LINC00035 Transcriptional Regulation of SLC16A3 via CEBPB Affects Glycolysis and Cell Apoptosis in Ovarian Cancer

Objective. Ovarian cancer (OC) represents the most lethal gynecologic malignancy globally. Over the decades, lncRNAs have been considered as study focuses due to their genome-wide expression through multiple mechanisms in which regulation of target gene transcription through interaction with transcription factors or epigenetic proteins is proven. In the present work, we focus on the functional role of LINC00035 in OC and its regulation mechanism on gene expression. Methods. We collected OC tissues and adjacent tumor-free tissues surgically resected from 67 OC patients. Cultured human OC cell lines SKOV3 and A2780 were assayed for their viability, migration, invasion, apoptosis in vitro using CCK-8 assays, transwell assays, and flow cytometric analysis. OC cell tumorigenesis in vivo was evaluated by mouse xenograft experiments. Glycolysis was evaluated by glucose uptake, lactate release, and ATP production assays. Luciferase activity assay, RNA immunoprecipitation (RIP), and RNA pull-down were performed to confirm the interactions among LINC00035, CEBPB, and SLC16A3. Results. LINC00035 was upregulated in OC tissues. LINC00035 knockdown was shown to repress SKOV3 and A2780 cell viability, migration, invasion, induce their apoptosis, and reduce glucose uptake, lactate release, and ATP production. LINC00035 could recruit CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 transcription. SLC16A3 was upregulated in OC tissues. SLC16A3 knockdown exerted similar effects on SKOV3 and A2780 cells as LINC00035 knockdown. Rescue experiments found SLC16A3 overexpression resisting to LINC00035 knockdown on SKOV3 and A2780 cell viability, migration, invasion, apoptosis, glucose uptake, lactate release, and ATP production. Results also showed LINC00035 knockdown could inhibit OC cell tumorigenesis in vivo. Conclusion. The study reveals that LINC00035 promotes OC progression by regulating glycolysis and cell apoptosis through CEBPB-mediated transcriptional promotion of SLC16A3.

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Group III phospholipase A2 downregulation attenuated survival and metastasis in ovarian cancer and promotes chemo-sensitization

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01985-9 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Upasana Ray ◽

Debarshi Roy ◽

Ling Jin ◽

Prabhu Thirusangu ◽

Julie Staub ◽

...

Keyword(s):

Ovarian Cancer ◽

Cell Viability ◽

Phospholipase A2 ◽

Mtt Assay ◽

Metabolic Inhibitor ◽

Autophagic Flux ◽

Ciliated Cells ◽

Group Iii

Abstract Background Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. Methods CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited cancer progression. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. Conclusions Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.

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Berberine Improves Chemo-Sensitivity to Cisplatin by Enhancing Cell Apoptosis and Repressing PI3K/AKT/mTOR Signaling Pathway in Gastric Cancer

Frontiers in Pharmacology ◽

10.3389/fphar.2020.616251 ◽

2020 ◽

Vol 11 ◽

Author(s):

Yingying Kou ◽

Bending Tong ◽

Weiqing Wu ◽

Xiangqing Liao ◽

Min Zhao

Keyword(s):

Gastric Cancer ◽

Drug Resistance ◽

Multidrug Resistance ◽

Cell Viability ◽

Cancer Cells ◽

Cell Apoptosis ◽

Mtor Signaling ◽

Multi Drug Resistance ◽

Gastric Cancer Cells

Gastric cancer is one of the most common malignancies ranks as the second leading cause of cancer-related mortality in the world. Cisplatin (DDP) is commonly used for gastric cancer treatment, whereas recurrence and metastasis are common because of intrinsic and acquired DDP-resistance. The aim of this study is to examine the effects of berberine on the DDP-resistance in gastric cancer and explore the underling mechanisms. In this study, we established the DDP-resistant gastric cancer cells, where the IC50 values of DDP in the BGC-823/DDP and SGC-7901/DDP were significantly higher than that in the corresponding parental cells. Berberine could concentration-dependently inhibited the cell viability of BGC-823 and SGC-7901 cells; while the inhibitory effects of berberine on the cell viability were largely attenuated in the DDP-resistant cells. Berberine pre-treatment significantly sensitized BGC-823/DDP and SGC-7901/DDP cells to DDP. Furthermore, berberine treatment concentration-dependently down-regulated the multidrug resistance-associated protein 1 and multi-drug resistance-1 protein levels in the BGC-823/DDP and SGC7901/DDP cells. Interestingly, the cell apoptosis of BGC-823/DDP and SGC-7901/DDP cells was significantly enhanced by co-treatment with berberine and DDP. The results from animals also showed that berberine treatment sensitized SGC-7901/DDP cells to DDP in vivo. Mechanistically, berberine significantly suppressed the PI3K/AKT/mTOR in the BGC-823/DDP and SGC-7901/DDP cells treated with DDP. In conclusion, we observed that berberine sensitizes gastric cancer cells to DDP. Further mechanistic findings suggested that berberine-mediated DDP-sensitivity may be associated with reduced expression of drug transporters (multi-drug resistance-1 and multidrug resistance-associated protein 1), enhanced apoptosis and repressed PI3K/AKT/mTOR signaling.

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TRIAP1 Inhibition Activates the Cytochrome c/Apaf-1/Caspase-9 Signaling Pathway to Enhance Human Ovarian Cancer Sensitivity to Cisplatin

Chemotherapy ◽

10.1159/000501633 ◽

2019 ◽

Vol 64 (3) ◽

pp. 119-128 ◽

Cited By ~ 1

Author(s):

Tian-Mei Zhang

Keyword(s):

Ovarian Cancer ◽

Nude Mice ◽

Cell Apoptosis ◽

Caspase 9 ◽

Ovarian Carcinoma Cell Line ◽

Skov3 Cells ◽

Cyt C ◽

Sirna Group

Objective: To investigate whether TRIAP1inhibition affects the ovarian cancer cell resistance to cisplatin (DDP) via the Cyt c/Apaf-1/caspase-9 pathway by in vitro and in vivo experiments. Methods: CCK8 assay was performed to find out how treatment with both TRIAP1 siRNA and DDP affects the cell viability of SKOV3 cells and DDP-resistant human ovarian carcinoma cell line SKOV3/DDP. SKOV3/DDP cells were transfected with control siRNA or TRIAP1 siRNA before 24 h of treatment with DDP (5 μg/mL). Flow cytometry was employed to detect cell apoptosis and Western blot to examine the expressions of Cyt c/Apaf-1/caspase-9 pathway-related proteins. SKOV3/DDP cells transfected with control siRNA or TRIAP1 siRNA were subcutaneously injected into BALB/c-nu/nu nude mice followed by the intraperitoneal injection of DDP (4 mg/kg). Cyt c/Apaf-1/caspase-9 pathway in transplanted tumors was detected by immunohistochemistry. Results: TRIAP1 expression declined in SKOV3 cells when compared with SKOV3/DDP cells. The proliferation rate was lower in SKOV3/DDP cells transfected with TRIAP1 siRNA combined with treatment of DDP (1, 2, 4, 6, 8, 16, 32 μg/mL) than in those transfected with control siRNA. Moreover, the TRIAP1 siRNA group had an increased SKOV3/DDP cell apoptosis rate with the activation of the Cyt c/Apaf-1/caspase-9 pathway. During DDP treatment, nude mice in TRIAP1 siRNA group had slower growth and smaller size of transplanted tumor than those in control siRNA group, with increased expression of Cyt c, Apaf-1, and caspase-9. Conclusion: TRIAP1 inhibition may enhance the sensitivity of SKOV3/DDP cells to cisplatin via activation of the Cyt c/Apaf-1/caspase-9 pathway.

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Synergistic Induction of Erlotinib-Mediated Apoptosis by Resveratrol in Human Non-Small-Cell Lung Cancer Cells by Down-Regulating Survivin and Up-Regulating PUMA

Cellular Physiology and Biochemistry ◽

10.1159/000374030 ◽

2015 ◽

Vol 35 (6) ◽

pp. 2255-2271 ◽

Cited By ~ 40

Author(s):

Peipei Nie ◽

Weicheng Hu ◽

Tao Zhang ◽

Yiju Yang ◽

Benxin Hou ◽

...

Keyword(s):

Lung Cancer ◽

Cell Viability ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Cell Apoptosis ◽

Flow Cytometric Analysis ◽

Survivin Expression ◽

Small Cell ◽

Small Cell Lung ◽

Puma Expression

Background/Aim: Treatment of human non-small-cell lung cancer (NSCLC) often involves uses of multiple therapeutic strategies with different mechanisms of action. Here we found that resveratrol (RV) enhanced the anti-tumor effects of epidermal growth factor receptor (EGFR) inhibitor erlotinib in NSCLC cells. Methods: Cell viability was measured by MTT assay and clonogenicity assay. Western blot was applied to assess the protein expression levels of target genes. Cell apoptosis was monitored by AnnexinV-FITC assay and sub-G1 population assay. Intracellular ROS were measured by flow cytometric analysis. Cell caspase activities were carried out by fluorometric assays. Results: Exposure of H460, A549, PC-9 and H1975 cells to minimal or non-toxic concentrations of RV and erlotinib synergistically reduced cell viability, colony formation and induced cell apoptosis. Furthermore, RV synergistically enhanced erlotinib-induced apoptosis was involved in ROS production. Additionally, co-treatment with RV and erlotinib repressed the expressions of anti-apoptosis proteins, such as survivin and Mcl-1, whereas promoted p53 and PUMA expression and caspase 3 activity. Moreover, the combination was also more effective at inhibiting the AKT/mTOR/S6 kinase pathway. Subsequently, small interfering RNA (siRNA) depletion of PUMA and overexpression of survivin significantly attenuated NSCLC cells apoptosis induced by the combination of the two drugs. Conclusion: Our findings suggested that RV synergistically enhanced the anti-tumor effects of erlotinib in NSCLC cells were involved in decrease of survivin expression and induction of PUMA expression. In conclusion, based on the observations from our study, we indicated that the combined administration of these two drugs might be considered as a novel therapeutic regimen for treating NSCLC.

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Effect of therapeutic GAS6/AXL inhibition of tumor and stromal cells on DNA damage and response to chemotherapy in ovarian cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e14676 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e14676-e14676 ◽

Cited By ~ 2

Author(s):

Mary M Mullen ◽

Elena Lomonosova ◽

Hollie M Noia ◽

Lei Guo ◽

Lindsay Midori Kuroki ◽

...

Keyword(s):

Ovarian Cancer ◽

Dna Damage ◽

Neoadjuvant Chemotherapy ◽

Cell Viability ◽

Mouse Models ◽

Stromal Cells ◽

Predictive Biomarker ◽

Response To Chemotherapy

e14676 Background: Ovarian cancer is the leading cause of death due to gynecologic malignancy. Biomarkers to predict chemoresponse and novel therapies to target these proteins would be practice changing. We aim to establish serum and tissue GAS6 as a predictive biomarker of chemoresponse and to determine if AXL inhibition through sequestration of its ligand, GAS6, with AVB-S6-500 (AVB) can improve chemoresponse. Methods: AVB was supplied by Aravive Biologics. High grade serous ovarian cancer (HGSOC) tumor samples were obtained pre- and post-neoadjuvant chemotherapy. AXL and GAS6 expression were evaluated by immunohistochemistry and serum concentration. In vitro viability and clonogenic assays were performed on chemoresistant tumor (OVCAR8, OVCAR5, COV62, and POV71-hTERT) and stromal cells (CAF86) treated with chemotherapy +/- AVB. Mouse models (OVCAR8, PDX, OVCAR5) were used to determine if the combination of chemotherapy + AVB reduced tumor burden. Immunofluorescent assays targeting ɣH2AX were used to evaluate DNA damage. Results: Patients with high pretreatment tumor GAS6 expression ( > 85%, n = 7) or serum GAS6 concentrations ( > 25ng/mL, n = 13) were more likely to be resistant to neoadjuvant chemotherapy than those with low tumor GAS6 expression ( < 45%, n = 4) (P = 0.010) or low serum GAS6 concentrations ( < 15ng/mL, n = 5) (P = 0.002). Carboplatin plus AVB (2µM, 5µM) and paclitaxel plus AVB (1µM) resulted in decreased cell viability and clonogenic growth compared to chemotherapy alone (p < 0.05) in all tumor and stromal cell lines. Synergism was seen between carboplatin+AVB and paclitaxel+AVB with a weighted combination index < 1. In vivo tumor mouse models treated with chemotherapy+AVB had significantly smaller subcutaneous and intraperitoneal (IP) tumors than those treated with chemotherapy alone (3.1mg vs 64mg, P = 0.003 OVCAR8; 62mg vs 157mg, P = 0.0108 PDX subcutaneous model; 0.05mg vs 0.3669mg, P < 0.001 OVCAR5 IP model). Increased DNA damage was noted in tumor and stromal cells treated with carboplatin+AVB compared to carboplatin alone (OVCAR8, COV362, CAF86 P < 0.001). Conclusions: High GAS6 is associated with lack of neoadjuvant chemoresponse in HGSOC patients. The combination of chemotherapy with AVB decreases tumor cell viability, tumor growth, and an increase in DNA damage response.

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NDRG2 gene expression pattern in ovarian cancer and its specific roles in inhibiting cancer cell proliferation and suppressing cancer cell apoptosis

10.21203/rs.2.20090/v2 ◽

2020 ◽

Author(s):

Fenhong Kang ◽

Yanlong Wang ◽

Yaping Luo ◽

Yongjun Zhang

Keyword(s):

Cell Cycle ◽

Ovarian Cancer ◽

Cell Viability ◽

Cancer Cells ◽

Cancer Cell ◽

Cell Apoptosis ◽

Colony Formation ◽

G1 Phase ◽

Ovarian Cancer Cells ◽

Cycle Arrest

Abstract Background: The cancer cell metastasis and the acquisition of chemotherapy resistance remain huge challenge for ovarian cancer treatment. Previously, N-myc downstream-regulated gene 2 (NDRG2) serves as a tumor suppressor for many cancers. Here, we attempted to investigate the specific roles of NDRG2 in ovarian cancer. Methods: The expression levels of NDRG2 were detected by qRT-PCR or Immunoblotting. CCK-8 assay was employed to examine the cell viability of ovarian cancer cells. The colony formation ability was determined by colony formation assay. Flow cytometry analyses were performed to detect the cell apoptosis and cell cycle. Xenograft tumor assay was performed to detect the in vivo function of NDRG2. Results: We revealed that NDRG2 mRNA expression and protein levels were downregulated within both ovarian cancer tissues and cell lines. The overexpression of NDRG2 dramatically inhibited the cell viability and colony formation and tumor growth, whereas promoted the cell apoptosis, cell cycle arrest in G1 phase within ovarian cancer cells. More importantly, NDRG2 overexpression significantly enhanced the suppressive roles of cisplatin (DDP) in ovarian cancer cell viability. On the contrary, NDRG2 silence exerted opposing effects on ovarian cancer cells. Conclusions: In summary, we provide a solid experimental basis demonstrating the tumor-suppressive effects of NDRG2 in inhibiting the cell proliferation, enhancing the cell apoptosis, eliciting the cell cycle arrest in G1 phase, and promoting the suppressive effects of DDP on the viability of ovarian cancer cells. NDRG2 administration presents a potent adjuvant treatment for ovarian cancer therapy.

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NEAT1 Overexpression Indicates a Poor Prognosis and Induces Chemotherapy Resistance via the miR-491-5p/SOX3 Signaling Pathway in Ovarian Cancer

Frontiers in Genetics ◽

10.3389/fgene.2021.616220 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xinzhuan Jia ◽

Lan Wei ◽

Zhengmao Zhang

Keyword(s):

Ovarian Cancer ◽

Cell Lines ◽

Cell Apoptosis ◽

Expression Patterns ◽

Chemotherapy Resistance ◽

Direct Interaction ◽

Luciferase Reporter ◽

Tissue Samples ◽

Qrt Pcr

BackgroundAccumulated studies have reported that dysregulated long non-coding RNAs (lncRNAs) are crucial in ovarian cancer (OC) initiation and development. However, detailed biological functions of lncRNA NEAT1 during the progression of OC remains to be uncovered.PurposeOur aim was to identify the role of NEAT1 in cisplatin resistance of ovarian cancer and the underlying mechanisms.MethodsThe expression patterns of NEAT1 in OC cell lines and tissue samples were identified by qRT-PCR. The cisplatin (DDP) sensitivity of OC cells was detected by MTT and CCK8 assay, while OC cell apoptosis and cell cycle were detected using flow cytometer assays. In addition, effects of NEAT1 on tumor growth were determined by xenograft tumor model. Luciferase reporter assay was conducted to prove the regulatory relation of miR-491-5p, NEAT1, and SOX3. Importantly, the expression of NEAT1 in exosomes from cisplatin-resistant patients was also determined by using qRT-PCR.ResultsIn this study, upregulated NEAT1 was detected in OC cell lines and tissues. Meanwhile, NEAT1 was also increased in cisplatin-resistant OC cell lines and tissues. Upregulation of NEAT1 inhibited cisplatin-induced OC cell apoptosis and promoted cell proliferation, while knockdown of NEAT1 played the opposite role. These effects were also observed in vivo. Furthermore, direct interaction was observed between NEAT1 and miR-491-5p. NEAT1 led to the upregulation of miR-491-5p-targeted SOX3 mRNA. Importantly, this study also showed upregulated NEAT1 expression in serum exosomes derived from cisplatin-resistant patients.ConclusionNEAT1 is vital in the chemoresistance of ovarian cancer through regulating miR-491-5p/SOX3 pathway, showing that NEAT1 might be a potential target for OC resistance treatment.

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Mel Aggravates Pyroptosis Induced by Temozolomide Through Inhibiting NRF2-are Pathway in Glioblastoma

10.21203/rs.3.rs-35576/v1 ◽

2020 ◽

Author(s):

Hao Pan ◽

Handong wang ◽

Qiang Wang ◽

Wenhao Niu ◽

Qi Wu ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Combined Treatment ◽

Flow Cytometric Analysis ◽

Glioma Cells ◽

Mtor Pathway ◽

Tumor Growth Inhibition ◽

Dependent Manner ◽

Related Protein

Abstract Background:Glioblastoma(GBM) is a common malignant tumor of the brain. It has been verified that melatonin(MEL) can inhibit glioma proliferation. But its mechanism has not been fully elucidated. We intend to examine the mechanism of MEL on glioma cells from the perspective of pyroptosis and Nrf2. Methods:Expression of MEL receptor in glioma was detected by western blot. GBM cell viability treated with temozolomide(TMZ) plus MEL was detected by CCK-8. Pyroptosis rate was determinate by flow cytometric analysis. Western blot was used to detect the Nrf2 and pyroptosis related protein level after MEL treatment. Orthotopic tumor growth inhibition study was performed to further investigate the tumor inhibition effect of TMZ plus MEL.Results:We first confirmed MEL receptor was abundant in glioma tissue and cell lines. After combined treatment of TMZ and MEL, cell viability decreased significantly as compared to those of TMZ treatment alone. Also, the ratio of pyroptosis and ROS level increased, followed by elevated expression of pyroptosis related protein. Furthermore, MEL can induce a diminution of Nrf2 expression in glioma in dose- and time-dependent manner. TMZ can increase Nrf2-ARE pathway expression, which also can be deprived by MEL. Its inhibition of Nrf2 depends on dephosphorylation of IGF-1/AKT/mTOR pathway. More importantly, after overexpression of Nrf2 in glioma cells, the level of pyroptosis-related protein elevated by MEL decreased, suggesting that the effect of MEL on promoting pyroptosis is dependent on its inhibition of Nrf2. In vivo results further confirmed that MEL plus TMZ induced significantly decreased tumor size and increased pyroptosis rate, but had no significant effect on mouse body weight, ALT, AST.Conclusion:MEL can inhibit the phosphorylation of IGF-1/AKT/mTOR pathway at millimol level, which further reduces the expression of Nrf2 and promotes pyroptosis of glioma cells. Considering the modest efficacy of TMZ chemotherapy, MEL can be considered as a potential chemotherapy sensitizer to improve the chemotherapy effect of glioma.

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