Aberrant DNA methyltransferase expression in pancreatic ductal adenocarcinoma development and progression

Background

Altered gene methylation, regulated by DNA methyltransferases (DNMT) 1, 3a and 3b, contributes to tumorigenesis. However, the role of DNMT in pancreatic ductal adenocarcinoma (PDAC) remains unknown.

Methods

Expression of DNMT 1, 3a and 3b was detected in 88 Pancreatic ductal adenocarcinoma (PDAC) and 10 normal tissue samples by immunohistochemistry. Changes in cell viability, cell cycle distribution, and apoptosis of PDAC cell lines (Panc-1 and SW1990) were assessed after transfection with DNMT1 and 3b siRNA. Levels of CDKN1A, Bcl-2 and Bax mRNA were assessed by qRT-PCR, and methylation of the Bax gene promoter was assayed by methylation-specific PCR (MSP).

Results

DNMT1, 3a and 3b proteins were expressed in 46.6%, 23.9%, and 77.3% of PDAC tissues, respectively, but were not expressed in normal pancreatic tissues. There was a co-presence of DNMT3a and DNMT3b expression and an association of DNMT1 expression with alcohol consumption and poor overall survival. Moreover, knockdown of DNMT1 and DNMT3b expression significantly inhibited PDAC cell viability, decreased S-phase but increased G1-phase of the cell cycle, and induced apoptosis. Molecularly, expression of CDKN1A and Bax mRNA was upregulated, and the Bax gene promoter was demethylated. However, a synergistic effect of combined DNMT1 and 3b knockdown was not observed.

Conclusion

Expression of DNMT1, 3a and 3b proteins is increased in PDAC tissues, and DNMT1 expression is associated with poor prognosis of patients. Knockdown of DNMT1 and 3b expression arrests tumor cells at the G1 phase of the cell cycle and induces apoptosis. The data suggest that DNMT knockdown may be a novel treatment strategy for PDAC.     

MAP3K10 promotes the proliferation and decreases the sensitivity of pancreatic cancer cells to gemcitabine by upregulating Gli-1 and Gli-2

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human malignancies and is regulated by Sonic Hedgehog (Shh) signaling. Recently, MAP3K10 has been shown to regulate Shh signaling, suggesting a role for MAP3K10 in the tumorigenesis of PDAC. We determined the expression status of MAP3K10 in PDAC tissues and cell lines, and analyzed the viability and cell proliferation of PDAC cells with an overexpression or knockdown of MAP3K10 in vitro. MAP3K10 was upregulated in PDAC tissues and cell lines. Overexpression of MAP3K10 promoted the proliferation and decreased the gemcitabine sensitivity of pancreatic cancer cells. In contrast, knockdown of MAP3K10 significantly decreased cell proliferation and sensitized cells to gemcitabine. However, neither overexpression nor knockdown of MAP3K10 affected cell migration. Moreover, overexpression of MAP3K10 resulted in upregulation of Gli-1 and Gli-2 in PDAC cells. Our results indicate a novel and important role for MAP3K10 in the proliferation and chemoresistance of PDAC. Our study suggests that targeting MAP3K10 is a potential strategy for the development of alternative therapies for pancreatic cancers.     

KRAS promotes tumor metastasis and chemoresistance by repressing RKIP via the MAPK–ERK pathway in pancreatic cancer

Oncogenic KRAS plays a crucial role in pancreatic ductal adenocarcinoma (PDAC) development and progression. However, the mechanism has not been clearly elucidated. RKIP is a tumor repressor, and loss of RKIP has been shown in PDAC. Here, we found that KRAS expression was inversely correlated with RKIP expression in PDAC fresh tissue regardless of the KRAS mutant status. The negative correlation between KRAS and RKIP was further confirmed in our PDAC tissue microarray. KRAS overexpression and RKIP downregulation were associated with poor clinical outcomes. Knockdown or overexpression of KRAS in PDAC cell lines robustly increased or decreased, respectively, RKIP protein and mRNA levels. Furthermore, the MAPK–ERK pathway was involved in the regulation of RKIP. KRAS‐regulated RKIP expression, which in turn affected the expression of pivotal epithelial–mesenchymal transition (EMT) and apoptosis factors. The biological function of the KRAS–RKIP axis was demonstrated in human pancreatic cancer cells in vitro and in vivo. KRAS knockdown increased RKIP expression and inhibited metastasis and chemoresistance. Moreover, the feature of metastasis and chemoresistance was rescued in the KRAS‐knockdown cells through the inhibition of RKIP by RNA interference. In conclusion, our studies demonstrate how KRAS inhibits the tumor suppressor RKIP, thus offering novel justification for targeting RKIP as a strategy to overcome KRAS‐induced tumor metastasis and chemoresistance in PDAC.     

The role of lncRNA MSC-AS1/miR-29b-3p axis-mediated CDK14 modulation in pancreatic cancer proliferation and Gemcitabine-induced apoptosis

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related death due to the failure of traditional therapies. In the present study, we attempted to construct a lncRNA-miRNA-mRNA network which may modulate PDAC cell proliferation and Gemcitabine-induced cell apoptosis starting from CDK14, a new member of the CDK family and an oncogene in many cancers. Based on TCGA data, a significant positive correlation was observed between lncRNA MSC-AS1 and CDK14. Moreover, MSC-AS1 expression was upregulated in PDAC tissues. Higher MSC-AS1 expression was correlated with poorer prognosis in patients with PDAC. MSC-AS1 knockdown in Panc-1 and BxPC-3 cells significantly inhibited the cell proliferation. Moreover, miR-29b-3p, which has been reported to act as a tumor suppressor, was predicted to bind to both MSC-AS1 and CDK14. Contrary to MSC-AS1, higher miR-29b-3p expression was correlated to better prognosis in patients with PDAC. In both PDAC cell lines, miR-29b-3p negatively regulated MSC-AS1 and CDK14. As confirmed using luciferase reporter gene and RIP assays, MSC-AS1 served as a ceRNA for miR-29b-3p to counteract miR-29b-mediated CDK14 repression. MSC-AS1 knockdown inhibited CDK14 protein levels and PDAC proliferation and enhanced gemcitabine-induced cell death and apoptosis while miR-29b-3p inhibition exerted an opposing effect; the effect of MSC-AS1 knockdown was partially attenuated by miR-29b-3p inhibition. Taken together, we demonstrated that MSC-AS1/miR-29b-3p axis modulates the cell proliferation and GEM-induced cell apoptosis in PDAC cell lines through CDK14. We provided a novel experimental basis for PDAC treatment from the perspective of lncRNA-miRNA-mRNA network.     

Verticillin A overcomes apoptosis resistance in human colon carcinoma through DNA methylation-dependent upregulation of BNIP3

Drug resistance is a major cause of failure in cancer chemotherapy. Therefore, identification and combined use of adjuvant compounds that can overcome drug resistance may improve the efficacy of cancer therapy. We screened extracts of Verticillium species-infected mushrooms for antitumor compounds and identified the compound Verticillin A as an inducer of hepatoma cell apoptosis in vitro and an inhibitor of tumor xenograft growth in vivo. Verticillin A exhibited a potent apoptosis-sensitizing activity in human colon carcinoma cells exposed to TRAIL or Fas in vitro. Furthermore, Verticillin A effectively sensitized metastatic human colon carcinoma xenograft to TRAIL-mediated growth inhibition in vivo. At the molecular level, we observed that Verticillin A induces cell-cycle arrest in the G? phase of the cell cycle in human colon carcinoma cells, markedly upregulating BNIP3 in both hepatoma and colon carcinoma cells. Notably, silencing BNIP3 decreased the sensitivity of tumor cells to Verticillin A-induced apoptosis in the absence or presence of TRAIL. We found that the BNIP3 promoter is methylated in both human hepatoma and colon carcinoma cells and tumor specimens. Verticillin A upregulated the expression of a panel of genes known to be regulated at the level of DNA methylation, in support of the concept that Verticillin A may act by demethylating the BNIP3 promoter to upregulate BNIP3 expression. Taken together, our findings identify Verticillin A as a potent apoptosis sensitizer with great promise for further development as an adjuvant agent to overcome drug resistance in human cancer therapy.     

Inhibition of oncogenic Pim-3 kinase modulates transformed growth and chemosensitizes pancreatic cancer cells to gemcitabine

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a 5-year survival rate of only 6%. Although the cytosine analog gemcitabine is the drug commonly used to treat PDAC, chemoresistance unfortunately renders the drug ineffective. Thus, strategies that can decrease this resistance will be essential for improving the dismal outcome of patients suffering from this disease. We previously observed that oncogenic Pim-1 kinase was aberrantly expressed in PDAC tissues and cell lines and was responsible for radioresistance. Furthermore, members of the Pim family have been shown to reduce the efficacy of chemotherapeutic drugs in cancer. Therefore, we attempted to evaluate the role of Pim-3 in chemoresistance of PDAC cells. We were able to confirm upregulation of the Pim-3 oncogene in PDAC tissues and cell lines vs. normal samples. Biological consequences of inhibiting Pim-3 expression with shRNA-mediated suppression included decreases in anchorage-dependent growth, invasion through Matrigel and chemoresistance to gemcitabine as measured by caspase-3 activity. Additionally, we were able to demonstrate that Pim-1 and Pim-3 play overlapping but non-identical roles as it relates to gemcitabine sensitivity of pancreatic cancer cells. To further support the role of Pim-3 suppression in sensitizing PDAC cells to gemcitabine, we used the pharmacological Pim kinase inhibitor SGI-1776. Treatment of PDAC cells with SGI-1776 resulted in decreased phosphorylation of the proapoptotic protein Bad and cell cycle changes. When SGI-1776 was combined with gemcitabine, there was a greater decrease in cell viability in the PDAC cells vs. cells treated with either of the drugs separately. These results suggest combining drug therapies that inhibit Pim kinases, such as Pim-3, with chemotherapeutic agents, to aid in decreasing chemoresistance in pancreatic cancer.     

Caveolin-1 mediated radioresistance of 3D grown pancreatic cancer cells

Background and purpose

Resistance of pancreatic ductal adenocarcinoma (PDAC) to chemo- and radiotherapy is a major obstacle. The integral membrane protein Caveolin-1 (Cav-1) has been suggested as a potent target in human pancreatic carcinoma cells.

Materials and methods

Human pancreatic tumor cells were examined in a three-dimensional (3D) cell culture model with regard to clonogenic survival, apoptosis, radiogenic DNA-double strand breaks and protein expression and phosphorylation under siRNA-mediated knockdown of Cav-1 without and in combination with irradiation (X-rays, 0-6 Gy). Immunohistochemistry was used to assess Cav-1 expression in biopsies from patients with PDAC.

Results

Tumor cells in PDAC showed significantly higher Cav-1 expression relative to tumor stroma. Cav-1 knockdown significantly reduced β1 integrin expression and Akt phosphorylation, induced Caspase 3- and Caspase 8-dependent apoptosis and enhanced the radiosensitivity of 3D cell cultures. While cell cycling and Cav-1 promoter activity remained stable, Cav-1 knockdown-induced radiosensitization correlated with elevated numbers of residual DNA-double strand breaks.

Conclusions

Our data strongly support the concept of Cav-1 as a potent target in pancreatic carcinoma cells due to radiosensitization and Cav-1 overexpression in tumor cells of PDAC. 3D cell cultures are powerful and useful tools for the testing of novel targeting strategies to optimize conventional radio- and chemotherapy regimes for PDAC.     

Dihydroartemisinine Enhances Dictamnine-induced Apoptosis via a Caspase Dependent Pathway in Human Lung Adenocarcinoma A549 Cells

Dictamnine (Dic) has the ability to exert cytotoxicity in human cervix, colon, and oral carcinoma cells anddihydroartemisinin (DHA) also has potent anticancer activity on various tumour cell lines. This report exploresthe molecular mechanisms by which Dic treatment and combination treatment with DHA and Dic cause apoptosisin human lung adenocarcinoma A549 cells. Dic treatment induced concentration- and time-dependent cell death.FCM analysis showed that Dic induced S phase cell cycle arrest at low concentration and cell apoptosis at highconcentration in which loss of mitochondrial membrane potential (ΔΨm) was not involved. In addition, inhibitionof caspase-3 using the specific inhibitor, z-DQMD-fmk, did not attenuate Dic-induced apoptosis, implying thatDic-induced caspase-3-independent apoptosis. Combination treatment with DHA and Dic dramatically increasedthe apoptotic cell death compared to Dic alone. Interestingly, pretreatment with z-DQMD-fmk significantlyattenuated DHA and Dic co-induced apoptosis, implying that caspase-3 plays an important role in Dic and DHAco-induced cell apoptosis. Collectively, we found that Dic induced S phase cell cycle arrest at low concentrationand cell apoptosis at high concentration in which mitochondria and caspase were not involved and DHA enhancedDic induced A549 cell apoptosis via a caspase-dependent pathway.     

Significance of S100P as a biomarker in diagnosis,prognosis and therapy of opisthorchiasis‐associated cholangiocarcinoma

Cholangiocarcinoma (CCA) is a malignancy of bile duct with the difficulty in early diagnosis, poor prognosis and less alternation in therapy. S100P is a member of S100 family proteins and plays important roles in cancers. We investigated the S100P expression and its correlation with clinicopathology in 78 cases of opisthorchiasis‐associated CCA, and the effects of S100P knockdown with shRNA interference on the proliferation, cell cycle, migration, apoptosis and sensitivity to anti‐cancer drug. Extremely high expression of S100P mRNA was detected in the CCA tumor tissues. The increased S100P protein expression was immunohistochemically confirmed and localized in the CCA cytoplasm and/or nuclei as well as in the hyperneoplasia and dysplasia bile ducts, but not in normal bile ducts. The intensity of immunostaining was correlated with survival, tumor stage and metastasis, and the high expression could be an independent prognostic factor. High levels of S100P were detected in the serum and bile fluid of CCA patients. The shRNA‐mediated knockdown of S100P expression inhibited the proliferation in vitro and in vivo, and migration of CCA cells, arrested cell cycle with the up‐regulated expression of cell cycle arrest related factors, p21, p27, GADD45A, and 14‐3‐3 zeta. S100P knockdown also promoted CCA cell apoptosis by up‐regulating expression of apoptosis related factors, DR5, TRADD, caspase 3 and BAX, and increased the sensitivity of CCA cells to the chemotherapeutic agents sunitinib and apigenin. Taken together, this study indicates that S100P might be a promising biomarker for the diagnosis, prognosis and therapy of CCA.     

Silencing of the hypoxia-inducible cell death protein BNIP3 in pancreatic cancer

Hypoxic conditions exist within pancreatic adenocarcinoma, yet pancreatic cancer cells survive and replicate within this environment. To understand the mechanisms involved in pancreatic cancer adaptation to hypoxia, we analyzed expression of a regulator of hypoxia-induced cell death, Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). We found that BNIP3 was down-regulated in nine of nine pancreatic adenocarcinomas compared with normal pancreas despite the up-regulation of other hypoxia-inducible genes, including glucose transporter-1 and insulin-like growth factor-binding protein 3. Also, BNIP3 expression was undetectable even after hypoxia treatment in six of seven pancreatic cancer cell lines. The BNIP3 promoter, which was remarkably activated by hypoxia, is located within a CpG island. The methylation status of CpG dinucleotides within the BNIP3 promoter was analyzed after bisulfite treatment by sequencing and methylation-specific PCR. Hypermethylation of the BNIP3 promoter was observed in all BNIP3-negative pancreatic cancer cell lines and eight of 10 pancreatic adenocarcinoma samples. Treatment of BNIP3-negative pancreatic cancer cell lines with a DNA methylation inhibitor, 5-aza-2' deoxycytidine, restored hypoxia-induced BNIP3 expression. BNIP3 expression was also restored by introduction of a construct consisting of a full-length BNIP3 cDNA regulated by a cloned BNIP3 promoter. Restoration of BNIP3 expression rendered the pancreatic cancer cells notably more sensitive to hypoxia-induced cell death. In conclusion, down-regulation of BNIP3 by CpG methylation likely contributes to resistance to hypoxia-induced cell death in pancreatic cancer.     

Dulxanthone A induces cell cycle arrest and apoptosis via up-regulation of p53 through mitochondrial pathway in HepG2 cells

Natural products derived from plants provide a rich source for development of new anticancer drugs. Dulxanthone A was found to be an active cytotoxic component in Garcinia cowa by bioactivity-directed isolation. Studies to elucidate the cytotoxic mechanisms of dulxanthone A showed that dulxanthone A consistently induced S phase arrest and apoptosis in the most sensitive cell line HepG2. Furthermore, p53 was dramatically up-regulated, leading to altered expression of downstream proteins upon dulxanthone A treatment. Cell cycle related proteins, such as cyclin A, cyclin B, cyclin E, cdc-2, p21 and p27 were down-regulated. Some apoptosis correlated proteins were also altered following the drug treatment. Bcl-2 family members PUMA was up-regulated while Bcl-2 and Bax were down-regulated. However, the expression ratio of Bax/Bcl-2 was increased. This resulted in the release of cytochrome C from the mitochondria to the cytosol. Concurrently, Apaf-1 was stimulated with p53 by dulxanthone A. In result, cytochrome C, Apaf-1 and procaspase-9 form an apoptosome, which in turn triggered the activation of caspase-9, caspase-3 and downstream caspase substrates. Lamin A/C and PARP were down-regulated or cleaved, respectively. Moreover, cell cycle arrest and apoptosis in HepG2 cells induced by dulxanthone A were markedly inhibited by siRNA knockdown of p53. In summary, dulxanthone A is an active cytotoxic component of G. cowa. It induces cell cycle arrest at lower concentrations and triggers apoptosis at higher concentrations via up-regulation of p53 through the intrinsic mitochondrial pathway in HepG2 cells. Dulxanthone A is therefore likely a promising preventive and/or therapeutic agent against Hepatoma.     

Histone deacetylase inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis in human malignant glioma cells

Histone deacetylase (HDAC) inhibitors have both apoptotic and differentiating effects on various tumor cells. However, the mechanisms underlying the effect of HDAC inhibitors remain unclear. In this study, we investigated the function of anti-proliferative effects of HDAC inhibitors, N-butyric acid and trichostatin A, on human malignant glioma cell lines, U251-MG and D54. MTT assay showed a dose-dependent inhibition of cellular proliferation in both cell lines. Cell cycle analysis revealed increased sub-G1 population in both lines, and G1 arrest only in U251-MG cells. Induction of apoptosis was also supported by the occurrence of DNA fragmentation in tumor cells treated with HDAC inhibitors. Furthermore, caspase inhibition assay indicated that HDAC inhibitor-induced apoptosis was caspase-dependent. Neither mitochondrial membrane potential nor the expression of caspase-9 was changed by treatment with HDAC inhibitors, suggesting the possibility that HDAC inhibitor-induced apoptosis was not mediated by the mitochondrial cell death pathway. On the other hand, immunoblot assay confirmed increased expression of caspase-8 in both lines, and elevation of p21 but not p27 protein in U251-MG cells following HDAC inhibitor treatment. Taken together, the HDAC inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis with or without p21-mediated G1 arrest in human malignant glioma cells.     

shRNA against CD44 inhibits cell proliferation, invasion and migration, and promotes apoptosis of colon carcinoma cells

CD44 is a causal factor for tumor invasion, metastasis and acquisition of resistance to apoptosis. CD44 knockdown using inducible short hairpin RNA (shRNA) significantly reduces cell growth and invasion. Short hairpin RNA against CD44 and pGFP-V-RS-vector was used for knockdown of CD44 expression in SW620 colon cancer cells. Cell growth, invasion and migration assay, immunofluorescence for β-catenin expression and western blotting for Wnt signaling molecules were analyzed. Cell cycle analysis and western blot analysis for apoptotic molecules were evaluated. Short hairpin RNA against CD44 reduced the expression of CD44. Cell proliferation, migration and invasion were markedly inhibited and apoptosis was increased in shRNA CD44-transfected cells. Knockdown of CD44 decreased the phosphorylation of PDK1, Akt and GSK3β, and β-catenin levels. Decreased phosphorylated Akt led to an increase in phosphorylated FoxO1 and induced cell cycle arrest in the G0-G1 phase and a decrease in the S phase. The levels of Bcl-2 and Bcl-xL expression were down-regulated, while the levels of BAX expression and cleaved caspase-3, -8 and -9 were increased. CD44 knockdown by way of shRNA inhibited cell proliferation and induced cell apoptosis. This can be used as a therapeutic intervention with the anti-survival/pro-apoptotic machinery in human colon cancer.     

Silencing of CXCR4 sensitizes triple-negative breast cancer cells to cisplatin

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer for which there is no effective treatment. Previously, we and others demonstrated that CXCR4 surface expression is an independent prognostic factor for disease relapse and survival in breast cancer. In this study, we investigated the effects of CXCR4 gene silencing on cisplatin chemosensitivity in human triple-negative breast cancer cell lines. We found that CXCR4 silencing significantly inhibited cell growth, decreased colony formation, and enhanced cisplatin sensitivity while overexpression of CXCR4 rendered cells more resistant to cisplatin. Moreover, the percentage of apoptosis and cell cycle arrest at the G2/M phase of cisplatin-treated CXCR4 knockdown cells was significantly higher than control cells. Furthermore, we demonstrated CXCR4 knockdown cells showed lower levels of mutant p53 and Bcl-2 protein than the control group, while also having higher levels of caspase-3 and Bax. However overexpression of CXCR4 had the reverse effect. In vivo experiments confirmed that downregulation of CXCR4 enhanced cisplatin anticancer activity in tumor-bearing mice, and that this enhanced anticancer activity is attributable to tumor cell apoptosis. Thus, this study indicates that CXCR4 can modulate cisplatin sensitivity in TNBC cells and suggests that CXCR4 may be a therapeutic target for TNBC.     

FR901228, a novel histone deacetylase inhibitor, induces cell cycle arrest and subsequent apoptosis in refractory human pancreatic cancer cells

Histone deacetylase (HDAC) inhibitors have antiproliferative activity against human cancer cells via cell cycle arrest, differentiation, and apoptosis. However, no report has focused on the apoptotic potential of HDAC inhibitors in refractory human pancreatic cancer. This study was designed to examine the apoptotic potential of FR901228, a novel HDAC inhibitor, in five human pancreatic cancer cell lines: Capan-1, BxPC-3, HPAF, Panc-1, and MIAPaCa-2. FR901228 markedly inhibited the proliferation of all five cell lines (IC50: 1-500 nM), with the greatest effect in MIAPaCa-2 cells. Treatment of each cell line with FR901228 (10-100 nM) caused cell cycle arrest at the G1 or G2/M phase and subsequent apoptosis. FR901228 induced expression of hyperacetylated histone H3 after 3 h of treatment and overexpression of p21Waf-1 after 6 h. In addition, FR901228 induced apoptosis by activating caspase-3, which led to cleavage of p21Waf-1 into a 15-kDa breakdown product and drove cancer cells from cell cycle arrest into apoptosis. FR901228 also decreased the protein level of survivin dramatically. Our results show that FR901228 markedly inhibits the growth of pancreatic cancer cells, not only through cell cycle arrest, but also through subsequent apoptosis; this was accompanied by caspase-3 activation, survivin degradation, and p21Waf-1 cleavage. FR901228 may prove clinically useful as an agent for refractory pancreatic cancers.     

Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis

Altered expression of apoptosis-regulating genes plays an important role in the aggressive growth behavior and chemoresistance of pancreatic ductal adenocarcinoma. In the present study, the hypoxia-inducible proapoptotic gene, BNIP3, was analysed in terms of expression, effect on patient survival, and chemo-responsiveness in pancreatic cancer cell lines. cDNA microarray, real-time light cycler quantitative polymerase chain reaction, laser-capture microdissection, and immunohistochemistry analyses were used to evaluate BNIP3 expression in normal and diseased pancreatic specimens. Modulation of BNIP3 expression was achieved using specific siRNA molecules. The effect of chemotherapeutic agents on pancreatic cancer cells was assessed utilizing 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide assays. BNIP3 mRNA levels were 3.0- and 6.3-fold lower in chronic pancreatitis and pancreatic cancer compared to the normal pancreas, respectively. Microdissection analysis confirmed the reduction of BNIP3 expression in pancreatic cancer cells compared to normal duct cells. By immunohistochemistry, BNIP3 was predominantly expressed in the acinar cells of the normal and diseased pancreas. Interestingly, while BNIP3 was undetectable in the cancer cells of 59% of the cases, 75-100% of PanIN2/3 lesions displayed BNIP3 immunoreactivity. Loss of BNIP3 expression correlated with poorer survival of patients (8 vs 14 months for BNIP3 negative vs positive tumors). Hypoxia induced BNIP3 expression in four out of eight pancreatic cancer cell lines, while it was absent under normoxic and hypoxic conditions in the remaining four. Downregulation of BNIP3 resulted in increased resistance to 5-fluoro-uracil and gemcitabine. In conclusion, loss of BNIP3 expression occurs late in pancreatic cancer, contributes to resistance to chemotherapy, and correlates with a worsened prognosis.     

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and apoptosis in colorectal cancer cells via p53-dependent and -independent pathways

Many chemotherapeutic agents induce apoptosis via a p53-dependent pathway. However, up to 50% of human cancers have p53 mutation and loss of p53 function. Histone deacetylase inhibitors (HDACIs) are emerging as a potentially important new class of anticancer agents. Here, we report that, Trichostatin A (TSA), a pan-HDAC inhibitor, could induce G2/M cell cycle arrest and apoptosis in both colorectal cancer cell lines with wild-type p53 (HT116 cells) and mutant p53 (HT29 cells), although HCT116 cells had more apoptotic cells than HT29 cells. TSA induces apoptosis in both cell lines via the mitochondrial pathway as indicated by decrease of the mitochondrial membrane potential (MMP) and activation of caspase-3. Additionally, TSA induces expression of the pro-apoptotic protein Bax and decreases the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL in both cell lines. Bax knockdown by siRNA significantly impaired TSA-induced apoptosis in both cell lines. These data suggest that TSA induces G2/M cell cycle arrest and Bax-dependent apoptosis in colorectal cancer cells (HCT116 cells and HT29 cells) by both p53-dependent and -independent mechanisms. However, cells with normal p53 function are more sensitive to TSA-induced apoptosis.