Buzhong Yiqitang Regulates Apoptosis via Nrf2/ROS Pathway to Alleviate Cisplatin Resistance in A549/DDP Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Buzhong Yiqitang-containing serum in alleviating the cisplatin resistance in human non-small cell lung cancer （A549/DDP） cells via regulating the nuclear factor E2-related factor 2 （Nrf2）/reactive oxygen species （ROS） signaling pathway. MethodThe serum containing Buzhong Yiqitang was prepared and A549/DDP cells were cultured and randomly grouped： blank （10% blank serum）， cisplatin （10% blank serum+20 mg·L-1 cisplatin）， Buzhong Yiqitang （10% Buzhong Yiqitang-containing serum+20 mg·L-1 cisplatin）， ML385 （10% blank serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， Buzhong Yiqitang+ML385 （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 ML385+20 mg·L-1 cisplatin）， tertiary butylhydroquinone （TBHQ） （10% blank serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）， and Buzhong Yiqitang+TBHQ （10% Buzhong Yiqitang-containing serum+5 μmol·L-1 TBHQ+20 mg·L-1 cisplatin）. The median inhibitory concentration （IC50） of cisplatin in each group was determined by the cell counting kit-8 （CCK-8） method and the resistance index （RI） was calculated. The apoptosis rate was detected by flow cytometry. The ROS content of each group was determined with the DCFH-DA fluorescence probe. Western blot was employed to determine the protein levels of Nrf2， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）， cytochrome C （Cyt C）， and B-cell lymphoma-2 （Bcl-2）. ResultCompared with those in the cisplatin group， the IC50 and RI of A549/DDP cells to cisplatin in Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups decreased （P˂0.05）. Compared with the blank group， the cisplatin， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 groups showed increased apoptosis rate of A549/DDP cells （P˂0.05）. Compared with the blank group， cisplatin promoted the expression of Nrf2 （P˂0.05）. Compared with the cisplatin group， Buzhong Yiqitang， ML385， and Buzhong Yiqitang+ML385 inhibited the expression of Nrf2 （P<0.05）， elevated the ROS level （P˂0.05）， up-regulated the protein levels of cleaved Caspase-3 and Cyt C， and down-regulated the protein level of Bcl-2 （P<0.05）， which were the most significant in the Buzhong Yiqitang+ML385 group. Compared with the cisplatin group， the TBHQ group showed increased IC50 and RI of cisplatin （P<0.05）， decreased apoptosis rate of A549/DDP cells （P<0.05）， up-regulated protein levels of Nrf2 and Bcl-2 （P<0.05）， lowered level of ROS （P˂0.05）， and down-regulated protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. Compared with the TBHQ group， Buzhong Yiqitang+TBHQ decreased the IC50 and RI of cisplatin in A549/DDP cells （P<0.05）， increased the apoptosis rate （P<0.05）， down-regulated the protein levels of Nrf2 and Bcl-2 （P<0.05）， increased ROS （P˂0.05）， and up-regulated the protein levels of cleaved Caspase-3 and Cyt C （P<0.05）. ConclusionBuzhong Yiqitang induced apoptosis by inhibiting Nrf2/ROS pathway to alleviate cisplatin resistance in A549/DDP cells.

High expression of MYH9 inhibits apoptosis of non-small cell lung cancer cells through activating the AKT/c-Myc pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC).@*METHODS@#Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice.@*RESULTS@#MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05).@*CONCLUSION@#High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.

Research progress on the relationship between mitochondrial function and oral squamous cell carcinoma / 口腔疾病防治

@#Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck. In recent years, the incidence rate has been increasing. Mitochondria are dynamic organelles involved in various cell behaviors in eukaryotic cells. Mitochondrial dysfunction is closely related to tumor development. As a switch that determines cancer cell death, targeting mitochondria has become the focus of OSCC treatment. This article reviews the relationship between mitochondria and tumorigenesis and development, OSCC treatment, and cisplatin resistant OSCC. Current studies have found that mitochondrial dysfunction promotes cell carcinogenesis, and the mitochondrial morphology and function of cancer cells are significantly changed. The increase of mitochondrial fission improves the invasiveness of cancer cells, and mitophagy dysfunction can induce cancer cell apoptosis. The emergence of drugs and the development of nanotechnology in targeted drug delivery systems have opened up new methods for targeting mitochondria to treat OSCC, reducing the side effects of systemic medication. The cisplatin resistance of OSCC is generated through the mitochondrial pathway, and the mitochondrial function and mutation mechanism of mitochondrial DNA are clarified in order to provide new ideas for targeting mitochondria to treat cisplatin resistant OSCC.

Effects of miR-485-5p on cisplatin-resistant ovarian cancer cell lines via targeting EGFR / 中华内分泌外科杂志

Objective:To explore the effects of miR-485-5p on cisplatin-resistant ovarian cancer cells and it’s mechanism.Methods:RT-qPCR was used to detect the expression of miR-485-5p in human normal ovarian epithelial cell line (IOSE-80) and ovarian cancer cell lines (A2780,SKOV3,OVCAR3,OVCA433) . Cisplatin (DDP) -resistant ovarian cancer cells were constructed and the expression of miR-485-5p and EGFR was detected. CCK8 assay was used to detect cell proliferation ability in each group. Cell apoptosis was measured by flow cytometry.Results:Compared with IOSE-80 cell, miR-485-5p expression was decreased in each ovarian cancer cell lines, while the expression of EGFR was increased (all P<0.05) .The expression of miR-485-5p in SKOV3/DDP cell lines was further decreased while EGFR expression was further increased than that in SKOV3 cells ( P<0.05) . Transfection of miR-485-5p mimic into SKOV3/DDP cells could inhibit cell proliferation and induce apoptosis, but the results were reversed when cells were transfected with miR-485-5p inhibitor (all P<0.05) . The proliferation was increased while apoptosis was decreased in EGFR transfected SKOV3/DDP cells, but this effects was partially offseted by miR-485-5pmimic. Conclusion:miR-485-5p participates in the regulation of cisplatin resistance of ovarian cancer cells, and overexpression of miR-485-5p can promote the chemosensitivity of ovarian cancer, which may be achieved through negative regulation of EGFR.

Screening of Molecular Markers of Cisplatin Resistance in Lung Adenocarcinoma and Functional Verification Based on TCGA Database / 肿瘤防治研究

Objective To explore the related genes that play a key regulatory role in cisplatin resistance in lung adenocarcinoma. Methods Bioinformatics methods were used to download the differentially-expressed genes between cisplatin sensitive group and drug resistant group in patients with lung adenocarcinoma in TCGA database and GDSC database. GO function analysis and KEGG pathway enrichment analysis were carried out to analyze the differentially-expressed genes. The protein-protein interaction network was constructed and hierarchical cluster analysis was carried out to screen the key genes. The key genes were verified at the cell level by real-time fluorescence quantitative PCR and ELISA. Then the expression of the selected key gene in A549/DDP cells was silenced by siRNA and its sensitivity to cisplatin was detected. Results We screened out 178 differentially-expressed genes. After cluster analysis, CXCL9, CXCL10, NKX2-1 and SFTPA1 were regarded as the key genes of cisplatin resistance in lung adenocarcinoma. CXCL10 was temporarily selected for subsequent verification and function experiment. The mRNA expression of CXCL10 in A549/DDP cells was significantly higher than that in A549 cells (P < 0.001), and the expression of CXCL10 protein in the supernatant of A549/DDP cells was higher than that in A549 cells, which were consistent with the prediction of bioinformatics. The sensitivity of A549/DDP cells to DDP increased after silencing CXCL10 expression. Conclusions CXCL10 is a key gene to regulate cisplatin resistance in lung adenocarcinoma. Downregulating the expression of CXCL10 can become a potential target for reversing cisplatin resistance in lung adenocarcinoma.

Astragalus Polysaccharides Improve Cisplatin Resistance by Inhibiting EMT of Lung Adenocarcinoma A549/DDP Cells Transplanted into Nude Mice / 中国实验方剂学杂志

ObjectiveTo investigate the inhibitory effect of Astragalus polysaccharide （APS） on epithelial-mesenchymal transition （EMT） induced by transforming growth factor-β1 （TGF-β1） in cisplatin （DDP）-resistant lung adenocarcinoma cell line A549/DDP cells transplanted into nude mice and the molecular mechanism in improving DDP resistance. MethodBALB/c nude mice were randomly divided into a blank group， a model group， a DDP group， and a combination group （APS combined with DDP）. A549/DDP cells were infected with TGF-β1-overexpressed lentiviral vector and the negative control. The infected cells were inoculated subcutaneously in nude mice. The A549/DDP cells with TGF-β1 gene overexpression were inoculated into all groups except the control group with negative TGF-β1 gene overexpression. The drug intervention was performed eight days after cell inoculation. The mice in the combination group received intragastric administration of APS （0.3 g·kg-1·d-1） and intraperitoneal injection of cisplatin （0.003 5 g·kg-1）， and those in the cisplatin group received intraperitoneal injection of cisplatin （0.003 5 g·kg-1）. After 32 days of cell inoculation， the nude mice were killed and the tumor tissues and lungs were collected. The tumor weight was recorded and the inhibition rate was calculated. The number of metastatic nodules of the lung tumor on the whole slide was counted under the microscope. Immunohistochemistry， Western blot， and real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） were used to detect the protein and gene expression of EMT molecular markers α-catenin and N-cadherin， and tumor drug resistance markers human lung resistance protein （LRP）， multidrug resistance-associated protein （MRP）， and P-glycoprotein （P-gp） in the transplanted tumor. ResultCompared with the blank group， the model group showed increased tumor weight and metastatic nodules of the lung tumor （P<0.05）， decreased protein and mRNA expression of α-catenin （P<0.05）， and elevated protein and mRNA expression of N-cadherin， LRP， MRP， and P-gp （P<0.05）. Compared with the model group and the cisplatin group， the combination group showed reduced tumor weight and metastatic nodules of the lung tumor （P<0.05）， increased protein and mRNA expression of α-catenin （P<0.05）， and decreased protein and mRNA expression of N-cadherin， LRP， MRP， and P-gp （P<0.05）. ConclusionAPS can inhibit the growth and metastasis of the transplanted tumor of lung adenocarcinoma and improve cisplatin resistance， which may be related to the inhibition of EMT of tumor cells.

Potential mechanism of cisplatin resistance in non-small cell lung cancer A549 cells analyzed by the whole-transcriptome / 中国胸心血管外科临床杂志

@#Objective    To reveal the potential mechanism of cisplatin resistance in non-small cell lung cancer A549 cells by comparing the expression profiles of wild-type A549 cells and cisplatin-resistant A549 cells (A549/DPP) through whole transcriptome sequencing analysis. Methods    The cisplatin resistant A549 (A549/DDP) cell line was first established. Then, the whole-transcriptome analysis was conducted both on A549 and A549/DDP cells. Next, the differentially expressed RNAs of lncRNA-seq, circRNA-seq, and miRNA-seq data were identified, respectively, followed by functional enrichment analysis. Finally, a comprehensive analysis based on the whole transcriptome data was performed and the construction of the ceRNA network was carried out. Results    A total of 4 517 lncRNA, 123 circRNA, and 145 miRNA were differentially expressed in A549/DDP cells compared with the A549 cell line. These different RNAs were significantly enriched in cancer-related pathways. The ceRNA network contained 12 miRNAs, 4 circRNAs, 23 lncRNAs, and 9 mRNA nodes, of which hsa-miR-125a-5p and hsa-miR-125b-5p were important miRNAs based on the topological analysis. Conclusion    Tumor necrosis factor signaling pathway and p53 signaling pathway are involved in A549/DPP resistance. Hsa-miR-125a-5p and hsa-miR-125b-5p may be potential targets for reversing cisplatin resistance.

Inhibitory effects of polyphyllins I and VII on human cisplatin-resistant NSCLC via p53 upregulation and CIP2A/AKT/mTOR signaling axis inhibition / 中国天然药物

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a human oncoprotein that is overexpressed in multiple kinds of cancers including non-small cell lung cancer (NSCLC). CIP2A plays an 'oncogenic nexus' to participate in the tumorigenesis and chemoresistance in several cancer types. AKT and mTORC1 overactivation are detected in NSCLC and many other cancers. Previous studies found that the CIP2A/AKT/mTOR pathway controls cell growth, apoptosis, autophagy process. Polyphyllin I (PPI) and polyphyllin VII (PPVII) are natural components extracted from Paris polyphylla that display anti-cancer properties. In the present study, we investigated whether PPI and PPVII can be used in the cisplatin (DDP)-resistant human NSCLC cell line A549/DDP. Results demonstrated that PPI and PPVII treatment significantly suppressed A549/DDP cell proliferation, migration, invasion and EMT, induced apoptosis and autophagy. Further examination of the mechanism revealed that the PPI and PPVII significantly upregulated the p53, induced caspase-dependent apoptosis and suppressed the CIP2A/AKT/mTOR pathway. The activation of autophagy was mediated through PPI and PPVII induced inhibition of mTOR. We propose that PPI and PPVII might be developed as candidate drugs for DDP-resistant NSCLC.

Effect of Buzhong Yiqi Tang on Cisplatin Resistance A549/DDP Cells in Nude Mice with Lung Adenocarcinoma / 中国实验方剂学杂志

Objective: To investigate the effect of Buzhong Yiqi Tang on cisplatin resistance tumor cells A549/DDP in nude mice with lung adenocarcinoma, and determine the optimal concentration. Method: The 36 BALB/C nude mice were randomly divided into no-load group, model group, cisplatin group, low-dose Buzhong Yiqi Tang group (1.46 g · kg-1), medium-dose Buzhong Yiqi Tang group (2.92 g · kg-1) and high-dose Buzhong Yiqi Tang group (5.84 g · kg-1). The no-load group was inoculated with A549/DDP cells transfected by uninfected lentiviral transforming growth factor-β1(TGF-β1), and the other groups were inoculated with TGF-β1 over-expression A549/DDP cells. After the intervention, the nude mice were put to death. The tumors of mice were weighed, and the inhibition rate was calculated, liver, spleen and lung metastases of tumors were counted under microscope, and pathological observation was made for lung histomorphological changes. Immunohistochemistry, Western blot and Real-time polymerase chain reaction(Real-time PCR) were used to detect the expressions of lung resistance-related protein (LRP), multidrug resistance-related protein (MRP), P-glycoprotein (P-gp) in transplantation tumor. Result: Compared with the model group, the weight of tumors and the number of pulmonary metastatic nodules decreased in the Buzhong Yiqi Tang intervention group. With the increase of the dosage of Buzhong Yiqi Tang, the weight of tumors gradually decreased, while the inhibition rate increased. The effect of the high-dose group was the most significant (PPPConclusion: Buzhong Yiqi Tang can inhibit the growth and metastasis of transplanted tumors, alleviate lung injury and improve the drug resistance of A549/DDP to cisplatin, with a certain dose-effect relationship. High-dose concentration (5.84 g ·kg-1) was the best concentration in nude mice.

Expression of lncRNA PVT1 in colorectal cancer tissues and cells and its effect on chemo-sensitivity to cisplatin and the possible mechanisms / 中国肿瘤生物治疗杂志

@#Objective: To study the regulatory effects and possible mechanism of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) on chemotherapy sensitivity to cisplatin (DDP) of colorectal cancer (CRC)．Methods: A total of 112 pairs of matched cancer and adjacent non-cancerous tissues were obtained from the CRC patients who underwent surgical resection in the First Affiliated Hospital of Xinxiang Medical University betweenApril 2006 and March 2011.All specimens were confirmed by pathological examinations. Tumor tissues and corresponding adjacent non-cancerous tissues from 30 cisplatin-sensitive CRC patients and 30 cisplatin-resistant patients were selected. Human CRC cell lines (HT29, SW480, HCT116, RKO and LoVo) and normal colonic epithelial cell line NCM460 were also collected for this study; and DDP-resistant RKO/DDP and LoVo/DDP cell lines were constructed. siPVT1, siNC, LV-PVT1 and LV-NC were transfected into LoVo and RKO cells or LoVo/DDP and RKO/DDP cells using lipofectamineTM2000. The expression of lncRNA PVT1 in CRC tissues and cells was tested by Real-time qPCR. CCK-8 assay, flow cytometry and WB were performed to test the effect of PTV1 knockout or enforcement on cell proliferation, apoptosis and expressions of apoptosis-related proteins, respectively. The CRC subcutaneous transplanted xenograft model was established on athymic nude mice to study the effect of PVT1 over-expression on tumor growth and DDP resistance. Results: PVT1 was highly expressed in the cancer tissues and CRC cells, and its expression was positively associated with cisplatin resistance of CRC. After knockdown of PVT1, the proliferation of cisplatinresistant CRC cells was significantly suppressed, while the apoptosis was significantly enhanced (P<0.05 or P<0.01); Mechanically, the levels of drug resistance-associated molecules, including MDR1 and MRP1, as well as the expression of anti-apoptotic Bcl-2 were significantly downregulated whereas the levels of pro-apoptotic Bax and cleaved caspase-3 were increased in PVT1-silenced DDP-resistant CRC cells. Over-expression of PVT1 reversely increased proliferation and decreased apoptosis of CRC cells (P<0.05 or P<0.01). In addition, PVT1 over-expression in CRC cells significantly promoted DDP-resistance in vivo (P<0.05). Conclusion: Collectively, knockdown of PVT1 expression can significantly suppress cell proliferation and promote apoptosis of DDP-resistant CRC cells. Overexpression of PVT1 can significantly promote the growth of CRC cells in vitro and transplanted xenograft in vivo. PVT1 regulates endogenous apoptosis pathways and further promotes the sensitivity of CRC cells to cisplatin chemotherapy via inhibiting the expressions of MDR1 and MRP1.

Preliminary mechanism study on overexpression of PDCD4 in reversing cisplatin resistance in gastric cancer / 医学研究生学报

Objective Gastric cancer is the most common cancer in the world. In China, Patients with gastric cancer are mostly treated with platinum-based chemotherapy. Programmed cell death 4 (PDCD4) was found as an important proapoptosis recently, the aim of the present study was to investigate the role of PDCD4 reversed the apoptosis induced by cisplatin in gastric cancer cell. The study will provide the target marker for treatment and diagnosis of cisplatin resistance in gastric cancer.Methods Stable transfection with pCMV-PDCD4 vector into human cisplatin resistance gastric cancer cell line-SGC7901/DDP; the cells were divided into control group, over-expression group, control with cisplatin group, over-expression with cisplatin group for following experiments. Hoechst dying with immunofluorescence and flow cytometry were used to measure the cell apoptosis in vitro; Real-time PCR was used to detect the mRNA expression levels of PDCD4, and the protein levels of PDCD4, pAK, pGSK3β, BCL-2 and Bak were detected by Western blot. The cells were divided into vector group, PDCD4 group, PDCD4 with activator group for detect the level of PARP(C) by Western blot.Results Compared with control group, the Results of real-time PCR and western blot were showed the level of PDCD4 was augmented in over-expression group (also in the over-expression with cisplatin group), which was indicated stable transfection with PDCD4 was successful. Immunofluorescence (with hoechst dying) and flow cytometry demonstrated that PDCD4 facilitated cell apoptosis exposed to cisplatin. PDCD4 overexpression attenuated the protein levels of pAkt, pGSK3β and BCL-2, but increased the protein levels of BAK. Furthermore, incubation with SC-79 (the activator of Akt) reversed cell apoptosis induced by PDCD4.Conclusion Overexpression of PDCD4 promotes the apoptosis induced by cisplatin through pAKT/pGSK3β pathway, which is favorable to reverse cisplatin resistance in gastric cancer.

Exogenous hydrogen sulfide promotes the proliferation, invasion and cisplatin resistance of human ovarian cancer cells through EGFR/PI3K/Akt signaling pathway / 上海交通大学学报（医学版）

Objective·To investigate the effects and mechanisms of exogenous hydrogen sulfide on the proliferation, apoptosis, invasion and cisplatin resistance of human ovarian cancer cells. Methods·The human ovarian cancer cell line SKOV3 cells and human cisplatin resistant cell line SKOV3/DDP cells were studied. The effects of NaHS on cell proliferation, apoptosis and invasion in SKOV3 cells were detected respectively by CCK-8, flow cytometry and Transwell invasion assay. The effect of NaHS on cisplatin resistance in SKOV3 and SKOV3/DDP cells was detected by calculating the IC50 and IR. The phosphorylation levels of EGFR, PI3K and Akt in SKOV3 and SKOV3/DDP cells were assayed by Western blotting. After treated with erlotinib (EGFR inhibitor), LY294002 (PI3K inhibitor) and MK-2206 (Akt inhibitor), the phosphorylation levels of EGFR, PI3K and Akt in SKOV3 and SKOV3/DDP cells, as well as cell proliferation, invasion in SKOV3 cells and cisplatin resistance in SKOV3/DDP cells were detected. Results·Compared with the control group, NaHS could significantly promote the proliferation (P=0.000) and invasion (P=0.033) in SKOV3 cells; increase IC50 (P=0.027, P=0.009) and decrease IR of cisplatin (P=0.001, P=0.009) in SKOV3 and SKOV3/DDP cells. NaHS could activate EGFR (P=0.000, P=0.037), PI3K (P=0.009, P=0.013)and Akt(P=0.000,P=0.023)in SKOV3 and SKOV3/DDP cells.Erlotinib,LY294002 and MK-2206 could block the effects of NaHS on the proliferation (all P=0.000) and invasion (all P<0.01) in SKOV3 cells, and also reverse the effect of NaHS on the cisplatin resistance in SKOV3/DDP cells (all P=0.000). Conclusion·Exogenous hydrogen sulfide can induce the proliferation and invasion in SKOV3 cells, and promote the cisplatin resistance in SKOV3 and SKOV3/DDP cells, which mechanisms are related to activation of EGFR/PI3K/Akt signaling pathway.

Long non-coding RNA LINC00261 sensitizes human colon cancer cells to cisplatin therapy

Colon cancer is one of the most common digestive tumors. The present study aimed to explore the functional role, as well as the underlying mechanism of long non-coding RNA LINC00261 in colon cancer. Expression of LINC00261 was analyzed in colon cancer cell lines and human normal cell lines. Acquired resistance cell lines were then built and the acquired resistance efficiency was detected by evaluating cell viability. Thereafter, the effects of LINC00261 overexpression on cisplatin-resistant colon cancer cells were measured, as well as cell apoptosis, viability, migration, and invasion. Subsequently, we investigated the interaction of LINC00261 and β-catenin. The results showed that the LINC00261 gene was down-regulated in colon cancer cell lines and tissues, and in cisplatin-resistant cells. LINC00261 overexpression might relieve cisplatin resistance of colon cancer cells via promoting cell apoptosis, and inhibiting cell viability, migration, and invasion. Moreover, LINC00261 might down-regulate nuclear β-catenin through restraining β-catenin from cytoplasm into nuclei or it could also promote β-catenin degradation and inhibit activation of Wnt pathway. Finally, LINC00261 reduced cisplatin resistance of colon cancer in vivo and enhanced the anti-colon cancer effect of cisplatin through reducing tumor volume and weight.

Exogenous hydrogen sulfide promotes the proliferation, invasion and cisplatin resistance of human ovarian cancer cells through EGFR/PI3K/Akt signaling pathway / 上海交通大学学报（医学版）

Objective: To investigate the effects and mechanisms of exogenous hydrogen sulfide on the proliferation, apoptosis, invasion and cisplatin resistance of human ovarian cancer cells. Methods: The human ovarian cancer cell line SKOV3 cells and human cisplatin resistant cell line SKOV3/DDP cells were studied. The effects of NaHS on cell proliferation, apoptosis and invasion in SKOV3 cells were detected respectively by CCK-8, flow cytometry and Transwell invasion assay. The effect of NaHS on cisplatin resistance in SKOV3 and SKOV3/DDP cells was detected by calculating the IC50 and IR. The phosphorylation levels of EGFR, PI3K and Akt in SKOV3 and SKOV3/DDP cells were assayed by Western blotting. After treated with erlotinib (EGFR inhibitor), LY294002 (PI3K inhibitor) and MK-2206 (Akt inhibitor), the phosphorylation levels of EGFR, PI3K and Akt in SKOV3 and SKOV3/DDP cells, as well as cell proliferation, invasion in SKOV3 cells and cisplatin resistance in SKOV3/DDP cells were detected. Results: Compared with the control group, NaHS could significantly promote the proliferation (P=0.000) and invasion (P=0.033) in SKOV3 cells; increase IC50 (P=0.027, P=0.009) and decrease IR of cisplatin (P=0.001, P=0.009) in SKOV3 and SKOV3/DDP cells. NaHS could activate EGFR (P=0.000, P=0.037), PI3K (P=0.009, P=0.013) and Akt (P=0.000, P=0.023) in SKOV3 and SKOV3/DDP cells. Erlotinib, LY294002 and MK-2206 could block the effects of NaHS on the proliferation (all P=0.000) and invasion (all P<0.01) in SKOV3 cells, and also reverse the effect of NaHS on the cisplatin resistance in SKOV3/DDP cells (all P=0.000). Conclusion: Exogenous hydrogen sulfide can induce the proliferation and invasion in SKOV3 cells, and promote the cisplatin resistance in SKOV3 and SKOV3/DDP cells, which mechanisms are related to activation of EGFR/PI3K/Akt signaling pathway.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Up-regulation of Keap-1 inhibits the nuclear import of Nrf-2 and decreases cisplatin resistance in ovarian cancer Skov3/DDP cells / 基础医学与临床

Objective To test the status of Keap-1/Nrf-2 pathway in cisplatin-resistant Skov3/DDP cells,and the contribution of these molecular in cisplatin (cis-pt) resistance.Methods Skov3/DDP was divided into four groups:blank,cis-pt,Keap-1 up-regulated and Keap-1 plus cis-pt.Keap-1 was up-regulated in Skov3/DDP cells by trans-faction with pFlag-CMV-Keap-1 plasmid which was validated by expression of flag and increase of Keap-1 level using Western blot.The effect of Keap-1 up-regulation on Nrf-2 re-distribution was analyzed by Western blot.FCM was employed to detect apoptosis.ROS level was measured by DHE staining.Results The expression of Keap-1 was decreased obviously in Skov3/DDP cells compared with Skov3 cells (P＜0.05),Nrf-2 in Skov3/DDP cells detected mainly in nucleus.Then,the Keap-1 was successfully up-regulated (3.5 fold) by exogenous expression of pFlag-CMV-Keap-1 plasmid,as a result,nucleus portion of Nrf-2 was significantly reduced (0.2 fold).Next,the FCM results showed significant increase of apoptosis in Keap-1 up-regulated Skov3/DDP cells after cis-pt administration (P＜0.05).Finally,ROS level was significantly raised in Skov3/DDP cells treated with cis-pt plus Keap-1 up-regulation.Conclusions Up-regulation of Keap-1 reduced the nucleus portion of Nrf-2 and reverses cisplatin resistance in Skov3/DDP cells.

Role of Fas overexperssion in reversing cisplatin resistance in stomach cancer cells / 中国病理生理杂志

AIM: To study the effect of Fas on cisplatin resistance in stomach cancer cells and its possible mechanisms.METHODS: The expression of Fas at mRMA and protein levels in SGC-7901 cells and SGC-7901 /DDP cells was determined by RT-qPCR and Western blot.Fas-containing adenovirus vector was transfected into the SGC-7901 /DDP cells to upregulate Fas expression.The cell viability was detected by CCK-8 assay.The cell cycle and cell apoptosis were analyzed by flow cytometry.The protein levels of Fas, P38 /p-P38, JNK/p-JNK, cleaved caspase-8/caspase-8 and cleaved caspase-3 /caspase-3 were detected by Western blot.RESULTS: The expression of Fas at both mRNA and protein levels was significantly downregulated in the SGC-7901 /DDP cells.Fas expression was decreased by cisplatin in a dose-dependent manner in the SGC-7901 cells.Overexpression of Fas suppressed the viability and induced apoptosis in the SGC-7901 /DDP cells, and upregulated the protein levels of p-P38, p-JNK, cleaved caspase-8 and cleaved caspase-3.CONCLUSION:Overexpression of Fas increases the sensitivity of the SGC-7901 /DDP cells to cisplatin, and inhibits the cell growth and promotes cell apoptosis.The mechanism may be related to the activation of JNK and P38 pathway.

The role of Twist on cisplatin resistance of Hela cervical cancer cells in hypoxia microenvironment and its mechanism / 医学研究生学报

Objective Drug resistance is a major problem for the successful chemotherapeutic treatment and prognosis of cervi -cal cancer .The article investigated the role of Twist on cisplatin resistance of Hela cervical cancer cells in hypoxia microenvironment and its possible mechanism to provide an experimental basis to improve the therapeutic efficacy for cervical cancer . Methods Hela Cells were divided into two groups, normal oxygen group A (no CoCl2) and hypoxia group A (addition of 150μmol/L CoCl2 medium).6 hours later, different degrees(10-3, 10-4, 10-5, 10-6, 10-7 mol/L) of cisplatin (10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 mol/L) were added into two groups .24 hours later , MTT were added to measure IC 50 and growth inhibition ratio .CoCl2 were used to mimic hypoxia environ-ment.The working concentration and the treatment duration of Cispla-tin was optimized by MTT method.The expressions of Twist and MDR1 in normal oxygen group , cisplatin group , hypoxic group B and hypoxic cisplatin group B were determined by immunofluores-cence and western blot . Results The IC50 values were 10 -5.3 mol/L and 10 -4.5 mol/L of cisplatin respectively in normal oxygen group and hypoxia group, and there was significantly difference between them (P<0.05).The optimized working concentration and work time of cisplatin was 10 -5 mol/L and 24 h.Compare to the normal oxygen group (13.08 ±2.39, 29.57 ±12.80), the expres-sions of Twist (20.81 ±2.07, 24.25 ±4.51, 33.14 ±4.24) and MDR1 (35.26 ±8.41, 60.13 ±22.32, 76.00 ±9.96) was signifi-cantly higher than those in other three groups and which were the highest in hypoxic cisplatin group (P<0.05).There were significant positive correlations among them in hypoxic group and hypoxic cisplatin group (r =0.686,P <0.05;r =0.546,P <0.05). Conclusion The hypoxia microenvironment may be related to the cisplatin resistance of Hela cervical cancer cells .The possible mechanism is hypoxia activates Twist and upregulates MDR 1 expression , resulting in cisplatin resistance of Hela cells .

Characteristics of cisplatin resistance in human gastric cancer cell line SGC-7901 / 安徽医科大学学报

Objective To investigate the characteristics of cisplatin resistance in human gastric cancer cell line SGC-7901 . Methods Single cell gel electrophoresis ( SCGE ) was used to measure the level of DNA damage and repair in gastric cancer cell line SGC-7901 and gastric cancer cisplatin resistance cell line SGC-7901/DDP by ob-serving the tail length. Morphological changes of SGC-7901 and SGC-7901/DDP were recorded to evalutate the differences between the two lines. The degree of migration of SGC-7901/DDP and SGC-7901 measured by cell wound scratch assay was used to estimate the ability of invasion. MTT assay was performed to determine the drug sensitivity, IC50 values and cross-resistance of SGC-7901/DDP treated with 5-FU, VP-16, ADM, TAX and LOHP separately. Results The level of DNA damage and repair in SGC-7901/DDP was higher than that in SGC-7901 ac-cording to the tail length of SCGE tests ,suggesting the relationship between cisplatin resistance and the abiity of DNA damage and repair. There was a much smaller volume in SGC-7901/DDP compared with SGC-7901,and clone aggregation always appeared in SGC-7901/DDP. Cell wound scratch assay showed that the migration of SGC-7901/DDP was weaker than that of SGC-7901. MTT showed the significant increase of IC50 and cross resitance in SGC-7901/DDP compared with SGC-7901 treated with 5-FU, VP-16, ADM, TAX and LOHP simultaneously. Conclu-sion The ability of DNA damage and repair in gastric cancer cisplatin resistance cell line SGC-7901 is enhanced significantly. The SGC-7901/DDP shows a notable promotion on multidrug resistance in vivo, and the migration of SGC-7901/DDP is weaker than that of SGC-7901 .

Modulation effect of chromosome kinesin protein KIF4A on cisplatin resistance of A549/DDP cells / 国际生物医学工程杂志

Objective To investigate the process that chromosome kinesin KIF4A promote cisplatin resistance in lung cancer cells.Methods Reverse transcription PCR (RT-PCR) and Western Blot experiments were performed to analyze the expression of KIF4A in lung cancer cells A549 and cisplatin (DDP) resistant cells A549/ DDP.Cell transfection, RNA interference (RNAi) experiments and thiazolyl blue tetrazolium bromide (MTT) assays were carried out to examine cell proliferation of A549 cells with overexpression of exogenous KIF4A and A549/DDP cells with depletion of endogenous KIF4A after cisplatin treatment.Results Expression of KIF4A in A549/DDP cells was higher than that in A549 cells.With overexpression of exogenous KIF4A, A549 cells displayed drug resistance to cisplatin.On the contrary, depletion of endogenous KIF4A in A549/DDP cells resulted in cisplatin sensitivity.Conclusions Chromosome kinesin KIF4A involves in the regulation of cisplatin resistance in lung cancer cells and KIF4A may be a potential and effective new biological target for treatment of lung cancer cisplatin resistance.