Gemcitabine-resistant Cells Research Articles

Lactic acidosis promotes aggressive features of cholangiocarcinoma cells via upregulating ALDH1A3 expression through EGFR axis

AimsLactic acidosis (LA) generated in tumor microenvironment promotes tumor metastasis and drug resistance. This study aimed to demonstrate the impacts and the mechanisms of LA on aldehyde dehydrogenase1A3 (ALDH1A3) in promoting aggressiveness and gemcitabine resistance in cholangiocarcinoma (CCA) cell lines. The clinical relevance and the molecular pathway related to the upregulation of ALDH1A3 in LA cells will be revealed. Main methodsALDH1A3 expression and its clinical significances in CCA tissues were analyzed using the GEO databases. Human CCA cell lines, KKU-213A-LA and KKU-213B-LA maintained in the LA medium were studied and compared with its parental cells cultured in normal medium. Aggressive features—proliferation, colony formation, migration, invasion, and gemcitabine response were determined. Expression of ALDH1A3, EGFR and the downstream effectors were analyzed using real-time PCR and Western blotting. Key findingsALDH1A3 was upregulated in patient CCA tissues and correlated with LDHA and shorter survival of CCA patients. mRNA and protein of ALDH1A3 were increased in LA cells. Attenuation of ALDH1A3 expression by siRNA significantly reduced cell proliferation, colony formation, migration, invasion, and gemcitabine resistance of LA cells, and gemcitabine resistant cells. The EGF/EGFR signaling via Erk and STAT3 was pinned to be involved in the induction of ALDH1A3 expression in LA cells. The transcriptomic analysis from TCGA dataset supported the links between LDHA, EGFR and ALDH1A3 in several tumor tissues. SignificanceLactic acidosis upregulated EGFR and ALDH1A3 expression, leading to the aggressiveness of CCA cells. The EGFR/ALDH1A3 axis could be a novel therapeutic target to eradicate metastatic CCA.

Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling pathway regulates ABC transporters to reverse gemcitabine resistance in pancreatic cancer

BackgroundAlthough gemcitabine has been considered as the first-line drug for advanced pancreatic cancer (PC), development of resistance to gemcitabine severely limits the effectiveness of this chemotherapy, and the underlying mechanism of gemcitabine resistance remains unclear. Various factors, such as ATP binding cassette (ABC) transporters, microRNAs and their downstream signaling pathways are included in chemoresistance to gemcitabine. This study investigated the potential mechanisms of microRNAs and ABC transporters related signaling pathways for PC resistance to gemcitabine both in vivo and in vitro.MethodsImmunohistochemistry and Western blotting were applied to detect the expression of ABC transporters. Molecular docking analysis was performed to explore whether gemcitabine interacted with ABC transporters. Gain-of-function and loss-of-function analyses were performed to investigate the functions of hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and dual-luciferase reporter assay were used to confirm the downstream regulatory mechanisms of hsa-miR-3178.ResultsWe found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant PC tissues and cells. Molecular docking analysis revealed that gemcitabine can bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These results were also verified by animal experiments. RhoB was down-regulated in gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178. Kaplan–Meier survival curve showed that lower RhoB expression was significantly associated with poor overall survival in PC patients. Rescue assays demonstrated that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating the PI3K/Akt pathway-mediated upregulation of ABC transporters.ConclusionsOur results indicate that hsa-miR-3178 promotes gemcitabine resistance via RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These findings suggest that hsa-miR-3178 could be a novel therapeutic target for overcoming gemcitabine resistance in PC.

MP40-17 LNCRNA SNHG16 MEDIATE CISPLATIN AND GEMCITABINE RESISTANCE IN UPPER TRACT UROTHELIAL CARCINOMA CELLS BY SPONGING OF MIR-31-5P

You have accessJournal of UrologyCME1 May 2022MP40-17 LNCRNA SNHG16 MEDIATE CISPLATIN AND GEMCITABINE RESISTANCE IN UPPER TRACT UROTHELIAL CARCINOMA CELLS BY SPONGING OF MIR-31-5P Wei-Chi Hsu, Wei-Ming Li, A-Mei Huang, Lin-Li Chang, Hui-Hui Lin, Wen-Jeng Wu, Ching-Chia Li, and Hung-Lung Ke Wei-Chi HsuWei-Chi Hsu More articles by this author , Wei-Ming LiWei-Ming Li More articles by this author , A-Mei HuangA-Mei Huang More articles by this author , Lin-Li ChangLin-Li Chang More articles by this author , Hui-Hui LinHui-Hui Lin More articles by this author , Wen-Jeng WuWen-Jeng Wu More articles by this author , Ching-Chia LiChing-Chia Li More articles by this author , and Hung-Lung KeHung-Lung Ke More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002600.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chemotherapy is suggested perioperatively for advanced upper tract urothelial carcinoma (UTUC) to improve recurrence-free and cancer-specific survivals. The standard regimens of chemotherapy are gemcitabine (GEM) and cisplatin (CDDP). After repeated chemotherapy, 30% of the patients will develop drug resistance and the disease will progress. Therefore, it is very important to clarify the mechanism of chemotherapy resistance and overcome it. This study aimed to investigate whether miR-31-5p can enhance the chemosensitivity of CDDP and GEM and the molecular mechanisms. METHODS: In this study, we established BFTC909 derived CDDP-resistant (BFTC909-CR) and GEM-resistant cells (BFTC909-GR). MiR-31-5p expression was analyzed by real-time qPCR in UTUC cell lines. The effects of miR-31-5p combined with CDDP or GEM on the chemosensitivity of cells were analyzed by WST-1. Western blot was applied to detect anti-apoptotic and multidrug resistant protein expression. Silencing of lncRNA was to evaluate CDDP and GEM response by cytotoxicity assay and miR-31-5p levels by real-time PCR. RESULTS: We found that the levels of miR-31-5p were lower in UTUC cell lines than immortalized normal urothelium cells. MiR-31-5p may direct targeted to 3′-UTR of CAMK2D mRNA. The combination of miR-31-5p with CDDP or GEM significantly down-regulated the expression of Bcl-2, Calcium2+/Calmodulin Dependent Protein Kinase II Delta (CaMKIIδ) and MDR1 as compared with drug treatment alone. Bcl-2, CaMKIIδ and MDR1 had higher expression in CDDP and GEM resistant cells than parental cells. In addition, we found that lncRNA SNHG16 may direct target to miR-31-5p. SNHG16 knockdown induced miR-31-5p levels in BFTC909 cells and indicated that SNHG16 may negatively regulate the expression of miR-31-5p. Importantly, SNHG16 silencing increased CDDP sensitivity in BFTC909 cells. CONCLUSIONS: Taken together, our study indicated that SNHG16 and miR-31-5p might modulate CDDP or Gem-resistance through regulating CaMKIIδ, Bcl-2 and MDR1 in UTUC cells. Source of Funding: This study was supported by grants from the Ministry of Science and Technology (MOST 109-2314-B-037-105-MY3) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e681 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wei-Chi Hsu More articles by this author Wei-Ming Li More articles by this author A-Mei Huang More articles by this author Lin-Li Chang More articles by this author Hui-Hui Lin More articles by this author Wen-Jeng Wu More articles by this author Ching-Chia Li More articles by this author Hung-Lung Ke More articles by this author Expand All Advertisement PDF DownloadLoading ...

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.

Increased m6A modification of lncRNA DBH-AS1 suppresses pancreatic cancer growth and gemcitabine resistance via the miR-3163/USP44 axis.

BackgroundGemcitabine is among the most commonly utilized chemotherapeutic agents for treating pancreatic cancer (PC), yet patients ultimately develop chemoresistance and thus exhibit a poor prognosis. Long noncoding RNAs (lncRNAs) can function as key regulators of PC progression and may serve as prognostic biomarkers in individuals with gemcitabine-resistant PC. This study sought to explore the role of the lncRNA DBH-AS1 in this oncogenic setting.MethodsBased on public databases and qRT-PCR analyses the expression of lncRNA DBH-AS1 in PC tissues and cell lines. The effects of lncRNA DBH-AS1 on proliferation and gemcitabine resistance were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA DBH-AS1, miR-3163 and USP44.ResultsWe found that PC tissues exhibited DBH-AS1 downregulation that was particularly pronounced in gemcitabine-resistant PC tissues and cells. This DBH-AS1 downregulation was negatively correlated with the malignancy of PC tumors and with patient survival outcomes. Additionally, decreased DBH-AS1 expression in PC was found to be linked to the METTL3-dependent m6A methylation of the lncRNA, with functional analyses revealing that DBH-AS1 was able to suppress the growth of PC cells. Mechanistically, DBH-AS1 was able to increase PC cell sensitivity to gemcitabine by sequestering miR-3163 and thus upregulating USP44 in these tumor cells. Clinically, patient-derived PC tumor xenografts exhibiting high levels of DBH-AS1 expression were found to be responsive to gemcitabine treatment.ConclusionsOverall, these data underscore a key role for DBH-AS1 as a regulator of PC tumor growth and a promising therapeutic target capable of predicting PC patient responsiveness to gemcitabine treatment.

Chemoresistance Transmission via Exosome-Transferred MMP14 in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies. Gemcitabine is the most commonly used chemotherapy for the treatment of PDAC, but the development of drug resistance still remains challenging. Recently, exosomes have emerged as important mediators for intercellular communication. Exosomes affect recipient cells’ behavior through the engulfed cargos, however the specific cargos responsible for gemcitabine resistance in PDAC are poorly understood. Here, we reported that exosomes could transfer gemcitabine resistance via a metalloproteinase 14 (MMP14)-dependent mechanism. MMP14 was identified as a major differentially secreted protein from the gemcitabine-resistant PDAC cells by comparative secretome. It was packaged into the exosomes and transmitted from the chemoresistant cells to the sensitive ones. The exosome-transferred MMP14 could enhance drug resistance and promotes the sphere-formation and migration abilities of the recipient sensitive PDAC cells. Mechanically, exosome-transferred MMP14 promotes the stability of CD44, the cancer stem cell marker in the recipient cells. Our results indicate that MMP14 is a key player for exosome-mediated transfer of gemcitabine resistance, thus targeting MMP14 in exosomes may represent a novel strategy to limit gemcitabine resistance in PDAC.

MiR-624-5p enhances NLRP3 augmented gemcitabine resistance via EMT/IL-1β/Wnt/β-catenin signaling pathway in ovarian cancer

The current study investigates the NLRP3’s cytotoxicity inhibitory effect among ovarian cancer cells and how it interacts with Wnt/β-catenin in vitro conditions. Further, the study also analyzed the regulatory role of NLRP3 in resistance to gemcitabine among ovarian cancer cells and its underlying interaction mechanisms with Wnt/β-catenin in vitro. The current in vitro study detailed that when downregulating NLRP3, it could enhance the gemcitabine sensitivity in GRC cells. In case of gemcitabine-resistant cells, the up-regulation of NLRP3 can increase the drug-resistance through the activation of IL-1β, EMT and Wnt/β-catenin signaling pathways. High expression of miR-624-5p was recorded in ovarian drug resistant cancer cells and it also boosted the cell viabilities. NLRP3 can reinstate the functioning of miR-624-5p in drug resistant cells. This phenomenon concludes that NLRP3 is a promising therapeutic target and can be implemented in traditional chemotherapy to increase the efficacy of the treatment. The current study conducted in vitro experiments and the findings infer that the downregulation of NLRP3 can enhance the sensitivity of gemcitabine among GRC cells. This mechanism will increase the treatment efficacy by inhibiting the drug resistance in GRC. These two entities are the new promising biomarkers that can be used in the detection of platinum resistance in ovarian cancer patients and conduct novel clinical research.

Olive Mill Wastewater Inhibits Growth and Proliferation of Cisplatin- and Gemcitabine-Resistant Bladder Cancer Cells In Vitro by Down-Regulating the Akt/mTOR-Signaling Pathway.

Bladder cancer patients whose tumors develop resistance to cisplatin-based chemotherapy often turn to natural, plant-derived products. Beneficial effects have been particularly ascribed to polyphenols, although their therapeutic relevance when resistance has developed is not clear. The present study evaluated the anti-tumor potential of polyphenol-rich olive mill wastewater (OMWW) on chemo-sensitive and cisplatin- and gemcitabine-resistant T24, RT112, and TCCSUP bladder cancer cells in vitro. The cells were treated with different dilutions of OMWW, and tumor growth and clone formation were evaluated. Possible mechanisms of action were investigated by evaluating cell cycle phases and cell cycle-regulating proteins. OMWW profoundly inhibited the growth and proliferation of chemo-sensitive as well as gemcitabine- and cisplatin-resistant bladder cancer cells. Depending on the cell line and on gemcitabine- or cisplatin-resistance, OMWW induced cell cycle arrest at different phases. These differing phase arrests were accompanied by differing alterations in the CDK-cyclin axis. Considerable suppression of the Akt-mTOR pathway by OMWW was observed in all three cell lines. Since OMWW blocks the cell cycle through the manipulation of the cyclin-CDK axis and the deactivation of Akt-mTOR signaling, OMWW could become relevant in supporting bladder cancer therapy.

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.

Acquired chemoresistance can lead to increased resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus

Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV) against different malignancies, including pancreatic ductal adenocarcinoma (PDAC). Our previous studies have demonstrated that VSV-based OVs are effective against the majority of tested human PDAC cell lines. However, some PDAC cell lines are resistant to VSV. PDAC is one of the deadliest types of human malignancies in part due to intrinsic or acquired chemoresistance. Here, we investigated how acquired chemoresistance impacts the efficacy of VSV-based OV therapy. Using an experimental evolution approach, we generated PDAC cell lines with increased resistance to gemcitabine and examined their responsiveness to oncolytic virotherapy. We found that gemcitabine-resistant PDAC cells become more resistant to VSV. The cross-resistance correlated with upregulated levels of a subset of interferon-stimulated genes, resembling the interferon-related DNA damage resistance signature (IRDS), often associated with resistance of cancer cells to chemotherapy and/or radiation therapy. Analysis of ten different PDAC cell lines showed that four PDAC cell lines most resistant to VSV were also highly resistant to gemcitabine, and they all displayed IRDS-like expression in our previous reports. Our study highlights a possible interaction between two different therapies that should be considered in the future for the development of rational treatment regimens.

Effect of Photodynamic Therapy on Gemcitabine-Resistant Cholangiocarcinoma in vitro and in vivo Through KLF10 and EGFR.

Cholangiocarcinoma is a relatively rare neoplasm with increasing incidence. Although chemotherapeutic agent such as gemcitabine has long been used as standard treatment for cholangiocarcinoma, the interindividual variability in target and drug sensitivity and specificity may lead to therapeutic resistance. In the present study, we found that photodynamic therapy (PDT) treatment inhibited gemcitabine-resistant cholangiocarcinoma cells via repressing cell viability, enhancing cell apoptosis, and eliciting G1 cell cycle arrest through modulating Cyclin D1 and caspase 3 cleavage. In vivo, PDT treatment significantly inhibited the growth of gemcitabine-resistant cholangiocarcinoma cell-derived tumors. Online data mining and experimental analyses indicate that KLF10 expression was induced, whereas EGFR expression was downregulated by PDT treatment; KLF10 targeted the EGFR promoter region to inhibit EGFR transcription. Under PDT treatment, EGFR overexpression and KLF10 silencing attenuated the anti-cancer effects of PDT on gemcitabine-resistant cholangiocarcinoma cells by promoting cell viability, inhibiting apoptosis, and increasing S phase cell proportion. Importantly, under PDT treatment, the effects of KLF10 silencing were significantly reversed by EGFR silencing. In conclusion, PDT treatment induces KLF10 expression and downregulates EGFR expression. KLF10 binds to EGFR promoter region to inhibit EGFR transcription. The KLF10/EGFR axis participates in the process of the inhibition of PDT on gemcitabine-resistant cholangiocarcinoma cells.

Curcumin enhances drug sensitivity of gemcitabine-resistant lung cancer cells and inhibits metastasis.

This study aimed to investigate the effects of curcumin (Cur) on the proliferation, migration, and invasion of gemcitabine (GEM) resistant lung cancer A549 cells (A549/GEM), and the potential mechanism. After treating with GEM, individually or combined with Cur, the inhibition, migration, and invasion of A549/GEM were tested by the CCK8, transwell, and cell wound healing assays, respectively. QRT-PCR and Western blot were used to detect mRNA and protein markers. Finally, the therapeutic effects of GEM, individually or combined with Cur, were verified in nude mice. The results indicated that the combined application of Cur and GEM can improve the sensitivity of A549/GEM to the GEM. Compared with the GEM, GEM plus Cur significantly decreased the migration and invasion of A549/GEM cells. The expression levels of MMP9 , Vimentin, and N-cadherin were significantly decreased, while the E-cadherin expression was increased. In vivo experiments showed a better therapeutic effect of GEM combined with Cur than that of GEM alone, and the combination therapy did not cause more toxicity to animals. In summary, Cur reversed GEM resistance and inhibited the EMT process in A549/GEM cells. GEM, combined with Cur, is safe and more effective in the treatment of non-small cell lung cancer.

Long noncoding RNA NORAD acts as a ceRNA mediates gemcitabine resistance in bladder cancer by sponging miR-155–5p to regulate WEE1 expression

BackgroundIncreasing evidences have proved that long noncoding RNAs (lncRNAs) regulate the occurrence of bladder cancer (BC) and participate in various pathophysiology processes. However, little is unknown about the role of lncRNAs in drug resistance of BC cells. In this study, we explored the role of non-coding RNA activated by DNA damage (NORAD) in the gemcitabine (GEM) resistant of BC cells and explored its potential mechanism. MethodsReal-time quantitative PCR (RT-qPCR) was used to detect the expression of NORAD and miR-155–5p of BC cells. Cell counting kit-8 (CCK-8) and Western blot were used to detect cell inhibition rate and the expression of WEE1 G2 checkpoint kinase (WEE1), P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). Flow cytometry detected cell cycle and apoptosis. Dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay were used to confirm the targeting relationship between miR-155–5p, NORAD and WEE1. The xenograft model was used to observe the function of NORAD in vivo. immunohistochemistry (IHC) assay was used to detect the expression of WEE1, caspase-3 and Ki67 in tumor tissues. ResultsNORAD highly expressed in GEM-resistant BC cell lines. Knockdown of NORAD significantly inhibited the proliferation of T24/GEM cells, the expression of drug-resistant proteins P-gp and MRP1, inhibit the G0/G1 phase of cells, and induce cell apoptosis. Knockdown of NORAD reversed the promotion effect of miR-155–5p on WEE1 expression and promoted the sensitivity of T24/GEM cells to GEM. In vivo, knockdown of NORAD inhibited the tumor growth, and enhanced the GEM-sensitivity in mice. ConclusionThese data highlight the potential of NORAD acts as a therapeutic target for BC GEM resistance. It revealed the vital roles of NORAD/miR-155–5p/WEE1 axis in GEM resistant BC cells, providing a novel therapeutic strategy for BC.

MicroRNA-34a Alleviates Gemcitabine Resistance in Pancreatic Cancer by Repression of Cancer Stem Cell Renewal.

This study aimed to enhance the sensitivity of pancreatic ductal adenocarcinoma cells by microRNA-34a (miR-34a)-mediated targeting of Notch 1. Cell viability was determined by using an MTT (3-(4,5)-dimethylthiahiazo(-2)-3,5-diphenytetrazoliumromide) assay. The expression levels of miR-34a and relevant mRNAs were determined using quantitative polymerase chain reaction. Protein levels were measured by Western blotting. Cellular stemness was assessed by cell invasiveness and sphere formation assays. A transplanted tumor model was established for in vivo experiments. MicroRNA-34a enhanced gemcitabine sensitivity both in vivo and in vitro. MicroRNA-34a suppressed the stemness and proliferation of pancreatic cancer stem cells. MicroRNA-34a directly associated with Notch 1, which lies upstream of epithelial-mesenchymal transition signaling pathways. MicroRNA-34a sensitized pancreatic cancer cells to gemcitabine treatment by inhibiting Notch 1 signaling in pancreatic cancer stem cells, indicating that miR-34a has the potential to be developed as a novel therapeutic agent for the treatment of gemcitabine-resistant pancreatic ductal adenocarcinoma cells.

Ginsenoside Rg3 increases gemcitabine sensitivity of pancreatic adenocarcinoma via reducing ZFP91 mediated TSPYL2 destabilization

BackgroundGinsenoside Rg3 and gemcitabine have mutual enhancing antitumor effects. However, the underlying mechanisms are not clear. This study explored the influence of ginsenoside Rg3 on Zinc finger protein 91 homolog (ZFP91) expression in pancreatic adenocarcinoma (PAAD) and their regulatory mechanisms on gemcitabine sensitivity. MethodsRNA-seq and survival data from The Cancer Genome Atlas (TCGA)-PAAD and Genotype-Tissue Expression (GTEx) were used for in-silicon analysis. PANC-1, BxPC-3, and PANC-1 gemcitabine-resistant (PANC-1/GR) cells were used for in vitro analysis. PANC-1 derived tumor xenograft nude mice model was used to assess the influence of ginsenoside Rg3 and ZFP91 on tumor growth in vivo. ResultsGinsenoside Rg3 reduced ZFP91 expression in PAAD cells in a dose-dependent manner. ZFP91 upregulation was associated with significantly shorter survival of patients with PAAD. ZFP91 overexpression induced gemcitabine resistance, which was partly conquered by ginsenoside Rg3 treatment. ZFP91 depletion sensitized PANC-1/GR cells to gemcitabine treatment. ZFP91 interacted with Testis-Specific Y-Encoded-Like Protein 2 (TSPYL2), induced its poly-ubiquitination, and promoted proteasomal degradation. Ginsenoside Rg3 treatment weakened ZFP91-induced TSPYL2 poly-ubiquitination and degradation. Enforced TSPYL2 expression increased gemcitabine sensitivity of PAAD cells and partly reversed induced gemcitabine resistance in PANC-1/GR cells. ConclusionGinsenoside Rg3 can increase gemcitabine sensitivity of pancreatic adenocarcinoma at least via reducing ZFP91 mediated TSPYL2 destabilization.

BMI1 activates P-glycoprotein via transcription repression of miR-3682-3p and enhances chemoresistance of bladder cancer cell.

Chemoresistance is the most significant reason for the failure of cancer treatment following radical cystectomy. The response rate to the first-line chemotherapy of cisplatin and gemcitabine does not exceed 50%. In our previous research, elevated BMI1 (B-cell specific Moloney murine leukemia virus integration region 1) expression in bladder cancer conferred poor survival and was associated with chemoresistance. Herein, via analysis of The Cancer Genome Atlas database and validation of clinical samples, BMI1 was elevated in patients with bladder cancer resistant to cisplatin and gemcitabine, which conferred tumor relapse and progression. Consistently, BMI1 was markedly increased in the established cisplatin- and gemcitabine-resistant T24 cells (T24/DDP&GEM). Functionally, BMI1 overexpression dramatically promoted drug efflux, enhanced viability and decreased apoptosis of bladder cancer cells upon treatment with cisplatin or gemcitabine, whereas BMI1 downregulation reversed this effect. Mechanically, upon interaction with p53, BMI1 was recruited on the promoter of miR-3682-3p gene concomitant with an increase in the mono-ubiquitination of histone H2A lysine 119, leading to transcription repression of miR-3682-3p gene followed by derepression of ABCB1 (ATP binding cassette subfamily B member 1) gene. Moreover, suppression of P-glycoprotein by miR-3682-3p mimics or its inhibitor XR-9576, could significantly reverse chemoresistance of T24/DDP&GEM cells. These results provided a novel insight into a portion of the mechanism underlying BMI1-mediated chemoresistance in bladder cancer.

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 2403: Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer

Abstract Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to current treatment regimens contributes to the dismal prognosis for patients diagnosed with the disease. Accumulated evidence indicates that metabolic alterations may play a role in chemo-resistance. In this project, we investigated the role of pyruvate dehydrogenase Alpha1 (PDHA1) in chemo-resistance. Methods: Wild type and Gemcitabine-resistant pancreatic cancer cell lines MIA PaCa-2, HPAF-II, and UN-KPC-961 were used. Mitochondrial respiration, ATP production and glycolytic activities were measured by seahorse. PDHA1 was overexpressed or its activity inhibited by over-expressing PDHA1 kinase in these cells and cancer cell survival was measured by MTT and cancer stemness measured by Western and RT-PCR. PDHA1 level was measured in human and mouse PDAC tissues. Results: Seahorse analysis showed a significantly lower mitochondrial activity, ATP production, and increased glycolytic activity and lactate production in gemcitabine-resistant compared to non- resistant pancreatic cancer cells. PDHA1 mRNA and protein levels were less in gemcitabine-resistant cancer cells compared to non-resistant cells. PDHA1 levels were less in human PDAC tissues from patients and mice who developed resistance to chemotherapy. PDHA1 over-expression in gemcitabine-resistant cancer cells reversed resistance to chemotherapy, decreased cancer cell survival and decreased expression of cancer stemness markers. Whereas, PDHA1 inhibition by over-expressing PDHA1 kinase, in non-resistant cancer cells, induced resistance to chemotherapy, increased cancer cell survival and increased expression of cancer stemness markers. We found that inhibition of histone deacetylase (HDAC) (class 1 and 2) increased transcription level of PDHA1 and reversed chemo-resistance. Conclusion: We show that PDHA1 is a regulator of chemo-resistance in pancreatic cancer cells. PDHA1is a novel target for treating chemo-resistant PDAC. Citation Format: Fouzia Zayou, Chintan Chheda, Adrian Lim, Omer H. Elmadbouh, Stephen J. Pandol, Mouad Edderkaoui. Pyruvate dehydrogenase mediates chemo-resistance in pancreatic cancer [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 2403.

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.

An experimental study on the malignant characteristics of gemcitabine resistant cells by Transferring MiRNA-222-3p via exosomes.

article: An experimental study on the malignant characteristics of gemcitabine resistant cells by Transferring MiRNA-222-3p via exosomes - Minerva Surgery 2021 October;76(5):488-90 - Minerva Medica - Journals