Effect of vitamin K3 on apoptosis induced by androgen-independent prostate cancer cell PC-3M

AIM: To study the effect of vitamin K₃ (VK₃) on the induction of apoptosis in androgen-independent prostate cancer cell PC-3M in vitro.METHODS: Cell viability was estimated by MTT assay. AO/EB staining was performed to detect apoptotic cells. Apoptosis and the changes of cell cycle were detected by flow cytometry. NAC was used to observe the effect of growth inhibition by VK₃. RT-PCR was used to confirm the changes in gene expression. Levels of intracellular peroxides were estimated by using an oxidation-sensitive fluorescent probe DCFH-DA. RESULTS: PC-3M cells growth was significantly inhibited by VK₃ (≥60 μmol/L, P<0.05). The inhibitory effect was time and dosage dependent. The result of AO/EB staining showed that apoptosis of PC-3M cells were induced by VK₃. A typical subdiploid peak before G₀/G₁ phase was observed after treated for 12 h with VK₃ (60 μmol/L) by flow cytometry. The effect of growth inhibition treated with VK₃ was antagonized by antioxygen NAC (5, 10, 20, 40, 80 μmol/L). An increase in the level of DCF fluorescence after PC-3M cells were treated for 1-2 h with VK₃ was observed. Antioxidase GSH-Px and CAT were run-down after treated with VK₃. CONCLUSION: The results indicate that apoptosis in PC-3M cells is induced through oxidative stress by VK₃.     

Role of mtCLIC/CLIC4 in injured C6 glioma cells induced by hydrogen peroxide

AIM: To explore the changes and the possible function of mtCLIC/CLIC4 (mitochondrial chloride intracellular channel 4) proteins in malignant C6 glioma cells treated with hydrogen peroxide (H₂O₂). METHODS: The viability of C6 cells was measured by MTT assay, LDH release rate was detected by ultraviolet spectrophotometry, CLIC4 mRNA level was determined by RT-PCR and CLIC4 protein level was measured by Western blotting. RESULTS: Compared with the control group, the cell viability was constant, the LDH release rate increased obviously, and the CLIC4 protein level also increased significantly in 500 μmol/L H2O2 treated group (P<0.05, respectively). However, the cell viability decreased, LDH release rate increased significantly (P<0.01, respectively), and the CLIC4 protein level increased obviously in 1 000 μmol/L H₂O₂ treated group (P<0.05). CONCLUSION: The CLIC4 protein may be involved in the process of C6 injuries induced by H₂O₂.     

Apoptosis induction in gastric carcinoma cells by celecoxib combined with adriamycin

AIM:To study the apoptosis induction of cyclooxygenase-2 (COX-2) inhibitor，celecoxib and adriamycin (ADM) on tumor apoptosis of gastric carcinoma MGC-803 cells, and to explore their possible molecular mechanism(s) and interactions.METHODS:The number of MGC-803 cells was observed by MTT assay. Tumor apoptosis was studied by fluorescence microscopy, flow cytometry (FCM), and DNA ladder. RESULTS:MGC-803 cell number was significantly decreased with increasing dose of ADM. Cells were accumulated in G₀/G₁ phase and the number of cells in S phase was decreased. ADM (5 mg/L) combined with celecoxib (25 μmol/L) markably inhibited the growth of MGC-803 cells. Significant morphological changes of typical apoptosis were observed after treatment with combined use of celecoxib and ADM. Compared with ADM or celecoxib alone, ADM plus celecoxib obviously enhanced the DNA ladder fragment revealed by agarose gel electrophoresis of DNA. After exposure to combined celecoxib and ADM treatment for 48 h, MGC-803 cells were accumulated in G₀/G₁ phase. There was a decrease in the number of cells in S phase as compared to celecoxib or ADM alone. CONCLUSION:Celecoxib and ADM appear to have synergistic effects for the apoptosis induction. This may be an important prospect for applying COX-2 inhibitors to assist chemical therapy of ADM in clinical use.     

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.     

Mcl-1 involves in G2/M arrest of HL-60 cells induced by diallyl disulfide

AIM: To investigate the role of Mcl-1 in the G₂/M arrest induced by diallyl disulfide (DADS) in leukemic HL-60 cells.METHODS: The inhibitory effect of DADS on human leukemic HL-60 cells was detected by CCK-8 method in vitro. Flow cytometry analysis was employed to observe the cycle arrest in HL-60 cells and the effect of DADS-induced G₂/M arrest on HL-60 cells with Mcl-1 gene knockdown by RNAi silencing. The expression of Mcl-1, PCNA and CDK1 in HL-60 cells treated with DADS was determined by Western blotting. The binding of Mcl-1 with PCNA and CDK1 was detected by coimmuno-precipitation. RESULTS: HL-60 cells were treated with DADS at concentration of 15, 30, 60, 120 or 240 μmol/L for 48 h. The inhibition rates of HL-60 cell proliferation were 31.15%, 55.88%, 66.14%, 75.29% and 80.35%, respectively, and gradually enhanced with the increase in the concentration of DADS (P<0.05). Flow cytometry analysis revealed that the proliferation of HL-60 cells was blocked by DADS in the G₂/M phase. Treatment with DADS for 24 h and 48 h at concentrations of 60 μmol/L and 120 μmol/L, the percentage of G₂/M phase cells increased as compared to the untreated cells (P<0.05). DADS induced arrest of HL-60 cells in G₂/M phase in a time- and dose- dependent manner (P<0.05). The results of Western blot analysis indicated that Mcl-1, PCNA and CDK1 in HL-60 cells were significantly reduced after treated with DADS (P<0.05). HL-60 cell cycle progression delayed by silencing Mcl-1 gene with siRNA technique, suggesting that silence of Mcl-1 gene led to G₂/M arrest. Compared to the cells treated with DADS only, the percentage of G₂/M cells raised in the cells with Mcl-1 gene silencing and treated with DADS (P<0.05), indicating that Mcl-1 gene silencing enhanced the effect of DADS-induced G₂/M arrest in HL-60 cells. The binding of Mcl-1 with PCNA and CDK1 was detected by coimmuno-precipitation and the formation of heterodimers was observed, which was decreased after treated with DADS for 4 h.CONCLUSION: DADS inhibits the proliferation of HL-60 cells and induces its G₂/M phase arrest. The decreased expression of PCNA is related to inhibiting the proliferation of leukemic cells. Knockdown of Mcl-1 gene enhances the effect of DADS-induced G₂/M arrest.     

Tubuloside B rescues the PC12 neuronal cells from H2O2-induced apoptosis

AIM:To investigate the neuroprotective effect of tubuloside B, one of the phenylethanoids isolated from the stems of Cistanche salsa, on H₂O₂-induced injury in PC12 cells.METHODS:PC12 cells were exposed to various doses of tubuloside B for 12 h, then treated with H₂O₂ at concentration of 100 μmol/L for 24 h. The cell viability was observed with MTT assay. Reactive oxygen species and the mitochondrial membrane potential were measured with laser scanning confocal microscopy (LSCM). The DNA content and percentage of apoptosis were assayed by DNA agarose gel electrophoresis and flow cytometry. The activation of caspase-3 was detected with the caspase-3 activity assay kit. RESULTS:Following treatment with H₂O₂ for 24 h, H₂O₂ induced a significant decrease in cell viability; DNA ladder was observed and apoptosis percentage was as high as 48.0%. Accumulation of intracellular ROS, increase in caspase-3 activity and the decrease in mitochondrial membrane potential as indicated with the decrease of red/green ratios (from 5.97 to 0.41) were detected. However, pretreatment with tubuloside B (1, 10 or 100 mg·L^-1) for 12 h exhibited cytoprotective effects in a dose-dependent manner. Tubuloside B obviously enhanced the cell viability, reduced formation of the DNA ladder, and significantly reduced the number of cells labeled with Annexin-V. The percentage of apoptosis/necrosis neurons was significantly decreased to 30.9%, 18.3% and 6.2%, respectively. LSCM showed that the tubuloside B attenuated the accumulation of ROS and the H₂O₂-induced collapse of mitochondrial membrane potential in PC12 cells. The significant decrease in caspase-3 activity was detected, compared to the H₂O₂-treated cells at the same time point. CONCLUSION:Tubuloside B has the neuroprotective capacity to antagonize H₂O₂-induced apoptosis and injury in PC12 cells, indicating it may be useful for treating some neurodegenerative diseases.     

Double TdR blocking induces synchronization of cell cycle in human gastric cancer SGC-7901 cells

AIM: To investigate the effects of double thymidine deoxyribonucleoside (TdR) blocking on the cell cycle of human gastric cancer SGC-7901 cells.METHODS: SGC-7901 cells in the logarithm period were selected in the study and treated with TdR at concentration of 2 mmol/L, 4 mmol/L or 8 mmol/L for 15 h as the first blocking. After incubation in TdR-free medium for 10 h, the cells were treated with TdR at same concentrations again for another 15 h as the second blocking. The blocked cells were released by washing in fresh medium twice and incubation in TdR-free medium containing 10% fetal bovine serum for 12 h. The cells were collected and the cell cycle was detected by flow cytometry.RESULTS: By double TdR (2 mmol/L, 4 mmol/L and 8 mmol/L) blocking to synchronize the cell cycle, the cells in G₀/G₁ phase accounted for 77.3%, 77.5% and 77.0%, respectively. After further incubation for 12 h in TdR-free medium, the proportion of the cells in each phase of the cell cycles returned to normal range. CONCLUSION: The method of double TdR blocking is an ideal access in short term to acquire a large number of cells in G₀/G₁ phase.     

H2S inhibits neuron apoptosis induced by anoxia-reoxygenation through cAMP-mediated PI3-K/Akt/P70S6K kinase cell-survival signaling pathways

AIM: To investigate the effect of hydrogen sulfide on neuron apoptosis through PI₃-K/Akt/P70S6K cell-survival signal transduction pathways after neuron anoxia-reoxygenation.METHODS: Newborn (24-48 h) Wistar rats were decapitated.The hippocampus tissue was dissected and cells were suspended.Cells were plated at 1.0×10⁸ cells/L on poly-dlysine-treated 96-well (100 μL/well) plates and 6-well (2 mL/well) plates.Cells were used after 7 days.For anoxia-reoxygenation (oxygen glucose deprivation,OGD) experiments,cells were washed three times in a glucose-free balanced salt solution (BSS).They were then placed in deoxygenated glucose-free medium and cultured under 95% N₂,5% CO₂ in an anaerobic chamber equilibrated to 37 ℃ and 100% humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 95% air,5% CO₂.In experimental group,cells were respectively carried out OGD,OGD+150 μmol/L NaHS,OGD+150 μmol/L NaHS+10 μmol/L triciribin,OGD+150 μmol/L NaHS+10 nmol/L rapamycin and OGD+150 μmol/L NaHS+10 μmol/L triciribin+10 nmol/L rapamycin.Control cells were cultured normally.24 h later,neuron viability and apoptosis were measured.The level of cAMP and protein expression of PI₃-K,Akt and P70S6K were detected.RESULTS: NaHS enhanced concentration of cAMP and expression of PI₃-K,Akt and P70S6K.Meanwhile,increased neuron viability and decreased neuron apoptosis (P<0.01 vs group C or group I/R) were observed.Triciribin inhibited Akt and P70S6K,as well as increased neuron apoptosis and decreased neuron viability (P<0.05,P<0.01 vs group NaHS).Rapamycin inhibited P70S6K,as well as increased neuron apoptosis and decreased neuron viability (P<0.05,P<0.01 vs group NaHS).CONCLUSION: H₂S inhibits hippocampus neuron apoptosis and protects neuron from anoxia-reoxygenation injury through cAMP-mediated PI₃-K/Akt/P70S6K kinase cell-survival signaling pathways.     

Effects of curcumin analogue B50 on proliferation and apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM: To compare the effects of B50, a mono-carbonyl analogue of curcumin, on the proliferation and apoptosis between homologous nasopharyngeal carcinoma cells CNE-2R and CNE-2 with different radioresistance.METHODS: The effects of B50 on cell viability and cell growth were detected by MTT assay and colony-forming experiment, respectively. The changes of cell cycle, apoptosis and mitochondrial membrane potential (MMP) were determined by flow cytometry.RESULTS: B50 inhibited the cell viability of CNE-2R cells in a time-and dose-dependent manner with the IC₅₀ of (8.06±0.14) μmol/L (24 h), (2.49±0.02)μmol/L (48 h) and (1.42±0.02) μmol/L (72 h), which was more effective than that in CNE-2 cells . The inhibitory effect of B50 on CNE-2R cell growth was more effective than that on CNE-2 cells . After treated with B50 for 48 h, the proportion of CNE-2R cells in G₂/M stage was increased from 7.1% to 34.9%, which was better than that of CNE-2 cells (from 12.4% to 35.7%). After treated with B50 for 24 h, the early apoptotic rate in CNE-2R cells was increased from 3.7% to 19.5%, which was better than that in CNE-2 cells (from 4.4% to 14.8%), and the MMP in CNE-2R cells was decreased by (43.17±3.11)%, which was better than that in CNE-2 cells .CONCLUSION: B50 is more effective on inhibiting the cell viability and cell growth, blocking the cell cycle at G₂/M stage, inducing apoptosis and decreasing MMP in CNE-2R cells than those in CNE-2 cells, indicating that B50 may enhance the radio-sensitivity of CNE-2R cells by blocking the cell cycle and inducing apoptosis through mitochondrial pathway.     

Relationship between zinc and zinc transporter expression in breast cancer MCF-7 cells

AIM: To study the changes of zinc transporter gene expression in MCF-7 cell line exposed to ZnCl₂ and TPEN. METHODS: Human breast cancer cell line MCF-7 was exposed to different concentrations of ZnCl₂ (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT and levels of zinc transporter mRNA by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations. RESULTS: MCF-7 cells viability rate was significantly decreased when exposed to ZnCl₂ (with 150 μmol/L and 200 μmol/L) and TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl₂ and decreased when exposed to TPEN. ZnT-1 mRNA level was increased along with the increasing concentration of ZnCl₂ but decreased when exposed to TPEN. The expressions of ZIP2 and ZIP10 were increased along with the increasing concentration of TPEN. CONCLUSION: ZnT-1 gene expression is induced by zinc supplement and repressed by zinc deficiency. ZIP2 and ZIP10 gene expressions are induced by zinc deficiency.     

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.     

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.     

Effect of pregnancy-associated glycoproteins on E-selectin-medi ated endothelial cell adhesion

AIM:To explore the effect of high purified human chorionic gonadotropin (hCGs) and glycodelins isolated from amniotic flui d and serum of pregnant women on E-selectin mediated endothelial cell adhesion.METHODS:The hCGs and glycodelins in human amniotic fluid and se rum collected from pregnant women were purified by using chromatography.The bin ding of[51Cr］-labelled HepG2 cells to IL-1β stimutated human umbilical vein endothelial cells (HUVECs) in the presence of increasing concentrations of hCGs and glycodelins was quantified.The effect of hCG and glycodelin on E-selec tin-mediated endothelial cell adhesion was observed.RESULTS:HCGs isolated from amniotic fluid and serum of pregnant women inhibited the E-selectin mediated adhesion of HepG2 cells to HUVECs in a dose-dependent manner,the IC₅₀ was 3.2×10-6 mol/L and 6.3×10 -6 mol/L,respectively and the relative inhibition was 1.5×104 and 1 .1×104,respectively,compared to sialyl-Lewisx.The IC₅₀ of amnioti c fluid glycodelin and serum glycodelin was 3.2×10-6 mol/L and 6.3×10 -6 mol/L respectively,and the relative inhibition was 7.2×102 and 3.7×102,respectively,compared with sialyl-Lewisx.CONCLUSION:Pregnancy-associated glycoproteins,hCG and glycodel in,are the powerful inhibitors for E-selectin-mediated endothelial cell adhesion.     

Photoactivation promotes curcumin to induce apoptosis of human gastric cancer MGC-803 cells

AIM: To investigate the effect of photoactivated curcumin on apoptosis of human gastric cancer MGC-803 cells. METHODS: The effect of photoactivated curcumin on the growth inhibitory rate of gastric cancer MGC-803 cells was detected by MTT assay. The changes of nuclear morphology were observed under optical microscope with Hoechst 33258 fluorescent staining. The apoptotic rate, mitochondrial membrane potential, intracellular reactive oxygen species and Ca²⁺ level was determined by flow cytometry. The activity of caspase-3, caspase-8 and caspase-9 was detected by colorimetry. The protein levels of cytochrome C, Bcl-2, Bax and heat-shock protein 70 (HSP70) were analyzed by Western blotting. RESULTS: The growth inhibitory rate of MGC-803 cells treated with curcumin at concentration of 5.0 μmol/L was (29.74±2.30)%.Some apoptotic cells were observed under optical microscope, and the apoptotic rate was (12.54±1.75)%. The growth inhibitory rate of MGC-803 cells treated with photoactivated curcumin was (44.93±3.61)%.Significant morphological changes in the nucleus, such as chromatin condensation and apoptotic body formation, were observed under light microscope, and the apoptotic rate was (26.58±2.67)%. The cell cycle was arrested at G₀/G₁ phase. Compared with curcumin group, significant reduction in mitochondrial membrane potential,significant increase in cytochrome C, intracellular reactive oxygen species, Ca²⁺ level and the activity of caspase-3, caspase-8 and caspase-9 were observed in photoactivated curcumin group (P<0.01). Photoactivated curcumin also significantly inhibited the protein expression of Bcl-2 and HSP70 in the cells. CONCLUSION: Photoactivated curcumin enhances the apoptosis of gastric cancer MGC-803 cells by inhibiting Bcl-2 expression and promoting the mitochondrial pathway.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Effects of luteolin on endothelial cell injury induced by H2O2

AIM: To study the effect of luteolin on endothelial cell injuries induced by H₂O₂. METHODS: Endothelial cells were cultured in vitro and divided into seven groups: control; solvent; H₂O₂; quercetin+H₂O₂; luteolin-L+H₂O₂; luteolin-M+H₂O₂; luteolin-H+H₂O₂ group, respectively. Endothelial cells were incubated with 750 μmol/L H₂O₂ for 18 h or 14 h in the absence or presence of various concentrations of luteolin and quercetin. The expression of caspase-3, the pro-apoptotic factors, was detected by immunohistochemical technique, and the apoptotic index was detected by flow cytometry. Meanwhile, the amount of malondialdehyde (MDA), nitric oxide (NO), lacate dehydrogenase (LDH) in serum, and cell viability were measured. RESULTS: Incubation of endothelial cells with H₂O₂ for 18 h elicited the increase in the extent of immunostaining for caspase-3, the rate of apoptosis, the release of LDH, and the number of dead cells accompanied by the decrease in the content of NO in the medium, which were inhibited by pretreatment of luteolin at concentrations of 0.5-50 μmol/L in a concentration-dependent manner (P<0.01). CONCLUSION: Luteolin possesses a protective effect against endothelial cell injuries induced by H₂O₂, which may be related with anti-oxidation, increasing the concentration of NO and inhibiting the activation of caspase-3.     

Upregulatory effect of isopsoralen on expression of estrogen receptor in human lens epithelial cells

AIM: To investigate the effect of isopsoralen(ISR) on the expression of estrogen receptor (ER) in human lens epithelial cells (HLECs). METHODS: HLECs were cultured and sub-cultured in vitro. The cultured HLECs pretreated with E₂ or ISR were exposed to H₂O₂ at the concentration of 300 μmol/L. The expression of ERα and ERβ in HLECs was analyzed by flow cytometry. RESULTS: The expression of ERα and ERβ in H₂O₂ group was obviously decreased as compared to control group (P<0.01). The expression of ERα and ERβ in the cells treated with E₂ and with ISR at the concentration of 10^-5 mol/L, 10^-6 mol/L or 10^-7 mol/L plus H₂O₂ was obviously increased as compared to the cells treated with H₂O₂ only (P<0.01). A concentration-dependent effect of ISR was observed. CONCLUSION: H₂O₂ decreases the expression of ERα and ERβ in HLECs.E₂ and ISR increase the expression of ERα and ERβ in HLECs treated with H₂O₂ in a concentration-dependent manner, which may account for their antioxidative effect.     

Effects of DHEA and G6PD antisense oligodeoxynucleotides on Raji cells

AIM:To investigate the suppressive effects of dehydroepiandrosterone (DHEA) and glucose-6-phosphate dehydrogenase (G6PD) antisense oligodeoxynucleotides on Raji cells. METHODS:Raji cell line was cultured in vitro in the presence of DHEA at different concentrations ranged from 0.05 μmol/L to 500 μmol/L or G6PD antisense oligodeoxynucleotides. The viability and proliferation of the cells pretreated with dehydroepiandrosterone or G6PD antisense oligodeoxynucleotides were evaluated. Meanwhile, intracellular activities and mRNA expression of G6PD were analyzed. RESULTS:DHEA and G6PD antisense oligodeoxynucleotides does not influence the viability of cells in culture. Raji cells treated with DHEA at concentration of 50 μmol/L or 500 μmol/L for 72 h or with 10.0 μmol/L G6PD antisense oligodeoxynucleotides for 48 h had significant lower cell numbers compared with control (P<0.01). Raji cells treated with DHEA at concentration more than 5.0 μmol/L for 72 h had significant decreased G6PD activities (P<0.01) but no change in mRNA expression levels was observed. With 10.0 μmol/L G6PD antisense oligodeoxynucleotides pretreatment for 48 h, the G6PD mRNA expression levels and activities were significantly decreased (P<0.01). CONCLUSION:DHEA or G6PD antisense oligodeoxynucleotides at specific concentration have suppressive effects on G6PD activities and proliferation in Raji cells to a certain extent, but the suppressive mechanisms are different.     

Effect of norcantharidin on proliferation and invasion of human breast cancer cell line SKBR3 in vitro

AIM: To investigate the effect of norcantharidin（NCTD）on proliferation and invasion of human breast cancer cell line SKBR3 in vitro and its anticancer mechanisms.METHODS: MTT assay was used to determine SKBR3 cell proliferation. Light and FACScan were used to detect apoptosis and cell cycle. The invasiveness of SKBR3 was evaluated by the adhesion test，Matrigel experiment and the crossing-river test.RESULTS: NCTD had inhibitive effects on growth of SKBR3 cells in a dose and time-dependent manner, with the IC₅₀ value of 12.5 mg/L at 24 h．The cells treated with 10 mg/L NCTD for 24 h and 48 h showed typical apoptotic morphology and hypodiploid peak before G₁ phase. The cell cycle was arrested at G₂/M phase. The apoptosis percentage was up to 3.44% and 6.17%, and the G₂/M percentage was up to 35.82% and 38.70%. NCTD also could inhibit obviously the adhesion, movement and invasive capability simulating human basement membrane of SKBR3. Its effect was also in a dose-dependent manner. In the NCTD-treated group, crossing-river time was prolonged significantly and passing-membrane cells markedly decreased. CONCLUSION: NCTD in vitro inhibits not only the proliferation and growth of human breast cancer cells but also invasion and metastasis of the cells at relatively low concentration. NCTD shows prominent anti-tumor effects.     

Role of ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H2O2 preconditioning

AIM:To explore the role of extracellular signal-regulated kinases ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H₂O₂ preconditioning in PC12 cells. METHODS:In PC12 cells, the experimental model of cytoprotection by H₂O₂ preconditioning against oxidative stress-induced injury was set up. The morphological changes in the apoptotic cells were observed by using of chromatin dye Hoechst 33258. The percent of apoptotic cells was determined by flow cytometry (FCM) with propidium iodide staining. The levels of p-ERK1/2 and p-STAT3 expression were detected by Western blotting assay. RESULTS:Preconditioning with H₂O₂ at concentration of 100 μmol/L for 90 min obviously inhibited apoptosis induced by 300 μmol/L H₂O₂, and both ERK1/2 and STAT3 were activated. UO126 (10 μmol/L, an inhibitor of ERK1/2) or AG-490 (10μmol/L, an inhibitor of JAK2) significantly blocked the cytoprotection effect of H₂O₂ preconditioning. Moreover, UO126 (10 μmol/L) also markedly inhibited the up-regulation of p-STAT3 expression by H₂O₂ preconditioning. CONCLUSION:H₂O₂ preconditioning activates ERK1/2-STAT3 signal pathway, which may be one of the mechanisms underlying H₂O₂ preconditioning-induced cytoprotection.