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.     

Effects of gemcitabine on inhibiting proliferation and inducing apoptosis of CD34+CD38- myeloid leukemia stem cells

AIM：To investigate the effects of gemcitabine (GEM), a novel analog of deoxycytidine and nucleoside reductase inhibitor similar to cytarabine (Ara-C) in structure, on the proliferation and apoptosis of myeloid leukemic stem cells (LSCs), CD34+CD38-KG1a cells. METHODS：The expression of CD34 and CD38 on the surface of acute myeloid leukemia KG1a cells was detected by flow cytometry. The effects of GEM at various concentrations for 24 h and sustained medication for 14 d and 21 d on the proliferation and colony-forming ability of KG1a cells were analyzed by soft agar colony-forming experiment. The changes of the cell cycle of KG1a cells treated with various concentrations of GEM were tested by flow cytometry. The apoptosis of KG1a cells was determined by flow cytometry with the staining of Annexin V-FITC and propidium iodide (PI). RESULTS：The percentage of CD34+CD38- cells in acute myeloid leukemia KG1a cells was (98.02±0.72)%.Treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell cycle distribution of the KG1a cells, whereas KG1a cells treated with 0.5 mg/L GEM for 24 h were arrested at G0/G1 phase. After treatment with 0.1 mg/L and 0.5 mg/L GEM for 24 h, the colony numbers at 14 d and 21 d were lower than that in saline control group. No difference of the colony numbers between the cells treated with normal saline and 0.05 mg/L GEM for 14 d and 21 d was observed. After sustained medication with 0.05 mg/L, 0.1 mg/L and 0.5 mg/L GEM and Ara-C for 14 d and 21 d, the colony numbers decreased as compared to saline control group. Treatment with 0.5 mg/L GEM for 24 h increased the apoptotic rate of KG1a cells compared with saline control group, while treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell apoptosis. CONCLUSION：GEM inhibits the proliferation and colony-forming ability, arrests the cell cycle at G0/G1 phase and induces apoptosis of CD34+CD38- acute myeloid leukemia cells.     

Effect of miR-375 on viability,cell cycle and apoptosis of colon cancer HCT116 cells

AIM: To investigate the effect of microRNA-375 (miR-375) on the viability, cell cycle and apoptosis of HCT116 cells.METHODS: The expression of miR-375 in different colorectal cancer cell lines was detected by real-time PCR. The miR-375 mimics was transfected into HCT116 cells by Lipofectamine^TM 2000. The mRNA expression of miR-375 and AEG-1 was detected by real-time PCR. The HCT116 cell viability was detected by MTT assay. The changes of apoptosis and cell cycle distribution were analyzed by flow cytometry.RESULTS: Real-time PCR showed that miR-375 expression was the lowest in HCT116 among 4 colorectal cancer cell lines. The expression level of miR-375 significantly increased in miR-375 mimics group compared with that in the negative control group. The high expression level of miR-375 significantly inhibited the mRNA expression of AEG-1. After transfection with miR-375 mimics, the cell viability was inhibited, the apoptotic rate was increased, the proportion of G₁-stage cells was increased, and the proportion of S-stage cells was decreased.CONCLUSION: miR-375 inhibits the viability, mediates the cell cycle arrest and promotes the apoptosis of colon cancer HCT116 cells. miR-375 may act as a tumor suppressor in colorectal cancer by inhibiting AEG-1.     

Inhibition of cell proliferation and arrest of cell cycle progression by blocking Cl- channels in nasopharyngeal carcinoma cells

AIM: The roles of Cl^-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 μmol/L arrested cells in G₁ phase (G₁ population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G₁ phase. The results suggest that activation of Cl^-channels and RVD is necessary for facilitating cells to proceed to the S phase from G₁ phase and maintaining cell proliferation.     

Pseudolaric acid B induces glioblastoma cell line U87 mitotic arrest and apoptosis

AIM: To investigate the effects of pseudolaric acid B on the growth and apoptosis of glioblastoma cell line U87. METHODS: The cell morphological changes were observed under inverted microscope. The cell viability was evaluated by MTS assay. The cell cycle was analyzed by flow cytometry and Western blot. The cell apoptosis was detected by flow cytometry. The changes of apoptosis-related proteins cleaved PARP, caspase-3, procaspase-9 and caspase-8 were determined by Western blot. RESULTS: Pseudolaric acid B inhibited the viability of U87 cells, arrested U87 cells in mitosis. Apoptosis of U87 cells was induced by pseudolaric acid B. The caspase pathway was activated. CONCLUSION: Pseudolaric acid B induces glioblastoma cell line U87 mitotic arrest and apoptosis.     

Effects of p21siRNA on cell cycle uncoupling and apoptosis

AIM:To investigate the effects of P21 protein on cell cycle uncoupling and cell apoptosis with RNA interference assay. METHODS:The expression of P21 protein in HeLa cells was induced by mitomycin (MMC). Lipofect transfection assay was used to take the p21 siRNA into HeLa cells and MMC was given 48 h after transfection. FCM assay was applied to detect the expression of P21 and ratio of polyploid cells and apoptosis. RESULTS:p21 siRNA plasmid interfered the expression of P21 protein in HeLa cells. The number of 2 haploid cells was decreased obviously (P<0.01). The number of 4 haploid and 8 haploid cells was increased significantly (P<0.01) compared with control plasmid 24 and 48 h after MMC was given. CONCLUSION:p21 siRNA silenced the P21 protein and cell death in HeLa cells was induced by p53-independent pathway in the condition of lower expression of P21 protein. The mechanism may be related to cell cycle uncoupling and apoptosis by p53-independent pathway.     

Role of ClC-3 chloride channel in inhibition of nasopharyngeal carcinoma cell cycle by metformin

AIM: To study the roles of ClC-3 chloride channel in the inhibition of nasopharyngeal carcinoma cell cycle by metformin. METHODS: The CNE-2Z cells were treated with metformin at different concentrations. The viability of CNE-2Z cells was measured by CCK-8 assay. The cell cycle distribution was detected by flow cytometry. The protein expression of ClC-3 was determined by Western blot. The Cl^- currents was record by the patch-clamp technique. In addition, the cell cycle distribution was analyzed in the nasopharyngeal carcinoma CNE-2Z cells which over-expressed ClC-3 by pEZ-M03-ClC-3 plasmid transfection. RESULTS: Metformin inhibited the viability of CNE-2Z cells at 5, 10 and 20 mmol/L. Metformin at 10 mmol/L prevented the activation of chloride currents induced by hypotonicity, inhibited the protein expression of ClC-3 chloride channel and arrested the nasopharyngeal carcinoma CNE-2Z cells at G₀/G₁ phases. ClC-3 chloride channel protein over-expression reversed the effect of metformin on the cell cycle distribution of CNE-2Z cells. CONCLUSION: Metformin inhibits the CNE-2Z cell cycle, which may be related to the inhibition of ClC-3 chloride channel function and protein expression.     

Zoledronic acid inhibits proliferation and induces apoptosis in U937 cells

AIM: To study the antiproliferation and proapoptotic effects of zoledronic acid(ZOL) on human acute myeloid leukemia cell line U937. METHODS: The viability of U937 cells was detected by CCK-8 assay. The cell cycle of the U937 cells was analyzed by flow cytometry with PI staining. Apoptotic rate was assessed by flow cytometry with Annexin V-PI and Hoechst 33342 staining. Mitochondrial membrane potential was detected by JC-1 assay. Methylcellulose was used to assess colony formation. The protein levels of p21, Bcl-2 and Bax were determined by Western blot. RESULTS: ZOL inhibited the viability of U937 cells. ZOL induced S-phase cell cycle arrest in the U937 cells. The results of flow cytometry analysis with Annexin V-PI and Hoechst 33342 staining showed that ZOL also induced apoptosis in a dose- and time-dependent manner. Mitochondrial membrane potential assay was also used to verify the apoptosis. The apoptotic rate was consistent with the reduction of mitochondrial membrane potential. Colony formation assay showed that ZOL significantly inhibited the colony formation capacity of the U937 cells. This was achieved by the induction of S-phase cell cycle arrest, and up-regulation of Bax and p21, and down-regulation of Bcl-2. CONCLUSION: ZOL inhibits cell proliferation by regulating the expression of cell cycle-related protein, and induces apoptosis via the mitochondrial apoptotic pathway.     

Effects of azathioprine on proliferation,cell cycles and apoptosis of me-senchymal stem cells from rat bone marrow in vitro

AIM: To investigate the effects of azathioprine (AZA) on the proliferation, cell cycle and apoptosis of mesenchymal stem cells (MSCs) from the bone marrow of Sprague-Dawley rats in vitro. METHODS: MSCs were cultured in low-glucose DMEM containing 10% FBS,and treated with AZA at concentrations of 50 mg/L, 100 mg/L, 200 mg/L and 300 mg/L for 24 h, 48 h and 72 h. The effects of AZA on the growth curve and proliferation of MSCs were tested by cell counter under microscope. The apoptosis and cell cycle was determined by flow cytometry. RESULTS: Pure MSCs were gained by 3 times of passages. No significant effect of AZA at concentration of less than 100 mg/L on the proliferation, cell cycle and apoptosis of MSCs was observed (P>0.05). Under the condition of more than 200 mg/L for 72 h, AZA inhibited the growth of MSCs.The inhibitory rate was more than 66%, and the rate of apoptosis was increased (P<0.05). However, at the concentration of 300 mg/L for 72 h, AZA decreased the apoptotic rate and the necrotic rate of MSCs was obviously increased (P<0.05). Using AZA at concentration of more than 200 mg/L, as the action time prolonged, the MSCs in G₀/G₁ phase were increased, and those in S phase were decreased (P<0.05). CONCLUSION: At some concentrations, AZA significantly affects the proliferation, apoptosis and cell cycle of MSCs. Large dose of AZA may cause MSCs to death.     

T63 enhances radiotherapeutic sensitivity of nasopharyngeal carcinoma cells through apoptosis and G2/M arrest

AIM: To explore the antitumor effect of a curcumin analogue T63 in human nasopharyngeal carcinoma (NPC) cell lines CNE-2 and CNE-2R. METHODS: Cell viability was monitored by the methods of MTT and colony formation assay. Cell cycle distribution was detected by flow cytometry. Apoptosis was examined using the annexin V-FITC/PI staining assay. RESULTS: A growth inhibitory effect was observed with T63 treatment in a dose-dependent manner. Either T63 or ionizing radiation (IR) significantly induced G2/M arrest and apoptosis in NPC cells. In addition, T63 treatment combined with IR induced significantly higher apoptosis and G2/M arrest in NPC cells. CONCLUSION: T63 exhibits potent inhibitory activity on NPC cells and induces the radiotherapeutic sensitivity. Therefore, T63 has a potential as a preventive or therapeutic agent for treating NPC.     

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.     

Effect of p19ARF on growth and apoptosis of leukemia cells

AIM:To study the effect of p19ARF on the biological behavior of human leukemia cells. METHODS:p19ARF was cloned in eukaryotic expression vector pcDNA3.1 and transferred into INK4a/ARF locus-deficient leukemia cells HEL and K562. The changes in biological characteristics of the two p19ARF-transfected cells were observed.RESULTS:The growth of the p19ARF-transfected HEL cells was significantly inhibited compared with the vector-transfected cells; Cell cycle analysis showed that the expression of foreign p19ARF gene resulted in G1 phase cell cycle arrest and apoptosis cell death in some of HEL cells. However, p19ARF had no marked effects on the growth of K562 cells with p53 gene mutation and did not induce apoptosis in K562 cells.CONCLUSION:p19ARF suppressed the growth of leukemia cells by p53-dependent pathway.     

Effect of fucoxanthin on growth and apoptosis of HSC-T6 cells

AIM: To explore the effect of fucoxanthin (Fu) on the growth and apoptosis of HSC-T6 cells. METHODS: HSC-T6 cells were divided into blank control group, negative control group and drug groups (treated with different concentrations of Fu). The cell viability was detected by CCK-8 assay at 24 h, 48 h and 72 h after Fu treatment. The cell cycle distribution and apoptotic rate were analyzed by flow cytometry. The protein expression of Bcl-2 and Bax were detected by Western blot. RESULTS: Compared with blank control group, the viability of HSC-T6 cells was inhibited by Fu at concentrations of 15~75 μmol/L in a dose- and time-dependent manner (P < 0.01). The cell ratio of G₁ phase was significantly decreased (P < 0.01) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.01) in 60 μmol/L Fu group after 24 h. The cell ratio of G₁ phase was significantly decreased (P < 0.05) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.05) in 15 μmol/L and 30 μmol/L Fu groups in a dose-dependent manner after 48 h. The early cell apoptotic rates and total cell apoptotic rates were significantly increased in the Fu treatment groups in a dose-dependent manner (P < 0.05). The protein expression of Bax was significantly increased in the Fu treatment groups and the protein expression of Bcl-2 was significantly decreased in 30 μmol/L and 60 μmol/L Fu groups (P < 0.05).CONCLUSION: Fu inhibits the growth of HSC-T6 cells possiblely via arresting the cell cycle at S phase and G₂ phase. The apoptosis of HSC-T6 cells induced by Fu might be via down-regulating the protein expression of Bcl-2 and up-regulating the protein expression of Bax.     

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.     

Curcumin analogue B67 induces apoptosis and G2/M arrest to suppress radioresistant nasopharyngeal carcinoma cells

AIM: To study the effect of curcumin analogues B67 on radioresistant nasopharyngeal carcinoma cells (CNE-2R). METHODS: The effects of B67 on the cell viability and proliferation of CNE-2R and the parent cells CNE-2 were detected by MTT assay and colony formation assay, respectively. The changes of cell cycle, apoptosis and mitochondrial membrane potential were determined by flow cytometry. The morphological changes of the cells were observed under fluorescence microscope. Node mice were subcutaneously inoculated with the cells to determine the tumorigenic ability. RESULTS: The IC₅₀ of B67 on the viability of CNE-2R cells after treatment for 24 h, 48 h and 72 h were 3.96,2.59 and 0.89 μmol/L, respectively, and those of CNE-2 cells were 8.84, 3.55 and 1.10 μmol/L,respectively. The IC₅₀ of B67 on the proliferation of CNE-2R cells after treatment for 48 h was 0.55 μmol/L, and that of CNE-2 cells was 0.73 μmol/L. After treated with B67 for 24 h, CNE-2R and CNE-2 cells at G₂/M stage increased from 5.32% to 40.01% and from 9.07% to 15.73%,respectively. After treated with B67 for 48 h, the apoptosis of CNE-2R and CNE-2 cells increased from 5.49% to 38.06% and from 4.99% to 35.74%, respectively. The mitochondrial membrane potential in CNE-2R and CNE-2 cells was decreased by 66.76% and 72.09%, respectively. After treated with B67 for 24 h, the tumorigenic rate of CNE-2R cells was 0%, while the rates of CNE-2 cells in low- and high-concentration groups were 100% and 0%, respectively.CONCLUSION: Curcumin analogue B67 exhibits enhanced suppressive activity on radioresistant nasopharyngeal carcinoma cells by inducing G₂/M-phase arrest, promoting cell apoptosis and changing mitochondrial membrane potential.     

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.     

Jagged1 regulates STAT3 signaling pathway and affects growth and apoptosis of multiple myeloma cells

AIM:To investigate the effect of Jagged1 on the growth and apoptosis of multiple myeloma cells. METHODS:Transfection with small interfering RNA targeting Jagged1 and negative control was carried out in multiple myeloma cell line U266, and the mRNA and protein levels of Jagged1 in the cells were determined by RT-qPCR and Wes-tern blot. The cells without transfection were used as blank control. Trypan blue staining was used to draw the cell growth curve. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein levels of STAT3, p-STAT3 and Bax in the cells were determined by Western blot. STAT3 signaling pathway inhibitor AG490 was used to detect the activation level of STAT3 signaling in the cells. RESULTS:Compared with the U266 cells without transfection, the expression of Jagged1 at mRNA and protein levels decreased in the U266 cells transfeced with small interfering RNA targeting Jagged1 (P<0.05). However, the expression of Jagged1 at mRNA and protein levels did not change in the U266 cells transfected with small interfering RNA negative control. Knockdown of Jagged1 expression decreased the cell viability, increased the apoptotic rate, increased Bax levels, and decreased the protein level of p-STAT3 in the U266 cells (P<0.05). AG490 treatment decreased the protein level of p-STAT3, blocked the activation of STAT3 signaling pathway, promoted the cell apoptosis induced by Jagged1 knockdown, and inhibited the viability of the U266 cells. CONCLUSION:Knock-down of Jagged1 expression promotes the apoptosis of multiple myeloma cells by inhibiting STAT3 signaling pathway, thus suppressing cell growth.     

Effect of ClC-3 siRNA on cell cycle of HeLa cells

AIM: To investigate the roles of ClC-3 chloride channels in the regulation of cell cycle and the relationship between ClC-3 chloride channels and the cell cycle regulators, such as cyclin D1, cyclin-dependent kinase (CDK)4, CDK6, P21 and P27 in the HeLa cells.METHODS: ClC-3 genes were silenced by the siRNA technique in the HeLa cells. The transfection efficiency of ClC-3 siRNA was detected by real-time PCR. The cell cycle distribution was analyzed by the flow cytometry. The protein expression of ClC-3, P21, P27, CDK4, CDK6 and cyclin D1 was determined by Western blot.RESULTS: ClC-3 was knocked down by ClC-3 siRNA in the HeLa cells. Transfection of the cells with ClC-3 siRNA arrested the cells at G₀/G₁ phases, decreased the expression of cyclin D1, CDK4 and CDK6, and increased the expression of P21 and P27.CONCLUSION: ClC-3 plays an important role in the cell cycle of HeLa cells through the G₁-S transition point. ClC-3 may regulate the cell cycle progression by up-regulation of cyclin D1, CDK4 and CDK6 expression and/or by down-regulation of P21 and P27 expression.