Effects of Buzhong Yiqi decoction-medicated serum on drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin

AIM:To investigate the effects of Buzhong Yiqi decoction-medicated serum on the drug resistance of human lung adenocarcinoma cell line A549/DDP to cisplatin. METHODS:Medicated serum containing Buzhong Yiqi decoction was prepared. The optimal dose of the medicated serum was selected by MTT assay. The A549 cells and A549/DDP cells were treated with the optimal medicated serum and cisplatin at different concentrations. The IC 50, resistance index and reversal index were determined. The cells were divided into control serum group, optimal medicated serum group, LY294002 group, LY294002 + optimal medicated serum group (combination group) and negative group. The expression of PI3K and Akt at mRNA and protein levels was detected by the methods of immunocytochemistry, Western blotting and real-time PCR. RESULTS:Treatment with 10% middle dose of medicated serum for 48 h was the optimal dose and time for medicated serum. The sensitivity of A549/DDP cells to cisplatin was obviously enhanced when the cells were exposed to the optimal medicated serum with the reversal index of 2.46. The expression of PI3K and Akt at mRNA and protein levels in optimal medicated serum group and combination group was significantly decreased. CONCLUSION:Buzhong Yiqi decoction decreases the resistance index by reducing the expression of PI3K in A549/DDP cells, thus increasing the sensitivity of A549/DDP cells to cisplatin.     

Reversal effect of shikonin on cisplatin resistance of ovarian cancer SKOV3/DDP cells

AIM:To explore the reversal effect of shikonin on cisplatin resistance of ovarian cancer SKOV3/DDP cells and its potential mechanism. METHODS:The proper conditions of treatment with shikonin and cisplatin were determined by CCK-8 assay. The cell cycle and apoptotic rate were analyzed by flow cytometry. The protein levels of cell cycle-and apoptotic-related molecules, such as cyclin D1, cyclin-dependent kinases 2 (CDK2), P18, p-Rb, Bcl-2, Bax and cleaved caspase-3, were determined by Western blot. RESULTS:The results of CCK-8 assay showed that compared with cisplatin group, combined treatment with shikonin and cisplatin had a better inhibitory effect on the growth of cisplatin-resistant SKOV3/DDP cells. The cell cycle G₁/S transition was inhibited, while early apoptotic rate was increased after combined use of shikonin and cisplatin. The results of Western blot showed that compared with cisplatin group, the cells in combination group had lower protein levels of cyclin D1, CDK2, p-Rb and Bcl-2, accompanied with higher protein levels of P18, Bax and cleaved caspase-3. CONCLUSION:Shikonin reverses the cisplatin resistance of ovarian cancer SKOV3/DDP cells. The mechanism may be related to the regulation of cell cycle-and apoptotic-related molecules, and further inhibition of cell viability and promotion of cell apoptosis.     

The effect of Shenmai injection on cardiac myocyte apoptosis after hypoxia

AIM: To observe the effect of Shenmai injection, a chinese medicine, on apoptosis of cardiac myocytes after hypoxia. METHODS: Cardiac myocytes were separated from neonate rat heart and cultivated in vitro. Hypoxia condition was induced by mixture of 95%N2 and 5%CO2. Cells were exposed to hypoxia for 6 h or 12 h and treated with Shenmai injection (5 mL/L) from 24 h before hypoxia until the end of hypoxia. First, apoptosis was detected with Annexin V-FITC and PI staining by flowcytometry. Then, the activity of cardiac myocyte mitochondria was observed by MTT method. Mitochondria membrane potential and the activity of caspase 3,7 were also measured by laser scan microscopy and multi-detection microplate reader, respectively. RESULTS: The apoptotic cells became more and more with prolonged hypoxia. Shenmai injection enhanced mitochondria activity, kept membrane potential, inhibited the activation of caspase3,7 and then decreased apoptotic cells (P<0.01). CONCLUSION: Hypoxia induces apoptosis in cardiac myocytes by mitochondria pathway. Shenmai injection can decrease cardiomyocyte apoptosis after hypoxia, the mechanism is related to mitochondria membrane potential stabilization and caspase inhibition.     

Effect of rapamycin on proliferation,invasion, adhesion,apoptosis and autophagy of human lung adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum

AIM: To study the effect of rapamycin (Rap) on the proliferation, invasion, adhesion, apoptosis and autophagy of human adenocarcinoma A549 and resistant A549/DDP cells treated with cis-diamminedichloroplatinum (DDP). METHODS: Human adenocarcinoma A549 and resistant A549/DDP cell lines were cultured. The inhibitory effects of Rap alone or combined with DDP on A549 and resistant A549/DDP cells were detected by MTT assay. The in vitroinvasion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by Transwell methods. The in vitroadhesion abilities of the 2 cell lines treated with Rap alone or combined with DDP were detected by adhesion experiments. The apoptosis of A549 and resistant A549/DDP cells induced by Rap alone or combined with DDP was analyzed by flow cytometry. The cell autophagy marker proteins beclin-1 and LC3 in A549 and resistant A549/DDP cells treated with Rap alone or combined with DDP were detected by Western blotting.RESULTS: Compared with Rap or DDP alone group, the combination of Rap and DDP significantly inhibited the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells in vitro, and promoted the cell apoptosis and autophagy marker proteins beclin-1 and LC3 expression (all P＜0.05).CONCLUSION: Rap enhances the effect of DDP through promoting the cell autophagy, thereby inhibiting the proliferation, invasion and adhesion of A549 and resistant A549/DDP cells and inducing the cell apoptosis with a synergistic effect.     

Effects of cisplatin on glucose-6-phosphate dehydrogenase expression and activity in HeLa cells

AIM: To investigate the effect of cisplatin (DDP) on the expression of glucose-6-phosphate dehydrogenase (G6PD) mRNA and G6PD activity in HeLa cells in vitro. METHODS: After treated with the concentration of 5 mg/L DDP for several hours, HeLa cells were harvested. The growth inhibition was detected by methyl thiazolyl tetrazolium (MTT) assay. The content of glutathione (GSH) was determined by Beutler method. The activity of G6PD was determined by velocity method. The expression of G6PD mRNA was assayed by fluorescent quantitative real-time PCR. RESULTS: DDP significantly inhibited the proliferation of HeLa cells in vitro. At both 4 h and 24 h, the expressions of G6PD mRNA were up-regulated and G6PD activity were higher than that in control group (P<0.05). The content of GSH in 4 h group was lower than that in control group. CONCLUSION: DDP up-regulates the expression of G6PD mRNA and increases G6PD activity. These maybe contributes to the resistance of oxygen stress.     

Effect of LPS on intracellular localization of p38 protein kinase in Raw264.7 cells

AIM: To investigate the activation dynamics and the intracellular localization of p38 protein kinase in Raw264.7 cells after lipopolysaccharide (LPS) stimulation.METHODS: Protein kinase assay and immunogold electron microscope technique were used to check the activation dynamics and distribution of p38 MAPK in Raw264.7 cells before and after LPS stimulation. RESULTS: The kinase assay results showed that a marked increase in p38 activity was detected 15 min after LPS treatment, and reached maximal activity 30 min post stimulation, then dropped down and got closed to the pre-stimulated level 2 h later. The optimal LPS concentration for treatment was 100 μg/L. The immunogold electron microscope data showed that p38 spread evenly in every part of the cytosol of the non-stimulated and EGF stimulated Raw264.7 cells, such as endoplasmic reticulum, mitochondria, lysosome, while the golden granules intensity in the cytosol area decreased and in the nuclear area increased significantly after LPS stimulation.CONCLUSION: p38 MAPK moves to the nuclei of Raw264.7 cells on account of stimulation by LPS.     

Role of Fas overexperssion in reversing cisplatin resistance in stomach cancer cells

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.     

Effect of PARP-1 on cisplatin resistance in breast cancer MCF-7 cells

AIM: To study the effect of poly(ADP-ribose) polymerase-1 (PARP-1) on cisplatin resistance of human breast cancer MCF-7 cells and its possible mechanisms.METHODS: The expression of PARP-1 at mRNA and protein levels in MCF-7 cells and MCF-7/DDP cells was determined by real-time PCR and Western blot. The expression of PARP-1 in the MCF-7/DDP cells was blocked by PARP-1 siRNA. The cell viability and apoptosis were detected by the CCK-8 assay and flow cytometry analysis, respectively. Furthermore, the protein levels of PARP-1, Bcl-2, Bax, cleaved caspase-3, caspase-3, cytochrome C (Cyto-C), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) were detected by Western blot.RESULTS: The expression of PARP-1 at both mRNA and protein levels was significantly up-regulated in the MCF-7/DDP cells. The expression of PARP-1 was increased in the MCF-7 cells treated with cisplatin. Knockdown of PARP-1 induced the apoptosis of MCF-7/DDP cells with an increased sensitivity to cisplatin. Meanwhile, knockdown of PARP-1 down-regulated the protein levels of Bcl-2/Bax and p-ERK, but up-regulated the protein levels of cleaved caspase-3 and Cyto-C. After incubated with a specific ERK inhibitor U0126, the cell viability in PARP-1 siRNA group was down-regulated significantly.CONCLUSION: Knockdown of PARP-1 increases the sensitivity of MCF-7/DDP cells to cisplatin, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of ERK signaling pathway by inhibiting phosphorylation of ERK.     

Effect of p38 MAPK on cisplatin-induced renal tubular cell apoptosis

AIM:To investigate the role of p38 MAPK in cisplatin-induced rat renal proximal tubular cell (RPTC) apoptosis. METHODS:To determine the optimal concentration of cisplatin to induce RPTC apoptosis, the cells were treated with 0, 5, 10 and 20 μmol/L cisplatin for 24 h, and then the cell lysates were collected for Western blot analysis of cleaved PARP, p38 and phosphor ylated p38 (p-p38). To determine the role of p38 MAPK in cisplatin-induced RPTC apoptosis, the cells were divided into control group, cisplatin group (the cells were treated with cisplatin for 24 h) and cisplatin+p38 MAPK inhibitor group (the cells were treated with p38 MAPK inhibitor SB203580 for 1 h, and then treated with cisplatin for another 24 h). The morphological changes of apoptotic cells were observed under phase-contrast fluorescence microscope. The apoptotic rate of the cells were analyzed by flow cytometry. The caspase activity of RPTC lysates was examined using Ac-DEVD-AFC kit. The protein levels of p-p38, p38, cleaved PARP and cleaved caspase-3 were determined by Western blot. The pH value of extracellular environment of the cells was measured by pH meter. RESULTS:Cisplatin at 20 μmol/L obviously induced apoptosis of RPTC. The p38 MAPK was phosphorylated and its phosphorylation peaked at 15 min after cisplatin treatment. The apoptotic rate of RPTC was 12.08% after cisplatin induction. Cisplatin treatment also enhanced caspase activity, and increased cleavage of PARP and caspase-3 proteins (P<0.05). The p38 MAPK inhibitor SB203580 effectively inhibited the phosphorylation of p38 MAPK, down-regulated the RPTC apoptosis rate and caspase activity, and reduced the cleavage of PARP and caspase-3 proteins. The pH value change in RPTC culture medium was also inverted by SB203580. CONCLUSION:The phosphorylation of p38 MAPK is involved in cisplatin-induced apoptosis of RPTC. The apoptosis induced by cisplatin results in the change of acidic extracellular environment, which is inhibited by p38 MAPK inhibitor SB203580.     

Effect of miR-24 on chemotherapy sensitivity in lung cancer A549 cells

AIM: To study the effect of microRNA (miR)-24 on chemotherapy sensitivity and its possible mechanisms in human lung adenocarcinoma A549 cells. METHODS: The expression of miR-24 in the A549 cells and A549/DDP cells was determined by real-time PCR. Transfection of miR-24 inhibitor was used to down-regulate the miR-24 level in the A549/DDP cells. The viability and apoptosis rate were measured by CCK-8 assay and flow cytometry, respectively. The protein levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cytochrome C (Cyt C), phosphorylated extracellular signal regulated kinase (p-ERK) and P53 were detected by Western blot. Luciferase reporter assay was used to predict and identify the target genes of miR-24. RESULTS: The expression of miR-24 was significantly higher in the A549/DDP cells than that in the A549 cells (P<0.05). miR-24 inhibitor induced cell apoptosis and increased the sensitivity of the A549/DDP cells to cisplatin. Furthermore, miR-24 inhibitor down-regulated the ratio of Bcl-2/Bax, while up-regulated the protein levels of P53, p-ERK, cleaved caspase-9, cleaved caspase-3 and Cyt C. Incubation with U0126, a specific ERK inhibitor, partly reversed the viability of miR-24 inhibitor transfected A549/DDP cells. Bioinformatics analysis demonstrated that p53 was a potential target gene of miR-24. Co-teansfection of miR-24 inhibitor and P53 siRNA in A549/DDP cells partially reversed the effect of miR-24 inhibitor on cell viabiltiy. CONCLUSION: Down-regulation of miR-24 increases the sensitivity of A549/DDP cells to cisplatin. The mechanism may be related to directly targeting p53 gene and over-activation of ERK/P53 signaling pathway, thus promoting apoptosis via mitochondrial apoptosis pathway.     

Cisplatin enhances proliferation-inhibitory and apoptosis-inducing effects of ECRG2 protein on human esophageal cancer EC9706 cells

Abstract: AIM: To investigate the effects of esophageal cancer-related gene 2 (ECRG2) protein combined with cisplatin (DDP) on the proliferation and apoptosis of human esophageal cancer EC9706 cells. METHODS: Methylthiazolyl tetrazolium (MTT) assay was used to examine the effects of single ECRG2 protein and ECRG2 protein combined with DDP on the proliferation of EC9706 cells. Hoechest 33258 staining was applied to analyze the effect of single ECRG2 protein and ECRG2 protein combined with DDP on apoptosis of EC9706 cells. The expression of p53 in EC9706 cells was detected by Western blotting. RESULTS: ECRG2 protein inhibited the proliferation of EC9706 cells. ECRG2 protein combined with DDP enhanced the inhibitory effect time- and dose-dependently in a certain range of concentrations. The number of apoptotic cells after treated with ECRG2 protein combined with DDP for 24 h was larger than those treated with single ECRG2 protein. The proliferation-inhibitory and qpoptosis-inducing expression of p53 was up-regulated in ECRG2 protein and DDP combination group compared with single ECRG2 protein group. CONCLUSION: DDP enhances the proliferation-inhibitory and apoptosis-inducing effects of ECRG2 protein on EC9706 cells. The apoptosis-inducing mechanism may be related to the up-regulation of p53 expression.     

Mitochondrial membrane potential and caspase-3 are involved in apoptosis of RD cells induced by TRAIL and cisplatin

AIM: To study the synergistic induction of apoptosis by TRAIL and cisplatin in rhabdomyosarcoma cells and investigate the role of mitochondrial membrane potential and caspase-3 in this process.METHODS: Rhabdomyosarcoma cells were treated with TRAIL, cisplatin for 3 days, respectively or combination. The cytotoxicity was observed by MTT assay. The apoptotic rates and change of mitochondrial membrane potential and caspase-3 were determined by flow cytometry (FCM). The obvious morphological changes in rhabdomyosarcoma cells were confirmed by electron microscope. RESULTS: Rhabdomyosarcome cells were treated with TRAIL (1.0, 10.0 and 100.0 μg/L), the cytotoxicity indices were 18.9%, 20.8% and 43.5%, respectively. With cisplatin (1.0, 5.0 and 10.0 mg/L), the indices of cytotoxicity were 9.8%, 23.4% and 43.8%, respectively. When TRAIL and cisplatin treatment used simultaneously, the cytotoxicity index increased obviously. The activity of caspase-3 in rhabdomyosarcoma cells was upregulated and the mitochondrial membrane potential was downregulated with cisplatin, which were paralleled by the apoptotic rates. The obvious apotosis morphological changes in rhabdomyosarcoma cells were shown by electron microscope. CONCLUSION: TRAIL and cisplatin are able to kill rhabdomyosarcoma cells. TRAIL in combination with cisplatin shows synergistic effect on rhabdomyosarcoma cells by increasing the caspase-3 activity and suppressing mitochondrial membrane potential.     

Mitochondria associated endoplasmic reticulum membranes in cisplatin induced ovarian cancer cell apoptosis

AIM: To explore the structural change of mitochondria associated endoplasmic reticulum membranes (MAMs) in SKOV3 cells exposed to cisplatin. METHODS: The SKOV3 cells were treated with cisplatin at concentration of 6 mg/L. The protein levels of active caspase-3, as well as the colocalization of B-cell receptor-associated protein 31 (BAP31) and voltage-dependent anion channel protein 1 (VDAC1) in the SKOV3 cells were determined by the method of indirect immunofluorescence. The apoptotic rate of the SKOV3 cells was analyzed by flow cytometry. The structural change of MAMs was observed under transmission electron microscope. RESULTS: Under the confocal microscope, we found that cisplatin increased the protein levels of active caspase-3 as well as colocalization of BAP31 and VDAC1 in the SKOV3 cells. The results of flow cytometry demonstrated that cisplatin increased the apoptotic rate of the SKOV3 cells (P<0.05). The results of transmission electron microscopy showed that cisplatin induced increase in mitochondrial-associated membrane structures (P<0.05). CONCLUSION: Cisplatin induces SKOV3 cell apoptosis with increased MAMs contacts. MAMs may play a role in cisplatin induced SKOV3 cell apoptosis.     

Effects of berberine on the proliferation of human pulmonary carcinoma cells

AIM: To study the effect of berberine on the proliferation of human pulmonary carcinoma cells (PG cells). METHODS: The proliferation of PG cells was determined by using MTT assay. Cell cycle and apoptosis of PG cells were determined by using flow cytometry. Confocal scanning imaging system was used to assay the ROS-releasing level of PG cells. RESULTS: Berberine was shown to inhibit proliferation of PG cells directly and in a concentration-dependent manner (P<0.05，P<0.01). Berberine blocked the cell cycle of PG cells， and beberine at concentration of 40 mg/L induced the apoptosis of PG cells. After 6 or 12 h incubation, berberine began to induce ROS-releasing in PG cells at 10 mg/L. While incubated with berberine for 24 h, PG cells were induced to release ROS at lower concentration. CONCLUSION: Berberine has an inhibitory effect on the proliferation of PG cells, and these effect may be carried out by regulating the production of intracellular ROS and the process of cell cycle.     

miR-125a-5p reverses cisplatin resistance of non-small-cell lung cancer A549/DDP cells by targeting LIMK1

AIM:To explore the effect of microRNA-125a-5p (miR-125a-5p) on cisplatin (DDP) resistance of non-small-cell lung cancer A549/DDP cells and its related mechanisms. METHODS:The expression levels of miR-125a-5p and LIM kinase 1 (LIMK1) in non-small-cell lung cancer tissues, A549 cells and A549/DDP cells were detected by RT-qPCR. The A549/DDP cell viability, apoptotic rate and expression of drug resistance-related proteins after over-expression or knockdown of miR-125a-5p and/or LIMK1 expression were detected by MTT assay, flow cytometry and Western blot, respectively. The targeting relationship between miR-125a-5p and LIMK1 was verified by TargetScan online prediction and dual-luciferase reporter system. The cell viability, apoptotic rate and expression of drug resistance-related proteins after co-expression of miR-125a-5p and LIMK1 were also determined. RESULTS:The expression level of miR-125a-5p was down-regulated and LIMK1 expression was up-regulated in non-small-cell lung cancer tissues and cell lines (P<0.05). The results of dual-luciferase assay indicated that miR-125a-5p negatively regulated the expression of LIMK1. The expression of drug resistance-related proteins and the viability of A549/DDP cells were inhibited after over-expression of miR-125a-5p or knockdown of LIMK1, while the apoptosis was enhanced. Over-expression of LIMK1 attenuated the inhibitory effect of miR-125a-5p on A549/DDP cell viability and drug resistance-related protein expression (P<0.05). CONCLUSION:miR-125a-5p reverses the resistance of A549/DDP cells to DDP by inhibiting the expression of LIMK1 and drug resistance-related proteins.     

Effects of nitric oxide on mitochondrial damage caused by exogenous calcium

AIM: To study the effects of nitric oxide (NO) on mitochondrial damage caused by exogenous calcium. METHODS: Normal myocardial mitochondria were divided into three groups; L-arginine control group (CG), Ca^2＋-damaged group (DG) and L-NAME-preserved group (PG). Mitochondria of all groups were incubated at 30 ℃ with reaction medium containing 20 μmol/L EDTA, 100 μmol/L CaCl₂ and 1 μmol/L L-NAME with 100 μmol/L CaCl₂ respectively. Then the NO₂^-/NO₃^- contents, mitochondrial viability and membrane potential were investigated. RESULTS: The NO₂^-/NO₃^- contents of DG was obviously higher than that of CG and PG, meanwhile, there was no obvious difference between CG and PG. Mitochondrial viability and membrane potential of DG were significantly lower than that of CG and PG, and negatively related to NO^-₂/NO^-₃ contents (r=-0.5297, P＜0.01; r=-0.6041, P＜0.01). But, the mitochondrial viability and membrane potential of PG were still lower than that of CG. CONCLUSION: Exogenous calcium could activate mitochondrial nitric oxide synthase resulting in NO production and the latter play an important role in decreasing mitochondrial viability and membrane potential.     

Pantoprazole sodium inhibits epithelial-mesenchymal transition and cisplatin resistance in lung cancer cells and underlying mechanism

AIM: To explore the inhibitory effects of pantoprazole sodium on epithelial-mesenchymal transition and cisplatin resistance in lung cancer cells and the underlying mechanism.METHODS: Using MTT method, wound healing assay, Transwell experiment, Western blot, the differences of morphology, invasion ability, migration ability, drug sensitivity and protein expression between A549/DDP cells and A549 cells were determined. The effect of pantoprazole sodium on morphology, invasion ability, migration ability, drug sensitivity and protein expression in A549/DDP cells were also observed.RESULTS: Compared with A549 cells, A549/DDP cells had higher invasion and migration abilities, and lower drug sensitivity, exhibited mesenchymal phenotype and activated c-Met/AKT/mTOR pathway. Pantoprazole sodium inhibited the abilities of invasion and migration, and reversed the mesenchymal phenotype, drug resistance and the c-Met/AKT/mTOR pathway activation in A549/DDP cells. Treatment with c-Met inhibitor SU11274, PI3K inhibitor LY294002 and mTOR inhibitor rapamycin had the same effects on A549/DDP cells as that of pantoprazole sodium.CONCLUSION: Pantoprazole sodium inhibits invasion, migration, epithelial-mesenchymal transition and cisplatin resistance in lung cancer cells by down-regulating c-Met/AKT/mTOR pathways.     

Mechanism of Herceptin enhancing apoptosis and improving sensitivity to chemotherapy in Ishikawa cells

AIM: To investigate the role of Herceptin in the apoptosis and drug sensitivity of endometrial cancer Ishikawa cells.METHODS: The IC₅₀ values of Herceptin, adriamycin(ADR), cisplatin(DDP) and paclitaxel(PTX) for Ishikawa cells were detected by MTT method. Ishikawa cells were treated with single drug and combined chemotherapy for 24 h, the cell cycle and the apoptosis ratio were determined by flow cytometry.RESULTS: The IC₅₀ values of Herceptin, ADR, DDP and PTX were 57.12 mg/L, 0.572 μmol/L, 67.4 μmol/L and 719.5 nmol/L, respectively. Herceptin significantly enhanced the cytotoxicity of the chemotherapeutic drugs, and increased apoptosis ratio statistically.CONCLUSION: Herceptin enhances the apoptosis-inducing ability of the chemotherapeutic drugs and improves the chemotherapeutic sensitivity in Ishikawa cells.     

Reversing effect of naringin on cisplatin resistance in human lung cancer A549/DDP cells

AIM: To investigate the effect of naringin (NRG) on cisplatin (DDP) resistance in human lung cancer A549/DDP cells and its possible mechanism. METHODS: A549/DDP cells were cultured in vitro and treated with NRG and/or DDP at different concentrations for 24 h, and then the cell viability were measured by CCK-8 assay. The combination index (CI) of NRG and DDP were analyzed by Chou-Talalay method. The apoptosis rate was analyzed by flow cytometry. Western blot was performed to detect the protein levels of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), p-Akt, CXC chemokine receptor 4 (CXCR4), cleaved caspase-3, Bcl-2 and Bax.RESULTS: The protein levels of P-gp, MRP1, p-Akt and CXCR4 in the A549/DDP cells were higher than those in the A549 cells (P<0.05). The cell viability was remarkably reduced in a dose-dependent manner when A549/DDP cells were exposed to NRG and/or DDP (P<0.05), and the IC₅₀ values of NRG and DDP were 36.92 μmol/L and 129.77 μmol/L, respectively. When the inhibition rate exceeded 15%, NRG in combination with DDP produced a synergistic effect (CI<1). Combination treatment with NRG and DDP significantly induced apoptosis (P<0.05), up-regulated the protein levels of cleaved caspase-3 and Bax, and down-regulated the protein level of Bcl-2 (P<0.05). Meanwhile, NRG remarkably down-regulated the protein levels of P-gp, MRP1, p-Akt and CXCR4 in a dose-dependent manner (P<0.05). CONCLUSION: NRG may enhance the sensibility of A549/DDP cells to DDP most likely via up-regulating the protein level of Bax and down-regulating the protein levels of Bcl-2, P-gp, MRP1, p-Akt and CXCR4.     

Effect of CLCN2 antisense oligonucleotide on U251 cells injury by cisplatin

AIM: To investigate the effect of chloride channel CLCN2 antisense oligonucleotide on the cell injury of malignant U251 glioma cells induced by cisplatin (DDP). METHODS: The experiment was divided into 4 groups: control group (nonsense oligonucleotide), CLCN2 antisense oligonucleotide group, DDP group (DDP＋nonsense oligonucleotide), DDP＋CLCN2 antisense oligonucleotide group. The viability of U251 cells was measured by MTT assay, CLCN2 mRNA level was determined by RT-PCR, cell apoptosis was measured by TUNEL assay. RESULTS: Compared to the control group, the cell viability, CLCN2 and cyclinD1 mRNA decreased in CLCN2 antisense oligonucleotide group, DDP treated group and CLCN2 antisense oligonucleotide with DDP treated group, cells apoptosis increased. Compared to DDP group, the cell viability (P<0.05) and CLCN2 mRNA decreased in CLCN2 antisense oligonucleotide with DDP treated group, and cells apoptosis increased (P<0.01). Compared to CLCN2 antisense oligonucleotide group, CLCN2 mRNA significantly decreased (P<0.01) in CLCN2 antisense oligonucleotide with DDP treated group. CONCLUSION: CLCN2 antisense oligonucleotide inhibits the expression of CLCN2 mRNA in U251 cells. Inhibition of CLCN2 mRNA facilitates the cell injury of U251 cells induced by DDP. The decrease in CLCN2 mRNA is involved in the mechanism of cell injury by DDP.