Gemcitabine-resistant Pancreatic Cancer Cells Research Articles

Astaxanthin inhibits gemcitabine-resistant human pancreatic cancer progression through EMT inhibition and gemcitabine resensitization.

Pancreatic cancer rapidly acquires resistance to chemotherapy resulting in its being difficult to treat. Gemcitabine is the current clinical chemotherapy strategy; however, owing to gemcitabine resistance, it is only able to prolong the life of patients with pancreatic cancer for a limited number of months. Understanding the underlying molecular mechanisms of gemcitabine resistance and selecting a suitable combination of agents for the treatment of pancreatic cancer is required. Astaxanthin (ASX) is able to resensitize gemcitabine-resistant human pancreatic cancer cells (GR-HPCCs) to gemcitabine. ASX was identified to upregulate human equilibrative nucleoside transporter 1 (hENT1) and downregulate ribonucleoside diphosphate reductase (RRM) 1 and 2 to enhance gemcitabine-induced cell death in GR-HPCCs treated with gemcitabine, and also downregulates TWIST1 and ZEB1 to inhibit the gemcitabine-induced epithelial-mesenchymal transition (EMT) phenotype in GR-HPCCs and to mediate hENT1, RRM1 and RRM2. Furthermore, ASX acts through the hypoxia-inducible factor 1α/signal transducer and activator of transcription 3 signaling pathway to mediate TWIST1, ZEB1, hENT1, RRM1 and RRM2, regulating the gemcitabine-induced EMT phenotype and gemcitabine-induced cell death. Co-treatment with ASX and gemcitabine in a tumor xenograft model induced by GR-HPCCs supported the in vitro results. The results of the present study provide a novel therapeutic strategy for the treatment of gemcitabine-resistant pancreatic cancer.

Gemcitabine resistant pancreatic cancer cells are sensitive to paclitaxel treatment

Gemcitabine resistant pancreatic cancer cells are sensitive to paclitaxel treatment

MiR-153 enhances the therapeutic effect of gemcitabine by targeting Snail in pancreatic cancer.

Pancreatic cancer (PC) is one of the most lethal cancers, with an overall 5 years survival rate of <5%. The clinical benefit of gemcitabine based chemotherapeutic strategy on PC was limited by its high drug resistance rate. Snail, one of the master regulators of epithelial-mesenchymal transition, has been implicated in the progression of various cancers. However, whether it is also linked to the development of chemosensitivity to gemcitabine in PC is unknown, and the regulatory pathways controlling Snail also need to be explored. Cell apoptosis analysis was performed using flow cytometry assay. Quantitative real-time PCR was used to investigate the level of microRNA and the mRNA expression of its target, Snail. Snail expression was measured by immunoblotting and immunohistochemistry. A xenografted tumor model was used to test the in vivo effects of miR-153 on chemosensitivity to gemcitabine. The results of this study demonstrated the decrease of miR-153 expression in PC tumor tissue, which is correlated with a poor prognosis. miR-153 mimic transfection enhanced gemcitabine sensitivity in gemcitabine-resistant PC cells, while downregulation of miR-153 decreased gemcitabine sensitivity. In addition, miR-153 was found to target the 3'-UTR of Snail mRNA. Furthermore, we found that the increase of apoptosis in gemcitabine-resistant PC cells resulted from miR-153 mimic transfection was reversed by overexpression of Snail. miR-153 reverses the resistance of PC cells to gemcitabine by directly targeting Snail, and it may be a potential novel therapeutic target for overcoming gemcitabine resistance in human PC.

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.

A novel HDAC inhibitor, CG200745, inhibits pancreatic cancer cell growth and overcomes gemcitabine resistance

Pancreatic cancer is predominantly lethal, and is primarily treated using gemcitabine, with increasing resistance. Therefore, novel agents that increase tumor sensitivity to gemcitabine are needed. Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents, since HDAC plays an important role in cancer initiation and progression. We evaluated the antitumor effect of a novel HDAC inhibitor, CG200745, combined with gemcitabine/erlotinib on pancreatic cancer cells and gemcitabine-resistant pancreatic cancer cells. Three pancreatic cancer-cell lines were used to evaluate the antitumor effect of CG200745 combined with gemcitabine/erlotinib. CG200745 induced the expression of apoptotic proteins (PARP and caspase-3) and increased the levels of acetylated histone H3. CG200745 with gemcitabine/erlotinib showed significant growth inhibition and synergistic antitumor effects in vitro. In vivo, gemcitabine/erlotinib and CG200745 reduced tumor size up to 50%. CG200745 enhanced the sensitivity of gemcitabine-resistant pancreatic cancer cells to gemcitabine, and decreased the level of ATP-binding cassette-transporter genes, especially multidrug resistance protein 3 (MRP3) and MRP4. The novel HDAC inhibitor, CG200745, with gemcitabine/erlotinib had a synergistic anti-tumor effect on pancreatic cancer cells. CG200745 significantly improved pancreatic cancer sensitivity to gemcitabine, with a prominent antitumor effect on gemcitabine-resistant pancreatic cancer cells. Therefore, improved clinical outcome is expected in the future.

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.

PI3K inhibitor LY294002, as opposed to wortmannin, enhances AKT phosphorylation in gemcitabine-resistant pancreatic cancer cells.

LY294002 and wortmannin are chemical compounds that act as potent inhibitors of phosphoinositide 3-kinases (PI3Ks). Both of them are generally used to inhibit cell proliferation as cancer treatment by inhibiting the PI3K/protein kinaseB (AKT) signaling pathway. In this study, LY294002 (but not wortmannin) showed an abnormal ability to enhance AKT phosphorylation (at Ser472) specifically in gemcitabine (GEM)-resistant pancreatic cancer (PC) cell lines PK59 and KLM1-R. LY294002 was shown to activate AKT and accumulate phospho-AKT at the intracellular membrane in PK59, which was abolished by treatment with AKTi-1/2 or wortmannin. Inhibiting AKT phosphorylation by treatment with AKTi-1/2 or wortmannin further enhanced LY294002-induced cell death in PK59 and KLM1-R cells. In addition, treatment with wortmannin alone failed to inhibit cell proliferation in both PK59 and KLM1-R cells. Thus, our results reveal that LY294002 displays the opposite effect on PI3K-dependent AKT phosphorylation, which maintains cell survival from the cytotoxicity introduced by LY294002 itself in GEM-resistant pancreatic cancer cells. We suggest that targeting the PI3K/AKT signaling pathway with inhibitors may be counterproductive for patients with PC who have acquired GEM-resistance.

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.

Retraction.

This article has been retracted at the request of: Editor-in-Chief and Co-author 'Restoring sensitivity to oxaliplatin by a novel approach in gemcitabine-resistant pancreatic cancer cells in vitro and in vivo' by Banerjee, S., Kong, D., Azmi, A. S., Wang, Z., Ahmad, A., Sethi, S. and Sarkar, F. H. The above article, published online on 16 November 2010, in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal Editor-in-Chief, Professor Peter Lichter, and Wiley Periodicals, Inc. A university investigation involving the first and corresponding author determined that several figures in this paper were mislabeled, manipulated, or duplicated and relabeled while processing/compiling the final figures assembled from the original data sources. Therefore, the authors are retracting the paper in its entirety although they maintain that these issues did not affect the major conclusions. They apologize for any inconvenience this may have caused. Reference Banerjee, S., Kong, D., Azmi, A. S., Wang, Z., Ahmad, A., Sethi, S. and Sarkar, F. H. (2011), Restoring sensitivity to oxaliplatin by a novel approach in gemcitabine-resistant pancreatic cancer cells in vitro and in vivo. Int. J. Cancer, 128: 1240-1250. doi: 10.1002/ijc.25658.

Abstract 2154: TX1111: a peptide homologue of Topoisomerase-1 sensitizes pancreatic cancer cells to gemcitabine

Abstract Pancreatic Ductal Adeno Carcinoma (PDAC) is the fourth most common cause of cancer deaths in the United States and accounts for over 95% of all pancreatic cancers. The combined 1- and 5-year survival rates for PDAC are very poor, at 25% and 6% respectively. A major hallmark of pancreatic cancer is tumor recurrence and extremely poor response to chemotherapy. The current standard of care for patients with advanced pancreatic cancer is a chemotherapy regimen that includes gemcitabine. This treatment results in a modest benefit i.e., an increase in survival of only 5 weeks. The high mortality in patients diagnosed with this pancreatic cancer is primarily due to its drug-resistant nature and the lack of effective therapeutic strategies to overcome drug resistance. This disappointing situation strongly suggests that an improved and increased understanding of drug resistance mechanisms could lead to the development of novel therapeutic strategies for the successful treatment of patients with pancreatic cancer. Our studies have shown that one of the contributing factors that lead to chemotherapy resistance is the protease activator urokinase plasminogen activator (uPA). To validate the contribution of uPA in chemoresistance, we overexpressed uPA in MIAPaCa-2 and PANC-1 cells and determined chemoresistance. We observed that uPA overexpressed cells showed increased chemoresistance in both Mia PaCa-2 (&amp;gt;25 fold) and PANC-1 (&amp;gt;6 fold). Mass-Spec analysis of nuclear uPA-IP from pancreatic cancer cells revealed that uPA interacts with Topoisomerase-1 (TOPO-1). This was further validated by western blot analysis of nuclear co-IP in pancreatic cancer cells. Further the binding domains of TOPO-1 to uPA were identified using overlapping a peptides array (PepSpot-JPT) followed by synthesis of these peptides. We observed that a TOPO-1 homologous peptide designated TX1111 was capable of significantly suppressing gemcitabine resistance in pancreatic cancer cells. Our observations show that the specific peptide TX1111 suppresses chemoresistance by 45% in Mia Paca-2 cells (p&amp;lt;0.001) and by 40% in PANC cells (p = 0.002). These results demonstrate that: 1. Overexpression of uPA promotes gemcitabine resistance in pancreatic cancer cells 2. uPA and TOPO-1 interact in the nucleus of pancreatic cancer cells 3. The peptide homologue of TOPO-1, TX1111 sensitizes gemcitabine resistant pancreatic cancer cells. Citation Format: Manu Gnanamony, Victoria Stepanova, Lily Criscione, Jerusha Boyineni, Stephen J. Marshall, AiXuan Holterman, Christopher S. Gondi. TX1111: a peptide homologue of Topoisomerase-1 sensitizes pancreatic cancer cells to gemcitabine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2154.

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.

Toward targeted therapy in chemotherapy-resistant pancreatic cancer with a smart triptolide nanomedicine.

Chemoresistance is the major impediment for treating pancreatic cancer. Herb-derived compound triptolide (TP) can inhibit proliferation of chemo-resistant pancreatic cancer (CPC) cell lines through multiple mechanisms, which exhibited superior anticancer efficacy compared with gemcitabine. However, toxicity due to non-specific exposure to healthy tissues hindered its clinical translation. Herein we successfully achieved targeting CPC cells and avoiding exposure to healthy tissues for TP by nucleolin-specific aptamer (AS1411) mediated polymeric nanocarrier. We conjugated AS1411 aptamer to carboxy terminated poly(ethylene glycol)–block–poly(d, l-lactide) (HOOC-PEG-PDLLA), then prepared AS1411-PEG-PDLLA micelle loading TP (AS-PPT) through solid dispersion technique. AS-PPT showed more antitumor activity than TP and equivalent specific binding ability with gemcitabine-resistant human pancreatic cancer cell (MIA PaCa-2) to AS1411 aptamer in vitro. Furthermore, we studied the distribution of AS-PPT (Cy3-labed TP) at tissue and cellular levels using biophotonic imaging technology. The results showed AS1411 facilitated TP selectively accumulating in tumor tissues and targeting CPC cells. The lifetime of the MIA PaCa-2 cell-bearing mice administrated with AS-PPT was efficiently prolonged than that of the mice subjected to the clinical anticancer drug Gemzar® in vivo. Such work provides a new strategy for overcoming the drug resistance of pancreatic cancer.

Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells.

In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.

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.

321 Zidovudine, an anti-viral drug, resensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine by inhibition of the Akt-GSK3beta-Snail pathway

321 Zidovudine, an anti-viral drug, resensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine by inhibition of the Akt-GSK3beta-Snail pathway

Abstract 1764: Galeterone and its novel analogs induce profound anti-cancer activities in human pancreatic cancer cell lines: Implications for pancreatic cancer therapy

Abstract Pancreatic cancer is a lethal disease with a 3-5% survival rate. Histologically, 90% of human pancreatic cancer is presented as pancreatic ductal adenocarcinoma (PDAC). The aggressive nature of the disease and its high metastatic potential makes the development of efficacious therapeutic strategies very significant. Current therapeutic strategies are ineffective as the deregulated RAS/MAPK and PI3K/AKT/mTOR pathways to enhance drug resistance. Gemcitabine alone or in combination with erlotinib, the currently used drugs for PDAC, elicit only marginal survival benefits. The modest efficacy of gemcitabine therapy has been attributed to activation of the NF-κB pathway which activates genes involved in gemcitabine resistance. Recent studies have implicated the eIF4F (specifically, eIF4E-Mnk1/2 axis) that control gene expression at the translational level towards PDAC gemcitabine drug resistant. Several groups have also implicated the androgen receptor in PDAC progression/metastasis and have investigated the use anti-androgens in several in vitro and vivo models of PDAC and in a few clinical trials with mixed results. In the course of our studies to develop more potent androgen receptor degrading agents (ARDAs), we discovered that galeterone (gal) and its novel analogs also effectively target oncogenic eukaryotic protein translation. These novel small molecule inhibitors significantly depleted Mnk1/2 protein expression and decreased eIF4E phosphorylation. Interestingly, our studies revealed that the phosphorylation level of Nf-kB (p65) was significantly decreased after exposing pancreatic cancer cells to ARDAs. We note that these targets are implicated in PDAC progression and drug resistance. Here we report for the first time, the efficacy of galeterone and its lead ARDA analogs in inhibiting cell viability of both gemcitabine-naïve and gemcitabine/erlotinib-resistant human pancreatic cancer cells in vitro. Gal/ARDAs alone or in combination with gemcitabine significantly reduced the colony forming potential of gemcitabine-naïve and gemcitabine resistant pancreatic cancer cells. Decreased invasive potential of PDAC cells was shown through wound-healing and matrigel-coated Boyden chambers assays. Gal/ARDAs also caused significant depletion of matrix metalloproteinases (MMPs). Ezh2, a prominent metastatic enhancing gene in pancreatic cancer was also downregulated at the protein level. Immunoblot analysis of treated lysates also revealed induction of apoptosis via caspase 3 and PARP cleavage. Flow cytometric analysis showed that gal/ARDAs did not only deplete cyclins but also inhibited cell cycle progression. Our results highlight the unique ability of gal and its novel analogs to inhibit the growth, colonization and metastatic potential of a variety of PDAC cells through modulation of several oncogenic targets. Citation Format: Andrew K. Kwegyir-Afful, Puranik Purushottamachar, Marlena S. Martin, Vincent C.o. Njar. Galeterone and its novel analogs induce profound anti-cancer activities in human pancreatic cancer cell lines: Implications for pancreatic cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1764. doi:10.1158/1538-7445.AM2015-1764

Pyruvate kinase isoenzyme M2 is a therapeutic target of gemcitabine-resistant pancreatic cancer cells

Despite its wide use as a first-line therapeutic agent, gemcitabine has shown limited efficacy in advanced pancreatic cancer due to chemoresistance by as yet unidentified mechanisms. Our goal here was to identify molecular features involved in gemcitabine chemoresistance. Pyruvate kinase M2 (PKM2), a key enzyme of aerobic glycolysis, has recently emerged as an important therapeutic target for cancer treatment. It is involved in the metabolic reprogramming of cancer cells and has previously unexpected non-metabolic functions that are heavily involved in tumor growth and survival. Herein, we report that the chemoresistance of pancreatic cancer to gemcitabine was dependent on PKM2 expression and its non-metabolic function. Knocking-down of PKM2 significantly enhanced gemcitabine-induced cell apoptosis through the activation of caspase 3/7 and PARP cleavage, and this inhibitory activity was associated with p38-mediated activation of p53 phosphorylation at serine 46. Our findings support the potential of PKM2 as a novel target for gemcitabine chemoresistance and suggest the feasibility of combining gemcitabine and PKM2 inhibition for the improved chemotherapy of pancreatic cancer.

Zidovudine, an anti-viral drug, resensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine by inhibition of the Akt-GSK3β-Snail pathway.

Pancreatic cancer is one of the most difficult malignancies to treat owing to the rapid acquisition of resistance to chemotherapy. Gemcitabine, a first-line treatment for pancreatic cancer, prolongs patient survival by several months, and combination treatment with gemcitabine and other anti-cancer drugs in the clinic do not show any significant effects on overall survival. Thus, identification of a drug that resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine and a better understanding of the molecular mechanisms of gemcitabine resistance are critical to develop new therapeutic options for pancreatic cancer. Here, we report that zidovudine resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine as shown by screening a compound library, including clinical medicine, using gemcitabine-resistant cells. In analyzing the molecular mechanisms of zidovudine effects, we found that the epithelial-to-mesenchymal transition (EMT)-like phenotype and downregulation of human equilibrative nucleoside transporter 1 (hENT1) are essential for the acquisition of gemcitabine resistance, and zidovudine restored these changes. The chemical biology investigations also revealed that activation of the Akt-GSK3β-Snail1 pathway in resistant cells is a key signaling event for gemcitabine resistance, and zidovudine resensitized resistant cells to gemcitabine by inhibiting this activated pathway. Moreover, our in vivo study demonstrated that co-administration of zidovudine and gemcitabine strongly suppressed the formation of tumors by gemcitabine-resistant pancreatic cancer and prevented gemcitabine-sensitive pancreatic tumors from acquiring gemcitabine-resistant properties, inducing an EMT-like phenotype and downregulating hENT1 expression. These results suggested that co-treatment with zidovudine and gemcitabine may become a novel therapeutic strategy for pancreatic cancer by inhibiting chemoresistance-specific signaling.

The Histone Deacetylase Inhibitor Valproic Acid Sensitizes Gemcitabine-Induced Cytotoxicity in Gemcitabine-Resistant Pancreatic Cancer Cells Possibly Through Inhibition of the DNA Repair Protein Gamma-H2AX

Gemcitabine (GEM) remains a major chemotherapeutic drug for pancreatic cancer, but resistance to GEM has been a big problem, as its response rate has been decreasing year by year. The effect of the histone deacetylase inhibitor (HDAI) valproic acid (VPA) was compared with tranilast and RI-1 as a combinatorial treatment with GEM in four pancreatic cancer cell lines, BxPC-3, PK45p, MiaPaCa-2 and PK59. Cell viability assays were carried out to check the cytotoxic effects, western blotting was carried out for DNA repair mechanisms, and localization was determined by immunofluorescence. The sensitization factors (i.e., the fold ratio of cell viability for GEM/GEM plus drug) reveal that VPA increases the cytotoxic sensitization to GEM at approximately 2.7-fold, 1.2-fold, 1.5-fold and 2.2-fold in BxPC-3, MiaPaCa-2, PK-45p and PK-59 cell lines, respectively. Moreover, GEM induces activation of the DNA repair protein H2AX proportional to the dosage. Interestingly, however, this effect can be abrogated by VPA. These results indicate that VPA enhances GEM-induced cytotoxicity in GEM-resistant pancreatic cancer cells, possibly through inhibition of DNA damage signaling and repair. Our study suggests VPA as a potential therapeutic agent for combinatorial treatment with GEM in pancreatic cancer.

Pachymic Acid Inhibits Growth and Induces Apoptosis of Pancreatic Cancer In Vitro and In Vivo by Targeting ER Stress

Pachymic acid (PA) is a purified triterpene extracted from medicinal fungus Poria cocos. In this paper, we investigated the anticancer effect of PA on human chemotherapy resistant pancreatic cancer. PA triggered apoptosis in gemcitabine-resistant pancreatic cancer cells PANC-1 and MIA PaCa-2. Comparative gene expression array analysis demonstrated that endoplasmic reticulum (ER) stress was induced by PA through activation of heat shock response and unfolded protein response related genes. Induced ER stress was confirmed by increasing expression of XBP-1s, ATF4, Hsp70, CHOP and phospho-eIF2α. Moreover, ER stress inhibitor tauroursodeoxycholic acid (TUDCA) blocked PA induced apoptosis. In addition, 25 mg kg-1 of PA significantly suppressed MIA PaCa-2 tumor growth in vivo without toxicity, which correlated with induction of apoptosis and expression of ER stress related proteins in tumor tissues. Taken together, growth inhibition and induction of apoptosis by PA in gemcitabine-resistant pancreatic cancer cells were associated with ER stress activation both in vitro and in vivo. PA may be potentially exploited for the use in treatment of chemotherapy resistant pancreatic cancer.