Gemcitabine-resistant Cell Lines Research Articles

Hypoxia-Induced PLOD2 is a Key Regulator in Epithelial-Mesenchymal Transition and Chemoresistance in Biliary Tract Cancer.

The prognosis of biliary tract cancer (BTC) is unfavorable due to its chemoresistance. Hypoxia triggers epithelial-to-mesenchymal transition (EMT), which is closely related to drug resistance. In this study, we focused on the functional roles of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), a hypoxia-induced gene, in BTC, and assessed the clinical significance of PLOD2. The expression of PLOD2 under hypoxia was assessed in BTC cell lines. Gemcitabine-resistant (GR) BTC cell lines were transfected with small interfering RNA (siRNA) against PLOD2, and EMT markers and chemoresistance were evaluated. PLOD2 expression was also characterized using immunohistochemistry in BTC clinical specimens following resection. Patient survival was analyzed and the role of PLOD2 expression was examined. The expression of PLOD2 was induced by hypoxia in vitro and was upregulated in BTC-GR cell lines, which had low expression of epithelial markers and high expression of mesenchymal markers. Downregulation of PLOD2 by siRNA resulted in improved chemoresistance, recovery of epithelial markers, and reduction of mesenchymal markers. In the resected BTC samples, PLOD2 expression was significantly correlated with lymph node metastasis (p = 0.037) and stage (p = 0.001). Recurrence-free survival (p = 0.011) and overall survival (p < 0.001) rates were significantly lower in patients with high expression of PLOD2. PLOD2 expression was an independent prognostic factor for overall survival (p = 0.019). The expression of PLOD2 influenced chemoresistance through EMT, and high expression of PLOD2 was a significant unfavorable prognostic factor in BTC patients. PLOD2 might be a potential therapeutic target for overcoming chemoresistance.

A novel chemo-resistant gene MSX2 discovered by establishment of two pancreatic cancer drug resistant cell lines JF305/CDDP and PANC-1/GEM

Objective: To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines. Methods: The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC(50)), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes. Results: The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day (P<0.05); the proportion of S phase was decreased from (45±2)% to (30±2)% (P<0.05), and the migration ability was enhanced from (32 ±1) cells per field to (158±5) cells per field (P<0.01). The expression of multidrug resistance-related genes MRP2, MDR1, LRP and MSX2 was increased in JF305/CDDP cells (P<0.05). Knockdown of MSX2 in JF305 cells reduced the expression of MRP2, whereas overexpression of MSX2 in PANC-1 cells upregulated MRP2 level (P<0.05). Conclusions: Two stable multidrug resistant cell lines of pancreatic cancer, JF305/CDDP and PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.

Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line.

Cholangiocarcinoma (CCA) is a deleterious bile duct tumor with poor prognosis and is relatively resistant to chemotherapy. Therefore, alternative or supplementary agents with anticancer and chemosensitizing activities may be useful for the treatment of CCA. A novel anthocyanin complex(AC) nanoparticle, developed from extracts of cobs of purple waxy corn and petals of blue butterfly pea, has exhibited chemopreventive potential invivo. In the present study, the anti-CCA activities of AC and their underlying molecular mechanisms were investigated further invitro using a CCA cell line (KKU213). The potential use of AC as a chemosensitizer was also evaluated in a gemcitabine-resistant CCA cell line (KKU214GemR). It was demonstrated that AC treatment suppressed proliferation of KKU213 CCA cells in dose- and time-dependent manners. AC treatment also induced apoptosis and mitochondrial superoxide production, decreased clonogenicity of CCA cells, and downregulated forkhead box proteinM1(FOXM1), nuclear factor-κB (NF-κB) and pro-survival protein B-cell lymphoma-2 (Bcl-2). The expression of endoplasmic reticulum (ER) stress-response proteins, including protein kinase RNA-like ER kinase, phosphorylated eIF2α, eukaryotic initiation factor2α and activating transcription factor4, also decreased following AC treatment. It was also identified that AC treatment inhibited KKU214GemR cell proliferation in dose- and time-dependent manners. Co-treatment of KKU214GemR cells with low doses of AC together with gemcitabine significantly enhanced efficacy of the latter against this cell line. Therefore, it is suggested that AC treatment is cytotoxic to KKU213 cells, possibly via downregulation of FOXM1, NF-κB, Bcl-2 and the ER stress response, and by induction of mitochondrial superoxide production. AC also sensitizes KKU214GemR to gemcitabine treatment, which may have potential for overcoming drug resistance of CCA.

Silencing of ECHDC1 inhibits growth of gemcitabine-resistant bladder cancer cells.

Combined gemcitabine and cisplatin (GC) treatment is a first line chemotherapy for bladder cancer. However, acquired resistance to GC has been a major problem. To address the mechanism of gemcitabine resistance, and to identify potential biomarkers or target proteins for its therapy, we aimed to identify candidate proteins associated with gemcitabine resistance using proteomic analysis. We established gemcitabine-resistant human bladder cancer cell lines (UMUC3GR and HT1376GR) from gemcitabine-sensitive human bladder cancer cell lines (UMUC3 and HT1376). We compared the protein expression of parental and gemcitabine-resistant cell lines using isobaric tags for relative and absolute quantification (iTRAQ) and liquid chromatography tandem mass spectrometry. Among the identified proteins, ethylmalonyl-CoA decarboxylase (ECHDC1) expression was significantly increased in both of the gemcitabine-resistant cell lines compared to the respective parental cell lines. Silencing of ECHDC1 reduced ECHDC1 expression and significantly inhibited the proliferation of UMUC3GR cells. Furthermore, silencing of ECHDC1 induced upregulation of p27, which is critical for cell cycle arrest in the G1 phase, and induced G1 arrest. In conclusion, ECHDC1 expression is increased in gemcitabine-resistant bladder cancer cells, and is involved in their cell growth. ECHDC1, which is a metabolite proofreading enzyme, may be a novel potential target for gemcitabine-resistant bladder cancer therapy.

A Concise Synthesis of Three Branches Derived from Polysaccharide RN1 and Anti-Pancreatic Cancer Activity Study.

RN1, a polysaccharide from flowers of Panax pseudo-ginsieng Wall. Var. notoginseng (Burkill) Hoo & Tseng, is a potential multi-targeting drug candidate for pancreatic cancer treatment. However, the active targeting domain of RN1 is still unknown. Herein, three RN1 derived branches were synthesized via [3+2] or [2+2] strategies, efficiently. Two pentasaccharides, 18 and 27, showed similar inhibition effect on pancreatic cancer BxPC-3 cells to that of RN1 at same concentration. Interestingly, tetrasaccharide 21 potently inhibited gemcitabineresistant cell line Panc-1 at high concentration. These suggest that the branches of RN1 might be the active targeting domain and tetrasaccharide 21 might be a potential leading compound for pancreatic cancer with gemcitabine resistance.

MiR-3656 expression enhances the chemosensitivity of pancreatic cancer to gemcitabine through modulation of the RHOF/EMT axis

The highly refractory nature of pancreatic cancer (PC) to chemotherapeutic drugs is one of the key reasons contributing to the poor prognosis of this disease. MicroRNAs (miRNAs) are key regulators of gene expression and have been implicated in a variety of processes from cancer development through to drug resistance. Herein, through miRNA profiling of gemcitabine-resistant (GR) and parental PANC-1 cell lines, we found a consistent reduction of miR-3656 in GR PANC-1 cells. miR-3656 overexpression enhanced the antitumor effect of gemcitabine, whereas silencing of miR-3656 resulted in the opposite effect. By performing mechanistic studies using both in vitro and in vivo models, we found that miR-3656 could target RHOF, a member of the Rho subfamily of small GTPases, and regulate the EMT process. Moreover, enforced EMT progression via TWIST1 overexpression compromised the chemotherapy-enhancing effects of miR-3656. Finally, we found significantly lower levels of miR-3656 and higher levels of RHOF in PC tissues compared with adjacent noncancerous pancreatic tissues, and this was also associated with poor PC patients’ prognosis. Taken together, our results suggest that the miR-3656/RHOF/EMT axis is an important factor involved in regulating GR in PC, and highlights the potential of novel miR-3656-based clinical modalities as a therapeutic approach in PC patients.

Effect of arenobufagin on human pancreatic carcinoma cells.

Pancreatic carcinoma (PC) is a deadly form of cancer with poor overall survival. Currently, chemotherapy such as gemcitabine and 5-fluorouracil (5-FU) are the most popular medications that can improve survival, but rapid drug-resistance makes the search for more effective drugs urgent. Upon looking for natural components to treat PC, it was found that arenobufagin, a cardiac glycosides-like compound, showed significant effects on the gemcitabine-resistant pancreatic carcinoma cell line Panc-1 and the gemcitabine-sensitive cell line ASPC-1 at nanomolar concentrations. The present study used MTT and clonogenic survival assays to examine survival and proliferation, and western blotting to assess changes in the associated mitogen activated protein kinase and phosphoinositide 3-kinase pathways and expression of apoptosis-related proteins. The current study also detected the cell cycle by flow cytometry. Arenobufagin inhibited cell survival and proliferation, decreased the phosphorylation of key downstream proteins of K-Ras, including protein kinase B and extracellular signal related kinase, induced cell cycle G2/M phase arrest and apoptosis, and downregulated the level of phosphorylated epidermal growth factor receptor. Notably, the present data also showed that arenobufagin can enhance the sensitivity of PC cells to gemcitabine and 5-FU. In conclusion, arenobufagin could enhance the effect of gemcitabine and 5-FU on PC cells by targeting multiple key proteins. Therefore, arenobufagin has potential as anadjuvant therapy for the treatment of PC.

Curcumin sensitizes pancreatic cancer cells to gemcitabine by attenuating PRC2 subunit EZH2, and the lncRNA PVT1 expression

Development of resistance to chemotherapeutic drugs is a major challenge in the care of patients with pancreatic ductal adenocarcinoma (PDAC). Acquired resistance to chemotherapeutic agents in PDAC has been linked to a subset of cancer cells termed 'cancer stem cells' (CSCs). Therefore, an improved understanding of the molecular events underlying the development of pancreatic CSCs is required to identify new therapeutic targets to overcome chemoresistance. Accumulating evidence indicates that curcumin, a phenolic compound extracted from turmeric, can overcome de novo chemoresistance and re-sensitize tumors to various chemotherapeutic agents. However, the underlying mechanisms for curcumin-mediated chemosensitization remain unclear. The Enhancer of Zeste Homolog-2 (EZH2) subunit of Polycomb Repressive Complex 2 (PRC2) was recently identified as a key player regulating drug resistance. EZH2 mediates interaction with several long non-coding RNAs (lncRNAs) to modulate epithelial-mesenchymal transition and cancer stemness, phenomena commonly associated with drug resistance. Here, we report the re-sensitization of chemoresistant PDAC cells by curcumin through the inhibition of the PRC2-PVT1-c-Myc axis. Using gemcitabine-resistant PDAC cell lines, we found that curcumin sensitized chemoresistant cancer cells by inhibiting the expression of the PRC2 subunit EZH2 and its related lncRNA PVT1. Curcumin was also found to prevent the formation of spheroids, a hallmark of CSCs, and to down-regulate several self-renewal driving genes. In addition, we confirmed our in vitro findings in a xenograft mouse model where curcumin inhibited gemcitabine-resistant tumor growth. Overall, this study indicates clinical relevance for combining curcumin with chemotherapy to overcome chemoresistance in PDAC.

Src Inhibition Can Synergize with Gemcitabine and Reverse Resistance in Triple Negative Breast Cancer Cells via the AKT/c-Jun Pathway.

PurposeGemcitabine-based chemotherapy remains one of the standards in management of metastatic breast cancer. However, intrinsic and acquired resistance to gemcitabine inevitably occurs. The aims of this study were to assess the efficacy of the combination of src inhibition and gemcitabine in gemcitabine-resistant breast cancer cells.Methods and ResultsBy using colony formation, sphere forming, flow cytometry, cell counting kit-8 and transwell assays, 231/GEM-res (gemcitabine-resistant) cell line, which was 10 times more resistant, was shown to have elevated drug tolerance, enhanced proliferative and self-renewal abilities, compared with its parental cells. Inhibition of src by both saracatinib (AZD0530) and siRNA could partially reverse gemcitabine resistance and attenuate resistance-associated anti-apoptosis, migration and stem cell capacities. In addition, the combination of src inhibition and gemcitabine had synergistic antitumor effects. Western blot analysis revealed up-regulation of pro-apoptotic protein BAX, along with the down-regulation of anti-apoptotic proteins (BCL-XL, Survivin), migration associated proteins (p-FAK, MMP-3) and cancer stem cell (CSC) markers (CD44, Oct-4), which was probably mediated by AKT/c-Jun pathway.ConclusionIn highly gemcitabine-resistant 231 cells, src inhibition can synergize with gemcitabine, reverse drug resistance, inhibit tumor growth/metastasis/stemness of cancer stem cells, possibly via the AKT/c-Jun pathway.

Abstract 2946: Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines

Abstract Background: Aldehyde dehydrogenase (ALDH) forms a superfamily of enzymes that catalyze conversion of aldehydes into carboxylic acids, and they are categorized into 19 families in human. Increased ALDH activity was reported to be a potential marker of cancer stem cell (CSC) in various solid tumors including lung cancer. Furthermore, High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs. Recent studies revealed that ALDH7A1 is highly expressed in prostate cancer and associated with recurrence in patients with surgically resected non-small-cell lung cancer. However, there is not a fundamental link between ALDH7A1 expression and malignant phenotype, including drug resistance and colony formation. Material and methods: Anticancer drug resistant cell lines were established from lung cancer cell lines (PC-6, DMS53, PC-14, PC-9, NCI-H23 and NCI-H2228 cells) by exposure to various anticancer drug, including 7-ethyl-10-hydroxycamptothesin (SN-38), gemcitabine (GEM), cisplatin (CDDP), etoposide, paclitaxel (TXL), pemetrexed, amrubicin, erlotinib and crizotinib. Protein expression was determined by western blotting and gene expression was examined by RT-PCR. ALDH enzyme activity was measured by ALDEFLOUR TM assay. Proliferation was measured using MTS assay. ALDH7A1encoding plasmid was stably transfected into PC-14 cell. These transfected cells displayed sphere formation on ultralow binding plates and survived for more than 3 weeks. Results: We found that the levels of ALDH7A1 expression were higher in various anticancer drug-resistant lung cancer cell lines compared with the parental cells. ALDH enzyme activity was about 4-9 fold higher in SN-38, TXL, CDDP and GEM resistant cell lines (PC6/SN38, PC6/TXL, PC14/CDDP and PC14/GEM) than the parents cells. Up-regulation of ALDH7A1 expression by using a forced expression vector in PC14 cell altered cytotoxicity to SN-38 and TXL. Furthermore, overexpression of ALDH7A1in PC-14 cells enhanced sphere formation relative to the cells transfected with control vector. Conclusion: These results suggested that increased ALDH7A1 expression in lung cancer cell lines might be one of the mechanisms of acquired resistance to anticancer agents and enhance colony formation. Citation Format: Yuichi Sakamori, Hiroaki Ozasa, Eiji Kunii, Yoshitaka Yagi, Takahiro Tsuji, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines. [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 2946.

Abstract 2114: Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells

Abstract ST6Gal-I is a sialyltransferase that adds α2-6 linked sialic acids to cell surface proteins as they pass through the trans-Golgi. Sialic acids, being negatively charged, are able to alter the function of selected cell surface receptors, which leads to dysregulation of various downstream cellular pathways. Overexpression of ST6Gal-I has been observed in various cancers including ovarian and pancreatic cancer. We have previously shown that knockdown of ST6Gal-I expression increases ovarian cancer cell susceptibility to the chemotherapeutic drug cisplatin. In the present study we further investigate whether resistance to additional drugs such as gemcitabine, the front line treatment for pancreatic cancer, is affected by ST6Gal-I activity. MiaPaCa-2 and BxPC3 pancreatic cancer cell lines, having high ST6Gal-I expression, were employed for our studies. To elucidate the mechanistic role of ST6Gal-I in gemcitabine resistance we created stable ST6Gal-I knockdown lines of MiaPaCa-2 and BxPC3 cells. Gemcitabine induced cell death was more pronounced in the knockdown cell lines, indicated by heightened activation of caspase-3. Gemcitabine is metabolized to a nucleoside analogue, gemcitabine triphosphate, which induces apoptosis by promoting DNA damage. Increased single stand DNA damage in the knockdown cells was confirmed using alkaline comet assay. Gemcitabine treatment led to greater activation of DNA damage markers and response elements (γH2AX, phospho CHK1, phospho CHK2), along with increased levels of cleaved caspase-3 in the knockdown cells as compared to empty vector control cells. We next developed a stable gemcitabine resistant MiaPaCa-2 cell line by growing parental cells in gemcitabine. We selected for the population of cells that survived and were able to replicate in gemcitabine containing media. ST6Gal-I levels were found to be increased in the stable gemcitabine resistant lines relative to parental cell lines, suggesting that cells with high ST6Gal-I expression selectively survive gemcitabine treatment. By measuring the levels of genes involved in activation over those responsible for inactivation of gemcitabine, we were able to obtain a gemcitabine sensitivity predictive ratio for MiaPaCa-2 and BxPC3 ST6Gal-I knockdown, empty vector, parental and stable gemcitabine resistant cell lines. This ratio has been described previously in literature as a metric for gauging gemcitabine resistance. According to the ratios obtained, ST6Gal-I knockdown enhanced drug sensitivity whereas high expression of ST6Gal-I offered protection against gemcitabine induced apoptosis. Collectively these data indicate that upregulation of ST6Gal-I imparts tumor cell survival through prevention of gemcitabine induced DNA damage. Citation Format: Asmi Chakraborty, Matthew Schultz, Hoa Q. Trummell, James A. Bonner, Susan Bellis. Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells. [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 2114.

Abstract 256: Analysis of combined drug effects on hENT1 and hENT4 in pancreatic cancer cells

Abstract Pancreatic cancer is the 4th leading cause of cancer death and diagnosis usually occurs at late stages due to the lack of symptoms and early detection, making surgical intervention almost unfeasible due to low survival rates. Pancreatic cancer patients have one of the worst prognoses among all cancer types with a 5 year survival rate of less than 5%. Despite significant improvement in understanding molecular and epigenetic changes of this disease, the prognosis and management remained unchanged. Gemcitabine, a deoxycytidine nucleoside analog, is the golden standard of advanced pancreatic cancer treatment for patients with locally advanced or metastatic cancer of the pancreas. Patients treated with gemcitabine can, however, eventually develop resistance to this drug. Previously published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C) though up-regulation of the human equilibrative nucleoside transporter 1 (hENT1). hENT1 (SLC29A1) is the major drug transporter for gemcitabine. One of the drugs currently being investigated for treatment of pancreatic cancer is metformin. Metformin is most commonly used for the treatment of type 2 diabetes mellitus and has exhibited both chemopreventive and chemotherapeutic activities in preclinical human pancreatic cancer cells and animal models. Metformin has been found to be transported by another member of the equilibrative nucleoside transporter (ENT) family named hENT4 (SLC29A4). The current study examined the combined drug effects of gemcitabine, metformin and I3C on hENT1 and hENT4 in pancreatic cancer cells. Several pancreatic cell lines were examined for cell viability, drug synergy and modulation of hENT1 and hENT4 expression when treated with either gemcitabine, metformin, I3C or in combination for 24h and 72h. The results varied for each of the cell lines. After 24h and 72h treatment there was a significant decrease in cell viability when pancreatic cancer cells were treated with metformin or gemcitabine or 500μM I3C alone or in combination. The decrease in cell viability for these treatment conditions was also time dependent, where pancreatic cancer cells treated for 72h exhibited lower cell viability compared to 24h treatment. Pancreatic cancer cells treated with 250μM I3C alone or in combination did not decrease cell viability after 24h; however after 72h the cell viability varies based on the cell line. In most cases the drug activity exhibited antagonistic effect in combination therapy treatment. hENT1 and hENT4 expression levels varied between cell lines, where gemcitabine resistant cell lines exhibited lower hENT levels. Our initial findings showed that gemcitabine may also modulate hENT4 expression in gemcitabine sensitive cell lines and I3C modulations hENT4 expression levels. Citation Format: Stancy J. Joseph, Beverly Word, Beverly Lyn-Cook. Analysis of combined drug effects on hENT1 and hENT4 in pancreatic cancer cells. [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 256.

A Histone Deacetylase Inhibitor Suppresses Epithelial-Mesenchymal Transition and Attenuates Chemoresistance in Biliary Tract Cancer.

Epithelial-mesenchymal transition (EMT) is involved in the characteristics of malignancy, such as invasion, metastasis, and chemoresistance. In biliary tract cancer (BTC), EMT is induced by transforming growth factor-beta 1 (TGF-β1). The EMT is reversible; therefore, it is conceivable that it could be related to some epigenetic changes. We focused on histone deacetylase (HDAC) inhibitors as regulators of TGF-β1 signaling, and investigated their effect on EMT and chemoresistance. We employed four BTC cell lines (MzChA-1, gemcitabine-resistant MzChA-1, TFK-1, and gemcitabine-resistant TFK-1) and used vorinostat as the HDAC inhibitor. The relative mRNA expression of an epithelial marker (CDH1) and mesenchymal markers (CDH2, vimentin, SNAI1) were measured by qRT-PCR to evaluate factors associated with EMT. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the chemoresistance of each cell line. In addition, NOD/SCID mice were used to evaluate the effect of vorinostat in vivo. In the parent MzChA-1 and TFK-1 cell lines, TGF-β1 induced EMT and chemoresistance; while vorinostat inhibited the EMT and chemoresistance induced by TGF-β1. In gemcitabine-resistant cell lines that highly expressed TGF-β1, vorinostat inhibited EMT and attenuated chemoresistance. We showed that vorinostat inhibits nuclear translocation of SMAD4 which is a signaling factor of TGF-β1, and this is one of the mechanisms by which vorinostat regulates EMT. We also showed that vorinostat attenuates the binding affinity of SMAD4 to the CDH1-related transcription factors SNAI1, SNAI2, ZEB1, ZEB2, and TWIST. Furthermore, combination therapy with vorinostat and gemcitabine improved survival time in the mice xenografted with gemcitabine resistant MzChA-1 cells. In conclusion, vorinostat regulated TGF-β1-induced EMT and chemoresistance through inhibition of SMAD4 nuclear translocation.

TM4SF1 Promotes Gemcitabine Resistance of Pancreatic Cancer In Vitro and In Vivo.

BackgroundTM4SF1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and affects the development of this cancer. Also, multidrug resistance (MDR) is generally associated with tumor chemoresistance in pancreatic cancer. However, the correlation between TM4SF1 and MDR remains unknown. This research aims to investigate the effect of TM4SF1 on gemcitabine resistance in PDAC and explore the possible molecular mechanism between TM4SF1 and MDR.MethodsThe expression of TM4SF1 was evaluated in pancreatic cancer cell lines and human pancreatic duct epithelial (HPDE) cell lines by quantitative RT-PCR. TM4SF1 siRNA transfection was carried out using Hiperfect transfection reagent to knock down TM4SF1. The transcripts were analyzed by quantitative RT-PCR, RT-PCR and western blotting for further study. The cell proliferation and apoptosis were obtained to investigate the sensitivity to gemcitabine of pancreatic cancer cells after silencing TM4SF1 in vitro. We demonstrated that cell signaling of TM4SF1 mediated chemoresistance in cancer cells by assessing the expression of multidrug resistance (MDR) genes using quantitative RT-PCR. In vivo, we used orthotopic pancreatic tumor models to investigate the effect of proliferation after silencing TM4SF1 by a lentivirus-mediated shRNA in MIA PaCa-2 cell lines.ResultsThe mRNA expression of TM4SF1 was higher in seven pancreatic cancer cell lines than in HPDE cell lines. In three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86), the expression of TM4SF1 was lower than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1). We evaluated that TM4SF1 was a putative target for gemcitabine resistance in pancreatic cancer cells. Using AsPC-1, MIA PaCa-2 and PANC-1, we investigated that TM4SF1 silencing affected cell proliferation and increased the percentages of cell apoptosis mediated by treatment with gemcitabine compared with cells which were treated with negative control. This resistance was associated with the expression of multidrug resistance genes including ABCB1 and ABCC1. In vivo, silencing of TM4SF1 in MIA PaCa-2 cell lines increased the effectiveness of gemcitabine-based treatment in orthotopic pancreatic tumor models evaluated using noninvasive bioluminescent imaging.ConclusionThese findings suggest that TM4SF1 is a surface membrane antigen that is highly expressed in pancreatic cancer cells and increases the chemoresistance to gemcitabine. Thus, TM4SF1 may be a promising target to overcome the chemoresistance of pancreatic cancer.

Abstract 4436: Metformin upregulates hENT1 expression and enhances gemcitabine efficacy in pancreatic cancer cells

Abstract Pancreatic cancer is the 4th leading cause of cancer death and is normally diagnosed at a late stage due to the lack of symptoms and early detection. Pancreatic cancer patients have one of the worst prognoses among all cancer types with a 5 year survival rate of less than 5%. Despite significant improvement in understanding molecular and epigenetic changes of this disease, the prognosis and management remained unchanged. The golden standard of advanced pancreatic cancer treatment is gemcitabine or gemcitabine with more targeted therapy. Patients treated with gemcitabine can, however, eventually develop resistance to this drug. Previously published data from our laboratory demonstrated enhanced efficacy of gemcitabine with the dietary agent, indole-3-carbinol (I3C) though up-regulation of the human equilibrative nucleoside transporter (hENT1). hENT1 is the major drug transporter for gemcitabine. One of the drugs currently being investigated for treatment of pancreatic cancer is metformin. Metformin is one of the most widely prescribed drugs for the treatment of type 2 diabetes mellitus. Metformin has exhibited both chemopreventive and chemotherapeutic activities in preclinical human pancreatic cancer cells and animal models. Currently this drug is in clinical trial for pancreatic cancer. Metformin has been found to be transported by a member of the equilibrative nucleoside transporter (ENT) family named ENT4.The current study examined the potential efficacy and regulation of hENT1 in pancreatic cancer cells when treated with gemcitabine and/or metformin. Several pancreatic cell lines (Mia PaCa-2, AsPC-1, Su86.86 and PANC-1) were examined for modulation of hENT1 expression when treated with gemcitabine or metformin or in combination for 24h and 72h. The results varied for each of the cell lines. After 24 h treatment, hENT1 expression analysis of Su86.86 and PANC-1 gemcitabine resistant cell lines showed no change in hENT1 expression levels. After 72h of treatment with gemcitabine and/or metformin hENT1 expression was up-regulated (p&amp;lt;0.05); however, metformin did not enhance the efficacy of gemcitabine. In AsPC-1, a cell line with moderate gemcitabine resistance, hENT1 expression was down-regulated (p&amp;lt;0.05) after 24 h treatment with gemcitabine and/or metformin; however, after 72h of treatment with gemcitabine and metformin, metformin increase the efficacy of gemcitabine (p&amp;lt;0.05). In MIA Paca-2, a gemcitabine sensitive cell line, the expression of hENT1 was up-regulated (p&amp;lt;0.05) after 24h treatment with gemcitabine. Metformin and combination with gemcitabine treatments also upregulated the expression of hENT1 (p&amp;lt;0.05) but metformin did not increase the efficacy of gemcitabine. There was no change in expression for MIA Paca-2 cells treated for 72h. Based on initial studies, the sensitivity of pancreatic cancer cell lines to gemcitabine/metformin combination is through up-regulation of hENT1. Citation Format: Stancy J. Joseph, Taylor Osborne, Beverly Word, Beverly Lyn-Cook. Metformin upregulates hENT1 expression and enhances gemcitabine efficacy in pancreatic cancer cells. [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 4436. doi:10.1158/1538-7445.AM2015-4436

Abstract 2696: Arenobufagin targets K-Ras downstream to enhance the efficiency of gemcitabine in human pancreatic carcinoma cells

Abstract Pancreatic carcinoma (PC) is a deadly form of cancer with poor overall survival. Currently, chemotherapeutic such as gemcitabine and 5-fluorouracil (5-FU) are the most popular medication that can slightly improve survival, but rapid drug-resistance makes it urgent to search for more effective drugs. In hunting for natural components that cure PC, we found that arenobufagin, a cardiac glycosides-like compound, showed significant effects on the gemcitabine-resistant cell line Panc-1, the gemcitabine-sensitive cell line ASPC-1 and K-Ras activating HPNE cells at nanomolar concentrations. We used MTT and clonogenic survival assays to examine survival and proliferation and western blotting to assess changes of K-Ras downstream and apoptosis related proteins. We also detected the cell cycle and migration by flow cytometry and wound healing assay. Arenobufagin inhibited cellular survival and proliferation, decreased the phosphorylation of key proteins of K-Ras downstream such as Akt, Erk and nuclear factor kappa B (NF-κB), induced cell cycle G2/M arrest and apoptosis, and downregulated the level of phosphorylated epidermal growth factor receptor (EGFR). Most importantly, our data showed that arenobufagin can enhance the sensitivity of PC cells to gemcitabine and 5-fluorouracil (5-FU). Arenobufagin could enhance the effect of gemcitabine and 5-FU on PC cells through targeting multiple key proteins. All above makes arenobufagin a novel agent for combination therapy of pancreatic carcinoma. Citation Format: Tianjiao Wang, Yuhui Yuan. Arenobufagin targets K-Ras downstream to enhance the efficiency of gemcitabine in human pancreatic carcinoma cells. [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 2696. doi:10.1158/1538-7445.AM2015-2696

Acquisition of chemoresistance to gemcitabine is induced by a loss-of-function missense mutation of DCK

The anti-tumor activity of gemcitabine (GEM) has been clinically proven in several solid tumors, including pancreatic cancer, biliary tract cancer, urinary bladder cancer, and non-small cell lung cancer. However, problems remain with issues such as acquisition of chemoresistance against GEM. GEM is activated after phosphorylation by deoxycytidine kinase (DCK) inside of the cell; thus, DCK inactivation is one of the important mechanisms for acquisition of GEM resistance. We previously investigated the DCK gene in multiple GEM resistant cancer cell lines and identified frequent inactivating mutations. In this study, we identified two crucial genetic alteration in DCK. (1) A total deletion of DCK in RTGBC1-TKB, an acquired GEM resistant cell line derived from a gall bladder cancer cell line TGBC1-TKB. (2) An E197K missense alteration of DCK in MKN28, a gastric cancer cell line; its acquired GEM resistant cancer cell line, RMKN28, showed a loss of the normal E197 allele. We introduced either normal DCK or altered DCK_E197K into RMKN28 and proved that only the introduction of normal DCK restored GEM sensitivity. Furthermore, we analyzed 104 healthy volunteers and found that none of them carried the same base substitution observed in MKN28. These results strongly suggest that (1) the E197K alteration in DCK causes inactivation of DCK, and that (2) loss of the normal E197 allele is the crucial mechanism in acquisition of GEM resistance in RMKN28.

Abstract A81: A microRNA signature identified for chemoresistance and mesenchymal phenotype is also found in advanced pancreatic cancer

Abstract Recent studies link cancer cells that possess an epithelial to mesenchymal transition (EMT) and cancer stem cell properties with chemoresistance and metastatic potential. Here, we investigated the molecular bases of the chemoresistant mesenchymal phenotype (CRMP) with emphasis on identifying a micro-RNA (miRNA) signature in advanced pancreatic ductal adenocarcinoma (PDAC). We developed a gemcitabine resistant cell model from a PDAC cell line, BxPC3. This gemcitabine resistant cell line (BxPC3-GZR) showed a pronounced mesenchymal phenotype and expressed high levels of CD44 a marker found on stem cells and showed a highly significant increase in expression of nine miRNAs and decrease in eight miRNAs. Based on relevance to cancer and on validation by q-RT-PCR we chose six of the miRNAs (miR-100, miR-125b, miR-21, miR-205, miR-27b and miR-455-3p) as a signature to determine whether they were similarly deregulated in 43 advanced PDAC tumor specimens previously analyzed as part of the Total Cancer Genome Atlas (TCGA). A strong correlation was observed for five of the six miRNAs in the tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both cellular CRMP and advanced PDAC. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13). Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. The results of this study suggests that CRMP is caused in part by deregulation of a specific set of miRNAs and that these miRNAs are also deregulated in advanced PDAC. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread. Citation Format: James W. Freeman, Alakesh Bera, Venkatasubbarao Kolaparthi. A microRNA signature identified for chemoresistance and mesenchymal phenotype is also found in advanced pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A81.

Drug-Resistant Urothelial Cancer Cell Lines Display Diverse Sensitivity Profiles to Potential Second-Line Therapeutics

Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUPrGEMCI20), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer.

Role of integrin-linked kinase in drug resistance of lung cancer.

ObjectiveThe objective of the present investigation was to investigate the role of integrin-linked kinase (ILK) in the gemcitabine-resistant lung cancer cell line A549 and explore the underlying mechanism.Materials and methodsGemcitabine-resistant A549 (A549/GemR) cell line was established by pulse-exposed to moderate concentration of gemcitabine (Gem), and the drug resistant index was measured by MTT assay. Expression of ILK in A549/GemR cell line was detected by Western blot and real-time PCR. An ILK gene-silencing cell line was constructed using lentivirus-coated ILK shRNA. MTT assay was used to detect the drug sensitivity of the A549/GemR cell line to Gem after the ILK gene silencing. Western blot was used to measure the expression of E-cadherin, fibronectin, and MRP1 (multidrug resistance-associated protein 1) after silencing the ILK gene.ResultThe drug resistance index of A549/GemR was 13.5, and the messenger RNA and protein level of ILK was increased in A549/GemR. IC50 (half maximal inhibitory concentration) decreased from 14.69 to 4.13 mg/L when ILK was knocked down in A549/GemR. The expression of fibronectin and MRP1 was upregulated and E-cadherin expression was downregulated in A549/GemR, and these changes were reversed after ILK was knocked down.ConclusionILK was involved in drug resistance to Gem in lung cancer, and this function may be mediated by epithelial–mesenchymal transition and the MRP1 pathway.