**1. Introduction**

In the modern society, we are surrounded by an amazing variety of materials, such as polymers, ceramics, composites, high strength alloys, superalloys, and so on. Naturally, existing materials possess some properties. However, one can change or modify the naturally occurring properties by altering the microstructure of the materials. The resultant materials are also called as the engineered materials. Recently, the engineered materials have gained much attention by researchers. The engineered materials exhibit better performance, such as large cyclic life, broad temperature ranges, lighter in weight, etc., than conventional materials. There exist numerous engineered materials such as metal-matrix composites, polymer-based composites, piezoelectric materials, magnetostrictive materials, metamaterials, etc. Owing to their large lifetime and high performance, these materials are significantly used almost in all fields of science and engineering. In this article, we have focused on metamaterials and their applications.


**4.2.2 How to calculate the blood glucose value from the spectrum**

We calculated the blood glucose value by using absorption spectra and PLSR. The invasion type blood glucose sensor (Antsense-2, DAIKIN) by enzyme electrode method is used for the measuring the reference blood glucose value.

First, we developed the calibration curve by PLSR, the measuring absorption spectra were used as the explanatory variable, measuring the blood glucose values by Antsense-2 were used as the objective variable. In calibration curve, the horizontal axis is the Antsense blood glucose value, and the vertical axis is the estimated blood glucose value by PLSR. Next, we measured to the new absorption spectra to measure the blood glucose value. Then, the blood glucose was calculated by using the measured absorption spectrum and calibration curve. And, we calculated the SEC and the SEP. Their values were used as the evaluated the accuracy of the calibration curve.

#### **4.2.3 Error Grid Analysis (EGA)**

EGA was developed by William L. Clarke in the University of Virginia. EGA is an indicator of clinical efficacy of blood glucose sensor. In fact, an error grid has been assembled to stay in the ideal range of 70 ~ 180 mg/dl blood glucose value.

The Clarke grid of EGA is shown in Fig.4. The horizontal axis is the actual blood glucose, and the vertical axis is the blood glucose value obtained from the developed blood glucose sensors. In this study, when measuring the blood glucose must be clinically effective. We judged the efficacy as a blood glucose sensor using the EGA.

#### **4.3 Absorption spectra of glucose, squalene oil, and finger**

In measuring the blood glucose value, must know the absorption peak of glucose spectrum.

In addition, we need to know the absorption wavelengths of squalene oil to be used as an internal standard method. Each sample was measured by ATR method (plate type prism). The absorption spectra of glucose powder and squalene oil are shown in Fig.5. The absorption spectrum of glucose has some absorption peaks in 1030 cm-1 (Fig.5-A, C-OH stretching vibration), 1130cm-1 (Fig.5-B, C-O-C antisymmetric stretching vibration), and 1450 cm-1 (Fig.5-C, CH2 scissoring vibration). The absorption spectrum of squalene oil has some

Fig. 4. Clarke grid

In measuring the subject's blood glucose value, the good accuracy calibration curve is essential. To improve the accuracy of the calibration curve must be accurately extract

In the ATR method, it is important to stick a sample to the prism. Many people of diabetes are elderly, person with dry skin on the fingertips are also often elderly. Squalene oil is used as internal standard method for the different dry skin of the subject's finger surface. To eliminate the effects of dry skin by applying the squalene oil, we can measure the subjects under the same conditions. And, there is no effect of squalene oil to apply normalization correction in the absorption peak of squalene oil. Furthermore, the S/N ratio of the spectrum is better, because to block the air from between the finger and prism by the oil.

We calculated the blood glucose value by using absorption spectra and PLSR. The invasion type blood glucose sensor (Antsense-2, DAIKIN) by enzyme electrode method is used for

First, we developed the calibration curve by PLSR, the measuring absorption spectra were used as the explanatory variable, measuring the blood glucose values by Antsense-2 were used as the objective variable. In calibration curve, the horizontal axis is the Antsense blood glucose value, and the vertical axis is the estimated blood glucose value by PLSR. Next, we measured to the new absorption spectra to measure the blood glucose value. Then, the blood glucose was calculated by using the measured absorption spectrum and calibration curve. And, we calculated the SEC and the SEP. Their values were used as the evaluated the

EGA was developed by William L. Clarke in the University of Virginia. EGA is an indicator of clinical efficacy of blood glucose sensor. In fact, an error grid has been assembled to stay

The Clarke grid of EGA is shown in Fig.4. The horizontal axis is the actual blood glucose, and the vertical axis is the blood glucose value obtained from the developed blood glucose sensors. In this study, when measuring the blood glucose must be clinically effective. We

In measuring the blood glucose value, must know the absorption peak of glucose spectrum. In addition, we need to know the absorption wavelengths of squalene oil to be used as an internal standard method. Each sample was measured by ATR method (plate type prism). The absorption spectra of glucose powder and squalene oil are shown in Fig.5. The absorption spectrum of glucose has some absorption peaks in 1030 cm-1 (Fig.5-A, C-OH stretching vibration), 1130cm-1 (Fig.5-B, C-O-C antisymmetric stretching vibration), and 1450 cm-1 (Fig.5-C, CH2 scissoring vibration). The absorption spectrum of squalene oil has some

**4.2.1 Internal standard method by squalene oil**

the measuring the reference blood glucose value.

in the ideal range of 70 ~ 180 mg/dl blood glucose value.

judged the efficacy as a blood glucose sensor using the EGA.

**4.3 Absorption spectra of glucose, squalene oil, and finger**

accuracy of the calibration curve.

**4.2.3 Error Grid Analysis (EGA)**

glucose information on the infrared absorption spectrum.

**4.2.2 How to calculate the blood glucose value from the spectrum**

Fig. 5. The absorption spectra of glucose powder and squalene oil

absorption peaks in 1377 cm-1 (Fig.5-D), 1462cm-1 (Fig.5-E), and 2922 cm-1 (Fig.5-F). The absorption peak of glucose and oil were not overlap.

The absorption spectra of the finger in before and after coating to squalene oil are shown in Fig.6. In absorption spectrum of finger, absorption peaks in 1377 cm-1 and 1462 cm-1 did not appear. But, absorption spectrum of finger coated with squalene oil has absorption peaks in 1377 cm-1 (Fig.6-D) and 1462 cm-1 (Fig.6-E). Therefore, it is possible to reduce individual differences in skin surface conditions due to the absorption spectra normalized at the absorption peak of squalene oil. We confirmed that the squalene oil is suitable as an internal standard method, and can measure the blood glucose value by absorption peaks of glucose.

#### **4.4 Non-invasive blood glucose measurement in subjects**

#### **4.4.1 Development of calibration curve**

The subjects were healthy eight men in their 20s. When developing a calibration curve, glucose tolerance test were performed on each subjects. In glucose tolerance test, first, the

Introduction of Non-Invasive Measurement Method by Infrared Application 87

E zone

(CC : Correlation coeficience, SEC : mg/dl)

All 0.79 21 77% 22% 0% 1% 0% #1 0.90 13 90% 7% 0% 3% 0% #2 0.95 16 90% 10% 0% 0% 0% #3 0.71 22 68% 32% 0% 0% 0% #4 0.71 14 83% 17% 0% 0% 0% #5 0.90 7 100% 0% 0% 0% 0% #6 0.84 16 90% 10% 0% 0% 0% #7 0.67 28 60% 35% 0% 5% 0% #8 0.96 9 95% 5% 0% 0% 0%

measurement accuracy of 6 among 8 subjects. In addition, the EGA results have improved in the same 6 subjects. The individual differences are eliminated by the individual calibration curve, and may be considered to be developed the most suitable calibration curve of each subject. Therefore, we measure blood glucose value of subjects by using the all subject

Each subject was measured again 20 times the infrared absorption spectra. We measured the blood glucose value by these absorption spectra and developing calibration curve in section 4.4.1. The result by calibration curve in all subjects and individual calibration curve are

In the result of blood glucose value calculated by all subjects calibration curve in Fig.8-(A), the SEP was ±32 mg/dl. This result was not good. On the other hand, in the result of blood glucose value calculated by individual calibration curve in Fig.8-(B) the SEP was ±13 mg/dl,

(A) The result by all subjects calibration curve (B) The result by #8 calibration curve

0

0 50 100 150 200 250

C

B

E B A

C

E

D

A

Reference value (mg/dl)

50

100

D

150

Estimated value (mg/dl)

200

250

Fig. 8. The result by developed calibration curve in all subjects and individual

E

D

A

0 50 100 150 200 250

C

B

E B A

C

Reference value (mg/dl)

Table 1. The results of all subjects and individual calibration curves by PLSR

**4.4.2 Non-invasive blood glucose measurement by calibration curve**

shown in Fig.8. The average blood glucose value was 100 mg/dl.

calibration curve and individual calibration curve.

0

50

100

D

150

Estimated value (mg/dl)

200

250

CC SEC A zone B zone C zone D zone

Fig. 6. The absorption spectra of the finger in before and after coating to squalene oil

subjects fasted for 12 hours. Then, subjects are ingested 75 g glucose, temporary increases in blood glucose value of subject. After the glucose tolerance test, we measured to the infrared absorption spectra and the invasive blood glucose measurement in every three minutes 20 times. Furthermore, after breakfast, before and after lunch, before and after dinner, we measured for four days (five times measurement in a day). We measured the total 40 times in one subject. The calibration curve is developed using these infrared absorption spectra.

Fig.7 shows the calibration curve developed in 320 infrared absorption spectra by all 8 subjects. In these results, the average blood glucose of the subjects was 120 mg/dl, correlation coefficient (CC) was 0.79, and measurement accuracy (SEC) was ± 21 mg / dl. This result in this sample scale has been obtained a significant correlation, but, this result was not satisfactory as a blood glucose measurement system. As the cause of this, it is considered to the individual differences by moisture content of each subject. Therefore, we developed each individual calibration curve using the infrared absorption spectrum of each subjects. The results of individual calibration curves are shown in Table 1. Comparing the individual calibration curves and all subjects calibration curve has improved the

Fig. 7. The calibration curve developed by all subjects

E

Uncorting oil

Coating oil D

Fig. 6. The absorption spectra of the finger in before and after coating to squalene oil

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

0

1

Absorbance

2

Fig. 7. The calibration curve developed by all subjects

0

50

100

D

150

Estimated value (mg/dl)

200

250

subjects fasted for 12 hours. Then, subjects are ingested 75 g glucose, temporary increases in blood glucose value of subject. After the glucose tolerance test, we measured to the infrared absorption spectra and the invasive blood glucose measurement in every three minutes 20 times. Furthermore, after breakfast, before and after lunch, before and after dinner, we measured for four days (five times measurement in a day). We measured the total 40 times in one subject. The calibration curve is developed using these infrared absorption spectra. Fig.7 shows the calibration curve developed in 320 infrared absorption spectra by all 8 subjects. In these results, the average blood glucose of the subjects was 120 mg/dl, correlation coefficient (CC) was 0.79, and measurement accuracy (SEC) was ± 21 mg / dl. This result in this sample scale has been obtained a significant correlation, but, this result was not satisfactory as a blood glucose measurement system. As the cause of this, it is considered to the individual differences by moisture content of each subject. Therefore, we developed each individual calibration curve using the infrared absorption spectrum of each subjects. The results of individual calibration curves are shown in Table 1. Comparing the individual calibration curves and all subjects calibration curve has improved the

0 50 100 150 200 250

C

B

E B A

C

E

D

A

Reference value (mg/dl)

(CC : Correlation coeficience, SEC : mg/dl)

Table 1. The results of all subjects and individual calibration curves by PLSR

measurement accuracy of 6 among 8 subjects. In addition, the EGA results have improved in the same 6 subjects. The individual differences are eliminated by the individual calibration curve, and may be considered to be developed the most suitable calibration curve of each subject. Therefore, we measure blood glucose value of subjects by using the all subject calibration curve and individual calibration curve.

#### **4.4.2 Non-invasive blood glucose measurement by calibration curve**

Each subject was measured again 20 times the infrared absorption spectra. We measured the blood glucose value by these absorption spectra and developing calibration curve in section 4.4.1. The result by calibration curve in all subjects and individual calibration curve are shown in Fig.8. The average blood glucose value was 100 mg/dl.

In the result of blood glucose value calculated by all subjects calibration curve in Fig.8-(A), the SEP was ±32 mg/dl. This result was not good. On the other hand, in the result of blood glucose value calculated by individual calibration curve in Fig.8-(B) the SEP was ±13 mg/dl,

Fig. 8. The result by developed calibration curve in all subjects and individual

Introduction of Non-Invasive Measurement Method by Infrared Application 89

matched by changes in infrared absorption spectra of blood glucose variability. From the above, it has been shown to accurately measure the blood glucose value from the glucose information in this method. Therefore, we can be proposed as a non-invasive blood glucose

We have measured the diabetics that have been actually measured blood glucose. Subject is four diabetics. We were measured before and after meals for 4 to 5 days in each subject. First, we developed the individual calibration curve of each subjects by using the PLSR analysis and infrared absorption spectra obtained in the three-day measurement. Then, the measured infrared absorption spectra in the 1-2 day were substituted in the developed

Table 2 shows the blood glucose data for each subject obtained by the conventional method for three days from the first day, and the results of the developed calibration curve for each subject by using the measured infrared absorption spectra. And Fig.10 shows the calibration curve of subject #1. From these results, this measurement system can be constructed to calibration curve for measuring blood glucose of diabetics, because, significant correlation has been obtained in this sample scale. The result of subject #4 show very good results than the other subjects. As this cause, the number of samples is very few for construct calibration

Subject # Number Ave. Max. Min. S.D. C.C SEC 1 15 226 339 156 56 0.758 ± 35 2 16 216 330 150 44 0.519 ± 37 3 10 157 221 108 41 0.645 ± 29 4 9 113 203 58 44 0.997 ± 3

Table 2. The blood glucose data and the results of the calibration curve for each subject

C

0 50 100 150 200 250 300 350 400

A E B

E

D

B

A

Reference value (mg/dl)

C

measurement by the infrared spectral measurements.

**4.5 Clinical application of measurement system**

Fig. 10. The calibration curve of subject #1

D

Estimated value (mg/dl)

calibration curve, and the blood glucose value was estimated.

this result was dramatically improved. And, by the EGA result, A-zone is 90%, and B-zone is 10%, this result was shown to be clinically effective. It is also considered to the impact of individual differences as this reason. Evanescent light is measured to invasion into the finger of about 0.3~2 μm in the ATR method, and, it is considered the infrared light not get to the blood vessel inside finger. Therefore, we are considered measured the interstitial tissue fluid. When the glucose comes out in tissue fluid from blood vessel, there is the time difference in between individual. So, if the subject measure the blood glucose by the calibration curve including other subjects information, the result have come to greatly affect these individual differences. For the above reasons, we measure the blood glucose value by using individual calibration curve of each subject.

#### **4.4.3 Verification of the calibration curve by loading vector**

In order to verify the validity of the calibration curve, we focused on the weighting of the loading vector obtained from the developed calibration curve. The loading vector is the calibration curve of data (infrared absorption spectra). In other words, the loading vector shows the wavenumber band to the impact to the calibration curve. The weighting of the loading vector by individual calibration curve in section 4.4.1 is shown in Fig.9-(A). The horizontal axis is the wavenumber, and the vertical axis is the weight of the loading vector.

There are absorption spectra three weighting curves because the number of PLS factors (principal components) obtained are three factors. In Fig.9-(A), in this loading vector, the wavenumber in 1220 cm-1 (Fig.9-(A)-A), 1150 cm-1 (Fig.9-(A)-B), 1020 cm-1 (Fig.9-(A)-C) were found to significant impact on the calibration curve.

Fig. 9. The weighting of the loading vector and each blood glucose value

Fig.9-(B) shows the infrared absorption spectra in large discrepancy between the subjects blood glucose value. In Fig.9-(B), characteristic absorption peaks can be found in 1220 cm-1 (Fig.9-(B)-A'), 1150 cm-1 (Fig.9-(B)-B'), 1020 cm-1 (Fig.9-(B)-C'). It is proportional to the blood glucose value and the absorbance in 1220 cm-1 and 1020 cm-1, and found a negative proportional relationship in 1150 cm-1. The weighting of the loading vector is almost matched by changes in infrared absorption spectra of blood glucose variability. From the above, it has been shown to accurately measure the blood glucose value from the glucose information in this method. Therefore, we can be proposed as a non-invasive blood glucose measurement by the infrared spectral measurements.

#### **4.5 Clinical application of measurement system**

88 Infrared Spectroscopy – Life and Biomedical Sciences

this result was dramatically improved. And, by the EGA result, A-zone is 90%, and B-zone is 10%, this result was shown to be clinically effective. It is also considered to the impact of individual differences as this reason. Evanescent light is measured to invasion into the finger of about 0.3~2 μm in the ATR method, and, it is considered the infrared light not get to the blood vessel inside finger. Therefore, we are considered measured the interstitial tissue fluid. When the glucose comes out in tissue fluid from blood vessel, there is the time difference in between individual. So, if the subject measure the blood glucose by the calibration curve including other subjects information, the result have come to greatly affect these individual differences. For the above reasons, we measure the blood glucose value by

In order to verify the validity of the calibration curve, we focused on the weighting of the loading vector obtained from the developed calibration curve. The loading vector is the calibration curve of data (infrared absorption spectra). In other words, the loading vector shows the wavenumber band to the impact to the calibration curve. The weighting of the loading vector by individual calibration curve in section 4.4.1 is shown in Fig.9-(A). The horizontal axis is the wavenumber, and the vertical axis is the weight of the loading

There are absorption spectra three weighting curves because the number of PLS factors (principal components) obtained are three factors. In Fig.9-(A), in this loading vector, the wavenumber in 1220 cm-1 (Fig.9-(A)-A), 1150 cm-1 (Fig.9-(A)-B), 1020 cm-1 (Fig.9-(A)-C) were

(A) The weighting of the loading vector (B) Absorption spectra Fig. 9. The weighting of the loading vector and each blood glucose value

C

Fig.9-(B) shows the infrared absorption spectra in large discrepancy between the subjects blood glucose value. In Fig.9-(B), characteristic absorption peaks can be found in 1220 cm-1 (Fig.9-(B)-A'), 1150 cm-1 (Fig.9-(B)-B'), 1020 cm-1 (Fig.9-(B)-C'). It is proportional to the blood glucose value and the absorbance in 1220 cm-1 and 1020 cm-1, and found a negative proportional relationship in 1150 cm-1. The weighting of the loading vector is almost

1300 1200 1100 1000 900

B'

75 mg/dl 134 mg/dl 171 mg/dl

C'

Wavenumber (cm-1)

0.0

0.2

Absorbance

0.4

0.6

A'

using individual calibration curve of each subject.

found to significant impact on the calibration curve.

A B

1750 1500 1250 1000 750

Wavenumber (cm-1)

vector.

0.0

Weighted factor

0.1

**4.4.3 Verification of the calibration curve by loading vector**

We have measured the diabetics that have been actually measured blood glucose. Subject is four diabetics. We were measured before and after meals for 4 to 5 days in each subject. First, we developed the individual calibration curve of each subjects by using the PLSR analysis and infrared absorption spectra obtained in the three-day measurement. Then, the measured infrared absorption spectra in the 1-2 day were substituted in the developed calibration curve, and the blood glucose value was estimated.

Table 2 shows the blood glucose data for each subject obtained by the conventional method for three days from the first day, and the results of the developed calibration curve for each subject by using the measured infrared absorption spectra. And Fig.10 shows the calibration curve of subject #1. From these results, this measurement system can be constructed to calibration curve for measuring blood glucose of diabetics, because, significant correlation has been obtained in this sample scale. The result of subject #4 show very good results than the other subjects. As this cause, the number of samples is very few for construct calibration

Table 2. The blood glucose data and the results of the calibration curve for each subject

Fig. 10. The calibration curve of subject #1

Introduction of Non-Invasive Measurement Method by Infrared Application 91

number of the PLS factors. From the comparison of the SEP and SEC, the optimal number of PLS factors for this measurement system is considered to be 2 or 3. Thus, with less data in after the start of measurement, we shall construct the calibration curve careful in the number of PLS factors. To improve measurement accuracy, we need to consider improving the system. If increased in the amount of information obtained from absorption spectra, we can be measured blood glucose value more accurately. That is, if increased the amount of infrared light obtained

**4.6 Conclusion of non-invasive measurement for blood glucose by IR spectroscopy**  We can confirm non-invasive blood glucose measurement system developed in this study is effective tools in clinical practice. In this measurement system, there is no pain which was felt by many diabetics so far. Because measurement time is 1 minute, it can be measured very easily and quickly. There is no stress to the patient, can measure to blood glucose value

Future challenges of this study are shown below. The number of subjects and the number of measurement times must be increasing. We must show in this measurement reproducibility. And, we must show that this system can be applied to any subject. If the blood glucose measurement sensor has been developed in this measurement method, future, this research

**5. Non-destructive analysis of the composition and mixture ratio for textile**

This section describes a Non-destructive analysis of the composition and mixture ratio for textile products. In Japan, the composition display of textile goods is obligated by the "Household Goods Labeling law". Therefore, analysis of the composition and mixture ratio for textile products in accordance with JIS method (L 1030-1,-2) are executed. But, existing methods is the destructive inspection, and a lot of time (six hours or more) and proficiency is necessary for the analysis. Also has concern about the safety and the environmental impact of such tests to use chemicals such as organic solvents. In addition, if the amount of remaining dirt on the sample is very small, if the remaining dirt color is same color of

In here, it proposes the application of the infrared spectroscopy for improve those problems. The method that we propose is non-destructive measurement, quickly and easy. We measured an infrared absorption spectrum of a textile by using a FT-IR. And we examined the possibility of a composition classification and a mixture ratio calculation by chemometrics. These experiments showed that the measurement system was effective.

Measuring system in this study, FT-IR (IRPrestige-21) and ATR method (DuraSampler-IR) was used. Measuring system is shown in Fig.12. The measuring yarn is folded, and set to cover on the prism. After, the measurement part of the prism and yarns were contacted by

**5.2 Analysis of the composition and mixture ratio for yarn products by ATR**

from the subjects' finger, the measurement accuracy is improved.

several times a day because the measurement time is very short.

will contribute significantly in community of increasing diabetics.

**products based on infrared spectroscopy**

sample, the dirt cannot be verified visually.

**5.2.1 Measurement system for yarn products**

**5.1 Background**

curves, and the average blood glucose value is small. In addition, it is considered to overfitting, because the PLS factor number is 5.

The spectra of diabetics as measured by the fourth day and fifth day were substituted in the individual calibration curve of each subject. Table 3 shows the accuracy of predicted blood glucose value and the EGA result. In addition, the scatter diagram of the Clark grid result of each subject is shown in Fig.11. From the EGA result, all data with one exception of subject #4 data are plotted in A and B zone of clinically safety ranges. Improvement of measurement accuracy is better, but, the important in clinical practice is give first aid when the blood glucose value showed abnormal. Therefore, from a good EGA results, this system can be treated as a reasonable measurement method of blood glucose value in diabetics in clinical practice.

Table 3. The accuracy of predicted blood glucose value and the EGA result

Fig. 11. The Clark grid result of each subject

To measure simply place a finger on the prism, this system has the advantage of measuring blood glucose value very easily for diabetics in many elderly. In addition, the burden on nurses is reduced, because the patient can measure in their own. Since the measurement time is about 1 minute, it is possible to measure the many patients in a short time. This is a worthwhile part in the clinical practice. When this system is used as a self-monitor blood glucose sensor of diabetic patients, the measurement error is a large. In particular, SEC of the subject #4 was ± 3 mg/dl, but SEP has become extremely large forecast error in ± 59 mg/dl. This cause is considered to the over-fitting, when building a calibration curve as previously described. In PLSR, if the increasing the number of the PLS factor, SEC is a smaller, SEP becomes large. Therefore, it is necessary to construct a standard curve with the optimal number of the PLS factors. From the comparison of the SEP and SEC, the optimal number of PLS factors for this measurement system is considered to be 2 or 3. Thus, with less data in after the start of measurement, we shall construct the calibration curve careful in the number of PLS factors. To improve measurement accuracy, we need to consider improving the system. If increased in the amount of information obtained from absorption spectra, we can be measured blood glucose value more accurately. That is, if increased the amount of infrared light obtained from the subjects' finger, the measurement accuracy is improved.

### **4.6 Conclusion of non-invasive measurement for blood glucose by IR spectroscopy**

We can confirm non-invasive blood glucose measurement system developed in this study is effective tools in clinical practice. In this measurement system, there is no pain which was felt by many diabetics so far. Because measurement time is 1 minute, it can be measured very easily and quickly. There is no stress to the patient, can measure to blood glucose value several times a day because the measurement time is very short.

Future challenges of this study are shown below. The number of subjects and the number of measurement times must be increasing. We must show in this measurement reproducibility. And, we must show that this system can be applied to any subject. If the blood glucose measurement sensor has been developed in this measurement method, future, this research will contribute significantly in community of increasing diabetics.


*3.2. Non-Conventional Manufacturing Processes—Electrical Discharge Machining (EDM)*

This review also presents some new insights into a non-conventional process, which is Electrical Discharge Machining (EDM). The process is a non-conventional machining method that allows the production of pieces with complex shapes, and it can be used in materials such as INCONEL® 718 and 625. This particular manufacturing technique removes material from the wrought-stock thanks to melting and vaporising cavities using electrical discharges that come from a scrolling wire [95], as illustrated in Figure 13.

**Figure 13.** Schematic representation of the EDM experiment setup (adapted from [96]).

Table 9 presents the latest experimental challenges and developments in the machining of INCONEL® alloys with EDM.

**Table 9.** Critical challenges and developments in the EDM process of INCONEL®.

#### **Table 9.** *Cont.*

#### *3.3. Tool Wear*

With the tool operation in machining, wear starts to be a key factor in quality and productivity, namely with INCONEL® alloys, such as 718 and 625. To identify a worn milling tool, the ISO 8688-2 [101] standard predicts that a tool presenting either *VB* = 300 μm or *VB*max = 500 μm on the flank is considered a worn tool [69]. For turning tools or inserts, the ISO 3685 standard is the one to consult [102]. Taking into account a novel lubrication method, Bartolomeis et al. [69] observed that the tool wear behaviour mechanisms during EL conditions were abrasion and microchipping on the cutting edge, due to the tendency of INCONEL® 718 to develop BUE.

Figure 14 packs the initial causes of wear, the various wear mechanisms that lead to different types of wear and the final consequences from the tool-wear due to INCONEL® machining. As a complement to Figure 14, Table 10 presents the typical TW mathematical models, used to characterize the numerous TW mechanisms. It is suggested to consult the work of Wang et al. [37] to better understand the additional variables described in Table 10.

**Table 10.** Typical TW mathematical models (adapted from [37]).


**Miscellaneous**

290 Squamous Cell Carcinoma

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*levels in lung, prostate and breast cancer patients.* Neuro Endocrinol Lett, 2008 Neuro

*Cancer Patients.* Journal of Health Science, 2008. 54(4): p. 493-496.

*antitrypsin genotype analysis.* Eur Respir J, 2003. 21: p. 215-219.

*inhibitor TATI.* Scand J Clin Lab Invest, 1991. 207: p. 5-7.

*malignancies.* Clinical Biochemistry 2004. 37(1): p. 56-60

*cancer.* Surgery Today, 1990. 941(1291): p. 1436-2813.

*as a cancer marker.* Cancer Lett, 1995. 89: p. 15-21.

*esophagus in Iran.* Oncogene, 2001. 20: p. 7368-7374.

*malignancies.* Clinical Biochemistry, 2004. 37(1): p. 56-60

Gastroenterol, 2006. 28: p. 7104-7112.

Dis Esophagus, 2010. 23(7): p. 597-602.

Endocrinol Lett(4): p. 18766166.

Humana Press, 2008.

1991. 11: p. 1-6.

2006. 5: p. 79-92.

*cancer development and progression.* Lancet Oncol, 2004. 5: p. 182-190. [25] Andolfatto S, et al., *Genomic DNA extraction from small amounts of serum to be used for a1-*

**17**

*USA*

**Vitamin D3 and Its Role in the Treatment of**

**Head and Neck Squamous Cell Carcinoma**

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common type of malignancy worldwide, and represents over 6% of the global cancer burden 1. Each year, HNSCC accounts for nearly 650,000 new cases of cancer, and over 35,000 deaths worldwide 1, 2. Historically, HNSCC has been a challenging disease to manage, with locally advanced disease often requiring a multidisciplinary approach of surgery, chemotherapy and radiation. However, despite significant advances among the aforementioned fields, the current 5-year survival rate of approximately 50% has improved very little over the last 30 years 2, 3. This poor improvement in prognosis is a reflection of the unique treatment challenges presented by HNSCC, which include advanced stages at diagnosis, high recurrence rate after surgical removal and second primary tumor development 4. Moreover, not only are current treatment options often non-curative, but they are associated with significant morbidity, including substantial physical deformity and functional deficits. Together, these challenges underscore the importance of developing novel anti-neoplastic treatment strategies which may improve survival and quality of life among HNSCC

One of the more intriguing novel anti-neoplastic agents is 1,25-dihydroxyvitamin D3 [(1,25(OH)2D3), calcitriol], the hormone first identified as an effective treatment for rickets, which has recently garnered wide-spread support as a possible anti-neoplastic agent in a number of different cancer subtypes 5. Epidemiological observational studies, performed mainly in the United States, have long postulated a correlation between higher latitude residence and overall cancer incidence and mortality. The first such theory was put forth in 1937 by Peller and Stephenson, who together hypothesized that increased sunlight exposure lowered the risk of cancer 6. Four years later, observational studies defined the relationship further, demonstrating a significant association between geographic latitude and cancer mortality 7. Since that time, scientists have continued to hypothesize that inadequate serum vitamin D levels at higher latitudes increases one's risk for a number of different cancers including colon*,* breast, prostate and ovarian cancers 8-11. Large cohort studies have

**1. Introduction**

patients.

\*Corresponding Author

M. Rita I. Young**\*** and David D. Walker *Ralph H. Johnson VA Medical Center, Charleston, Department of Otolaryngology – Head and Neck Surgery*

*Medical University of South Carolina, Charleston*

## **Vitamin D3 and Its Role in the Treatment of Head and Neck Squamous Cell Carcinoma**

M. Rita I. Young**\*** and David D. Walker

*Ralph H. Johnson VA Medical Center, Charleston, Department of Otolaryngology – Head and Neck Surgery Medical University of South Carolina, Charleston USA*

## **1. Introduction**

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common type of malignancy worldwide, and represents over 6% of the global cancer burden 1. Each year, HNSCC accounts for nearly 650,000 new cases of cancer, and over 35,000 deaths worldwide 1, 2. Historically, HNSCC has been a challenging disease to manage, with locally advanced disease often requiring a multidisciplinary approach of surgery, chemotherapy and radiation. However, despite significant advances among the aforementioned fields, the current 5-year survival rate of approximately 50% has improved very little over the last 30 years 2, 3. This poor improvement in prognosis is a reflection of the unique treatment challenges presented by HNSCC, which include advanced stages at diagnosis, high recurrence rate after surgical removal and second primary tumor development 4. Moreover, not only are current treatment options often non-curative, but they are associated with significant morbidity, including substantial physical deformity and functional deficits. Together, these challenges underscore the importance of developing novel anti-neoplastic treatment strategies which may improve survival and quality of life among HNSCC patients.

One of the more intriguing novel anti-neoplastic agents is 1,25-dihydroxyvitamin D3 [(1,25(OH)2D3), calcitriol], the hormone first identified as an effective treatment for rickets, which has recently garnered wide-spread support as a possible anti-neoplastic agent in a number of different cancer subtypes 5. Epidemiological observational studies, performed mainly in the United States, have long postulated a correlation between higher latitude residence and overall cancer incidence and mortality. The first such theory was put forth in 1937 by Peller and Stephenson, who together hypothesized that increased sunlight exposure lowered the risk of cancer 6. Four years later, observational studies defined the relationship further, demonstrating a significant association between geographic latitude and cancer mortality 7. Since that time, scientists have continued to hypothesize that inadequate serum vitamin D levels at higher latitudes increases one's risk for a number of different cancers including colon*,* breast, prostate and ovarian cancers 8-11. Large cohort studies have

<sup>\*</sup>Corresponding Author

Vitamin D3 and Its Role in the Treatment of Head and Neck Squamous Cell Carcinoma 295

Today, what is commonly referred to as "Vitamin D" is actually a combination of ergocalciferol (vitamin D2) and cholicalciferol (vitamin D3). Both compounds are readily available in diet form, with vitamin D2 being found in plant products and vitamin D3 found predominately in fatty fish, fish liver oil, and eggs with smaller amounts found in cheese and meat products. In either case, the total amount of either vitamin found within food items is relatively small; thus, many developed countries, including the United States, have turned to fortifying numerous food items, such as milk and juices, with both vitamin D2 and vitamin D3. Despite food fortification, diet is not the body's primary means for acquiring vitamin D3 stores. Instead, the majority of the body's cholecalciferol is synthesized *in vivo*  through a photosynthic process occurring in the epidermal layer of sun-exposed skin. Upon exposure to ultraviolet radiation, the epidermis is capable of transforming a cholesterol derivative (7-dehydrocholesterol) into vitamin D3 (cholecalciferol). The catalysis, however, is not uniform among all populations and is subject to significant and intense variations based

After synthesis, cholecalciferol enters the blood stream and is transported to the liver. There it undergoes intra-hepatic hydroxylation at the 25 position to create 25-hydroxyvitamin D3 [25(OH)D3 or calcidiol]. 25(OH)D3 is the circulating form of the hormone within the plasma as well as the most accurate biomarker of overall Vitamin D3 status 19. 25(OH)D3 is next catalyzed into one of two metabolites: the biologically inert 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] or the biologically active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Exactly which metabolite is created depends entirely on what enzyme is expressed within the local tissue. If the tissue expresses 24-hydroxylase (also known as CYP24A1), then 25(OH)D3 is modified to become 24,25(OH)2D3. This particular metabolite has poor affinity and avidity toward the VDR and is considered the first step in vitamin D decomposition. Alternatively, if the tissue expresses 1- hydroxylase (also known as CYP27B1), the biologically active metabolite 1,25(OH)2D3 is formed. This particular metabolite is most well known for its regulation of calcium homeostasis and is the

The majority of Vitamin D's hormonal action involves direct interaction with a single VDR. An intranuclear regulator of gene transcription, the VDR is a member of the class II steroid hormone receptors and is closely related to the retinoic acid and thyroid hormone receptors 17. The human receptor is located on chromosome 12q12q14 and consists of nine exons. Fully transcribed, the receptor is a 427-amino acid peptide with relatively simple binding configuration consisting of a DNA-binding domain termed the C-domain, a ligand-binding domain called the E-domain and an activating domain called the F-domain. The receptor acts by interacting with portions of DNA called vitamin D-responsive elements (VDREs). Located in the promoter region of target genes, VDRES are commonly found within 1 kilobase from the gene start sequence. Such areas are composed of hexanucleotide repeats separated by 3 nonspecified nucleotide bases (for example CGACTA-NNN-CGACTA where

Once 1,25(OH)2D3 is transported into the cell, it enters the nucleus where it is capable of binding to VDR. After binding, the 1,25(OH)2D3-VDR complex synergistically

on skin pigmentation, sunscreen use, age, gender and even obesity 18.

metabolite capable of exerting anti-neoplastic effects on cancer cells.

**3. Vitamin D mechanism of action**

N represents any nucleotide).

confirmed similar findings among HNSCC patients as well. Such studies have not only demonstrated significantly reduced 25-hydroxyvitamin D [25(OH)D] serum levels among HNSCC patients, but also found disease-free survival time to be significantly associated with 25(OH)D serum levels 12.

However, a definitive causal relationship is perhaps not as clear as it seems. Though these reports have evoked widespread enthusiasm within the scientific community, such zeal has remained tempered. Most of the data regarding an association between 25(OH)D and cancer is derived from retrospective ecological observational studies. To date, no large-scale randomized control trials have ever been done examining serum 25(OH)D levels and a primary outcome of cancer. In fact, smaller randomized control studies are extremely limited and yield conflicting results as to the significance of this relationship 13. There are several prospective studies examining the relationship, but such studies have offered no consensus findings regarding the association 14, 15.

The continued optimism regarding vitamin D's role as an anti-neoplastic agent is sustained, however, by a number of molecular studies which support the biologic feasibility of the theory. In particular, proponents cite the widely expressed vitamin D receptor (*VDR*), which to date has been found in over 30 different human tissues including lymphocytes, muscle, skin and cancer cells 16, 17. Furthermore, the terminal enzyme in the synthesis of active Vitamin D, 1--hydroxylase, also exhibits widespread expression allowing for synthesis of the hormonally active metabolite in a number of different cell types. Not only is vitamin D capable of ubiquitous synthesis and action, but *in vivo* studies within both human and animal models suggest that the active metabolite of vitamin D (calcitriol) is capable of eliciting a number of non-calcemic downstream effects. Many of these actions can be considered anti-neoplastic in nature, including anti-inflammatory, proapoptotic, and antiangiogenic properties, the promotion of cell differentiation, and the inhibition of cancercell proliferation.

In this chapter we describe the relevant molecular and biological mechanisms regarding Vitamin D metabolism. We also describe the basic and inherent anti-cancerous properties of vitamin D metabolites, with a specific emphasis on its role in the management of HNSCC.

## **2. Overview of vitamin D biological activity**

In 1919, Sir Edward Mellanby conducted a series of elegantly designed experiments on rachitic canines. By placing strict restrictions on both diet and sunlight exposure, he was able to establish a causal relationship between the bone disease rickets and a deficiency of an unidentified trace dietary substance. Mellanby concluded, at that time, that the unknown entity was most likely a previously undescribed fat-soluble vitamin. It was not until 1932, however, that Vitamin D's chemical structure was formally characterized, revealing that Vitamin D was not actually a vitamin, but a steroid hormone. In the late 1960s it was discovered that vitamin D was actually a precursor of a new steroid hormone, 1,25(OH)2D3, produced intrarenally. As both the physical structure and physiologic actions of 1,25(OH)2D3 continued to be better characterized, it was soon found to be a critical component of both calcium homeostasis and overall bone health. As research progressed into the 21st century, scientists discovered that the hormone impacted a number of important non-calcemic cell regulatory functions.

Today, what is commonly referred to as "Vitamin D" is actually a combination of ergocalciferol (vitamin D2) and cholicalciferol (vitamin D3). Both compounds are readily available in diet form, with vitamin D2 being found in plant products and vitamin D3 found predominately in fatty fish, fish liver oil, and eggs with smaller amounts found in cheese and meat products. In either case, the total amount of either vitamin found within food items is relatively small; thus, many developed countries, including the United States, have turned to fortifying numerous food items, such as milk and juices, with both vitamin D2 and vitamin D3. Despite food fortification, diet is not the body's primary means for acquiring vitamin D3 stores. Instead, the majority of the body's cholecalciferol is synthesized *in vivo*  through a photosynthic process occurring in the epidermal layer of sun-exposed skin. Upon exposure to ultraviolet radiation, the epidermis is capable of transforming a cholesterol derivative (7-dehydrocholesterol) into vitamin D3 (cholecalciferol). The catalysis, however, is not uniform among all populations and is subject to significant and intense variations based on skin pigmentation, sunscreen use, age, gender and even obesity 18.

After synthesis, cholecalciferol enters the blood stream and is transported to the liver. There it undergoes intra-hepatic hydroxylation at the 25 position to create 25-hydroxyvitamin D3 [25(OH)D3 or calcidiol]. 25(OH)D3 is the circulating form of the hormone within the plasma as well as the most accurate biomarker of overall Vitamin D3 status 19. 25(OH)D3 is next catalyzed into one of two metabolites: the biologically inert 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] or the biologically active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Exactly which metabolite is created depends entirely on what enzyme is expressed within the local tissue. If the tissue expresses 24-hydroxylase (also known as CYP24A1), then 25(OH)D3 is modified to become 24,25(OH)2D3. This particular metabolite has poor affinity and avidity toward the VDR and is considered the first step in vitamin D decomposition. Alternatively, if the tissue expresses 1- hydroxylase (also known as CYP27B1), the biologically active metabolite 1,25(OH)2D3 is formed. This particular metabolite is most well known for its regulation of calcium homeostasis and is the metabolite capable of exerting anti-neoplastic effects on cancer cells.

#### **3. Vitamin D mechanism of action**

294 Squamous Cell Carcinoma

confirmed similar findings among HNSCC patients as well. Such studies have not only demonstrated significantly reduced 25-hydroxyvitamin D [25(OH)D] serum levels among HNSCC patients, but also found disease-free survival time to be significantly associated

However, a definitive causal relationship is perhaps not as clear as it seems. Though these reports have evoked widespread enthusiasm within the scientific community, such zeal has remained tempered. Most of the data regarding an association between 25(OH)D and cancer is derived from retrospective ecological observational studies. To date, no large-scale randomized control trials have ever been done examining serum 25(OH)D levels and a primary outcome of cancer. In fact, smaller randomized control studies are extremely limited and yield conflicting results as to the significance of this relationship 13. There are several prospective studies examining the relationship, but such studies have offered no

The continued optimism regarding vitamin D's role as an anti-neoplastic agent is sustained, however, by a number of molecular studies which support the biologic feasibility of the theory. In particular, proponents cite the widely expressed vitamin D receptor (*VDR*), which to date has been found in over 30 different human tissues including lymphocytes, muscle, skin and cancer cells 16, 17. Furthermore, the terminal enzyme in the synthesis of active Vitamin D, 1--hydroxylase, also exhibits widespread expression allowing for synthesis of the hormonally active metabolite in a number of different cell types. Not only is vitamin D capable of ubiquitous synthesis and action, but *in vivo* studies within both human and animal models suggest that the active metabolite of vitamin D (calcitriol) is capable of eliciting a number of non-calcemic downstream effects. Many of these actions can be considered anti-neoplastic in nature, including anti-inflammatory, proapoptotic, and antiangiogenic properties, the promotion of cell differentiation, and the inhibition of cancer-

In this chapter we describe the relevant molecular and biological mechanisms regarding Vitamin D metabolism. We also describe the basic and inherent anti-cancerous properties of vitamin D metabolites, with a specific emphasis on its role in the management of HNSCC.

In 1919, Sir Edward Mellanby conducted a series of elegantly designed experiments on rachitic canines. By placing strict restrictions on both diet and sunlight exposure, he was able to establish a causal relationship between the bone disease rickets and a deficiency of an unidentified trace dietary substance. Mellanby concluded, at that time, that the unknown entity was most likely a previously undescribed fat-soluble vitamin. It was not until 1932, however, that Vitamin D's chemical structure was formally characterized, revealing that Vitamin D was not actually a vitamin, but a steroid hormone. In the late 1960s it was discovered that vitamin D was actually a precursor of a new steroid hormone, 1,25(OH)2D3, produced intrarenally. As both the physical structure and physiologic actions of 1,25(OH)2D3 continued to be better characterized, it was soon found to be a critical component of both calcium homeostasis and overall bone health. As research progressed into the 21st century, scientists discovered that the hormone impacted a number of

with 25(OH)D serum levels 12.

cell proliferation.

consensus findings regarding the association 14, 15.

**2. Overview of vitamin D biological activity**

important non-calcemic cell regulatory functions.

The majority of Vitamin D's hormonal action involves direct interaction with a single VDR. An intranuclear regulator of gene transcription, the VDR is a member of the class II steroid hormone receptors and is closely related to the retinoic acid and thyroid hormone receptors 17. The human receptor is located on chromosome 12q12q14 and consists of nine exons. Fully transcribed, the receptor is a 427-amino acid peptide with relatively simple binding configuration consisting of a DNA-binding domain termed the C-domain, a ligand-binding domain called the E-domain and an activating domain called the F-domain. The receptor acts by interacting with portions of DNA called vitamin D-responsive elements (VDREs). Located in the promoter region of target genes, VDRES are commonly found within 1 kilobase from the gene start sequence. Such areas are composed of hexanucleotide repeats separated by 3 nonspecified nucleotide bases (for example CGACTA-NNN-CGACTA where N represents any nucleotide).

Once 1,25(OH)2D3 is transported into the cell, it enters the nucleus where it is capable of binding to VDR. After binding, the 1,25(OH)2D3-VDR complex synergistically

Vitamin D3 and Its Role in the Treatment of Head and Neck Squamous Cell Carcinoma 297

integrity of the theory, paving the way for the initiation of many phase I and phase II clinical

Thus far, 1,25(OH)2D3has been presented as a secosteroid whose fundamental intra-nuclear action is critical for appropriate regulation of calcium homeostasis and proper bone mineralization. Vitamin D, however, has other non-calcemic regulatory functions, including many which make it an intriguing anti-neoplastic agent. Four separate cellular functions tend to be compromised in order for cancer to thrive within a normal host environment: 1) aberrant regulation of differentiation, 2) dysregulated cellular proliferation, 3) newly acquired ability permitting invasion and metastasis, 4) dysregulated cell destruction of altered self-cells. Numerous published reports have demonstrated that 1,25(OH)2D3 is capable of restoring or repairing each of the aforementioned hallmarks of carcinogenesis. It does so in a number of ways, including inhibition of cellular proliferation, disruption and inhibition of angiogenesis, promoting apoptosis, and improving tumor immunosurveillence.

The processes of cellular proliferation and differentiation are intimately intertwined, and the aberrant regulation of each is critically important to the prosperity of growing cancer cells. The normal cell grows along a continuum of cellular differentiation. As a cell proceeds toward terminal differentiation, its ability to divide and proliferate is decreased. This feature is exploited and utilized by many newer anti-neoplastic agents, including 1,25(OH)2D3. A promising feature of 1,25(OH)2D3 is its ability to manipulate the cell's state of differentiation. *In vitro* studies in human cell lines demonstrated that 1,25(OH)2D3 is capable of reducing cell proliferation by inducing cell cycle arrest in G0/G1 phase while simultaneously promoting

These results were replicated within murine SCC cell lines which showed that administration of 1,25(OH)2D3to cells induces G0/G1 cell-cycle arrest via the transcriptional activation of *CDKN1B* and subsequent dephosphorylation of pRB 26. The study demonstrated that in both *in vivo* and *in vitro* studies, the increases seen in CDKN1B were the result of downregulated p21 activity, a potent CDK inhibitor 26. Human SCC cell lines that positively express VDR showed a potent decrease in cellular proliferation when exposed to 1,25(OH)2D3 and its analogues 27. Vitamin D's ability to force cells into differentiation may be of critical importance, as such actions can aid in the inhibition of

Cancer cells will often acquire the ability to escape the confines of the original tumor nidus. The tumor cell often relies heavily on its angiogenic properties to spread. In theory, limiting the tumor's ability to aberrantly generate rogue blood supplies may limit its ability to thrive and metastasize, offering a potential avenue to achievement of loco-regional control. Several studies have demonstrated that 1,25(OH)2D3 contains potent antiangiogenic properties capable of reducing the invasiveness of cancer cells. *In vitro* studies demonstrated that

trials.

**4. Vitamin D and cancer**

cellular differentiation 24, 25.

tumor cell proliferation 28.

**4.2 Angiogenesis**

**4.1 Cellular proliferation and differentiation**

heterodimierizes with retinoid-X receptor (RXR). The 1,25(OH)2D3-VDR-RXR complex then binds specifically to a VDRE sequence complex, with the VDR occupying the 3' region and the RXR positioned at the 5' end. Conformational changes which occur after 1,25(OH)2D3 binding results in the release of coreppressors and the simultaneous exposure of binding sites for transcriptional coactivators. VDR coactivators are then capable of binding the complex, which results in the acetylation and subsequent release of histones from the DNA strand. Without histones in place, the appropriate transcription factors are capable of binding to the naked DNA strand and transcription of the target gene is able to initiate.

Certain variables that construct this transcriptional machinery apparatus may have a direct role within the formation of cancer. Recent resequencing of *VDR* gene identified 194 singlenucleotide polymorphisms (SNPs). It has been hypothesized that *VDR* SNPs themselves may have a direct impact on formation and prognosis of certain types of cancer, including HNSCC. The two most widely investigated receptors regarding their impact and role within cancer include a *FokI* restriction fragment-length polymorphism (RFLP) in exon 2 and an adjacent *TaqI* RFLP in exon 9. These two polymoprhisms were recently investigated with respect to their association with HNSCC incidence. Such studies yielded mixed results. Findings from one study demonstrated that the heterozygous genotype of *Taq I* (Tt) polymorphism may be associated with an increased susceptibility to HNSCC 20. In a separate study, however, both homozygous variant genotypes, *TaqI* (tt) and *FokI* (ff), were associated with a reduced risk of HNSCC as compared to the more common *TaqI (TT)* and *FokI (FF)* genotypes. Thus, it remains uncertain as to whether or not specific *VDR* SPNs provide a protective or deleterious effect regarding HNSCC risk 21. However, demonstrations of both increased and decreased HNSCC risk warrant continued research into how *VDR* SNPs may not only impact cancer incidence, but also mortality and treatment response.

#### **3.1 Biological plausibility within cancer**

The general biologic characteristics and mechanisms of action detailed above presented two main molecular hurdles for scientists considering 1,25(OH)2D3 as a plausible anti-neoplastic agent. First, there was the seemingly restricted localization of 1- hydroxylase. The enzyme responsible for converting vitamin D into its active form is highly expressed in renal proximal tubules and was once thought to be solely expressed intra-renally However, it is now known that many non-renal cells, including bone, placenta, prostate, keratinocytes, macrophages, T-lymphocytes, dendritic cells and several cancer cells all express the converting enzyme capable of producing the hormonally active metabolite 1,25(OH)2D3 22. This particular formation of Vitamin D contains a high affinity for the VDR and is capable of eliciting an anti-neoplastic response. It is this formation with which we concern ourselves for the remainder of the discussion.

The second major impasse was the restrictive localization of VDR to skeletal tissues. However, since VDRs were first discovered their localization has been confirmed on a variety of tissues. With respects to SCC, it has been demonstrated that not only do human cancer cell lines (SCL-1, SCL-2 ) exhibit VDR expression, but in fact they express the receptor in higher amounts than normal human skin cell lines 23. The discovery of extra-renally expressed 1- hydroxylase and extra-skeletally expressed VDR was paramount in vitamin D's acceptance as a possible anticancer agent. Such findings sustained the molecular integrity of the theory, paving the way for the initiation of many phase I and phase II clinical trials.

## **4. Vitamin D and cancer**

296 Squamous Cell Carcinoma

heterodimierizes with retinoid-X receptor (RXR). The 1,25(OH)2D3-VDR-RXR complex then binds specifically to a VDRE sequence complex, with the VDR occupying the 3' region and the RXR positioned at the 5' end. Conformational changes which occur after 1,25(OH)2D3 binding results in the release of coreppressors and the simultaneous exposure of binding sites for transcriptional coactivators. VDR coactivators are then capable of binding the complex, which results in the acetylation and subsequent release of histones from the DNA strand. Without histones in place, the appropriate transcription factors are capable of binding to the naked DNA strand and transcription of the target gene is able to initiate.

Certain variables that construct this transcriptional machinery apparatus may have a direct role within the formation of cancer. Recent resequencing of *VDR* gene identified 194 singlenucleotide polymorphisms (SNPs). It has been hypothesized that *VDR* SNPs themselves may have a direct impact on formation and prognosis of certain types of cancer, including HNSCC. The two most widely investigated receptors regarding their impact and role within cancer include a *FokI* restriction fragment-length polymorphism (RFLP) in exon 2 and an adjacent *TaqI* RFLP in exon 9. These two polymoprhisms were recently investigated with respect to their association with HNSCC incidence. Such studies yielded mixed results. Findings from one study demonstrated that the heterozygous genotype of *Taq I* (Tt) polymorphism may be associated with an increased susceptibility to HNSCC 20. In a separate study, however, both homozygous variant genotypes, *TaqI* (tt) and *FokI* (ff), were associated with a reduced risk of HNSCC as compared to the more common *TaqI (TT)* and *FokI (FF)* genotypes. Thus, it remains uncertain as to whether or not specific *VDR* SPNs provide a protective or deleterious effect regarding HNSCC risk 21. However, demonstrations of both increased and decreased HNSCC risk warrant continued research into how *VDR* SNPs may not only impact cancer incidence, but also mortality and treatment

The general biologic characteristics and mechanisms of action detailed above presented two main molecular hurdles for scientists considering 1,25(OH)2D3 as a plausible anti-neoplastic agent. First, there was the seemingly restricted localization of 1- hydroxylase. The enzyme responsible for converting vitamin D into its active form is highly expressed in renal proximal tubules and was once thought to be solely expressed intra-renally However, it is now known that many non-renal cells, including bone, placenta, prostate, keratinocytes, macrophages, T-lymphocytes, dendritic cells and several cancer cells all express the converting enzyme capable of producing the hormonally active metabolite 1,25(OH)2D3 22. This particular formation of Vitamin D contains a high affinity for the VDR and is capable of eliciting an anti-neoplastic response. It is this formation with which we concern ourselves

The second major impasse was the restrictive localization of VDR to skeletal tissues. However, since VDRs were first discovered their localization has been confirmed on a variety of tissues. With respects to SCC, it has been demonstrated that not only do human cancer cell lines (SCL-1, SCL-2 ) exhibit VDR expression, but in fact they express the receptor in higher amounts than normal human skin cell lines 23. The discovery of extra-renally expressed 1- hydroxylase and extra-skeletally expressed VDR was paramount in vitamin D's acceptance as a possible anticancer agent. Such findings sustained the molecular

response.

**3.1 Biological plausibility within cancer**

for the remainder of the discussion.

Thus far, 1,25(OH)2D3has been presented as a secosteroid whose fundamental intra-nuclear action is critical for appropriate regulation of calcium homeostasis and proper bone mineralization. Vitamin D, however, has other non-calcemic regulatory functions, including many which make it an intriguing anti-neoplastic agent. Four separate cellular functions tend to be compromised in order for cancer to thrive within a normal host environment: 1) aberrant regulation of differentiation, 2) dysregulated cellular proliferation, 3) newly acquired ability permitting invasion and metastasis, 4) dysregulated cell destruction of altered self-cells. Numerous published reports have demonstrated that 1,25(OH)2D3 is capable of restoring or repairing each of the aforementioned hallmarks of carcinogenesis. It does so in a number of ways, including inhibition of cellular proliferation, disruption and inhibition of angiogenesis, promoting apoptosis, and improving tumor immunosurveillence.

#### **4.1 Cellular proliferation and differentiation**

The processes of cellular proliferation and differentiation are intimately intertwined, and the aberrant regulation of each is critically important to the prosperity of growing cancer cells. The normal cell grows along a continuum of cellular differentiation. As a cell proceeds toward terminal differentiation, its ability to divide and proliferate is decreased. This feature is exploited and utilized by many newer anti-neoplastic agents, including 1,25(OH)2D3. A promising feature of 1,25(OH)2D3 is its ability to manipulate the cell's state of differentiation. *In vitro* studies in human cell lines demonstrated that 1,25(OH)2D3 is capable of reducing cell proliferation by inducing cell cycle arrest in G0/G1 phase while simultaneously promoting cellular differentiation 24, 25.

These results were replicated within murine SCC cell lines which showed that administration of 1,25(OH)2D3to cells induces G0/G1 cell-cycle arrest via the transcriptional activation of *CDKN1B* and subsequent dephosphorylation of pRB 26. The study demonstrated that in both *in vivo* and *in vitro* studies, the increases seen in CDKN1B were the result of downregulated p21 activity, a potent CDK inhibitor 26. Human SCC cell lines that positively express VDR showed a potent decrease in cellular proliferation when exposed to 1,25(OH)2D3 and its analogues 27. Vitamin D's ability to force cells into differentiation may be of critical importance, as such actions can aid in the inhibition of tumor cell proliferation 28.

#### **4.2 Angiogenesis**

Cancer cells will often acquire the ability to escape the confines of the original tumor nidus. The tumor cell often relies heavily on its angiogenic properties to spread. In theory, limiting the tumor's ability to aberrantly generate rogue blood supplies may limit its ability to thrive and metastasize, offering a potential avenue to achievement of loco-regional control. Several studies have demonstrated that 1,25(OH)2D3 contains potent antiangiogenic properties capable of reducing the invasiveness of cancer cells. *In vitro* studies demonstrated that

Vitamin D3 and Its Role in the Treatment of Head and Neck Squamous Cell Carcinoma 299

immunoinhibitory effect of the cancer. Such findings suggest that 1,25(OH)2D3 may be

Tissues of HNSCC patients who received treatment with 1,25(OH)2D3 contained increased levels of CD4+ cells and, more significantly, CD8+T cells. Such findings indicate an increased infiltration of immune effector cells into the tumor microenvironment. Also prominent was an increase in cells expressing the lymphoid activation marker CD69, which represent mainly T cells and monocytes. The same study showed that HNSCC patients who received 1,25(OH)2D3 prior to treatment had a lengthier time to tumor recurrence compared with patients who were not treated before surgery. The treatment arm exhibited a median time to recurrence that was almost 3.5-fold longer than control patients who were untreated before surgery 42. These findings are complemented by separate studies suggesting that treatment

helpful in overcoming the profound immunosuppression associated with HNSCC 41.

with 1,25(OH)2D3 may be an appropriate method of restarting the immune system 43.

One of the most discussed aspects regarding the implementation of vitamin D as an anticancer medication is dosing regimen. The Institute of Medicine recently addressed vitamin D dosing regimens in its newly published Dietary Referenced Intakes for Vitamin D. Their review of the literature yielded the suggested dietary intake of 600 IU per day for persons 1 to 70 years of age and 800 IU per day for persons over 70. Such intakes corresponded to serum 25(OH)D levels of at least 20 ng per milliliter (50 nmol per liter) with recommended daily ingestion not to exceed 4000 IU per day 44. The same review concluded that the prevalence of vitamin D deficiency in North America, which previously published reports had suggested to be as high as 54% in some populations, has been drastically overestimated 45. The authors concluded that the vast majority of North American individuals have serum 25(OH)D concentrations above 20 ng per milliliter, which is adequate for bone health in at

Another area of debate is the concept of vitamin D screening as a predictor for cancer incidence. Since its inception, many have taken disagreed with such practices. One of the main areas of contention rests in the number of confounding variables associated with low serum 25(OH)D levels. These variables, which include obesity, lack of physical activity, and poor diet or supplementation practices, have the ability to impact cancer risk. Critics also cite reverse-causation bias, noting that in certain individuals poor health reduces one's

It is the opinion of the authors that the role of 1,25(OH)2D3 as an anti-cancer agent warrants continued clinical and laboratory exploration. Further investigation into the relationship calls for randomized control trials of vitamin D for cancer prevention. As noted by the recently published IRAC review, observational studies themselves are unlikely to "disentangle the complex relationships between vitamin D and known cancer risk factors 46." Such reports, however, have paved the way for numerous ongoing phase I and II clinical trials which should begin to elucidate what role, if any, 1,25(OH)2D3 may play in the

ability to participate in outdoor activities, resulting in lower vitamin D levels.

**5. Future clinical considerations**

least 97.5% of the population

treatment of HNSCC.

**6. Conclusion and future perspective**

1,25(OH)2D3 and its analogs directly inhibit tumor-derived endothelial cell proliferation in addition to disrupting angiogenic signaling in cancer cells 29.

Within HNSCC cell-lines, treatment with 1,25(OH)2D3 resulted in a significantly lower production of pro-angiogenic cytokines, VEGF and PDGF 27. Meanwhile, the proinflammatory and proangiogenic cytokine, IL-8, exhibits no change after administration of 1,25(OH)2D3 27. The finding of decreasing concentrations of VEGF has prompted considerable interest, as it has not only been cited as a marker for tumor metastasis, but also used as a prognostic factor in HNSCC patients 30, 31.

## **4.3 Apoptosis**

Apart from its actions on the cell cycle, 1,25(OH)2D3 can also suppress cancer growth via induction of apoptosis 32-34. Results carried out on human HNSCC cell lines demonstrated that exposure to 1,25(OH)2D3 results in the downregulaion of several anti-apoptotic genes. In particular, 1,25(OH)2D3 represses the expression of the anti-apoptotic, pro-survival gene BCL-2 while simultaneously increasing pro-apoptotic gene products of BAX and BAK 35. Studies completed on mouse SCC tumor cells with 1,25(OH)2D3 revealed a form of caspase-dependent apoptosis involving the growth-promoting/pro-survival signaling molecule, mitogen-activated protein kinase kinase (MEK) 36. The study was able to demonstrate that exposure to 1,25(OH)2D3 resulted in increased VDR expression and concomitant cleavage of the pro-survival signaling molecule MEK via a caspasedependent manner.

#### **4.4 Immune escape**

Key to the survival of cancer cells is the evasion of destruction, a critically fundamental step of which is the ability to evade the immune system. HNSCC, like all cancers, is capable of evading the host's immune system via a number of different mechanisms. Cancer can evade antigenic recognition via tissue sequestration, improper lymphocytic homing due to improper expression of adhesion molecules, and antigenic shedding and modulation. In certain instances, however, antigenic recognition remains intact, despite a poor immune response. In such cases, cancers continue to thrive by releasing immunosuppressive cytokine, downregulating MHC I complex, or instilling dysregulated or improper costimulation

Numerous studies performed on HNSCC patients have investigated the effects of 1,25(OH)2D3 on the immune profile of patients. Within the past 10 years, it has been shown that not only do monocytes, B-cells, and T-cells express VDR, but also contain the enzyme 1- -hydroxylase capable of converting 25(OH)D3 into 1,25(OH)2D3 37. Such findings confirmed long-standing speculation that vitamin D is critical for the appropriate management and integrity of the immune system. Numerous studies have since attempted to understand exactly how vitamin D modulates the host immune profile.

HNSCC itself is associated with profound immunosuppression 38. This is in part due to tissue infiltration by immunosuppressive immature progenitor cells with surface marker CD34 39, 40. In one study, HNSCC patients treated with 1,25(OH)2D3 exhibited decreased levels of intratumoral CD34+ cell populations and diminished the preexisting profound immunoinhibitory effect of the cancer. Such findings suggest that 1,25(OH)2D3 may be helpful in overcoming the profound immunosuppression associated with HNSCC 41.

Tissues of HNSCC patients who received treatment with 1,25(OH)2D3 contained increased levels of CD4+ cells and, more significantly, CD8+T cells. Such findings indicate an increased infiltration of immune effector cells into the tumor microenvironment. Also prominent was an increase in cells expressing the lymphoid activation marker CD69, which represent mainly T cells and monocytes. The same study showed that HNSCC patients who received 1,25(OH)2D3 prior to treatment had a lengthier time to tumor recurrence compared with patients who were not treated before surgery. The treatment arm exhibited a median time to recurrence that was almost 3.5-fold longer than control patients who were untreated before surgery 42. These findings are complemented by separate studies suggesting that treatment with 1,25(OH)2D3 may be an appropriate method of restarting the immune system 43.

## **5. Future clinical considerations**

298 Squamous Cell Carcinoma

1,25(OH)2D3 and its analogs directly inhibit tumor-derived endothelial cell proliferation in

Within HNSCC cell-lines, treatment with 1,25(OH)2D3 resulted in a significantly lower production of pro-angiogenic cytokines, VEGF and PDGF 27. Meanwhile, the proinflammatory and proangiogenic cytokine, IL-8, exhibits no change after administration of 1,25(OH)2D3 27. The finding of decreasing concentrations of VEGF has prompted considerable interest, as it has not only been cited as a marker for tumor metastasis, but also

Apart from its actions on the cell cycle, 1,25(OH)2D3 can also suppress cancer growth via induction of apoptosis 32-34. Results carried out on human HNSCC cell lines demonstrated that exposure to 1,25(OH)2D3 results in the downregulaion of several anti-apoptotic genes. In particular, 1,25(OH)2D3 represses the expression of the anti-apoptotic, pro-survival gene BCL-2 while simultaneously increasing pro-apoptotic gene products of BAX and BAK 35. Studies completed on mouse SCC tumor cells with 1,25(OH)2D3 revealed a form of caspase-dependent apoptosis involving the growth-promoting/pro-survival signaling molecule, mitogen-activated protein kinase kinase (MEK) 36. The study was able to demonstrate that exposure to 1,25(OH)2D3 resulted in increased VDR expression and concomitant cleavage of the pro-survival signaling molecule MEK via a caspase-

Key to the survival of cancer cells is the evasion of destruction, a critically fundamental step of which is the ability to evade the immune system. HNSCC, like all cancers, is capable of evading the host's immune system via a number of different mechanisms. Cancer can evade antigenic recognition via tissue sequestration, improper lymphocytic homing due to improper expression of adhesion molecules, and antigenic shedding and modulation. In certain instances, however, antigenic recognition remains intact, despite a poor immune response. In such cases, cancers continue to thrive by releasing immunosuppressive cytokine, downregulating MHC I complex, or instilling dysregulated or improper

Numerous studies performed on HNSCC patients have investigated the effects of 1,25(OH)2D3 on the immune profile of patients. Within the past 10 years, it has been shown that not only do monocytes, B-cells, and T-cells express VDR, but also contain the enzyme 1- -hydroxylase capable of converting 25(OH)D3 into 1,25(OH)2D3 37. Such findings confirmed long-standing speculation that vitamin D is critical for the appropriate management and integrity of the immune system. Numerous studies have since attempted to understand

HNSCC itself is associated with profound immunosuppression 38. This is in part due to tissue infiltration by immunosuppressive immature progenitor cells with surface marker CD34 39, 40. In one study, HNSCC patients treated with 1,25(OH)2D3 exhibited decreased levels of intratumoral CD34+ cell populations and diminished the preexisting profound

exactly how vitamin D modulates the host immune profile.

addition to disrupting angiogenic signaling in cancer cells 29.

used as a prognostic factor in HNSCC patients 30, 31.

**4.3 Apoptosis**

dependent manner.

**4.4 Immune escape**

costimulation

One of the most discussed aspects regarding the implementation of vitamin D as an anticancer medication is dosing regimen. The Institute of Medicine recently addressed vitamin D dosing regimens in its newly published Dietary Referenced Intakes for Vitamin D. Their review of the literature yielded the suggested dietary intake of 600 IU per day for persons 1 to 70 years of age and 800 IU per day for persons over 70. Such intakes corresponded to serum 25(OH)D levels of at least 20 ng per milliliter (50 nmol per liter) with recommended daily ingestion not to exceed 4000 IU per day 44. The same review concluded that the prevalence of vitamin D deficiency in North America, which previously published reports had suggested to be as high as 54% in some populations, has been drastically overestimated 45. The authors concluded that the vast majority of North American individuals have serum 25(OH)D concentrations above 20 ng per milliliter, which is adequate for bone health in at least 97.5% of the population

Another area of debate is the concept of vitamin D screening as a predictor for cancer incidence. Since its inception, many have taken disagreed with such practices. One of the main areas of contention rests in the number of confounding variables associated with low serum 25(OH)D levels. These variables, which include obesity, lack of physical activity, and poor diet or supplementation practices, have the ability to impact cancer risk. Critics also cite reverse-causation bias, noting that in certain individuals poor health reduces one's ability to participate in outdoor activities, resulting in lower vitamin D levels.

#### **6. Conclusion and future perspective**

It is the opinion of the authors that the role of 1,25(OH)2D3 as an anti-cancer agent warrants continued clinical and laboratory exploration. Further investigation into the relationship calls for randomized control trials of vitamin D for cancer prevention. As noted by the recently published IRAC review, observational studies themselves are unlikely to "disentangle the complex relationships between vitamin D and known cancer risk factors 46." Such reports, however, have paved the way for numerous ongoing phase I and II clinical trials which should begin to elucidate what role, if any, 1,25(OH)2D3 may play in the treatment of HNSCC.

Vitamin D3 and Its Role in the Treatment of Head and Neck Squamous Cell Carcinoma 301

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## *Edited by Xiaoming Li*

This book points to some new areas for investigation on squamous cell carcinoma (SCC). Firstly, the features and management of some specific SCC is discussed to give the readers the general principles in dealing with these uncommon and sophisticated conditions. Some new concepts in adjuvant therapy including neoadjuvant therapy and gold nanoparticle-based photo dynamic therapy are introduced. Secondly, a detailed discussion of molecular aspects of tumor invasion and progression in SCC is provided with the emphasis on the roles of some important factors. The role of tumor microenvironment in head and neck SCC is specifically discussed. Thirdly, the roles of cancer stem cells (CSC) in cancer therapy of SCC are described. Molecular mechanisms involving therapeutic resistance and new therapeutic strategies targeting CSC are discussed in detail. Finally, other aspects concerning SCC are included, which involve the assessment, genetic manipulation and its possible clinical implications for the treatment of SCC.

> ISBN 978-953-51-0024-9 ISBN 978-953-51-6805-8

Squamous Cell Carcinoma

Squamous Cell Carcinoma

*Edited by Xiaoming Li*

Photo by Manuel Faba Ortega / iStock