Dendritic cells transfected with recombinant adenovirus carrying IL-24 gene induces CTLs to promote apoptosis of cervical cancer cell line CaSki

AIM：To investigate the therapeutic potential of interleukin-24 (IL-24) in cancer treatment, we observed the inductive effect of cytotoxic T-lymphocytes (CTLs) against human cervical cancer cell line CaSki using dendritic cells (DCs) transfected with recombinant adenovirus carrying IL-24 gene (Ad-IL-24-DCs). METHODS：Immature mouse DCs were isolated and cultured. The DCs were infected with recombinant adenovirus carrying IL-24 gene. The CaSki cell lysate-loaded autologous DCs vaccine was also prepared. The cell proliferation was analyzed by MTT assay. The cell apoptosis was determined by flow cytometry with PE-Annexin V staining. The cleaved caspase-3 in the Ad-IL-24-DCs was measured by Western blotting. Colony-forming assay was used to detect the colony number of Ad-IL-24-DCs. The tumorigenic capacity of CaSki cells in vivo in the presence of Ad-IL-24-DCs was observed by tumor-burdened model. RESULTS：IL-24 was highly expressed in the Ad-IL-24-DCs. In the presence of Ad-IL-24-DCs, the apoptosis of CaSki cells was significant increased. Additionally, the protein level of cleaved caspase-3 was increased in CaSki cells in the presence of Ad-IL-24-DCs, suggesting that the CaSki cells with Ad-IL-24-DCs induced apoptosis. CTLs induced by Ad-IL-24-DCs inhibited the tumorigenic capacity of CaSKi cells in vivo. CONCLUSION：Genetically modified DCs are successfully prepared by infection with Ad-IL-24 and show a significant effect on triggering specific CTLs against CaSki cells.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.     

Expression of SCD-1 in cervical carcinoma and effect of its down-regulation on proliferation and apoptosis of cervical carcinoma cells

AIM:To examine the expression of stearoyl-CoA desaturase-1 (SCD-1) in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki, and to investigate the effect of down-regulation of SCD-1 on the proliferation and apoptosis of cervical carcinoma cells. METHODS:The expression of SCD-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki. SCD-1 siRNA and control siRNA were utilized to transfect CaSki cells by Lipofectamine 2000, and SCD-1 protein level was determined by Western blotting after transfection. Furthermore, CCK-8 and flow cytometry were utilized to investigate the changes of cell proliferation and apoptosis after transfection with SCD-1 siRNA in CaSki cells. Subsequently, the activities of caspase-3 and caspase-9 were analyzed by Caspase-Glo3/7 and 9 detection kit after transfection with SCD-1 siRNA in CaSki cells. Finally, the protein expression of Bcl-2 and Bax was detected by Western blotting. RESULTS:The protein expression of SCD-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues, and the protein expression of SCD-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which CaSki cells displayed the highest SCD-1 protein level. In addition, the protein expression of SCD-1 in SCD-1 siRNA group was significantly lower than that in untreated group and control siRNA group. Compared with untreated group and control siRNA group, the proliferation of CaSki cells was markedly inhibited in SCD-1 siRNA group. Early apoptotic rate in SCD-1 siRNA group was evidently higher than that in untreated group and control siRNA group. The activities of caspase-3 and caspase-9, and the level of Bax protein were significantly elevated, and the protein level of Bcl-2 was obviously reduced after transfection with SCD-1 siRNA in CaSki cells. CONCLUSION: SCD-1 may play an important role in the occurrence and development of cervical carcinoma, and its down-regulation, which mediates cell proliferation inhibition and apoptosis, may be tightly associated with the activities of caspase-3 and caspase-9, and the protein expression of Bcl-2 and Bax.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.     

Notch-1 gene silencing promotes phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells

AIM: To investigate the effect of Notch1 gene silencing on phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells.METHODS: shRNA-Notch1 eukaryotic expression plasmid was constructed and transfected into MCF-7 cells. The expression of Notch1 and Hes-1 was observed by Western blotting after transfction. Apoptosis and mitochondrial membrane potential were detected by flow cytometry. Western blotting was also used to determine the protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 after Notch1 silencing was performed in MCF-7 cells.RESULTS: Silencing of Notch1 significantly reduced the expression of Notch1 and Hes-1 in MCF-7 cells (P<0.01). In shNotch1 group, the number of apoptotic cells was much higher (P<0.01) and mitochondrial membrane potential was much lower (P<0.05) than those in shControl group. The protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 increased obviously after silencing of Notch1 was performed in MCF-7 cells (P<0.05).CONCLUSION: Notch1 silencing induces apoptosis of human breast cancer MCF-7 cells through promoting phosphorylations of JNK1 and p53, and increasing the production of PUMA, NOXA and cleaved caspase-3.     

Sodium aescinate induces apoptosis of HeLa cells by inhibiting Akt/ERK signaling pathways and increasing death receptor expression

AIM:To study the effects of sodium aescinate on the apoptosis of cervical cancer HeLa cells and its molecular mechanism. METHODS:MTT assay was used to detect the growth and proliferation of HeLa cells. The morphological alteration was observed under inverted microscope. Annexin V-FITC/PI double staining and DAPI nuclear staining were used to determine the apoptosis of HeLa cells induced by sodium aescinate. The apoptosis-related proteins PARP, cleaved caspase-8 and pro-caspase-3, and the proliferation-associated molecules Akt and ERK, as well as TRAIL receptors DR4 and DR5 were detected by Western blotting. RESULTS:Sodium aescinate inhibited the growth of HeLa cells in a concentration-dependent manner. Treatment with sodium aescinate induced the typical morphology of apoptotic cells and increased the apoptotic rate significantly. The cleaved PARP, cleaved caspase-8 and cleaved caspase-9 protein expression was observed. The expression of DR4 and DR5 was up-regulated. Meanwhile, pro-caspase-3 was decreased, and the levels of p-Akt and p-ERK were down-regulated by sodium aescinate in a dose-dependent manner. CONCLUSION:Sodium aescinate inhibits the proliferation and promotes the apoptosis of HeLa cells by increasing death receptor expression and repressing proliferation-associated signaling pathways.     

Et-DHA induces apoptosis of HepG2 cells through activation of mitochondrial pathway and induction of granzyme B expression in T cells

AIM：To investigate the effect of ethyl docosahexaenoate (Et-DHA) on the apoptosis of human hepatocarcinoma HepG2 cells. METHODS：HepG2 cells were used to test the anticarcinogenicity of Et-DHA. The direct inhibition of HepG2 cells by Et-DHA was detected by MTT. Nuclear morphological features of the HepG2 cells were observed under fluorescence microscope after staining with Hochest 33258. The levels of Bax, Bak, Bid, Bcl-2, Smac and cytochrome C (Cyt C) in mitochondria and cytosol, the cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 in cytosol, as well as the release of reactive oxygen species (ROS), total superoxide dismutase (SOD) and caspase-9 activity in the Et-DHA-treated HepG2 cells were determined by Western blotting and ELISA. Furthermore, by co-culturing the HepG2 cells with T cells, the effects of proliferation of Et-DHA-treated T cells on the activity of HepG2 cells were observed, and the level of granzyme B was detected. RESULTS：Et-DHA significantly inhibited the growth of HepG2 cells in a concentration- and time-dependent manner. The ROS release and caspase-9 activity increased markedly in Et-DHA-treated HepG2 cells, and no significant change of the total SOD activity was observed. The levels of the pro-apoptotic proteins Bax, Bak and Bid in mitochondria increased, the anti-apoptotic protein Bcl-2 as well as mitochondrial Cyt C and Smac levels decreased, and the cytoplasmic Cyt C, Smac, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3 and cleaved Bid levels showed dose-dependent increases. Additionally, the degree of Et-DHA-induced apoptosis in HepG2 cells in the co-culture group (T cells+HepG2 cells) showed a further increase as compared with the HepG2 cells treated with Et-DHA alone. Due to Et-DHA inducing elevation of granzyme B level in the T cells, the granzyme B released into HepG2 cells was significantly increased. CONCLUSION:Et-DHA might induce the apoptosis of HepG2 cells through activation of caspase-3 mainly via a mitochondrial intrinsic pathway and a caspase-8 pathway, and promote the increase in granzyme B indirectly by activating T cells, thus enhancing the cytotoxic effect on HepG2 cells.     

Effects of p21-activated protein kinase 2 down-regulation on proliferation and apoptosis of human breast cancer cells

AIM：To study the effect of p21-activated protein kinase 2 (PAK2) knockdown by RNA interference on the proliferation and apoptosis of human breast cancer cells. METHODS：The short hairpin RNA (shRNA) targeting PAK2 gene was designed and used for packing lentivirus in 293T cells.Human breast cancer MCF-7 cells were infected by the virus particles and PAK2 knockdown stable cell line was established by puromycin selection. The knockdown efficiency was assessed by Western blotting. The proliferation ability of MCF-7 cells was evaluated by CellTiter 96 AQueous and anchorage-independent growth assays. The cell apoptosis induced by staurosporine was detected by flow cytometry. RESULTS：The protein level of PAK2 was significantly suppressed after silencing of PAK2 gene in MCF-7 cells (P<0.01). Furthermore, knockdown of PAK2 caused remarkable inhibition of the cell proliferation and colony formation (P<0.01). Staurosporine induced more apoptosis in the PAK2 knockdown cells compared with the control cells (P<0.01). CONCLUSION：Knockdown of PAK2 inhibits the proliferation of MCF-7 cells and increases the sensitivity of chemotherapeutic drug-induced cell apoptosis, suggesting that PAK2 might be a new therapeutic target in breast cancer treatment.     

Effects of down-regulation of X-linked inhibitor of apoptosis protein gene on docetaxel-induced apoptosis in bladder cancer cells

AIM: To observe the effects of down-regulation of X-linked inhibitor of apoptosis protein ( XIAP ) gene on the chemotherapeutic sensitivity of bladder cancer cells. METHODS: The shRNA and shRNA-XIAP were transfected into bladder cancer T24T cells. The fluorescence microscopy was used to detect the transfection effects. XIAP gene expression was detected by RT-PCR and Western blotting. Docetaxel was administrated to T24T, T24T shRNA and T24T shRNA-XIAP bladder cancer cells. ATPase methods was performed to measure in vitro cell viability. Morphological changes and apoptotic rates were determined by phase contrast microscopy and flow cytometry assay. Cellular poly(ADP-ribose) polymerase (PARP) and caspase-3 protein expression and their cleavage were assayed by Western blotting. RESULTS: The fluorescence was obvious in the T24T shRNA and T24T shRNA-XIAP cells under the fluorescence microscope. Using Western blotting and RT-PCR methods, the protein and mRNA levels of XIAP gene in T24T shRNA-XIAP cells were significantly decreased,respectively. After treated with various concentrations of docetaxel for 24 h, the IC₅₀ of T24T shRNA-XIAP cells was (1.23?0.62) nmol/L, lower than that of T24T and T24T shRNA cells, which were (8.22?1.23) and (8.35?0.98) nmol/L (P<0.01), respectively. Compared with control group, the typical morphological changes of apoptosis were observed in T24T shRNA-XIAP cells. Detected by flow cytometry assay, the apoptotic rates in shRNA-XIAP group were (41.45?6.23)% and (74.82?5.46)% after exposed to docetaxel at the concentrations of 2 nmol/L and 5 nmol/L for 24 h, which were significantly higher than that in T24T shRNA group with (25.34?3.81)% and (34.14?6.25)%, respectively (P<0.01). Compared with T24T shRNA cells, the cleavage of PARP and caspase-3 proteins in the cells transfected with shRNA-XIAP was significantly increased. CONCLUSION: XIAP gene is significantly down-regulated via shRNA-XIAP, which could increase the docetaxel-induced apoptosis and cytotoxic activity.     

Dexmedetomidine reduced isoflurane-induced neuroapoptosis by inhibiting activation of p38 and JNK proteins in hippocampus of neonatal rats

AIM：To investigate the effect of dexmedetomidine (Dex) on neuronal apoptosis induced by isoflurane (Iso) and its relationship with the expression of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) proteins in the hippocampus of neonatal rats. METHODS：Forty-eight neonatal SD rats at postnatal day 7 were randomly divided into control group (Con), Dex group, Iso group and Iso combined with Dex (Iso+Dex) group. Rats in Iso and Iso+Dex groups were exposed to 0.75% Iso for 6 h, while rats in Con and Dex groups were exposed to air for 6 h. Rats were intraperitoneally injected with 25 μg·kg-1 Dex (Dex and Iso+Dex groups) or 150 μL saline (Con and Iso groups) 20 min before exposure and 2 and 4 h after exposure. After the termination of anesthesia, the neuronal apoptosis in hippocampal CA1 region was detected by TUNEL staining, and the protein expression of cleaved caspase-3, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK) and JNK in hippocampal tissues was detected by Western blotting. RESULTS：The number of TUNEL positive cells in hippocampal CA1 region of the rats in Iso group was increased by 447.57% (P<0.01) compared with Con group, while Dex significantly inhibited the increased TUNEL positive cells in Iso group by 75.18% (P<0.01). The expression of cleaved caspase-3 protein in Iso group was increased by 126.29% (P<0.01) compared with Con group, while Dex reversed the increased cleaved caspase-3 protein expression (P<0.01). Iso significantly increased the phosphorylation of p38 and JNK proteins (P<0.01), while Dex reversed the increased p-p38 and p-JNK proteins (P<0.01). CONCLUSION：Dex attenuates Iso-induced neuroapoptosis in the hippocampus of neonatal rats through inhibiting the phosphorylation of p38 and JNK proteins.     

Effect of HDAC1 silencing on apoptosis of squamous cell carcinoma of skin via regulating STAT3 signaling pathway

AIM: To investigate the effect of histone deacetylase 1 (HDAC1) silencing on apoptosis of squamous cell carcinoma of skin. METHODS: Skin squamous cell carcinoma A431 cells were transfected with HDAC1 small interfering RNA (HDAC1 siRNA) or small interfering RNA negative control (siRNA NC). The expression levels of HDAC1 in transfected cells were detected by RT-PCR and Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. The inhibitor of STAT3 signaling pathway was used to treat the A431 cells transfected with HDAC1 siRNA. The cell viability was detected by MTT assay, the apoptosis was analyzed by flow cytometry, and the protein levels of STAT3, p-STAT3 and cleaved caspase-3 were determined by Western blot. RESULTS: HDAC1 siRNA inhibited the expression of HDAC1 at mRNA and protein levels in the A431 cells. After interfering with the expression of HDAC1, the cell viability and the protein level of p-STAT3 in the cells decreased, while the apoptotic rate and the protein level of cleaved caspase-3 in the cells were increased. After treatment with the inhibitor of STAT3 pathway, the viability of A431 cells transfected with siRNA and the protein level of p-STAT3 decreased, while the apoptotic rate and the protein le-vel of cleaved caspase-3 in the cells were increased. CONCLUSION: Interference with HDAC1 expression may regulate the STAT3 signaling pathway to inhibit the viability of skin squamous cell carcinoma cells, thus promoting the apoptosis of squamous cell carcinoma of skin.     

Effect of gambogic acid on proliferation and apoptosis of human diffuse large B-cell lymphoma cell line OCI-LY-1

AIM: To investigate the effects of gambogic acid (GA) on the proliferation and apoptosis of human diffuse large B-cell lymphoma cells. METHODS: Diffuse large B-cell lymphoma cell line OCI-LY-19 was treated with GA. The cell viability and proliferation were evaluated by MTS assay. The morphological changes of the cells were observed under inverted microscope and fluorescence microscope with PI staining. The cell apoptosis was detected by flow cytometry. The changes of proteasome-related proteins Ubs, Bax, HSP90 and IκB-α, the apoptosis-related proteins PARP, pro-caspase-3 and caspase-8, and the proliferation-associated molecules ERK and STAT5 were detected by Western blotting. RESULTS: The proliferation of OCI-LY-19 cells was inhibited and the apoptosis was induced by GA. The expression of Ubs, Bax and HSP90 was up-regulated by GA treatment in a dose-dependent manner. The PARP protein was cleaved. The levels of apoptosis-related proteins pro-caspase-3 and pro-caspase-8, and proliferation-associated molecules p-Akt, p-ERK and p-STAT5 were down-regulated by GA treatment in a dose- and time-dependent manner. CONCLUSION: Gambogic acid down-regulates the activity of proteasome, inhibits cell proliferation and promotes cell apoptosis of diffuse large B cell lymphoma.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbonate on the proliferation and apoptosis in K562 cells

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB on the proliferation and apoptosis of K562 cells and to explore the anti-tumor mechanism of PDTC.METHODS: Trans AMTM NF-κB p65 kit was used to detect the activity of p65 in K562 cells treated by PDTC. The effect of PDTC on the proliferation of K562 cells was measured by WST-1 method. DNA damage was detected by single cell gel electrophoresis (comet assay). The procaspase-3 and activated protein level of caspase-3 were detected by Western blotting.RESULTS: The activity of p65 in K562 cells was inhibited after treated by PDTC (P<0.01). Simultaneously the cell proliferation was significantly inhibited in a dose-and time-dependent manner (P<0.01). The degree of DNA damage in K562 cells treated with PDTC at concentrations of 25 μmol/L, 50 μmol/L or 100 μmol/L was more severe than that in control. The rates of comet cells in the PDTC-treated groups (43.50％, 84.00％, 95.63％) were significantly higher than those in control (9.75％, P<0.01), and it was also dose-dependent. The expression of procaspase-3 and activated caspase-3 protein were detected in the cytoplasm of the K562 cells treated by PDTC by Western blotting.CONCLUSION: NF-κB plays an important role in regulating cell proliferation and apoptosis in K562 cells. PDTC inhibits NF-κB activity and elevates the expression of caspase-3, which is related to increase in cell apoptosis.     

Silencing of FoxM1 induces apoptosis of oral squamous-cell carcinoma cells by promoting mitochondrial release of cytochrome C

AIM: To study the effect of forkhead box protein M1 (FoxM1) silencing on apoptosis of oral squamous-cell carcinoma. METHODS: Oral squamous-cell carcinoma SCC9 cells were infected with FoxM1-shRNA lentivirus or negative control lentivirus. The silencing effect was measured by RT-qPCR and Western blot. The changes of cell viability effect was measured by MTT saay. The cell colony formation ability was measured by plate experiment. Flow cytometry was used to analyze the changes of apoptotic rate. Western blot was used to measure the protein levels of cleaved caspase-3 and cleaved caspase-9 in the cells. The changes of mitochondrial membrane potential were measured by JC-1 method. Western blot was used to measure the protein level of cytochrome C in the mitochondria and cytoplasm. RESULTS: Infection with FoxM1-shRNA lentivirus successfully reduced the expression of FoxM1 in oral squamous-cell carcinoma cells (P<0.05). Negative control lentivirus had no effect on the expression level of FoxM1 in the cells. The cell viability was reduced by FoxM1 silencing, and the ability of cell colony formation was also decreased. The apoptotic rate and the protein levels of cleaved caspase-3 and cleaved caspase-9 were all increased (P<0.05), and the mitochondrial membrane potential was decreased. The protein level of cytochrome C in the cytoplasm was increased, while the protein level of cytochrome C in the mitochondria was decreased (P<0.05). CONCLUSION: Silencing of FoxM1 induces the apoptosis of oral squamous-cell carcinoma cells by decreasing the mitochondrial membrane potential and promoting the release of cytochrome C from mitochondria.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.     

Artesunate induces apoptosis of human hepatocellular carcinoma HepG2 cells by increasing generation of reactive oxygen species

AIM: To investigate the effect of reactive oxygen species (ROS) on the apoptosis of HepG2 cells induced by artesunate. METHODS: The effect of artesunate on the viability of HepG2 cells was measured by MTT assay. The morphological changes of the apoptotic cells were observed by the method of Hoechst 33258 fluorescence staining.The apoptosis of HepG2 cells was analyzed by flow cytometry. DCFH-DA was used to detect the changes of ROS generation during the process of apoptosis. The protein levels of Bax, Bcl-2, cleaved caspase-3 and cytochrome C (Cyt C) were determined by Western blot. HepG2 cells were pretreated with apocynin and then Western blot was used to detect the expression of p47^phox and p22^phox, and ROS changes were analyzed by flow cytometry. RESULTS: Compare with control group, the cell viability was obviously inhibited after treatment with artesunate for 24 h (P<0.05). The nuclei were densely stained, and the proportion of apoptotic cells was increased (P<0.05). ROS was increased significantly (P<0.05). The results of Western blot demonstrated that the expression level of Bax was increased, Bcl-2 was decreased, the ratio of Bax/Bcl-2 was increased, and the protein levels of cleaved caspase-3 and Cyt C were increased. Pretreatment with apocynin reduced the expression of p47^phox and p22^phox and the generation of ROS in the artesunate treatment group. CONCLUSION: Artesunate induces the apoptosis of HepG2 cells. The possible mechanism may be related to the increase in the generation of ROS.     

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.     

Berberine promotes anti-proliferative effects of doxorubicin in T24 bladder cancer cells by enhancing apoptosis

AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.