Molecular Targets of Benzyl Isothiocyanates in Pancreatic Cancer

as compared to Kras mutated cells (PanC-1 and MIA PaCa-2). Further studies are needed to elucidate the role of Kras mutation in TRAIL or BITC-induced apoptosis. This book provides the reader with an overall understanding of the biology of pancreatic cancer, hereditary, complex signaling pathways and alternative therapies. The book explains nutrigenomics and epigenetics mechanisms such as DNA methylation, which may explain the etiology or progression of pancreatic cancer. Book also summarizes the molecular control of oncogenic pathways such as K-Ras and KLF4. Since pancreatic cancer metastasizes to vital organs resulting in poor prognosis, special emphasis is given to the mechanism of tumor cell invasion and metastasis. Role of nitric oxide and Syk kinase in tumor metastasis is discussed in detail. Prevention strategies for pancreatic cancer are also described. The molecular mechanisms of the anti-cancer effects of curcumin, benzyl isothiocyante and vitamin D are discussed in detail. Furthermore, this book covers the basic mechanisms of resistance of pancreatic cancer to chemotherapy drugs such as gemcitabine and 5-flourouracil.


Introduction
Addiction of cancer cells to survival pathways has been well documented in most of the cancer models including the pancreatic cancer. Pancreatic cancer is one of the most aggressive tumors with an average five year survival rate of less than 5% (Jemal A et al., 2010). It is associated with high expression levels of various survival pathways, such as KRAS, STAT3, AKT, NFkB, HDAC etc. Furthermore, pancreatic cancer acquires resistance to various apoptosis signals such as FasL, TRAIL. In addition, pancreatic cancer gets resistance to various chemo-drugs including gemcitibine by altering survival pathways.
Currently, there is no effective treatment for pancreatic cancer because conventional chemotherapy including the gemcitabine and 5-FU, and radiation treatment has shown very limited success in improving the patient survival. Therefore, the development of novel approaches to prevent and treat pancreatic cancer is an important mission.
Evidence from epidemiological, pharmacological, and case-control studies continue to support the notion that isothiocyanates (ITCs) present in cruciferous vegetables may have substantial chemopreventive activity against various human malignancies including pancreatic cancer (Zhang Y et al., 1992); Stoner GD & Morse MA, 1997). Benzyl isothiocyanate (BITC), an agent that is present in cruciferous vegetables such as, watercress, cabbage, cauliflower, mustard, and horseradish, is widely consumed as part of a routine diet. BITC has been reported to inhibit initiation, growth, and metastasis of human cancers in rodents (Batra S et al., 2010;Boreddy SR et al., 2011a;Boreddy SR etal., 2011b;Kim EJ et al., 2011;Sahu RP & Srivastava SK, 2009;Zhang Y et al., 1992). The structure of BITC is shown in Fig.1. Our laboratory has shown that BITC potentially suppresses the growth and induces apoptosis in pancreatic cancer cells by targeting various key survival molecules (Batra S et al., 2010;Boreddy SR et al., 2011a;Sahu RP & Srivastava SK, 2009). In the present chapter, we discuss the current understanding of BITC, its targets and mechanism of action in pancreatic cancer.

Effect of BITC on STAT3 signalling pathway
Signal Transducer Activator Transcription (STAT3) transcription factors are latent proteins that bind to the genome on activation to either induce or to repress gene expression (Bromberg et al., 1999). STAT3 is aberrantly activated in majority of the cancers including pancreatic cancer (Wei et al., 2003). Clinical specimens have revealed that more than 50% of the breast and lung cancer, and over 95% of head and neck cancers have hyperactive STAT3 signaling (Darnell, 2005). Interestingly, STAT3 deficient mice in a chemical carcinogenesis model have shown the reduced proliferation of epithelial cells due to inability to pass through G1-S-G2 cell cycle progression (Chan et al., 2004). Furthermore, Chiarle et al. have demonstrated that disruption of STAT3 signaling by anti-sense oligoneclosides was sufficient to impair the growth of solid tumors (Chiarle, 2005), highlighting the potential of anti-STAT3 therapy in clinical medicine. Recently, numerous natural and synthetic compounds have been discovered to target STAT3 signaling. Results from our laboratory showed that benzyl isothiocyanate (BITC) targets STAT3 signaling to induce apoptosis in pancreatic cancer (Sahu & Srivastava, 2009).
Our laboratory showed that BITC significantly suppress the phosphorylation of STAT3 at both Tyr-705 and Ser-727 to induce apoptosis in BxPC-3 (Fig. 2), MIA PaCa-2, Capan-1 and PanC-1 pancreatic cancer cell lines, in a dose and time dependent manner (Sahu & Srivastava, 2009). Interestingly, BITC also down regulated the protein levels of STAT3 in these cell lines, although its functional implications are yet to be explored. Furthermore, down regulation of STAT3 protein expression by BITC was transcriptional, as evidenced by RT-PCR analysis of BITC treated BxPC-3 cells (Fig. 2). BITC-induced apoptosis was further substantiated by IL-6 treatment, which specifically phosphorylates STAT3 at Tyr-705 (Berishaj, 2007) and STAT3α overexpression. IL-6 prewww.intechopen.com treated BxPC-3 cells showed significant resistance to BITC-induced apoptosis (Fig. 3). Similarly, when STAT3α was over expressed in BxPC-3 cells, BITC-induced apoptosis was severely abrogated, indicating that BITC targets STAT3 to induce apoptosis in pancreatic cancer cells.

Effect of BITC on AKT/FOXO/Bim signaling pathway
Phosphotidyl inositol 3phosphate (PI3K)/AKT signaling plays a critical role in cell survival and growth during embryonic development as well as during normal cell survival (Finkielsztein & Kelly, 2009). However, cancer cells exploit the same pathway to overcome apoptosis induced by either therapeutic drugs or internal stimuli such as oxidative stress. Upon binding of growth factors to Tyrosine Kinase Receptor (TKR), PI3K is directly or indirectly activated by TKRs by inhibiting or removing the P85 regulatory unit of PI3K (Vivanco & Sawyers, 2002). Activated PI3K phosphorylates phosphatidylinositol and converts inositol 4,5biphosphate (PIP2) into PIP3. Consequently, AKT and PDK translocate to membrane and interact with PIP3 through PH domain leading to conformational changes in AKT to expose phosphorylation sites. AKT is phosphorylated by PDK1 at Ser-308 leading to stabilization of AKT. Yet another phosphorylation takes place at Tyr-473, which is required for full activation of AKT. In addition, another protein complex mTOR has been shown to be required for the phosphorylation of AKT (Sarbassov et al., 2005). This pathway is negatively regulated by phosphatases, such as PTEN, which dephosphorylates PIP3 thus limiting its availability (Osaki et al., 2004).
Recently, FOXO transcription factors received ample of attention in cancer because of direct involvement in apoptosis and drug resistance (Salih & Brunet, 2008). FOXO1 and FOXO3a are the members of FOXO transcription factors, which operate right under the AKT signaling. Upon growth signal stimulation, AKT is activated by phosphorylation at Ser-473, which further phosphorylates FOXO1 or FOXO3a transcription factors. Phosphorylated FOXOs bind to 14-3-3 chaperons and transported out of nucleus and subjected to proteosomal degradation (Tzivion et al., 2011). But during oxidative stress or growth factor withdrawal, AKT is dephosphorylated leading to nuclear import of FOXOs and induction of pro-apoptotic proteins such as Bim and PUMA (Obexer, 2011).
A recent report has shown that 59% of the pancreatic tumors harbor aberrantly activated AKT signaling (Schlieman et al., 2003). One of the possible reasons behind hyperactive AKT signaling in pancreatic cancer is due to mutation or deletion of PTEN gene (Sawai et al., 2008). Indeed, strategies aimed at blocking AKT activation could be a promising treatment for pancreatic cancer. Interestingly BITC significantly inhibited AKT signaling in vitro and in vivo. BITC suppressed the phosphorylation of AKT at both Ser-308 and Ser-473 in BxPC-3 and PanC-1 cells, in dose dependent manner (Boreddy et al., 2011a). Furthermore, BITC also suppressed the phosphorylation of various other key molecules of PI3K/AKT pathway such as PI3K (Tyr-458), PDK1 (Ser-241), mTOR (Ser-2448) etc. (Fig. 4), indicating that BITC targets PI3K/AKT signaling to induce apoptosis in pancreatic cancer cells. Interestingly, BITC was almost ineffective in human pancreatic ductal epithelial (HPDE-6) cells (Fig. 4). Over expression of AKT blocked the apoptosis inducing effects of BITC in pancreatic cancer cells.
Recently, FOXO transcription factor received plenty of attention as a potential target for cancer therapy, as they are directly involved in apoptosis induction. Interestingly, BITC significantly suppressed the phosphorylation of FOXO1 (Ser-256) and FOXO3a (Ser-253), without effecting the protein levels in both BxPC-3 and PanC-1 cells (Fig. 5). Moreover, immunoprecipitation studies showed that BITC treatment significantly masked 14-3-3 binding motif on FOXO proteins indicating that more of FOXO proteins were retained in the nucleus (Fig. 5B). Furthermore, BITC significantly increased the expression FOXO1 transactive genes such as P21, P27 and Bim in both the cell lines, BxPC-3 and PanC-1 (Fig. 5C).

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Apart from phosphorylation, another tier of FOXO transcription factor regulation is acetylation. Interestingly, BITC also reduced the acetylation of FOXO proteins. Probably, inhibition of acetylation by BITC was due to down regulation of CBP protein expression, since SIRTs were not altered by BITC treatment. Role of AKT in BITC-induced apoptosis was further confirmed by using PI3K inhibitor LY-294002 and overexpression of wild type AKT in BxPC-3 cells. Interestingly, when BxPC-3 cells were pre-treated with LY-294002 followed BITC (10µM) for 24h, apoptosis induction was potentiated, as compared to BITC alone treated cells (Fig. 6), whereas AKT overexpression severely abrogated BITC-induced apoptosis in BxPC-3 cells (Fig. 6). In line with apoptosis results, phosphorylation of FOXO proteins were increased with AKT overexpression, whereas Bim, P27, P21 expression was reduced. However, BITC partially blocked these effects, indicating that BITC targets AKT pathway to induce apoptosis in pancreatic cancer cells lines (Fig. 6).

BITC Regulates NFkB Activity by Inhibiting HDACs
NFkB transcription factors are mainly involved in the regulation of immune and inflammatory response, apart from cell proliferation and apoptosis Ghosh et al., 1998;Hart et al., 1998). NFkB is normally located in the cytoplasm sequestered by its endogenous inhibitor IkB. Upon cellular stimulation, IkB proteins are phosphorylated at Ser-32/36 liberating NFkB, which translocates to the nucleus and gets involved in the transcription of responsive genes such as Cyclin D1 (Sun & Andersson, 2002). NFkB may activate various survival signals to promote cell survival. NFkB is known to interfere with inducers of extrinsic apoptosis pathway by up regulating the FLIP-like inhibitory protein (Kreuz et al., 2001). NFkB also induces the expression of inhibitors of apoptosis proteins such as IAP (Deveraux et al., 1998) and some members of the Bcl2 (Shou et al., 2002) family proteins, thereby protecting the cells from various apoptosis stimuli. NFkB is also known to play critical role in drug resistance is various cancer models (Arlt et al., 2003). Hence, inhibiting of NFkB activation may potentiate the clinical efficacy of the drugs.
BITC significantly inhibits the phosphorylation of NFkB at both Ser-276 and Ser-536 in both BxPC-3 and Capan-2 pancreatic cancer cells, in a dose and time dependent manner (Fig.  7A&B). Interestingly, BITC down regulated the expression of NFkB in BxPC-3 cells but not in Capan-2 cells, indicating that BITC differentially act on different cells (Batra et al., 2010). Furthermore, BITC drastically inhibited the nuclear localization of NFkB in BxPC-3 cells (Fig. 7C). BxPC-3 cells that were transfected with a luciferase gene containing NFkBpromoter and treated with BITC demonstrated around 90% decrease in luciferase activity, as compared to control cells (Fig. 7D). Furthermore, BITC also decreased Cyclin D1 expression and transcriptional activity, as it is one of the target genes of NFkB (Fig. 7E & Fig. 7F). Interestingly expression of IKK was decreased with BITC treatment, but neither phosphorylation (Ser32/36) nor protein levels of IkB were altered in BITC treated BxPC-3 cells (Fig 7A), indicating that down regulation of IKK by BITC treatment could be the reason for inhibition of NFkB phosphorylation (Ser-536).
Apart from the phosphorylation, NFkB is known to be regulated by acetylation. Interestingly, BITC also inhibited the acetylation of NFkB on lysine residue in BxPC-3 cells. BITC suppressed the acetylation of NFkB by altering the expression of HDAC1 and HDAC3 www.intechopen.com (Fig. 8A&B), as these molecules play critical role in NFkB acetylation. In agreement with other HDAC inhibitors' data, such as veronistat (SAHA) and tricostatin A (TSA), BITC also up regulated the expression of p21 in BxPC-3 and Capan-2 cells, in a dose dependent manner (Fig. 8C).

Role of HDACs in BITC-induced NFkB deacetylation was further substantiated by HDAC overexpression in BxPC-3 cells. HDAC1/3 overexpression significantly outweighed the effects of BITC in BxPC-3 cells. Furthermore, overexpression of HDACs protected BxPC-3 cells from BITC-induced apoptosis, as indicated by the reduced cleavage of caspase-3, PARP and increased survival in HDACs overexpressing
BxPC-3 cells, as compared to BITC alone treated cells (Fig. 9).

BITC induces ROS generation, DNA damage and cell cycle arrest in pancreatic cancer cells
As many drugs induce cell death in cancer cells by triggering ROS generation, it was quite obvious to see whether BITC could induce ROS generation in pancreatic cancer cells. In agreement with other drugs, BITC caused significant generation of H2O2 in Capan-2 cells in a dose and time dependent manner (Fig. 10). On the contrary, BITC induced a modest increase in the generation of hROS, such as singlet oxygen, superoxide, nitric oxide, hydroxyl and alkyl peroxide radicals in response to BITC treatment.

B I T C -i n d u c e d R O S g e n e r a t i o n a l s o r e s u l t e d i n D N A d a m a g e a s e v i d e n c e d b y t h e
phosphorylation of H2A.X at Ser-139, which is considered to be the hall mark of DNA double strand breaks (Sedelnikova et al., 2003). Interestingly, when BITC-treated cells were cultured in fresh medium without BITC for additional 48h cells showed persistent H2A.X phosphorylation (Fig. 12), indicating that BITC induce permanent DNA damage in Capan-2 cells (Zhang et al., 2006). As protective mechanism, DNA damage lead to cell cycle arrest to obtain brief window of time to compensate/repair the damage that occurred due to ROS www.intechopen.com production. Accordingly, treatment of Capan-2 cells with BITC (10µM) for 24h resulted in the increased accumulation of cells in G2/M phase (42%) (Srivastava, 2004). The increased expression and phosphorylation of Chk2 (Thr-68) by BITC treatment caused G2/M arrest. Furthermore, BITC also decreased the phosphorylation and expression of Cdc25C (ser-216), Cdc2 (Tyr-15) and Cyclin B1in apan-2 cells, as compared to control cells (Fig.12).   Interestingly, although ERK, JNK and P38 were activated in response to BITC treatment, they had different roles in BITC-induced cell cycle arrest and apoptosis. MEK-1 inhibitor PD98059 significantly abrogated BITC induced G2/M cell cycle arrest and apoptosis (Fig.  13A, B&D). Whereas, both JNK (SP600125) and P38 (SB202190) inhibitors failed to protect the cells from BITC-mediated G2/M cell cycle arrest. Further, MEK-1 inhibitor blocked BITC-mediated activation of ERK as well as down-regulation of G2/M regulatory proteins such as cyclin-dependent kinase-1 (Cdk1), cyclin B1, Cdc25C and cleavage of caspase-3 and PARP, suggesting the involvement of ERK in BITC-induced G2/M cell cycle arrest and apoptosis (Fig. 13C). BITC-mediated apoptosis was almost completely blocked in the cells pre-treated with ERK, JNK or P38 inhibitors as evaluated by cell death apoptosis ELISA assay (Fig.13D). Similar results were obtained with MAPK8-shRNA in Capan-2 cells, indicating that all the MAPK were involved in BITC-induced apoptosis but only ERK was involved in BITC-induced cell cycle arrest.
Involvement of BITC-induced ROS generation in cell cycle arrest and apoptosis was further confirmed by treatment with antioxidants such as NAC, tiron, GSH and SOD. BITC-induced phosphorylation of MAPK and down regulation of cell cycle proteins such as GSH, Cdk1, Cdc25C, Cyclin B1 were significantly blocked by the treatment also with NAC (Fig.14). Furthermore, BITC-induced apoptosis was inhibited when cells were pre-treated with antioxidants, such as tiron, GSH and SOD. These results indicate that BITC induces ROS in pancreatic cancer cells which leads to DNA damage, cell cycle arrest and apoptosis. www.intechopen.com

BITC sensitizes human pancreatic cancer cells to radiation and TRAIL treatment
Increased systemic toxicity and resistance are the major drawbacks of radiation therapy in pancreatic cancer treatment. Interestingly, BITC potentiated therapeutic effect of γirradiation in BxPC-3 cells. BxPC-3 cells were pre-treated with 2.5 or 5 µM BITC for 24h, followed by treatment with different doses of γ-irradiation (2.5, 5, 10 and 20Gy) at a dose of 4Gy/minute. The cells were allowed for 24 or 48h before being analyzed for survival assay. BxPC-3 cells pre-treated with BITC and treated with 5Gy γ-irradiation show intense cell death, as compared to either treatment alone, indicating that BITC sensitizes the cells to γirradiation (Sahu et al., 2009c). Furthermore, as shown in Table 1, more cells were accumulated in G2/M arrest in response to combination treatment, as compared to either treatment alone. In addition, expression of cell cycle proteins Chk2 and Cdc25 was increased in combination treated cells, as compared to control cells. Interestingly, DNA damage markers H2A.X (Ser-139) and ATR (Ser-428) also increased in combination treatment, indicating that BITC sensitizes cells to γ-irradiation. In agreement with cell cycle data, apoptosis induction was more in combination treated cells.
Similarly, BITC also potentiated the apoptosis inducing activity of TRAIL in pancreatic cancer cells. BxPC3 cells had a 3.84 fold increase in apoptosis upon treatment with BITC alone, an 8.65 fold increase was observed with TRAIL alone, and a 12.39 fold increase was seen when cells were treated with BITC combined with TRAIL. Similarly, Panc-1 cells underwent a 1.49 fold increase in apoptosis upon treatment with BITC, a 1.82 fold increase with TRAIL alone, and a 3.45 fold increase with BITC combined with TRAIL compared to vehicle. Interestingly, sensitization of pancreatic cancer cells to TRAIL by BITC was more in Kras wild type cells (BxPC-3) as compared to Kras mutated cells (PanC-1 and MIA PaCa-2). Further studies are needed to elucidate the role of Kras mutation in TRAIL or BITC-induced apoptosis.

BITC inhibits pancreatic cancer angiogenesis
Pancreatic tumors can acquire substantial development of new blood vessels in a process called angiogenesis (Philip, 2008). This vascular development is a necessary component of solid tumor growth and progression. Numerous reports have shown that disrupting tumor angiogenesis could effectively inhibit tumor growth and metastasis. BITC has shown promising potentials as anti-angiogenesis agent for pancreatic cancer vitro and in vivo.
In a rat aorta ring assay model, treatment with 5 µM BITC reduced sprouting of new blood vessels by 67% as compared to control aortic rings (Fig. 15A). Furthermore, 5 µmol BITC treatment drastically (70%) suppressed new embryonic blood vessel growth in each egg as compared to control eggs in a CAM assay model (Fig. 15B), indicating that BITC has potential to inhibit tumor angiogenesis (Boreddy et al., 2011b).
BITC was also effective in suppressing the secretion of pro-angiogenic factors from pancreatic cancer cells under both, normoxia and hypoxia conditions. Hypoxia alone induced the secretion of both MMP-2 and VEGF around 2-4 folds in both BxPC-3 and PanC-1 cells; however, BITC significantly inhibited the secretion of both VEGF and MMP-2 from the both BxPC-3 and PanC-1 cells under normoxia and hypoxia conditions (Fig. 16A-D). Interestingly, BITC significantly inhibited the migration and invasion of both, BxPC-3 and PanC-1 cells in a dose dependent manner. These steps are critical for the migration of the tumor cells in vivo.

BITC suppresses pancreatic tumor growth and angiogenesis in vivo
BITC exhibited similar results in vivo as observed in culture. Tumor growth in BITC-fed mice was substantially retarded, as compared to control mice. Tumors appeared to grow more slowly in BITC-fed mice as compared with control mice. For example, 6 weeks after treatment with12 μmol BITC, the average tumor volume in control mice was about 1.92-fold higher than that in BITC-treated mice (mean tumor volume, control vs BITC treated: 334 vs172 mm 3 , difference = 162 mm 3 , 95% CI = 118 to 204 mm 3 ;P = .008; Fig.18A). Furthermore, average tumor weight in BITC-treated mice was 225mg, whereas in control mice tumor weight was 425mg (Fig. 18B), indicating that BITC potentially suppress the growth of pancreatic tumors in vivo. Interestingly, BITC-treated mice did not show any toxicity symptoms such as weight loss (Fig. 18C). It is noteworthy that when animals were orally gavaged with 12µmol/day BITC for 46 days, mean BITC concentration in plasma after1 hour of BITC administration was 6.5 ± 0.1 mmol/ L (n=10), whereas accumulated BITC concentration in the tumors after 46 days was 7.5 ± 0.3 µmol/g (n=10). These results indicate that the therapeutic concentration of BITC could be achieved in vivo by oral feeding.
A 76% reduction in hemoglobin content was observed in BITC-treated matrigel plugs that were implanted in Nu-Nu athymic nude mice as compared to untreated plugs (Fig. 19A). Similarly, BITC-treated tumor xenografts showed 61% reduced hemoglobin content as compared to untreated xenografts (Fig. 19B).

Does BITC have multiple targets in pancreatic cancer?
Since, BITC inhibits the phosphorylation and protein levels of various key survival molecules such as STAT3, AKT and NFkB, indicating that BITC has multiple targets in pancreatic cancer. However, at this time, it is not clear whether BITC is targeting various survival pathways individually or it is the tandem effect upstream regulators. Since previous reports showed that STAT3 is being regulated by AKT through FOXO1 (Kortylewski et al., 2003) and NFkB is a direct target of AKT (Dan, 2008), presently we assume that AKT is the main target of BITC and other targets are obligated events but further studies are needed to conclude interaction of these pathways (Fig. 21).