Gemcitabine-resistant Pancreatic Cancer Cell Line Research Articles

Antiproliferative Activity of Piceamycin by Regulating Alpha-Actinin-4 in Gemcitabine-Resistant Pancreatic Cancer Cells.

Although gemcitabine-based regimens are widely used as an effective treatment for pancreatic cancer, acquired resistance to gemcitabine has become an increasingly common problem. Therefore, a novel therapeutic strategy to treat gemcitabine-resistant pancreatic cancer is urgently required. Piceamycin has been reported to exhibit antiproliferative activity against various cancer cells; however, its underlying molecular mechanism for anticancer activity in pancreatic cancer cells remains unexplored. Therefore, the present study evaluated the antiproliferation activity of piceamycin in a gemcitabine-resistant pancreatic cancer cell line and patient-derived pancreatic cancer organoids. Piceamycin effectively inhibited the proliferation and suppressed the expression of alpha-actinin-4, a gene that plays a pivotal role in tumorigenesis and metastasis of various cancers, in gemcitabine-resistant cells. Long-term exposure to piceamycin induced cell cycle arrest at the G0/G1 phase and caused apoptosis. Piceamycin also inhibited the invasion and migration of gemcitabine-resistant cells by modulating focal adhesion and epithelial-mesenchymal transition biomarkers. Moreover, the combination of piceamycin and gemcitabine exhibited a synergistic antiproliferative activity in gemcitabine-resistant cells. Piceamycin also effectively inhibited patient-derived pancreatic cancer organoid growth and induced apoptosis in the organoids. Taken together, these findings demonstrate that piceamycin may be an effective agent for overcoming gemcitabine resistance in pancreatic cancer.

Synergistic effects of resveratrol with gemcitabine in pancreatic cancer chemotherapy by inhibiting the c-Met/PARP1 axis

Objective: Pancreatic cancer is a highly malignant tumor of the digestive tract with a dismal prognosis. A key challenge of pancreatic cancer is its resistance to chemotherapy. The c-Met/PARP1 axis plays an important role in the therapeutic resistance of breast cancer and hepatocellular carcinoma. Therefore, this study aims to explore potential therapeutic targets for improving chemotherapy for pancreatic cancer and the underlying mechanisms. Methods: Gemcitabine-resistant pancreatic cancer cell lines were constructed by our laboratory using a continuous low-concentration gemcitabine induction method. The proliferation and apoptosis of combination therapy were examined using flow cytometry and comet assay. Synergistic effects of two drugs were determined by Chou-Talalay's combination index (CI). The interactions between proteins were predicted using the AutoDock model and detected through Coimmunoprecipitation assays. Results: The combination of resveratrol and gemcitabine inhibited proliferation and promoted apoptosis of pancreatic cancer cells. We found that c-Met and PARP1 were highly expressed in gemcitabine-resistant pancreatic cancer cells. However, resveratrol inhibited their expression and improved the effectiveness of gemcitabine-induced DNA damage. In addition, our data demonstrated that resveratrol and gemcitabine had synergistic effects (CI < 1). Furthermore, the protein interactions between c-Met and PARP1 were attenuated after the early stage of resveratrol intervention. Using AutoDock models, we predicted the potential binding sites where resveratrol could impact the protein interaction between c-Met (tyrosine kinase domain) and PARP1 (CAT domain). Conclusion: Our results suggested that the synergistic effects of resveratrol with gemcitabine depend on the c-Met/PAPR1 axis. Using resveratrol as a combined chemotherapy agent may have clinical benefits for patients with refractory pancreatic cancer.

Prognostic evaluation of pancreatic cancer based on the model of chemo-radiotherapy resistance-related genes.

The incidence and mortality of pancreatic cancer are almost the same, and the 5-year survival rate is less than 10%. The high mortality of pancreatic cancer is related to chemo-radiotherapy. The present study aimed to establish a prognostic signature of pancreatic cancer based on chemo-radiotherapy resistant-related genes (CRRGs). In this study, we explored the radiation-resistant and chemotherapy-resistant pancreatic cancer cell lines by colony formation and a subcutaneous tumor model in nude mice. Next, we obtained CRRGs from radiation- and gemcitabine-resistant pancreatic cancer cell lines in the Gene Expression Omnibus (GEO) database. Based on univariate Cox and least absolute shrinkage and selection operator (LASSO) Cox regression analyses, a prognostic model of the pancreatic adenocarcinoma (PAAD) cohort in The Cancer Genome Atlas (TCGA) database (N=177) was established and verified using the GEO cohort data set (N=112). Finally, the functions of candidate target genes were verified by a methyl thiazolyl tetrazolium (MTT) assay, a colony formation assay, and a subcutaneous tumor model in nude mice. Through the in vitro and in vivo experiments, we found that radiotherapy- and chemotherapy-resistant pancreatic cancer cells were cross-resistant to chemotherapy and radiotherapy. We constructed a risk model consisting of nine CRRGs (SNAP25, GPR87, DLL1, LAD1, WASF3, ARHGAP29, ZBED2, GAD1, and JAG1) by using public databases. According to the Kaplan-Meier curve analysis, the survival of the high-risk group was worse than that of the low-risk group. We then used nomograms to predict the 1/3/5-year overall survival (OS) in pancreatic cancer patients. We chose JAG1 as a candidate target since it has been proven to be involved in the stemness maintenance of cancer cells, and found that JAG1 silencing inhibited the proliferation and chemo-radiotherapy tolerance of pancreatic cancer cells. This study established and validated a prognostic signature of pancreatic cancer using nine CRRGs. The in vitro and in vivo experiments showed that JAG1 could promote the proliferation and chemoradiotherapy tolerance of pancreatic cancer cell lines. These findings may offer new insights into the role of CRRGs in pancreatic cancer and provide novel prognostic biomarkers for the treatment of pancreatic cancer.

Identification and Clinical Significance of Pancreatic Cancer Stem Cells and Their Chemotherapeutic Drug Resistance.

Pancreatic cancer ranks in the 10th-11th position among cancers affecting men in Taiwan, besides being a rather difficult-to-treat disease. The overall 5-year survival rate of pancreatic cancer is only 5-10%, while that of resectable pancreatic cancer is still approximately 15-20%. Cancer stem cells possess intrinsic detoxifying mechanisms that allow them to survive against conventional therapy by developing multidrug resistance. This study was conducted to investigate how to overcome chemoresistance and its mechanisms in pancreatic cancer stem cells (CSCs) using gemcitabine-resistant pancreatic cancer cell lines. Pancreatic CSCs were identified from human pancreatic cancer lines. To determine whether CSCs possess a chemoresistant phenotype, the sensitivity of unselected tumor cells, sorted CSCs, and tumor spheroid cells to fluorouracil (5-FU), gemcitabine (GEM), and cisplatin was analyzed under stem cell conditions or differentiating conditions. Although the mechanisms underlying multidrug resistance in CSCs are poorly understood, ABC transporters such as ABCG2, ABCB1, and ABCC1 are believed to be responsible. Therefore, we measured the mRNA expression levels of ABCG2, ABCB1, and ABCC1 by real-time RT-PCR. Our results showed that no significant differences were found in the effects of different concentrations of gemcitabine on CSCs CD44+/EpCAM+ of various PDAC cell line cultures (BxPC-3, Capan-1, and PANC-1). There was also no difference between CSCs and non-CSCs. Gemcitabine-resistant cells exhibited distinct morphological changes, including a spindle-shaped morphology, the appearance of pseudopodia, and reduced adhesion characteristics of transformed fibroblasts. These cells were found to be more invasive and migratory, and showed increased vimentin expression and decreased E-cadherin expression. Immunofluorescence and immunoblotting experiments demonstrated increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells showed activation of the receptor protein tyrosine kinase c-Met and increased expression of the stem cell marker cluster of differentiation (CD) 24, CD44, and epithelial specific antigen (ESA). We concluded that the expression of the ABCG2 transporter protein was significantly higher in CD44+ and EpCAM+ CSCs of PDAC cell lines. Cancer stem-like cells exhibited chemoresistance. Gemcitabine-resistant pancreatic tumor cells were associated with EMT, a more aggressive and invasive phenotype of numerous solid tumors. Increased phosphorylation of c-Met may also be related to chemoresistance, and EMT and could be used as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Abstract 390: A comparison of the activity of the quadruplex-targeting experimental drugs QN-302 and CX-5461 (pidnarulex) in wild-type and gemcitabine-resistant pancreatic cancer cell lines

Abstract The compound QN-302, a tetra-substituted naphthalene diimide (ND) derivative has been designed as a potent compound for targeting quadruplex sequences in cancer genes. It has single-digit nM anti-proliferative activity in a panel of human pancreatic cancer (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It also has significant anti-tumor activity in xenograft, orthotopic and genetic (KPC) models for PDAC. Its mode of action involves the targeting of quadruplex (G4)-forming sites in promoter regions of cancer-associated genes, where their prevalence is over-represented compared to other genes. The structurally unrelated compound CX-5461 has also been reported to be a G4-binding agent, with selective activity in BRCA1/2 deficient cells and tumors (Xu and Hurley, Bioorg Med Chem Lett. 2022; Xu et al., Nat Commun, 2017). It is currently in early-stage clinical trials in breast and other solid cancers with deficiencies in DNA repair (Hilton et al., Nat Commun. 2022). We have previously reported that a precursor to QN-302, the tri-substituted naphthalene diimide compound CM03, is highly active in both wild-type and gemcitabine-resistant PDAC cell lines (Ahmed et al, Sci Rep, 2020). This is explained by the finding using whole-genome transcriptome analysis that the CM03-sensitive genes are largely unaffected in the resistant lines. We now report that the clinical candidate compound QN-302, which is 10-fold more potent than CM03, retains its potency in both MIA-PACA2 and PANC-1 PDAC cell lines with >1000-fold increase in gemcitabine resistance. This is also the case for CX-5461, although this compound is 50-100 times less potent, with GI50 values of 83-95 nM in cell proliferation assays treated for 96 h. Gemcitabine either alone or in combination is still a mainstay of current PDAC treatment. Resistance to gemcitabine is common and is a major contributor to the poor outcomes for most PDAC patients. The ability of QN-302 to retain activity in chemo-resistant PDAC cell lines suggests that it will offer significant advantage in the clinic over gemcitabine-based therapies. QN-302 is bio-available at therapeutic doses and is well tolerated at these levels in animal models. It is being developed for clinical evaluation by Qualigen Therapeutics Inc and is currently at the pre-IND stage. Citation Format: Ahmed Ahmed, Tariq Arshad, Stephen Neidle. A comparison of the activity of the quadruplex-targeting experimental drugs QN-302 and CX-5461 (pidnarulex) in wild-type and gemcitabine-resistant pancreatic cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 390.

Pralatrexate mediates effective killing of gemcitabine-resistant pancreatic cancer: role of mTOR/4E-BP1 signal pathway

Gemcitabine is the first-line chemotherapeutic agent for pancreatic cancer. However, gemcitabine-resistance frequently leads to poor prognosis. Exploring new chemotherapeutic agents is important for patients with gemcitabine-resistant pancreatic cancer. In this study, we established a new acquired gemcitabine-resistant pancreatic cancer cell line BxPC-GEM-20 from parental BxPC-3. We found that pralatrexate significantly inhibited the growth of BxPC-GEM-20. The half-maximal inhibitory concentration of pralatrexate on BxPC-GEM-20 cell was about 3.43 ± 0.25 nM. Pralatrexate was found to effectively inhibit the clonal growth of BxPC-GEM-20 cell. Additionally, pralatrexate at 20 mg/kg had an excellent tumor inhibitory effect with an inhibitory rate of 76.92% in vivo. This pralatrexate therapy showed good safety profile that with little to no additional influence on the hepatic, renal function as well as body weight changes in nude mice. Pralatrexate was confirmed to prevent cells from entering the G2/M phase, leading to the promotion of apoptosis and autophagy. Further analysis demonstrated that the reduced phosphorylation of mTOR played a significant role in the tumor cell damage caused by pralatrexate. Pralatrexate effectively inhibited the mTOR/4E-BP1 pathway. Activation of mTOR pathway can further obstruct the repressive effect of pralatrexate on gemcitabine-resistant pancreatic cancer. In summary, pralatrexate induces effective inhibition of gemcitabine-resistant pancreatic cancer. This may lead to the expansion of pralatrexate's application and offer benefit to gemcitabine-resistant pancreatic cancer patients in the future.

Histone acetyltransferase 1 promotes gemcitabine resistance by regulating the PVT1/EZH2 complex in pancreatic cancer

The poor prognosis of pancreatic cancer is primarily due to the development of resistance to therapies, including gemcitabine. The long noncoding RNA PVT1 (lncRNA PVT1) has been shown to interact with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), promoting gemcitabine resistance in pancreatic cancer. In this study, we found histone acetyltransferase 1 (HAT1) enhanced the tolerance of pancreatic cancer cells to gemcitabine and HAT1-mediated resistance mechanisms were regulated by PVT1 and EZH2. Our results showed that the aberrant HAT1 expression promoted gemcitabine resistance, while silencing HAT1 restored gemcitabine sensitivity. Moreover, HAT1 depletion caused a notable increase of gemcitabine sensitivity in gemcitabine-resistant pancreatic cancer cell lines. Further research found that HAT1 increased PVT1 expression to induce gemcitabine resistance, which enhanced the binding of bromodomain containing 4 (BRD4) to the PVT1 promoter, thereby promoting PVT1 transcription. Besides, HAT1 prevented EZH2 degradation by interfering with ubiquitin protein ligase E3 component n-recognin 4 (UBR4) binding to the N-terminal domain of EZH2, thus maintaining EZH2 protein stability to elevate the level of EZH2 protein, which also promoted HAT1-mediated gemcitabine resistance. These results suggested that HAT1 induced gemcitabine resistance of pancreatic cancer cells through regulating PVT1/EZH2 complex. Given this, Chitosan (CS)-tripolyphosphate (TPP)-siHAT1 nanoparticles were developed to block HAT1 expression and improve the antitumor effect of gemcitabine. The results showed that CS-TPP-siHAT1 nanoparticles augmented the antitumor effects of gemcitabine in vitro and in vivo. In conclusion, HAT1-targeted therapy can improve observably gemcitabine sensitivity of pancreatic cancer cells. HAT1 is a promising therapeutic target for pancreatic cancer.

Abstract 2403: Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer

Abstract Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to current treatment regimens contributes to the dismal prognosis for patients diagnosed with the disease. Accumulated evidence indicates that metabolic alterations may play a role in chemo-resistance. In this project, we investigated the role of pyruvate dehydrogenase Alpha1 (PDHA1) in chemo-resistance. Methods: Wild type and Gemcitabine-resistant pancreatic cancer cell lines MIA PaCa-2, HPAF-II, and UN-KPC-961 were used. Mitochondrial respiration, ATP production and glycolytic activities were measured by seahorse. PDHA1 was overexpressed or its activity inhibited by over-expressing PDHA1 kinase in these cells and cancer cell survival was measured by MTT and cancer stemness measured by Western and RT-PCR. PDHA1 level was measured in human and mouse PDAC tissues. Results: Seahorse analysis showed a significantly lower mitochondrial activity, ATP production, and increased glycolytic activity and lactate production in gemcitabine-resistant compared to non- resistant pancreatic cancer cells. PDHA1 mRNA and protein levels were less in gemcitabine-resistant cancer cells compared to non-resistant cells. PDHA1 levels were less in human PDAC tissues from patients and mice who developed resistance to chemotherapy. PDHA1 over-expression in gemcitabine-resistant cancer cells reversed resistance to chemotherapy, decreased cancer cell survival and decreased expression of cancer stemness markers. Whereas, PDHA1 inhibition by over-expressing PDHA1 kinase, in non-resistant cancer cells, induced resistance to chemotherapy, increased cancer cell survival and increased expression of cancer stemness markers. We found that inhibition of histone deacetylase (HDAC) (class 1 and 2) increased transcription level of PDHA1 and reversed chemo-resistance. Conclusion: We show that PDHA1 is a regulator of chemo-resistance in pancreatic cancer cells. PDHA1is a novel target for treating chemo-resistant PDAC. Citation Format: Fouzia Zayou, Chintan Chheda, Adrian Lim, Omer H. Elmadbouh, Stephen J. Pandol, Mouad Edderkaoui. Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2403.

Suppression of Wnt/β-catenin and RAS/ERK pathways provides a therapeutic strategy for gemcitabine-resistant pancreatic cancer

Pancreatic cancer is a major malignant tumor without an effective treatment. KRAS mutations occur in 90% of the pancreatic cancer patients and are a major obstacle for treatment of pancreatic cancer. Pancreatic cancer patients have been treated with limited chemotherapeutic agents such as gemcitabine. However, patients often develop resistance to gemcitabine that is attributed to KRAS mutations. Gemcitabine treatment activates both the Wnt/β-catenin and RAS/ERK pathways. These signaling pathways are also activated in the gemcitabine-resistant pancreatic cancer cell lines, suggesting that they play an important role in gemcitabine resistance in pancreatic cancer. The gemcitabine-resistant cell lines show enhanced migratory and invasive capabilities than their parental lines. Therefore, we investigated the effects of a small molecule, KYA1797K that degrades both β-catenin and RAS, on pancreatic cancer. KYA1797K decreased the expression level of both β-catenin and KRAS in pancreatic cancer cell lines expressing either wild-type or mutant KRAS. It also suppressed migration and invasion of gemcitabine-resistant and parental pancreatic cancer cells. Overall, we demonstrated that inhibiting the Wnt/β-catenin and RAS/ERK pathways by destabilizing β-catenin and RAS could be a therapeutic approach to overcome gemcitabine resistance in pancreatic cancer.

LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/\u03b2-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes

BackgroundPancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood.MethodsThe viability of pancreatic cancer cells was assessed by MTT assay in vitro and xenograft tumor formation assay in vivo. The expression levels of PVT1 and miR-619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting analysis and qRT-PCR were performed to assess the protein and mRNA levels of Pygo2 and ATG14, respectively. Autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of PVT1 were further investigated by gain- and loss-of-function assays in vitro.ResultsIn the present study, we demonstrated that PVT1 was up-regulated in gemcitabine-resistant pancreatic cancer cell lines. Gain- and loss-of-function assays revealed that PVT1 impaired sensitivity to gemcitabine in vitro and in vivo. We further found that PVT1 up-regulated the expression of both Pygo2 and ATG14 and thus regulated Wnt/β-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p. Moreover, we discovered three TCF/LEF binding elements (TBEs) in the promoter region of PVT1, and activation of Wnt/β-catenin signaling mediated by the up-regulation of Pygo2 increased PVT1 expression by direct binding to the TBE region. Furthermore, PVT1 was discovered to interact with ATG14, thus promoting assembly of the autophagy specific complex I (PtdIns3K-C1) and ATG14-dependent class III PtdIns3K activity.ConclusionsThese data indicate that PVT1 plays a critical role in the sensitivity of pancreatic cancer to gemcitabine and highlight its potential as a valuable target for pancreatic cancer therapy.

Effect of microRNA-762 on the drug resistance to gemcitabine of pancreatic cancer PANC-1 cell line

Objective To explore the effect of microRNA (miRNA, miR)-762 on the drug resistance to gemcitabine (GEM) of pancreatic cancer PANC-1 cells. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of miR-762 mRNA in gemcitabine-resistant pancreatic cancer cell line PANC-1/GEM and the non-drug-resistant cell line PANC-1 of pancreatic cancer. The miR-762 mimics, miR-762 inhibitors and scramble sequences were transfected into PANC-1/GEM cells respectively with Lipofectamine™ 2000. The proliferation and the drug sensitivity of PANC-1/GEM cells to GEM were measured by cell counting kit-8 (CCK-8) assay. The apoptosis of PANC-1/GEM cells was examined by flow cytometry. The expression of apoptosis related proteins was detected by Western blotting. Results The miR-762 expression was significantly up-regulated in PANC-1/GEM cells (2.88±0.37) compared to that in PANC-1 cells (1.22±0.32) (t=7.340, P<0.01), and the difference is statistically significant. The miR-762 mRNA expression in PANC-1/GEM cells were obviously increased after transfection with miR-762 mimics in mimics group (4.96±0.67) as compared with negative control group (2.87±0.45) (t=-4.920, P<0.01), but markedly decreased after transfection with miR-762 inhibitors in mimics group (0.72±0.18) as compared with negative control group (2.87±0.45) (t=8.430, P<0.01). Meanwhile, A450, half maximal inhibitory concentration (IC50) and B cell lymphoma/leukemia-2 (bcl-2), phosphorylated protein kinase B (p-Akt) and phosphorylated glycogen synthase kinase 3β (p-GSK-3β) protein expression in miR-762 mimics group were significantly higher than those in negative control group after transfection, and apoptosis rate and bcl-2 associated X protein (bax) protein expression were significantly lower than those in negative control group after transfection. While A450, IC50 and bcl-2, p-Akt and p-GSK-3β protein expression in miR-762 inhibitors group were significantly lower than those in negative control group after transfection, and apoptosis rate and bax protein expression were significantly higher than those in negative control group after transfection (P<0.01). Conclusion The expression of miR-762 is up-regulated in pancreatic cancer drug resistant cells and can induce the cell proliferation and inhibit cell apoptosis in PANC-1/GEM cells, thus reducing the sensitivity of PANC-1/GEM cells to GEM, which may be associated with the inhibition of pro-apoptotic factor bax expression and the promotion of bcl-2, p-Akt and p-GSK-3β expression. Key words: Pancreatic cancer; MicroRNA-762; Gemcitabine; Chemoresistance

Cathepsin D Expression and Gemcitabine Resistance in Pancreatic Cancer.

BackgroundCathepsin-D (CatD), owing to its dual role as a proteolytic enzyme and as a ligand, has been implicated in cancer progression. The role of CatD in pancreatic ductal adenocarcinoma is unknown.MethodsCatD expression quantified by immunohistochemistry of tumor-tissue microarrays of 403 resected pancreatic cancer patients from the ESPAC-Tplus trial, a translational study within the ESPAC (European Study Group for Pancreatic Cancer) trials, was dichotomously distributed to low and high H scores (cut off 22.35) for survival and multivariable analysis. The validation cohort (n = 69) was recruited based on the hazard ratio of CatD from ESPAC-Tplus. 5-fluorouracil-, and gemcitabine-resistant pancreatic cancer cell lines were employed for mechanistic experiments. All statistical tests were two-sided.ResultsMedian overall survival was 23.75 months and median overall survival for patients with high CatD expression was 21.09 (95% confidence interval [CI] = 17.31 to 24.80) months vs 27.20 (95% CI = 23.75 to 31.90) months for low CatD expression (χ2LR, 1DF = 4.00; P = .04). Multivariable analysis revealed CatD expression as a predictive marker in gemcitabine-treated (z stat = 2.33; P = .02) but not in 5-fluorouracil-treated (z stat = 0.21; P = .82) patients. An independent validation cohort confirmed CatD as a negative predictive marker for survival (χ2LR, 1DF = 6.80; P = .009) and as an independent predictive marker in gemcitabine-treated patients with a hazard ratio of 3.38 (95% CI = 1.36 to 8.38, P = .008). Overexpression of CatD was associated with a concomitant suppression of the acid sphingomyelinase, and silencing of CatD resulted in upregulation of acid sphingomyelinase with rescue of gemcitabine resistance.ConclusionsAdjuvant gemcitabine is less effective in pancreatic ductal adenocarcinoma with high CatD expression, and thus CatD could serve as a marker for biomarker-driven therapy.

Establishment and characterization of the gemcitabine-resistant human pancreatic cancer cell line SW1990/gemcitabine.

Due to its rapid progression, metastasis and resistance to chemotherapy, pancreatic cancer is one of the most malignant tumor types to affect the digestive system. Gemcitabine chemotherapy is typically the first choice of treatment for advanced pancreatic cancer; however, chemoresistance is a major obstacle to successful treatment. In order to elucidate the underlying mechanisms of gemcitabine resistance in pancreatic cancer, the drug-resistant cell line SW1990-gemcitabine (SW1990-GZ) was established using the human pancreatic cancer cell line SW1990. The IC50, resistance index and growth of SW1990 and SW1990-GZ cells were also assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays. The cellular uptake of gemcitabine in SW1990 and SW1990-GZ was measured using high performance liquid chromatography (HPLC). The protein expression of p53 was also assessed by western blot analysis. The results demonstrated that the IC50 of SW1990 and SW1990-Gz was 0.07±0.0021 and 87.5±3.24 µg/ml, respectively, and that the resistance index ratio of SW1990-Gz was 1,250. The growth rate of SW1990-GZ cells was low compared with that of SW1990 cells. The HPLC results indicated that gemcitabine uptake was markedly reduced in SW1990-GZ cells compared with in SW1990 cells at different time points. The protein expression of p53 was significantly higher in GEM-resistant SW1990-GZ cells compared with that in SW1990 cells (P<0.01). These results suggest that a human gemcitabine-resistant pancreatic cancer cell line was successfully established, with stable and significant drug resistance. The results of the present study suggest that the decreased cellular uptake of gemcitabine may serve an important role in gemcitabine chemoresistance in SW1990-GZ cells; thus, this cell line may be used as an effective in vitro model to improve our understanding of gemcitabine-resistance in pancreatic cancer.

Enzyme-treated Asparagus Extract Down-regulates Heat Shock Protein 27 of Pancreatic Cancer Cells.

From the standpoint of cancer therapy, it is valuable to enhance the anticancer effects of chemotherapy. Our previous reports revealed that up-regulation of heat-shock protein 27 (HSP27) has been linked to gemcitabine resistance of pancreatic cancer cells. Enzyme-treated asparagus extract (ETAS) is an extract that is produced from asparagus. The purpose of this study was to investigate the effect of ETAS on the expression of HSP27 and other HSPs in the gemcitabine-resistant pancreatic cancer cell line KLM1-R. KLM1-R cells were treated with ETAS, and expression levels of HSPs, including HSP27, were investigated by western blotting. ETAS down-regulated HSP27 and pHSP27 (serine 78) in KLM1-R cells, but, HSP70 and GRP78 levels were not altered. This study suggests the potential therapeutic benefit of ETAS in enhancing anticancer effects by its combination with gemcitabine for patients with pancreatic cancer.

All-trans retinoic acid enhances gemcitabine cytotoxicity in human pancreatic cancer cell line AsPC-1 by up-regulating protein expression of deoxycytidine kinase

We previously showed that gemcitabine resistance in pancreatic cancer chemotherapy correlates with suppressed expression of deoxycytidine kinase (dCK), which catalyzes the rate-limiting step of gemcitabine activation. The purpose of the present study was to find a drug that might be useful to enhance the cytotoxicity of gemcitabine by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cell line AsPC-1. Screening of 40 prescription drugs identified 35 with no intrinsic cytotoxicity towards AsPC-1 cells. When AsPC-1 cells were pre-incubated with these drugs and then incubated with gemcitabine, we found that all-trans retinoic acid (ATRA) significantly decreased the viability by 28% compared with that of non-treated cells. Luciferase assay showed that ATRA transactivated the DCK promoter in AsPC-1 cells by about 2-fold compared with the untreated control, and an increase of dCK protein expression was confirmed by immunoblotting. ATRA decreased the half-maximal inhibitory concentration (IC50) of gemcitabine by 2.8-fold (ATRA-non-treated cells, 28.8nM; ATRA-treated cells, 10.0nM). The ATRA concentration of 0.03μM was sufficient to enhance gemcitabine cytotoxicity, and the effect was well maintained in the concentration range from 0.03 to 50μM. These results indicate that ATRA enhances gemcitabine cytotoxicity by increasing dCK expression in gemcitabine-resistant human pancreatic cancer cells.

MicroRNA-1285 inhibits malignant biological behaviors of human pancreatic cancer cells by negative regulation of YAP1.

Pancreatic ductal adenocarcinoma is a most deadly malignancy, with a 5-year survival rate of ~7%. Chemotherapy is the main treatment strategy of this disease. However, the high rate of resistance to chemotherapeutic agent contributes to poor prognosis. MicroRNAs are essential for the initiation, progression and chemoresistance of human malignancies. Previous studies have shown that miRNA-1285 participates in renal cell carcinoma and hepatocellular carcinoma. However, its roles in pancreatic ductal adenocarcinoma are poorly understood. In this study, we confirmed that miR-1285 was significantly down-regulated in gemcitabine-resistant pancreatic cancer cell lines by qRT-PCR. We found that miR-1285 suppressed cell proliferation as well as increased the sensitivity of PDAC cells to gemcitabine by CCK8 assays in vitro. Results from transwell assay indicated that miR-1285 inhibited pancreatic cancer cell migration and invasion. Experiments using different cell lines got identical results. All those results demonstrated that miR-1285 act as tumor suppressor of pancreatic cancer. To our knowledge, this study is the first to elucidate the function of miR-1285 in pancreatic cancer. Western blotting analysis verified that miR-1285 negatively regulated YAP1 protein level, together with EGFR and β-catenin. YAP1 is a known oncoprotein of pancreatic cancer. As silencing of YAP1 activity might be beneficial in cancer prevention and treatment, our results suggest that miR-1285 might serve as a novel therapeutic target for miRNA-based therapy in pancreatic cancer. Further research elucidating the exact mechanisms of miRNA-1285 function and the correlation between miR-1285 levels in tissues or serum and clinical characteristics of pancreatic cancer is needed later.

Aptamer-Drug Conjugates of Active Metabolites of Nucleoside Analogs and Cytotoxic Agents Inhibit Pancreatic Tumor Cell Growth.

Aptamer-drug conjugates (ApDCs) have the potential to improve the therapeutic index of traditional chemotherapeutic agents due to their ability to deliver cytotoxic drugs specifically to cancer cells while sparing normal cells. This study reports on the conjugation of cytotoxic drugs to an aptamer previously described by our group, the pancreatic cancer RNA aptamer P19. To this end, P19 was incorporated with gemcitabine and 5-fluorouracil (5-FU), or conjugated to monomethyl auristatin E (MMAE) and derivative of maytansine 1 (DM1). The ApDCs P19-dFdCMP and P19-5FdUMP were shown to induce the phosphorylation of histone H2AX on Ser139 (γ-H2AX) and significantly inhibited cell proliferation by 51%–53% in PANC-1 and by 54%–34% in the gemcitabine-resistant pancreatic cancer cell line AsPC-1 (p ≤ 0.0001). P19-MMAE and P19-DM1 caused mitotic G2/M phase arrest and inhibited cell proliferation by up to 56% in a dose-dependent manner when compared to the control group (p ≤ 0.001). In addition, the cytotoxicity of P19-MMAE and P19-DM1 in normal cells and the control human breast cancer cell line MCF7 was minimal. These results suggest that this approach may be useful in decreasing cytotoxic side effects in non-tumoral tissue.

RNA sequencing of pancreatic adenocarcinoma tumors yields novel expression patterns associated with long-term survival and reveals a role for ANGPTL4

BackgroundPancreatic adenocarcinoma patients have low survival rates due to late-stage diagnosis and high rates of cancer recurrence even after surgical resection. It is important to understand the molecular characteristics associated with survival differences in pancreatic adenocarcinoma tumors that may inform patient care. ResultsRNA sequencing was performed for 51 patient tumor tissues extracted from patients undergoing surgical resection, and expression was associated with overall survival time from diagnosis. Our analysis uncovered 323 transcripts whose expression correlates with survival time in our pancreatic patient cohort. This genomic signature was validated in an independent RNA-seq dataset of 68 additional patients from the International Cancer Genome Consortium. We demonstrate that this transcriptional profile is largely independent of markers of cellular division and present a 19-transcript predictive model built from a subset of the 323 transcripts that can distinguish patients with differing survival times across both the training and validation patient cohorts. We present evidence that a subset of the survival-associated transcripts is associated with resistance to gemcitabine treatment in vitro, and reveal that reduced expression of one of the survival-associated transcripts, Angiopoietin-like 4, impairs growth of a gemcitabine-resistant pancreatic cancer cell line. ConclusionsGene expression patterns in pancreatic adenocarcinoma tumors can distinguish patients with differing survival outcomes after undergoing surgical resection, and the survival difference could be associated with the intrinsic gemcitabine sensitivity of primary patient tumors. Thus, these transcriptional differences may impact patient care by distinguishing patients who would benefit from a non-gemcitabine based therapy.

The Effects of HSP27 on Gemcitabine-Resistant Pancreatic Cancer Cell Line Through Snail

To evaluate the regulation mechanism of heat shock protein 27 (HSP27) on gemcitabine (GEM) resistance of pancreatic cancer cell. The expression vectors pEGFP-C1-HSP27 and the vectors of MicroRNA targeting Snail were introduced into GEM-sensitive pancreatic cancer SW1990 cells, and the vectors of small hairpin RNA targeting HSP27 were transfected into SW1990 and GEM-resistant SW1990/GEM cells. The expressions of HSP27, p-HSP27 (Ser82), Snail, ERCC1, and E-cadherin were evaluated by Western blotting. The sensitivity of transfected cells to GEM was detected by CCK-8 assay and Annexin V-FITC apoptosis assay. As compared to SW1990, SW1990/GEM showed significantly increased expressions of HSP27, p-HSP27, Snail and ERCC1 with decreased expression of E-cadherin. By increasing HSP27 expression, we found increase of Snail and ERCC1 with reduction of E-cadherin expressions, while reduction of HSP27 expression caused reduction of Snail and ERCC1 but increase of E-cadherin expressions. Downregulation of Snail resulted in the reduction of ERCC1 expression and increase of E-cadherin. Furthermore, downregulation of HSP27 or snail caused increased GEM sensitivity of pancreatic cancer cells, and upregulation of HSP27 showed the opposite results. There is an inverse correlation between HSP27 expression and GEM sensitivity of SW1990 cells, which might be realized by regulating E-cadherin and ERCC1 expressions through Snail.

Ribonucleotide reductase is an effective target to overcome gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells with dual resistant factors

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.