Salinomycin inhibited proliferation and induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP

AIM: To investigate the effect of salinomycin on the proliferation and apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. METHODS: The inhibitory effect of salinomycin on the growth of A549/DDP cells was tested by MTT method in vitro . The apoptosis and mitochondrial membrane potential (ΔΨ_m) of A549/DDP cells were assayed by flow cytometry. The activity of caspase-3, 8 and 9 was determined by the method of colorimetry. The levels of cytochrome C, Bcl- 2, Bax, β-catenin, and phosphorylated low-density lipoprotein receptor-related protein 6(p-LRP6) were measured by Western blotting. RESULTS: Salinomycin inhibited the growth of A549/DDP cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L decreased ΔΨ_m level, and increased reactive oxygen species (ROS), cytochrome C and cytosolic Ca²⁺ release in the cells. Salinomycin also increased the acti-vity of caspase-3, 8, and 9 in the cells, reduced the ratio of Bcl-2/Bax, and decreased the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin depresses the cell growth by inhibiting Wnt signaling, and induces the apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP via mitochondria-dependent and Bcl-2/Bax pathways.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

Valproate enhances imatinib-induced apoptosis and down-regulates Bcr/Abl mRNA and phosphorylated protein kinase B in chronic myeloid leukemia cell line K562

AIM：To investigate the effects of valproate and imatinib on the apoptosis of chronic myeloid leukemic cell line K562. METHODS：K562 cells were divided into 3 groups and treated with valproate, imatinib and cotreatment, respectively. Cell cycle, apoptosis, the mRNA expression of Bcr/Abl, total protein kinase B (PKB) and phosphorylated PKB (p-PKB) were analyzed. RESULTS：The apoptotic rates in valproate group, imatinib group and cotreatment group were (11.47±0.25)%, (28.43±1.70)% and (57.73±4.38)%, respectively (P<0.05). No obvious difference was observed in cell cycle between cotreatment group and monodrug group. Bcr/Abl mRNA and p-PKB in the above 3 groups were (0.00±0.00), (64.17±12.27), and (0.00±0.00) ×10 9 copies/(g total mRNA), respectively (P<005), and 0.25±0.02, 0.17±0.01 and 0.08±0.01, respectively (P<0.05). No apparent difference of PKB was found in the 3 groups. CONCLUSION：Valproate enhances imatinib-induced apoptosis and may link to the down-regulation of Bcr/Abl mRNA and p-PKB in chronic myeloid leukemic cell line K562.     

Transition of WT1 isoforms inhibits proliferation and promotes apoptosis of human leukemia cell line HL-60

AIM: To study the potential effects of exogenous Wilms tumor 1 (WT1) isoforms on the proliferation and apoptosis of human leukemia cell line HL-60. METHODS: WT1 (17AA－/KTS－) gene obtained by RT-PCR was cloned into a PCDH1-MCS1-EF1-copGFP plasmid. The recombinant plasmid was confirmed by enzyme digestion and sequencing, and was transfected into HL-60 cells by LipofectamineTM 2000. The stable transformants were selected by G418 screening. WT1(17AA－/KTS－) expression was identified by real-time fluorescence quantitative RT-PCR and Western blotting. The proliferation of the cells was measured by MTT assay. The apoptosis of the cells was determined by morphological observation and flow cytometry analysis. RESULTS: The eukaryotic expression vector PCDH1-MCS1-EF1-copGFP-WT1 (17AA－/KTS－) was successfully constructed. The recombinant cells exhibited high mRNA and protein levels of WT1(17AA－/KTS－). The growth of recombinant cells was slower than that of HL-60 cells transfected with control vector and normal HL-60 cells. After exposed to As2O3 at 2 μmol/L for 48 h, both recombinant cells and control cells exhibited the morphological characteristics of apoptosis, but the former was more typical than the latter. The apoptosis was enhanced in the recombinant cells after the cells were exposed to As2O3 for 24 h. CONCLUSION: Exogenous WT1(17AA-/KTS-) isoform inhibits the proliferation and promotes the apoptosis of leukemic cells.     

Knockdown of astrocyte elevated gene-1 by siRNA inhibits cell proliferation and induces apoptosis in human neuroblastoma cell line

AIM: To investigate the effect of siRNA-induced astrocyte elevated gene-1 ( AEG-1 ) down-regulation on the proliferation, apoptosis and cell cycle of neuroblastoma cells. METHODS: An siRNA targeting to AEG-1 mRNA (AEG-1 siRNA) was constructed and transfected into neuroblastoma cells with Lipofectamine 2000. A non-specific siRNA (control siRNA) and non-treatment were used as negative control and blank control,respectively . The cell proliferation was detected by MTT method and colony formation assay. The apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Compared with control groups, the expression of AEG-1 mRNA was evidently declined in the cells transfected with AEG-1 siRNAs (P<0.05). AEG-1 siRNA significantly decreased the cell proliferation. After treated with AEG-1 siRNA for 48 h, the percentage of apoptotic cells was significant increased and the cell cycle was arrested in G₀/G₁ phase compared with the control cells (P<0.05). CONCLUSION: The mRNA expression of AEG-1 is down-regulated by AEG-1 siRNA in neuroblastoma cells. Knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibits cell proliferation and apoptosis, and induces cell arrest in G₀/G₁ phase of the cell cycle.     

FAK shRNA inhibits growth of leukemic cell line BCR/ABL-BaF3

AIM: To investigate the effect of focal adhesion kinase (FAK) shRNA on the growth of leukemic cells.METHODS: Lentiviral-FAK-shRNA was transfected into BCR/ABL-BaF3 cells, while empty vector was transfected into the same cells for control. The proteins of FAK and other molecules were detected by Western blotting. Cell growth was observed by culturing the leukemic cells in RPMI-1640 medium in vitro, and colony formation was observed by culturing the leukemic cells in methylcellulose medium. To establish a murine model of leukemia, BCR/ABL-BaF3 cells were injected into BALB/c mice through tail vein. Survival time of the leukemic mice was monitored, and the distribution of the leukemic cells in spleen of the mice was also detected. RESULTS: FAK shRNA inhibited the protein expression of FAK, reduced STAT5 phosphorylation and induced caspases-3 activation in BCR/ABL-BaF3 cells. FAK shRNA inhibited the cell growth in vitro. Colony formation experiment showed that the number of colony in vector control group and FAK shRNA group was 215.60±13.01 and 125.00±9.06, respectively, and the difference was statistically significant (P<0.01). The mice in vector control group died between day 21 and day 27, while the mice in FAK shRNA group died between day 52 and day 60, and the difference was statistically significant (P<0.05). Moreover, 25 days after injection of leukemic cells, the percentage of leukemic cells in spleen of the leukemic mice in vector control group and FAK shRNA group was (82.40±6.13)% and (14.50±3.70)%, respectively (P<0.01). CONCLUSION: FAK shRNA inhibits the growth of leukemic cells in vitro and in vivo, indicating that FAK gene silencing might be a new therapeutic strategy for leukemia.     

ARHI gene inhibits cell growth,induces G2/M phase arrest and apoptosis of acute myeloid leukemia cell line U937

AIM: To investigate the expression of aplasia rashomolog member I (ARHI) gene in acute myeloid leukemia cells (AML) and to study the effects of ARHI on the growth of AML cell line U937.METHODS: The mRNA expression of ARHI in AML cells, 293FT cells, AML primary cells and healthy volunteer blood cells were detected by RT-PCR. After transfection with the MSCV-IRES-GFP-ARHI plasmid to the U937 cells, the growth curve was analyzed by MTT assay. U937 cells were re-suspended by fresh medium and cultured for 24 h, then the cell cycle distribution and apoptotic rate were determined. RESULTS: The mRNA of ARHI was positively detectable in 293FT cells and healthy volunteer blood cells instead of AML cell line and AML primary cells. The growth curve showed that cell viability in U937 cells with high expression of ARHI (U937-ARHI) was lower than that in the control cells (U937-GFP) on 6th~8th day. The ratio of G₂/M phase and apoptotic rate in the U937-ARHI cells were increased compare with control group(P<0.05). CONCLUSION: The mRNA level of ARHI is low in AML cells. High expression of ARHI gene in U937 cells inhibits cell growth, arrests the cells at G₂/M phase and induces apoptosis.     

Sodium valproate induces cycle arrest,apoptosis and elevates p21WAF1 mRNA expression in chronic myeloid leukemia cell line K562

AIM:To investigate the effects of sodium valproate (VPA) on the cell cycle and apoptosis of chronic myeloid leukemia cell line K562, and to explore the possible mechanisms. METHODS:K562 cells were treated with VPA. Cell cycle and apoptosis were analyzed by flow cytometry. The expression of p21WAF1 mRNA was detected by RT-PCR. RESULTS:After treatment with VPA, cell cycle was arrested obviously at G0/G1 phase ［(82.30±9.41)% vs (40.13±2.12)%, P<0.05］. The apoptotic rate was significantly higher in the cells treated with VPA than that in untreated cells ［(11.47%±0.25%) vs (4.77%±0.40%), P<0.05］. The level of p21WAF1 mRNA was increased ［(1.65±0.91) vs (0.25±0.04), P<0.05］. CONCLUSION:VPA induces elevated expression of p21WAF1 mRNA in K562 cells, resulting in G0/G1 phase arrest and apoptosis in vitro.     

Celecoxib inhibits viability,induces apoptosis and inhibits autophagy in acute myeloid leukemia cell lines HL-60 and HL-60A

AIM: To investigate the effects of celecoxib on viability, apoptosis and autophagy in acute myeloid leukemia (AML) cell lines HL-60 and HL-60A. METHODS: The HL-60 cells and HL-60A cells were cultured with various concentrations (0, 20, 40, 60, 80 and 100 μmol/L) of celecoxib. The inhibitory effect of celecoxib on the cell viability was evaluated by MTT assay. Apoptosis was analyzed by Annexin-V/PI staining. Apoptosis-related and autophagy-related proteins were determined by Western blot. RESULTS: IC₅₀ of celecoxib were 49.4 μmol/L, 32.0 μmol/L and 25.1 μmol/L for HL-60 cells treated with celecoxib for 24 h, 48 h and 72 h, respectively. For HL-60A cells, the corresponding IC₅₀ were 69.1 μmol/L, 42.5 μmol/L and 29.6 μmol/L, respectively. The results of flow cytometry analysis showed the proportions of Annexin-Ⅴ⁺ PI^-, Annexin-Ⅴ⁺ PI⁺ and Annexin-Ⅴ^-PI⁺ cells were increased in the HL-60 cells, and those of Annexin-Ⅴ⁺PI^- and Annexin-Ⅴ⁺ PI⁺ cells were increased in the HL-60A cells treated with celecoxib for 24 h. After treated with celecoxib, the induction of apoptosis was observed, the apoptosis-related proteins cleaved caspase-3 and cleaved PARP were upregulated, the autophagy-related proteins LC3 II and P62 were both increased, and mTOR, p-mTOR, 4-EBP and p-4-EBP were not changed, indicating that celecoxib inhibited autophagy in the AML cells without the mTOR pathway involvement. CONCLUSION: Celecoxib inhibits the viability of HL-60 cells and HL-60A cells in a time-and dose-dependent manner by its effects of inducing apoptosis and necrosis. Celecoxib inhibits mTOR-independent autophagy in AML cells, indicating a possible way of using celecoxib for enhancing the antitumor activity of therapeutic agents to induce cytoprotective autophagy in the AML cells.     

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM:To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3K/AKT signaling pathway in HL-60 cells in vitro.METHODS:Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3K, AKT and p-AKT were determined by Western blot. After treatment with PI3K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS:The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners (P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan (15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner (P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time (P<0.05), but caspase-8 did not show any change. The protein levels of PI3K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration (P<0.05). Treatment of HL-60 cells with LY294002, a PI3K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan (P<0.05). CONCLUSION:Lentinan induces HL-60 cell apoptosis by inhibiting PI3K/AKT signaling pathway.     

Reversal of apoptosis resistance of doxorubicin-resistant human myelogenous leukemia cell line K562/DOX by a cyclosporin D analogue PSC833

AIM:To study the reversal effect of a cyclosporin D analogue PSC833 on multidrug resistance of doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. METHODS:The reversal effects of PSC833 on resistance to doxorubicin (DOX)/vincristine (VCR) in K562/DOX cells were observed by MTT assay. The cell cycle analysis was performed by flow cytometry. Annexin V/PI staining was used to identify PSC833-induced apoptosis in K562/ DOX cells. These cells underwent incubation with DCFH-DA, JC-1 and Fluo-3/AM followed by flow cytometry for the measurement of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and intracellular calcium, respectively. The protein levels of cytochrome C (Cyt C), Bcl-2, Bax, and cleaved caspase-3 were detected by Western blotting. RESULTS:The DOX/VCR-induced cytotoxicity was significantly potentiated by PSC833. PSC833 arrested the cells in G2/M phase and increased the apoptosis induced by DOX in K562/DOX cells. During the apoptosis, the level of ROS and intracellular calcium increased, while the level of ΔΨm decreased. Furthermore, the release of Cyt C, activation of caspase-3, up-regulation of Bax and down-regulation of Bcl-2 were observed in K562/DOX cells treated with PSC833 and DOX. CONCLUSION: The reversal effect of PSC833 on multidrug resistance in K562/DOX cells is associated with the induction of apoptosis through a mitochondria-dependent pathway.     

Establishment of arsenic trioxide-resistant K562/AS2 cell line and the mechanism of multidrug resistance

AIM:To establish a arsenic trioxide (As₂O₃ )-resistant leukemic cell line to explore the mechanism of resistance to As₂O₃, and the relationship between the resistant cell line and the multidrug resistance was also investigated. METHODS:The arsenic trioxide (As₂O₃ )-resistant leukemic cell line was established by exposing the cells to the increasing concentration of As₂O₃. MTT assay was used to detect the cytotoxicity. Cell cycle was detected by PI assay. Flow Cytometry was used to detect the P-glycoprotein on the surface of the cells, the intracellular concentration of DNR, and the immuetype of the cells. RESULTS:The cell doublings time and the cell cycle of the arsenic trioxide (As₂O₃ )-resistant leukemic cell line, K562/AS2, is similar to that of K562. The relative resistant fold of K562/AS2 to As₂O₃, DNR, VP16 and Ara-C was 7.4, 2.9, 3.8 and 1.1, respectively. The relative resistant fold of multidrug resistant cell line, K562/ A02, to As₂O₃, DNR, VP16 and Ara-C was 0.8、94、2.5 and 0.9, respectively. The fluorescence of the P-glycoprotein on the surface or of the DNR inside the cells detected was not significantly different between the K562 and the K562/AS2 cell lines. CONCLUSIONS:A cell line, K562/AS2, resistant to clinical achieving level (2 μmol/L) of As₂O₃ has been established. The relative resistant fold of K562/ AS2 to As₂O₃ is about 7.4 fold to the parent K562 line sensitive to As₂O₃. Partial resistance of K562/AS2 to DNR and VP16 is observed , the mechanism of which is unrelated to the P-gp, the expression product of multidrug resistance gene 1 (mdr1).     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.     

Marsdenia tenacissima extract inhibits viability and induces apoptosis of melanoma cells via regulating PI3K/AKT/mTOR signaling pathway

AIM:To evaluate the effects of Marsdenia tenacissima extract (MTE) on the viability and apoptosis of mouse skin melanoma cell line B16-F10. METHODS:B16-F10 cells were treated with MTE at different doses for 24 h or at different doses for different time, and the cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels were determined by Western blot. Meanwhile, the cells were treated with insulin-like growth factor-1 (IGF-1) and the protein levels were measured again. RESULTS:The cells were treated with MTE for 72 h for further study according to the results of pre-experiments. MTE at 100 and 200 mg/L inhibited the viability of B16-F10 cells and decreased the protein expression of Ki67 and PCNA significantly. MTE induced the apoptosis of B16-F10 cells as demonstrated by increasing cleaved caspase-3 and cleaved caspase-9. Meanwhile, MTE down-regulated the protein levels of p-PI3K, p-AKT and mTOR. In addition, IGF-1, the activator of PI3K/AKT/mTOR pathway, alleviated the effects of MTE on the viability and apoptosis markedly. CONCLUSION:MTE inhibits the viability and induces the apoptosis of melanoma cells by down-regulating PI3K/AKT/mTOR signaling pathway.     

PMA specifically inhibits mRNA expression of BCR/ABL and Fyn in K562 cells

AIM: To study the effects of phorbol 12-myristate 13-acetate (PMA) on the mRNA expression of BCR/ABL and Fyn in K562 cells, and to explore their relationship. METHODS: The K562 cells were stimulated by PMA at a series of concentrations (1~250 μg/L) for 24 h, and the mRNA expression levels of BCR/ABL and Fyn in K562 cells were detected by real-time fluorescence quantitative PCR. The relative changes of both mRNA expression were measured using 2^-ΔΔCt formula. RESULTS: PMA significantly inhibits the mRNA levels of BCR/ABL and Fyn in a dose-dependent manner, and the correlation of these inhibitory effects were significant. Compared with gene Molt-4 cells, the inhibition by PMA was specific for K562 cells. The K562 cells were induced to differentiate to be pseudopodium-like cells. CONCLUSION: The PMA downregulates the mRNA level of Fyn by inhibiting BCR/ABL fusion gene.     

Knock-down of NEDD1 expression inhibits lung adenocarcinoma cell proliferation and migration

AIM: To explore the role of neural precursor cell expression developmentally down-regulated protein 1 (NEDD1) in the development and progression of lung cancer. METHODS: The differences of NEDD1 expression levels between lung cancer tissues and tumor-adjacent tissues were analyzed by the method of immunohistochemistry and TCGA database. Kaplan-Meier curve was used to analyze the correlation between lung cancer prognosis and the expression level of NEDD1. The proliferation of A549 cells was tested by plate colony formation experiment after knock-down of NEDD1 expression. The apoptosis rate and cell cycle distribution were examined by flow cytometry. The migration ability of the A549 cells was detected by Transwell assay. The protein levels of cell cycle-related molecules were determined by Western blot. Database analysis was performed to evaluate the relationship between the expression of NEDD1 and cyclin-dependent kinases 2 (CDK2). RESULTS: Compared with the tumor-adjacent tissues, the expression level of NEDD1 in the lung cancer tissues was increased, so as the database analysis, and the higher expression of NEDD1 showed a poorer prognosis. Under light microscope, the A549 cells showed a low proliferation rate after silencing the NEDD1 expression, and the colony formation ability of the cells was also reduced; knock-down of NEDD1 expression induced the apoptosis and inhibited the cell migration; knock-down of NEDD1 expression blocked the cells in G₁/S phase, and the protein levels of p-Rb and cyclinD1 were decreased, while the protein levels of p-Chk1, p-Chk2 and p-p53 were increased (P<0.05). A positive correlation between the expression of NEDD1 and CDK2 was noted by database analysis. CONCLUSION: NEDD1 plays an crucial role in promoting cell proliferation via inhibiting apoptosis and accelerating cell cycle, high expression of NEDD1 in lung adenocarcinoma tissue is related to poor prognosis, thus NEDD1 may be used as a candidate marker molecule for the diagnosis and prognosis of lung cancer.     

Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

Effects of shRNA targeting SIAH2 gene on proliferation and apoptosis of human hepatoma cell line HepG2

AIM：To investigate the effects of shRNA-mediated seven in absentia homolog 2 (SIAH2; one of ubiquitin ligases) gene silencing on cell cycle and apoptosis of human hepatoma cell line HepG2. METHODS：The specific recombinant vector pGenesil-SIAH2 was transiently transfected into HepG2 cells with Lipofectamine 2000. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of SIAH2. MTS assay was employed to evaluate the cell proliferation. Flow cytometry was used to determine the cell cycle and apoptosis of the transfected cells. RESULTS：Compared with control groups, the mRNA and protein levels of SIAH2 were reduced by pGenesil-SIAH2 transfection in HepG2-SIAH2 group. The proliferation of HepG2-SIAH2 cells was significantly inhibited. The percentage of G1-phase cells and the early apoptotic rate were significantly higher in HepG2-SIAH2 cells. CONCLUSION: Tansfection of pGenesil-SIAH2 effectively inhibits the proliferation of HepG2 cells, arrests the cells in G1 phase and induces apoptosis, indicating an experimental basis of SIAH2-targeting gene therapy for hepatocellular carcinoma.