Human Pancreatic Cancer Cell BxPC-3 Research Articles

Combined treatment with Denbinobin and Fas ligand has a synergistic cytotoxic effect in human pancreatic adenocarcinoma BxPC‐3 cells

Human pancreatic carcinoma is a highly malignant cancer. Previous studies have shown that the decoy receptor 3 (DcR3) for Fas ligand (FasL) plays significant roles in tumour progression and immune suppression. In the present study, we evaluated the anti-cancer activity of a natural compound, denbinobin (5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone), through decreasing DcR3 levels in human pancreatic adenocarcinoma cell lines. We used immunoprecipitation and ELISA assays to examine DcR3 levels, and used FACS to determine the percentage of cells with a sub-G1 DNA content. AsPC-1 and BxPC-3 human pancreatic cancer cells express high levels of DcR3. Denbinobin concentration-dependently decreased DcR3 levels in BxPC-3 cells. MTT and flow cytometry assays indicated that BxPC-3 was FasL-resistant because high concentrations (100 ng.mL(-1)) of soluble FasL did not inhibit cell growth. However, combinations of denbinobin (3 micromol.L(-1)) with lower concentrations of soluble FasL (10, 30 and 50 ng.mL(-1)) or membrane-bound FasL, were synergistic on cell growth inhibition and apoptosis. Exogenous excess DcR3 reversed this synergistic effect. We observed no significant increase in the levels of surface Fas, cleaved forms of caspase-8, -3, -9, Bax, Bid, Bcl-xL, cytochrome c or mitochondrial membrane potentials following denbinobin treatment. However, denbinobin treatment increased the levels of apoptosis-inducing factor. Denbinobin and FasL trigger a synergistic cytotoxic effect in human pancreatic adenocarcinoma cells. Denbinobin mediated a decrease in levels of DcR3, which played a major role in this synergistic effect, and also increased caspase-independent apoptosis, via apoptosis-inducing factor.

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.

RNA interference of MBD1 in BxPC-3 human pancreatic cancer cells delivered by PLGA-poloxamer nanoparticles

Methyl-CpG binding domain protein 1 (MBD1) is a transcriptional regulator that binds methylated CpG islands of tumor suppressor genes and represses their transcription. In a former study, we found high expression of MBD1 in pancreatic cancer cell lines and tissues which may play an important role in the development of pancreatic cancer. In the present study, we incorporated the siRNA sequence of MBD1 plasmid into a PLGA:Poloxamer carrier to test the therapeutic effect of this compound on BxPC-3 human pancreatic cancer cells. We found that an MBD1 siRNA plasmid can be successfully transfected into tumor cells and the MBD1 nanoparticle compound can inhibit cell growth and induce apoptosis. The MBD1 nanoparticle is a promising candidate for gene therapy of pancreatic cancer in vitro.

Dihydroartemisinin inhibits growth of pancreatic cancer cells in vitro and in vivo

Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has recently shown antitumor activity in various cancer cells. Its effect on pancreatic cancer is, however, unknown and the mechanism is unclear. The study aims to investigate its antitumor activity and underlying mechanisms in human pancreatic cancer BxPC-3 and AsPC-1 cells in vitro and subcutaneous BxPC-3 xenograft tumors in mice. The MTT assay was used to evaluate cell viability, and flow cytometry and laser scanning confocal microscopy were used to detect apoptosis, for cultured cells. Pancreatic tumors were established by subcutaneous injection of BxPC-3 cells in nude BALB/c mice, and DHA was administered intraperitoneally to the mice. The size of tumors was monitored and they were harvested after the mice had been killed. Tumor sections were immunostained with an anti-Ki-67 Ab to assess the proliferation index, or stained with TUNEL to evaluate in-situ cell apoptosis. The gene expression in cells and tumors was evaluated by western blot analysis. In the cultured cells, DHA inhibited cell viability, downregulated the expression of proliferating cell nuclear antigen and cyclin D1, and upregulated p21(WAF1/CIP1); and induced apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9, in a dose-dependent manner. Similarly, in mice bearing BxPC-3 xenograft tumors, administration of DHA inhibited tumor growth in a dose-dependent manner, and modulated tumoral gene expression consistent with the in-vitro observations. This study indicates that DHA may be a potent and promising agent to combat pancreatic cancer.

Effect of MBD1 siRNA on growth of pancreatic cancer cell line BxPC-3

Objective To observe the effect of MBD1 siRNA on the growth of pancreatic cancer cell line BxPC-3, and explore the role of MBD1 in the carcinogenesis of the pancreatic carcinoma. Methods One siRNA sequence targeting MBD1 was designed by software and cloned into the expres-sion plasmid pGCsi-U6/Neo/GFP. DNA sequencing was used to confirm that the recombinant plasmid was constructed correctly. The constructed plasmid and the control plasmid were stably transfected in-to human pancreatic cancer cell line BxPC-3. RT-PCR and Western blot were used to detect the MBD1 mRNA and protein expression after RNA interference. The cell growth curve was measured by MTT assay. The three kind of cells (BxPC-3, BxPC-3/vector, BxPC-3/MBD1-siRNA) were injected subcu-taneously to nucle mice, which were observed for 8 weeks for tumor formation respectively. Results RT-PCR and Western blot showed that the MBD1 mRNA and protein expression were significantly lower in the BxPC-3/MBD1-siRNA group than in BxPC-3 and BxPC-3/veetor group (P<0. 01). In vivo and vitro, the growth of BxPC-3 cell was suppressed after transfected with MBD1 siRNA. The MBD1 mRNA in tumor of BxPC-3/MBD1-siRNA group could not De detected by RT-PCR. The mRNA levels of some tumor suppressor genes including CDH1 and RB were up-regulated significantly after RNA interference targeting MBD1. Conclusion The stable MBD1-siRNA recombinant plasmid can decrease MBD1 expression in human pancreatic cancer cell BxPC-3 and inhibit the growth of the cell notably in vivo and vitro, the mechanism is still unclear. MBD1 mediating transcriptional repres-sion may play an important part in the development of pancreatic cancer. Key words: Pancreatic neoplasms; Transfection; RNA interference; Tumor suppressor gene

Compound MMH01 possesses toxicity against human leukemia and pancreatic cancer cells

MMH01 is a compound isolated from Antrodia cinnamomea. MMH01 markedly inhibited growth of human leukemia U937 and pancreatic cancer BxPC3 cells. It resulted in distinct patterns of cell cycle distribution in U937 (G2/M, sub-G1 and polyploidy) and BxPC3 cells (G0/G1 and sub-G1). The modes of cell death in U937 cells include apoptosis and mitotic catastrophe, whereas apoptosis-associated events or necrosis in BxPC3 cells. Neither mitochondrial membrane permeabilization nor caspase dependence was noted. Proteins involving mitotic catastrophe-associated cell death such as cyclin B1 and checkpoint kinase 2 were activated in U937 cells. Only slight to moderate viability inhibition was noted to human monocytes, the normal counterpart of these myeloid leukemic cells. In conclusion, MMH01 possesses cytotoxicity against human leukemia and pancreatic cancer cells.

Decreased Expression of Angiotensin-Converting Enzyme 2 in Pancreatic Ductal Adenocarcinoma Is Associated with Tumor Progression

Angiotensin II (ANG II), the biologically active peptide of the renin-angiotensin system (RAS), is generated by angiotensin-converting enzyme (ACE) and is a regulator of cardiovascular homeostasis. Recently, there has been increasing evidence that ANG II is involved in the regulation of cell proliferation and migration, as well as angiogenesis via the ANG II-type 1 receptor (AT1R). These findings suggest that the ACE-ANG II-AT1R pathway is related to cancer biology. Previous reports have shown that ACE is preferentially expressed in pancreatic ductal adenocarcinoma (PDAC) tissues. Recently a homologue of ACE, angiotensin-converting enzyme 2 (ACE2), was reported to counterbalance the function of ACE, but the expression and role of ACE2 in PDAC are still unclear. In the present study, we analyzed the expression of ACE2 in invasive human PDAC and surrounding non-malignant tissues by Western blot analysis and immunohistochemistry. The ANG II concentration in homogenates of pancreatic tissues was measured with ELISA, and ACE2 protein was detected by Western blot analysis in BxPC3 and SW1990 human pancreatic ductal cancer cells. We have shown for the first time that the expression of ACE2 is decreased in PDAC tissues, in which ANG II was accumulated. Treatment of BxPC3 and SW1990 cells with ANG II decreased the expression of ACE2. Therefore, ANG II may contribute to the down-regulation of ACE2. Moreover, reduction of ACE2 expression by RNA interference promoted the proliferation of cultured pancreatic cancer cells. These findings suggest that ACE2 may have clinical potential as a novel molecular target for the treatment of PDAC.

Suppression of growth of pancreatic cancer cell and expression of vascular endothelial growth factor by gene silencing with RNA interference

To explore the anti-angiogenesis and tumor cell growth suppressive effects resulted from gene silencing by RNAi in BxPC-3 human pancreatic cancer cells. The designation and transfection of vascular endothelial growth factor (VEGF)-siRNA lentivirus was carried out in vitro. Real-time PCR and western blot were conducted to measure the expression levels of VEGF mRNA and protein. Flow cytometry was employed to evaluate cell apoptosis and cell death. A lactate dehydrogenase (LDH) assay was used to assess the cytotoxicity of VEGF-siRNA. A 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to picture the cellular growth. For the in vivo study, BxPC-3 cells were injected subcutaneously into nude mice to form xenografts. The mice were divided into three groups according to the intervention used. The control group, the negative control group and the knockdown group of mice were injected with saline, an empty lentivirus vehicle and lentivirus carrying VEGF-siRNA, respectively. None of the mice died during the study. When these mice were killed, the xenografts were collected and the tumor sizes of the different groups were compared. Finally, immunohistochemistry was used to assess the VEGF expression level and microvascular density. After the transfection of VEGF-siRNA lentivirus, the cellular expression of VEGF mRNA decreased to 50% of the control and the VEGF protein in the BxPC-3 cells decreased to 30% of the control. Apoptosis and cell death increased after transfection of the VEGF-siRNA lentivirus. The LDH assay showed high cytotoxicity induced by VEGF-siRNA lentivirus transfection. The MTT assay showed slower cellular growth in the knockdown cells. Tumor growth suppression was observed in nude mice that had received the VEGF-siRNA lentivirus transfection, and the tumor sizes of the xenografts in this group were clearly smaller than those in other two groups. VEGF expression and microvascular density were significantly decreased. Vascular endothelial growth factor gene silencing via VEGF-siRNA can effectively inhibit the production of VEGF and exert an anti-angiogenesis and tumor cell growth suppressive effect both in vitro and in vivo.

Nitric oxide-donating aspirin inhibits the growth of pancreatic cancer cells through redox-dependent signaling

The novel chemopreventive nitric oxide-donating aspirin (NO-ASA) prevents nearly 90% of ductal adenocarcinomas in a animal tumor model. To decipher the mechanism of this effect, we studied in BxPC-3 human pancreatic cancer cells the sequence of signaling events leading from NO-ASA treatment to cell growth inhibition. NO-ASA inhibited the growth of BxPC-3 cells (IC 50 = 13 μM), by inhibiting proliferation modestly and inducing apoptosis, necrosis and G 1/S cell cycle block. At 15 min of treatment with NO-ASA, the intracellular levels of reactive oxygen species (ROS) began increasing (peak at 8 h, baseline levels by 24 h). ROS activated almost immediately in a time- and concentration-dependent manner the MAPK pathways p38, ERK and JNK (their activation was abrogated by the antioxidant N-acetylcysteine). MAPK activation induced p21 cip-1, which suppressed the levels of cyclin D1 that controls the G 1/S cell cycle transition. NO-ASA induced COX-2 expression starting 90 min after p21 cip-1 was induced. When COX-2 expression was knocked down using siRNA against cox-2, the expression of p21 cip-1 was induced by NO-ASA, regardless of the level of expression of COX-2, suggesting a marginal, if any, role for COX-2 in the growth inhibitory effect of NO-ASA. These findings along with the temporal sequence of individual changes indicate a signaling sequence that involves ROS → MAPKs → p21 cip-1 → cyclin D1 → cell death. Our findings establish the critical role of ROS as proximal signaling molecules in the action of anticancer compounds and may be useful in designing mechanism-driven approaches to cancer control.

Radiation-induced Nuclear Translocation of IkB-beta and Transcription of the Alpha 5 Integrin Gene may Upregulate Surface Alpha 5 Beta 1 to Cause Invasion by Human Pancreatic Cancer Cells

Ionizing radiation rapidly induces Panc-1 and BxPC-3 human pancreatic cancer cell invasion, which is mediated by the alpha 5 beta 1 integrin fibronectin receptor (FnR) and requires MMP 1 activity. Here, we report the effects of radiation on the levels and localization of alpha 5 beta 1 FnR and MMP 1 proteins, on alpha 5 integrin mRNA upregulation, and on the nuclear localization of the NFkB chaperone, IkB-beta.

S1885 TGFβ Utilizes NF-κB to Mediate PTEN Suppression in a Smad-Independent Manner in Pancreatic Cancer Cells

Background: Transforming growth factor-β (TGFβ) is a key tumor suppressor that is involved in cell differentiation and normal pancreatic cell homeostasis. In pancreatic cancer, the suppressor function is lost in part by events that affect the TGFβ-SMAD signaling pathway, such as bi-allelic loss of SMAD4 and oncogenic K-RAS activation that slows nuclear translocation of receptor-activated SMADs. With loss of TGFβ-SMAD signaling, TGFβ promotes cellular proliferation via SMAD-independent signaling pathways. We have shown that TGFβ downregulates the tumor suppressor PTEN in a SMAD-independent manner via activation of PKCα in pancreatic cancer cells, but the mechanisms leading to PKCα activation by TGFβ are unexplored. We examined if TGFβ could activate a Ca2+-dependent pathway, particularly Ca2+ entry through the plasmamembrane store-operated Ca2+ channel (SOC).We hypothesized that TGFβ increases cytoplasmic free Ca2+ ([Ca]cyt) via SOC after activation of the IP3 receptor (IP3R) on the endoplasmic reticulum membrane. Methods: BxPc-3 human pancreatic cancer cells were treated with 10 ng/ml TGFβ, 100 mM 2-APB (inhibitor for both IP3R and SOC), 30 mM La3+ (a selective SOC inhibitor), and the effect on [Ca]cyt were measured by a digital MetaFluor Imaging System using the fluorescent Ca2+-sensitive dye, fura 2-AM. Separated membrane and cytosolic fraction proteins were probed for PKCα by Western blotting. Results: TGFβ activates PKCα by 15 minutes after treatment, as evidenced by translocation from the cytosolic to the membrane fraction. TGFβ increased [Ca]cyt within 100 seconds after treatment (∆F 0.25 ± 0.02, n=38, P<0.01), while pretreatment with 2-APB inhibited increase in [Ca]cyt (∆F 0.01 ± 0.01, 98% inhibition, n=46, P<0.001). Pre-treatment of the cells with 2-APB prevented TGFβ-induced PKCα activation. Pretreatment with La3+ totally abolished the TGFβ-induced increase in [Ca]cyt (n=34, P<0.01). Conclusion: TGFβ may activate IP3R that triggers release of intracellular Ca2+ and Ca2+ entry via SOC. These intracellular Ca2+ stores are important for TGFβ-induced PKCα activation and subsequent tumor cell proliferation. Our data is the first to demonstrate that TGFβ can mobilize intracellular Ca2+ stores to activate proliferative PKCα signaling in pancreatic cells. Future treatments of pancreatic cancer and other cancers may involve manipulation of intracellular Ca2+ levels by interventions towards Ca2+-dependent pathways.

125: Protein Kinase Cβ Inhibition Induces Radiosensitization in Pancreatic Cancer

125: Protein Kinase Cβ Inhibition Induces Radiosensitization in Pancreatic Cancer

Sensitization of pancreatic cancer cells to ApoG2 induced killing by curcumin

13152 Background: In pancreatic cancer, several important survival molecules such as NF-kappa B and Bcl-2 or Bcl-XL are highly activated. Thus agents that inhibit NF-KappaB activation, together with agents that inhibit Bcl-2 or Bcl-XL protein function, may lead to enhanced cell killing. ApoG2, a new derivative of natural product gossypol, is a potent small molecule inhibitor of Bcl-2 and Bcl-XL proteins. Curcumin, an active ingredient from the rhizome of the plant, Curcuma longa, has anti-inflammatory and anti-cancer activities and works by inactivation of NF-Kappa B activity. In this study we have investigated the effects of curcumin on ApoG2 induced apoptosis. Methods: The BxPC-3 human pancreatic cancer cell line was used in this study. ApoG2 was dissolved in DMSO at 1 mM stock solution, and curcumin was dissolved in DMSO at 20 mM stock solution. Cell viability was determined by the standard MTT assay and apoptosis was measured by histone/DNA ELISA method. Results: Pretreatment of human pancreatic cell line BxPC-3 with curcumin sensitizes ApoG2 induced cell killing. In the dose escalation with ApoG2 at 48 hrs on MTT assay, 250 nM showed 75% cell viability, while 2.5 uM of curcumin at 72 hrs showed 80% cell viability. Pretreatment with curcumin for 24 hrs followed by ApoG2 at 48 hrs showed 55% cell viability (p = 0.0005). Apoptosis by histone/DNA ELISA showed 4-fold induction of cell death with ApoG2 at 48 hrs, 7-fold induction of cell death with curcumin at 72 hrs. However, pretreatment with curcumin for 24 hrs followed by ApoG2 at 48 hrs showed 10-fold induction in apoptosis compared to the control. Conclusions: From these results, we conclude that curcumin enhanced ApoG2 induced cell growth inhibition and apoptosis of BxPC-3 cells. No significant financial relationships to disclose.

Regulatory Effects of X-linked Inhibitor of Apoptosis Protein and Pro-apoptotic Protein Smac on Apoptosis Resistance to Chemotherapy in Pancreatic Cancer Cells

Objective: To investigate the relation of X-linked inhibitor of apoptosis (XIAP) and second mitochondria-derived activator of caspase (Smac) signaling pathway to chemoresistance in human pancreatic cancer Panc-1 and BXPC-3 cells. Methods: Apoptosis and the changes of XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil (FU) were measured by flow cytometry. The cytosolic expression of XIAP, Smac and caspase-3 was detected by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into of Panc-1 cells. The effect of cytosolic overexpression of Smac on apoptosis of Panc-1 cells was evaluated by flow cytometry. Results: Panc-1 was more resistant to cisplatin or 5-FU induced apoptosis than BXPC-3. Western blot revealed that chemoresistant Panc-1 highly expressed XIAP, and increased cytosolic expression of Smac might be responsible for the marked down-regulation of XIAP in chemo-sensitive BXPC-3 cells after exposure to cisplatin or 5-FU. Furthermore, cytosolic overexpression of Smac could significantly down-regulate the levels of XIAP and promote the activity of caspase-3, as well as sensitize Panc-1 cells to anticancer drug-induced apoptosis. Conclusion: Anticancer drug-induced apoptosis requires mitochondrial release of Smac and downregulation of XIAP, which may be an important determinant of chemo-sensitivity in pancreatic cancer cells. Up-regulation of cytosolic expression of Smac may act as an effective modifying signal to overcome apoptosis resistance to chemotherapy in pancreatic cancer cells.

Restoration of Smad4 in BxPC3 Pancreatic Cancer Cells Attenuates Proliferation without Altering Angiogenesis

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive human malignancy in which the transforming growth factor beta (TGF-beta) signal transducer, Smad4, is commonly mutated or deleted. BxPC3 human pancreatic cancer cells exhibit a homozygous deletion of the Smad4 gene, yet are growth inhibited by TGF-beta1. In the present study, we sought to determine whether reintroduction of Smad4 into BxPC3 cells alters their behavior in vitro and in vivo. Sham transfected and Smad4 expressing BxPC3 cells exhibited similar responses to TGF-beta1 with respect to p21 upregulation, hypophosphorylation of the RB protein, Smad2 phosphorylation, and Smad2/3 nuclear translocation. TGF-beta1 did not alter p27 expression, and silencing of p21 with an appropriate siRNA markedly attenuated TGF-beta1-mediated growth inhibition. Nonetheless, the presence of Smad4 was associated in vitro with a more prolonged doubling time, enhanced sensitivity to the growth inhibitory actions of exogenous TGF-beta1, and a more flattened cellular morphology. In vivo, Smad4 expression resulted in delayed tumor growth and decreased cellular proliferation, without effects on either apoptosis or angiogenesis. These findings indicate that, in spite of the absence of Smad4, growth inhibition in BxPC3 cells by TGF-beta1 is dependent on p21 upregulation and maintenance of RB in a hypophosphorylated, active state. Moreover, the presence of a functional Smad4 attenuates the capacity of BxPC3 cells to proliferate in vivo. However, this effect is transient, indicating that Smad4 growth inhibitory actions are circumvented in the later stages of pancreatic tumorigenicity.

Apoptosis of pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid in combination with horseradish peroxidase

To explore the mechanisms underlying the apoptosis of human pancreatic cancer BXPC-3 cells induced by indole-3-acetic acid (IAA) in combination with horseradish peroxidase (HRP). BXPC-3 cells derived from human pancreatic cancer were exposed to 40 or 80 micromol/L IAA and 1.2 microg/mL HRP at different times. Then, MTT assay was used to detect the cell proliferation. Flow cytometry was performed to analyze cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to detect apoptosis. 2,7-Dichlorofluorescin diacetate uptake was measured by confocal microscopy to determine free radicals. Level of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. IAA/HRP initiated growth inhibition of BXPC-3 cells in a dose- and time-dependent manner. Flow cytometry revealed that the cells treated for 48 h were arrested at G1/G0. After exposure to 80 micromol/L IAA plus 1.2 microg/mL HRP for 72 h, the apoptosis rate increased to 72.5 per thousand, which was nine times that of control. Content of MDA and activity of SOD increased respectively after treatment compared to control. Meanwhile, IAA/HRP stimulated the formation of free radicals. The combination of IAA and HRP can inhibit the growth of human pancreatic cancer BXPC-3 cells in vitro by inducing apoptosis.

Cell cycle arrest, apoptosis induction and inhibition of nuclear factor kappa B activation in anti-proliferative activity of benzyl isothiocyanate against human pancreatic cancer cells.

Benzyl isothiocyanate (BITC), a cruciferous vegetable-derived compound, has been shown to inhibit chemically induced cancer in animal models. Moreover, epidemiological studies have provided compelling evidence to suggest that cruciferous vegetables may be protective against cancer risk. Here, we report that BITC significantly inhibits growth of human pancreatic cancer BxPC-3 cells in a concentration-dependent manner with an IC(50) of approximately 8 micro M, a concentration that can be generated through dietary intake of cruciferous vegetables. Treatment of BxPC-3 cells with growth suppressive concentrations of BITC resulted in G(2)/M phase cell cycle arrest that was associated with a marked decline in protein levels of G(2)/M regulatory proteins including cyclin-dependent kinase 1 (Cdk1), cyclin B1 and cell division cycle 25B (Cdc25B). Further, BITC-mediated growth inhibition of BxPC-3 cells correlated with apoptosis induction that was characterized by an increase in Bax/Bcl-2 ratio, cleavage of procaspase-3 and poly(ADP-ribose)polymerase (PARP), and an increase in cytoplasmic histone-associated DNA fragmentation. Interestingly, BITC treatment caused inhibition of nuclear factor kappaB (NF-kappaB) activation, which is constitutively activated in human pancreatic cancer. Western blotting revealed concentration-dependent decrease in NF-kappaB/Rel-p65 protein level in BxPC-3 cells upon exposure to BITC. An increase in protein level of inhibitory subunit kappaB (IkappaBa) in association with reduced serine-32 phosphorylation was also observed in BITC-treated BxPC-3 cells. Consistent with these findings, BITC treatment caused a decrease in nuclear translocation of NF-kappaB as reflected by reduced DNA-binding capacity of NF-kappaB. Furthermore, the protein level of cyclin D1, a transcriptional target of NF-kappaB, was reduced significantly in BITC-treated BxPC-3 cells. To the best of our knowledge, this study is the first published report to implicate suppression of NF-kappaB activation as a potential mechanism for anti-proliferative activity of BITC against human pancreatic cancer cells.

Suicide Gene/Prodrug Therapy for Pancreatic Adenocarcinoma by E. Coli Purine Nucleoside Phosphorylase and 6-Methylpurine 2′-deoxyriboside

Recent advances in diagnostics, staging, and therapy for pancreatic cancer have not resulted in significant improvements in long-term survival, and development of new approaches is particularly urgent. The use of prodrug-activating genes is a possible approach for cancer gene therapy. The aim of this study was to evaluate the efficacy of Escherichia coli purine nucleoside phosphorylase (ePNP) on pancreatic tumors. ePNP activates the prodrug 6-methylpurine deoxyribose (MePdR) into methyl purine (MeP), which is highly toxic to dividing and nondividing cells. A recombinant pCAG-ePNP vector was constructed and used to establish pancreatic cancer cells expressing constitutively ePNP (ePNP+). The ePNP/MePdR system effects were tested in vitro on HA-RPC (rat) and BxPC3 (human) pancreatic cancer cell lines and then in vivo on tumors established in nude mice with BxPC3 ePNP+ cells. MePdR treatment of ePNP+ tumor cells induced cytotoxic and antiproliferative effects in a concentration-dependent manner with a 100% cell death since 5 x 10 mol/L. Bystander effect was strong in vitro as more than 50% of tumor cells were killed by MePdR with only 1%-2% of ePNP+ cells. In vivo, tumor growth was completely abolished with a prodrug treatment initiated 2 days after tumor cell inoculation, and mice remained tumor free. In addition, even if MePdR treatment was applied to large tumors, tumors significantly regressed. These preliminary results support the therapeutic potential of the MePdR/ePNP system, which induces a highly cytotoxic effect with a potent bystander effect on pancreatic tumors.

Concurrent chemoradiotherapy with gemcitabine and cisplatin for pancreatic cancer: from the laboratory to the clinic

Purpose : We have reported that gemcitabine and concurrent radiation is a promising therapy for patients with pancreatic cancer. We investigated whether the addition of cisplatin, which may increase the systemic efficacy of gemcitabine, would be synergistic with gemcitabine and/or radiation in human pancreatic cancer cell lines. Methods and Materials : BxPc3 and Panc-1 human pancreatic cancer cells were treated with three different schedules before radiation: (A) a sequential incubation of gemcitabine for 2 h followed by cisplatin for 2 h, (B) gemcitabine for 2 h, followed by washout of drug, replenishment of media for a 24-h incubation, followed by cisplatin for 2 h, and (C) gemcitabine for 24 h with a concurrent incubation of cisplatin for the last 2 h. Cells were assessed for clonogenic survival using a standard assay. Synergism was evaluated by the median effect analysis. Results : The schedule shown to be maximally synergistic for both cell lines was the consecutive 2-h gemcitabine, 2-h cisplatin exposure, particularly at surviving fractions of <0.5. Cisplatin did not produce radiosensitization nor did it affect gemcitabine-mediated radiosensitization. Conclusion : Cisplatin produces synergistic cytotoxicity with gemcitabine without compromising gemcitabine-mediated radiosensitization. On the basis of these laboratory and previous clinical observations, we have initiated a Phase I trial of cisplatin plus gemcitabine and radiotherapy in patients with unresectable pancreatic cancer.

26S proteasome inhibition induces apoptosis and limits growth of human pancreatic cancer.

The 26S proteasome degrades proteins that regulate transcription factor activation, cell cycle progression, and apoptosis. In cancer, this may allow for uncontrolled cell division, promoting tumor growth, and spread. We examined whether selective inhibition of the 26S proteasome with PS-341, a dipeptide boronic acid analogue, would block proliferation and induce apoptosis in human pancreatic cancer. Proteasome inhibition significantly blocked mitogen (FCS) induced proliferation of BxPC3 human pancreatic cancer cells in vitro, while arresting cell cycle progression and inducing apoptosis by 24 h. Accumulation of p21(Cip1-Waf-1), a cyclin dependent kinase (CDK) inhibitor normally degraded by the 26S proteasome, occurred by 3 h and correlated with cell cycle arrest. When BxPC3 pancreatic cancer xenografts were established in athymic nu/nu mice, weekly administration of 1 mg/kg PS-341 significantly inhibited tumor growth. Both cellular apoptosis and p21(Cip1-Waf-1) protein levels were increased in PS-341 treated xenografts. Inhibition of tumor xenograft growth was greatest (89%) when PS-341 was combined with the tumoricidal agent CPT-11. Combined CPT-11/PS-341 therapy, but not single agent therapy, yielded highly apoptotic tumors, significantly inhibited tumor cell proliferation, and blocked NF-kappaB activation indicating this systemic therapy was effective at the cancer cell level. 26S proteasome inhibition may represent a new therapeutic approach against this highly resistant and lethal malignancy.