Effects of 2-deoxy-D-glucose and oxaliplatin on proliferation and apoptosis of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of 2-deoxy-D-glucose (2-DG) or oxaliplatin (L-OHP) alone and the combination of both on the proliferation and apoptosis of hepatoma cell line SMMC-7721 in vitro. METHODS: Human hepatoma cell line SMMC-7721 was treated with 2-DG or L-OHP alone, or both. The inhibitory effect on the proliferation of SMMC-7721 cells was estimated by MTT method. The q value, which represents synergistic effect, was determined. Apoptotic rate and cell cycle were assayed by flow cytometry. The activity of caspase-3 was detected by a caspase-3 activity assay kit. RESULTS: 2-DG or L-OHP at different concentrations inhibited the growth of SMMC-7721 cells obviously and the inhibitory effect on SMMC-7721 cell growth strongly depended on the exposure time and dose. When the 2 drugs worked together, the inhibitory effect was improved (P<0.05). 2-DG induced cell apoptosis and arrested the cells at G₂/M phase. When combined with L-OHP,the 2 drugs induced more severe apoptosis and arrested the cells at S and G₂/M phase. Meanwhile, the activity of caspase-3 increased when the 2 drugs used together. CONCLUSION: 2-DG inhibits the growth of hepatoma cell line SMMC-7721. The combination of 2-DG and L-OHP improves the ability of L-OHP to attack the tumor cells. The mechanism might be related to increasing the activity of caspase-3.     

Salinomycin promotes the apoptosis-inducing effect of gefitinib on human lung adenocarcinoma cell line A549

AIM: To investigate the effect of salinomycin alone or in combination with gefitinib (an inhibitor of epidermal growth factor receptor tyrosine kinase) on the growth and apoptosis of human non-small-cell lung cancer cell line A549. METHODS: The inhibitory effect of salinomycin on the growth of A549 cells was tested by MTT assay. The cell apoptosis and the level of mitochondrial membrane potential were determined by flow cytometry. The activity of caspase-3, -8, and -9 was measured by the method of colorimetry. The protein levels of cytochrome C, Bcl- 2, p-EGFR, p-Akt and p-ERK were detected by Western blotting. RESULTS: Salinomycin or gefitinib alone inhibited the growth of A549 cells in a dose-dependent manner. Salinomycin or gefitinib also induced apoptosis of the cells. Salinomycin combined with gefitinib produced stronger inhibitory effect on the cell proliferation, and a significant increase in cell apoptosis was also observed. Compared with control group, salinomycin alone significantly reduced mitochondrial membrane potential, transitorily increased the levels of intracellular reactive oxygen species (ROS), cytoplasmic cytochrome C and Ca2+, and increased the activity of caspase-3, -8 and -9 in A549 cells. Gefitinib alone inhibited the protein expression of p-EGFR, p-Akt and p-ERK, but no obvious effect on the release of cytochrome C and the activity of caspase-3, -8 and -9 was found. The combination of salinomycin and gefitinib significantly reduced the protein levels of Bcl-2, p-EGFR, p-Akt and p-ERK, but the protein levels of EGFR, Akt and ERK were not obviously changed. CONCLUSION: The synergy of salinomycin and gefitinib is observed. Salinomycin inhibits the growth and induces apoptosis of human lung carcinoma A549 cells through Bcl-2 pathway and mitochondrial apoptosis pathway. Salinomycin also increases the sensitivity of A549 cells to gefitinib.     

Preparation of recombinant human soluble TRAIL and its inducing effect on apoptosis of tumor cells by TRAIL combined with bortezomib

AIM: To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to verify the biological activity of TRAIL. METHODS: The prokaryotic expression plasmid pET-28a (+)-TRAIL_114-281 was constructed. Human soluble TRAIL was obtained through optimized inducing protein expression and purification conditions. The biological activity of TRAIL was verified by CCK-8 assay. The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib (Velcade, PS-341) on the tumor cell lines H460(TRAIL-sensitive) and K562(TRAIL-resistance) for 24 h was determined. The apoptotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining. The activities of caspase-8, -9 and -3 in the cells were detected by colorimetric method. The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot. The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry. RESULTS: The recombinant human soluble TRAIL protein with stable bioactivity was successfully acquired, which induced apoptosis in H460 cells and K562 cells. After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL (P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration. Apoptotic rate in combination group was obviously higher than that in single group (P<0.05). In the process of apoptosis, the activities of caspase-8, -9 and -3 in H460 cells and K562 cells were both increased. The expression of Bcl-2 and cFLIP in treatment groups (especially the combination group) was decreased compared with control group. No significant change of the Bax expression level was observed. The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regulated after treated with bortezomib (P<0.05). CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.     

miR-155-specific siRNA enhances chemosensitivity of Burkitt lymphoma Raji cells to cytosine arabinoside by inducing apoptosis

AIM：To investigate the effect of miR-155-specific siRNA alone or in combination with cytosine arabinoside (Ara-C) on the growth and apoptosis of Burkitt lymphoma Raji cells. METHODS：miR-155-specific siRNA and/or Ara-C were used to treat the cells. Quantitative real-time polymerase chain reaction was used to detect the expression of miR-155. The growth of the cells was analyzed by CKK-8 assay. The cell apoptosis was determined by flow cytometry. RESULTS：The miR-155 expression level of the cells transfected with miR-155 siRNA was significantly lower than that in the 2 control groups. Ara-C or miR-155 siRNA alone inhibited the growth of Raji cells in a dose-depend manner. miR-155 siRNA combined with Ara-C produced more inhibition of cell proliferation (P<0.05). After treatment for 48 h, the apoptotic rate of Raji cells in miR-155 siRNA+Ara-C group ［(38.4±1.4)%］ was higher than that in Ara-C group ［(16.5±0.3)%］ and miR-155 siRNA group ［(14.6±0.3)%］, with statistically significant difference (P<0.05). The expression of caspase-3 in Ara-C+miR-155 siRNA group was increased significantly as compared with Ara-C group and miR-155 siRNA group. CONCLUSION：miR-155-specific siRNA enhances the chemosensitivity of Raji cells to Ara-C by inducing apoptosis through the caspase-3 pathway.     

Effect of metformin combined with paclitaxel on apoptosis and AMPK signaling in human breast cancer cells

AIM:To investigate the effect of metformin combined with paclitaxel on the viability and apoptosis of breast cancer cell line MCF-7 and its possible mechanism. METHODS:MCF-7 cells were treated with metformin at different concentrations (2, 5, 10, 20, 40 and 80 mmol/L) and in vitro cultured. The viability of MCF-7 cells was measured by MTT assay. Metformin at 2 mmol/L or paclitaxel at 2.4 mg/L alone or in combination was used to treat the cells, and compound C, an inhibitor of adenosine monophosphate-activated protein kinase (AMPK) signaling transduction pathway, was also used. The cells were divided into control group, metformin group, paclitaxel group, combination group, and combination +compound C group. The apoptosis of the cells was analyzed by flow cytometry. The expression of Bax, Bcl-2 and caspase-3 at mRNA and protein levels was determined by RT-qPCR and Western blot. The protein levels of AMPK and P21 were examined by Western blot. RESULTS:Metformin at different concentrations (2, 5, 10, 20, 40 and 80 mmol/L) significantly inhibited the cell viability in a concentration-dependent manner (P<0.05). Compared with control group, treatment with metformin at 2 mmol/L or paclitaxel at 2.4 mg/L alone or in combination significantly inhibited the cell viability, induced apoptosis (P<0.05), decreased the level of Bcl-2 (P<0.05), increased the levels of Bax and caspase-3 (P<0.05), and promoted the protein expression of AMPK and P21 (P<0.05). The effects of metformin and paclitaxel in combination were better than those of single drug treatment, while AMPK inhibitor weaken these effects. CONCLUSION:Metformin combined with paclitaxel inhibits the viability and induces the apoptosis of breast cancer MCF-7 cells by activating AMPK signaling pathway and regulating apoptosis signaling pathway.     

NAC-1 -specific siRNA enhances paelitaxel-induced apoptosis of ovarian cancer cell line HO8910

AIM: To observe the effect of NAC-1 -specific siRNA alone, or in combination with paelitaxel on proliferation and apoptosis of human ovarian cancer cell line HO8910. METHODS: Ovarian cancer cells were treated with NAC-1 siRNA alone or in combination with paelitaxel. The level of NAC-1 mRNA was assessed by real-time quantitative PCR. Western blotting analysis was used to detect NAC-1 protein and the activation of epidermal growth factor receptor(EGFR) downstream signals,Akt and ERK. The cell proliferation rate was measured by MTT assay, and the cell cycle and apoptosis were determined by flow cytometry. RESULTS: After treated with NAC-1 -specific siRNA for 48 h, the expression of NAC-1 at mRNA and protein levels in HO8910 cells decreased by 71.1% and 80.5%, respectively. The cells in G₁ phase increased. The protein levels of p-Akt and p-ERK were decreased by 43.7% and 49.8%, respectively. After treated with NAC-1 -specific siRNA for 72 h, the proliferation inhibitory rate of the cells was increased to 45.6% as compared with the cells treated with negative siRNA. Apoptotic rate of the cells treated with NAC-1 siRNA (0.5 μmol/L combined with 2 μmol/L of paelitaxel) for 72 h was (30.93±4.57)%,higher than that of the cells treated with paelitaxel alone[(23.85±3.65)%]. CONCLUSION: NAC-1 siRNA suppresses NAC-1 gene expression and EGFR downstream signaling activation, inhibits cell proliferation and enhances the responsiveness of ovarian cancer cells to paelitaxel. The combination treatment produces synergistic inhibition.     

Proliferation-inhibitory and apoptosis-inducing effects of cinnamic acid combined with cisplatin on human hepatocellular carcinoma cell line MHCC97

AIM：To investigate the effects of cinnamic acid (CA) combined with cisplatin on the proliferation and apoptosis of human hepatocellular carcinoma cell line MHCC97. METHODS：Human hepatocellular carcinoma cell line MHCC97 was culured and divided into CA group, cisplatin group, CA+cisplatin group and control group. MTT assay, inverted microscopy, annexin V-FITC/PI staining and flow cytometry were applied to identify the viability, morphology and apoptosis of the cells. The apoptosis-related signaling protein caspase-3 was detected by Western blotting. RESULTS：CA and cisplatin either alone or in combination significantly inhibited the proliferation and induced obvious apoptosis of MHCC97 cells, while CA alone or combined with cisplatin had no significant inhibitory effect on normal human liver L-02 cells. The rates of mid-and late apoptosis or necrosis were higher in cisplatin group than that in CA group or combination group, but the early apoptotic rate was just the opposite. Pro-apoptotic activity in combination group was much stronger than that in CA group or cisplatin group at lower concentration, and combination group promoted apoptosis and decreased the cytotoxic side effects of cisplatin. CA and cisplatin either alone or in combination also up-regulated the cleaved caspase-3 expression in a time-dependent manner, and the effects in CA group and combination group were higher than that in cisplatin group. CONCLUSION：CA and cisplatin either alone or in combination inhibit the growth of MHCC97 cells by inducing apoptosis, and the activation of caspase-3 may play important roles in these processes.     

DIM enhances effect of cis-diamminedichloroplatinum on growth inhibition and apoptosis in human prostate cancer cell PC-3

AIM: To study the effects of the combination of cis-diamminedichloroplatinum(DDP) and 3, 3-diindolylmethane (DIM) on the growth and apoptosis of human prostate cancer cell PC-3. METHODS: MTT method was applied to detect the cell growth inhibitory rate. The cell apoptosis was measured by the flow cytometry and acridine orange staining method. The expression of the anti-oncogene p21 was detected by RT-PCR technique. RESULTS: The combination of 60 μmol·L^-1 DIM and 0.4 mg·L^-1 DDP effectively inhibited the growth and induced apoptosis in PC-3 cells. This result was the same as the effect of using 4 mg·L^-1 DDP only. The cell growth inhibitory and apoptosis rates for the combination of DIM and DDP were much higher than those for the individual effect. Both the combination and the single effect of these two medicines (i.e., DIM and DDP) all strengthen p21 mRNA expression significantly, and the effect of combination was more significant. CONCLUSION: DIM significantly enhances the effects of DDP on the growth inhibition and apoptosis induction in PC-3 cells.     

Effects of salinomycin combined with L-asparaginase on proliferation and apoptosis of human acute T-cell leukemia Jurkat cells

AIM:To investigate the effects of salinomycin alone or in combination with L-asparaginase on the growth and apoptosis of human acute T-cell leukemia Jurkat cells, and the possible mechanism. METHODS:The growth of Jurkat cells was tested by Cell Counting Kit-8 in vitro. The levels of cytochrome C, Bcl-2, caspase-3, caspase-8 and caspase-9 were measured by Western blotting. Flow cytometry was used to assay cell apoptosis. RESULTS:Salinomycin or L-asparaginase alone inhibited the growth of Jurkat cells in a dose-dependent manner. The IC50 value of L-asparaginase was 8.12 IU/L, while that of salinomycin was 0.75 μmol/L. Salinomycin combined with L-asparaginase induced more significant inhibition of cell proliferation (P<0.05). Western blotting showed that the expression of Bcl-2 protein in combination group was significantly reduced, and the expression of caspase-3, caspase-8, caspase-9 and cytochrome C was significantly increased (P<0.05). Flow cytometry showed that the apoptotic rates of Jurkat cells incubated with salinomycin (0.5 μmol/L), L-asparaginase (2.5 IU/L) and both drugs for 48 h were (7.11±0.23)%, (25.43±0.47)% and (39.12±1.97)%, respectively, and significantly higher than that in control group ［(6.67±0.13)%, P<0.05］.CONCLUSION: Salinomycin synergizes with L-asparaginase-induced cytotoxicity in vitro, and the combined treatment with salinomycin and L-asparaginase induces the apoptosis of Jurkat cells.     

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.     

Apoptosis induction in gastric carcinoma cells by celecoxib combined with adriamycin

AIM:To study the apoptosis induction of cyclooxygenase-2 (COX-2) inhibitor，celecoxib and adriamycin (ADM) on tumor apoptosis of gastric carcinoma MGC-803 cells, and to explore their possible molecular mechanism(s) and interactions.METHODS:The number of MGC-803 cells was observed by MTT assay. Tumor apoptosis was studied by fluorescence microscopy, flow cytometry (FCM), and DNA ladder. RESULTS:MGC-803 cell number was significantly decreased with increasing dose of ADM. Cells were accumulated in G₀/G₁ phase and the number of cells in S phase was decreased. ADM (5 mg/L) combined with celecoxib (25 μmol/L) markably inhibited the growth of MGC-803 cells. Significant morphological changes of typical apoptosis were observed after treatment with combined use of celecoxib and ADM. Compared with ADM or celecoxib alone, ADM plus celecoxib obviously enhanced the DNA ladder fragment revealed by agarose gel electrophoresis of DNA. After exposure to combined celecoxib and ADM treatment for 48 h, MGC-803 cells were accumulated in G₀/G₁ phase. There was a decrease in the number of cells in S phase as compared to celecoxib or ADM alone. CONCLUSION:Celecoxib and ADM appear to have synergistic effects for the apoptosis induction. This may be an important prospect for applying COX-2 inhibitors to assist chemical therapy of ADM in clinical use.     

Combination therapy of FK228 with rapamycin synergistically promotes human breast cancer cell apoptosis by DNA damage and cell cycle arrest

AIM: To investigate the depressant effect of FK228 combined with rapamycin on the human breast cancer cell line MCF-7 and MDA-MB-435.METHODS: FK228, a new histone deacetylase inhibitor, and rapamycin, the specific inhibitor of the mammalian target of rapamycin (mTOR) protein, were used in the study. MCF-7 cells and MDA-MB-435 cells were exposed to different concentrations of FK228 and rapamycin. The inhibitory rate of cell growth was determined by SRB assay. Combination index (CI) was used to evaluate the interaction between FK228 and rapamycin. The expression of the apoptotic proteins, cycle proteins and nucleic acid proteins were detected by Western blotting. The cell cycle was analyzed by flow cytometry.RESULTS: Both FK228 and rapamycin showed growth inhibitory effects on the breast cancer cell lines in a time-and dose-dependent manner. CI of the 2 drugs was less than 1 when the inhibitory rate of the cell growth was 50% effective dose (ED50)~ED70, indicating a synergistic effect. The combination therapy of FK228 with rapamycin increased the apoptotic proteins, and induced the down-regulation of phosphorylated Akt and over-expression of caspase-3 compared with a single use of the drugs. The combination therapy of FK228 with rapamycin reduced the cycle proteins, and the cell cycle was arrested in G₂/M. The levels of phosphorylated H2AX and acetylated H3 were ob-viously increased after combination therapy.CONCLUSION: The combination therapy of FK228 with rapamycin inhibits the cell proliferation and increases apoptosis with a synergistic effect, which may become a new trend for treating endometrial cancer.     

Brucine induces apoptosis in colon cancer SW480 cells via inhibiting IL-6/STAT3 signaling pathway

AIM: To observe the effects of brucine on the viability and apoptosis of colon cancer SW480 cells.METHODS: The SW480 cells were divided into control group, 1 μmol/L brucine treatment group, 100 μg/L IL-6 treatment group and IL-6＋brucine treatment group. The cell viability was detected by CCK-8 assay. The apoptotic rate was measured by flow cytometry using fluorescein-labeled Annexin V/PI. The changes of apoptosis-related proteins were determined by Western blot. The protein level of p-STAT3 was also detected by immunofluorescence staining. RESULTS: Brucine inhibited SW480 cell growth, and the viability inhibition rate of the SW480 cells treated with brucine alone was more efficient than using brucine combined with IL-6 (P < 0.05). The apoptotic SW480 cells increased significantly after 1 μmol/L brucine treatment as compared with brucine treatment alone (P < 0.05). The apoptotic SW480 cells were significantly reduced in brucine and IL-6 combination treatment group (P < 0.05). Brucine inhibited the protein level of p-STAT3 significantly. The protein level of p-STAT3 was significantly increased in 100 μg/L IL-6 treatment group. Compared with 1 μmol/L brucine treatment alone, the expression of Bcl-2 was increased and the protein levels of p-STAT3, Bax and cleaved PARP were reduced in brucine and IL-6 combination treatment group (P < 0.05).CONCLUSION: Brucine may inhibit the activation of STAT3 phosphorylation in IL-6/STAT3 pathway to exert an antitumor effect on SW480 cells in vitro.     

Effect of 3,5-hydroxy-6,7,3’,4’-tetramethoxyflavone isolated from Laggera pterodonta on apoptosis of Hep-2 cells in vitro

AIM: To investigate the effect of 3,5-hydroxy-6,7,3’,4’- tetramethoxyflavone (HTMF) isolated from Laggera pterodonta on Hep-2 cell apoptosis and the underlying mechanism. METHODS: The MTT assay was used to observe the cytotoxicity of HTMF to the normal cells and the inhibitory effect of HTMF on the proliferation of tumor cells. The apoptosis was determined by flow cytometry. Western blotting was used to detect the expression of caspase-3 and caspase-9. RESULTS: HTMF significantly inhibited the growth of Hep-2 cells in dose and time dependent manners. HTMF exhibited weak cytotoxicity to the two normal cell lines Vero and EVC304, while showed low effect of anti-proliferation on HepG2 cells and A549 cells. The increase in apoptosis of Hep-2 cells by HTMF was observed with dose and time dependent manners. Western blotting showed that HTMF time dependently increased the expression of caspase-3 and caspase-9 in Hep-2 cells. CONCLUSION: HTMF has high inhibitory effect on the proliferation of Hep-2 cells by induction of apoptosis in the tumor cells through caspase-9 and caspase-3 activation. However, the cytotoxicity of HTMF to the normal cells is low.     

Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells

AIM: To investigate the effect of plumbagin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemic Kasumi-1 cells. METHODS: Kasumi-1 cells were treated with plumbagin alone, recombinant soluble TRAIL(rsTRAIL) alone or the combination of plumbagin with rsTRAIL to induce apoptosis. The cell proliferation was analyzed by CCK-8 assay. Apoptosis was determined by flow cytometry with AnnexinⅤ/PI double staining and TUNEL staining. The expression of DR4 and DR5 at mRNA level was measured by real-time PCR. The expression of signal transduction proteins, such as DR5, caspase-3, caspase-8, caspasep-9, Bid, Bax and c-FLIP was detected by Western blotting. RESULTS: Both rsTRAIL and plumbagin induced the apoptosis in Kasumi-1 cells, and combination of plumbagin with rsTRAIL enhanced the apoptosis. The ratios of Annexin V-positive Kasumi-1 cells were (27.7±2.9)%, (25.6±3.1)% and (52.1±3.3)% in 100 μg/L rsTRAIL group, 2 μmol/L plumbagin group and the combination group, respectively, and the positive rate in combination group was significantly higher than those in other 2 groups. TUNEL assay demonstrated that the number of apoptotic cells in combination group was higher than that in the cells treated with rsTRAIL or plumbagin alone. Plumbagin up-regulated the expression of DR5 at mRNA level in Kasumi-1 cells, and up-regulation of DR5, activation of caspase-8 and down-regulation of c-FLIP at protein level were detected in the cells treated with plumbagin alone and the combination of plumbagin with rsTRAIL. CONCLUSION: Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells by up-regulating DR5, activating caspase-8 and down-regulating c-FLIP.     

Effect of trastuzumab (herceptin) on the apoptosis induced by ZD1839 (iressa) in lung cancer A549 cells

AIM: ZD1839 and trastuzumab are reported to improve the therapeutic efficacy of treatment for non-smallcell lung cancer (NSCLC) and breast cancer, respectively, although the effectiveness of either drug alone is not satisfactory. NSCLC cells often express both EGFR and HER2. We therefore investigated whether a combination of ZD1839 and trastuzumab had an additive or synergistic antitumor effect. METHODS: MTT was used to measure the inhibitory effects of ZD1839 (iressa) and trastuzumab (herceptin) on the growth of A549 cells. The cell apoptosis was studied by DAPI staining, and Annexin V/PI double labeling. RESULTS: The inhibitory action of cell growth was seen in A549 cells dealing with ZD1839 and trastuzumab. They inhibited the growth of the human lung cancer cell line A549 in a concentration and time dependent manners. Compared with either ZD1839 or trastuzumab alone, combination with curcumin respectively increased the growth inhibition rate and increased apoptosis of A549 cells (P<0.05) significantly, suggesting the synergistic actions of the two drugs. CONCLUSION: The results suggest that combination treatment with ZD1839 and trastuzumab might have improved therapeutic efficacy against NSCLC cells expressing both EGFR and HER2.     

Potentiation of the action of phorbol ester by heparin in human CNE2 cells: Association with enhanced expression of c-jun,p53 and p21

AIM: To measure the effect of addition of heparin to TPA on cell proliferation and apoptosis in CNE2 cells and investigate the possible molecular mechanisms underlying heparin and TPA interaction on cell proliferation and apoptosis. METHODS: Cell viability and cell cycle were determined by cell counting and flow cytometry.Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL)and agarose gel electrophoresis. The expression of c-jun, c-fos, p21 and p53 was examined by Western blot. RESULTS: TPA alone inhibited CNE2 cell proliferation and evoked apoptosis associated with typical morphological changes and DNA fragmentation,which was augmented when heparin was added. Compared with TPA or heparin alone, TPA plus heparin obviously enhanced the number of TUNEL-positive cells from 23%±1.2% to 51%±0.9%. After exposure to different concentrations of heparin (with or without TPA) for 24 h, CNE2 cells were accumulated G₀/G₁ phase. There was a decrease in the number of cells in S phase by the combined heparin and TPA treatment compared to heparin or TPA alone. Western blot analysis revealed that TPA induced the increases in c-jun and p53, p21 protein expression and the levels were remarkably increased following heparin in combination with TPA treatment,whereas no significant change in c-fos was detected. CONCLUSION: These results suggest that heparin synergistically potentiates the action of TPA in CNE2 cells, which may be associated with the increase in c-jun protein level and the upregulation of p21, p53 protein expression.     

Relationship between apoptosis and differentiation from ESCs into cardiomyocytes induced by TGF-β1 and co-culture with visceral endoderm like END-2 cells

AIM: To provide the reference for optimizing the seed cells for tissue engineering,the relationship between apoptosis and differentiation in the process of induction on embryonic stem cells(ESCs) was investigated.METHODS: Day 2-3 embryoid bodys (EBs) were derived from ESCs,and then tissue growth factor-β₁(TGF-β₁) was added,and co-cultured with visceral endoderm like END-2 cell conditioned medium.The induced cells were evaluated by using immunofluoresence and transmission electron micrography.Cell apoptosis was analyzed using flow cytometry.RESULTS: The total percentage of beating EBs treated with TGF-β₁ was 43%.All the beating cardiomyocytes derived from ESCs expressed cardiac-specific proteins for TnT,and were observed the cardiac-specific ultrastructure.Interestingly,the total percentage of beating EBs treated with TGF-β₁ combined with END-2 cell conditioned medium was 88% (P＜0.01),and the beating areas were bigger.After 3 days of induction with different conditions,the apoptotic levels were (5.58%±0.65% and 9.60%±0.75%,P＜0.05),respectively.CONCLUSION: combination of TGF-β₁ with co-cultured visceral endoderm like END-2 cell conditioned medium could get a higher induction efficiency on the differentiation of ESCs into the cardiomyocytes.The partly inducible effect mechanisms may induce EDCs differentiation toward a cardiac phenotype and enhance apoptosis in cells not committed to cardiac differentiation.     

Adeno-associated virus type 9-mediated p65 silencing inhibits angiotensin II-induced apoptosis of H9c2 cells

AIM: To study the effect of p65 gene silencing by adeno-associated virus type 9 (AAV9)-mediated RNA interference on angiotensin Ⅱ (Ang Ⅱ; 10^-6 mol/L for 24 h)-induced apoptosis of rat ventricular H9c2 myocytes, and to elucidate the possible mechanism. METHODS: The H9c2 cells were transfected with rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA at multiplicity of infection (MOI)=4×10⁶ vg/cell. eGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of eGFP positive cells was determined by flow cytometry. The expression of p65 was determined by Western blot. CCK-8 assay was used to measured the viability of transfected H9c2 cells. The apoptosis of the cells transfected with the virus and with Ang Ⅱ stimulation was analyzed by flow cytometry. RESULTS: The cells began to exhibit eGFP expression on the 2nd day after transfection. The fluorescence intensity was increased over the time of transfection. eGFP expression reached the maximum on the 5th day, and the transfection efficiency was (52.7±1.9)% at this time point. Compared with blank control group, no significant effect of AAV9 on the viability of H9c2 cells was observed. In resting state, p65 in the H9c2 cells had a certain activity. After Ang Ⅱ stimulation, the activity of p65 was obviously increased, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited the expression of p65. The apoptosis of H9c2 cells in Ang Ⅱ stimulation group was significantly higher than that in blank control group, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited apoptosis of H9c2 cells. CONCLUSION: Transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibits the expression of p65 gene of NF-κB pathway in the H9c2 cells without causing cell growth inhibition, and reduces the apoptosis induced by Ang Ⅱ.