OBJECTIVETo investigate the effect of PD98059 on the proliferation and cell cycle of rat hepatic stellate cells (HSCs) stimulated by acetaldehyde and explore its mechanism.

METHODSRat HSCs stimulated by acetaldehyde were incubated with different concentrations of PD98059. Cell proliferation was assessed by MTT colorimetric assay. Cell cycle was analysed by flow cytometry. The mRNA of cyclin D1 and CDK4 were examined by RT-PCR.

RESULTS20, 50, 100 micromol/L PD98059 could significantly inhibit the proliferation of HSCs stimulated by acetaldehyde in a does-dependent manner (0.109+/-0.020, 0.081+/-0.010 and 0.056+/-0.020 vs 0.146+/-0.030, F=31.385, P<0.05) and provoke G0/G1 phase arrest of HSCs stimulated by acetaldehyde in a does-dependent manner (61.9%+/-6.3%, 64.1%+/-3.3% and 70.9%+/-4.8% vs 55.2%+/-4.4%, F=16.402, P<0.05). 50, 100 micromol/L PD98059 could markedly inhibit cyclin D1 mRNA expression of HSC stimulated by acetaldehyde (0.56+/-0.04 and 0.46+/-0.03 vs 0.65+/-0.07, F=68.758, P<0.05) and CDK4 mRNA expression (0.39+/-0.07 and 0.33+/-0.05 vs 0.50+/-0.06, F=29.406, P<0.05).

CONCLUSIONThe Erk signal transduction pathway plays an important role in regulating the proliferation and cell cycle of rat hepatic stellate cells stimulated by acetaldehyde, which may be partly related to its regulative effect on the expression of cyclin D1 gene and CDK4 gene

Acetaldehyde ; pharmacology ; Animals ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinases ; metabolism ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; Hepatocytes ; drug effects ; Proto-Oncogene Proteins ; Rats

OBJECTIVETo investigate the effect of Oviductus Ranae (OR) on the expressions of CyclinD1, CDK6 and P15 in the liver of aged male rats.

METHODSEighteen male SD rats were randomly divided into 3 equal groups, namely the OR group, VE group and ageing model group. The rats received subcutaneous injection of D-galactose for 6 weeks to establish the aging models, and another 6 rats were injected daily with normal saline (NS) to serve as the normal control group. From the third week of the experiment, the rats were given oral OR or Vitamin E (VE) accordingly till the sixth week. After completion of the drug administration, all the rats were sacrificed for detecting the expressions of CyclinD1, CDK6 and P15 in the liver tissue by Western blotting.

RESULTSThe relative expression levels of CyclinD1, CDK6 and P15 in the liver of the rats in the OR group were 41.73-/+0.54, 23.29-/+0.30 and 1.49-/+0.30, respectively, significantly up-regulated as compared with those in the ageing model group (P<0.01). The expressions of the proteins were obviously down-regulated in the model group in comparison with those in the normal control group.

CONCLUSIONSOR treatment can lower the expressions of Cyclin D1 and CDK6 in the liver to enhance the liver cell proliferation in aged male rats. OR also promotes the expression of P15 through a feedback mechanism to prevent excessive proliferation of the cells. The effect of OR against ageing is mediated possibly by up-regulation of the proteins associated with the cell proliferation in the liver, a mechanism different from that of VE.

Aging ; metabolism ; Animals ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 6 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Liver ; metabolism ; Male ; Materia Medica ; pharmacology ; Rats

Glioblastoma is a common brain tumor and the overall survival rate of the patients is very low, so it is an effective way to develop the potential chemotherapy or adjuvant chemotherapy drugs in glioblastoma treatment. As a well-known antimalarial drug, artesunate(ARTs) has clear side effects, and recently it has been reported to have antitumor effects, but rarely reported in glioblastoma. Different concentrations of ARTs were used to treat the glioblastoma cells, and then the inhibitory effect of ARTs on glioblastoma proliferation was detected by MTT assay; Ki67 immunofluorescence assay was used to detect the proliferation of cells; Soft agar experiment was used to explain the clonal formation abilities ; Flow Cytometry was used to detect the cell cycle; and Western blot assay was used to determine the expression of key cell cycle protein. MTT assay results indicated that ARTs-treated glioblastoma cell A172, U251, U87 were significantly inhibited in a time-and-dose dependent manner as compared to the control group(DMSO treatment group). Soft agar experiment showed that ARTs could significantly reduce the clonal formation ability of glioblastoma. Furthermore, Flow cytometry analysis showed that ARTs could obviously increase the cell proportion in G₀/G₁ phase and reduce the cell proportion in S phase. Western blot results showed that the expressions of cell cycle-related proteins CDK2, CDK4, cyclin D1 and cyclin B1 were all obviously down-regulated. Above all, ARTs may inhibit the proliferation of glioblastoma cells by arresting cell cycle in G₀/G₁ phase through down-regulating the expression of CDK2, CDK4, cyclin D1, cyclin B1. These results may not only provide a novel method for rediscovering and reusing ARTs but also provide a new potential drug for treating glioblastoma.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Artesunate ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Glioblastoma ; drug therapy ; pathology ; Humans

OBJECTIVETo explore the effects of sodium selenite on the expressions of beta-catenin and cyclin D1 in colorectal cancer cells HCT 116 and SW480.

METHODSHCT 116 and SW480 cells were treated by 10 micromol/L sodium selenite at different time points. The expressions and transcription of beta-catenin and cyclin D1 were detected by Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Meanwhile, the impact of MG132 (a proteasome inhibitor) pretreatment on the expressions of beta-catenin and cyclin D1 was observed through Western blot analysis. The interaction between beta-catenin and T cell factor 4 (TCF4) after selenite treatment was evaluated using co-immunoprecipitation assay.

RESULTSSodium selenite inhibited the expression of beta-catenin and transcription of its target such as cyclin D1. MG132 pretreatment prevented the inhibition of beta-catenin signaling triggered by selenite in HCT 116 and SW480 cells. Furthermore, selenite treatment disrupted the interaction between beta-catenin and TCF4 in HCT 116 and SW480 cells.

CONCLUSIONSSodium selenite can lower the expression levels of beta-catenin and its target cyclin D1, during which the proteasome-mediated degradative pathway may be involved. The decreased interaction between beta-catenin and TCF4 due to sodium selenite may be also involved in the regulation of beta-catenin signaling.

Cell Line, Tumor ; Colorectal Neoplasms ; metabolism ; Cyclin D1 ; metabolism ; HCT116 Cells ; Humans ; Sodium Selenite ; pharmacology ; beta Catenin ; metabolism

OBJECTIVETo explore the possible mechanism(s) of taurine-inhibiting the proliferation of hepatic stellate cells (HSC), this study investigated the effect of taurine on the HSC cell cycle and its regulatory protein expression.

METHODSCell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. Cell cycle regulatory protein Cyclin D1 and P21waf1 expression were determined by immunocytochemistry and image-analysis system, and real-time quantitative PCR.

RESULTSHSC proliferation was markedly inhibited when HSC were treated with taurine at concentrations of 5, 10, 20, 30, 40 and 50 mmol/L for 48 hours, and the inhibition rates were 6.7%, 14.4%, 23.3%, 32.2%, 36.7% and 45.6% respectively (P < 0.05-0.01). In the flow cytometry analysis, it was found that taurine could block HSC in the G0/G1 phase from entering the S phase, resulting in more cells in the G0/G1 phase and fewer in the S phase. The percentage of the cells in the G0/G1 phase and the S phase at the dosage of 40 mmol/L were 68.2%+/-1.4% and 26.2+/-1.3% respectively, which was significantly different in comparison to the controls (56.2%+/-1.7% and 38.5%+/-0.8% respectively, P < 0.01). HSC expressed cyclin D1 and P21waf1. Taurine inhibited cyclin D1 expression and induced P21waf1 expression. The cyclin D1 protein and mRNA in the HSC treated with 40 mmol/L taurine were significantly reduced compared with the controls [protein (optical density value): 0.13+/-0.02 versus 0.18+/-0.02, P < 0.01; mRNA: 5776.7+/-3345.0 versus 18,400.6+/-1374.8 copies/10(6) GAPDH, P < 0.01]; and the P21waf1 protein and mRNA were markedly increased compared with the controls [protein (optical density value): 0.19+/-0.02 versus 0.14+/-0.01, P < 0.01; mRNA: 44,866.7+/-3910.7 versus 16,933.3+/-960.9 copies/10(6) GAPDH, P less than 0.05].

CONCLUSIONSCyclin D1 and P21waf1 were cell cycle regulatory proteins in HSC, and taurine can inhibit the HSC cyclin D1 expression and stimulate P21waf1 expression, facilitate arresting cells in G0/G1 phase, and suppress cell proliferation.

Animals ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line ; Cell Proliferation ; Cyclin D1 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Depression, Chemical ; Hepatocytes ; cytology ; Rats ; Taurine ; pharmacology

This study was aimed to investigate the effect of Honokiol (HNK) on proliferation and apoptosis of acute myeloid leukemia HL-60 cells and its potential mechanism. Inhibitory effect of HNK on the HL-60 cell proliferation was detected by MTT assay. Flow cytometry was used to detect the change of cell cycle and AnnexinV/PI staining was used to detect apoptosis. Western blot was applied to analyze the cell cycle protein (cyclins), cyclin-dependent kinase (CDK), P53, P21, P27, BCL-2, BCL-XL, Bax, caspase-3/9 and proteins for MAPK signal pathway. The results showed that HNK could inhibit the proliferation of HL-60 cells in time- and dose dependent ways. HNK arrested HL-60 cells in G0/G1 phase, and S phase cells decreased significantly (P < 0.05). The expression of cyclin D1, cyclin A, cyclin E and CDK2/4/6 were significantly down-regulated (P < 0.05), the expression of P53 and P21 was significantly upregulated after treating for 24 h with HNK (P < 0.05). After 24 h treatment with HNK, HL-60 cell apoptosis increased significantly with the upregulation of activated caspase-3, -9, BAX expression and the downregulation of BCL-2, BCL-XL expression. The MAPK subfamily, P38 and JNK were not significantly changed, but the expression of MEK1/2-ERK1/2 was significantly downregulated (P < 0.05). It is concluded that HNK arrestes the cells at G0/G1 phase and induces HL-60 cell apoptosis through the intervention of MEK1/2-ERK1/2 signaling pathway.
Apoptosis ; drug effects ; Biphenyl Compounds ; pharmacology ; Caspase 3 ; Cell Cycle ; Cell Proliferation ; drug effects ; Cyclin D1 ; Cyclin E ; Cyclin-Dependent Kinase 2 ; HL-60 Cells ; Humans ; Lignans ; pharmacology ; Oncogene Proteins ; Signal Transduction ; bcl-2-Associated X Protein

OBJECTIVETo study the effects of NS398, a selective cyclooxygenase-2 (COX-2) inhibitor, on the transcription and translation of BCL-3 and its regulatory gene cyclin D1 in colon cancer cell line SW480.

METHODSHuman colon cancer cells SW480 were divided into two groups: SW480 cells in experimental group were treated with NS398 in different concentrations(25 micromol/L, 50 micromol/L, 100 micromol/L and 200 micromol/L) for 48 h or 72 h. SW480 cells in control group were treated with media which did not contain NS398. Then the expressions of BCL-3 and cyclin D1 were detected by RT-PCR, Western blot, and immunocytochemistry.

RESULTSAt 48 hours RT-PCR showed that BCL-3 mRNA and cyclin D1 mRNA decreased in a dose-dependent manner in the experimental group. However, there were no significant differences in the levels of BCL-3 protein and cyclin D1 protein between two groups (P>0.05). At 72 hours, BCL-3 protein and cyclin D1 protein also decreased in a dose-dependent manner in the experimental group. When the concentration of NS398 reached 100 micromol/L, the differences between the two groups in the expression of BCL-3 mRNA and protein became statistically significant (P<0.01). When the concentration of NS398 reached 50 micromol/L, the differences in the expression of cyclin D1 mRNA and protein were statistically significant (P<0.05).

CONCLUSIONSBCL-3 is expressed in colon cancer cell line SW480. COX-2 inhibitor can inhibit the expression of BCL-3 and cyclin D1 in a dose-dependent manner. NS398 may down-regulate the expression of cyclin D1 through BCL-3.

Cell Line, Tumor ; drug effects ; Colonic Neoplasms ; metabolism ; Cyclin D1 ; metabolism ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Humans ; Nitrobenzenes ; pharmacology ; Proto-Oncogene Proteins ; metabolism ; Sulfonamides ; pharmacology ; Transcription Factors ; metabolism

OBJECTIVETo investigate the effects of total flavonoids of Astragali Radix (TFA) and calycosin on apoptosis induction and cell cycle in human erythroleukemia cell line K562.

METHODMTT assay was used to measure the inhibition effect on the proliferation of K562 cells cultured with TFA and calycosin. The effect of TFA and calycosin on cell cycle in K562 was detected by PI staining. The apoptosis induction effect was measured by Annexin V/PI double staining. RT-PCR was used to determine the level of Cyclin D1 mRNA in K562 cells after treated with TFA and calycosin.

RESULTTFA and calycosin could inhibit the proliferation of K562 cells, the 50% inhibiting concentration of TFA and calycosin were 98.63 mg x L(-1) and 130.32 mg x L(-1) respectively. TFA and calycosin could not induce apoptosis in K562 cells, but could increase the number of cells in the G0/G1 phase. The level of Cyclin D1 mRNA in K562 cells decreased after treated with TFA and calycosin.

CONCLUSIONTFA could inhibit the proliferation of K562 cells, and attribute to arrest them in the G0/G1 phase and decrease Cyclin D1 mRNA.

Apoptosis ; drug effects ; Astragalus Plant ; Cell Proliferation ; drug effects ; Cyclin D1 ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Humans ; Isoflavones ; pharmacology ; K562 Cells

OBJECTIVETo investigate the anti-cancer efficacy and mechanism of sorafenib and 5-fluorouracil (5-FU) therapy in vitro using the HCC cell line MHCCLM3.

METHODSThe effects of sorafenib and 5-FU, alone or in combination, on the proliferation of MHCCLM3 cells were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle established by Chou and Talalay. Effects on cell cycle distributions were tested by flow cytometry and expression of proteins related to the RAF/MEK/ERK and STAT3 signaling pathways and cyclinD1 were tested by western blotting.

RESULTSSorafenib and 5-FU alone or in combination displayed significant efficacy in inhibiting proliferation of the MHCCLM3 cells, with the following inhibition rates: sorafenib: 46.16% +/- 2.52%, 5-FU: 28.67% +/- 6.16%, and sorafenib + 5-FU: 22.59% +/- 6.89%. The sorafenib + 5-FU combination did not provide better results than treatment with either drug alone. The combination index values of the sorafenib and 5-FU treatments were mainly greater than 1, indicating that the two agents induced antagonistic, instead of synergistic, effects on the MHCCLM3 cells. In addition, the MHCCLM3 cells were less sensitive to 5-FU when administrated in combination with sorafenib, as evidenced by the half inhibitory concentration (IC50) significantly increasing from (102.86 +/- 27.84) mg/L to (178.61 +/- 20.73) mg/L (P = 0.003). Sorafenib alone induced G1 phase arrest (increasing from 44.73% +/- 1.63% to 65.80% +/- 0.56%; P less than 0.001) and significantly decreased the proportion of cells in S phase (decreasing from 46.63% +/- 0.65% to 22.83% +/- 1.75%; P less than 0.01), as well as down-regulated cyclinD1 expression (0.57 +/- 0.03-fold change vs. untreated control group; P less than 0.01). 5-FU alone up-regulated cyclinD1 expression (1.45 +/- 0.12-fold change vs. untreated control group; P less than 0.01). Moreover, sorafenib alone significantly inhibited the RAF/MEK/ERK and STAT3 pathways, with the fold-changes of p-C-RAF, p-ERK1/2 and p-STAT3 being 0.56 +/- 0.05, 0.54 +/- 0.02 and 0.36 +/- 0.02, respectively (all P less than 0.01); 5-FU alone produced no significant effects on these pathways.

CONCLUSIONAdministered alone, both sorafenib and 5-FU exert anti-tumoral activity on in vitro cultured HCC cells. The sorafenib + 5-FU combination treatment produces antagonistic, rather than synergistic, effects. Sorafenib-inhibited RAF/MEK/ERK and STAT3 signaling and cyclinD1 expression may have induced the observed G1phase arrest and S phase reduction, thereby reducing the cells' sensitivity to 5-FU.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Drug Antagonism ; Fluorouracil ; pharmacology ; Humans ; Niacinamide ; analogs & derivatives ; pharmacology ; Phenylurea Compounds ; pharmacology ; STAT3 Transcription Factor ; metabolism ; Signal Transduction

This study was purposed to investigate the proliferation, differentiation and apoptosis of human promyelocytic leukemia HL-60 cells induced by proanthocyanidin (PAC). HL-60 cells were incubated with 20 mg/L PAC for 24 h, the cell growth was evaluated by CCK-8 assay. the effect of PAC on HL-60 cells was evaluated and the cells morphology was observed by optical microscopy. Expression of CD14 and CD11b, and cell cycle were analyzed by flow cytometry. The results showed that the growth of HL-60 cells was inhibited after treatment with PAC of different concentration in a dose-dependent manner (P < 0.05). 20 mg/L PAC displayed significant effect on HL-60 cells with inhibition ratio (72.3 ± 1.8)% for 24 h. Microscopy displayed that some cells differentiated to relative mature cells after treating for 48 h. Expression of CD14 increased and the expression of CD11b increased a little after treating with 20 mg/L PAC for 24 h, the ratio of cells in G0/G1 phase increased, but the ratio of cells in S phase decreased. The mRNA and protein expression of P21 gene increased, but the protein expression of CDK4 and Cyclin D1 decreased. It is concluded that PAC may inhibit the proliferation of HL-60 cells in vitro, induces the differentiation of HL-60 cells, and arrests the cells in G0/G1 phase. The possible mechanism may be related to up-regulation of P21 gene expression and down-regulation of the protein expression of CDK4 and Cyclin D1.
Cell Cycle Checkpoints ; drug effects ; Cell Differentiation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Proanthocyanidins ; pharmacology