Gemcitabine-resistant Cells Research Articles

Rubicon can predict prognosis in patients with pancreatic ductal adenocarcinoma after neoadjuvant chemoradiotherapy.

Despite previous therapeutic studies on autophagy in cancer, its role in the treatment of pancreatic ductal adenocarcinoma remains controversial, especially regarding its effect on chemotherapy, radiotherapy, and both combined. We focused on RUN domain Beclin-1 interacting and cysteine-rich-containing protein (Rubicon) to reveal its contribution to pancreatic ductal adenocarcinoma after chemoradiotherapy. To evaluate the clinical significance of Rubicon, immunohistochemistry was performed, and Rubicon expression was analyzed across 81 specimens resected from patients with pancreatic ductal adenocarcinoma after neoadjuvant chemoradiotherapy. A gemcitabine-resistant pancreatic ductal adenocarcinoma cell line was established followed by Rubicon expression and autophagy flux estimation. Finally, gemcitabine sensitivity, invasion ability, and cell viability were evaluated using Rubicon-targeting small interfering RNA. Rubicon expression in resected pancreatic ductal adenocarcinoma samples after chemoradiotherapy revealed significantly worse overall survival and recurrence-free survival in the Rubicon-high expression group than in the Rubicon-low expression group (overall survival: median [years] 2.02 vs. 3.21, p = 0.0359; recurrence-free survival: median [years] 0.90 vs. 1.90, p = 0.0146). In vitro, gemcitabine-resistant pancreatic ductal adenocarcinoma cell lines exhibited higher Rubicon expression and lower autophagy flux than the parental cell line (p < 0.01). Transduction with small interfering RNA downregulated the expression without affecting gemcitabine sensitivity, but it reduced invasion ability and cell viability (p < 0.01) in the gemcitabine-resistant pancreatic ductal adenocarcinoma cell line. High Rubicon expression is a significant, unfavorable prognostic factor in pancreatic ductal adenocarcinoma after neoadjuvant chemoradiotherapy. Downregulation of Rubicon expression improves invasion ability and cell viability in gemcitabine-resistant pancreatic ductal adenocarcinoma.

Elevated PAF1-RAD52 axis confers chemoresistance to human cancers.

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.

Calycosin inhibits gemcitabine-resistant lung cancer cells proliferation through modulation of the LDOC1/GNL3L/NFκB.

Lung cancer is the most common malignant cancer worldwide. Combination therapies are urgently needed to increase patient survival. Calycosin is a phytoestrogen isoflavone that has been reported previously to inhibit tumor cell growth, although its effects on lung cancer remain unclear. The aim of this study was to investigate the effects of calycosin on cell proliferation and apoptosis of gemcitabine-resistant lung cancer cells. Using calycosin to treat human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) and examine the effects on the cells. Cultured human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) were treated with increasing concentrations of calycosin. Cell viability and apoptosis were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, flow cytometry, and TUNEL assays. Western blots were used to measure the expression levels of proliferation-related proteins and cancer stem cell proteins in CL1-0 GEMR cells. The results showed that calycosin treatment inhibited cell proliferation, decreased cell migration ability, and suppressed cancer stem cell properties in CL1-0 GEMR cells. Interestingly, in CL1-0 GEMR cells, calycosin treatment not only increased LDOC1 but also decreased GNL3L/NFκB protein levels and mRNA levels, in concentration-dependent manners. We speculate that calycosin inhibited cell proliferation of the gemcitabine-resistant cell line through regulating the LDOC1/GNL3L/NFκB pathway.

Polymolecular botanical drug of Orthosiphon stamineus extract (C5OSEW5050ESA) as a complementary therapy to overcome gemcitabine resistance in pancreatic cancer cells.

Gemcitabine remains the cornerstone of pancreatic cancer treatment, despite exhibiting a modest effect on patient survival due to the development of drug resistance. Nuvastatic™ polymolecular botanical drug Orthosiphon stamineus (O.stamineus) is a folklore Asian herbal medicine that is used for the treatment of a variety of ailments. However, little is known about the mechanism of actions of the Nuvastatic™ polymolecular botanical drug of O.stamineus as a complementary therapy in resistant pancreatic cancer. It is postulated that the proprietary O.stamineus extract formulation (ID: C5EOSEW5050ESA) in Nuvastatic™ may sensitise resistant pancreatic cancer cells to gemcitabine. This study was conducted to assess the cytotoxic activity and synergistic effects of C5EOSEW5050ESA in gemcitabine-resistant pancreatic cancer cells. The effects of C5EOSEW5050ESA treatment on cell viability, multidrug-resistant genes, epithelial-mesenchymal transition, cellular senescence, cell death, and Notch signalling pathway were evaluated in gemcitabine-resistant Panc-1cells. C5EOSEW5050ESA sensitised gemcitabine resistant cells towards C5EOSEW5050ESA-gemcitabine combination treatment by reducing the expression of multidrug-resistant genes and epithelial-mesenchymal transition markers in gemcitabine-resistant cells compared to the control group, possibly through the inhibition of Notch signalling. This study provides valuable insight into using C5EOSEW5050ESA as a potential complementary treatment for resistant pancreatic cancer.

Mucin 4 Confers Gemcitabine Resistance and an Unfavorable Prognosis in Patients with Cholangiocarcinoma via AKT Activation.

Cholangiocarcinoma (CCA) exhibits aggressive biological behavior and a poor prognosis. Gemcitabine (GEM)-based chemotherapy is the first-line chemotherapy for advanced CCA but has a response rate of only 20-30%. Therefore, investigating treatments to overcome GEM resistance in advanced CCA is crucial. Among mucin (MUC) family members, MUC4 showed the greatest increase in the resistant versus parental sublines. MUC4 was upregulated in whole-cell lysates and conditioned media from gemcitabine-resistant (GR) CCA sublines. MUC4 mediated GEM resistance by activating AKT signaling in GR CCA cells. The MUC4-AKT axis induced BAX S184 phosphorylation to inhibit apoptosis and downregulated GEM transporter human equilibrative nucleoside transporter 1 (hENT1) expression. The combination of AKT inhibitors and GEM or afatinib overcame GEM resistance in CCA. In vivo, capivasertib (an AKT inhibitor) increased GEM sensitivity in GR cells. MUC4 promoted EGFR and HER2 activation to mediate GEM resistance. Finally, MUC4 expression in patient plasma correlated with MUC4 expression. Paraffin-embedded specimens from non-responders expressed significantly more MUC4 than did those from responders, and this upregulation was associated with poor progression-free survival and overall survival. In GR CCA, high MUC4 expression promotes sustained EGFR/HER2 signaling and AKT activation. The combination of AKT inhibitors with GEM or afatinib might overcome GEM resistance.

Wogonin increases gemcitabine sensitivity in pancreatic cancer by inhibiting Akt pathway.

Pancreatic cancer has a high degree of malignancy and a low 5-year survival rate, and drug resistance is one of the main factors leading to poor prognosis of pancreatic cancer. Wogonin is a flavonoid drug isolated from Scutellaria baicalensis, which has certain antitumor activity. Hence the purpose of this study was to investigate whether wogonin can be used to enhance the sensitivity of pancreatic cancer to gemcitabine chemotherapy, and investigate its possible sensitization mechanism. In vitro, MTT assay showed that wogonin increased gemcitabine cytotoxicity in gemcitabine-resistant pancreatic cancer cells. In vivo, Wogonin combined with gemcitabine was found to inhibit tumor growth in orthotopic pancreatic cancer mouse model. In order to explore the sensitization mechanism, the differentially expressed genes (DEGs) of the gemcitabine-resistant cell line Panc-1 and the gemcitabine-sensitive cell line Bxpc-3 were screened through the GEO database, and 15 differentially expressed genes were obtained by intersecting with the potential targets of wogonin. Gene Ontology and KEGG enrichment analysis was performed. Bioinformatics results predicted that wogonin promoted pancreatic cancer cell apoptosis by inhibiting protein kinase B (Akt) signaling, thereby enhancing the sensitivity of gemcitabine to Pancreatic cancer. The above results were also verified by flow cytometry and Western blotting experiments. In conclusion, wogonin may enhance the sensitivity of gemcitabine by inhibiting Akt pathway.

ADAM12 promotes gemcitabine resistance by activating EGFR signaling pathway and induces EMT in bladder cancer.

Gemcitabine (GEM)-based chemotherapy regimens is widely used in bladder cancer (BC) patients. However, GEM resistance may occur and result in treatment failure and disease progression. A disintegrin and metalloprotease 12 (ADAM12) plays a critical role in many cancers. However, the role of ADAM12 in GEM resistance of BC remains unclear. We analyzed the relationship between ADAM12 expression and tumor characteristics using the data downloaded from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. Then, we established GEM resistant BC cell lines and used quantitative real-time PCR, western blot, cell counting kit-8, immunohistochemistry, and xenograft mouse model to investigate the role of ADAM12 in GEM resistance. In general, ADAM12 was found to be upregulated in GEM resistant BC cells. ADAM12 knockdown increased the chemosensitivity of BC cells. We further proved that ADAM12 could promote GEM resistance by activating the epidermal growth factor receptor (EGFR) signaling pathway in BC. Furthermore, the epithelial-mesenchymal transition (EMT) phenotype was observed in GEM resistant BC cells. ADAM12 induced EMT process and promotes tumor progression in BC. Our findings suggested that ADAM12 was a key gene for GEM resistance and positively correlated with malignancy of BC. It might serve as a novel and valuable therapeutic target for BC.

Periplocin exerts antitumor activity by regulating Nrf2-mediated signaling pathway in gemcitabine-resistant pancreatic cancer cells

Although gemcitabine-based chemotherapy is common and effective for pancreatic cancer (PC), acquired drug resistance is one of the major reasons for treatment failure. Therefore, a novel therapeutic approach for gemcitabine-resistant PC is required. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an oxidative stress-responsive transcription factor regulating antioxidant responses and plays a crucial role in chemoresistance. In the present study, the antitumor activity of periplocin, a natural cardiac glycoside, was evaluated in an established gemcitabine-resistant PC cell line (PANC-GR). Nrf2 was overexpressed in gemcitabine-resistant cells, and Nrf2 knockdown recovered gemcitabine sensitivity in PANC-GR cells. The antiproliferative activity of periplocin was highly associated with Nrf2 downregulation and Nrf2-mediated signaling pathways in PANC-GR cells. Periplocin also increased reactive oxygen species production inducing G0/G1 cell cycle arrest and apoptosis in PANC-GR cells. Periplocin and gemcitabine combined significantly inhibited tumor growth in a PANC-GR cells-implanted xenograft mouse model via Nrf2 downregulation. Overall, these findings suggest that periplocin might be a novel therapeutic agent against gemcitabine resistance, as it could recover sensitivity to gemcitabine by regulating Nrf2-mediated signaling pathways in gemcitabine-resistant PC cells.

Abstract B045: Evaluating the role of altered RNA metabolism in pancreatic cancer therapy resistance

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal, mostly because they quickly develop resistance to current chemotherapies: Gemcitabine (GEM)/paclitaxel or cocktail FOLFIRINOX. Both chemotherapies rely on nucleoside analogues (GEM and 5-fluorouracil [5-FU], and resistance is developed without acquiring mutations, suggesting the role of non-mutational mechanisms for therapy resistance. Our recent findings demonstrated that PDACs depend on RNA splicing proteins to maintain tumors and are exquisitely susceptible to a range of therapies directed at RNA splicing, supporting the role of aberrant RNA splicing in PDAC therapy resistance. Here we evaluated the role of altered RNA splicing in PDAC therapy resistance. Three murine and human isogenic lines with either sensitivity or resistance to GEM were developed. We performed deep RNA sequencing (RNASeq, 80-100 million reads, traditional is 20-40 million reads), differential gene expression, and splicing analyses. GEM resistant PDAC cells demonstrated &amp;gt;40% of non-canonical RNA splice variants and altered expression of 30% of RNA-binding proteins encoded in the human/murine genome, compared to GEM sensitive ones. Specific alternatively spliced exons with common cis-elements were identified in therapy resistant cells, suggesting that these splicing changes are guided by a splicing factor or other RNA-binding protein(s) (RBPs). Top splicing changes were associated to mRNAs encoding ether lipid and pyruvate metabolism. This finding is important as previous studies have linked these metabolic pathways to therapy resistance, however, they have overlooked if protein isoforms, resulting from aberrant splicing, have different impacts in therapy resistance. In addition, GEM and H3B-8800, a novel spliceosome inhibitor showed higher synergistic activity in GEM resistant PDAC cells compared to GEM sensitive cells, suggesting that the development of resistance in PDAC is highly dependent on RNA splicing. Combinedly, these data strongly suggest that resistance to chemotherapeutic agents in PDAC cells requires altered RNA splicing and aberrant expression of associated proteins. Further investigation is ongoing to identify the splicing events and RBPs related to chemotherapy resistance, which will uncover novel mechanisms of therapy resistance and therapeutic modalities by targeting RNA splicing in pancreatic cancer. Citation Format: Md Afjalus Siraj, Xinning Shan, Robert Tseng, Luisa Escobar-Hoyos. Evaluating the role of altered RNA metabolism in pancreatic cancer therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B045.

Abstract PR023: Targeting gemcitabine resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a devastating 5-year survival rate of 11%. A lack of durable responses to standard-of-care chemotherapy combinations renders its treatment particularly challenging and largely contributes to the devastating prognosis. Gemcitabine, a pyrimidine anti-metabolite, is a cornerstone in PDAC therapy, but resistance remains a major hurdle. Multiple mechanisms of chemoresistance have been suggested to be mediated by rewired metabolism in PDAC cells. Accordingly, we hypothesize that metabolic reprogramming can be targeted to re-sensitize PDAC tumors to anti-metabolite chemotherapy. To define the spectrum of targetable metabolic and transcriptomic programs that drive cell-intrinsic resistance to gemcitabine, we established organoids from treatment-naïve PDAC patients. Gemcitabine high versus low responders, as assessed by dose response viability assays, were selected for metabolomic profiling and RNA sequencing. To define real-time reprogramming during the acquisition of resistance, we generated gemcitabine resistant murine pancreatic cancer cell lines and collected a time course series of metabolomic and transcriptomic datasets of sensitive, intermediate resistant and resistant cells. Integration of these dataset through a systems biology approach to define primary and de novo resistance is being used to characterize metabolic gemcitabine resistance mechanisms. These are functionally validated to provide novel approaches to re-sensitize resistant PDAC cells to gemcitabine in vitro and in vivo. The improvement of current chemotherapy combinations represents a promising approach, with potential to immediately translate into a clinical benefit and improve survival in this deadly disease. Citation Format: Alica Katrin Beutel, Rima Singh, Cecily Anaraki, Gregory Tong, Thomas Martinez, Alexander Kleger, Zeljka Jutric, Christopher James Halbrook. Targeting gemcitabine resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR023.

Tumor and stroma COL8A1 secretion induces autocrine and paracrine progression signaling in pancreatic ductal adenocarcinoma

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; p48-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

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.

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.

Plant-Derived Sulforaphane Suppresses Growth and Proliferation of Drug-Sensitive and Drug-Resistant Bladder Cancer Cell Lines In Vitro.

Simple SummaryThe natural compound sulforaphane is highly popular among tumor patients, since it is suggested to prevent oncogenesis and cancer progression. However, knowledge about its precise mode of action, particularly when drug resistance has been established, remains poor. The present study demonstrates the proliferation-blocking effects of SFN on a panel of drug-resistant bladder cancer cell lines.Combined cisplatin–gemcitabine (GC) application is standard for treating muscle-invasive bladder cancer. However, since rapid resistance to treatment often develops, many patients turn to supplements in the form of plant-based compounds. Sulforaphane (SFN), derived from cruciferous vegetables, is one such compound, and the present study was designed to investigate its influence on growth and proliferation in a panel of drug-sensitive bladder cancer cell lines, as well as their gemcitabine- and cisplatin-resistant counterparts. Chemo-sensitive and -resistant RT4, RT112, T24, and TCCSUP cell lines were exposed to SFN in different concentrations, and tumor growth, proliferation, and clone formation were evaluated, in addition to apoptosis and cell cycle progression. Means of action were investigated by assaying cell-cycle-regulating proteins and the mechanistic target of rapamycin (mTOR)/AKT signaling cascade. SFN significantly inhibited growth, proliferation, and clone formation in all four tumor cell lines. Cells were arrested in the G2/M and/or S phase, and alteration of the CDK–cyclin axis was closely associated with cell growth inhibition. The AKT/mTOR signaling pathway was deactivated in three of the cell lines. Acetylation of histone H3 was up-regulated. SFN, therefore, does exert tumor-suppressive properties in cisplatin- and gemcitabine-resistant bladder cancer cells and could be beneficial in optimizing bladder cancer therapy.

Allyl-, Butyl- and Phenylethyl-Isothiocyanate Modulate Akt-mTOR and Cyclin-CDK Signaling in Gemcitabine- and Cisplatin-Resistant Bladder Cancer Cell Lines.

Combined cisplatin–gemcitabine treatment causes rapid resistance development in patients with advanced urothelial carcinoma. The present study investigated the potential of the natural isothiocyanates (ITCs) allyl-isothiocyanate (AITC), butyl-isothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC) to suppress growth and proliferation of gemcitabine- and cisplatin-resistant bladder cancer cells lines. Sensitive and gemcitabine- and cisplatin-resistant RT112, T24, and TCCSUP cells were treated with the ITCs, and tumor cell growth, proliferation, and clone formation were evaluated. Apoptosis induction and cell cycle progression were investigated as well. The molecular mode of action was investigated by evaluating cell cycle-regulating proteins (cyclin-dependent kinases (CDKs) and cyclins A and B) and the mechanistic target of the rapamycin (mTOR)-AKT signaling pathway. The ITCs significantly inhibited growth, proliferation and clone formation of all tumor cell lines (sensitive and resistant). Cells were arrested in the G2/M phase, independent of the type of resistance. Alterations of both the CDK–cyclin axis and the Akt–mTOR signaling pathway were observed in AITC-treated T24 cells with minor effects on apoptosis induction. In contrast, AITC de-activated Akt–mTOR signaling and induced apoptosis in RT112 cells, with only minor effects on CDK expression. It is concluded that AITC, BITC, and PEITC exert tumor-suppressive properties on cisplatin- and gemcitabine-resistant bladder cancer cells, whereby the molecular action may differ among the cell lines. The integration of these ITCs into the gemcitabine-/cisplatin-based treatment regimen might optimize bladder cancer therapy.

Artemisia argyi extract induces apoptosis in human gemcitabine-resistant lung cancer cells via the PI3K/MAPK signaling pathway

Ethnopharmacological relevance: Artemisia argyi H. Lév. & Vaniot (Asteraceae), also called “Chinese mugwort”, is frequently used as a herbal medicine in China, Japan, Korea, and eastern parts of Russia. It is known as “ai ye” in China and “Gaiyou” in Japan. In ancient China, the buds and leaves of A. argyi were commonly consumed before and after Tomb-sweeping Day. It is used to treat malaria, hepatitis, cancer, inflammatory diseases, asthma, irregular menstrual cycle, sinusitis, and pathologic conditions of the kidney and liver. Although A. argyi extract (AAE) has shown anti-tumor activity against various cancers, the therapeutic effect and molecular mechanism of AAE remains to be further studied in lung cancer.Aim of the study: This study aimed to demonstrate the anti-tumor effect of AAE and its associated biological mechanisms in CL1-0 parent and gemcitabine-resistant (CL1-0-GR) lung cancer cells.Experimental procedure: Human lung cancer cells CL1-0 and CL1-0-GR cells were treated with AAE. Cell viability was assessed using the MTT, colony, and spheroid formation assays. Migration, invasion, and immunofluorescence staining were used to determine the extent of epithelial– mesenchymal transition (EMT). JC-1 and MitoSOX fluorescent assays were performed to investigate the effect of AAE on mitochondria. Apoptosis was detected using the TUNEL assay and flow cytometry with Annexin V staining.Result: We found that A. argyi significantly decreased cell viability and induced apoptosis, accompanied by mitochondrial membrane depolarization and increased ROS levels in both parent cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0-GR). AAE-induced apoptosis is regulated via the PI3K/AKT and MAPK signaling pathways. It also prevents CL1-0 and CL1-0-GR cancer cell invasion, migration, EMT, colony formation, and spheroid formation. In addition, AAE acts cooperative with commercial chemotherapy drugs to enhance tumor spheroid shrinkage.Conclusion: Our study provides the first evidence that A. argyi treatment suppresses both parent and gemcitabine-resistant lung cancer cells by inducing ROS, mitochondrial membrane depolarization, and apoptosis, and reducing EMT. Our finding provides insights into the anti-cancer activity of A. argyi and suggests that A. argyi may serve as a chemotherapy adjuvant that potentiates the efficacy of chemotherapeutic agents.

Long non-coding RNA SNHG16 decreased SMAD4 to induce gemcitabine resistance in pancreatic cancer via EZH2-mediated epigenetic modification.

Gemcitabine resistance (GR) in pancreatic cancer (PC) results in poor patient outcomes. SMAD family member (Smad4) dysregulation is a significant role of GR in PC, and EZH2 is involved in Smad4 expression in tumor progression. Interestingly, lncRNA small nucleolar RNA host gene 16 (SNHG16) might interact with EZH2, indicating a potential pathway to overcome gemcitabine-resistant PC progression. We investigated the role of the SNHG16/EZH2/Smad4 pathway in gemcitabine-resistant PC cells (PANC-1/GR and SW1990/GR). First, we found that SNHG16 was upregulated both in wild-type PC cells and in gemcitabine-resistant PC cells. SNHG16 overexpression reduced gemcitabine cytotoxicity and apoptosis in PC cells. Meanwhile, SNHG16 upregulation caused p-Akt elevation and Smad4 reduction. However, SNHG16 silencing induced the opposite trend. Then, we found that EZH2 was enriched in SNHG16 based on RIP and RNA pulldown. In particular, SNHG16 overexpression promoted the interaction between EZH2 and the Smad4 promoter according to Chromatin immunoprecipitation-quantitative polymerase chain reaction. Finally, both EZH2 inhibition and Smad4 upregulation increased gemcitabine cytotoxicity and apoptosis in PC cells during SNHG16 overexpression. Moreover, both treatments decreased p-Akt and increased Smad4. Collectively, lncRNA SNHG16 decreased Smad4 to induce GR in PC via EZH2-mediated epigenetic modification.

Gemcitabine resistance of pancreatic cancer cells is mediated by IGF1R dependent upregulation of CD44 expression and isoform switching

Chemoresistance in pancreatic cancer cells may be caused by the expansion of inherently resistant cancer cells or by the adaptive plasticity of initially sensitive cancer cells. We investigated how CD44 isoforms switching contributed to gemcitabine resistance. Treating CD44 null/low single-cell clones with increasing amounts of gemcitabine caused an increase in expression of CD44 and development of gemcitabine resistant (GR) cells. Drug sensitivity, invasiveness, and EMT process was evaluated by MTT, Matrigel invasion assays, and western blots. Genetic knockdown and pharmacological inhibitors were used to examine the roles of CD44 and IGF1R in mediating gemcitabine resistance. CD44 promoter activity and its interactive EMT-related transcription factors were evaluated by luciferase reporter assay and chromatin immunoprecipitation assay. Kaplan–Meier curve was created by log-rank test to reveal the clinical relevance of CD44 and IGF1R expression in patients. We found silence of CD44 in GR cells partially restored E-cadherin expression, reduced ZEB1 expression, and increased drug sensitivity. The gemcitabine-induced CD44 expressing and isoform switching were associated with an increase in nuclear accumulation of phosphor-cJun, Ets1, and Egr1 and binding of these transcription factors to the CD44 promoter. Gemcitabine treatment induced phosphorylation of IGF1R and increased the expression of phosphor-cJun, Ets1, and Egr1 within 72 h. Stimulation or suppression of IGF1R signaling or its downstream target promoted or blocked CD44 promoter activity. Clinically, patients whose tumors expressed high levels of CD44/IGF1R showed a poor prognosis. This study suggests that IGF1R-dependent CD44 isoform switching confers pancreatic cancer cells to undergo an adaptive change in response to gemcitabine and provides the basis for improved targeted therapy of pancreatic cancer.

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.

A ferrocene-containing nucleoside analogue targets DNA replication in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a disease that remains refractory to existing treatments including the nucleoside analogue gemcitabine. In the current study we demonstrate that an organometallic nucleoside analogue, the ferronucleoside 1-(S,Rp), is cytotoxic in a panel of PDAC cell lines including gemcitabine-resistant MIAPaCa2, with IC50 values comparable to cisplatin. Biochemical studies show that the mechanism of action is inhibition of DNA replication, S-phase cell cycle arrest and stalling of DNA-replication forks, which were directly observed at single molecule resolution by DNA-fibre fluorography. In agreement with this, transcriptional changes following treatment with 1-(S,Rp) include activation of three of the four genes (HUS1, RAD1, RAD17) of the 9-1-1 check point complex clamp and two of the three genes (MRE11, NBN) that form the MRN complex as well as activation of multiple downstream targets. Furthermore, there was evidence of phosphorylation of checkpoint kinases 1 and 2 as well as RPA1 and gamma H2AX, all of which are considered biochemical markers of replication stress. Studies in p53-deficient cell lines showed activation of CDKN1A (p21) and GADD45A by 1-(S,Rp) was at least partially independent of p53. In conclusion, because of its potency and activity in gemcitabine-resistant cells, 1-(S,Rp) is a promising candidate molecule for development of new treatments for PDAC.