Gemcitabine-resistant Cell Lines Research Articles

Stepwise molecular mechanisms responsible for chemoresistance in bladder cancer cells

Chemotherapy resistance is an obstacle to cancer therapy and is considered a major cause of recurrence. Thus, understanding the mechanisms of chemoresistance is critical to improving the prognosis of patients. Here, we have established a stepwise gemcitabine-resistant T24 bladder cancer cell line to understand the molecular mechanisms of chemoresistance within cancer cells. The characteristics of the stepwise chemoresistance cell line were divided into 4 phases (parental, early, intermediate, and late phases). These four phase cells showed increasingly aggressive phenotypes in vitro and in vivo experiments with increasing phases and revealed the molecular properties of the biological process from parent cells to phased gemcitabine-resistant cell line (GRC). Taken together, through the analysis of gene expression profile data, we have characterized gene set of each phase indicating the response to anticancer drug treatment. Specifically, we identified a multigene signature (23 genes including GATA3, APOBEC3G, NT5E, MYC, STC1, FOXD1, SMAD9) and developed a chemoresistance score consisting of that could predict eventual responsiveness to gemcitabine treatment. Our data will contribute to predicting chemoresistance and improving the prognosis of bladder cancer patients.

NXPH4 Promotes Gemcitabine Resistance in Bladder Cancer by Enhancing Reactive Oxygen Species and Glycolysis Activation through Modulating NDUFA4L2.

Simple SummaryBladder cancer is one of the most common malignant tumors of the urinary system, and its treatment is mainly surgical resection, supplemented by chemotherapy after surgery. Postoperative chemotherapy can significantly reduce the tumor recurrence rate and improve the prognosis of bladder cancer patients. However, chemotherapy resistance is one of the major challenges in the treatment of bladder cancer. Therefore, we conducted a series bioinformatic analyses and functional experiments to reveal the novel role of NXPH4 in bladder cancer. We found NXPH4 not only influenced the proliferation, migration, invasion ability of cancer cells, but also affected the level of glycolysis and reactive oxygen species and further promoted the gemcitabine resistance of bladder cancer. Our research has found a novel molecule that may play an important part in the neoplasia, which may provide another angle in the treatment of bladder cancer.Bladder cancer is one of the most prevalent kinds of cancer worldwide, and resistance to gemcitabine is a major problem for patients. The pathogenesis of bladder cancer and mechanism of resistance to chemotherapy remain to be explored. Through bioinformatics analysis, we first found that NXPH4 was independently related to the prognosis of patients with bladder cancer. Through wound healing assays, transwell invasion assays, and plate clone formation assays, we found that NXPH4 promoted the proliferation, migration, and invasion of bladder cancer cells. The induced gemcitabine resistance cell line also showed a higher expression of NXPH4. A glycolytic activity assay demonstrated that the expression of NXPH4 was positively related to glycolysis. A higher level of reactive oxygen species caused by enhanced levels of NXPH4 was found in gemcitabine-resistant cell lines. NDUFA4L2, glycolysis, and reactive oxygen species were shown to be essential for NXPH4-regulated functions through rescue assays in cell lines. The roles of NXPH4-regulated glycolysis, gemcitabine resistance, and NDUFA4L2 were validated in vivo as well. Our results imply that NXPH4 contributes to the proliferation, migration, and invasion of bladder cancer by maintaining the stability of NDUFA4L2 and consequently activating reactive oxygen species and glycolysis.

Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

BackgroundResistance to standard therapy is a major reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Developing novel therapy to overcome PDAC drug-resistance is urgently needed. CRABP-II was highly expressed in all PDAC but not expressed in normal pancreatic tissues and chronic pancreatitis. CRABP-II was shown to promote PDAC migration and metastasis while its potential role in promoting PDAC drug-resistance was not known.MethodsA paired cohort of human primary and relapsing PDAC tissues was assessed for CRABP-II expression by immunohistochemistry. CRISPR/cas9 gene editing was used to establish CRABP-II knockout cell lines and MTT assays were performed to assess gemcitabine sensitivity in vitro. Cleaved caspase-3/PARP blots and Annexin V staining were conducted to detect cell apoptosis. Gene expression microarray, Q-PCR, western blots, Co-IP and RNA-IP were used to study the molecular function of CRABP-II. Sucrose gradient ultracentrifugation was applied to isolate lipid rafts and LC–MS-MS was used to assess cholesterol content. Both subcutaneous CDX models and orthotopic PDX models were established to examine the efficacy of SNIPER-11 and the synergistic effect between SNIPER-11 and gemcitabine in vivo.ResultsA higher expression of CRABP-II was found in relapsing PDAC tissue and was associated with poor prognosis. Gemcitabine-resistant cell lines exhibited increased level of CRABP-II, while CRABP-II knockout resensitized PDAC cells to gemcitabine. Mechanistically, aberrant expression of CRABP-II increased the stability of SREBP-1c mRNA through cooperation with HuR and upregulated the downstream genes of SREBP-1c to favor cholesterol uptake and accumulation in lipid rafts. Increased lipid raft cholesterol accumulation facilitated ATK survival signaling and PDAC drug resistance. The small compound SNIPER-11 treatment effectively induced CRABP-II protein degradation, induced apoptosis, and suppressed tumor growth. Combination of SNIPER-11 and gemcitabine significantly reduced the lipid raft cholesterol content in CDX/PDX and profoundly inhibited tumor progression.ConclusionsThese findings identified CRABP-II as a novel regulator of cholesterol metabolism and suggested that CRABP-II is a selective target for overcoming PDAC drug resistance.

ACAT-1-Regulated Cholesteryl Ester Accumulation Modulates Gemcitabine Resistance in Biliary Tract Cancer.

Biliary tract cancer (BTC) has few choices of chemotherapy, including gemcitabine, therefore exploring the mechanisms of gemcitabine resistance is important. We focused on lipid metabolism because biliary tract epithelial cells are essential in cholesterol and bile acid metabolism and the messenger RNA (mRNA) microarray analysis showed high acyl coenzyme A: cholesterol acyltransferase 1 (ACAT-1) expression in BTC gemcitabine-resistant (GR) cell lines. We hypothesized that aberrant accumulation of cholesteryl ester (CE) regulated by ACAT-1 could modulate GR in BTC. CE accumulations were measured in human BTC cell lines, and the relationships between CE levels, ACAT-1 expressions, and gemcitabine sensitivity were analyzed. We performed a small-interfering RNA (siRNA)-mediated knockdown and biochemical inhibition of ACAT-1 in BTC cell lines and alterations of gemcitabine sensitivity were evaluated. To evaluate the clinical significance of ACAT-1 in regard to GR, immunohistochemistry was performed and ACAT-1 expressions were analyzed in resected BTC specimens. CE levels were correlated with ACAT-1 expressions and GR in four human BTC cell lines. siRNA-mediated knockdown of ACAT-1 in two independent GR cell clones as well as ACAT-1 inhibitor treatment significantly increased gemcitabine sensitivity; knockdown of ACAT-1: 5.63- and 8.02-fold; ACAT-1 inhibitor: 8.75- and 9.13-fold, respectively. ACAT-1 expression in resected BTC specimens revealed that the disease-free survival of the ACAT-1 low-intensity group (median 2.3 years) had a significantly better outcome than that of the ACAT-1 high-intensity group (median 1.1years) under gemcitabine treatment after surgery (*p<0.05). Our findings suggest that CE and ACAT-1 might be a novel therapeutic target for GR in BTC.

Abstract 3196: Soluble factors released by pancreatic cancer cells enhance neutrophil survival

Abstract Pancreatic cancer (PC) remains a challenge to modern-day cancer therapeutics, with a dismal five-year survival rate of 10%. Due to the pancreas' tricky location and the dense stroma surrounding it, patients have late diagnosis and fail to respond to chemotherapy regimens. The genetic stability of the tumor microenvironment (TME) in comparison with rapidly evolving tumor cells makes it an attractive target for the development of cancer therapeutics. Neutrophils, the most abundant immune cell, is an essential player in the TME. These neutrophils can become pro-tumorigenic based on signals they receive in the TME by regulating immune suppression, tumor metastasis, and angiogenesis. Previous studies from our laboratory demonstrate an increase in the number of neutrophils in the pancreatic tumor with the PC disease's progression in the KPC Model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre). Based on our previous observation and accumulating literature, we hypothesize that pancreatic tumor cells can increase neutrophil proliferation and/or survival in circulation and tumor in situ. We investigated the effect of soluble factors released by pancreatic tumor cells on neutrophil proliferation/survival with this rationale. For this objective, we utilized murine promyelocytes cells (MPRO), a mouse neutrophil cell line, and a human leukemia cell line (HL60), a human neutrophil cell line. We treated MPRO and HL60 cells with a gradient of supernatants collected from T3M-4- and CD18/HPAF-parent and gemcitabine-resistant cells. By utilizing the WST assay, we observed an increase in MRPO and HL60 cells' viability with an increasing gradient of supernatant collected from both T3M-4, CD18/HPAF-parent gemcitabine-resistant cells with serum-free media as a control. We also observed a slight increase in neutrophil viability when treated with the supernatant collected from parent cell-lines supernatant compared to supernatant collected from the gemcitabine resistant cell-lines. Next, to delineate the effect of direct interaction between pancreatic tumor cells and neutrophils, we utilized a co-culture assay between MPRO cells and CD18/HPAF cells. We observed an increase in the viability of CD18/HPAF cells when placed in contact with the neutrophils; however, we observed inhibition in the viability of neutrophils placed in contact with the tumor cells. Our results showing positive viability of neutrophils with the treatment of soluble tumor factors explain higher neutrophil to lymphocyte ratio in PC patients' blood circulation. Neutrophils in the TME release pro-tumorigenic cytokines that increase tumor cell proliferation, albeit resulting in neutrophil death as observed by neutrophil growth inhibition. Along with neutrophil survival, a more mechanistic understanding of neutrophil recruitment and polarization in TME will facilitate designing novel therapeutics for PC. Citation Format: Paran Goel, Sugandha Saxena, Caitlin Molczyk, Rakesh Singh. Soluble factors released by pancreatic cancer cells enhance neutrophil survival [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 3196.

Abstract 1087: CXCR2 and its ligands modulate chemotherapy resistance in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest survival rates of cancers, with a five-year relative survival of 9%. The significant factor contributing to low survivorship is the intrinsic resistance to chemotherapy. In the present study, we elucidate a targetable pathway that facilitates resistance in these cells. One inflammatory pathway used in cancer progression is CXCR2, which binds chemokines CXCL 1-3 and 5-8. Our group and others have shown an association of CXCR2 with tumor progression, angiogenesis and metastasis in PDAC, and chemotherapy resistance in other cancers. In this study, we test the hypothesis that CXCR2 is integral in PDAC chemotherapy resistance. We generated gemcitabine resistant cell lines (GemR) from the parent cell lines of T3M4 (WT KRAS) and CD18/HPAF (Mutant KRAS) with 40- and 67-fold higher resistance in comparison to the parent cell lines, respectively. Using qRT-PCR and ELISA, we profiled chemokines' expression, CXCL1, 5, and 8, on parent- and GemR-cell lines. We observed a higher basal level expression of CXCL1, 5, and 8 ligands in GemR cells than the parent cell lines. Further treatment with gemcitabine, the parent cell lines had a higher increase in CXCL1 and 8 ligand expression in comparison with GemR cell lines. With further investigation of T3M4, we found a dose-dependent and time-dependent increase for CXCL1 and CXCL8. CXCL5 had a constant high expression with all doses of gemcitabine. With our initial observations of gemcitabine treatment enhancing CXCR2 ligands in PDAC cells, we evaluated the effects of the small molecule CXCR2 antagonist SCH 479833 (SCH 47) in combination with gemcitabine treatment on the parent and GemR cell lines. We used the relative IC50 and half the relative IC50 for both the gemcitabine and SCH 47 treatment doses. We observed that a lower concentration of gemcitabine in the presence of the SCH 47 was more effective than the higher concentration of gemcitabine without SCH 47 for the GemR-cell lines. Together, our observations show chemotherapy-resistant PDAC cell lines express higher CXCR2 ligand levels at baseline, with parent cell lines increasing expression with chemotherapy treatment. We also show an advantageous combination treatment of CXCR2 antagonist with lower gemcitabine concentration. Overall, these results show a promising future for CXCR2 antagonism in the treatment of resistant PDAC tumors. Citation Format: Caitlin Molczyk, Dipakkumar R. Prajapati, Sugandha Saxena, Paran Goel, Michelle Varney, Rakesh K. Singh. CXCR2 and its ligands modulate chemotherapy resistance in pancreatic ductal adenocarcinoma [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 1087.

Zebularine suppressed gemcitabine-induced senescence and improved the cellular and plasma pharmacokinetics of gemcitabine, augmented by liposomal co-delivery

Chemoresistance is a major factor driving cancer recurrence. This study investigated the potential of zebularine, a dual cytidine deaminase (CDA)/epigenetic inhibitor, to circumvent gemcitabine-resistance in pancreatic cancer using a nanomedicine co-delivery approach. The mRNA expression of key metabolic enzymes, including CDA for gemcitabine deactivation in a gemcitabine-resistant cell line Gr2000 and its parental MIA PaCa-2 was compared using quantitative reverse transcription polymerase chain reaction. A highly gemcitabine-resistant population (HRP) in Gr2000 were characterised for their growth pattern, β-galactosidase activity (a hallmark of senescence) and chemosensitivity to zebularine after isolation. The CDA inhibition effects of zebularine on the intracellular gemcitabine accumulation and pharmacokinetics in rats when co-delivered with pH-sensitive liposomes (pSL) were investigated. Gr2000 had a 3-time upregulated mRNA expression and enzyme activity for CDA. The HRP (28% of bulk Gr2000) were predominately senescent cells which re-proliferated following a growth arrest for a week. Zebularine suppressed the regrowth of senescent cells, meanwhile enhanced cellular gemcitabine concentration by 2-fold. When co-delivered with pSL, zebularine increased cellular gemcitabine concentration by 4-fold, and extended the half-life of gemcitabine in plasma by 22-fold in rats. In conclusion, multiple mechanisms including therapy-induced senescence were identified with gemcitabine-resistance. Co-delivery of zebularine using liposomes could provide multifaceted benefits in gemcitabine therapy for pancreatic cancer treatment.

Effects of Low Dose Fractional Radiation on Chemosensitivity of Gemcitabine-Resistant Human Pancreatic Cancer SW1900/GZ Cell

Objective: To investigate whether low-dose fractionated radiation (LDFRT) could enhance gemcitabine sensitivity in drug-resistant human pancreatic cancer SW1900/GZ cell, and to further explore the underlying mechanism. Methods: Gemcitabine-resistant human pancreatic cancer SW1900 cell line (SW1900/GZ) was induced by high concentration gemcitabine intermittent shock in vitro. The cell counting kit 8 (CCK8) was used to determine SW1900/GZ cell lines. SW1900/GZ cells were divided into six groups as follows: control, LDFRT, high dose radiation (HDRT), gemcitabine (GEM), low dose fractional radiation plus gemcitabine (LDFRT+ GEM) and high dose radiation plus gemcitabine (HDRT+ GEM) groups. The rate of apoptosis was determined by flow cytometry (FCM). Protein levels of multidrug resistance gene (MDR) and multidrug resistance-related protein gene (MRP) were examined by Western blotting. Results: The results of CCK8 test showed that the half-maximal inhibitory concentration (IC50) of non-drug-resistant cell line SW1900 and drug-resistant cell line SW1990/GZ were 230.4ng/ml and 856.6ug/ml respectively. The IC50 of SW1990/GZ was 3700 times more than the former. LDFRT significantly promoted apoptosis in SW1900 cells. Moreover, in the LDFRT group, protein levels of MDR and MRP were markedly decreased. Conclusion: This study established an effective gemcitabine-resistant cell line SW1900 of human pancreatic cancer (SW1900/GZ cell line). LDFRT sensitizes resistant SW1900/GZ pancreatic cancer cell to gemcitabine through down-regulation the expression of MDR and MPR proteins.

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

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

MicroRNA-136-5p regulates gemcitabine resistance in pancreatic cancer via down-regulating ZNF32.

We explored the regulation of microRNA-136-5p on gemcitabine resistance of pancreatic cancer (PCa) and further highlighted the crosstalk between microRNA-136-5p and ZNF32, so as to provide an effective theoretical basis for target treatment of PCa. MicroRNA-136-5p and ZNF32 levels in tumor specimens of 48 patients with PCa were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) method, and the interplay between microRNA-136-5p and clinicopathological parameters, as well as prognosis of those patients was also analyzed. Meanwhile, in gemcitabine-resistant PCa cell lines PANC-1 and CFPAC-1, microRNA-136-5p overexpression model was constructed, and cell counting kit-8 (CCK-8), transwell, as well as cell wound healing assays were carried out to assess the impact of microRNA-136-5p on PCa cell functions. Finally, Luciferase assay and recovery experiments were conducted to specify the precise underlying mechanism. qRT-PCR results revealed a significant low expression of microRNA-136-5p both in tumor tissues of PCa patients and in PCa cell lines compared to the normal control groups. In addition, microRNA-136-5p mimics markedly attenuated the proliferation and migration abilities of the gemcitabine-resistant PCa cell lines. The Luciferase assay verified certain binding sites between microRNA-136-5p and ZNF32, while qPCR results indicated a negative correlation between the two in PCa tissues. Moreover, recovery experiments demonstrated that ZNF32 overexpression reversed the inhibitory effect on the malignant progression of gemcitabine-resistant PCa cells induced by microRNA-136-5p. MicroRNA-136-5p can reduce the proliferation rate and metastasis ability of PCa cells via regulating ZNF32, and thus alleviates gemcitabine resistance in PCa cells.

In silico identification of therapeutic compounds against microRNA targets in drug-resistant pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a major health issue that has been eluding efforts to identify viable therapeutic treatment options. Besides having the lowest survival rate among all types of cancer, almost all conventional methods of treatment are futile against this condition, leaving patients to succumb to this ailment faster than ever. As it is increasingly becoming difficult to come up with new compounds for the treatment of various diseases, alternative solutions are required for tackling these problems. In this study, publically available miRNA and gene expression data were used to identify common elements that were present in gemcitabine-resistant PDAC cell lines. By selecting overexpressed genes involved in pancreatic cancer and cancer pathways in general, potential drug candidates for the treatment of PDAC were identified. In this study, 21 differentially expressed miRNAs were identified from PANC-1 cell line treated with gemcitabine. Pathway analysis revealed that MET and PPARG were overexpressed in cancer-related pathways, including pancreatic cancer, and could be targeted for PDAC treatment. Using CMap, fisetin was identified a likely candidate drug for the treatment of PDAC. Docking studies indicated that fisetin was bound to c-Met and PPARG with an XP G score of –12.819 and –7.021 kcal/mol, respectively. As miRNAs have increasingly been shown to part take in important cancer-related processes and pathways, researching drug development methods based on miRNA targets could be beneficial for pharmaceutical industries. Communicated by Ramaswamy H. Sarma

PCL20-117: Can Green Tea Extract Inhibit the Tumor Growth and Invasiveness of 5-FU and Gemcitabine-Resistant Metastatic and Remetastatic Cell Lines in a Hamster Pancreatic Cancer Model?

PCL20-117: Can Green Tea Extract Inhibit the Tumor Growth and Invasiveness of 5-FU and Gemcitabine-Resistant Metastatic and Remetastatic Cell Lines in a Hamster Pancreatic Cancer Model?

Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition.

SummaryIn situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and β-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

Activation of GLP-1 receptor enhances the chemosensitivity of pancreatic cancer cells.

This study aimed to determine whether and how the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide affects the chemoresistance and chemosensitivity of pancreatic cancer cells to gemcitabine in vitro and in vivo. The GLP-1R and protein kinase A (PKA) levels were compared between the human pancreatic cancer cell line PANC-1 and the gemcitabine-resistant cell line PANC-GR. The in vitro effects of liraglutide on the cell proliferation and apoptosis as well as the nuclear factor-kappa B NF-κB expression levels of PANC-GR cells were evaluated. In addition, a mouse xenograft model of human pancreatic cancer was established by s.c. injection of PANC-1 cells, and the effects of liraglutide on the chemosensitivity were evaluated in vitro and in vivo. In contrast to PANC-1 cells, PANC-GR cells exhibited lower expression levels of GLP-1R and PKA. Incubation with liraglutide dose dependently inhibited the growth, promoted the apoptosis, and increased the expression of GLP-1R and PKA of PANC-GR cells. Similar effects of liraglutide were observed in another human pancreatic cancer cell line MiaPaCa-2/MiaPaCa-2-GR. Either the GLP-1R antagonist Ex-9, the PKA inhibitor H89, or the NF-κB activator lipopolysaccharide (LPS) could abolish the antiproliferative and proapoptotic activities of liraglutide. Additionally, each of these agents could reverse the expression of NF-κB and ABCG2, which was decreased by liraglutide treatment. Furthermore, liraglutide treatment increased the chemosensitivity of pancreatic cancer cells to gemcitabine, as evidenced by in vitro and in vivo experiments. Thus, GLP-1R agonists are safe and beneficial for patients complicated with pancreatic cancer and diabetes, especially for gemcitabine-resistant pancreatic cancer.

LncRNA SLC7A11-AS1 Promotes Chemoresistance by Blocking SCFβ-TRCP-Mediated Degradation of NRF2 in Pancreatic Cancer

Drug resistance is the major obstacle of gemcitabine-based chemotherapy for the treatment of pancreatic ductal adenocarcinoma (PDAC). Many long non-coding RNAs (lncRNAs) are reported to play vital roles in cancer initiation and progression. Here, we report that lncRNA SLC7A11-AS1 is involved in gemcitabine resistance of PDAC. SLC7A11-AS1 is overexpressed in PDAC tissues and gemcitabine-resistant cell lines. Knockdown of SLC7A11-AS1 weakens the PDAC stemness and potentiates the sensitivity of resistant PDAC cells toward gemcitabine in vitro and in vivo. SLC7A11-AS1 promotes chemoresistance through reducing intracellular reactive oxygen species (ROS) by stabilizing nuclear factor erythroid-2-related factor 2 (NRF2), the key regulator in antioxidant defense. Mechanically, SLC7A11-AS1 is co-localized with β-TRCP1 in the nucleus. The exon 3 of SLC7A11-AS1 interacts with the F-box motif of β-TRCP1, the critical domain that recruits β-TRCP1 to the SCFβ-TRCP E3 complex. This interaction prevents the consequent ubiquitination and proteasomal degradation of NRF2 in the nucleus. Our results demonstrate that the overexpression of SLC7A11-AS1 in gemcitabine-resistant PDAC cells can scavenge ROS by blocking SCFβ-TRCP-mediated ubiquitination and degradation of NRF2, leading to a low level of intracellular ROS, which is required for the maintenance of cancer stemness. These findings suggest SLC7A11-AS1 as a therapeutic target to overcome gemcitabine resistance for PDAC treatment.

Long non-coding RNA SSTR5-AS1 facilitates gemcitabine resistance via stabilizing NONO in gallbladder carcinoma

Gallbladder carcinoma (GBC) is the most aggressive carcinoma of the biliary tract, effective chemotherapy was critical for the patients with unresectable GBC. However, chemotherapy resistance is still problematic for clinicians. Here, we identified a specific long non-coding RNA, SSTR5-AS1, in GBC patient that facilitates gemcitabine resistance. SSTR5-AS1 is significantly increased in GBC samples and cell lines, especially in gemcitabine-resistant cell lines, and higher SSTR5-AS1 expression was correlated with poorer overall survival rate in GBC patients. Our data revealed that upregulated SSTR5-AS1 facilitates gemcitabine resistance via inhibiting apoptosis. Knockdown of SSTR5-AS1 sensitized drug resistant GBC cells to gemcitabine in vitro and strongly inhibited xenografts formed by drug resistant GBC cells in vivo. Moreover, we found via streptavidin pull down assay that NONO specifically binds to sense sequence of SSTR5-AS1 and prevented proteasome mediated NONO degradation, which resulted in increased NONO protein level without affecting the transcription of NONO. NONO functions as the downstream effector of SSTR5-AS1 and is required for SSTR5-AS1 mediated gemcitabine resistance. Collectively, our data provided novel insights into lncRNA-mediated chemotherapy resistance and suggested a novel therapeutic target to improve chemotherapy strategies for unresectable GBC patients.

Anti-parasitic Drug Ivermectin Exhibits Potent Anticancer Activity Against Gemcitabine-resistant Cholangiocarcinoma In Vitro

The antiparasitic drug, ivermectin (IVM), exerts anticancer activities in diverse cancer types. However, its anticancer activity against cholangiocarcinoma (CCA), especially the drug-resistant phenotype, has not yet been explored. IVM was tested for its anticancer activity against gemcitabine-sensitive (KKU214) and gemcitabine-resistant (KKU214GemR) CCA cell lines in vitro using the sulforhodamine B and clonogenic assays as well as cell-cycle analysis. IVM treatment inhibited cell proliferation and colony formation of both KKU214 and KKU214GemR in a dose- and time-dependent manner. KKU214GemR cells were more sensitive than KKU214 to IVM treatment. IVM treatment caused S-phase cell-cycle arrest and also cell death as indicated by an increase of sub-G0/G1 population in KKU214GemR cells treated with IVM for 48 h. IVM exerts anti-CCA activities and gemcitabine-resistant KKU214GemR cells are more sensitive to IVM treatment. Thus, IVM might be useful as an alternative treatment for CCA, especially in patients who do not respond to gemcitabine.

Screening miRNAs associated with resistance gemcitabine from exosomes in A549 lung cancer cells.

PurposeTo establish a gemcitabine-resistant lung adenocarcinoma cell line, A549/G+, and to screen the differences of miRNA expression in exosomes from A549 and A549/G+ cells.MethodsA549 cells were exposed in gemcitabine until they were resistant to gemcitabine, and extracted exosomes from A549 and A549/G+. The RNAs from exosomes were subjected to miRNA expression microarray experiments.ResultsAfter 39 weeks of continuous induction, we induced drug resistance in A549 cells. The resistance index was 6. Via GeneChip miRNA 4.0 analysis, there were 446 differential miRNAs between A549 and A549/G+. Target gene prediction and pathway analysis discovered the microRNAs in the intersections may participate in drug resistance.ConclusionThese differential miRNAs help to do in-depth research to elucidate the mechanism of resistance to gemcitabine in non-small cell lung cancer.

Abstract 3021: The role of survivin splice variants in pancreatic ductal adenocarcinoma Gemcitabine resistance

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis and an increased chemoresistance. Dysfunction in alternative splicing has been associated with chemoresistance in many cancers. Alternative splicing of survivin mRNA generates six splicing isoforms some of which are associated with cancer progression and chemoresistance. Survivin is a member of the inhibitors of apoptosis (IAPs) family and a cell cycle-associated oncoprotein overexpressed in almost all cancers. Interestingly, some of these survivin splice variants are proapoptotic (2α and 2β) while others are antiapoptotic (survivin, Δex3, 3α and 3β). It has been reported that increased levels of survivin 2β and 3β play a role in chemoresistance in ovarian cancers and other tumors, but this has not been studied in pancreatic cancer. Our recent RT-qPCR data also show an overexpression of all survivin splice variants in chemoresistant PDAC cell lines compared to the chemo-sensitive cell line. We, therefore, hypothesize that increased expression of survivin splice variants 2β and 3β contribute to chemo-resistance in PDAC. To test this hypothesis, we repeatedly selected the resistant cells from the normally chemosensitive MiaPaca2 cell line after exposure to gemcitabine. We then assessed the levels of survivin splice variants in the new cell line (gemcitabine-resistant MiaPaca2). Using the Alamar blue assay, we confirmed that Panc1 and gemcitabine-resistant MiaPaca2 are more resistant to gemcitabine compared to MiaPaca2 cells. We also observed a statistically significant overexpression of survivin 2β and 3β in gemcitabine-resistant cell lines compared to the more sensitive MiaPaca2 cells. To further test our hypothesis, we will knockdown survivin 2β and 3β in Panc1 and gemcitabine-resistant MiaPaca2, and/or overexpress these splice variants in MiaPaca2 cells then assess response to gemcitabine. The outcomes of this study might create new biomarkers for predicting the clinical outcomes of gemcitabine treatment and generate new therapeutic targets for pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Janviere Kabagwira, Amber Gonda, Mei Li Kwong. The role of survivin splice variants in pancreatic ductal adenocarcinoma Gemcitabine resistance [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 3021.

Long non-coding RNA GHET1 contributes to chemotherapeutic resistance to Gemcitabine in bladder cancer.

Bladder cancer (BC) ranks first in the incidence of urogenital tumors in China and second only to prostate cancer in the West. This study will clarify the roles and mechanism of lncRNA GHET1 in chemotherapeutic resistance of BC to Gemcitabine. The expression of GHET1 was examined using real-time quantitative PCR. Cell Counting Kit-8 assay was applied to analyze cell proliferation and Gemcitabine sensitivity. Cell apoptosis was detected using Annexin V-FITC/PI double-stained flow cytometry. The expression of ABCC1 protein was examined using Western blotting. Firstly, the expression of GHET1 was up-regulated in BC, its high expression was relevant to high grade and muscle invasion of BC patients. Secondly, high expression of GHET1 was related to low Gemcitabine sensitivity of BC patients, and GHET1 was highly expressed in Gemcitabine-resistant BC cell lines. Thirdly, knockdown of GHET1 decreased the IC50 of Gemcitabine in Gemcitabine-resistant BC cell lines and advanced the Gemcitabine-induced cytotoxicity; GHET1 promoted Gemcitabine resistance in BC. Finally, knockdown of GHET1 also inhibited the expression of ABCC1 protein in Gemcitabine-resistant BC cells. High expression of GHET1 was related with the low sensitivity to Gemcitabine of BC; GHET1 contributed to chemotherapeutic resistance to Gemcitabine in BC through up-regulating ABCC1 expression. Our findings are helpful to expound the molecular mechanism of chemotherapeutic resistance in BC.