GEM-resistant Cell Research Articles

Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/β-catenin axis and activating dCK

BackgroundPreviously, our investigations have underscored the potential of hyperthermia to improve the therapeutic efficacy of gemcitabine (GEM) in pancreatic cancer (PC). Nonetheless, the precise underlying mechanisms remain elusive. MethodsWe engineered two GEM-resistant PC cell lines (BxPC-3/GEM and PANC-1/GEM) and treated them with GEM alongside hyperthermia. The impact of hyperthermia on the therapeutic potency of GEM was ascertained through MTT assay, assessment of the concentration of its active metabolite dFdCTP, and evaluation of deoxycytidine kinase (dCK) activity. Lentivirus-mediated dCK silencing was further employed to validate its involvement in mediating the GEM-sensitizing effect of hyperthermia. The mechanism underlying hyperthermia-mediated dCK activation was explored using bioinformatics analyses. The interplay between hyperthermia and the ephrin A4 (EFNA4)/β-catenin/dCK axis was investigated, and their roles in GEM resistance was further explored via the establishment of xenograft tumor models in nude mice. ResultsHyperthermia restored dCK expression in GEM-resistant cell lines, concurrently enhancing GEM sensitivity and fostering DNA damage and cell death. These observed effects were negated by dCK silencing. Regarding the mechanism, hyperthermia activated dCK by downregulating EFNA4 expression and mitigating β-catenin activation. Overexpression of EFNA4 activated the β-catenin while suppressing dCK, thus diminishing cellular GEM sensitivity—a phenomenon remediated by the β-catenin antagonist MSAB. Consistently, in vivo, hyperthermia augmented the therapeutic efficacy of GEM on xenograft tumors through modulation of the ephrin A4/β-catenin/dCK axis. ConclusionThis study delineates the role of hyperthermia in enhancing GEM sensitivity of PC cells, primarily mediated through the suppression of the EFNA4/β-catenin axis and activation of dCK.

Lovastatin Treatment Inducing Apoptosis in Human Pancreatic Cancer Cells by Inhibiting Cholesterol Rafts in Plasma Membrane and Mitochondria

Resistance to anticancer drugs is a problem in the treatment of pancreatic ductal carcinoma (PDAC) and overcoming it is an important issue. Recently, it has been reported that statins induce apoptosis in cancer cells but the mechanism has not been completely elucidated. We investigated the antitumor mechanisms of statins against PDAC and their impact on resistance to gemcitabine (GEM). Lovastatin (LOVA) increased mitochondrial oxidative stress in PDAC cells, leading to apoptosis. LOVA reduced lipid rafts in the plasma membrane and mitochondria, suppressed the activation of epithelial growth factor receptor (EGFR) and AKT in plasma membrane rafts, and reduced B-cell lymphoma 2 (BCL2)-Bcl-2-associated X protein (BAX) binding and the translocation of F1F0 ATPase in mitochondrial rafts. In the three GEM-resistant cell lines derived from MIA and PANC1, the lipid rafts in the cell membrane and the mitochondria were increased to activate EGFR and AKT and to increase BCL2-BAX binding, which suppressed apoptosis. LOVA abrogated these anti-apoptotic effects by reducing the rafts in the resistant cells. By treating the resistant cells with LOVA, GEM sensitivity improved to the level of the parental cells. Therefore, cholesterol rafts contribute to drug resistance in PDAC. Further clinical research is warranted on overcoming anticancer drug resistance by statin-mediated intracellular cholesterol regulation.

Lauric Acid Overcomes Hypoxia-Induced Gemcitabine Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Although gemcitabine (GEM) is widely used in chemotherapy for pancreatic ductal adenocarcinoma (PDA), drug resistance restricts its clinical effectiveness. To examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced energy production and decreased mitochondrial reactive oxygen species levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. Inhibition of hypoxia-inducible factor-1α in both cell lines did not restore the GEM sensitivity. In contrast, treatment of both cell types with lauric acid (LAA), a medium-chain fatty acid, restored GEM sensitivity. These results suggest that decreased energy production, decreased mitochondrial reactive oxygen species levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of oxidative phosphorylation by LAA could be a tool to overcome GEM resistance. Clinical verification of the effectiveness of LAA in GEM resistance is necessary in the future.

Abstract 1678: Overcoming treatment resistance in advanced bladder cancers using a neo-adjuvant combination with a natural product analog

Abstract Urinary bladder cancer (BC) is a common cancer worldwide, with approximately 573,278 new cases and 212,536 deaths arising annually. Tumors detected beyond the transitional zone of the bladder (advanced BC) account for most deaths. In advanced BC, tumor heterogeneity and resistance to successive chemotherapy regimens are major contributors to morbidity and mortality. Toxicity associated with the combination of Gemcitabine and Cisplatin (G+C) treatment limits the efficacy of chemotherapy. We tested the potential antitumor activity of a novel combination therapy involving a semisynthetic derivative of the pentacyclic triterpene (PTA) Ursolic Acid (N-methyl piperazine conjugated Ursolate, UA4) and Gemcitabine (Gem) in two Gem resistant BC cell lines developed in the laboratory. UA4 was synthesized from the PTA Ursolic Acid and Gem was purchased from LC-Labs. We tested the response to Gem and UA4 on naïve (WT) and Gem Resistant (GemR) cells. We generated Gem resistant BC cell lines HTB-4 (T24) and HTB-9 (5637) by repeated selection in increased Gem doses in vitro. The surviving cells were tested for a stable resistant phenotype. We measured the sensitivity to Gem and UA4 alone, and in adjuvant and neo-adjuvant combinations by colorimetric, cell counting, or clonogenic assays. The antitumor efficacy of UA4 and Gem were tested in vivo by xenograft studies on Fox N1-/- athymic mice (both sexes). The expression of markers associated with drug resistance was determined by qPCR, immunoblotting, and immunofluorescence studies. The 50% growth inhibition (IC50) dose in Gem treated cells increased significantly in 5637 and T24 GemR cells - 10-fold higher in GemR (100nM) than in WT cells (10 nM). Both WT and GemR cells showed equal sensitivity to UA4 (~5 µM). GemR cells showed elevated mRNA expression of a pyrimidine salvaging enzyme, cytidine deaminase (CDA), which degrades intracellular Gem, MRP2, and ENT1, all related to limiting intracellular drug availability. Tetrahydrouridine (THU), a competitive inhibitor of CDA, reduced the resistance significantly, indicating CDA as the principal mediator of resistance in GemR cells. Oral administration of UA4 was highly effective in reducing tumor growth of 5637 WT and GemR xenografts, whereas Gem (25mg/kg, 2x per week, i.p.) did not significantly reduce growth of GemR xenografts. Gem antitumor activity on 5637 WT tumors was predominantly due to apoptosis. Increased expression of autophagy markers, but not markers of apoptosis, was evident in vitro and in vivo treatments with UA4. Overall, treatment with UA4 was equally effective in decreasing viability in both WT and GemR advanced BC cells in vitro and reducing their tumor growth in vivo. Unlike Gem, UA4 did not induce significant levels of apoptosis but mainly autophagy-induced cell death. Ongoing studies indicate potential to use UA4 as an adjuvant or neo-adjuvant therapy in the treatment of advanced BC. Citation Format: Juliette R. Seremak, Sunilkanth Bonigala, Kunj B. Gupta, Siva S. Panda, Bal L. Lokeshwar. Overcoming treatment resistance in advanced bladder cancers using a neo-adjuvant combination with a natural product analog [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1678.

Abstract 3825: Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies due to the acquisition of intrinsic or acquired resistance to chemotherapeutic drugs, such as Gemcitabine (Gem). Recent evidence revealed that calcium signaling significantly contributes to Gem-associated chemoresistance (Gem resistance) in PDAC cells. Andrographis, a labdane diterpenoid isolated from the traditional herb Andrographis paniculate, is known to have a potent calcium channel-blocking potential. Accordingly, we hypothesized that Andrographis might facilitate the reversal of Gem resistance in PDAC. Methods: The critical regulatory pathways associated with the Gem resistance in PDAC were identified by analyzing publicly available cell line-based transcriptomic profiling. Gem resistant (Gem-R) PDAC cells (Gem-R MIA PaCa-2 and BxPC-3) were established by continuously culturing cells with Gem. A series of cell culture experiments were performed using Gem-R PDAC cells to evaluate the Andrographis-mediated reversal of Gem resistance in PDAC cells. At last, the cell culture findings were validated in patient-derived PDAC organoids. Results: Transcriptomic profiling identified the calcium signaling pathway as one of the key regulators of Gem resistance (Fold enrichment: 2.8, P =0.002). The combination of Andrographis and Gem revealed a superior anti-cancer potential in Gem-R PDAC cells on cell viability, clonogenicity, and migration compared to individual treatments with Andrographis or Gem. The combined treatment potentiated cellular apoptosis in Gem-R MIA PaCa-2 (Andro, Gem vs. Combination, 20.3%, 10.9% vs. 31.4%) and Gem-R BxPC-3 (Andro, Gem vs. Combination, 11.3%, 6.8% vs. 16.1%). The fluo-4 assay demonstrated that Andrographis decreased intracellular calcium concentration in Gem-R PDAC cells, while the combined treatment with Andrographis and Gem decreased its concentration more in Gem-R MIA PaCa-2 (fold change [FC] =0.79 vs. Andrographis; FC =0.54 vs. Gem) and Gem-R BxPC-3 (FC =0.80 vs. Andrographis; FC =0.73 vs. Gem). Finally, the combined treatment with Andrographis and Gem showed anti-cancer activity in patient-derived PDAC organoids. Conclusions: We have firstly investigated the Andrographis-mediated reversal of chemoresistance against Gem in PDAC cells, possibly through the regulation of cellular apoptosis and calcium signaling, using a systematic series of Gem resistant cell cultures and patient-derived tumor organoid models. Our findings could provide evidence of the combined treatment with Andrographis and Gem as a complementary therapeutic modality against PDAC. Citation Format: Keisuke Okuno, Caiming Xu, Masanori Tokunaga, Cristina Fillat, Haiyong Han, Yusuke Kinugasa, Ajay Goel. Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3825.

Abstract 4801: Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma

Abstract Hypoxia is a feature of the pancreatic ductal adenocarcinoma (PDA). Although gemcitabine (GEM) is widely used in chemotherapy for PDA, drug resistance restricts its clinical effectiveness. We have found that hypoxia contributes to chemoresistance, however the mechanism is still unclear. The aim of this study is to clarify the mechanism and to develop a new therapeutic strategy. First, we examined the effect of hypoxia to GEM sensitivity, stemness and energy metabolism in three human pancreatic ductal carcinoma cell lines. The inhibition rate by GEM was lower under CoCl2-induced chemical hypoxia than normoxia. Reactive oxygen species (ROS) after GEM was increased under normoxia but not under hypoxia. Flux analysis showed that both oxidative phosphorylation (OXPHOS) and glycolysis were suppressed under hypoxia, indicating a quiescent state. Next, we investigated the relationship between hypoxia and stemness. Hypoxia increased stem cell markers mRNA (C-Myc, Oct-3/4, CD24, CD44, CD133) expression and tumorsphere formation efficiency (TFE). In addition, hypoxia and GEM administration reduced sphere size but increased the number of spheres and TFE. This indicated that hypoxia and GEM suppressed proliferation, however promoted stemness. Second, to examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced OXPHOS and decreased mitochondrial ROS levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. In addition, tetramethylrhodamine-stained mitochondrial membrane potential levels decreased, suggesting mitochondrial impairment. This indicated that long hypoxia and treatment with GEM induced mitochondrial impairment and acquisition of stemness, causing chemoresistance. We further investigated the effect of lauric acid (LAA), a medium-chain fatty acid, on GEM resistance. We have previously reported that LAA causes reprogramming of energy metabolism and induces cell death in cancer cells. Treatment with LAA restored GEM sensitivity in both GEM-resistant cell lines. Flux analysis showed that both OXPHOS and glycolysis were promoted, indicating displacement from quiescent state by LAA. These results suggest that decreased energy production, decreased mitochondrial ROS levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of OXPHOS by LAA could be a tool to overcome GEM resistance. Citation Format: Tadataka Takagi, Hiroki Kuniyasu, Masayuki Sho, Rina Tani, Shiori Mori, Shingo Kishi, Yukiko Nishiguchi, Yudai Hojo. Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4801.

Survivin Splice Variant 2β Enhances Pancreatic Ductal Adenocarcinoma Resistance to Gemcitabine.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis, as it is difficult to predict or circumvent, and it develops chemoresistance quickly. One cellular mechanism associated with chemoresistance is alternative splicing dysfunction, a process through which nascent mRNA is spliced into different isoforms. Survivin (Baculoviral IAP Repeat-Containing Protein 5 (BIRC5)), a member of the inhibitor of apoptosis (IAP) protein family and a cell cycle-associated oncoprotein, is overexpressed in most cancers and undergoes alternative splicing (AS) to generate six different splicing isoforms.MethodsTo determine if survivin splice variants (SSV) could be involved in PDAC chemoresistance, a Gemcitabine (Gem) resistant (GR) cell line, MIA PaCa-2 GR, was created and assessed for its SSV levels and their potential association with GR. Cross-resistance was assessed in MIA-PaCa-2 GR cells to FIRINOX (5-fluorouracil (5-FU), irinotecan, and oxaliplatin). Once chemoresistance was confirmed, RT-qPCR was used to assess the expression of survivin splice variants (SSVs) in PDAC cell lines. To confirm the effect of SSVs on chemoresistance, we used siRNA to knockdown all SSVs or SSV 2β.ResultsThe MIA PaCa-2 GR cell line was 40 times more resistant to Gem and revealed increased resistance to FIRINOX (5-fluorouracil (5-FU), irinotecan, and oxaliplatin); when compared to the parental MIA-PaCa-2 cells. RT-qPCR studies revealed an 8-fold relative expression increase in SSV 2β and a 2- to 8-fold increase in the other five SSVs in the GR cells. Knockdown of all SSV or SSV 2β only, using small inhibitory RNA (siRNA), sensitized the GR cells to Gem, indicating that these SSVs play a role in PDAC chemoresistance.ConclusionThese findings provide evidence for the potential role of SSV 2β and other SSVs in innate and acquired PDAC chemoresistance. We also show that the expression of SSVs is not affected by the type of chemoresistance, therefore targeting survivin splice variants in combination with chemotherapy could benefit a wide range of patients.

Gemcitabine Resistance in Pancreatic Ductal Carcinoma Cell Lines Stems from Reprogramming of Energy Metabolism

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis because it is often detected at an advanced stage, and drug resistance interferes with treatment. However, the mechanism underlying drug resistance in PDAC remains unclear. Here, we investigated metabolic changes between a parental PDAC cell line and a gemcitabine (GEM)-resistant PDAC cell line. We established a GEM-resistant cell line, MIA-G, from MIA-PaCa-2 parental (MIA-P) cells using continuous therapeutic-dose GEM treatment. MIA-G cells were also more resistant to 5-fluorouracil in comparison to MIA-P cells. Metabolic flux analysis showed a higher oxygen consumption rate (OCR) in MIA-G cells than in MIA-P cells. Notably, OCR was suppressed by GEM treatment only in MIA-G cells. GEM treatment increased mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS) in MIA-P cells, but not in MIA-G cells. Glutamine uptake and peroxidase levels were elevated in MIA-G cells. The antioxidants N-acetyl-L-cysteine and vitamin C increased the sensitivity to GEM in both cell lines. In MIA-G cells, the expression of the mitochondrial transcription factor A also decreased. Furthermore, rotenone reduced the sensitivity of MIA-P cells to GEM. These findings suggest that the suppression of oxidative phosphorylation contributes to GEM resistance by reducing ROS production. Our study provides a new approach for reducing GEM resistance in PDAC.

Abstract 3121: Characterization of nab-paclitaxel resistant and gemcitabine resistant BxPC3 and MIA PaCa-2 pancreatic cancer cells

Abstract The gemcitabine (Gem) and nab-paclitaxel (Nab-p) combination is currently the FDA approved standard of care for patients with Stage IV metastatic pancreatic cancer. The response rate of the Gem and Nab-p regimen ranges from 23% to 46%, with many patients eventually becoming refractory. The specific aim of this research project is to determine which genes and pathways differ in Gem resistant (GemR) or Nab-p resistant (Nab-pR) MIA PaCa2 (MP2) and BxPC3 cells when compared with their parental cell lines. Cell proliferation assays were carried out to confirm resistance of the cell lines. The IC50 values of gemcitabine in the Gem resistant BxPC3 (BxPC3-GemR) and MP2 (MP2-GemR) are 26 and 64 fold higher than those in the corresponding parental cell lines, respectively. The IC50 values of nab-paclitaxel in the Nab-p resistant BxPC3 (BxPC3-Nab-pR) and MP2 (MP2-Nab-pR) are 13 and 140 fold higher than those in the corresponding parental cell lines, respectively. RNA sequencing (RNA-seq) was then performed to compare the gene expression profiles of the parental and resistant cell lines. Gene set enrichment analysis (GSEA) revealed that differentially expressed genes between Gem resistant and parental BxPC3 cell lines are often enriched in pathways associated with endoplasmic reticulum (ER) stress response (e.g. ATF-6 and XBP1) whereas in MP2 cells, The AMPK signaling pathway (e.g. PRKAB1, and PRKAG1) seemed to be activated in Gem resistant cells. NFkB signaling pathway (e.g. RIPK2 and NFKB2) was elevated in both Nab-p resistant BxPC3 and MP2 cell lines compared to their corresponding parental cell lines. Genes from these pathways were selected for further validation of their differential expression using RT-PCR. ATF6 which is a stress sensor and transducer in the ER is upregulated in BxPC3-GemR. PRKAB1 and PRKAG1 which regulate AMPK activity are upregulated in MP2-GemR. RIPK2 and NFKB1 are both up in the Nab-p resistant cell lines. Results from this study could provide insights on how to devise new therapeutic regimens to overcome resistance to gemcitabine and nab-paclitaxel. Citation Format: Danielle M. Arias, John Collins, Pawan Noel, Daniel D. Von Hoff, Haiyong Han. Characterization of nab-paclitaxel resistant and gemcitabine resistant BxPC3 and MIA PaCa-2 pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3121.

TNNC1 Reduced Gemcitabine Sensitivity of Nonsmall-Cell Lung Cancer by Increasing Autophagy.

BackgroundAs we know, chemotherapy resistance is a critical factor leading to recurrence and metastasis of nonsmall-cell lung cancer (NSCLC). To clarify the key target and potential mechanism of resistance to gemcitabine (GEM) in NSCLC, we selected Gene Expression Omnibus Data Set and statistically analyzed a parent cell group and a GEM-resistant cell group. Results showed that the expression of troponin C1, slow skeletal and cardiac type (TNNC1) in GEM-resistant cells was higher than in parent cells, which implies that TNNC1 was associated with GEM resistance in lung cancer cells.Material/MethodsTNNC1 expression level was detected by reverse transcription-quantitative polymerase chain reaction or western blot in GEM-resistant patient serum and cell lines. It could reduce or increase autophagy response and GEM resistance accordingly by inhibition of the short interfering ribonucleic acid or by forced overexpression of TNNC1 viruses in A549 cell line and GEM-resistant cell line (A549/GemR) respectively. Blocking autophagy with 3-methyladenine increased the sensitivity of chemotherapy confirmed by flow cytometry and microtubule-associated protein 1A/1B – light chain 3 punctate assay. What’s more, in a loss-of-function model, silencing of forkhead box 03 (FOXO3) in A549/GemR cells could rescue the autophagy weakened by TNNC1.ResultsTNNC1 promoted GEM chemoresistance of NSCLC by activating cytoprotective autophagy, regulated negatively by FOXO3. This research may provide a completely new strategy for NSCLC treatment.ConclusionsTargeting the TNNC1/FOXO3 signaling pathway in NSCLC may be a novel strategy to combat GEM resistance.

Oblongifolin C reverses GEM resistance via suppressing autophagy flux in bladder cancer cells.

A number of previous studies have demonstrated that inhibiting autophagy can increase the cellular cytotoxicity of chemotherapeutic agents in urothelial cancer cells. However, the mechanistic roles of autophagy in gemcitabine (GEM) resistant bladder cancer cells have not been thoroughly investigated. In the present study, immunohistochemistry staining of autophagy marker LC3 was performed in bladder cancer and healthy control tissues and demonstrated an essential role of autophagy in cancer development. A GEM-resistant cell line was established to assess the effects of autophagy on the acquisition of GEM resistance. Western blotting of autophagy markers in GEM-resistant bladder cancer cells suggested that GEM resistance was caused, at least partially, by GEM-induced autophagy. GEM resistance was demonstrated to be reversed by the inhibition of autophagy by 3-methyladenine. In addition, oblongifolin C (OC), a novel autophagic flux inhibitor purified from traditional Chinese medicine, was found to enhance the efficiency of GEM in GEM-resistant bladder cancer cells by inhibiting autophagic flux. In conclusion, data from the present study suggest that autophagy serves an important role in bladder cancer development and GEM resistance. OC treatment has the ability to reverse GEM-resistance in bladder cancer cells by suppressing autophagic flux, thereby providing a potential adjunctive therapeutic option for bladder cancer GEM treatment.

Quercetin induces apoptosis and enhances gemcitabine therapeutic efficacy against gemcitabine-resistant cancer cells.

Quercetin, an abundant flavonoid found in various fruits and vegetables, displays multiple biological activities, including anticancer effects. Therefore, quercetin is receiving increasing attention as a potential adjuvant anticancer treatment. Gemcitabine (GEM) resistance is a major issue for clinicians and patients with advanced cancers, making it crucial to determine ways to bolster its effects. In this study, we explored the anticancer effects and mechanistic actions of quercetin in GEM-resistant cancer cells. Pancreatic cancer (BxPC-3, PANC-1) and hepatocellular carcinoma (HepG2, Huh-7) cell lines were studied. Proliferation assays showed that quercetin had cytotoxic effects on GEM-resistant cell lines (HepG2 and PANC-1), and flow cytometric analysis indicated a significant pro-apoptotic effect on these cell lines. GEM treatment, in combination with quercetin, resulted in increased anticancer effects compared with GEM alone. Quercetin led to S phase arrest in GEM-resistant cell lines, and western blot analysis revealed tumour protein p53 upregulation and cyclin D1 downregulation. This study provides mechanistic insight into the anticancer effects of quercetin and suggests that quercetin adjuvant treatment may benefit patients who are resistant to GEM therapy.

Identification of chemoresistance-related mRNAs based on gemcitabine-resistant pancreatic cancer cell lines.

Gemcitabine (GEM) alone and GEM‐based chemotherapy are the preferred regimens for treating advanced unresectable and metastatic pancreatic cancer (PC). However, these treatments have limited efficacy due to acquired resistance of cancer cells to chemotherapy, the mechanisms of which are not fully understood. In this study, we established two stable multidrug‐resistant cell lines, BxPC‐3‐GR and CFPAC‐1‐GR, from their corresponding parental cells through exposure to GEM following a stepwise incremental dosing strategy. The GEM IC50 values of BxPC‐3‐GR and CFPAC‐1‐GR increased 112‐fold and 210‐fold, respectively, compared to parental cell lines. In vitro and in vivo experiments confirmed that both GEM‐resistant cell subgroups declined in proliferative capacity, but were more resistant to GEM. Unlike CFPAC‐1‐GR, BxPC‐3‐GR exhibited enhanced migratory and invasive properties compared with BxPC‐3 in vitro. We also compared differentially expressed mRNA profiles between parental and GEM‐resistant cells using transcriptome sequencing. RRM1, STIM1, and TRIM21 were significantly upregulated in both GEM‐resistant cell lines and confirmed to be associated with the degree of GEM resistance by quantitative reverse‐transcription polymerase chain reaction and western blot analysis. These three genes were more highly expressed in PC tissues and potentially regarded as prognostic biomarkers through database mining. Thus, our findings provide chemo‐resistant cell models to better understand the underlying mechanisms of chemoresistance, and to explore potential biomarkers for GEM response in PC patients.

Synergistic and Pharmacotherapeutic Effects of Gemcitabine and Cisplatin Combined Administration on Biliary Tract Cancer Cell Lines.

Gemcitabine (GEM) and cisplatin (CDDP) combination therapy (GC) is the standard chemotherapy for advanced biliary tract cancer (BTC); however, its pharmacotherapeutic efficacy remains unclear. To investigate the effects of GC, we selected 11 from 17 BTC cell lines, according to their GEM sensitivity, to be assessed using the MTS assay. The presence of synergistic effects of GC was determined using the Bliss additivism model (BM) and the combination index (CI) at a GEM:CDDP molar ratio of 7:1; this ratio was based on the respective human renal clearances of the two drugs. The pharmacotherapeutic effects were evaluated by comparing the IC50 values for administrations of GEM alone and GC in combination. All cell lines showed synergistic effects when analyzed using the BM. Based on the CI values, strong synergism, synergism, and additive effects were seen in four, five, and two cell lines, respectively. For all four GEM-resistant cell lines, on which GC had strong synergistic effects, the pharmacotherapeutic effects of GC were disappointing, with all IC50 values > 1 µM. For the GEM-effective cell lines, on which GC had synergistic or additive effects, the IC50 values were all <1 µM, and the differences were small between the IC50s for administration of GEM alone and GC in combination. Our results suggest that GC has synergistic effects on BTC cell lines but that its pharmacotherapeutic effects are inadequate.

Abstract 5264: Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer

Abstract Background: Increased aerobic glycolysis (Warburg effect) leading to acidic microenvironment confers chemoresistance to weakly basic Gemcitabine, the standard treatment in Pancreatic ductal adenocarcinoma (PDAC). Metabolomic perturbations in two PDAC cell lines having differential sensitivity to Gemcitabine was investigated by profiling over 40 metabolites using 1H-NMR spectroscopy. Notably, a significant decrease in the concentration of β-alanine (βA) was observed in Gem resistant compared to Gem sensitive cell line, suggesting βA may have a role in chemotherapy response. We aimed to evaluate the role of βA supplementation in improving Gem efficacy in Gem resistant cell lines Methods: PDAC Panc-1 cell line extracts treated with Gem with and without βA supplementation were analysed via 1H-NMR spectroscopy. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using the Seahorse XF analyser platform. Global mRNA profiling was done by Next Generation Sequencing. Proliferation, migration and cell cycle analysis was measured by Xcelligence real time cell analysis system and flow cytometry, respectively. PANC-1 and BxPC-3 tumor xenografts treated with Gemcitabine with and without βA for 4 weeks and the tumor volumes and weights were measured. Results: βA supplementation in Gem resistant cells results in: (1) decreased basal glycolytic rate, proton leak and ATP production; (2) increased pyruvate, decreased lactic acid and NAD+ concentrations with decreased LDH-A transcription; also increased carnosine levels with a more basic pH in the conditioned media; (3) enhanced sensitivity to Gem treatment, with a significant reduction in proliferation, cell migration, and increased apoptosis and relative hENt-1 mRNA levels and (5) xenograft tumor volumes and weights were significantly reduced upon βA supplementation. Conclusion: βA supplementation reprograms cell metabolism by normalizing energy production and reducing microenvironment acidification, thus enhancing chemo-sensitivity in Gem resistant PDAC cells and xenografts. βA can be potentially used as a co-therapeutic in PDAC. Note: This abstract was not presented at the meeting. Citation Format: Danny Yakoub, Smitha T. Totiger, Alexandra Moran, Sujit Suwal, Julio Pimentel, Lauren O'Donell, Jamie Walls, Nagaraj Nagathihalli, Nipun Merchant. Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5264.

A novel low molecular weight nanocomposite hydrogel formulation for intra-tumoural delivery of anti-cancer drugs

Herein, an injectable formulation composed of a low molecular weight gelator (LMWG) based hydrogel and drug-loaded polymeric nanocapsules (NCs) is described. The NCs, made of hyaluronic acid and polyglutamic acid and loaded with C14-Gemcitabine (GEM C14), showed a size of 40 and 80 nm and a encapsulation efficiency >90%. These NCs exhibited a capacity to control the release of the encapsulated drug for >1 month. GEM C14-loaded NCs showed activity against various cancer cell lines in vitro; cell growth inhibition by 50% (GI50) values of 15 ± 6, 10 ± 9, 13 ± 3 and 410 ± 463 nM were obtained in HCT 116, MIA PaCa-2, Panc-1 and Panc-1 GEM resistant cell lines respectively. Nanocomposite hydrogels were prepared using the LMWG - N4-octanoyl-2'-deoxycytidine and loaded for the first time with polymeric NCs. 2% and 4% w/v nanocapsule concentrations as compared to 8% w/v NC concentrations with 2% and 3% w/v gelator concentrations gave mechanically stronger gels as determined by oscillatory rheology. Most importantly, the nanocomposite formulation reformed instantly into a gel after injection through a needle. Based on these properties, the nanocomposite gel formulation has potential for the intratumoural delivery of anticancer drugs.

Abstract B08: ER chaperone GRP78 increases chemoresistance in pancreatic ductal adenocarcinoma

Abstract Purpose: We show that expression of GRP78, the master regulator of the endoplasmic reticulum (ER) stress response, leads to chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC) and utilize a small molecule inhibitor to block the activity of GRP78, increasing the efficacy of currently available therapies in vitro and in vivo. Background: Gemcitabine (GEM) is the standard chemotherapeutic agent for adjuvant therapy of PDAC and a commonly used agent in other treatment settings, including neoadjuvant treatment of borderline resectable PDAC and palliative treatment of metastatic PDAC. However, recurrent disease is observed in an alarming number of patients treated with GEM or GEM-based treatment regimens. A molecular-level understanding of the mechanisms that contribute to chemoresistance is essential for the development of more effective treatment strategies. ER chaperone proteins protect cells from apoptosis in response to cellular insults, including inflammation and pancreatitis. Elevated expression of chaperone proteins also causes the activation of many “pro-survival” pathways that promote cell survival and resistance to routinely utilized therapeutic regimens. We hypothesize that expression of ER chaperone protein GRP78, is responsible for innate and acquired resistant to GEM and that therapies targeting GRP78 by blocking its activity with the small molecule inhibitor, IT-139, could potentially improve patient response to GEM-based treatment regimens. Methods: We examined if GRP78 overexpression correlated with chemoresistance in PDAC through establishing a patient-derived orthotopic xenograft (PDX) model. Treating tumor-bearing mice with GEM continuously or releasing mice from GEM treatment after 3-4 weeks of exposure identified PDX lines that were either sensitive or resistant to GEM. Tumors from these cohorts were analyzed for GRP78 expression. To examine the role of GRP78 in chemoresistance in vitro, GRP78 expression was induced in GEM-sensitive cell lines prior to GEM treatment. Further, to investigate the effect of GRP78 loss on chemoresistance, siRNA knockdown of GRP78, followed by GEM treatment, was used in GEM-resistant cell lines. Finally, the efficacy of combination treatment with GEM and IT-139, a ruthenium-based anticancer drug and GRP78 inhibitor, was determined in vitro and in vivo. Results: Here, we report that PDX lines that result in GEM resistant PDAC have higher GRP78 expression than that of GEM sensitive PDX lines. In vitro, increased GRP78 induces chemoresistance in GEM-sensitive PDAC cell lines while knockdown of GRP78 reduces chemoresistance in GEM-resistant PDAC cell lines. Finally, we found that IT-139 can overcome GRP78 mediated chemoresistance. In vitro, IT-139 restores sensitivity to cytotoxic drugs in drug resistant PDAC cells and induces twice as much cell death in combination treatment compared with GEM alone. In vivo, a single weekly IT-139 treatment in combination with GEM caused a 35% increase in median survival and a 25% increase in overall survival compared to GEM alone. Conclusions: Collectively, our data show that GRP78 expression promotes chemoresistance in PDAC and therapeutic strategies blocking the activity of GRP78 increase the efficacy of currently available therapies. Citation Format: Jenifer B. Gifford, Wei Huang, Ann E. Zeleniak, Antreas Hindoyan, Hong Wu, Timothy R. Donahue, Reginald Hill.{Authors}. ER chaperone GRP78 increases chemoresistance in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B08.

Abstract 5473: Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene

Abstract Gemcitabine (GEM) has shown clinical activity in several solid tumors such as pancreatic and biliary tract cancers, urinary cancer, and non-small cell lung cancers, but a substantial number of patients acquire GEM chemoresistance. GEM functions after phosphorylation by deoxycytidine kinase (DCK) and its inactivation is one of the important mechanisms for acquisition of gemcitabine resistance. We established eight GEM resistant cell lines including four pancreatic, two biliary tract, and two gastrointestinal cancers and investigated mechanisms for acquisition of GEM resistances; six GEM resistant cell lines harbored complete inactivating mutations of DCK by protein-truncating mutation or complete or partial loss of the gene, and one gastric cancer cell line harbored a missense mutation that inactivate DCK activity. One pancreatic cancer cell line lost GEM sensitivity by unknown mechanism. Our present results strongly suggest that genetic alteration of DCK can be the major cause of acquisition of GEM resistance. Further studies analyzing primary tumors will give us (1) the fully understanding of acquisition of GEM resistance, and (2) desirable clues to overcome the acquisition of GEM resistance. Citation Format: Akira Horii, Yuriko Saiki, Tomohiro Nakano, Chiharu Kudo, Makoto Sunamura. Human cancer cells acquire chemoresistance to gemcitabine mainly through loss-of-function mutations in the DCK gene. [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 5473. doi:10.1158/1538-7445.AM2015-5473

MicroRNA-218 promotes gemcitabine sensitivity in human pancreatic cancer cells by regulating HMGB1 expression.

The purpose of this study was to examine the effect of gemcitabine (GEM) on microRNA-218 (miR-218) expression in human pancreatic cancer cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to examine the differences in miR-218 expression between the GEM-sensitive BxPC-3 pancreatic cancer cells and GEM-resistant PANC-1 cells. The effect of GEM on the expression of miR-218 in PANC-1 cells was also investigated. PANC-1 cells were transfected either with HMGB1 siRNA to knock down the expression of HMGB1 or with the recombinant HMGB1 expression vector (pcDNA3.1-HMGB1) to overexpress HMGB1. The effect of ectopic expression of HMGB1 on the apoptosis of miR-218-transfected and GEM-treated PANC-1 cells was examined by flow cytometric analysis. The miR-218 expression level was lower in GEM-resistant PANC-1 cells compared to GEM-sensitive BxPC-3 cells (P<0.05). The percentage of apoptotic PANC-1 cells was significantly increased in the miR-218 mimic + GEM group compared to the mimic ctrl + GEM group and the normal control group (P<0.01). The HMGB1 expression level was markedly decreased in PANC-1 cells transfected with HMGB1 siRNA but was significantly increased in PANC-1 cells transfected with the recombinant HMGB1 expression vector, pcDNA3.1-HMGB1 (P<0.01). The proportion of apoptotic PANC-1 cells was significantly lower in the miR-218 mimic + GEM + pcDNA3.1-HMGB1 group compared to the miR-218 mimic + GEM + HMGB1 siRNA group (P<0.01). The expression level of miR-218 was downregulated in the GEM-resistant cell line. miR-218 promoted the sensitivity of PANC-1 cells to GEM, which was achieved mainly through regulating the expression of HMGB1 in PANC-1 cells.

Proteomic profiling of chemoresistance in pancreatic adenocarcinoma

Gemcitabine‐based chemotherapy is offered to patients with pancreatic adenocarcinoma (PC) per current clinical guidelines, with minimal benefit to disease free and overall survival due to rapid development of chemoresistance. Our objective in this study was to identify proteins involved in chemoresistance using proteomic profiling of GEM resistant (Panc‐1, MiaPaCa‐2) and GEM sensitive (BXPC‐3, Su8686) PC cell lines. Total protein lysates were prepared from the cell lines, separated by SDS‐PAGE and digested using standard protocols. Tryptic peptides were analyzed in duplicate on the mass spectrometer. Spectra were searched against the IPI protein database using the Sequest algorithm, then analyzed using Scaffold software and Power Law Global Error Model. We identified 19 proteins with statistically (p&lt;0.005) increased expression in drug resistance, and 57 proteins decreased in drug resistant cells. Various proteins known to be involved in epithelial to mesenchymal transition, which is associated with increased chemoresistance and progression of PC, are differentially expressed in the cell lines. We validated our findings using western blot. We conclude that proteomics can be used to identify chemoresistance related proteins, a crucial step in combating drug resistance in PC treatment.