing 61% of identity with the COX-1 gene, described in 1988 by Merlie et al. (Merlie *et al.*, 1988).

Fig. 1. Estructure of COX-2 gene regulatory region. Transcription regulatory elements are shown, their names (above) and upstream position (below with numbers) are indicated.

among others (Paz *et al.*, 2008; Trompezinski *et al.*, 2001). Some of these molecules are known to mediate an important outcome of UVB irradiation: immunosuppression. It has been vastly proved since Margaret Kripke's first description (Fisher & Kripke, 1977) that UVr-induced DNA damage is not sufficient to lead to skin cancer development; there must be a concomitant decrease in normal immunosurvillance (Aubin, 2003; Ullrich & Kripke, 1984). This immunosuppression is mainly mediated by IL-4, IL-10 and prostaglandin-E2

PGs -including PGE2- and thromboxanes (TXs), are subproducts of key enzymes in the inflammatory response: cyclooxygenases (COXs). There are two isoforms described for COX: COX-1, which is constitutively expressed in many tissues and the inducible isoform COX-2, which is up-regulated under physiological and pathophysiological conditions. The relationship between COX-2 over-expression and non-melanoma skin cancer development and progression has been very well documented and described. Along this chapter we will discuss different aspects concerning this relationship, including molecular pathways involved in COX-2 expression, cellular effects of COX-2 products and experimental treatments for non-melanoma skin cancer, which comprises the modulation of

COX-2 is a single copy gene, whose location was identified in chromosome 1 by a fluorescence in situ hybridization technique (locus 1q25.2-25.3) (Tay *et al.*, 1994). Its gene size is about 8 kb and it includes 10 exons with 9 introns, resulting in a primary mRNA of 4.5 kb. The expression of this gene, as it was mentioned before, is inducible. There are many factors which can contribute to the induction of COX-2 expression, including cytokines, growth

COX-2 was first described in 1991 by Kujubu et al. (Kujubu *et al.*, 1991) as a primary response gene, which encodes an amino acid sequence sharing 61% of identity with the

Fig. 1. Estructure of COX-2 gene regulatory region. Transcription regulatory elements are shown, their names (above) and upstream position (below with numbers) are

**2. Molecular pathways involved in COX-2 expression**

COX-1 gene, described in 1988 by Merlie et al. (Merlie *et al.*, 1988).

factors, hormones and bacterial components.

(PGE2) (Shreedhar *et al.*, 1998).

the COX-2 pathway.

**2.1 COX-2 gene structure**

indicated.

In 1995 Inoue et al. analyzed the sequence of the human COX-2 5'-flanking region, describing several transcription regulatory elements (Figure 1), which include a TATA box, a CRE motif, an E-box, a NF-IL6 motif, three Sp1 sites, two AP-2 sites and two NF-κB sites (Inoue *et al.*, 1995). Many animal COX-2 5'-flanking sequences have been characterized, exhibiting similar structures in mouse, horse, cow and rat.

All of these sequence elements have a potential role in the induction of the expression of COX-2. Some inducers and the molecular pathways involved in the activation of transcription factors, which finally lead to COX-2 expression, are described in the next section.

#### **2.2 Stimulus and mechanisms involved in COX-2 expression**

Prostaglandins and thromboxanes production are intimately related to inflammation, besides the fact that they produce a regulation of the overall inflammatory response (Ricciotti & FitzGerald, 2011). In this way, it is reasonable that many cytokines and molecules involved in the inflammatory process may play some role in the production of PGs and TXs. Certainly, TNF-α, IL-1 and Toll-like receptors agonists (like bacterial lipopolysaccharide -LPS-) can highly induce the expression of COX-2 gene in many cell types.

Besides the three mentioned molecules, there are other stimuli which are capable of inducing COX-2 expression. Other cytokines and different growth factors –like IL-6, EGF, FGF and PDGF- may lead to its expression (Tanabe & Tohnai, 2002). Keratinocytes, the most abundant cells in the skin, are a cell type that is particularly exposed to natural UVr. This stimulus is sufficient to induce the production of many inflammation-related molecules by the keratinocytes -such as TNF-α, IL-6 and COX-2 itself- among others (Paz *et al.*, 2008). Finally, there are many chemical agents used as tumor promoters (Lim *et al.*, 2011; Rundhaug *et al.*, 2007), which are able to induce the expression of COX-2.

There are two main molecular pathways involved in COX-2 expression (Figures 2 and 3). The first one includes the activation of a transcription factor (NF-κB, nuclear factor kappa B) and the second one involves the mitogen-activated protein kinases (MAPKs) cascade -with three subgroups: p38 MAPK, ERKs and JNK-.

NF-κB is normally inhibited by IκB binding; this inhibitor can be phosphorylated and degraded in the proteasome, releasing the nuclear factor to translocate to the nucleus and to start its transcriptional effects. In this pathway the trigger stimuli is related to the interaction of membrane receptors with their corresponding agonists, like TNF-α receptor (TNFR), IL-1 receptor (IL-1R) and Toll-like receptor 4 (TLR-4). As it is shown in Figure 2 there are several molecules involved in the pathway, relating the membrane receptors with the phosphorylation of IκB. TNFR recruits TRADD (TNFR-associated death domain protein), which recruits TRAF-2 (TNFR-associated factor 2), leading to the final common path: phosphorylation of NIK (NF-κB-inducing kinase), IKK (IκB kinase complex) and the final phosphorylation (and consequent inactivation) of IκB. The other side of this pathway comprises IL-1R and TLR-4. Both molecules use MyD88 (Myeloid differentiation factor 88) as a linker for the recruitment of IRAK (IL-1R-activated kinase) and the consequent phosphorylation of TRAF-6 (TNFR-associated factor 6), which leads to the same final common path above mentioned. Once released, NF-κB translocates to the nucleus activating the transcription of target genes, like COX-2.

Cyclooxygenase-2 Overexpression in Non-Melanoma Skin Cancer:

(Chun & Surh, 2004).

Molecular Pathways Involved as Targets for Prevention and Treatment 179

MEK-1/2 by Raf-1 (which in turn is activated by Ras oncogene) and MKK-3/6 by RIP. Besides these kinases, a family of proteins named SFKs -Src family kinases, including Fyn kinase- can directly activate MAPK kinases. Considering the beginning of the pathway, MAPKK kinases are activated by STE20 kinases or by GTP-binding proteins (Chang & Karin, 2001). Finally, at the end of the pathway MAPK activates transcription factors such as AP-1 (homodimers or heterodimers conformed by Jun, Fos, JDP and ATF families) leading to an increment in COX-2 expression. Another activated transcription factors can be C/EBPs, which play different roles in COX-2 expression, according to the cell type involved

Fig. 3. Mitogen-activated protein kinases –MAPKs- molecular pathway. Membrane receptors which receive the trigger stimuli are shown, also the different downstream molecules involved and their crosslinks. This cascade includes kinases which phosphorylate

and activate the next kinase. The common final path through the activation of the transcription factor AP-1, that finally leads to COX-2 expression, can also be seen.

phosphorylating IκB, releasing in this way an active NF-κB (Chun & Surh, 2004).

In addition to the described mechanisms, there is an intense crosstalk between NF-κB and MAPKs pathways (Figure 4). For example, MEKK 1 and RIP are activated by TRAF-2 (Wu & Zhou, 2010) and MEKK 1 and JNK can be activated in LPS-treated macrophages through and adapter protein, named ECSIT (Kopp *et al.*, 1999). Besides, MEKK 1 is capable of

Fig. 2. Nuclear factor kappa B -NF-κB- molecular pathway. Membrane receptors with their corresponding agonists as trigger stimuli (grey arrows) are shown, also the different downstream molecules involved. The common final path, that leads to NF-κB activation and subsequent COX-2 expression, can also be seen. P: phosphorylated molecule.

MAPKs pathway includes a cascade of kinases with specific activity, each of them phosphorylates (and activates) the next kinase in the path, ultimately leading to the activation of transcription factors which promote the transcription of target genes. This pathway is one of the most significant in the activation of COX-2 expression and prostaglandins biosynthesis. JNK (c-Jun N-terminal kinase) and p38 MAPK share some of the activation stimuli -like proinflammatory cytokines and bacterial endotoxins- also environmental stressors, such as heat shock, oxidative stress and ultraviolet light (highly relevant in the development of skin cancer). On the other hand, ERKs (extracellularregulated kinases) are mainly activated by growth factors and oncogenes, like Ras. As it is shown in Figure 3, the three MAPKs have individual upstream kinases involved in their activation (MAPK kinase). MKK-4/7 activate JNK, while MEK-1/2 activate ERKs and MKK-3/6 activate p38. MKKs and MEKs are known as MAPK kinases, and all of them are under the control of an upstream kinase (generally named as MAPKK kinase). MAPKK kinases are not specific, and in this step the pathways begin to crosslink. We describe only a few examples of these MAPKK kinases: the phosphorylation of MKK-4/7 by MEKK 1,

Fig. 2. Nuclear factor kappa B -NF-κB- molecular pathway. Membrane receptors with their corresponding agonists as trigger stimuli (grey arrows) are shown, also the different

downstream molecules involved. The common final path, that leads to NF-κB activation and

MAPKs pathway includes a cascade of kinases with specific activity, each of them phosphorylates (and activates) the next kinase in the path, ultimately leading to the activation of transcription factors which promote the transcription of target genes. This pathway is one of the most significant in the activation of COX-2 expression and prostaglandins biosynthesis. JNK (c-Jun N-terminal kinase) and p38 MAPK share some of the activation stimuli -like proinflammatory cytokines and bacterial endotoxins- also environmental stressors, such as heat shock, oxidative stress and ultraviolet light (highly relevant in the development of skin cancer). On the other hand, ERKs (extracellularregulated kinases) are mainly activated by growth factors and oncogenes, like Ras. As it is shown in Figure 3, the three MAPKs have individual upstream kinases involved in their activation (MAPK kinase). MKK-4/7 activate JNK, while MEK-1/2 activate ERKs and MKK-3/6 activate p38. MKKs and MEKs are known as MAPK kinases, and all of them are under the control of an upstream kinase (generally named as MAPKK kinase). MAPKK kinases are not specific, and in this step the pathways begin to crosslink. We describe only a few examples of these MAPKK kinases: the phosphorylation of MKK-4/7 by MEKK 1,

subsequent COX-2 expression, can also be seen. P: phosphorylated molecule.

MEK-1/2 by Raf-1 (which in turn is activated by Ras oncogene) and MKK-3/6 by RIP. Besides these kinases, a family of proteins named SFKs -Src family kinases, including Fyn kinase- can directly activate MAPK kinases. Considering the beginning of the pathway, MAPKK kinases are activated by STE20 kinases or by GTP-binding proteins (Chang & Karin, 2001). Finally, at the end of the pathway MAPK activates transcription factors such as AP-1 (homodimers or heterodimers conformed by Jun, Fos, JDP and ATF families) leading to an increment in COX-2 expression. Another activated transcription factors can be C/EBPs, which play different roles in COX-2 expression, according to the cell type involved (Chun & Surh, 2004).

Fig. 3. Mitogen-activated protein kinases –MAPKs- molecular pathway. Membrane receptors which receive the trigger stimuli are shown, also the different downstream molecules involved and their crosslinks. This cascade includes kinases which phosphorylate and activate the next kinase. The common final path through the activation of the transcription factor AP-1, that finally leads to COX-2 expression, can also be seen.

In addition to the described mechanisms, there is an intense crosstalk between NF-κB and MAPKs pathways (Figure 4). For example, MEKK 1 and RIP are activated by TRAF-2 (Wu & Zhou, 2010) and MEKK 1 and JNK can be activated in LPS-treated macrophages through and adapter protein, named ECSIT (Kopp *et al.*, 1999). Besides, MEKK 1 is capable of phosphorylating IκB, releasing in this way an active NF-κB (Chun & Surh, 2004).

Cyclooxygenase-2 Overexpression in Non-Melanoma Skin Cancer:

way (Ricciotti & FitzGerald, 2011).

shown.

Molecular Pathways Involved as Targets for Prevention and Treatment 181

All of these AA-derived molecules, which play different roles in many physiological and pathophysiological mechanisms, have a short half-life *in vivo* (from seconds to minutes), performing its actions in an autocrine and paracrine fashion, rather than in an endocrine

Fig. 5. Prostaglandins synthesis. a) Conversion of arachidonic acid –AA- into prostaglandin H2 -PGH2-, by the two enzymatic activities of COX-2. b) Conversion of PGH2 into different final products, by specific synthases. The chemical structures and names of each product are

Prostaglandins, particularly PGE2, have been related to cell growth in some cell types. The role of PGE2 has been widely described in keratinocytes, as a cell proliferation inducer. Indeed, it was described at the end of the 1970 decade, that there was an increase in the production of PGE2 in a mouse model of epidermal hyperproliferation -induced by 12-Otetradecanoylphorbol-13-acetate (TPA)- (Ashendel & Boutwell, 1979). In this TPA-induced proliferation model, keratinocyte growth was effectively blocked using the Non-Steroidal Anti-Inflammatory Drug indomethacin and successfully restored with an external supplementation of PGE2 (Furstenberger *et al.*, 1979; Furstenberger & Marks, 1978). A few years later, Pentland described not only the capacity of keratinocytes to produce their own PGs from an external source of AA, but also the autocrine effects of these PGs on the cellular

**3.1 Effects of PGE2 on cell cycle and proliferation**

growth rate (Pentland & Needleman, 1986).

Finally, it is worth to notice that there are other pathways involved in the activation of NF-κB or MAPKs (Figure 4). For example, Phosphoinositol triphosphate (PIP3, produced by active PI3K) activates Akt protein, which is able to phosphorilate IκB, therefore releasing active NF-κB (Ouyang *et al.*, 2006).

Fig. 4. Crosstalk between MAPKs, PIP3 and NF-κB pathways. Membrane receptors, the different downstream molecules involved and their crosslinks are shown. The final path, which leads to COX-2 transcription and expression, can also be seen. P: phosphorylated molecule.

#### **3. Molecular and cellular effects of COX-2 products**

COXs -also known as PGH synthases- are the enzymes responsible for the conversion of arachidonic acid (AA) into PGH2. AA is released from the cell membrane phospholipids by Phospholipase-A2. AA conversion chemical reaction is catalyzed by the two enzymatic activities of COXs: cyclooxigenase (or bis-dioxygenase) activity generates a cyclopentane hydroperoxy endoperoxide (PGG2) from AA. Peroxidase activity reduces the hydroperoxy group yielding the final product PGH2 (Figure 5.a). PGH2 by turns can be transform to different products (PGI2 or prostacyclin, PGD2, PGF2a, PGE2 and TXA2) by specific synthases (Figure 5.b).

Finally, it is worth to notice that there are other pathways involved in the activation of NF-κB or MAPKs (Figure 4). For example, Phosphoinositol triphosphate (PIP3, produced by active PI3K) activates Akt protein, which is able to phosphorilate IκB, therefore releasing

Fig. 4. Crosstalk between MAPKs, PIP3 and NF-κB pathways. Membrane receptors, the different downstream molecules involved and their crosslinks are shown. The final path, which leads to COX-2 transcription and expression, can also be seen. P: phosphorylated

COXs -also known as PGH synthases- are the enzymes responsible for the conversion of arachidonic acid (AA) into PGH2. AA is released from the cell membrane phospholipids by Phospholipase-A2. AA conversion chemical reaction is catalyzed by the two enzymatic activities of COXs: cyclooxigenase (or bis-dioxygenase) activity generates a cyclopentane hydroperoxy endoperoxide (PGG2) from AA. Peroxidase activity reduces the hydroperoxy group yielding the final product PGH2 (Figure 5.a). PGH2 by turns can be transform to different products (PGI2 or prostacyclin, PGD2, PGF2a, PGE2 and TXA2) by specific synthases

**3. Molecular and cellular effects of COX-2 products**

active NF-κB (Ouyang *et al.*, 2006).

molecule.

(Figure 5.b).

All of these AA-derived molecules, which play different roles in many physiological and pathophysiological mechanisms, have a short half-life *in vivo* (from seconds to minutes), performing its actions in an autocrine and paracrine fashion, rather than in an endocrine way (Ricciotti & FitzGerald, 2011).

Fig. 5. Prostaglandins synthesis. a) Conversion of arachidonic acid –AA- into prostaglandin H2 -PGH2-, by the two enzymatic activities of COX-2. b) Conversion of PGH2 into different final products, by specific synthases. The chemical structures and names of each product are shown.

#### **3.1 Effects of PGE2 on cell cycle and proliferation**

Prostaglandins, particularly PGE2, have been related to cell growth in some cell types. The role of PGE2 has been widely described in keratinocytes, as a cell proliferation inducer. Indeed, it was described at the end of the 1970 decade, that there was an increase in the production of PGE2 in a mouse model of epidermal hyperproliferation -induced by 12-Otetradecanoylphorbol-13-acetate (TPA)- (Ashendel & Boutwell, 1979). In this TPA-induced proliferation model, keratinocyte growth was effectively blocked using the Non-Steroidal Anti-Inflammatory Drug indomethacin and successfully restored with an external supplementation of PGE2 (Furstenberger *et al.*, 1979; Furstenberger & Marks, 1978). A few years later, Pentland described not only the capacity of keratinocytes to produce their own PGs from an external source of AA, but also the autocrine effects of these PGs on the cellular growth rate (Pentland & Needleman, 1986).

Cyclooxygenase-2 Overexpression in Non-Melanoma Skin Cancer:

cAMP/CREB pathway (Ansari *et al.*, 2007).

synthesizes more PGE2.

Molecular Pathways Involved as Targets for Prevention and Treatment 183

unknown. Nowadays, all of the Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are well-known inhibitors of COXs enzymes. There are different groups of NSAIDs each of them with diverse mechanisms of action and also selectivity for the two isoforms of the enzyme. As it was stated before, PGs and COXs implication in the inflammatory response have been described many decades ago. However, the positive feedback loop that involves these molecules (Figure 6) should be highlighted, since PGE2 (a sub-product of the enzyme) is able to regulate both COX isoforms expression, as it has been proved in keratinocytes. Amazingly, not only COX-2 expression was increased but also was COX-1 (Maldve *et al.*, 2000). The mechanism underlying this feedback involves EP2 receptor and the activation of

Fig. 6. COX-2 expression positive feedback loop. PGE2, through its binding to EP2 receptor,

Besides their direct effects on inflammation, PGs play key roles in the development of the immune response, being capable of modifying dendritic cells and T helper (Th) cells responses. During the activation of naive Th cells by dendritic cells, or other antigen presenting cells, they can differentiate into different phenotypes: Th1 cells (characterized by IFN-γ production), Th2 cells (characterized by IL-4 production), Th17 cells (characterized by IL-17 production) or Treg cells (characterized by IL-10 production). PGE2

activates the cAMP/CREB pathway that finally leads to COX-2 expression, which

Interestingly, the final effect of PGE2 in a specific cell type depends on the kind of receptor expressed in the target cell. There are four types of PGE2 receptors, identified as E Prostanoid receptors 1 to 4 (EP1-EP4) (Narumiya *et al.*, 1999; Negishi *et al.*, 1993). These receptors are G-protein-coupled receptors which can activate different molecular pathways. EP1 acts through the activation of Phospholipase C-β leading to an increase of intracellular calcium, while the other three receptors regulate adenylate cyclase activity. Meanwhile EP3 is able to down-regulate its activity, EP2 and EP4 can up-regulate it (Negishi *et al.*, 1995). Other PGs also exert their effects through G-protein-coupled receptor, according to Table 1.

Table 1. Molecular effects of prostaglandins –PGs-. The type of PG, subtype of prostanoid receptor involved, G-protein-coupled receptor activated and final molecular effect are shown. ↑: increase, ↓: decrease, ↑ ↓: both effects are possible.

Recently, in a study performed using primary mouse keratinocytes, Ansari et al. demonstrated that PGE2 is capable of inducing an indirect phosphorilation of epidermal growth factor receptor (EGFR), leading to the activation of Ras/MEK/ERK/AP-1 and PI3-K/Akt/NF-κB pathways; besides the direct activation of Adenylate cyclase/PKA/CREB through EP receptor. Transcription factors CREB, AP-1 and NF-κB lead to its proliferative effects through the activation of the expression of cyclin D1 and VEGF (Ansari *et al.*, 2008).

#### **3.2 Prostaglandins effects on inflammation and immune system**

The involvement of PGs in the inflammatory response has been largely known. The typical signs of inflammation -heat, redness, swelling and pain- result from PGs direct effects. Indeed, redness and edema are a consequence of an increased vasodilatation and vascular endothelium permeability, mediated by the interaction of PGI2 and PGE2 with vascular smooth muscle cells, respectively (Funk, 2001; Yamamoto *et al.*, 1999). Besides vascular modifications, inflammation-related pain depends on PGE2 stimulation of peripheral sensory neurons (Ricciotti & FitzGerald, 2011). Additionally, fever caused in many inflammatory processes is also due to the interaction of PGE2 with the nervous system, in this case with neurons of the organum vasculosa lamina terminalis at the midline of the preoptic area (Funk, 2001). In order to relieve one or all of these effects, inhibitors of PGs synthesis have been used as anti-inflammatory drugs for more than a century (acetylsalicylic acid was released in 1899 by Bayer), even when their molecular target was

Interestingly, the final effect of PGE2 in a specific cell type depends on the kind of receptor expressed in the target cell. There are four types of PGE2 receptors, identified as E Prostanoid receptors 1 to 4 (EP1-EP4) (Narumiya *et al.*, 1999; Negishi *et al.*, 1993). These receptors are G-protein-coupled receptors which can activate different molecular pathways. EP1 acts through the activation of Phospholipase C-β leading to an increase of intracellular calcium, while the other three receptors regulate adenylate cyclase activity. Meanwhile EP3 is able to down-regulate its activity, EP2 and EP4 can up-regulate it (Negishi *et al.*, 1995). Other PGs also exert their effects through G-protein-coupled receptor, according to Table 1.

PGE2 EP1 Gq ↑IP3 EP2 Gs ↑ cAMP EP3 Gi, G12, GRho ↓ cAMP, ↑ Ca2+ EP4 Gs ↑ cAMP PGD2 DP Gs ↑ cAMP CRTH2 Gi ↓ cAMP, ↑ Ca2+

PGF2α FPA, FPB Gq, GRho ↑IP3

shown. ↑: increase, ↓: decrease, ↑ ↓: both effects are possible.

**3.2 Prostaglandins effects on inflammation and immune system**

PGI2 IP-IP Gs, Gq, Gi ↑ ↓ cAMP, ↑IP3 IP-TPα Gs ↑ cAMP

Table 1. Molecular effects of prostaglandins –PGs-. The type of PG, subtype of prostanoid receptor involved, G-protein-coupled receptor activated and final molecular effect are

Recently, in a study performed using primary mouse keratinocytes, Ansari et al. demonstrated that PGE2 is capable of inducing an indirect phosphorilation of epidermal growth factor receptor (EGFR), leading to the activation of Ras/MEK/ERK/AP-1 and PI3-K/Akt/NF-κB pathways; besides the direct activation of Adenylate cyclase/PKA/CREB through EP receptor. Transcription factors CREB, AP-1 and NF-κB lead to its proliferative effects through the activation of the expression of cyclin D1 and VEGF (Ansari *et al.*, 2008).