Gemcitabine-resistant PANC-1 Research Articles

Abstract 7202: Strategy of targeting PKCβΙ protein to overcoming the acquired gemcitabine resistant pancreatic cancer cells

Abstract Pancreatic cancer (PC) is one of the most malignant cancers with limitation of treatment options such as resection surgery, radiation therapy and gemcitabine-based chemotherapy. To treating PC patients, gemcitabine-based chemotherapy is common and effective for PC, acquired gemcitabine resistance is one of the major reasons for treatment failure. Therefore, there is a need to suggest effective strategies and novel therapeutic approach for gemcitabine-resistant PC. Protein Kinase C β1 (PKC β1) is known as involved in multiple signal transduction related to oncogenic functions in cancer. We suggested that PKC β as one of the novel biomarker of drug resistance and further investigated the molecular mechanism. Among the natural products as small molecule inhibitor, we found that evodiamine has potent antitumor activity and various biological activities. Based on various biological activities of evodiamine, the scaffold was synthesized and investigated for antiproliferative and antitumor activities against gemcitabine resistant PANC-1 (PANC-GR) cells. Also, target validation experiment was employed to elucidate the molecular mechanism and verifying the target to overcoming gemcitabine resistance. Mechanistically, evodiamine derivatives have shown that inhibition of PKC β1 can modulate the proliferation of PANC-GR cells and exhibited tumor growth inhibitory activity in xenograft mouse model. Keywords: Pancreatic cancer cell, Gemcitabine resistance, Protein Kinase C β1 (PKC β1), Molecular mechanism Acknowledgements: This research was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (RS-2023-00273240). Citation Format: EunSeo Bae, Yijae Lim, Junhwa Hong, Woong Sub Byun, Simin Chun, Suckchang Hong, Sang Kook Lee. Strategy of targeting PKCβΙ protein to overcoming the acquired gemcitabine resistant pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7202.

Antitumor effect of 3-(quinolin-2-ylmethylene)-4,6-dimethyl-5-hydroxy-7-azaoxindole down-regulating the Gas6-Axl axis.

In this study, a new series of 3-arylidene-4,6-dimethyl-5-hydroxy-7-azaoxindole compounds with a wide range of functional groups were designed, synthesized, and evaluated for their antitumor activity. Among the 35 compounds, compound 6-15, with a quinoline moiety, showed cytotoxic IC50 values superior to those of sunitinib against the seven cancer cell lines (MCF-7, MDA-MB-231, HT-29, DU145, U937, A549, and PANC-1). However, its inhibitory activity against receptor tyrosine kinases (VEGFR2, PDGFRβ, c-KIT, FGFR1, FLT3, CSF1R, EGFR, Axl, and Axl mutant) was 100-3000-fold weaker than that of sunitinib. Interestingly, compound 6-15 exerted a 3.6-fold stronger cytotoxicity than sunitinib in the gemcitabine-resistant PANC-1cell line and significantly inhibited Axl, which was in contrast with the effect of sunitinib. Nonetheless, both compounds suppressed the expression of growth arrest-specific 6 (Gas6), the ligand of Axl. The inhibitory effect of compound 6-15 on the Gas6-Axl axis was similar to that of Gas6 knockdown by siRNA in PANC-1cells in terms of apoptosis induction, increase in Bax/Bcl-2 ratio, Axl down-regulation, and PI3K/Akt inhibition. The inhibitory effect of compound 6-15 on tumor growth in mouse tumor models with A549 and PANC-1 xenografts was much greater than that of cisplatin or gemcitabine. Taken together, the current findings demonstrate that compound 6-15 is a promising anticancer drug candidate that acts by inhibiting the Gas6-Axl axis.

Organic cation transporter 2 activation enhances sensitivity to oxaliplatin in human pancreatic ductal adenocarcinoma

Oxaliplatin, a third-generation platinum derivative, has become one of the main chemotherapeutic treatments for esophagus, gastric and colorectal cancer; however, it is still unclear the potential effectiveness for pancreatic ductal adenocarcinoma (PDAC) with gemcitabine resistance. Here, we observed that PDAC tumors have low level of organic cation transporter 2 (OCT2, also known as SLC22A2) compared with non-tumor tissues and identified that OCT2 expression is positively correlated with oxaliplatin sensitivity in PDAC cells. Treatment of OCT2 inhibitors or knockdown of OCT2 expression significantly decreased the sensitivity to oxaliplatin in PANC-1 cells. In addition, bisulfite sequencing polymerase chain reaction analysis revealed that higher methylation frequency represses OCT2 expression in gemcitabine-resistant PANC-1 (PANC-1/GR) cells. Moreover, we found that treatment of DNA methyltransferase (DNMT) inhibitors, decitabine or 5-azacytidine recover OCT2 expression and oxaliplatin sensitivity in PANC-1/GR cells, and DNMT1 level has inverse correlation with OCT2 expression in PDAC cells and tumors. Our findings jointly suggest that OCT2 expression is a potential and predictive marker for evaluating oxaliplatin sensitivity and developing alternative treatments for PDAC patients with gemcitabine resistance.

Abstract 6386: Phosphomimetic Dicer rewires glutamine metabolism and gemcitabine resistance in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a common malignancy that shows high cancer-mortality and poor prognosis, in the absence of obvious symptoms and biomarkers for early diagnosis. Gemcitabine is a type of chemotherapy drug which commonly used to treat PDAC, however, the rapid acquisition of resistance to gemcitabine treatment has been observed in many of PDAC patients. The miRNA-producing enzymes Dicer is crucial for the maturation of miRNAs, and we found previously that ERK/Sp1 signaling transactivate both gene and protein expressions of Dicer, leading an acquisition of gemcitabine resistance in pancreatic cancer cells both in vitro and in vivo. In this study, we further identified that phosphomimetic S1016E of Dicer may regulate miRNAs maturation, resulting an imbalance of glutaminase (GLS) and glutamine synthetase (GS/GLUL). Metabolism analysis revealed that gemcitabine-resistant PANC-1 (PANC-1/GEM) cells have high glutamine dependence and GLS/GLUL ratio. Our study suggests that phosphomimetic Dicer S1016E plays a critical role in glutamine metabolism reprogramming and gemcitabine resistance of pancreatic cancer. Citation Format: Ching-Feng Chiu, Ji Min Park, Yu-Shiuan Shen, Chia-Ying Lin, Tung-Wei Hsu, Yen-Hao Su, Hsin-An Chen. Phosphomimetic Dicer rewires glutamine metabolism and gemcitabine resistance in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6386.

The enhancement of Tetrandrine to gemcitabine-resistant PANC-1 cytochemical sensitivity involves the promotion of PI3K/Akt/mTOR-mediated apoptosis and AMPK-regulated autophagy

BackgroundIn the process of tumor development, the resistance of pancreatic cancer cells to gemcitabine (GEM) is mainly due to the suppression and dysregulation of apoptosis signals to a large extent. Therefore, it is very necessary to develop pro-apoptotic drugs for combined treatment of pancreatic cancer to increase the activity of GEM and improve the prognosis of pancreatic cancer. Methods and ResultsGEM-resistant PANC-1 cells were treated with increasing doses of GEM. The effects of GEM and TET on apoptosis were evaluated by flow cytometry and Hoechst 33258 staining. We also evaluated the expression of survivin by real-time PCR, and the expression levels of proteins involved in apoptosis, autophagy, and PI3K/Akt/mTOR signaling were detected by western blotting. The results showed that TET downregulated expression of survivin by inhibiting the PI3K/Akt/mTOR signaling pathway to promote pancreatic cancer cell apoptosis, thereby enhancing pancreatic cancer cell sensitivity to GEM. Moreover, TET enhanced cytotoxic and autophagy-dependent cell death by upregulating the AMPK-autophagy axis, and this effect was reversed by inhibition of AMPK. ConclusionsTET promotes apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway and promotes autophagy via up-regulating the AMPK signaling pathway to play an anti-tumor effect in GEM-resistant pancreatic cancer cells, which represents a new therapeutic strategy for the treatment of GEM-resistant pancreatic cancer.

ERK-mediated transcriptional activation of Dicer is involved in gemcitabine resistance of pancreatic cancer.

Gemcitabine has been a commonly used therapeutic agent for treatment of pancreatic cancer. In the clinic, agrowing resistance to gemcitabine has been observed in patients with pancreatic cancer, and investigation of the underlying mechanism of gemcitabine resistance is urgently required. The microRNA (miRNA)-producing enzyme, Dicer, is crucial for the maturation of miRNAs, andis involvedin clinical aggressiveness, poor prognosis, and survival outcomes in various cancers, however, the role of Dicer in acquired gemcitabine resistance of pancreatic cancer isstill not clear. Here, we found that Dicer expression was significantly increased in gemcitabine-resistant PANC-1 (PANC-1/GEM) cells compared with parental PANC-1 cells and observed a high level of Dicer correlated with increased risk of pancreatic cancer. Suppression of Dicer obviously decreased gemcitabine resistance in PANC-1/GEM cells; consistently, overexpression of Dicer in PANC-1 cells increased gemcitabine resistance. Moreover, we identified that transcriptional factor Sp1 targeted the promoter region of Dicer and found ERK/Sp1 signaling regulated Dicer expression in PANC-1/GEM cells, as well as positively correlated with pancreatic cancer progression and suggest that targeting the ERK/Sp1/Dicer pathway has potential therapeutic value for pancreatic cancer with acquired resistance to gemcitabine.

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells.

BackgroundAspirin (acetylsalicylic acid) and celecoxib have been used as potential anti-cancer therapies. Aspirin exerts its therapeutic effect in both cyclooxygenase (COX)-dependent and -independent pathways to reduce tumor growth and disable tumorigenesis. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, reduces factors that cause inflammation and pain. The question is whether aspirin and celecoxib have other molecular targets of equal or more therapeutic efficacy with significant anti-cancer preventive benefits.AimHere, we propose that aspirin and celecoxib exert their anti-cancer effects by targeting and inhibiting mammalian neuraminidase-1 (Neu-1). Neu-1 has been reported to regulate the activation of several receptor tyrosine kinases (RTKs) and TOLL-like receptors and their downstream signaling pathways. Neu-1 in complex with matrix metalloproteinase-9 (MMP-9) and G protein-coupled receptors (GPCRs) has been reported to be tethered to RTKs at the ectodomain.Materials and MethodsThe WST-1 cell viability assay, Caspase 3/7 assay, and Annexin V assay were used to evaluate the cell viability and detect apoptotic and necrotic cells following treatment in MiaPaCa-2, PANC-1 and the gemcitabine-resistant PANC-1 variant (PANC-1 GemR) cells. Microscopic imaging, lectin cytochemistry, and flow cytometry were used to detect levels of α-2,3 sialic acid. Epidermal growth factor (EGF)-stimulated live cell sialidase assays and neuraminidase assays were used to detect Neu-1 activity. Immunocytochemistry was used to detect levels of EGFR and phosphorylated EGFR (pEGFR) following treatment.ResultsFor the first time, aspirin and celecoxib were shown to significantly inhibit Neu-1 sialidase activity in a dose- and time-dependent manner following stimulation with EGF. Aspirin blocked Neu-1 desialylation of α-2,3-sialic acid expression following 30 min stimulation with EGF. Aspirin and celecoxib significantly and dose-dependently inhibited isolated neuraminidase (Clostridium perfringens) activity on fluorogenic substrate 2ʹ-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA). Aspirin inhibited phosphorylation of the EGFR in EGF-stimulated cells. Aspirin dose- and time-dependently induced CellEvent caspase-3/7+ cells as well as apoptosis and necrosis on PANC-1 cells.ConclusionThese findings signify a novel multimodality mechanism(s) of action for aspirin and celecoxib, specifically targeting and inhibiting Neu-1 activity, regulating EGF-induced growth receptor activation and inducing apoptosis and necrosis in a dose- and time-dependent manner. Repurposing aspirin and celecoxib as anti-cancer agents may also upend other critical targets involved in multistage tumorigenesis regulated by mammalian neuraminidase-1.SignificanceThese findings may be the missing link connecting the anti-cancer efficacy of NSAIDs to the role of glycosylation in inflammation and tumorigenesis.

Emodin alleviates gemcitabine resistance in pancreatic cancer by inhibiting MDR1/P-glycoprotein and MRPs expression.

Gemcitabine is a gold standard chemotherapeutic agent for pancreatic cancer. However, gemcitabine has limited effectiveness due to the short-term development of chemoresistance. Emodin, a natural anthraquinone derivative isolated from the roots of rheumatic palm leaves prevents immunosuppression and exerts anticancer effects. The present study aimed to evaluate the effect of emodin on gemcitabine resistance. Gemcitabine-resistant PANC-1 pancreatic cancer cell xenografts were established in athymic mice, which were randomly assigned into four treatments groups as follows: Gemcitabine group, Emodin group, Gemcitabine+Emodin group and Negative control group. Body weight, tumor volume and tumor weight were measured over the course of treatment. The effect of each treatment on tumor tissue proliferation and apoptosis from nude mice was evaluated by using immunohistochemistry. The effect of each treatment on the proliferation of gemcitabine-resistant PANC-1 cells was also determined by using the Cell Counting Kit-8. Then, reverse transcription-quantitative (RT-q) PCR and western blotting were used to detect the mRNA and protein expression, respectively, of multidrug resistance gene 1 (MDR1) and the drug resistance-related proteins MRP1 and MRP5. The function and expression level of DR1 gene product, p-glycoprotein, was also analysed by flow cytometry and RT-qPCR, respectively. The results demonstrated that the combination of gemcitabine and emodin significantly reduced xenograft volume and reduced tumor growth in mice compared with treatment with gemcitabine or emodin only. In addition, emodin treatment reduced resistance to gemcitabine, which was characterized by the downregulation of P-glycoprotein, MRP1 and MRP5 expression in the group receiving combination treatment. The level of P-glycoprotein was also decreased in the group treated with gemcitabine+emodin compared with the single treatment groups. Taken together, these results demonstrated that emodin enhanced gemcitabine efficacy in tumor treatment and alleviated gemcitabine resistance in PANC-1 cell xenografts in mice via suppressing MDR1/P-glycoprotein and MRP expression.

(3E,5E)-3,5-Bis(pyridin-3-methylene)-tetrahydrothiopyran-4-one enhances the inhibitory effect of gemcitabine on pancreatic cancer cells

Gemcitabine (GEM) is a commonly used treatment for advanced pancreatic cancer. However, chemoresistance and toxic side effect limits its clinical success. In an earlier study, our laboratory found that the curcumin analogue, (3E,5E)-3,5-Bis(pyridin-3-methylene)-tetrahydrothiopyran-4-one (FN2) had strong inhibitory effect on human pancreatic cancer cells. In the present study, we investigated the effects of FN2 in combination with GEM on growth inhibition and apoptosis in human pancreatic cancer Panc-1 cells. The results showed that the combination of FN2 and GEM synergistically inhibited the growth of Panc-1 cells. Panc-1 cells survived the GEM treatment became partially resistant to the drug. Treatment with FN2 in combination with GEM strongly inhibited the growth and stimulated apoptosis in the GEM resistant Panc-1 cells. Mechanistic studies showed that inhibition of cell growth and induction of apoptosis in the GEM resistant Panc-1 cells were associated with decreases in activation of NF-κB and Akt. FN2 in combination with GEM also decreased the level of Bcl-2 and increased the level of Bax. Results of the present study indicate that GEM in combination with FN2 may represent an effective strategy for improving the efficacy of GEM and decreasing the resistance of pancreatic cancer to GEM chemotherapy.

Gemcitabine‐induced epithelial‐mesenchymal transition‐like changes sustain chemoresistance of pancreatic cancer cells of mesenchymal‐like phenotype

Growing body of evidence suggests that epithelial-mesenchymal transition (EMT) is a critical process in tumor progression and chemoresistance in pancreatic cancer (PC). The aim of this study was to analyze the role of EMT-like changes in acquisition of resistance to gemcitabine in pancreatic cells of the mesenchymal or epithelial phenotype. Therefore, chemoresistant BxPC-3, Capan-2, Panc-1, and MiaPaca-2 cells were selected by chronic exposure to increasing concentrations of gemcitabine. We show that gemcitabine-resistant Panc-1 and MiaPaca-2 cells of mesenchymal-like phenotype undergo further EMT-like molecular changes mediated by ERK-ZEB-1 pathway, and that inhibition of ERK1/2 phosphorylation or ZEB-1 expression resulted in a decrease in chemoresistance. Conversely, gemcitabine-resistant BxPC-3 and Capan-2 cells of epithelial-like phenotype did not show such typical EMT-like molecular changes although the expression of the tight junction marker occludin could be found decreased. In pancreatic cancer patients, high ZEB-1 expression was associated with tumor invasion and tumor budding. In addition, tumor budding was essentially observed in patients treated with neoadjuvant chemotherapy. These findings support the notion that gemcitabine treatment induces EMT-like changes that sustain invasion and chemoresistance in PC cells.

Oridonin overcomes the gemcitabine resistant PANC-1/Gem cells by regulating GST pi and LRP/1 ERK/JNK signalling.

Background: Chemotherapy remains a primary treatment method for advanced pancreatic cancer. However, chemotherapy resistance can influence the therapeutic effect of pancreatic cancer. The resistance mechanism of chemotherapeutic agents such as gemcitabine, which is an agent typically used to treat pancreatic cancer, is complicated and can be influenced by genes and the environment. Oridonin is a tetracyclic diterpenoid compound extracted from the traditional Chinese herb Rabdosia labtea. Oridonin may overcome drug resistance in pancreatic cancer, but researching pancreatic cancer drug resistance of chemotherapy by oridonin is not completely understood.Purpose: The present study aimed to assess the impact of oridonin on multidrug resistance proteins, apoptosis-associated proteins and energy metabolism in gemcitabine-resistant PANC-1 (PANC-1/Gem) pancreatic cancer cells.Methods: Gemcitabine resistance in PANC-1/Gem cells was induced using a concentration gradient of gemcitabine. Cell Counting Kit-8 assays were used to detect the impact of gemcitabine and oridonin on the proliferation of PANC-1 and PANC-1/Gem cells. Western blot analysis and immunofluorescence were used to detect the expression of multidrug resistance proteins, apoptosis-associated proteins and low-density lipoprotein receptor protein 1 (LRP1) proteins in PANC-1/Gem cells. The effects of gemcitabine and oridonin on PANC-1/Gem cells apoptosis were detected using flow cytometry. Animal xenograft tumor assays were used to detect the effect of gemcitabine and oridonin on pancreatic cancer in vivo. Furthermore, the ATP Assay kit was used to determine the effects of gemcitabine and oridonin on ATP levels in PANC-1/Gem cells. Immunofluorescence assays were used to detect the effects of gemcitabine and oridonin on the expression of low-density lipoprotein receptor protein 1 (LRP1) in PANC-1/Gem cells. In addition, LRP1 expression was knocked down in PANC-1/Gem cells via lentiviral vector-mediated RNA silencing. Clone formation assays and Western blot analysis were used to detect the effect of LRP1 knockdown on the proliferation of PANC-1/Gem cells.Results: The present results demonstrate that oridonin overcomes PANC-1/Gem cells gemcitabine reistance by regulating GST pi and LRP1/ERK/JNK signaling.Conclusion: In conclusion, the present study indicated that oridonin could overcome gemcitabine resistance in PANC-1/Gem cells by regulating GST pi and LRP1/ ERK/JNK signaling, inducing cell apoptosis. Therefore, oridonin with gemcitabine may be a promising preoperative treatment for patients who suffer from pancreatic cancer.

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.

OSU-A9 inhibits pancreatic cancer cell lines by modulating p38-JAK-STAT3 signaling

Pancreatic cancer is an aggressive malignancy that is the fourth leading cause of death worldwide. Since there is a dire need for novel and effective therapies to improve the poor survival rates of advanced pancreatic cancer patients, we analyzed the antitumor effects of OSU-A9, an indole-3-carbinol derivative, on pancreatic cancer cell lines in vitro and in vivo. OSU-A9 exhibited a stronger antitumor effect than gemcitabine on two pancreatic cancer cell lines, including gemcitabine-resistant PANC-1 cells. OSU-A9 treatment induced apoptosis, the down-regulation of Akt phosphorylation, up-regulation of p38 phosphorylation and decreased phosphorylation of JAK and STAT3. Cell migration and invasiveness assays showed that OSU-A9 reduced cancer cell aggressiveness and inhibited BxPC-3 xenograft growth in nude mice. These results suggest that OSU-A9 modulates the p38-JAK-STAT3 signaling module, thereby inducing cytotoxicity in pancreatic cancer cells. Continued evaluation of OSU-A9 as a potential therapeutic agent for pancreatic cancer thus appears warrented.

Combined treatment with cotyleninA and phenethyl isothiocyanate induces strong antitumor activity mainly through the induction of ferroptotic cell death in human pancreatic cancer cells.

The treatment of pancreatic cancer, one of the most aggressive gastrointestinal tract malignancies, with current chemotherapeutic drugs has had limited success due to its chemoresistance and poor prognosis. Therefore, the development of new drugs or effective combination therapies is urgently needed. CotyleninA (CN-A) (a plant growth regulator) is a potent inducer of differentiation in myeloid leukemia cells and exhibits potent antitumor activities in several cancer cell lines. In the present study, we demonstrated that CN-A and phenethyl isothiocyanate (PEITC), an inducer of reactive oxygen species (ROS) and a dietary anticarcinogenic compound, synergistically inhibited the proliferation of MIAPaCa-2, PANC-1 and gemcitabine-resistant PANC-1 cells. A combined treatment with CN-A and PEITC also effectively inhibited the anchorage-independent growth of these cancer cells. The combined treatment with CN-A and PEITC strongly induced cell death within 1day at concentrations at which CN-A or PEITC alone did not affect cell viability. A combined treatment with synthetic CN-A derivatives (ISIR-005 and ISIR-042) or fusicoccinJ (CN-A-related natural product) and PEITC did not have synergistic effects on cell death. The combined treatment with CN-A and PEITC synergistically induced the generation of ROS. Antioxidants (N-acetylcysteine and trolox), ferroptosis inhibitors (ferrostatin-1 and liproxstatin), and the lysosomal iron chelator deferoxamine canceled the synergistic cell death. Apoptosis inhibitors (Z-VAD-FMK and Q-VD-OPH) and the necrosis inhibitor necrostatin-1s did not inhibit synergistic cell death. Autophagy inhibitors (3-metyladenine and chloroquine) partially prevented cell death. These results show that synergistic cell death induced by the combined treatment with CN-A and PEITC is mainly due to the induction of ferroptosis. Therefore, the combination of CN-A and PEITC has potential as a novel therapeutic strategy against pancreatic cancer.

RNA interference targeting ETS-1 downregulates RRM2 and enhances gemcitabine sensitivity in pancreatic cancer cells

Aberrant expression of the Ets-1 transcription factor is increasingly associated with the progression of several human cancers. Previously we reported Ets-1 over-expression in gemcitabine resistance pancreatic cancer cell lines. In the present study, we identified Ribonucleotide reductase–M2 subunit, a key enzyme involved in the DNA synthesis is elevated in gemcitabine-resistant PANC1 cells. Silencing of Ets-1 expression in resistant cell line diminishes the Ribonucleotide reductase–M2 expression. The RAS/ERK pathway is commonly activated in carcinomas and promotes oncogenesis by altering transcriptional programs. Ets-1 silencing abrogates the expression of Ribonucleotide reductase–M2 levels in parental cells treated with epidermal growth factor to further activate the RAS/ERK pathway. Our findings clearly demonstrate the involvement of RAS/ERK-mediated Ets-1 pathway in the development of gemcitabine resistance by over-expressing Ribonucleotide reductase–M2.

Synergistic combination of gemcitabine and dietary molecule induces apoptosis in pancreatic cancer cells and down regulates PKM2 expression.

Gemcitabine, an effective agent in treatment of cancer of pancreas, has undergone failures in many instances after multiple cycles of therapy due to emergence of drug resistance. Combination of dietary compounds with clinically validated drugs has emerged as an effective therapeutic approach to treat pancreatic tumors, refractory to gemcitabine therapy. In order to optimize a possible synergistic combination of Gemcitabine (GCB) with dietary molecules, Betuilnic acid (BA) and Thymoquinone (TQ), stand-alone IC50 dose of GCB, BA and TQ was calculated for pancreatic cancer cell lines. Fixed IC50 dose ratio of the dietary molecules in combination with reduced IC50 dose of GCB was tested on GCB resistant PANC-1 and sensitive MIA PaCa-2 cells for synergism, additive response and antagonism, using calcusyn. Combination index (CI) revealed that pre-treatment of BA and TQ along with GCB synergistically inhibited the cancer cell proliferation in in-vitro experiments. Pyruvate kinase (PK) M2 isoform, a promising target involved in cancer cell metabolism, showed down-regulation in presence of TQ or BA in combination with GCB. GCB with BA acted preferentially on tumor mitochondria and triggered mitochondrial permeability transition. Pre-exposure of the cell lines, MIA PaCa-2 and PANC-1, to TQ in combination with GCB induced apoptosis. Thus, the effectiveness of BA or TQ in combination with GCB to inhibit cell proliferation, induce apoptosis and down-regulate the expression of PKM2, reflects promise in pancreatic cancer treatment.

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer.

Background:Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance.Methods:We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining.Results:The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients.Conclusions:miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

Garcinol sensitizes human pancreatic adenocarcinoma cells to gemcitabine in association with microRNA signatures

Alterations in microRNA (miRNA/miR) genes are of biological importance in the pathophysiology of cancers, including pancreatic cancer (PaCa). Although growing evidence supports the role of miRNA in cancer, their response to dietary phytochemicals is less known. Previously, we showed that garcinol induces PaCa cell growth arrest and apoptosis in vitro. The present study, discusses chemo-sensitization by garcinol in synergism with first-line PaCa drug, gemcitabine. The miRNA expression profile of gemcitabine-resistant Panc-1 cells treated with garcinol and/or gemcitabine was also evaluated. Garcinol synergizes with gemcitabine to inhibit cell proliferation and induce apoptosis in PaCa cells with significant modulation of key cancer regulators including PARP, VEGF, MMPs, ILs, caspases, and NF-κB. In addition, biostatistical analyses, quantitative reverse transcription PCR data, and in silico modeling using TargetScan5, PicTar, and DNA intelligent analysis, microT-V.B4 database showed that these two agents modulated a number of microRNAs (miR-21, miR-196a, miR-495, miR-605, miR-638, and miR-453) linked to various canonical oncogenic signaling pathways. We identified garcinol-specific miRNA biomarkers that sensitize PaCa cells to gemcitabine treatment, thus attenuating the drug-resistance phenotype. These results prompt further interest in garcinol and gemcitabine combination strategy as a drug modality to improve treatment outcome in patients diagnosed with PaCa.

The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Induces Growth Inhibition and Enhances Gemcitabine-Induced Cell Death in Pancreatic Cancer

Pancreatic cancer is an aggressive human malignancy that is generally refractory to chemotherapy. Histone deacetylase inhibitors are novel agents that modulate cell growth and survival. In this study, we sought to determine whether a relatively new histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), inhibits pancreatic cancer cell growth. The effects of SAHA on the growth of three pancreatic cancer cell lines (BxPC3, COLO-357, and PANC-1) were examined with respect to cell cycle progression, p21 induction and localization, and interactions with the nucleoside analogue gemcitabine. SAHA induced a G(1) cell cycle arrest in BxPC-3 cells and COLO-357 cells but not in PANC-1 cells. This arrest was dependent, in part, on induction of p21 by SAHA, as p21 was not induced in PANC-1 cells, and knockdown of p21 using small interfering RNA oligonucleotides nearly completely suppressed the effects of SAHA on cell cycle arrest in COLO-357 and partly attenuated the effects of SAHA in BxPC-3. COLO-357 and BxPC-3 cells, but not PANC-1 cells, were also sensitive to gemcitabine. In the gemcitabine-resistant PANC-1 cells, a 48-h cotreatment with SAHA rendered the cells sensitive to the inhibitory and proapoptotic effects of gemcitabine. An additive effect on growth inhibition by SAHA and gemcitabine was observed in COLO-357 and BxPC-3 cells. Moreover, analysis of p21 distribution in COLO-357 cells revealed that SAHA induced the cytoplasmic localization of both p21 and phospho-p21. These data indicate that SAHA exerts proapoptotic effects in pancreatic cancer cells, in part, by up-regulating p21 and sequestering it in the cytoplasm, raising the possibility that SAHA may have therapeutic potential in the treatment of pancreatic cancer.