Kartick C. Pramanik

Molecular Targets of Benzyl Isothiocyanates in Pancreatic Cancer

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.

Abstract 2579: Capsaicin inhibits β-catenin signaling in pancreatic cancer cells by disrupting nuclear β-catenin/TCF-1 complex: critical role of STAT-3

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnβ-catenin/TCF signaling plays an important role in normal development. However, its aberrant activation is associated with several cancers including pancreatic cancer. In our previous study, we have shown that capsaicin-induced apoptosis in pancreatic cancer cells was associated with mitochondrial depolarization and oxidative stress. In the present study, we evaluated the effect of capsaicin on β-catenin/TCF signaling. In a concentration and time-dependent study, we observed that capsaicin treatment inhibits the activation of disheveled (Dsh) and phosphorylation of STAT-3 at Tyr705 in L3.6PL, Panc-1 and MiaPaCa-2 pancreatic cancer cells. Capsaicin treatment activated GSK-3β by inhibiting its phosphorylation and further activated APC and Axin multicomplex leading to the ubiquitination and degradation of β-catenin. Pre-treatment of cells with MG-132 blocked capsaicin-mediated proteasomal degradation of β-catenin. The expression of TCF-1 and β-catenin-responsive proteins c-myc and cyclin D1, were also decreased in response to capsaicin treatment. Significant cleavage of caspase-9, caspase-3 and PARP was observed in all the cell lines. To establish the involvement of β-catenin in capsaicin-induced apoptosis, cells were treated with LiCl or SB415286, inhibitors of GSK-3β. Our results reveal that capsaicin treatment suppressed LiCl-mediated activation of β-catenin signaling. Our results further showed that capsaicin blocked the nuclear translocation of β-catenin and TCF-1. The immunoprecipitation results also indicated that capsaicin treatment reduced the interaction of β-catenin with TCF-1 in the nucleus. Interestingly, STAT-3 over expression or STAT-3 activation by IL-6, significantly increased the levels of β-catenin and attenuated the effects of capsaicin in inhibiting β-catenin signaling. These observations indicate the direct regulation of β-catenin by STAT-3 in our model. Taken together, our results suggest that capsaicin-induced apoptosis in pancreatic cancer cells was associated with the inhibition of β-catenin signaling due to the dissociation of β-catenin/TCF-1 complex leading to apoptosis, and this process was orchestrated by STAT-3. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2579. doi:1538-7445.AM2012-2579

Abstract 2591: Caffeic acid phenethyl ester (CAPE) suppresses melanoma tumor growth by inhibiting AKT/XIAP pathway

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnThe incidence of melanoma is constantly on the rise indicating that current available therapies are not very effective. Moreover, melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppress the growth of melanoma cells and induce ROS generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here we determined the potential mechanism of CAPE against melanoma in vitro and in vivo. In a concentration and time-dependent study, CAPE treatment suppressed the activating phosphorylation of PI3K at Tyr-458, PDK1 at Ser-241, mTOR at Ser-2448 and AKT at Ser-473 in B16F0 and SK-MEL-28 melanoma cells. Furthermore, the expression of XIAP, survivin and BCl2 were down-regulated by CAPE treatment in both the cell lines. Significant apoptosis was observed in both the cell lines by CAPE treatment as indicated by cleavage of caspase-3 and PARP. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. Since Akt regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE treatment decreased the interaction of AKT with XIAP. To establish the involvement of Akt in the apoptosis-inducing effects of CAPE, cells were transfected with Akt. Our results reveal that AKT over-expression attenuated the decrease in XIAP and significantly blocked CAPE-mediated apoptosis. Similarly, over-expression of XIAP further decreased CAPE-induced apoptosis. To confirm the involvement of ROS in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant NAC prior to CAPE treatment. Our results indicate that NAC blocked CAPE mediated AKT/XIAP inhibition and protected the cells from apoptosis. In addition, oral administration of 10mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE treated mice showed reduced phosphorylation of PI3K, AKT, mTOR and protein level of XIAP and enhanced the cleavage of caspase-3 and PARP. Taken together, our results suggest that CAPE suppresses AKT/XIAP pathway leading to apoptosis in melanoma tumor cells in vitro and in vivo. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute].nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2591. doi:1538-7445.AM2012-2591

Abstract 2566: Critical role of autophagy proteins Beclin 1 and LC3B in deguelin-induced apoptosis in pancreatic tumor cells in vitro and in vivo: A new paradigm

Deguelin is known to inhibit cell growth by inducing apoptosis in various cancer models primarily by inhibiting AKT, but its role in autophagy is largely unknown. In the present study, we evaluated the role of autophagy in deguelin-induced apoptosis in pancreatic cancer. Our results reveal that PanC-1, AsPC-1 and BxPC-3 cells that were treated with deguelin show significant increase in autophagosome vacuoles (AV) formation and increase in the expression of Beclin1, ATG5 and LC3B. In general, induction of autophagy is considered to be a protective mechanism; however, few recent studies have indicated the pro-apoptotic role of autophagy as well. To address this question, cells were pretreated with specific inhibitors of autophagy such as chloroquine and 3-methyladenine prior to deguelin treatment and apoptosis was evaluated. Surprisingly, inhibition of autophagy by all the above-mentioned inhibitors significantly blocked deguelin-induced apoptosis, indicating that apoptosis induction by deguelin was dependent on autophagy. To establish that deguelin-mediated autophagy was involved in apoptosis, Beclin1, ATG5 and LC3B were silenced and treated with deguelin. Consistent with our inhibitor data, silencing of Beclin1 and LC3B made the cells resistant to deguelin-induced apoptosis, however silencing of ATG5 provided modest protection, indicating that Beclin1 and LC3B play a critical role in deguelin-induced apoptosis in pancreatic cancer cells. Previous studies have shown that Beclin1 is cleaved by caspases into N-terminal and C-terminal fragments. C-terminal translocates to mitochondria and cause cytochrome C release to trigger apoptosis. In agreement, our results show that the C-terminal fragment of Beclin1 was increased, whereas cytochrome C levels were decreased in mitochondrial fraction, indicating that Beclin1 acts as a proapoptotic protein in our model. Similarly, LC3B has been shown to induce apoptosis by releasing Fas from caveolin1 to form DISC and trigger extrinsic apoptosis. To test this possibility, DISC formation was evaluated in deguelin treated control and LC3B-silenced PanC-1 cells. Interestingly, DISC formation was significantly reduced in LC3B silenced PanC-1 cells, as compared to LC3B expressing cells. To further validate our observations in vivo, PanC-1-luc cells were orthotopically implanted into the pancreas of nu/nu mice and treated with 5mg/kg deguelin (i.p., daily). After 42 days of treatment, deguelin treated mice showed substantially reduced tumor growth as compared to control mice. In addition, tumors of deguelin treated mice exhibited increased levels of Beclin1 and LC3B. Taken together, our results demonstrate that deguelin-induced apoptosis in pancreatic tumor cells was mediated through autophagy. [Supported in part by R01 grants CA129038 and CA106953 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2566. doi:1538-7445.AM2012-2566

Abstract 1862: Capsaicin mediated activation of JNK/FOXO/BIM causes apoptosis in pancreatic cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnCapsaicin, a homovanillic acid derivative (N-vanillyl-8-methyl-nonenamide) is an active component of chili pepper. We previously reported that capsaicin mediated generation of reactive oxygen species (ROS) causes apoptosis in pancreatic cancer cells. In the present study, we investigated the potential mechanism of capsaicin mediated apoptosis in pancreatic cancer cells. In a time and concentration dependent study, capsaicin treatment activated c-Jun N terminal kinase (JNK) in BxPC-3, AsPC-1 and L3.6PL pancreatic cancer cells. Further, capsaicin induced activation of JNK in turn activated FOXO1 by phosphorylating at Ser 256 and pro-apoptotic protein BIM at Ser 69. The expression of BIM also increased in response to capsaicin treatment. Capsaicin treatment also caused cleavage of caspase-9, caspase-3 and PARP resulting in apoptosis. Blocking capsaicin mediated activation of JNK by JNK-specific inhibitor SP600125 attenuated nuclear localization of FOXO-1, activation of BIM and abrogated capsaicin-induced apoptosis. To confirm the involvement of ROS in the activation of JNK, FOXO and Bim, cells were treated with antioxidants catalase and EUK (mimetic of catalase) prior to capsaicin treatment. Our results reveal that both the antioxidants blocked capsaicin mediated JNK/FOXO/BIM activation and protected the cells from apoptosis. Furthermore, silencing FOXO1 by siRNA blocked capsaicin-mediated activation of BIM and apoptosis. To further confirm the involvement of Bim in capsaicin-induced apoptosis, cells were transfected with BIM siRNA. Silencing Bim drastically reduced capsaicin-mediated cleavage of caspase-3 and PARP indicating the role of Bim in inducing apoptosis in our model. Taken together, our results suggest that ROS generated by capsaicin activates JNK and FOXO-1 which in turn activates BIM resulting in the cleavage of caspase-9/3 and PARP leading to apoptosis in pancreatic cancer cells. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1862. doi:10.1158/1538-7445.AM2011-1862

Abstract 3694: Activation of ASK1 by capsaicin mediates apoptosis in pancreatic cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnApoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK) kinase kinase family plays an essential role in reactive oxygen species (ROS) induced cellular response. Thioredoxin (Trx), a redox sensitive protein binds to ASK1 and inhibits its activation. In our previous study, we have shown that capsaicin induced apoptosis in pancreatic cancer cells was associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. In the present study, we evaluated the interaction of thioredoxin/ASK1 in pancreatic cancer cells. In a concentration dependent study, we observed that capsaicin treatment down regulated thioredoxin and increased the phosphorylation (activation) of ASK1 at Thr845 in AsPC-1 and BxPC-3 cells. ASK1 has been shown to activate JNK, which in turn activates MKK4/7 resulting in the cleavage of caspase-9, and caspase-3. In agreement, our results reveal that capsaicin treatment activated JNK, MKK4/7, caspase-9 and caspase-3. Interestingly, treatment of cells with antioxidants such as tiron or catalase blocked the activation of ASK1 cascade by capsaicin and protected the cells from apoptosis indicating the involvement of ROS in the activation of ASK1. To confirm the interaction of Trx/ASK1 and the dissociation of ASK1 by capsaicin in our model, BxPC-3 cell were transiently transfected with contructs encoding ASK1 fused to C-terminal hemaggutinin (HA) and flag tagged Trx. Our results reveal that Trx over expression suppressed the effects of capsaicin whereas ASK1 over expression enhanced the apoptosis-inducing effects of capsaicin. Taken together, our results suggest that ROS generated by capsaicin reduce Trx expression resulting in the activation of ASK1 and downstream effectors leading to apoptosis in pancreatic cancer cells. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2011-3694

Abstract 953: Benzyl isothiocyanate suppresses pancreatic cancer angiogenesis and invasion by inhibiting Akt and MMP-2

We have shown in our previous studies that benzyl isothiocyante (BITC) suppress the growth of pancreatic cancer cells by inhibiting the activation of NF-kB and STAT-3 without affecting the viability of normal HPDE cells. Further, we demonstrated that oral administration of BITC significantly blocked the growth of pancreatic tumor xenografts in vivo. In the present study, we examined the effect of BITC on pancreatic cancer angiogenisis and invasion. Our results demonstrate that 5-10µM BITC almost completely blocked neovasculture formation in an ex vivo chick chorio-allantoic membrane (CAM) model. In agreement with these results, in vivo matrigel plug assay in athymic nude mice also reveal that the hemoglobin content in the implanted matrigel plug was reduced by 70% in the mice which were orally fed 12µmol BITC for 7 days as compared to controls, suggesting reduced blood vessel formation. In a matrigel tube formation assay using mouse brain endothelial cells, BITC treatment significantly inhibited VEGF-induced tube formation. As compared to controls, 5µM BITC treatment for 36h caused a drastic reduction in the number of cells that migrated into the wounding area in a wound healing assay. Our results also showed that the tumor cell invasion was effectively (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 953.

Abstract 3781: 3,3′-diindolylmethane(DIM)-induced apoptosis in ovarian cancer cells is mediated by inhibition of EGFR-ERK pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnOvarian cancer is one of the leading causes of gynaecological deaths in the United States and several other countries. Genetic alterations including overexpression of EGFR (about 70% of ovarian tumors) play a crucial role in the signal transduction pathways that regulate key cellular functions such as cell survival and proliferation and are responsible for comprising traditional chemotherapy. Several epidemiological studies suggested a decreased risk of ovarian cancer in the population consuming Brassica vegetables. 3,3′-diindolylmethane (DIM) is an indole compound present in Brassica vegetables. In our previous studies, we have demonstrated that BR-DIM, a formulated version of DIM suppress the growth of ovarian cancer cells by causing cell cycle arrest. However, the exact mechanism by which DIM suppress the growth of ovarian cancer cells was not clear. In the present study, we delineated the mechanism of DIM in SKOV3, OVCAR-3 and TOV-21G human ovarian cancer cells. Our results show that DIM treatment induces apoptosis in all the cell lines in a dose-dependent manner, as analyzed by flow cytometry. Our western blot analysis reveals that DIM treatment causes significant down regulation of constitutive EGFR and HER2 protein level as well as phosphorylation of EGFR at Tyr992, Tyr845 and Tyr1173 in SKOV-3 and OVCAR-3 cells. To determine whether DIM can suppress the activation of EGFR by activating phosphorylation, cells were treated with EGF after DIM treatment. Our results reveal that DIM treatment block the activation of EGFR induced by EGF in OVCAR-3 and TOV-21G cells. In addition, DIM treatment drastically reduces the phosphorylation of ERK without affecting the protein level. DIM treatment also inhibits the kinase activity of ERK as observed by the down regulation of p-ELK in all the three ovarian cancer cell lines. ERK is known to be regulated by EGFR. Taken together our results indicate that DIM induces apoptosis in ovarian cancer cells by inhibiting EGFR-ERK pathway. Further mechanistic studies are in progress. [Supported in part by RO1 grants CA 106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3781.

Abstract 1877: Involvement of mitochondrial respiratory complexes in capsaicin mediated ROS generation and apoptosis in pancreatic cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnCapsaicin (N-vanillyl-8-methyl-nonenamide) is a homovanillic acid derivative and the spicy component of chili pepper. In our previous study, we have shown that capsaicin induced apoptosis in pancreatic cancer cells was associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. However, the exact mechanism by which capsaicin causes ROS generation and cell death was not fully clear. We now evaluated the effect of capsaicin on mitochondrial respiratory complexes and ROS generation. The generation of ROS was about 3 fold and as early as 2h after capsaicin treatment in BxPC-3 cells and inhibited by rotenone and antimycin-A but not by 4-nitropropionic acid or sodium azide. Rotenone, 4-nitropropionic acid, antimycin A and sodium azide are the inhibitors of NADH-ubiquinone oxidoreductase (Complex I), succinate ubiquinone oxidoreductase (Complex II), ubiquinol cytochrome c reductase (Complex III) and cytochrome c oxidase (Complex IV) respectively. To further delineate the mechanism of ROS generation, effect of capsaicin was evaluated on the enzymatic activities of above-mentioned mitochondrial complexes in BxPC-3 cells. Our results show that capsaicin treatment significantly reduced complex I and complex III enzymatic activities suggesting the involvement of mitochondrial complex I and III in capsaicin mediated ROS generation. Capsaicin however did not affected complex II or complex IV activities. Our results further revealed that about 3 fold apoptosis induced by capsaicin in BxPC-3 cells was inhibited by rotenone and antimycin-A. Moreover, capsaicin mediated release of cytochrome c and AIF and cleavage of caspase-9 and caspase-3 were significantly inhibited by rotenone and antimycin-A. On the other hand, catalase or EUK (a mimetic of catalase) pretreatment block capsaicin-mediated ROS generation and apoptosis. Taken together, our results suggest the involvement of mitochondrial complex I and III in capsaicin mediated ROS generation and apoptosis. [Supported in part by RO1 grants CA 106953 and CA129038 (to SKS) awarded by National Cancer Institute].nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1877.