Gemcitabine-induced Cell Apoptosis Research Articles

Retraction Note to: Linc‑ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR‑124/PTBP1/PKM2 axis.

In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

Overexpression of microRNA-205-5p exerts suppressive effects on stem cell drug resistance in gallbladder cancer by down-regulating PRKCE.

Some microRNAs (miRs or miRNAs) have been reported to function as tumor suppressors in gallbladder cancer (GBC). However, the specific effect of miR-205-5p on GBC remains unclear. The objective of the present study was to unravel the effects of miR-205-5p on the drug resistance in GBC. For this purpose, the expression of miR-205-5p and protein kinase C ϵ (PRKCE) was quantified in the peripheral blood sample harvested from GBC patients and healthy volunteers. Then the relationship between miR-205-5p and PRKCE was validated. After isolating the GBC stem cells, ectopic expression and depletion experiments were conducted to analyze the effect of miR-205-5p and PRKCE on cell proliferation, drug resistance, apoptosis, and colony formation rate as well as the expression of apoptotic factors (Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), and cleaved caspase 3). Finally, the mouse xenograft model of GBC was established to verify the function of miR-205-5p in vivo. Intriguingly, our results manifested that miR-205-5p was down-regulated, while PRKCE was up-regulated in peripheral blood samples and stem cells of patients with GBC. Moreover, miR-205-5p targeted PRKCE and negatively regulated its expression. The overexpression of miR-205-5p or silencing of PRKCE inhibited the drug resistance, proliferation, and colony formation rate while promoting apoptosis of GBC stem cells. Additionally, the overexpression of miR-205-5p attenuated drug resistance to gemcitabine but promoted the gemcitabine-induced cell apoptosis by inhibiting the PRKCE in vivo. Overall, an intimate correlation between miR-205-5p and PRKCE is a key determinant of drug resistance of GBC stem cells, thus, suggesting a novel miR-205-5p-based clinical intervention target for GBC patients.

N,N-diethylaminobenzaldehyde targets aldehyde dehydrogenase to eradicate human pancreatic cancer cells.

Pancreatic cancer is a common cause of worldwide cancer-related mortality with a poor 5-year survival rate. Aldehyde dehydrogenase (ALDH) activity is a possible marker for malignant stem cells in solid organ systems, including the pancreas, and N,N-diethylaminobenzaldehyde (DEAB) is able to inhibit ALDH activity. In the present study, the role of DEAB in the treatment of pancreatic cancer cells and the potential underlying mechanisms were investigated. The ALDH activities of pancreatic cancer cell lines treated with or without DEAB were analyzed by an ALDEFLUOR™ assay. The Cell Counting Kit-8 and colony formation assays, and cell cycle analysis were used to evaluate the viability, colony-forming ability and cell quiescence of cell lines under DEAB treatment, respectively. DEAB and/or gemcitabine-induced cell apoptosis was assessed by flow cytometry. DEAB reduced ALDH activity and inhibited the proliferation, colony-forming ability and cell quiescence of pancreatic cancer cell lines. Compared with respective controls, DEAB alone and the combination of gemcitabine and DEAB significantly decreased cell viability and increased cell apoptosis. Moreover, reverse transcription-PCR and western blotting were used to measure the expressions of B cell lymphoma 2 (Bcl2) associated X protein (Bax) and Bcl2 mRNA and protein. The anti-cancer effect of DEAB was associated with upregulation of Bax expression. Therefore, targeting ALDH with DEAB may be a potential therapeutic choice for pancreatic cancer, demonstrating a synergic effect with gemcitabine.

The preparation of cell-containing microbubble scaffolds to mimic alveoli structure as a 3D drug-screening system for lung cancer

Cancer is the leading cause of mortality worldwide, and lung cancer is the most malignant. However, the high failure rate in oncology drug development from in vitro studies to in vivo preclinical models indicates that the modern methods of evaluating drug efficacies in vitro are not reliable. Traditional 2D cell culture has proved inadequate to mimic real physiological conditions. Current 3D cell culture methods do not represent the delicate structure of lung alveoli. To mimic lung alveoli structure, a cell-containing enzyme-crosslinked gelatin microbubble scaffold was produced by mixing surfactant-containing gelatin solution with microbial transglutaminase (mTGase)-mixed A549 cell suspension in a four-channel flow-focusing microfluidic device. With uniform pore size of about 100 μm in diameter, this gelatin microbubble scaffold resembled the lung alveoli in structure and in mechanical properties with good biocompatibility. Effective gemcitabine concentration required to induce cell death in microbubble scaffolds was significantly higher than in 2D culture together with a longer treatment time. Cell death mechanisms were confirmed to be gemcitabine-induced cell apoptosis through Western blotting and real-time polymerase chain reaction. H&E staining and TUNEL assay showed rounded cells with DNA damage in drug-treated scaffolds. Taken together, the cell-containing microbubble scaffolds successfully mimicked lung alveoli in structure and cellular responses after gemcitabine treatment were similar to clinical regimen of treating lung carcinoma. The microbubble scaffold is promising to facilitate anticancer drug discovery by providing more accurate preclinical predictions.

The role of lncRNA MSC-AS1/miR-29b-3p axis-mediated CDK14 modulation in pancreatic cancer proliferation and Gemcitabine-induced apoptosis

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related death due to the failure of traditional therapies. In the present study, we attempted to construct a lncRNA-miRNA-mRNA network which may modulate PDAC cell proliferation and Gemcitabine-induced cell apoptosis starting from CDK14, a new member of the CDK family and an oncogene in many cancers. Based on TCGA data, a significant positive correlation was observed between lncRNA MSC-AS1 and CDK14. Moreover, MSC-AS1 expression was upregulated in PDAC tissues. Higher MSC-AS1 expression was correlated with poorer prognosis in patients with PDAC. MSC-AS1 knockdown in Panc-1 and BxPC-3 cells significantly inhibited the cell proliferation. Moreover, miR-29b-3p, which has been reported to act as a tumor suppressor, was predicted to bind to both MSC-AS1 and CDK14. Contrary to MSC-AS1, higher miR-29b-3p expression was correlated to better prognosis in patients with PDAC. In both PDAC cell lines, miR-29b-3p negatively regulated MSC-AS1 and CDK14. As confirmed using luciferase reporter gene and RIP assays, MSC-AS1 served as a ceRNA for miR-29b-3p to counteract miR-29b-mediated CDK14 repression. MSC-AS1 knockdown inhibited CDK14 protein levels and PDAC proliferation and enhanced gemcitabine-induced cell death and apoptosis while miR-29b-3p inhibition exerted an opposing effect; the effect of MSC-AS1 knockdown was partially attenuated by miR-29b-3p inhibition. Taken together, we demonstrated that MSC-AS1/miR-29b-3p axis modulates the cell proliferation and GEM-induced cell apoptosis in PDAC cell lines through CDK14. We provided a novel experimental basis for PDAC treatment from the perspective of lncRNA-miRNA-mRNA network.

STAT1-mediated inhibition of FOXM1 enhances gemcitabine sensitivity in pancreatic cancer.

Forkhead box protein M1 (FOXM1) was identified as an oncogenic transcription factor and master regulator of tumor progression and metastasis. FOXM1 expression often correlates with poor prognosis and chemotherapy resistance. In the present study, we investigated the association of FOXM1 expression and chemoresistance in pancreatic cancer. Elevated FOXM1 protein levels were associated with gemcitabine chemoresistance in patients with pancreatic cancer. In gemcitabine resistance cell line models of pancreatic cancer, FOXM1 expression increased, which induced gemcitabine chemoresistance in vitro. In pancreatic cancer cells treated with gemcitabine, FOXM1 affected nuclear factor κB (NF-κB) signaling activity. Immunohistochemical analysis demonstrated a negative association of FOXM1 expression and the level of phosphorylated signal transducer and activator of transcription 1 (pSTAT1) in human pancreatic cancer tissues. Dual-luciferase reporter assays and chromatin-immunoprecipitation assays demonstrated that pSTAT1 directly binds to the FOXM1 promoter to down-regulate its transcription. Interferon γ (IFNγ) promoted gemcitabine-induced cell apoptosis and inhibited cell proliferation in vitro and in vivo by FOXM1 inhibition. These data suggested that FOXM1 enhances chemoresistance to gemcitabine in pancreatic cancer. IFNγ could be used to down-regulate the expression of FOXM1 through STAT1 phosphorylation, thereby increasing the sensitivity of pancreatic cancer cells to gemcitabine. These studies suggested the sensitization by IFNγ in pancreatic ductal adenocarcinoma (PDAC) chemotherapy, which requires further clinical studies.

Metformin Inhibits Gemcitabine Induced Apoptosis in Pancreatic Cancer Cell Lines.

Many preclinical and clinical studies are currently evaluating metformin in combination with classical therapeutic agents as anti-cancer therapy. In this study we used three distinct pancreatic cancer cell lines and evaluated cell death by trypan blue assay and Western Blots using antibodies directed against cleaved caspase 3 and PARP. Surprisingly, we observed that 20mM metformin did not enhance, but rather inhibited gemcitabine induced cell death in murine 7265PDA, 6606PDA and 6606l cells. Microenvironmental aspects such as oxygen supply or the pH value did not influence the inhibition of cancer cell apoptosis by metformin. Glucose concentration in the medium, however, had a major effect on the impact of metformin. Medium with 0.5g/L glucose strongly increased metformin induced apoptosis and also prevented the inhibitory effect of metformin on gemcitabine induced cell apoptosis, when compared with medium containing 4.5g/L glucose. We conclude that the combination of metformin with gemcitabine has inappropriate effects for a successful treatment of pancreatic cancer. Thus, it might be more promising to use metformin in combination with other drugs that reduce the uptake or the metabolism of glucose.

RETRACTED ARTICLE: Linc-ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR-124/PTBP1/PKM2 axis

In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

Pyruvate kinase isoenzyme M2 is a therapeutic target of gemcitabine-resistant pancreatic cancer cells

Despite its wide use as a first-line therapeutic agent, gemcitabine has shown limited efficacy in advanced pancreatic cancer due to chemoresistance by as yet unidentified mechanisms. Our goal here was to identify molecular features involved in gemcitabine chemoresistance. Pyruvate kinase M2 (PKM2), a key enzyme of aerobic glycolysis, has recently emerged as an important therapeutic target for cancer treatment. It is involved in the metabolic reprogramming of cancer cells and has previously unexpected non-metabolic functions that are heavily involved in tumor growth and survival. Herein, we report that the chemoresistance of pancreatic cancer to gemcitabine was dependent on PKM2 expression and its non-metabolic function. Knocking-down of PKM2 significantly enhanced gemcitabine-induced cell apoptosis through the activation of caspase 3/7 and PARP cleavage, and this inhibitory activity was associated with p38-mediated activation of p53 phosphorylation at serine 46. Our findings support the potential of PKM2 as a novel target for gemcitabine chemoresistance and suggest the feasibility of combining gemcitabine and PKM2 inhibition for the improved chemotherapy of pancreatic cancer.

PERK1/2 silencing sensitizes pancreatic cancer BXPC-3 cell to gemcitabine-induced apoptosis via regulating Bax and Bcl-2 expression.

BackgroundOur previous study has demonstrated that knockdown of activated ERK1/2(pERK1/2) sensitizes pancreatic cancer cells to chemotherapeutic drug gemcitabine (Gem) treatment. However, the details of this survival mechanism remain undefined. It has also shown that Bcl-2 confers resistance and Bax sensitizes to gemcitabine-induced apoptosis in pancreatic cancer cells. Furthermore, the extracellular signaling-regulated kinase (ERK) signaling pathway regulates Bcl-2/Bax expression ratio. We therefore tested the hypothesis that pancreatic cancer cells are resistant to gemcitabine and this resistance is due to activation of ERK1/2 and subsequent upregulation of Bcl-2 and downregulation of Bax.MethodsPancreatic cancer cell BXPC-3 was used in the study. The effect of pharmacological inhibition of ERK1/2 on resistance of pancreatic cancer cells to apoptosis induced by treatment with gemcitabine was analyzed. The following methods were utilized: TUNEL and ELISA were used to detect apoptosis. Western blot was used to detect the protein expression.ResultsGemcitabine treatment enhanced the activity of ERK1/2 in the BXPC-3 cells. Inhibition of the ERK1/2 by PD98059 could downregulate Bcl-2 and upregulate Bax and was associated with restoration of sensitivity to gemcitabine in BXPC-3 cells. Depletion of endogenous Bcl-2 expression by specific small interfering RNA transfection significantly increased gemcitabine-induced cell apoptosis. Combined treatment with PD98059 and Bax siRNA transfection could decrease gemcitabine-induced ERK1/2 and Bax activation, which subsequently resulted in decreased apoptosis.ConclusionsThe upregulation of ERK1/2-dependent Bcl-2 and downregulation of ERK1/2-dependent Bax can protect human pancreatic cancer cells from gemcitabine-induced apoptosis. Targeting the ERK1/2-Bax/Bcl-2 pathway may in part lead to sensitization of pancreatic cancer to gemcitabine.