Ravi P. Sahu

In vitro and in vivo induction of apoptosis by capsaicin in pancreatic cancer cells is mediated through ROS generation and mitochondrial death pathway

Pancreatic cancer is one of the most common invasive malignancies and the fourth leading cause of cancer related mortality in U.S., thus developing new strategies to control pancreatic cancer is an important mission. We investigated the mechanism of capsaicin, the major pungent ingredient of red-chili pepper, in inducing apoptosis in pancreatic cancer cells. Treatment of AsPC-1 and BxPC-3 cells with capsaicin resulted in a dose-dependent inhibition of cell-viability and induction of apoptosis which was associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. These effects were significantly blocked when the cells were pretreated with a general antioxidant N-acetyl cysteine (NAC). Exposure of AsPC-1 and BxPC-3 cells to capsaicin was also associated with increased expression of Bax, down-regulation of bcl-2, survivin and significant release of cytochrome c and AIF in the cytosol. On the contrary, above-mentioned effects were not observed in the normal acinar cells in response to capsaicin-treatment. Capsaicin-treatment resulted in the activation of JNK and JNK inhibitor SP600125 afforded protection against capsaicin-induced apoptosis. Furthermore, capsaicin when given orally markedly suppressed the growth of AsPC-1 pancreatic tumor xenografts in athymic nude mice, without side effects. Tumors from capsaicin treated mice demonstrated increased apoptosis, which was related to the activation of JNK and increased cytosolic protein expression of Bax, cytochrome c, AIF and cleaved caspase-3, as compared with controls. Taken together, these results show that capsaicin is an effective inhibitor of in vitro and in vivo growth of pancreatic cancer cells. These findings provide the rationale for further clinical investigation of capsaicin against pancreatic cancer.

Activation of ATM/Chk1 by curcumin causes cell cycle arrest and apoptosis in human pancreatic cancer cells

Curcumin has been shown to inhibit the growth of various types of cancer cells; however, at concentrations much above the clinically achievable levels in humans. The concentration of curcumin achieved in the plasma after oral administration in humans was estimated to be around 1.8 μM. Here, we report that treatment of BxPC-3 human pancreatic cancer cells with a low and single exposure of 2.5 μM curcumin for 24 h causes significant arrest of cells in the G2/M phase and induces significant apoptosis. Immunoblot studies revealed increased phosphorylation of H2A.X at Ser-139 and Chk1 at Ser-280 and a decrease in DNA polymerase-β level in curcumin-treated cells. Phosphorylation of H2A.X and Chk1 proteins are an indicator of DNA damage whereas DNA polymerase-β plays a role in the repair of DNA strand breaks. Normal immortalised human pancreatic ductal epithelial (HPDE-6) cells remained unaffected by curcumin treatment. In addition, we also observed a significant increase in the phosphorylation of Chk1 at Ser-345, Cdc25C at Ser-216 and a subtle increase in ATM phosphorylation at Ser-1981. Concomitant decrease in the expressions of cyclin B1 and Cdk1 were seen in curcumin-treated cells. Further, curcumin treatment caused significant cleavage of caspase-3 and PARP in BxPC-3 but not in HPDE-6 cells. Silencing ATM/Chk1 expression by transfecting BxPC-3 cells with ATM or Chk1-specific SiRNA blocked the phosphorylation of ATM, Chk1 and Cdc25C and protected the cells from curcumin-mediated G2/M arrest and apoptosis. This study reflects the critical role of ATM/Chk1 in curcumin-mediated G2/M cell cycle arrest and apoptosis in pancreatic cancer cells.

The Role of STAT-3 in the Induction of Apoptosis in Pancreatic Cancer Cells by Benzyl Isothiocyanate

BACKGROUND Benzyl isothiocyanate (BITC), a compound found in cruciferous vegetables, has been reported to have anticancer properties, but the mechanism whereby it inhibits growth of human pancreatic cancer cells is incompletely understood. METHODS Human pancreatic cancer cells (BxPC-3, AsPC-1, Capan-2, MiaPaCa-2, and Panc-1) and immortalized human pancreatic cells (HPDE-6) were treated with vehicle or with BITC at 5-40 microM, cell survival was evaluated by sulforhodamine B assay, and apoptosis by caspase-3 and poly-ADP ribose polymerase cleavage or by a commercial assay for cell death. Total and activated signal transducer and activator of transcription-3 (STAT-3) protein expression in the cells were examined by western blotting, STAT-3 mRNA levels by reverse transcription-polymerase chain reaction, and STAT-3 DNA-binding and transcriptional activity by commercially available binding and reporter assays. The effects of BITC treatment on tumor growth, apoptosis, and STAT-3 protein expression in vivo were studied in xenografts of BxPC-3 pancreatic tumor cells in athymic nude mice. All statistical tests were two-sided. RESULTS BITC treatment reduced cell survival and induced apoptosis in BxPC-3, AsPC-1, Capan-2, and MiaPaCa-2 cells, and to a much lesser extent in Panc-1 cells, but not in HPDE-6 cells. It also reduced levels of activated and total STAT-3 protein, and as a result, STAT-3 DNA-binding and transcriptional activities. Overexpression of STAT-3 in BxPC-3 cells inhibited BITC-induced apoptosis and restored STAT-3 activity. In mice that were fed BITC (60 micromol/wk, five mice, 10 tumors per group), growth of BxPC-3 pancreatic tumor xenografts was suppressed compared with control mice (at 6 weeks, mean tumor volume of control vs BITC-treated mice = 334 vs 172 mm3, difference =162 mm3, 95% confidence interval = 118 to 204 mm3; P = .008) and tumors had increased apoptosis and reduced STAT-3 protein expression. CONCLUSION BITC induces apoptosis in some types of pancreatic cancer cells by inhibiting the STAT-3 signaling pathway.

Benzyl isothiocyanate-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of MAPK in human pancreatic cancer cells

In our previous studies, we have shown that benzyl isothiocyanate (BITC) inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three (MAPK) family members [extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK) and P38] in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24 h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in MiaPaCa-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPKs and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G(2)/M arrest and apoptosis, Capan-2 cells were pre-treated with MAPK-specific inhibitors or MAPK8-short hairpin RNA (shRNA) prior to BITC treatment. Significant protection from BITC-induced G(2)/M arrest was observed in the cells pre-treated with MAPK kinase (MEK-1) but not JNK or P38 inhibitors. On the other hand, BITC-induced apoptosis was almost completely abrogated in the cells pre-treated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G(2)/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of reactive oxygen species (ROS) in Capan-2 and MiaPaCa-2 cells, which in part was orchestrated by depletion of reduced glutathione (GSH) level. Blocking ROS generation with N-acetyl-L-cysteine (NAC) significantly prevented GSH depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G(2)/M arrest by blocking G(2)/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together, our results suggest that BITC-mediated G(2)/M arrest is mediated through ERK activation, whereas apoptosis is via ERK, JNK and P38.

Benzyl isothiocyanate suppresses pancreatic tumor angiogenesis and invasion by inhibiting HIF-α/VEGF/Rho-GTPases: pivotal role of STAT-3.

Our previous studies have shown that benzyl isothiocyanate (BITC) suppresses pancreatic tumor growth by inhibiting STAT-3; however, the exact mechanism of tumor growth suppression was not clear. Here we evaluated the effects and mechanism of BITC on pancreatic tumor angiogenesis. Our results reveal that BITC significantly inhibits neovasularization on rat aorta and Chicken-Chorioallantoic membrane. Furthermore, BITC blocks the migration and invasion of BxPC-3 and PanC-1 pancreatic cancer cells in a dose dependant manner. Moreover, secretion of VEGF and MMP-2 in normoxic and hypoxic BxPC-3 and PanC-1 cells was significantly suppressed by BITC. Both VEGF and MMP-2 play a critical role in angiogenesis and metastasis. Our results reveal that BITC significantly suppresses the phosphorylation of VEGFR-2 (Tyr-1175), and expression of HIF-α. Rho-GTPases, which are regulated by VEGF play a crucial role in pancreatic cancer progression. BITC treatment reduced the expression of RhoC whereas up-regulated the expression of tumor suppressor RhoB. STAT-3 over-expression or IL-6 treatment significantly induced HIF-1α and VEGF expression; however, BITC substantially suppressed STAT-3 as well as STAT-3-induced HIF-1α and VEGF expression. Finally, in vivo tumor growth and matrigel-plug assay show reduced tumor growth and substantial reduction of hemoglobin content in the matrigel plugs and tumors of mice treated orally with 12 µmol BITC, indicating reduced tumor angiogenesis. Immunoblotting of BITC treated tumors show reduced expression of STAT-3 phosphorylation (Tyr-705), HIF-α, VEGFR-2, VEGF, MMP-2, CD31 and RhoC. Taken together, our results suggest that BITC suppresses pancreatic tumor growth by inhibiting tumor angiogenesis through STAT-3-dependant pathway.

Benzyl Isothiocyanate–Mediated Inhibition of Histone Deacetylase Leads to NF-κB Turnoff in Human Pancreatic Carcinoma Cells

NF-κB/p65 is constitutively activated in pancreatic cancers, where it plays a critical role in the transcriptional activation of multiple cell survival genes. We have previously shown the apoptosis-inducing effects of benzyl isothiocyanate (BITC) in pancreatic cancer cells. We hypothesized that inhibition of NF-κB/p65 could be the mechanism of BITC-induced apoptosis. Therefore, the effect of BITC on NF-κB/p65 was evaluated in BxPC-3, Capan-2, and normal HPDE-6 cells by Western blotting, transcriptional and DNA-binding activity, and immunohistochemistry in the xenografted tumors. Our results reveal a remarkable decrease in the phosphorylation of NF-κB/p65 at Ser536 in both BxPC-3 and Capan-2 cells by BITC treatment. The expression of NF-κB/p65 was downregulated significantly in BxPC-3 cells, whereas it remained unchanged in Capan-2 cells. BITC treatment caused a significant decrease in NF-κB transcriptional and DNA-binding activity in both BxPC-3 and Capan-2 cells. A drastic decrease was observed in the expression and reporter activity of cyclin D1 in both the cell lines. Moreover, BITC also caused a significant decrease in the expression and activity of histone deacetylase (HDAC) 1 and HDAC3 in BxPC-3 and HDAC3 in Capan-2 cells. Overexpression of HDAC1 or HDAC3 abrogated the effects of BITC. BITC treatment did not cause any change in HDAC expression in normal HPDE-6 cells. Immunohistochemical analysis of tumors from BITC-treated mice showed significantly reduced staining for NF-κB, cyclin D1, HDAC1, and HDAC3 compared with control. Our results suggest inhibition of HDAC1/HDAC3 by BITC as a plausible mechanism of NF-κB inactivation, resulting in the in vitro and in vivo growth suppression of pancreatic cancer cells. Mol Cancer Ther; 9(6); 1596–608. ©2010 AACR.

Triphala inhibits both in vitro and in vivo xenograft growth of pancreatic tumor cells by inducing apoptosis

BackgroundTriphala is commonly used in Ayurvedic medicine to treat variety of diseases; however its mechanism of action remains unexplored. This study elucidates the molecular mechanism of Triphala against human pancreatic cancer in the cellular and in vivo model.MethodsGrowth-inhibitory effects of Triphala were evaluated in Capan-2, BxPC-3 and HPDE-6 cells by Sulphoradamine-B assay. Apoptosis was determined by cell death assay and western blotting. Triphala was administered orally to nude mice implanted with Capan-2 xenograft. Tumors were analyzed by immunohistochemistry and western blotting.ResultsExposure of Capan-2 cells to the aqueous extract of Triphala for 24 h resulted in the significant decrease in the survival of cells in a dose-dependent manner with an IC50 of about 50 μg/ml. Triphala-mediated reduced cell survival correlated with induction of apoptosis, which was associated with reactive oxygen species (ROS) generation. Triphala-induced apoptosis was linked with phosphorylation of p53 at Ser-15 and ERK at Thr-202/Tyr-204 in Capan-2 cells. Above mentioned effects were significantly blocked when the cells were pretreated with an antioxidant N-acetylcysteine (NAC), suggesting the involvement of ROS generation. Pretreatment of cells with pifithrin-α or U0126, specific inhibitors of p53 or MEK-1/2, significantly attenuated Triphala-induced apoptosis. Moreover, NAC or U0126 pretreatment significantly attenuated Triphala-induced p53 transcriptional activity. Similarly, Triphala induced apoptosis in another pancreatic cancer cell line BxPC-3 by activating ERK. On the other hand, Triphala failed to induce apoptosis or activate ERK or p53 in normal human pancreatic ductal epithelial (HPDE-6) cells. Further, oral administration of 50 mg/kg or 100 mg/kg Triphala in PBS, 5 days/week significantly suppressed the growth of Capan-2 pancreatic tumor-xenograft. Reduced tumor-growth in Triphala fed mice was due to increased apoptosis in the tumors cells, which was associated with increased activation of p53 and ERK.ConclusionOur preclinical studies demonstrate that Triphala is effective in inhibiting the growth of human pancreatic cancer cells in both cellular and in vivo model. Our data also suggests that the growth inhibitory effects of Triphala is mediated by the activation of ERK and p53 and shows potential for the treatment and/or prevention of human pancreatic cancer.

The environmental stressor ultraviolet B radiation inhibits murine antitumor immunity through its ability to generate platelet-activating factor agonists

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr -/- mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3(EGFP) transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.

Chloroquine Promotes Apoptosis in Melanoma Cells by Inhibiting BH3 domain Mediated PUMA Degradation

The BH3-only protein PUMA counters Bcl-2 family anti-apoptotic proteins and promotes apoptosis. Although PUMA is a key regulator of apoptosis, the post-transcriptional mechanisms that control PUMA protein stability are not understood. We show that a lysosome-independent activity of chloroquine prevents degradation of PUMA protein, promotes apoptosis and reduces the growth of melanoma xenografts in mice. Compared to wild–type PUMA, a BH3 domain deleted PUMA protein showed impaired decay in melanoma cells. Fusion of the BH3 domain to a heterologous protein led to its rapid turnover that was inhibited by chloroquine. While both chloroquine and inhibitors of lysosomal proteases stalled autophagy, only choroquine stabilized PUMA protein and promoted apoptosis. Our results reveal a lysosomal protease independent activity of chloroquine that selectively promotes apoptosis in melanoma cells.

Cigarette Smoke Exposure Inhibits Contact Hypersensitivity via the Generation of Platelet-Activating Factor Agonists

Previous studies have established that pro-oxidative stressors suppress host immunity because of their ability to generate oxidized lipids with platelet-activating factor receptor (PAF-R) agonist activity. Although exposure to the pro-oxidative stressor cigarette smoke (CS) is known to exert immunomodulatory effects, little is known regarding the role of PAF in these events. The current studies sought to determine the role of PAF-R signaling in CS-mediated immunomodulatory effects. We demonstrate that CS exposure induces the generation of a transient PAF-R agonistic activity in the blood of mice. CS exposure inhibits contact hypersensitivity in a PAF-R–dependent manner as PAF-R–deficient mice were resistant to these effects. Blocking PAF-R agonist production either by systemic antioxidants or treatment with serum PAF-acetyl hydrolase enzyme blocked both the CS-mediated generation of PAF-R agonists and PAF-R–dependent inhibition of contact hypersensitivity (CHS) reactions, indicating a role for oxidized glycerophosphocholines with PAF-R agonistic activity in this process. In addition, cyclooxygenase-2 inhibition did not block PAF-R agonist production but prevented CS-induced inhibition of CHS. This suggests that cyclooxygenase-2 acts downstream of the PAF-R in mediating CS-induced systemic immunosuppression. Moreover, CS exposure induced a significant increase in the expression of the regulatory T cell reporter gene in Foxp3EGFP mice but not in Foxp3EGFP mice on a PAF-R–deficient background. Finally, regulatory T cell depletion via anti-CD25 Abs blocked CS-mediated inhibition of CHS, indicating the potential involvement of regulatory T cells in CS-mediated systemic immunosuppression. These studies provide the first evidence, to our knowledge, that the pro-oxidative stressor CS can modulate cutaneous immunity via the generation of PAF-R agonists produced through lipid oxidation.