Effects of carbon monoxide on hypoxia inducible factor-1 expression in hypoxia preconditioned mice

AIM:To study the effect of carbon monoxide (CO) on hypoxia inducible factor-1α(HIF-1α) expression and on hypoxic tolerance duration in hypoxia preconditioned mice. METHODS:Mice were injected with normal saline (NS) or hemin intraperitoneally. Western blot was used to detect HIF-1α in hippocampus. The tolerant duration of each hypoxic run was recorded. RESULTS:After 1 and 2 runs of hypoxia exposure, there were no significant differences in HIF-1α expression and hypoxic tolerance duration between N and hemin injection groups. After 3 and 4 runs of hypoxia exposure, the content of HIF-1α was lower and hypoxic tolerance duration was shorter (P＜0.05) in hemin injection groups than that in NS injection groups. CONCLUSION:CO could decrease the expression of HIF-1α, and decrease hypoxic tolerance duration in hypoxia preconditioned mice. The mechanism may be that CO binds to hemoprotein and inhibits hemoprotein oxygen sensor.     

Effects of hypoxia on the expression of heparanase and invasiveness of SKOV3 ovarian cancer cell line

AIM: To evaluate the effects of hypoxia on the expression of heparanase and the invasiveness of SKOV3 ovarial carcinoma cell line. METHODS: SKOV3 cells were incubated at either normoxia (37 ℃, 5％CO2, 21％O2) or hypoxia (37 ℃, 5％CO2, 1％O2) condition for 12 h, 24 h and 36 h. RT-PCR and Western blotting were used to detect mRNA and the protein expressions of heparanase under different conditions. Cell invasiveness was measured by matrigel invasion assay. RESULTS: Compared to normoxia group, the heparanase mRNA expression level in hypoxia group was increased and in 12 h hypoxia group was the highest. The heparanase protein expression in hypoxia group was also significantly increased (P<0.01) and the expression of heparanase in hypoxia group was also different (P<0.05). Compared to normoxia group, the level of cell invasion was markedly increased in 12 h, 24 h and 36 h groups (P<0.05). During 12-36 h hypoxia period, the increase in hypoxia-induced invasiveness in SKOV3 cell line showed a time-dependent manner (P<0.05). Meanwhile, there was a positive correlation between the expression of HPA and the invasiveness of SKOV3 cells (r=0.8530, P<0.05). CONCLUSION: Invasion of SKOV3 cells in hypoxia condition correlates with heparanase level. Hypoxia plays an important role in the augmentation of the heparanase expression and the invasiveness of human ovarian cancer.     

Effect of up-regulation of Shh-Gli1 signaling pathway on radioresistance and cell cycle in human esophageal cancer cell line Eca109

AIM:To investigate the relationship between Sonic Hedgehog (Shh) signaling pathway and cell cycle and radioresistance of esophageal cancer by up-regulating Gli1, a key factor in Shh signaling pathway. METHODS:The human esophageal cancer cell line Eca109 was transfected with plasmid to induce Gli1 over-expression, which served as Eca109-ox-Gli1 group. In addition, Eca109 cells transfected with empty plasmid served as negative control group and the untreated Eca109 cells were used as normal control group. The expression of Gli1 was confirmed by real-time PCR and Western blot. The radiosensitivity of the cells in the 3 groups was determined by colony formation assay. The effect of irradiation on the cell cycle was analyzed by flow cytometry. RESULTS:The expression of Gli1 in Eca109-ox-Gli1 group was higher than that in the other 2 groups (P<0.05). The survival fraction at dose of 2 Gy in Eca109-ox-Gli1 group was higher than that in normal control group, indicating that the radioresistance of the Eca109 cells transfected with Gli1 plasmid was increased. The cells in Eca109-ox-Gli1 group showed higher S phase proportion than that in normal control group and negative control group (P<0.01). After irradiation at dose of 6 Gy, all cells in the 3 groups found that the cell cycle was arrested at G₂/M phase, while the cells in normal control group showed higher G₂/M phase proportion than that in Eca109-ox-Gli1 group (P<0.01). CONCLUSION:The up-regulation of Gli1 may enhance the radioresistance of esophageal cancer by regulating the cell cycle.     

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.     

Effect of PTEN gene overexpression on cell cycle and tumor invasion in a human ovarian carcinoma SKOV3 cell line

AIM:To investigate the role of phosphatase and tensin homology deleted on chromosome(PTEN) gene in the cell cycle and invasion ability of human ovarian carcinoma SKOV3 cell line in vitro. METHODS:Human ovarian carcinoma SKOV3 cells were transfected with a eukaryotic expression plasmid vector containing PTEN gene in vitro,and then the positive cell clones were selected and amplified. MTT method was used to observe the inhibitory rate,flow cytometry was used to detect the cycle of transfected PTEN cells and apoptosis level. Western blotting analysis was used to determine PTEN gene expression. The invasiveness of transfected cells were measured quantitatively by Matrigel invasion assays (Transwell chamber). RESULTS:The expression of PTEN mRNA in SKOV3 cells increased after transfection with PTEN gene. Flow cytometry showed that the percentage of cells in S phase increased,but that in G₂/M phase decreased. Invasiveness of SKOV3 was significantly decreased. CONCLUSION:The transfection of PTEN gene into SKOV3 cells can inhibit human ovarian carcinoma SKOV3 cell proliferation,invasion and induce SKOV3 cell apoptosis.     

Influence of hypoxia-inducible factor-1 alpha on lung cancer cell A549 growth in vitro and in vivo

AIM: To study the role of hypoxia-inducible factor-1alpha(HIF-1α) on lung cancer cells A549 growth in vitro and in vivo. METHODS: To observe the growth rate of A549 cells after HIF-1α transfected, A549 cells (1×10⁶/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group (group A, n=10), the HIF-1α transfected group(group B, n=10). The weights of subcutaneous tumor were detected. The resected specimens were made into paraffin-embedded sections. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemistry(ISH). The expressions of HIF-1α、 apoptosis-related protein survivin and bcl-2 were analyzed by Western blot. RESULTS: The growth rates of the HIF-1α transfected lung cancer cells A549 were significantly increased, and more importantly, the HIF-1α transfected lung cancer cells A549 was able to enhance lung cancer growth in nude mice(P＜0.05). The PCNA were increased significantly in group B, compared with group A. The expressions of HIF-1α, survivin and bcl-2 in group B were increased significantly than that of group A. CONCLUSIONS: HIF-1α increases lung cancer cells A549 growth in vitro and in vivo and its mechanism may be due to promotion of proliferation and inhibition of apoptosis.     

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.     

Effects of isorhapontigenin on cell cycle arrest and down-regulation of cyclin D1 expression in bladder cancer cells

AIM：To explore the inhibitory mechanism of isorhapontigenin (ISO) on the proliferation, migration and invasion of UMUC3 bladder cancer cells. METHODS：Human UMUC3 bladder cancer cells were pretreated with ISO, and the proliferation of the cells was observed under phase-contrast microscope and by ATPase assay. The expression of cyclin D1 was determined by RT-PCR and Western blotting. The cell cycle alteration was detected by flow cytometry, and the cell migration was examined by wound-healing assay. RESULTS：Over 20 μmol/L of ISO significantly inhibited the proliferation of UMUC3 cells with the IC50 of (22.5±2.8) μmol/L. The mRNA and protein levels of cyclin D1 in UMUC3 cells were markedly decreased after treatment with ISO. Exposure of UMUC3 cells to low dose (5 μmol/L) of ISO led to significant induction of G0/G1 growth arrest at both 12 h (58.82%) and 24 h (63.94%), compared with the negative control cells (47.33%) without inducing obvious apoptosis. ISO at dose of 5 μmol/L also markedly inhibited the cell migration. CONCLUSION：ISO significantly exhibits inhibitory effects on the proliferation and migration of human bladder cancer cells by down-regulation of cyclin D1 expression accompanying with G0/G1 cell cycle arrest.     

Photodynamic action of hematoporphyrin on human pancreatic cancer cell line Panc-1

AIM: To study the killing effect of photodynamic therapy (PDT) with hematoporphrin on human pancreatic cancer cell line Panc-1 in vitro.METHODS: The Panc-1 cells were divided into several groups according to the concentrations of photosensitizer and light doses to which the cells were exposed.The absorbance values of CCK-8 test were used as the detection index and were converted to cell survival.To study the main mechanism of PDT, the ROS production, the apoptotic rate and necrotic rate, and the expression of activated caspase-3 in the cells were measured by fluorescence microscopy, flow cytometry and Western blotting, respectively.RESULTS: With the concentration of photosensitizer and light dose increased, the survival rates of Panc-1 cells accordingly decreased, and the killing effect of PDT reached the maximum when the concentration of photosensitizer was 10 mg/L and the light dose was 20 J/cm².The synergistic effect between concentration of photosensitizer and light dose was observed.Using photosensitizer or light alone had no PDT effect.Exposure to photosensitizer and light induced ROS production and caused apoptosis and necrosis in Panc-1 cells in a concentration-dependent manner, and the rate of apoptosis was always higher than the rate of necrosis.PDT also increased the expression of activated caspase-3 in Panc-1 cells in a concentration-dependent manner.CONCLUSION: PDT with hematoporphyrin has killing effect on human pancreatic cancer cell line Panc-1 by producing ROS, activating caspase-3 and inducing apoptosis and necrosis in a concentration-dependent manner.The concentration of photosensitizer and light dose show synergistic effect on killing the cancer cells.     

Changes of hypoxia inducible factor 1 alpha and heme oxygenase 1 in the process of hypoxic pulmonary hypertension development in rats

AIM: To observe the expression of hypoxia inducible factor-1α (HIF-1α) gene and heme oxygenase-1 (HO-1) gene in pulmonary arteries in hypoxic rats. METHODS: Forty male Wistar rats were exposed to hypoxia for 0, 3, 7, 14 or 21 days. Mean pulmonary pressure (mPAP), vessel morphometry, right ventricle hypertrophy index (RVHI) were measured. Lungs were either inflation fixed for immunohistochemistry and in situ hybridization or frozen for later measurement of HO-1 enzyme activity. RESULTS: During hypoxia, mPAP increased to significantly higher values than the control values after 7-day of hypoxia，reaching its peak after 14-day of hypoxia, then remained on the high level. Pulmonary artery remodeling developed significantly after 14-day of hypoxia. Expression of HIF-1α protein in control was poorly positive, but was up-regulated in pulmonary arterial tunica intimae of all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein were markedly up-regulated after 3-day and 7-day of hypoxia, then tended to decline after 14-day and 21-day of hypoxia. HIF-1α mRNA staining was poorly positive in control, hypoxia for 3 days and hypoxia for 7 days, but began to enhance significantly after 14-day of hypoxia, then remained stable. Expression of HO-1 protein began to increase after 7-day of hypoxia, reaching its peak after 14-day of hypoxia, then remained stable. Expression of HO-1 mRNA began to increase after 3-day of hypoxia, reaching its peak after 7-day of hypoxia, then declined. CONCLUSION: HIF-1α and HO-1 are both involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats. Furthermore, HIF-1α may inter-regulate with HO-1 gene in this process.     

Effects and mechanisms of shikonin on the radiation sensitization of human ovarian cancer cell line SKOV-3

Objective: To investigate the radiosensitizing?effect and the underlying mechanism of shikonin on the human ovarian cancer cell line SKOV-3. Methods: The viability of SKOV-3 cells after treating with different concentrations (0,5,10,20,40,8 0 and 120 μg/mL) of shikonin was measured by MTT assay; The survival rate of SKOV-3 cells after treating with different doses(0,2,4,6 and 8 Gy) of x-ray radiotherapy was testet by clone forming assay.The SKOV-3 cells were divided into 4 groups: the Control group (Control group), the Shk group (8μg/mL Shk treatment), the 8 Gy group (8 Gy X-ray radiotherapy treatment) and the Shk + 8 Gy group (8 Gy μg/mL Shk treatment for 48 hours, followed by 8 Gy X-ray radiotherapy treatment). The cell cycle was examined by PI staining using flow cytometry and the phosphorylation of PI3K and AKT levels were analyzed by western blotting in each group. Results: In the ranged of 0-80μg/mL, shikonin decreased SKOV-3 cell viability in a concentration-dependent manner（P<0.05）. The value of IC50 was 38.54±0.57 μg/mL. Compared with radiotherapy alone, the survival curve was markedly shit to the left after shikonin combined radiotherapy（P<0.05）. The value of radiotherapy sensitization ratio (SER) was 1.45±0.05. Moreover, Compared with 8 Gy alone group, the percentage of G2/M phase and the phosphorylation levels of PI3K and AKT were decreased in Shk+8 Gy group（P<0.05）. Conclusion: Shikonin could increase the radiosensitivity of SKOV-3, and the mechanism may be related to attenuat radiation-induced the G2/M phase arrest and inhibition of PI3K/AKT signaling pathway.     

Inhibitory effects of septin 2 gene silencing induced by siRNA on migration of human colorectal cancer cell line LoVo

AIM:To investigate the expression of septin 2 in human colorectal cancer cell lines and the effect of septin 2 on the capacity of migration of human colorectal cancer cell line LoVo. METHODS:Real-time fluorescence quantitative RT-PCR and Western blotting analysis were used to determine the mRNA and protein levels of septin 2 in different metastatic potential cell lines, respectively. The expression of septin 2 in LoVo cells was silenced by siRNA. The efficacy of siRNA was confirmed by real-time fluorescence quantitative RT-PCR. Septin 2 and its co-localization with F-actin were measured by immunofluorescence method. The migration of transfected cells was analyzed by scratch test. RESULTS:The expression of septin 2 in LoVo cells was significantly higher than that in low metastatic potential cell lines such as SW480, HCT-116 and HT-29 at both mRNA and protein levels. The mRNA expression of septin 2was successfully silenced in LoVo cells by siRNA. Cell wound closure rate was also decreased in septin 2 siRNA group as compared with control group(both P<0.05). The co-expression of septin 2 and F-actin formed the typical filamentous-granular organization, and down-regulation of septin 2 resulted in cell skeleton disturbance with less or shorter pseudopodium and decreased stress fibers. CONCLUSION:Septin 2 is highly expressed in LoVo cells. Partial deletion of septin 2 represses the capability of tumor cell migration.     

Effects of diallyl disulfide on checkpoint kinase Chk1/2 of cell cycle G2/M in human gastric cancer cell line MGC803

AIM: To investigate the change of cell cycle checkpoint kinases Chkl/2 in human gastric cancer MGC803 cells induced by diallyl disulfide (DADS). METHODS: mRNA expression was assayed by means of RT-PCR in MGC803 cells treated with DADS. Western blotting was used to measure the activation of Chk1/2 for elucidate the possible mechanism of DADS-induced human gastric cancer cell cycle arrest. Interaction between Chk1/2 and Cdc25C was studied by means of immunoprecipitation. RESULTS: The level of Chkl and Chk2 mRNA had no significant difference between the DADS treatment and control by RT-PCR (P>0.05). Western blotting analysis showed that phosphorylation of Chk1 was increased following the treatment of MGC803 cells with 30 mg/L DADS after 1 h in a time-dependent manner (P<0.01). Phosphorylation of Chk2 was weak in the untreated cells, furthermore, its expression did not changed by DADS (P>0.05). Expression of Chk1 and Chk2 had no change after treated with DADS. Coimmunoprecipitated Chk1 or Chk2 was detected by anti-Chk1 or anti-Chk2 immunoprecipitation followed by anti-Cdc25C immunoblotting. DADS enhanced the binding activity of Chk1 kinase to Cdc25C in MGC803 cells, but didn’t influence the binding activity of Chk2 kinase to Cdc25C. CONCLUSION: Activation of Chk1 is involved in cell cycle arrest in MGC803 cells treated by DADS.     

Effect of Rip2 overexpression on apoptosis in human pancreatic cancer cell line Panc-1

AIM: To observe the effect of receptor-interacting protein 2 (Rip2) overexpression on human pancreatic cancer cell line Panc-1. METHODS: pEGFP-C2 and pEGFP-Rip2 plasmids were respectively transfected into the Panc-1 cells using JetPRIME reagent. The cells were divided into control group, pEGFP-C2 group and pEGFP-Rip2 group. The apoptosis in the cells was detected 48 h after transfection by flow cytometry. Rip2 level and the expression of apoptosis-related proteins, Bax, cytoplasmic cytochrome c (Cyt-c) and Bcl-2, were analyzed by Western blot. The activity of caspase-3 was measured by colorimetric method. RESULTS: Rip2 protein expression significantly increased in the cells transfected with control and pEGFP-C2 plasmids. The apoptotic rate in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group, whereas no significant difference of apoptotic rate was observed between control group and pEGFP-C2 group. The protein expression of Bax and cytoplasmic Cyt-c was remarkably increased and the protein expression of Bcl-2 was obviously decreased in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group. The activity of caspase-3 in pEGFP-Rip2 group was obviously increased as compared with control group and pEGFP-C2 group. CONCLUSION: Overexpression of Rip2 is able to induce apoptosis in the Panc-1 cells, and the mechanism may be related to the up-regulation of Bax and cytoplasmic Cyt-c protein expression, down-regulation of Bcl-2 protein expression and enhancement of caspase-3 activity, thus activating intrinsic apoptotic pathway.     

Effect of over-expression of transcription factor CDX2 on proliferation and cell cycle of human gastric cancer cell line SGC-7901

AIM: To study the effect and the molecular mechanism of CDX2 over-expression on the proliferation, growth and cell cycle of human gastric cancer cell line SGC-7901. METHODS: The SGC-7901 cells in LV-CDX2-GFP group were transfected with the recombinant lentivirus vector LV-CDX2-GFP, the cells in LV-GFP group were transfected with the negative control lentiviral vector for the negative control, and the cells in blank control group were without any treatment. The cell proliferation was detected by CCK-8 assay. The cell cycle distribution was analyzed by flow cytometry. The expression of CDX2, Bax, Bcl-2, cyclin D1 and survivin was determined by semi-quantitative RT-PCR and Wes-tern blotting. RESULTS: Compared with LV-GFP group and blank control group, the proliferation activity of the SGC-7901 cells was significantly lower (P<0.05), the G0/G1 phase proportion increased (P<0.05), the mRNA and protein levels of Bcl-2, cyclin D1 and survivin were reduced (P<0.05), and the mRNA and protein levels of Bax were up-regulated (P<0.05) in LV-CDX2-GFP group. No statistically significant difference of the above indexes was observed (P>0.05) between LV-GFP group and blank control group. CONCLUSION: Over-expression of CDX2 mediated by lentivirus inhibits the proliferation and growth of human gastric cancer SGC-7901 cells and arrestes the cell cycle at G0/G1 phase, which may be related to down-regulation of Bcl-2, cyclin D1 and survivin and up-regulation of Bax.     

Cell cycle in aging model of WI-38 cells induced by tert-butylhydroperoxide

AIM: To investigate the mechanism of cell cycle in aging model induced by tert-butylhydroperoxide(t-BHP).METHODS: From the 30th population doubling(PD), WI-38 cells were exposed for 1 h to t-BHP at every two PDs and four stresses were induced. The cell morphology and ultrastructure, cell cycle assay and β-galactosidase cytochemistry staining were used to evaluate cell senescence. Then the levels of CDK4, CDK2, cyclin D1, cyclin E, p21 and p16 protein were detected by Western blotting.RESULTS: After four times treated with 100 μmol/L t-BHP, the proliferation and division in WI-38 cells were ceased, which showed that cell became larger and flat, the number of secondary lysosome and activity of SA-β-galactosidase were increased. In addition, the percentage of cells in the G1 phase was decreased. Furthermore, it showed that the protein levels of CDK4, CDK2 and cyclin E was reduced and cyclin D1, p21 and p16 was elevated.CONCLUSION: t-BHP induces senescence in WI-38 cells. The possible mechanism is that t-BHP can change the expression of proteins related with cell cycle.     

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

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

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.     

Berberine enhances mitomycin C-induced cell cycle arrest and apoptosis of T24 bladder cancer cells

AIM: To observe the effects of the combination of berberin (Ber) and mitomycin C (MMC) on the cell cycle arrest and apoptosis of T24 bladder cancer cells and the underlying mechanisms. METHODS: The T24 cells were exposed to MMC in the presence or absence of difference concentrations of Ber. The viability of the T24 cells was determined by CCK-8 assay. The cell cycle distribution was detected by flow cytometry. The apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI staining, and the protein expression levels of cyclin D1, survivin, CDK2, CDK4, p21 and p27 were determined by Western blot. RESULTS: CCK-8 experiments showed that Ber enhanced the inhibitory effect of MMC on the viability of T24 cells. The results of flow cytometry showed that Ber also enhanced the blockade effect of MMC on T24 cells in G₀/G₁ phase (P<0.05). Compared with the MMC group, Ber increased the expression of p21 and p27 up-regulated by MMC, and decreased the expression of cynlin D1, CDK2 and CDK4 (P<0.05). Meanwhile, Ber promoted MMC to inhibit the expression of survivin (P<0.05). Ber increased the apoptosis of T24 cells induced by MMC (P<0.05). CONCLUSION: Ber significantly enhances the inhibitory effect of MMC on the viability of T24 cells. The mechanism may be related to up-regulation of p21 and p27, thereby inhibiting the expression of cyclin D1, CDK-2 and CDK-4. At the same time, Ber inhibits the protein expression of survivin, which eventually leads to cell arrest in G₀/G₁ phase and promotes apoptosis.