Dysfunction of proteasome inhibits the proliferation of hBMSCs via activation of P53

AIM: To investigate the role of P53 in decreased cell proliferation and proteasomal activity during culture expansion of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: The proteasomal activity and expression level of P53 in early-passage (passage 3~4) and late-passage (passage≥14) hBMSCs were observed. Early-passage hBMSCs were divided into DMSO control group, MG132 group (treated with 10 μmol/L MG132 for 4 h) and pifithrin-α(PET-α) + MG132 group (pretreatment with 20 μmol/L PFT-α for 1 h then exposure to MG132 for 4 h). The proliferation and cell cycle distribution of the cells were measured by BrdU incorporation assay and PI staining. To further confirm the effect of P53 inhibitor on late-passage hBMSCs, the cells were incubated with 20 μmol/L PFT-α and the BrdU-positive cells were counted. RESULTS: Accompanied by reduced proteasomal activity, the expression level of P53 in late-passage hBMSCs was up-regulated by 16.89%±4.44% compared with early-passage cells. Application of the proteasome inhibitor MG132 reduced the proliferation of early-passage hBMSCs, and the percentage of BrdU-positive cells dropped to 33.36%±2.24%. However, MG132-induced decrease in cell proliferation was partially reversed by pretreatment with PFT-α. BrdU-positive cells in PFT-α + MG132 group were increased to 49.23%±2.67%. The cell cycle distribution had no significant difference between PFT-α + MG132 group and DMSO group, and the higher proliferation index was found in PFT-α + MG132 group but not in MG132 group. Inhibition of P53 activity in late-passage hBMSCs by PFT-α promoted the cell proliferation, and the percentage of BrdU-positive cells was higher than that in control group. CONCLUSION: Long-term expansion of hBMSCs in vitro reduces proteasomal activity, which in turn activates P53 to suppress cell proliferation through blocking cell cycle progression.     

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.     

Effect of MG132 on Aβ generation in SH-SY5Y cells

AIM: To observe the influences of different concentrations of MG132 on apoptosis and beta-amyloid protein(Aβ) generation in SH-SY5Y cells, and to explore the underlying mechanism.METHODS: SHSY-5Y cells were incubated with MG132 for 24 h. The final concentrations of MG132 were 2.5, 5 and 10 μmol/L. The cell viability was determined by MTT assay. The cell apoptosis was assessed by flow cytometry. The levels of Aβ were measured by ELISA. The relative protein levels were detected by Western blot.RESULTS: In the SH-SY5Y cells, MG132 reduced the cell viability, induced the cell apoptosis, increased the level of Aβ, and increased the expression of the related proteins for Aβ generation in a concentration-dependent manner.CONCLUSION: MG132 induces apoptosis and increases the levels of Aβ_1-42 and Aβ_1-40 by regulating the proteins related to Aβ generation in the SH-SY5Y cells.     

PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

Effect of camptothecin on the activation,proliferation and cell cycle distribution of the mouse T lymphocytes in vitro

AIM: To study the effects of camptothecin (CPT) on the activation, proliferation and cell-cycle distribution of the mouse T lymphocytes stimulated by concanvalin A (ConA) in vitro. METHODS: A model of T cell activation and proliferation was established by stimulated the cells with Con A. T cells were treated with different concentrations of CPT. The expression of CD69, the early marker of CD3+ T cell activation, was measured by FACS. The proliferation index was determined by carboxyl fluorescin diacetate succinmidyl ester by flow cytometry. The cell-cycle distribution was analyzed by propidium iodide staining. RESULTS: After stimulation with Con A for 6 h, the activation rate of CD69+ T cell in Con A group was (58.88±0.55)%. The percentages of CD69 positive cells were (55.48±0.98)%, (54.67±1.05)%, (50.40±0.82)%, (42.47±1.32)%, correspond to the treatments with different concentrations of CPT (10 nmol/L, 20 nmol/L, 50 nmol/L, 100 nmol/L), respectively. After 48 h treatment with Con A, the proliferation index in different concentrations of CPT treatment (10 nmol/L, 20 nmol/L, 50 nmol/L and 100 nmol/L) exerted a definite inhibitory effect on the proliferation (P<0.01). Moreover, the cell-cycle distribution analysis showed that apoptosis peak was observed in different concentrations of CPT treatment after 48 h cultured with Con A. CONCLUSION: CPT significantly inhibits the early stages of the Con A-induced T cell activation and proliferation, and detents the T lymphocytes in G₀/G₁ phase.     

Ubiquitin-dependent degradation of SnoN/Ski protein in glomerular mesangial cells induced by high glucose

AIM: To observe the protein expression of SnoN/Ski and ubiqutin ligase Arkadia in rat glomerular mesangial cells (GMCs) exposed to the high glucose. METHODS: Cultured rat glomerular mesangial HBZY-1 cells were divided into control group, 20 mmol/L glucose group, 30 mmol/L glucose group, 30 mmol/L glucose+MG132 group (culture medium containing 30 mmol/L glucose and 0.5 μmol/L specific proteasome inhibitor MG132), and mannitol group. The expression levels of SnoN, Ski and Arkadia were measured by Western blotting analysis, immunofluorescence and laser scanning confocal microscopy. RESULTS: In control GMCs, the expression of SnoN/Ski was rich and Arkadia was weak. After stimulated with high glucose, the expression of SnoN/Ski was decreased and Arkadia was gradually increased (P<0.05). Compared with high glucose group, the levels of SnoN/Ski and Arkadia were mostly reverted by adding the proteasome inhibitor MG132 at concentration of 0.5 μmol/L (P<0.01). The expression levels of SnoN/Ski and Arkadia were not significantly changed in mannitol group in comparison with control group (P>0.05). CONCLUSION: High glucose decreases the expression of SnoN/Ski through ubiquitin-dependent degradation of SnoN/Ski. The degradation of SnoN/Ski mediated by Arkadia may play an important role in the pathogenesis of diabetic nephropathy.     

Effects of proteasome inhibitor MG132 on proliferation of neural stem cells in subventricular zone

AIM: To investigate the role of proteasome in the degeneration of neuron in age-related diseases such as Parkinson disease (PD) by observing the effects of proteasome inhibitor MG132 on the proliferation of neural stem cells (NSCs) in the subventricular zone (SVZ).METHODS: Stereotaxic microinjection of proteasome inhibitor MG132 at a dose of 10 μg into the left lateral ventricle of 90-day-old mice was performed.The control mice were received the same volume of DMSO into the left lateral ventricle.Three days, 7 days and 14 days after injection, the total proteins isolat from SVZ were subject to proteasome activity assay using the fluorescence microplate reader.Meanwhile, the mice were administered with 5-bromo-2'-deoxyuridine (BrdU) by intraperitoneal injection.The numbers of BrdU-positive cells were counted to examine the effect of MG132 on the proliferation of NSCs.RESULTS: After microinjection of MG132 into the left lateral ventricle, the proteasome activity in SVZ was significantly decreased on day 3 and day 7 post-injection (P<0.05) as compared with the control animals.Furthermore, the numbers of BrdU⁺ cells in SVZ were significantly reduced to 21±4 on day 3 and to 22±3 on day 7 post-injection (P<0.05).After long periods of treatment with MG132, the proteasome activity in SVZ was restored to normal level on day 14 post-injection.At the same time, no statistical difference of BrdU⁺ cells between the mice treated with MG132 (82±4) and DMSO (67±6) was observed (P>0.05).CONCLUSION: Short-term injection of reversible proteasome inhibitor MG132 attenuates the proteasome activity in SVZ, which in turn inhibits the proliferation of NSCs, suggesting that the proteasome activity may be closely related to the proliferation potential of NSCs.     

Synergistic effect of decitabine and valproic acid on apoptosis and cell cycle arrest at G0/G1 phase in gastric cancer MGC-803 cells

AIM: To investigate the synergistic effect of decitabine (DCA) and valproic acid (VPA) on apoptosis and cell cycle arrest at G₀/G₁ phase in gastric cancer MGC-803 cells. METHODS: Gastric cancer MGC-803 cells were used in the study and divided into the following groups according to the treatment with different drugs for 72 h: DCA 1.5 μmol/L,DCA 3.0 μmol/L, VPA 1.5 mmol/L, DCA 1.5 μmol/L+VPA 1.5 mmol/L and DCA 3.0 μmol/L+VPA 1.5 mmol/L. The early and late apoptotic rates were detected by annexin V and PI staining. The cell cycle was also determined by flow cytometry. The relative nm23-H1 mRNA expression level was measured by real-time quantitative PCR. RESULTS: The apoptotic rates in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (early: 33.58%±3.88%; late: 31.52%±4.20%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (early: 42.61%±4.23%; late: 38.01%±3.86%), the percentages of the cells in G₀/G₁ phase in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (61.55%±2.38%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (66.75%±2.48%), and the relative nm23-H1 mRNA expression levels in VPA 1.5 mmol/L +DCA 1.5 μmol/L group (1.84±0.46) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (3.02±0.36) were all significantly higher than those in the corresponding concentrations of single drug treatment groups (P<0.01). CONCLUSION: Synergistic effect of VPA and DCA on apoptosis and cell cycle arrest in gastric cancer MGC-803 cells is possibly via inactivation of nm23-H1 gene expression.     

Inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in two human ovarian carcinoma cell lines

AIM: To investigate the inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in human ovarian carcinoma cells (OVCAR8 and SKOV3 cell lines). METHODS: The expression of MISIIR protein and the localization of MISIIR protein were analyzed by Western blotting and confocal spectral microscopy, respectively. Cell apoptosis and cell cycle were detected by flow cytometry (FCM). Cell viability was determined via MTT method. Clone formation test was used to detect oncogenicity in vitro.RESULTS: The MISIIR protein expression in OVCAR8 cells but not in SKOV3 cells was observed. MISIIR expression was seen on the OVCAR8 cell surface and in the cytoplasm with both antibodies. After treated with rhMIS for 48 h, the cell viability was significantly decreased in OVCAR8 cells. rhMIS inhibited the oncogenicity of OVCAR8 cells greatly. The cell apoptosis of OVCAR8 cell exposed to 10 mg/L rhMIS was (31.3±2.1)%, and OVCAR8 cells in the G1 phase were increased by (70.4±3.0)%. Compared to SKOV3 cells the differences were significant (P<0.01). CONCLUSION: Recombinant human Mullerian inhibiting substance suppresses the growth of MISIIR-positive ovarian cancer cells by inducing apoptosis and cell cycle arrest. We predict that rhMIS might be a new target to treat human ovarian malignancies.     

26S proteasome regulates differentiation of human bone marrow stromal cells into neural-like cells

AIM: To investigate the process of human bone marrow stromal cells (hBMSCs) differentiation into neural-like cells and to determine the role of 26S proteasome in neuronal differentiation. METHODS: Purified hBMSCs were treated with β-mercaptoethanol (β-ME) for 1 day and retinoic acid (RA) for 3 days, followed by growth factor (10 μg/L bFGF or 20 μg/L NGF) for another 3 days. Immunofluorescence was performed to detect the expression of nestin (a neural precursor cells marker), Tuj1 (a premature neuronal marker), and neurofilament (NF, a mature neuronal marker) at all stages of induced differentiation. Immunostaining and RT-PCR were used to analyze the expression of 26S proteasome during neuronal differentiation of hBMSCs. To further confirm the role of 26S proteasome in hBMSCs differentiation, cells were treated with β-ME/RA and then followed by protesome inhibitor MG132 and growth factor. Immunostaining was performed to detect NF-positive cells. RESULTS: Quantification results showed that the untreated cells were almost never positive for nestin, Tuj1 and NF. After treated with β-ME/RA, the numbers of nestin-positive cells (34.41%±1.27%) and Tuj1-positive cells (27.79%±1.27%) were increased. Notably, the numbers of NF-positive cells were significantly increased to 56.72%±2.4% after induction with β-ME/RA/GF. Immunofluorescence analysis showed that undifferentiated hBMSCs cells were weakly stained by antibody against 26S proteasome, but the numbers of cells with high-intensity of 26S proteasome were increased after treated with β-ME/RA. The RT-PCR result of 26S proteasome further confirmed that the mRNA level of the cells differentiated by β-ME/RA (1.33), as well as by β-ME/RA/GF (1.77), was significantly increased compared to the undifferentiated cells. Moreover, hBMSCs incubated with protesome inhibitor MG132 significantly decreased the numbers of NF-positive cells (37.59%±1.52%). CONCLUSION: After induction with β-ME/RA/GF, hBMSCs can be differentiated into neural-like cells, which is concomitant with the increase in 26S proteasome expression. Inhibitor of 26S protesome prevents hBMSCs differentiation, suggesting that 26S proteasome may be involved in the differentiation of hBMSCs into neural-like cells.     

Effect of ubiquitin-proteasome pathway on expression of Smad7 in cultured glomerular mesangial cells induced by high concentration of glucose

AIM:To observe the expression of Smad7 and Smad ubiquition regulatory factor-Smurf2 in rat glomerular mesangial cells (GMC) stimulated by the high concentration of glucose, and to investigate the effect of the ubiquition on Smad signaling by adding MG132 as a proteasome differential inhibitor.METHODS:Cultured rat GMC were divided into normal group (the concentration of glucose:5.6 mmol/L), high glucose group (20 mmol/L, 30 mmol/L, respectively), therapy group (30 mmol/L glucose with MG132).The expressions of Smurf2 and Smad7 in each group were measured by indirect immunofluorescence and laser scanning confocal microscope.RESULTS:(1) The expression of Smurf2 in GMC in normal group was weak (25.93±3.35) whereas the expression of Smad7 was strong (64.09±7.43).(2) The expression of Smurf2 in high glucose group was stronger than that in normal group (P<0.05), in a concentration-dependent manner, 20 mmol/L high glucose (56.99±7.00), 30 mmol/L high glucose (96.36±9.19), respectively.The expression of Smad7 in high glucose group was weakened (P<0.05), 20 mmol/L high glucose (45.33±6.67), 30 mmol/L high glucose (30.20±4.41), respectively.(3) In therapy group, the expression of Smurf2 was found weakened and expression of Smad7 was enhanced.CONCLUSION:(1) High glucose increases the expression of Smurf2 and decreases the expression of Smad7 in glomerular mesangial cells.(2) Ubiquition-proteasome pathway (UPP) is related with the regulation of Smad signal transduction pathways in diabetic nephropathy.     

Manumycin inhibits activity of pancreatic duct cancer cell line Panc-1 via inducing apoptosis

AIM: In this study, we investigated the anticancer effect and mechanisms of manumycin on pancreatic cancer cell line-Panc-1 and the role of p38MAPK pathway in apoptosis. METHODS: The test of anticancer effect was performed by MTT assay. Apoptosis was induced in the cells by manumycin and then treated with SB203580, a specific p38MAPK inhibitor. A quantitative caspase-3 activity assay kit was used in this experiment. RESULTS: Manumycin (6 μmol/L, 18 μmol/L, 54 μmol/L) significantly inhibited cell growth of pancreatic cancer cell line Panc-1. The inhibition rates 24 h after treatment with 6 μmol/L, 18 μmol/L and 54 μmol/L manumycin were 8.9%, 21.9% and 67.0%, respectively. Compared with the control group, the survival levels of the last two groups were of significant statistical difference (P<0.01). The anticancer effects also showed dosage-effect relationship, the value of IC50 24 h after treatment was 34.7 μmol/L. In addition, this reagent simultaneously activated caspase-3 protein, which was partly blocked by p38MAPK specific inhibitor, SB203580. CONCLUSION: Manumycin exerted anticancer effect on Panc-1 cell line via inducing cell apoptosis, which was partly regulated by p38MAPK.     

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 recombinant human CD40 ligand on biological behavior of ovarian cancer SKOV3 cells in vitro

AIM: To investigate the effect of recombinated human CD40 ligand (rhCD40L) on the biological behavior of ovarian cancer SKOV3 cell line in vitro.METHODS: After the SKOV3 cells were incubated with different concentrations of rhCD40L for various times, the cell proliferation was determined by MTT assay.The expression of the co-stimulatory molecules or adhesion molecules on SKOV3 cells and the changes of tumor necrosis factor receptor associated factor (TRAFs) inside the cells were measured by flow cytometry and direct immunofluorescence.Annexin V and PI dual color label assay were used to detect cell apoptosis or death in culture contained with rhCD40L.RT-PCR assay was employed to determine the change of apoptosis related gene c-myc, bcl-2 and bcl-xl expression in SKOV3 cells.RESULTS: rhCD40L inhibited proliferation of SKOV3 cells at concentration of 100 μg/L (0.65±0.10 vs 0.81±0.05) and reached a peak at concentration of 10 mg/L (0.13±0.12 vs 0.83±0.15, P＜0.01).The inhibitory effects showed a dose dependent manner.Cell cycle analysis showed that cell division was blocked in G1 phase.Increasing proportion of apoptosis of SKOV3 cells was related to up-regulation of CD95 expression (42.4% vs 59.2%, P＜0.05) and down-regulation of anti-apoptosis genes such as bcl-2 and bcl-xl expressions after incubation with rhCD40L.TRAF 2, 5 and 6 expressed highly in SKOV3 cells.The expression of TRAF 2 (81.3%±9.2% vs 50.4%±5.3%，P＜0.05), TRAF5 (47.2%±7.2% vs 7.2%±2.1%, P＜0.01) and TRAF6 (44.5%±6.3% vs 5.1%±1.1%, P＜0.01) was down-regulated and expression of TRAF 3 (25.2%±6.2% vs 68.8%±5.3%, P＜0.01) was up-regulated after co-culture with rhCD40L, but there was no effects found on the expression of TRAF 1 (4.3%±1.2% vs 5.1%±1.4%) and TRAF4 (7.4%±1.2% vs 8.1%±1.4%).CONCLUSION: By down-regulating expression of bcl-2, bcl-xl and changing expression profile of TRAF, rhCD40L inhibits the growth of SKOV3 cells by blocking the cell cycle progress in G1 and promotes the cells to apoptosis.     

Effects of oleanolic acid on apoptosis and cell cycle of HL-60 cells in vitro

AIM: To study the mechanism of oleanolic acid induced apoptosis and its influence on cell cycle in HL-60 cells in vitro. METHODS: The HL-60 cells were treated with different concentrations of oleanolic acid and then cultured for 12 h, 24 h, 48 h and 72 h, respectively. MTT assay was used to evaluate the inhibitory effect of oleanolic acid on HL-60 cells in vitro. The argarose gel electrophoresis was used to detect the chromatin DNA fragmentation. FACS was used to analyze the cell cycle of HL-60 cells. Western blotting was used to detect the activation of caspase-3 which has been confirmed the last execution of apoptosis pathway. RESULTS: MTT assay showed that oleanolic acid dramatically inhibited the growth of HL-60 cells in vitro, more than 50% HL-60 cells were inhibited when the cells were treated with 80 μmol/L oleanolic acid for 48 h; the apparent DNA ladder was detected after exposure of HL-60 cells to oleanolic acid for 48 h. FACS analysis showed that cell cycle of HL-60 cells was arrested in G₁ phase, the inhibition ratio of HL-60 cells achieved 63.24% and 67.90% after treated with oleanolic acid for 48 h and 72 h correspondingly. Western blotting detected the activation of caspase-3 after exposure of HL-60 cells to 80 μmol/L oleanolic acid for 48 h. CONCLUSION: These results suggest that oleanolic acid induces apoptosis and the cell cycle of HL-60 cells is arrested in G₁ phase.     

NIH3T3 cell proliferation and cell cycle in the conditions of hypoxia or low nutrition and its response to bFGF

AIM: To investigate NIH3T3 cell proliferation and cell cycle in the condition of hypoxia or low nutrition and its response to bFGF, and to explore the pathophysiological changes of fibroblast under hypoxic or low nutrional conditions.METHODS: The cells were placed in anaerobic workstation where the mixture gas was given to simulate hypoxic environment. Partial oxygen pressure (PO2) of medium was controlled in 27 mmHg, 44 mmHg and 175mmHg. NIH3T3 cells were cultured with low nutritional medium contained new bovine serum (NCS) less than 10% to simulate low nutritional environment. MTT assay was used for observing cell activity and flow cytometry for cell cycle analysis.RESULTS: Under 44 mmHg PO2, no obvious difference was shown between hypoxia group and normal group. Under 27 mmHg PO2, the proliferation activity of NIH3T3 cells was significantly lower than that in normal group (P<0.01), as well as the cell numbers in G0-G1 phase increased (P<0.05), S phase decreased (P<0.01). bFGF had no effect on cell proliferation. In 0.5% NCS medium, the NIH3T3 cell proliferation speed decreased (P<0.01) and cell cycle was arrested at G0-G1 (P<0.01). The proliferation speed was improved by bFGF (P<0.01).CONCLUSION: In lower PO2 or lower nutrinal condition, fibroblast proliferation activity is inhibited by cell cycle arrest in G0/G1 phase. However the decreasing proliferation in low nutritional medium could be improved by external bFGF.     

Probucol inhibits proliferation of rat aortic smooth muscle cells stimulated with oxidized low-density lipoprotein

AIM: To investigate the relationships between antiproliferative mechanisms of probucol and protein expressions of signaling molecules ERK1/2, MKP-1, HO-1 and Trx-1 in rat aortic smooth muscle cells (RASMCs) stimulated with ox-LDL. METHODS: The effects of probucol on cell cycle, cell proliferation and the expressions of ERK1/2, MKP-1, HO-1 and Trx-1 in the presence of ox-LDL were observed by means of MTT test, FCM and Western blotting. RESULTS: (1) Probucol significantly inhibited the proliferation of RASMCs stimulated with ox-LDL. A value in 100 μmol/L probucol+35 mg/L ox-LDL group was reduced by 34.9% as compared to ox-LDL group (P<0.01). (2) Probucol protected against ox-LDL-induced RASMCs proliferation through inducing cell growth arrest at G0/G1 phase and cell apoptosis. (3) ox-LDL increased the expression of p-ERK1/2 by 34.7% (P<0.01) and decreased MKP-1 by 60.0% (P<0.01), respectively, as compared to control. Probucol attenuated the increase in ox-LDL-stimulated p-ERK1/2 level by 15.7%, but increased MKP-1 expression by 2 times (P<0.01). (4)ox-LDL at concentration of 35 mg/L decreased the intracellular Trx-1 expression by 28.9% (P<0.05), and slightly increased the level of HO-1 expression as compared to control (P<0.05). Probucol enhanced the expression of Trx-1 by 91.6% (P<0.01) and HO-1 by 31.9% (P<0.01), respectively as compared to ox-LDL group. CONCLUSION: Probucol inhibits ox-LDL-stimulated the proliferation of RASMCs through increases in MKP-1/HO-1 expression, suppression of cell cycle progression and induction of cell apoptosis.     

Effects of erythromycin and its derivatives on the proliferation of T lymphocyte in vitro

AIM: To study the effects of erythromycin and its derivatives on the proliferation and the apoptosis of T lymphocytes and to evaluate the anti-inflammatory mechanism of erythromycin derivatives. METHODS: The effects of four erythromycin derivatives without antibacterial action on the proliferation T lymphocytes were evaluated by MTT. The effects of the erythromycin derivatives on apoptosis in T lymphocytes were observed by flow cytometry and TUNEL staining. RESULTS: The proliferation of T lymphocytes were inhibited by erythromycin derivative-Ⅰ and erythromycin, the IC50 was (425.2±32.1) μmol/L and (606.3±35.4) μmol/L, respectively. The IC50 value of erythromycin derivative-Ⅰ was lower than that of EM (P<0.01). Erythromycin derivative-Ⅰ (3-30 mg/L) and EM (30-100 mg/L) induced T lymphocytes apoptosis in a concentration-related manner by flow cytometry and TUNEL, while erythromycin derivative-Ⅰ induced cells to die at concentration of 100 mg/L. CONCLUSION: The anti-inflammatory activity of erythromycin derivatives may be due partly to their effects on the proliferation and apoptosis of T lymphocytes.