and migrate by lymphatic route to lymph nodes and vascular route to colonize in other organs including brain [11, 12]. Gene-expression profiling studies of breast cancer cells indicate that specific molecular pathways are associated with dissemination of primary tumor cells through a vascular route and not by lymphatic dissemination [12]. There is much interest in studying how and when the cancer cells initiate the metastatic cascade so that a therapeutic intervention can be developed to stop or delay the metastasis. Some cancer researchers [13] believe that targeted treatment of breast cancer with ER/PR modulators (Aromatase inhibitors) and targeted biologics such as Herceptin (Her-2 neu inhibitor) [14] and bevacizumab [15] (anti-vascular). Others argue that the metastasis of cancer cells is triggered by a dysregulated cellular Ca2+ homeostasis and altered Ca2+ signaling caused by imbalanced fluxes through ion channels and transporters [10, 16]. The BKCa channels are more sensitive to Ca2+ ions in cancer cells. In this regard, we studied whether the increased sensitivity of potentially new BKCa channel variant protein encoded by splice variants (**Figure 2**) *KCNMA1ΔE2* and *KCNMA1vE22* to intra and extra cellular Ca2+ in breast cancer [17]. In fact, a recent evidence indicates that KCa-Ca2+ channel complexes were found in cancer cells and contribute to cancerassociated functions such as cell proliferation, cell migration and the capacity to develop metastases [10]. The BKCa channels are unique since its activity is triggered by depolarization and enhanced by an increase in μM range of intracellular calcium

i]. In this regard, we recently showed that BKCa channel variant encoded by

progression to high grade glioblastoma multiforme (GBM) [18].  Epigenetics play an important role in cancer initiation, growth and progression. Understanding the precise mechanism helps us in developing diagnosis, prognosis and treatment strategies for affected cancer patients. For example, overexpression of Ezh2 plays a role in many cancers,

*BKCa channel is a 7-transmembrane tetramer of four monomeric pore-forming alpha-subunits encoded by KCNMA1. The cytoplasmic C-terminal domain has RCK1 and RCK2 (with calcium bowl) segments. We identified KCNMA1ΔE2 and KCNMA1E22 in human brain-specific metastatic breast cancer cells. Using relevant siRNA designs, we showed that these splice variants are formed by the deletion of exon 2 (E2) and 108*

i] and causes glioma

a new splice variant *KCNMA1vE22* is highly sensitive to [Ca2+

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

#### *Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain DOI: http://dx.doi.org/10.5772/intechopen.84957*

*Breast Cancer Biology*

in cancer cell biology [9, 10].

**2.1 Ion channels in breast cancer metastasis**

extremely difficult as brain provides a "safe haven" for cancer cells. Gene expression profiling has been used to predict metastatic gene-expression signature that is present in a subset of primary breast tumors [8]. However, a reliable profile has not yet been identified that specifically predicts brain metastases. Therefore, it is extremely important to study the genetic changes in breast cancer cells that metastasize to

Cancer research is not only focusing on understanding the possible role of transmembrane-BKCa channels in cancer development and progression but also on development of BKCa channel modulator drugs to attenuate cancer growth. Several researchers, including us have shown that brain tumor cells express BKCa and ATPsensitive potassium (KATP) channels that are highly responsive to minute changes in intracellular Ca2+ and ATP levels. This allows the brain tumor cells to develop pseudopodia for migration through constricted spaces in the brain parenchyma, as depicted in **Figure 1**. Several articles have described the efficacy of BKCa channelinhibiting drugs or molecules in reducing tumors in preclinical mouse tumor

models. A recent study has shown the role of intracellular BKCa channels (mitoBKCa)

Even now the metastatic breast cancers are incurable.  It is widely believed that breast cancer cells escape the primary site

*Anticipated role of BKCa and KATP channels in breast cancer cells that seek brain and colonize in brain parenchyma. The potassium ion channels expressed in breast cancer cells are extremely sensitive to minute surge*

 *efflux through BKCa channels. Similarly, slight imbalance*

 *efflux through KATP channels. Then the ion imbalance triggers the Ca2+*

brain and develop specific targeted therapeutic molecular agents.

**2. Channelopathy promote breast cancer metastasis**

**70**

**Figure 1.**

*of extracellular and intracellular Ca2+ and cause K+*

*entry, which promotes cancer cell migration though pseudopodia.*

*in ADP-ATP levels in the cell causes K<sup>+</sup>*

and migrate by lymphatic route to lymph nodes and vascular route to colonize in other organs including brain [11, 12]. Gene-expression profiling studies of breast cancer cells indicate that specific molecular pathways are associated with dissemination of primary tumor cells through a vascular route and not by lymphatic dissemination [12]. There is much interest in studying how and when the cancer cells initiate the metastatic cascade so that a therapeutic intervention can be developed to stop or delay the metastasis. Some cancer researchers [13] believe that targeted treatment of breast cancer with ER/PR modulators (Aromatase inhibitors) and targeted biologics such as Herceptin (Her-2 neu inhibitor) [14] and bevacizumab [15] (anti-vascular). Others argue that the metastasis of cancer cells is triggered by a dysregulated cellular Ca2+ homeostasis and altered Ca2+ signaling caused by imbalanced fluxes through ion channels and transporters [10, 16]. The BKCa channels are more sensitive to Ca2+ ions in cancer cells. In this regard, we studied whether the increased sensitivity of potentially new BKCa channel variant protein encoded by splice variants (**Figure 2**) *KCNMA1ΔE2* and *KCNMA1vE22* to intra and extra cellular Ca2+ in breast cancer [17]. In fact, a recent evidence indicates that KCa-Ca2+ channel complexes were found in cancer cells and contribute to cancerassociated functions such as cell proliferation, cell migration and the capacity to develop metastases [10]. The BKCa channels are unique since its activity is triggered by depolarization and enhanced by an increase in μM range of intracellular calcium [Ca2+ i]. In this regard, we recently showed that BKCa channel variant encoded by a new splice variant *KCNMA1vE22* is highly sensitive to [Ca2+ i] and causes glioma progression to high grade glioblastoma multiforme (GBM) [18].  Epigenetics play an important role in cancer initiation, growth and progression. Understanding the precise mechanism helps us in developing diagnosis, prognosis and treatment strategies for affected cancer patients. For example, overexpression of Ezh2 plays a role in many cancers,

#### **Figure 2.**

*BKCa channel is a 7-transmembrane tetramer of four monomeric pore-forming alpha-subunits encoded by KCNMA1. The cytoplasmic C-terminal domain has RCK1 and RCK2 (with calcium bowl) segments. We identified KCNMA1ΔE2 and KCNMA1E22 in human brain-specific metastatic breast cancer cells. Using relevant siRNA designs, we showed that these splice variants are formed by the deletion of exon 2 (E2) and 108 base pair deletion in exon 22 (E22), respectively.*

including breast cancer and brain tumors. H3K27M serves as an oncohistone and, if mutated it contributes to tumor development as Ezh2 is no longer able to methylate the histone and gene expression is aberrantly upregulated.

Furthermore, a recent computational analysis of human genomic sequence identified mutations that cause pathogenic splicing abnormalities in breast cancer susceptibility genes, BRCA1, BRCA2 and other genes [19]. Several investigations have reported that voltage gated ion channels are expressed in several cancers and contribute significantly to cell signaling, cell cycle progression and cell volume regulation, cancer cell proliferation, as well as metastasis. Hence, there is a great deal of interest in possible therapeutic potential of voltage gated ion channels as pharmacological targets [20, 21].

#### **2.2 Metastatic breast cancer in brain microenvironment**

Cancer cells have the innate ability to "exploit" the "chaotic" environmental challenges surrounding them and grow uninterrupted by manipulating transportome that regulate proliferation, apoptosis, metabolism, growth factor signaling, migration and invasion. Ion channels and transporters are some of the key modulators of cancer progression in hostile tumor microenvironment that includes hypoxia. It has been suggested that modulation of ion channels by the hypoxic environment may contribute to the aggressive phenotype observed in GBM cells residing in a hypoxic environment [22]. In hostile microenvironment such as hypoxia, BKCa channels are modulated to aid cancer cell invasion and neovascularization. Affymetrix Array analyses of brain tumor cell lines where KCNMA1 was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle, and invasion [18].

#### **2.3** *KCNMA1***/BKCa channel splice variants in breast cancer**

During the past decade, a number of genes associated with breast cancer have been cloned and identified. Gene expression levels alone cannot fully explain gene function as alternative splicing produce multiple mRNAs and protein isoforms. New molecular insights indicate that the metastatic capacity of breast tumors is an inherent feature, and not necessarily a late, acquired phenotype [23, 24]. Breast cancer cells show alternative mRNA splicing and have prognostic and therapeutic value [21]. Although there are many reports of alternative splicing events specific to breast cancer [25, 26], the biological activity of majority of alternatively spliced isoforms, and specifically their contribution to metastatic breast cancer biology, remains to be investigated. As many researchers are focusing on "Understanding and Preventing Brain Tumor Dispersal", we recently reported on a novel *KCNMA1* mRNA splice variant with a deletion of 108 base pairs (KCNMA1v) mostly overexpressed in high-grade gliomas [18]. In order to understand the role of alternative pre-mRNA splicing events of *KCNMA1/* BKCa channels, we employed specific inhibitors. We showed that the modulation of *KCNMA1/*BKCa channels in brain specific metastatic breast cancer cells (MDA-MB361) resulted in attenuation of migration, invasion [11, 17]. Further, we identified a hitherto unknown *KCNMA1* variant *KCNMA1vE22* (to be published) with a deletion of 108 base pairs of nucleotides and deletion of the entire exon-2 (*KCNMA1ΔE2*) expressed only in metastatic breast tumor cells seeking brain (**Figure 2**). However, biological function of *KCNMA1ΔE2* under different tumor microenvironment is yet to be elucidated. The *KCNMA1* splicing effects and the potential role of *KCNMA1ΔE2* as a critical posttranscriptional regulator of BKCa channel isoform resulting in diversified channel functions merit further investigation.

**73**

in **Table 1**.

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain*

lines with three different phenotypes (to be published).

**3. BKCa channels and neovascularization**

Identifying the most optimal and novel biomarker(s) for breast cancer metastasis to brain is ideal [27, 28] yet challenging because of the multi-factorial nature of the disease. The roles of roles of different ion channels in the development of cancer have been reported [29]. The identification of a potential new biomarker has relied heavily on an increase or decrease in gene expression, but these changes may not always result in altered protein expression. Growing evidence indicates that alternative or aberrant pre-mRNA splicing resulting in protein isoforms with diverse functions occurs during the development, progression, and metastasis of breast cancer [29]. Earlier, we have reported that the BKCa channels play a role in human breast tumor progression, cell proliferation, invasion, and micro-metastases [11, 17]. Nevertheless, the precise role of *KCNMA1* and its splice variants in modification of BKCa channel functions in promoting breast cancer metastasis to brain is still unclear.  In addition, using GeneChip Exon array (Affymetrix) we probed the presence of alternative splicing of *KCNMA1* in MDA-MB-361, MDA-MB-231 and MCF-7. The array data showed that *KCNMA1* splicing pattern is different among cell

**Invasion Trans-**

Untransfected 1 ± 0.09 1 ± 0.10 1 ± 0.17 1 ± 0.11 1 ± 0.21 Vector-transfected 0.98 ± 0.04 1.01 ± 0.12 0.95 ± 0.13 1.2 ± 0.15 1 ± 0.27

*Data shown are in SEM of n = 3 in triplicates. Biological function assays [11] and functional activity of BKCa channels were measured by membrane potential assay using FlexStation and in vivo mouse brain tumor models as*

*Effect of KCNMA1vE22 expression on biological functions of MDA-MB-231BR cell line.*

**endothelial migration**

1.4 ± 0.12 1.9 ± 0.15 1.5 ± 0.19 1.6 ± 0.21 3.8 ± 0.42

**Functional activity**

**Tumor volume at the 5th week**

The PCR results validated the findings of Exon array study. Two distinct splice

variants expressed in breast cell line (MDA-MB-361) metastatic to brain were identified (i) deletion of exon 2 (*KCNMA1ΔE2*) between S0-S1 protein subunit **(Figure 2**) corresponding to the cytoplasmic potential domain of BKCa channel α-subunit and (ii) deletion of 108 bp in exon 22 (*KCNMA1vE22*) between the S9 and S10 protein subunit (C-terminus). However, the biological function of these alternative splice variants in breast cancer remains to be investigated. To our knowledge we believe that our laboratory is the first to report the presence of these variants in metastatic breast cancers. We established that *KCNMA1vE22* plays a key role in several biological functions of MDA-MB-231BR cell line as represented

Altered ion channels could play a pivotal role in physiological angiogenesis in including cancer [30, 31]. BKCa channel inhibitor modulated the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway [32].

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

**(at 72 h)**

**Cell line Proliferation**

*KCNMA1vE22* transfected

*described by us earlier.*

**Table 1.**

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain DOI: http://dx.doi.org/10.5772/intechopen.84957*

*Data shown are in SEM of n = 3 in triplicates. Biological function assays [11] and functional activity of BKCa channels were measured by membrane potential assay using FlexStation and in vivo mouse brain tumor models as described by us earlier.*

#### **Table 1.**

*Breast Cancer Biology*

pharmacological targets [20, 21].

angiogenesis, cell cycle, and invasion [18].

including breast cancer and brain tumors. H3K27M serves as an oncohistone and, if mutated it contributes to tumor development as Ezh2 is no longer able to methylate

Furthermore, a recent computational analysis of human genomic sequence identified mutations that cause pathogenic splicing abnormalities in breast cancer susceptibility genes, BRCA1, BRCA2 and other genes [19]. Several investigations have reported that voltage gated ion channels are expressed in several cancers and contribute significantly to cell signaling, cell cycle progression and cell volume regulation, cancer cell proliferation, as well as metastasis. Hence, there is a great deal of interest in possible therapeutic potential of voltage gated ion channels as

Cancer cells have the innate ability to "exploit" the "chaotic" environmental challenges surrounding them and grow uninterrupted by manipulating transportome that regulate proliferation, apoptosis, metabolism, growth factor signaling, migration and invasion. Ion channels and transporters are some of the key modulators of cancer progression in hostile tumor microenvironment that includes hypoxia. It has been suggested that modulation of ion channels by the hypoxic environment may contribute to the aggressive phenotype observed in GBM cells residing in a hypoxic environment [22]. In hostile microenvironment such as hypoxia, BKCa channels are modulated to aid cancer cell invasion and neovascularization. Affymetrix Array analyses of brain tumor cell lines where KCNMA1 was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation,

During the past decade, a number of genes associated with breast cancer have been cloned and identified. Gene expression levels alone cannot fully explain gene function as alternative splicing produce multiple mRNAs and protein isoforms. New molecular insights indicate that the metastatic capacity of breast tumors is an inherent feature, and not necessarily a late, acquired phenotype [23, 24]. Breast cancer cells show alternative mRNA splicing and have prognostic and therapeutic value [21]. Although there are many reports of alternative splicing events specific to breast cancer [25, 26], the biological activity of majority of alternatively spliced isoforms, and specifically their contribution to metastatic breast cancer biology, remains to be investigated. As many researchers are focusing on "Understanding and Preventing Brain Tumor Dispersal", we recently reported on a novel *KCNMA1* mRNA splice variant with a deletion of 108 base pairs (KCNMA1v) mostly overexpressed in high-grade gliomas [18]. In order to understand the role of alternative pre-mRNA splicing events of *KCNMA1/* BKCa channels, we employed specific inhibitors. We showed that the modulation of *KCNMA1/*BKCa channels in brain specific metastatic breast cancer cells (MDA-MB361) resulted in attenuation of migration, invasion [11, 17]. Further, we identified a hitherto unknown *KCNMA1* variant *KCNMA1vE22* (to be published) with a deletion of 108 base pairs of nucleotides and deletion of the entire exon-2 (*KCNMA1ΔE2*) expressed only in metastatic breast tumor cells seeking brain (**Figure 2**). However, biological function of *KCNMA1ΔE2* under different tumor microenvironment is yet to be elucidated. The *KCNMA1* splicing effects and the potential role of *KCNMA1ΔE2* as a critical posttranscriptional regulator of BKCa channel isoform resulting in diversified channel functions

the histone and gene expression is aberrantly upregulated.

**2.2 Metastatic breast cancer in brain microenvironment**

**2.3** *KCNMA1***/BKCa channel splice variants in breast cancer**

**72**

merit further investigation.

*Effect of KCNMA1vE22 expression on biological functions of MDA-MB-231BR cell line.*

Identifying the most optimal and novel biomarker(s) for breast cancer metastasis to brain is ideal [27, 28] yet challenging because of the multi-factorial nature of the disease. The roles of roles of different ion channels in the development of cancer have been reported [29]. The identification of a potential new biomarker has relied heavily on an increase or decrease in gene expression, but these changes may not always result in altered protein expression. Growing evidence indicates that alternative or aberrant pre-mRNA splicing resulting in protein isoforms with diverse functions occurs during the development, progression, and metastasis of breast cancer [29]. Earlier, we have reported that the BKCa channels play a role in human breast tumor progression, cell proliferation, invasion, and micro-metastases [11, 17]. Nevertheless, the precise role of *KCNMA1* and its splice variants in modification of BKCa channel functions in promoting breast cancer metastasis to brain is still unclear.  In addition, using GeneChip Exon array (Affymetrix) we probed the presence of alternative splicing of *KCNMA1* in MDA-MB-361, MDA-MB-231 and MCF-7. The array data showed that *KCNMA1* splicing pattern is different among cell lines with three different phenotypes (to be published).

The PCR results validated the findings of Exon array study. Two distinct splice variants expressed in breast cell line (MDA-MB-361) metastatic to brain were identified (i) deletion of exon 2 (*KCNMA1ΔE2*) between S0-S1 protein subunit **(Figure 2**) corresponding to the cytoplasmic potential domain of BKCa channel α-subunit and (ii) deletion of 108 bp in exon 22 (*KCNMA1vE22*) between the S9 and S10 protein subunit (C-terminus). However, the biological function of these alternative splice variants in breast cancer remains to be investigated. To our knowledge we believe that our laboratory is the first to report the presence of these variants in metastatic breast cancers. We established that *KCNMA1vE22* plays a key role in several biological functions of MDA-MB-231BR cell line as represented in **Table 1**.

#### **3. BKCa channels and neovascularization**

Altered ion channels could play a pivotal role in physiological angiogenesis in including cancer [30, 31]. BKCa channel inhibitor modulated the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway [32].

Our work shows an association between the BKCa channel isoform expression and VEGF secretion by breast tumor cells, which might be exacerbated under hypoxia that has implications for vascular permeability and anticancer drug delivery (to be published). Understanding the underlying mechanism and splicing patterns of *KCNMA1* and expression of the splice variant *KCNMA1ΔE2* under normoxia and hypoxia alone and in coculture with brain endothelial cells will shed light on the role of *KCNMA1* alternative splicing in metastatic breast tumor biology. Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis [33]. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* that we have discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive exercise for the identification of a new generation of biomarkers. *KCNAM1*/BKCa channels are hypothesized to be involved in VEGF secretion and neovascularization in brain tumors. We tested this hypothesis by activation and suppression of *KCNMA1* in brain tumor cells and constructed a potential VEGF signaling pathway adapted from KEGG VEGF signaling pathway (**Figure 3**).

We rationalize that *KCNMA1ΔE2* is expressed specifically in metastatic breast tumors in brain, and this requires validation to confirm its role as a potential transformation biomarker of breast cancer metastasis to brain. In metastatic breast tumor cells seeking brain there is an upregulation and constitutive activation of *KCNMA1*, which correlates with increased malignancy [11]. In this context,

#### **Figure 3.**

*Adapted from KEGG-VEGF signaling pathway: we activated and suppressed KCNMA1 in brain tumor cells and constructed a probable VEGF signaling pathway affected by modified KCNAMA1 expression. The genes in rectangular boxes—red represents genes overexpressed by KCNMA1 overexpression and black represents genes downregulated by KCNMA1 inhibition in U-87 (glioma cells)*.

**75**

stimulating*.*

**Figure 4.**

*(brain Mets) cell lines.*

**4. Discussion**

**4.1 Splicing in health and disease**

aspects of tumor biology.

 MDA-MB-231 BR was established from the triple negative MDA-MB-231 cells, which are highly metastatic but have no organ specificity. The MDA-MB 231-BR cell line was derived from MDA-MB-231 cells following sequential rounds of implantation, resection from the brain, and re-injection into mice. Eventually a subline with selectivity for the brain was isolated [34], and exhibit higher KCNMA1 level than parental MDA-MB-231 cells, however the expression was far lower than the

*BKCa channels in breast cancer biology-expression of KCNMA1 by qPCR (A) and alternate splice variants (B) using Affymetrix Genechip Exon Array in MCF-7 (non Mets), MDA-MB-231 (Mets) and MDA-MB-361*

In addition, alternate splicing of *KCNMA1* [17] including *KCNMA1ΔE2* may provide a mechanism to generate a physiologically diverse complement of functionally and structurally diverse BKCa channel isoform that might affect cell proliferation, cell cycle, migration and micrometastases in brain. Future studies will validate the role of *KCNMA1ΔE2* in brain-specific metastatic process. Inhibiting *KCNMA1ΔE2 in in vitro* and *in vivo* models with shRNA or the variant BKCa channel using specific inhibitor like Iberiotoxin to attenuate breast tumor metastasis to brain using human metastatic breast tumor xenograft mouse models will be very

Many human diseases are implicated to errors in mRNA splicing. These aberrant splicing also provides an opportunity to develop targeted treatment to correct the faulty gene in some genetic disorders, or target aberrant protein encoded by these gene variants in human cancers. Breast cancer-specific biomarkers might generate specific epitopes that offer targets for developing diagnostic, prognostic and immunotherapy [35]. Articles on alternative pre-mRNA splicing regulation in cancer [36] and misregulation of mRNA splicing in cancer [29] highlights the important roles in promoting aberrant splicing, which in turn contributes to all

naturally-selected MDA-MB-361 cells (**Figure 4**).

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain DOI: http://dx.doi.org/10.5772/intechopen.84957*

**Figure 4.**

*Breast Cancer Biology*

ing pathway (**Figure 3**).

Our work shows an association between the BKCa channel isoform expression and VEGF secretion by breast tumor cells, which might be exacerbated under hypoxia that has implications for vascular permeability and anticancer drug delivery (to be published). Understanding the underlying mechanism and splicing patterns of *KCNMA1* and expression of the splice variant *KCNMA1ΔE2* under normoxia and hypoxia alone and in coculture with brain endothelial cells will shed light on the role of *KCNMA1* alternative splicing in metastatic breast tumor biology. Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis [33]. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* that we have discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive exercise for the identification of a new generation of biomarkers. *KCNAM1*/BKCa channels are hypothesized to be involved in VEGF secretion and neovascularization in brain tumors.  The genes in rectangular boxes—red represents genes overexpressed by KCNMA1 overexpression and black represents genes*

*downregulated by KCNMA1 inhibition in U-87 (glioma cells)*.

tumors in brain, and this requires validation to confirm its role as a potential transformation biomarker of breast cancer metastasis to brain. In metastatic breast tumor cells seeking brain there is an upregulation and constitutive activation of *KCNMA1*, which correlates with increased malignancy [11]. In this context,

**74**

**Figure 3.**

*BKCa channels in breast cancer biology-expression of KCNMA1 by qPCR (A) and alternate splice variants (B) using Affymetrix Genechip Exon Array in MCF-7 (non Mets), MDA-MB-231 (Mets) and MDA-MB-361 (brain Mets) cell lines.*

we showed (**Figure 4**) that the *KCNMA1* is overexpressed in breast cancer cells metastatic to brain (MDA-MB-361) and exhibit differences in expression levels in other non-metastatic (MCF-7) and metastatic to other organs (MDA-MB-231). MDA-MB-231 BR was established from the triple negative MDA-MB-231 cells, which are highly metastatic but have no organ specificity. The MDA-MB 231-BR cell line was derived from MDA-MB-231 cells following sequential rounds of implantation, resection from the brain, and re-injection into mice. Eventually a subline with selectivity for the brain was isolated [34], and exhibit higher KCNMA1 level than parental MDA-MB-231 cells, however the expression was far lower than the naturally-selected MDA-MB-361 cells (**Figure 4**).

In addition, alternate splicing of *KCNMA1* [17] including *KCNMA1ΔE2* may provide a mechanism to generate a physiologically diverse complement of functionally and structurally diverse BKCa channel isoform that might affect cell proliferation, cell cycle, migration and micrometastases in brain. Future studies will validate the role of *KCNMA1ΔE2* in brain-specific metastatic process. Inhibiting *KCNMA1ΔE2 in in vitro* and *in vivo* models with shRNA or the variant BKCa channel using specific inhibitor like Iberiotoxin to attenuate breast tumor metastasis to brain using human metastatic breast tumor xenograft mouse models will be very stimulating*.*

#### **4. Discussion**

#### **4.1 Splicing in health and disease**

Many human diseases are implicated to errors in mRNA splicing. These aberrant splicing also provides an opportunity to develop targeted treatment to correct the faulty gene in some genetic disorders, or target aberrant protein encoded by these gene variants in human cancers. Breast cancer-specific biomarkers might generate specific epitopes that offer targets for developing diagnostic, prognostic and immunotherapy [35]. Articles on alternative pre-mRNA splicing regulation in cancer [36] and misregulation of mRNA splicing in cancer [29] highlights the important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.

#### **4.2 BKCa channels as target to attenuate breast cancer metastasis**

*The* BKCa channels are known to function as oncogenes in certain cancers. These channels besides being sensitive to [Ca2+ i] are highly dependent on amounts of outward K<sup>+</sup> currents, which modulate the transmembrane potential of a cell. The BKCa channels are overexpressed in many types of cancers via gene amplification, alternative splicing or increased protein half-life. A recent study showed that by inhibiting BKCa channels with Iberiotoxin in breast cancer cells, tumorigenicity was reduced by downregulation of β-catenin and (phospho)Akt and HER-2/neu protein levels [37]. Evidence presented above clearly show that over expression of wild type BKCa channels or the presence of BKCa channel variant support breast cancer metastasis to brain. Understanding the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast cancers that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective diagnosis, prognosis and treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy. Activation of BKCa channels was shown to be a novel molecular pathway involved in zoledronic acidinduced apoptosis of MDA-MB-231 cells *in vitro* [32]. Du et al. [8] showed that BKCa promotes growth and metastasis of prostate cancer through facilitating the coupling between αvβ3 integrin and FAK. BKCa channels are shown to support cancer cell migration, invasion and tumorigenesis [11, 17, 18]. Hence it is extremely interesting to explore BKCa channels as putative targets for anti-breast cancer therapies.

#### **4.3 Alternate splicing of BKCa channels in diagnosis and prognosis**

Several articles have highlighted the use of alternative splicing as a promising source for new diagnostic, prognostic, predictive, and therapeutic tools [38–40]. The diversity of RNA species detected through RNA-seq holds the potential of extracellular RNAs as non-invasive diagnostic indicators of disease [41–44]. We recently reported that targeting the KCNMA1 variants may be a clinically beneficial strategy to prevent or at least slow down glioma transformation to GBM [18]. In both human and mouse lymphoma models, researchers have shown that MYC directly induced the transcription of genes encoding core splicing machinery components. They also showed that PRMT5 is involved in MYC-driven tumorigenesis in mice with lymphoma and discovered that tumor development was delayed [44]. Now due to high-throughput New Generation Sequencing (NGS) technologies the splicing diagnostic methodologies have improved. Hence NGS can be utilized in clinical diagnostics of splice variants in diagnosis, prognosis and treatment of breast cancers.

#### **4.4 Perspective of** *KCNMA1* **splice variants**

We believe that future therapies for metastatic breast cancer depend on further investigation into the mechanisms and cellular events caused by oncogene splicing such as *KCNMA1*. Such studies should lead to the development of future therapies for this deadly type of cancer. *KCNMA1* splice variants that are identified in breast tumor patients with brain metastasis will pave for accurate diagnosis and prognostication. Furthermore, they provide potential targets for anticancer drug development. Clinical outcome of *KCNMA1vE22* expression in breast metastasis is expected to reveal the variants' clinical importance. Quantifying the levels of *KCNMA1vE22* could be useful to identify biological process that increases the malignancy and affect prognosis of patients with breast cancer metastasis in the brain.

**77**

*Evidence of BKCa Channelopathy-Driven Breast Cancer Metastasis to Brain*

Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* and *KCNMA1E22* that we have recently discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive research to identify new generation of biomarkers for early detection and therapeutic intervention in breast cancer patients with high risk for

The authors thank the Scintilla Group, Bangalore, India; Anderson Cancer Institute and Mercer University Medical Center, Savannah, GA, USA; Vanderbilt-Ingram Cancer Center, Nashville, TN, USA; Cedars-Sinai Medical Center, Los Angeles, CA, USA; American Cancer Society, USA; Georgia Cancer Coalition, Atlanta, GA, USA; and NIH for providing opportunity and research grant support. We also thank Dr. Nagendra of MVIT, Bangalore, for assisting us with the STRING software for the analysis and Michigan State University Research Center, Grand Rapids, MI, USA, for generating KKEG pathway using Affymetrix analysis data.

*DOI: http://dx.doi.org/10.5772/intechopen.84957*

**5. Conclusions**

brain metastases.

**Author details**

Divya Khaitan1

and Nagendra Ningaraj1,2\*

\*Address all correspondence to<EMAIL_ADDRESS>
Education and Research, Bangalore, India

provided the original work is properly cited.

1 Department of Molecular Oncology, Scintilla Academy for Applied Sciences'

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Molecular Diagnostics Labs, Scintilla Bio-MARC, Bangalore, India

**Acknowledgements**

### **5. Conclusions**

*Breast Cancer Biology*

outward K<sup>+</sup>

channels besides being sensitive to [Ca2+

**4.2 BKCa channels as target to attenuate breast cancer metastasis**

*The* BKCa channels are known to function as oncogenes in certain cancers. These

BKCa channels are overexpressed in many types of cancers via gene amplification, alternative splicing or increased protein half-life. A recent study showed that by inhibiting BKCa channels with Iberiotoxin in breast cancer cells, tumorigenicity was reduced by downregulation of β-catenin and (phospho)Akt and HER-2/neu protein levels [37]. Evidence presented above clearly show that over expression of wild type BKCa channels or the presence of BKCa channel variant support breast cancer metastasis to brain. Understanding the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast cancers that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective diagnosis, prognosis and treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy. Activation of BKCa channels was shown to be a novel molecular pathway involved in zoledronic acidinduced apoptosis of MDA-MB-231 cells *in vitro* [32]. Du et al. [8] showed that BKCa promotes growth and metastasis of prostate cancer through facilitating the coupling between αvβ3 integrin and FAK. BKCa channels are shown to support cancer cell migration, invasion and tumorigenesis [11, 17, 18]. Hence it is extremely interesting

to explore BKCa channels as putative targets for anti-breast cancer therapies.

Several articles have highlighted the use of alternative splicing as a promising source for new diagnostic, prognostic, predictive, and therapeutic tools [38–40]. The diversity of RNA species detected through RNA-seq holds the potential of extracellular RNAs as non-invasive diagnostic indicators of disease [41–44]. We recently reported that targeting the KCNMA1 variants may be a clinically beneficial strategy to prevent or at least slow down glioma transformation to GBM [18]. In both human and mouse lymphoma models, researchers have shown that MYC directly induced the transcription of genes encoding core splicing machinery components. They also showed that PRMT5 is involved in MYC-driven tumorigenesis in mice with lymphoma and discovered that tumor development was delayed [44]. Now due to high-throughput New Generation Sequencing (NGS) technologies the splicing diagnostic methodologies have improved. Hence NGS can be utilized in clinical diagnostics of splice variants in diagnosis, prognosis and treatment of

We believe that future therapies for metastatic breast cancer depend on further investigation into the mechanisms and cellular events caused by oncogene splicing such as *KCNMA1*. Such studies should lead to the development of future therapies for this deadly type of cancer. *KCNMA1* splice variants that are identified in breast tumor patients with brain metastasis will pave for accurate diagnosis and prognostication. Furthermore, they provide potential targets for anticancer drug development. Clinical outcome of *KCNMA1vE22* expression in breast metastasis is expected to reveal the variants' clinical importance. Quantifying the levels of *KCNMA1vE22* could be useful to identify biological process that increases the malignancy and

affect prognosis of patients with breast cancer metastasis in the brain.

**4.3 Alternate splicing of BKCa channels in diagnosis and prognosis**

currents, which modulate the transmembrane potential of a cell. The

i] are highly dependent on amounts of

**76**

breast cancers.

**4.4 Perspective of** *KCNMA1* **splice variants**

Perhaps the discovery and validation of brain specific metastasis-associated *KCNMA1* alternate splice variants will serve as new tools for the diagnosis and classification of breast tumor patients with high risk of brain metastasis. In fact, splice variations in a number of genes have already been shown to correlate with malignancy and their occurrence could precede clinical cancer diagnosis. To date, however brain-specific alternate *KCNMA1* splice variants in breast cancer have not been reported. The variant *KCNMA1ΔE2* and *KCNMA1E22* that we have recently discovered potentially may fill the gap to serve as a biomarker of breast cancer metastasis to brain. Undoubtedly, the research on the putative association between *KCNMA1* splice variants and breast cancer metastases to brain will prove to be an extremely productive research to identify new generation of biomarkers for early detection and therapeutic intervention in breast cancer patients with high risk for brain metastases.

### **Acknowledgements**

The authors thank the Scintilla Group, Bangalore, India; Anderson Cancer Institute and Mercer University Medical Center, Savannah, GA, USA; Vanderbilt-Ingram Cancer Center, Nashville, TN, USA; Cedars-Sinai Medical Center, Los Angeles, CA, USA; American Cancer Society, USA; Georgia Cancer Coalition, Atlanta, GA, USA; and NIH for providing opportunity and research grant support. We also thank Dr. Nagendra of MVIT, Bangalore, for assisting us with the STRING software for the analysis and Michigan State University Research Center, Grand Rapids, MI, USA, for generating KKEG pathway using Affymetrix analysis data.

### **Author details**

Divya Khaitan1 and Nagendra Ningaraj1,2\*

1 Department of Molecular Oncology, Scintilla Academy for Applied Sciences' Education and Research, Bangalore, India

2 Molecular Diagnostics Labs, Scintilla Bio-MARC, Bangalore, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**81**

Section 3

Different Treatment

Strategies for Breast

Cancer

Section 3


3.2.1. Swelling Test

From a practical point of view, the membranes should absorb the exudate from the wound and at the same time provide a moist environment that promotes healing. For this reason, the percentage of swelling of five different formulations was determined by weight difference (Figure 2).

**Figure 2.** Percentage of swelling in alginate membranes as a function of time. Effect on swelling capacity by the addition of PVA + glycerin (**M1**), PVA + glycerin + propylene glycol (**M2**), PVP + glycerin (**M3**), PVP + glycerin + propylene glycol (**M4**), and CNp + PVP + glycerin + propylene glycol (**CNp-M4**) (mean ± SE; *n* = 3). There were significant differences when ANOVA was applied at 5, 20, 30, and 60 min (*p* < 0.05).

Figure 2 depicts a di fference at a time of 20 min, when propylene glycol is added to the formulation with PVA (M2) in comparison to M1; however, there is no significant di fference between using or not using propylene glycol in the formulation combined with PVP (M3 and M4, respectively). In order to determine the di fferences in the percentage of swelling according to each formulation as a function of time, an ANOVA was performed. It was demonstrated that both the formulation and the time during which the membrane was exposed to PBS exerted a statistically significant e ffect on swelling with a 95% confidence level.

The formulation with the highest swelling value was M1, at 30 min (802.8 ± 76.0%), while the addition of propylene glycol (M2) decreased the swelling percentage after 10 min. On the other hand, M2 showed the lowest value in the swelling test before dissolving. The addition of propylene glycol to the polyvinyl alcohol mixture could increase the intermolecular interactions between both excipients via hydrogen bonds in the –OH groups, favored by a wide steric disposition, but could decrease the entry capacity of water molecules and their interaction with alginate by saturation, decreasing the swelling ability. In the case of the interaction of propylene glycol and PVP, the situation could be the opposite. The interaction of both excipients is lower, resulting in a lower swelling capacity of the alginate; i.e., the excipient that determines the majority of the response. This phenomenon is confirmed with the profile observed for CNp-M4 in relation to M4.

Namely, when water enters the polymer matrix, the chains begin to relax, giving rise to the opening of the polymer networks. This promotes the penetration of more water; however, in the last stages of swelling, the di ffusion coe fficient is diminished because the chains are completely relaxed and near equilibrium [30].

The membranes began to dissolve after being exposed to PBS for a longer period of time. According to the composition of the medium, polymers undergo degradation and erosion processes. When a polymer degrades, the chains are cleaved into oligomers and subsequently monomers. The continuous loss of monomers will eventually lead to the phenomenon of erosion, which progressively changes the microstructure of the membrane through the formation of pores [31]. The combination of these processes could favor the possible application of our M4 and CNp-M4 membranes as wound dressings, because it would not be necessary to remove them from the application site, avoiding harm through injury and discomfort to the patient.

Moreover, the level of exudate from a wound (for example, an ulcer) can vary from absent (dry ulcer) to minimally exuding (<5 mL fluid per 24 h), to moderately exuding (5 to 10 mL fluid per 24 h), and finally to highly exuding (>10 mL fluid per 24 h) [32]. In this regard, the measurement of the swelling capacity of a wound dressing developed with alginate could be classified as dressings of low absorbance (alginate wound dressing that absorbs less than 6 g of liquid per g of dressing, or less than 12 g/100 cm2), and dressings of high absorbency (an alginate wound dressing that absorbs 6 g or more liquid per g of dressing, or 12 g or more/100 cm2) [33]. With this consideration, CNp-M4 possesses a value of 17.48 g/100 cm2, corresponding to high absorbency and similar to several commercial products.

## 3.2.2. Mechanical Test

Wound dressings must be resistant and flexible for ease of handling [17]. Thus, the mechanical properties of the membranes are depicted in Figure 3. Formulations with PVA (M1 and M2) did not demonstrate a significant di fference in %E with 73.54 ± 0.87% and 74.90 ± 1.67%, respectively. TS was similar for M1, M2, and M3 samples with 1.32 ± 0.02, 1.34 ± 0.01, and 1.96 ± 0.05 MPa, respectively. By way of comparison, M1 and M2 membranes exhibited a lower TS and %E than the remaining formulations, probably due to PVA being a polymer that has been characterized as possessing poor elasticity, a rigid membrane, and low hydrophilic characteristics [34].

**Figure 3.** Effect of plasticizers on the mechanical properties of alginate membranes. Effect on mechanical properties by the addition of PVA + glycerin (M1), PVA + glycerin + propylene glycol (M2), PVP + glycerin (M3), PVP + glycerin + propylene glycol (M4), and CNp + PVP + glycerin + propylene glycol (CNp-M4), respectively. (**a**) Elongation at break; (**b**) tensile stress, (mean ± SD; *n* = 3). \* indicates *p* < 0.05 as statistically significant.

The %E and TS ofM3 were 68.63±6.75% and 1.18±0.07MPa, respectively; these values significantly increased with the addition of propylene glycol (M4), to 120.01±5.97% and 1.96 ± 0.05 MPa, respectively. M4 exhibited the highest values in the assay and showed a significant difference with respect to the remaining formulations without CNp. This may be explained, at least in part, by the properties of propylene glycol, which is a plasticizer with a small molecular weight that is able to create multiple H-bonds with PVP and SA chains into a package and, consequently, aid in the formation of cross-linked networks [35].

Therefore, because of its greater swelling capacity and better mechanical properties, the M4 membrane (a mixture of sodium alginate, PVP, and propylene glycol) was chosen for the incorporation of CNp.

On the other hand, the mechanical properties of M4 were modified when CNp dispersion was added to the formulation. The %E of CNp-M4 showed the highest value, with 144.39 ± 14.52%; in contrast, TS decreased to 1.52 ± 0.16 MPa. This could be due to the addition of CNp dispersion to the formulation decreasing the number of hydrogen bonds between the polymer molecular chains; as a result, less strength is necessary to break the membrane [36]. In comparison, CNp-M4 showed the highest %E, which could be due to the effect of Pluronic® F-68. The latter is a surfactant that decreases the pore number, providing a membrane with a homogeneous structure; thus, it is more resistant to changes, rendering higher elasticity properties.

### 3.2.3. Thermogravimetric Analysis (TGA)

It is important to determine the thermal properties of a substance, because these provide useful information for their identification and the characterization of materials. In Figure 4, thermograms of curcumin, CNp, M4, and CNp-M4 membranes are presented. For curcumin, mass loss was observed at 173 ◦C by TGA (Figure 4a); due to the degradation of turmeric powder, water loss was not observed, possibly due to its high hydrophobicity [37]. The weight loss of CNp started at approximately 90 ◦C, corresponding to dehydration, and there was a second plateau from 280 to 350 ◦C, suggesting a better thermal stability for curcumin when it is inside PCL nanoparticles than when alone. However, CNp lost more weight in a smaller temperature range.

**Figure 4.** Thermal analysis of nanoparticle-coated alginate membrane using (**a**) thermogravimetric analysis (TGA) and (**b**) differential scanning calorimetry (DSC). The thermal properties of the M4 membrane, CNp-M4 membrane, CNp, and curcumin are represented as a, b, c, and d, respectively.

On the other hand, in the M4 membrane, the first mass loss occurred between 90 and 240 ◦C, whereas for the CNp-M4 sample, weight loss began between 100 and 240 ◦C. This could be due to the evaporation of water traces, the degradation of propylene glycol, PVP, glycerin (150-220 ◦C), and turmeric powder [38]. In the case of the CNp-M4 membrane, it presented a slighter weight loss compared to the M4 membrane. The second mass loss of both samples was between 270 and 425 ◦C; in this stage, the decomposition of the functional groups of SA polymer chains is presented. Finally, the last plateau in M4 and CNp-M4 membranes started at 425 ◦C, which corresponds to the degradation of the SA backbone [19]. In the same manner, the CNp-M4 membrane thermogram revealed a lower weight-loss temperature compared with that of CNp; this is probably because membrane formulation is a mixture that contains more substances than CNp. This is similar to the thermal behavior exhibited by curcumin and CNp.

### 3.2.4. Differential Scanning Calorimetry (DSC)

DSC is a technique used to determine the quantity of heat either absorbed or released when substances undergo physical or chemical changes [39]. In Figure 4b, DSC thermograms of curcumin, CNp, M4 membrane as vehicle, and CNp-M4 membrane are presented. The melting point of curcumin was found to be 174 ◦C (Table 2), which was expected with regard to the literature [40]. Furthermore, three thermal events were observed in the CNp: at 63.5; 101, and 212 ◦C. The first thermal event could correspond to the melting point of PCL (61 ◦C) [41], while events at 101 and 212 ◦C may indicate the presence of trehalose, which was employed as a cryoprotectant to lyophilize the CNp [42]. Interestingly, the melting point of curcumin was not detected in the CNp sample; this could be due to the high EE of curcumin inside PCL nanoparticles as a molecular dispersion.

**Table 2.** Thermal events of curcumin, CNp, and alginate membranes by DSC.

On the other hand, both M4 and CNp-M4 membranes revealed two thermal events. The first peak was around 87 ◦C for both formulations, whereas the second peak was around 233 ◦C for the M4 membrane and 249 ◦C for the CNp-M4 membrane. The latter peaks were due to the presence of SA in the formulation. A thermal peak prior to 100 ◦C was observed for both samples, possibly due to the presence of water in the membranes.


**Comparing the Efficacy of OnabotulinumtoxinA, Sacral Neuromodulation, and Peripheral Tibial Nerve Stimulation as Third Line Treatment for the Management of Overactive Bladder Symptoms in Adults: Systematic Review and Network Meta-Analysis**

**Chi-Wen Lo 1,2,3, Mei-Yi Wu 4, Stephen Shei-Dei Yang 2,3, Fu-Shan Jaw 1 and Shang-Jen Chang 2,3,\***

Received: 11 December 2019; Accepted: 5 February 2020; Published: 18 February 2020

**Abstract:** The American Urological Association guidelines for the management of non-neurogenic overactive bladder (OAB) recommend the use of OnabotulinumtoxinA, sacral neuromodulation (SNM), and peripheral tibial nerve stimulation (PTNS) as third line treatment options with no treatment hierarchy. The current study used network meta-analysis to compare the efficacy of these three modalities for managing adult OAB syndrome. We performed systematic literature searches of several databases from January 1995 to September 2019 with language restricted to English. All randomized control trials that compared any dose of OnabotulinumtoxinA, SNM, and PTNS with each other or a placebo for the management of adult OAB were included in the study. Overall, 17 randomized control trials, with a follow up of 3–6 months in the predominance of trials (range 1.5–24 months), were included for analysis. For each trial outcome, the results were reported as an average number of episodes of the outcome at baseline. Compared with the placebo, all three treatments were more efficacious for the selected outcome parameters. OnabotulinumtoxinA resulted in a higher number of complications, including urinary tract infection and urine retention. Compared with OnabotulinumtoxinA and PTNS, SNM resulted in the greatest reduction in urinary incontinence episodes and voiding frequency. However, comparison of their long-term efficacy was lacking. Further studies on the long-term effectiveness of the three treatment options, with standardized questionnaires and parameters are warranted.

**Keywords:** network meta-analysis; OnabotulinumtoxinA; overactive bladder; peripheral tibial nerve stimulation; sacral neuromodulation

**Key Contribution:** Updated systematic review to compare the efficacy of three existing third line treatments for the management of adult overactive bladder using network meta-analysis.


*Edited by Amar Patnaik*

Fillers - Synthesis, Characterization and Industrial Application comprises a set of chapters that brings an interdisciplinary perspective to accomplish a more detailed understanding of filler materials for the synthesis and characterization of different industrial applications. This book embraces all the chapters that are concerned with the effect of incorporating different fillers or particulates in fabricated composites. Significant research efforts all around the world are continuing to explore the properties of composite materials. Researchers are collectively focusing their efforts on the use of particulate fillers in composites for miscellaneous applications. This book delivers a comprehensive study associated with the sections of material science, polymer technology, anisotropic elasticity phenomena, fracture mechanics, applied mechanics, material synthesis, mechanical and thermo-mechanical characteristics, tribological behavior, etc.

ISBN 978-1-78985-791-7

Fillers - Synthesis, Characterization and Industrial Application

Published in London, UK © 2019 IntechOpen

© Dmytro Synelnychenko / iStock


**Author details**

**6.** Isosilybin A (ISBN) (extracted from *Silybum marianum*) activates apoptotic machinery in

**Plant Mode of action References**

14

33 37

54

Significantly induced caspase-3 mRNA and suppressed expression of the pro survival kinase Akt-1. Apoptosis was induced by activation of caspase-3 and

Significant block of G1 to S phase transition manifested by the increase of cell

Up-regulates DNA-protein kinase-dependent p53 activation to enhance UVB-

Activates apoptotic machinery in human prostate cancer cells via targeting Akt–

Inhibition of microtubule assembly 70

The topoisomerase-mediated DNA damage seems to be a candidate mechanism, by which some flavonoids may exert their cytotoxic potential

Inhibits active Stat3 phosphorylation, and causes caspase activation

**Table 4.** Mode of action of anticancer activity of phytochemicals present in selected North American medicinal plants

Currently, natural products, especially plant secondary metabolites such as isoprenoids, phenolics and alkaloids, have been demonstrated to be the leading providers of novel anticancer agents. Thiese important groups of phytochemicals represent a vast majority of chemical groups, including alkaloids, flavonoids, flavonols, flavanols, terpenes and terpe‐ noids, phenols, flavonolignans and steroids. Potential anticancer properties of these phyto‐ chemicals have been shown by both cell culture (*in vitro* methods) and animal (*in vivo* methods) studies. However, *in vitro* and *in vivo* findings should be strengthened by valid human clinical

trial data before introducing to the medicine cabinet as natural therapeutics or drugs.

**CEM**, Human drug-sensitive leukemia cells; **CEM/VLB**, Human multidrug-resistant-derived leukemia cells; **Jurkat clone E6-1**, Acute T-cell leukemia cells; **WiDr** and **HT29**, Human colon

human prostate cancer cells via targeting Akt–NF-kB–AR axis.

174 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

inhibiting Akt phosphorylation.

number in G0/G1 phase

induced apoptosis

Increases total p53 levels

NF-kB–AR axis

Activation of the mitochondrial apoptotic pathway

Induces G1 arrest in cell cycle progression

**7.** ISBN increases p53 protein levels.

Achyranthes aspera (Devil's Horsewhip)

Olea europaea (Olive)

Plantago lanceolata (Ribwort plantain)

Silybum marianum (Milkthistle)

Podophyllum peltatum

**6. Conclusion**

**Abbreviations**

(Mayapple)

Wasundara Fernando and H. P. Vasantha Rupasinghe\*

\*Address all correspondence to<EMAIL_ADDRESS>
Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia, Canada

## **References**

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Meet the editors

Ziyad S. Haidar, DDS, Cert Implantol, MSC, FRCSc, MBA, Ph.D., is a Full Professor of Biomaterials and Tissue Engineering and the scientific director of the Facultad de Odontología (Faculty of Dentistry), Universidad de los Andes (UAndes), Santiago, Chile. He is also the founder and head of the Biomaterials, Pharmaceutical Delivery, and Cranio-Maxillo-Facial Tissue Engineering Laboratory (BioMAT'X R&D&I Chile – HAIDAR

Lab). In addition, he serves as the head of innovation at the Centro de Investigación e Innovación Biomédica (CiiB), a faculty/theses member in the bioMedicine Doctoral (Ph.D. bioMedicina) Program at UAndes, and a visiting clinical and surgical professor at the MaxilloFacial Division of the Universidad de la Frontera and the Department of Head and Neck Surgery, Lautaru Hospital, both in Temuco, Chile. Dr. Haidar is a trained dentist, implantologist, and an oral and maxillofacial surgeon with a Ph.D. in Nanobiomaterials, Pharmaceuticals, and Tissue Engineering from McGill University, Montréal, Canada. He completed a post-doctoral training residency in orthopedics at the Montréal Shriners Hospital, McGill University Health Center, Montréal, Canada. Before moving to Chile, he served as Associate Professor of Bioceramics and the Chair of Excellence in BioEngineering at the Université de Limoges, Limoges, France and was an assistant professor in the Department of Pharmaceutics and Pharmaceutical Chemistry (cross-appointment with the Department of BioEngineering), University of Utah, Salt Lake City, UT, USA. Between 2010 and 2012 Dr. Haidar served as an adjunct professor of Head and Neck Surgery and the scientific director of the joint Utah–Inha R&D Center, Inha University Hospital, Incheon, Seoul, South Korea. He has won several prestigious awards from the International Bone and Mineral Society, Society for Biomaterials, Canadian Biomaterial Society, and the Canadian and Lebanese Societies of Plastic Surgeons, to name a few. His R&D&I focus on patient-oriented development and evaluation of bionanotechnology, biopolymers, bioceramics, and drug delivery systems for the repair, restoration, reconstruction, and regeneration of challenging craniofacial and orthopedic defects. Dr. Haidar is an international speaker with more than 125 publications, conference proceedings, textbooks, and patents to his credit. He is also an editorial board member of several national and international scientific journals and periodicals.

Ibrokhim Y. Abdurakhmonov received his B.S. (1997) *in biotechnology* from the National University, M.S. i*n plant breeding* (2001) from Texas A&M University of USA, Ph.D. (2002) *in molecular genetics*, Doctor of Science (2009) *in genetics*, and full professorship (2011) i*n molecular genetics and molecular biotechnology* from Academy of Sciences of Uzbekistan. He founded (2012) the Center of Genomics and Bioinformatics of Uzbekistan. He

received the 2010 TWAS prize, and "ICAC Cotton Researcher of the Year 2013" for his outstanding contribution to cotton genomics and biotechnology. He was elected as The World Academy of Sciences (TWAS) Fellow (2014) and as a member (2017) of the Academy of Sciences of Uzbekistan. He was appointed (2017) as a Minister of Innovative Development of Uzbekistan.

Abdelwahed Barkaoui is an Associate Professor of Mechanical Engineering at the International University of Rabat. He obtained his University habilitation from the University of Tunis El Manar-Tunisia in 2017 and his Ph.D. from the University of Orleans-France in 2012. he has a master's degree in mechanics obtained from the INSA of Lyon-France and a diploma of an engineer in electromechanical from ENI-Sfax Tunisia. Currently,

assistant director of LERMA laboratory and coordinator of Modelling & Simulation in Biomechanics & Biomaterials (MS2B) team. He is responsible for mechanical discipline and coordinator of the ABET accreditation project at the Higher School of Energy Engineering. his research mainly concerns problems in biomechanics, mechanobiology, and biomedical engineering. He was a member of the editorial board of several international scientific journals: Frontiers in Bioengineering and Biotechnology "biomechanics" (IF=3,66), Series on Biomechanics, and reviewer for several international journals known in the field of biomechanics and mechanical engineering. Besides, he is the author of more than 60 publications in international journals, books, and conferences.


*3.8. Texture Analysis*

A TA.XTplus texture analyzer (Stable Micro Systems Co. Ltd., Godalming, UK) equipped with a load cell of 5 kg was used to determine the texture properties of biscuits. Hardness was determined by the bend test, using the 3-point bend rig HDP/3PB (Stable Micro Systems Co. Ltd., Godalming, UK) with the following settings: pre-test speed of 5 mm/s, test speed of 3 mm/s, post-test speed of 10 mm/s, distance of 5 mm and distance between the supports of 2 cm [99]. At least 10 biscuit measurements were taken for each batch. Each biscuit was compressed once and maximum force was recorded.


**6. Conclusions**

**4.8. Anti-alpha-fodrin antibodies**

128 Autoantibodies and Cytokines

**development of MALT lymphoma**

**Auto-antibody Clinical findings** Anti-Ro52 kD Interstitial lung disease

dryness Anti-SS-B/La Liver (autoimmune disease) PBC

Anti-CENP-B Interstitial lung disease and fibrosis

Anti-CA6 Dryness and renal tubular acidosis

Anti-CEP-1 Arthritis and renal tubular dysfunction.

**Table 4.** Antibodies in pSS and their association with clinical manifestation.

ACPA Arthralgia and arthritis

Novel autoantibodies

Anti-PSP Dryness Anti-SP1 Dryness

Alpha fodrin is an actin-binding, organ-specific protein of the cytoskeleton. Antibodies against alpha-fodrin are detected in serum samples from patients with primary or secondary Sjögren's syndrome especially with sicca symptoms. Some authors suggest that they can be detected earlier in the course of pSS, sometimes before the emergence of anti SS-A or SS-B antibodies [64]. These antibodies, in the IgA and IgG class of immunoglobulins, are found in the serum and salivary glands of patients with pSS. However, other researchers did not describe any

Major salivary glands are the main extra endocrine glands targeted in pSS and saliva of patients with pSS is also a source of antibodies and cytokines. Large salivary glands are also the site for MALT lymphoma development. Investigators are interested in finding biomarkers in saliva, that allow for early pSS diagnosis, as well as the detection of mucosa associated lymphoma (MALT lymphoma). Cui et al. suggest set of three autoantibodies such as anti-cofilin-1 antibodies, anti-alpha enolase and anti-Rho GDP dissociation inhibitor 2(RGI2) antibodies, which, due to high specificity and sensitivity, may play a role as such biomarkers [68].

In the current pSS criteria, only anti-SS-A/Ro antibodies are taken into account as the most sensitive and specific for pSS diagnosis. Still, the multisymptom picture of this rheumatic disease compels the search for other immunologic markers of at least equal prognostic importance.

Anti-Ro60 kD Hematologic changes, photosensitivity, skin involvement, Raynaud's phenomena, and

significant sensitivity and specificity of these antibodies [65–67].

**5. Autoantibodies in course of pSS—summary**

RF Dryness, hypergammaglobulinemia, and leukopenia

**4.9. Autoantibodies in saliva that may be relevant in pSS associated with the**

pSS is a still not fully understood autoimmune disease, requiring doctor's vigilance. Even despite of a pSS having a mild course for a long time, there is a risk of organs and systems involvement. As it has been known for many years already, the risk of developing lymphomas is particularly increased in pSS patients compared to the healthy population. Although only one antibody (SS-A/Ro) has been included in the pSS diagnostic criteria, a lot of attention has been paid to new autoantibodies that can help clinicians in patient stratification in the early stages of diagnosis or may have a prognostic value.


**4. Discussion**

A 1/4 active suspension simulation model was established in MATLAB/Simulink, and a PSO-FNN-PID control algorithm was designed. By combining the PSO algorithm and the gradient descent method, the initial parameters of the FNN were optimized offline and then finetuned online, so as to obtain the optimal control rules and realize the real-time online adjustment of the PID control parameters. The combination of the two methods allowed the learning process to avoid falling into local minima while reaching the exact value at a later stage of learning. The simulation analysis indicated that the active suspension system with PSO-FNN-PID control had a preferable control effect compared with the passive suspension system and active suspension system with PID control, and could effectively attenuate the body vibration caused by external disturbance. Therefore, an examination of the graphs and data reveals that the PSO-optimized FNN-PID controller has significantly superior performance, and is capable of enhancing the vehicle's road adhesion and ensuring a smooth and comfortable ride. In the future, we will continue to explore the possibility of implementing this control system in the whole vehicle system and consider the development and implementation of related hardware and software. In addition, we also hope to make some changes to the basic PID control, such as including an integral anti-saturation control strategy, in order to obtain better results.

**Author Contributions:** Conceptualization, M.L. and J.L.; methodology, M.L.; software, J.L.; validation, J.L. and J.X.; formal analysis, J.L.; investigation, G.L.; resources, M.L.; data curation, J.L.; writing—original draft preparation, G.L.; writing—review and editing, M.L.; visualization, J.X.; supervision, M.L.; funding acquisition, M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Natural Science Foundation of Hainan Province, grant number 2019RC144, and supported by China Scholarship Council, grant number<PHONE_NUMBER>09.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.


**2.3 Privacy hazards in image analytics**

Image data analytics is widely used in health care, social media, and e-commerce applications. In social media large numbers of images are uploaded every day. An image is worth more than a thousand words and hence it may reveal the emotional state of a person also [6]. Some of the key privacy hazards in image data analytics include

Ethical code of conduct in digital data processing is required for privacy preserving data processing and it is possible only through stringent implementation of privacy regulations. More than hundred countries have passed legislations to

*Modern Privacy Threats and Privacy Preservation Techniques in Data Analytics DOI: http://dx.doi.org/10.5772/intechopen.99160*

protect individual privacy and GDPR of European Union was the pioneer. All other privacy policies and regulations are framed based on GDPR. The key features of GDPR and other privacy regulations are.

Failure to affirm the privacy compliance will attract serious consequences including huge amounts of fine and detriment to reputation. To ensure privacy preservation and abide by the local privacy regulations, firms are undergoing changes in their policies to incorporate privacy regulations. Following strategic changes were noticed in the year 2020 [10].