Anti-tumor effect and its related mechanisms of cinobufotalin combined with cisplatin on H22 liver cancer mice / 中国中药杂志

In order to observe the anti-tumor effect of cinobufotalin on H22 liver cancer mice and to explore its regulatory mechanism, 50 Kunming mice were subcutaneously inoculated with H22 intraperitoneal passage cells under the armpit to establish H22 hepatocellular carcinoma model. They were then randomly divided into model group, cinobufotalin low dose group, cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, which received 0.01% ethanol solution, 1 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cinobufotalin, 5 mg·kg~(-1) cisplatin, 5 mg·kg~(-1)cisplatin + 5 mg·kg~(-1) cinobufotalin respectively for 10 days. The general condition of mice during the intervention was observed, and the inhibition rate, tumor mass, thymus index, histopathological changes of the tumors, apoptotic rate of the tumors, the expressions of phosphatidylinositol 3-kinase(PI3 K), protein kinase B(Akt), apoptosis related gene(Fas), Fas ligand(FasL) mRNA and protein phosphorylated Akt(pAkt) protein in the tumors of each group were compared. The results showed that during the modeling period, the mice showed a decline in food intake, dark fur, poor mental status, and gradually worsened over time. The mental status of mice in each intervention group was improved gradually, especially in the cisplatin+cinobufotalin group. As compared with the model group, the tumor mass of each intervention group was lower(P<0.05). As compared with the cinobufotalin low dose group, the tumor mass was lower and inhibition rate was higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, the tumor mass was lower and the inhibition rate was higher in cisplatin+cinobufotalin group(P<0.05). As compared with the model group, the thymus index was higher in cinobufotalin high dose group and cisplatin + cinobufotalin group, while was lower in cisplatin group(P<0.05). As compared with the cinobufotalin low dose group, the thymus index was higher in the cinobufotalin high dose group and lower in the cisplatin group(P<0.05). As compared with the cinobufotalin high dose group, the thymus index was lower in cisplatin group(P<0.05). As compared with cisplatin group, the thymus index was higher in cisplatin+cinobufotalin group(P<0.05). Pathological staining showed that a large number of heterogeneous cells and mitotic phenomena were observed in the model group. Cell fragments and neutrophils were observed in the tumor tissues of the intervention groups, showing diffuse necrosis, and the diffuse necrosis was more obvious in the cisplatin+cinobufotalin group. As compared with the model group, the apoptotic rate of the tumors and the relative expressions of Fas mRNA and protein were higher in the intervention groups, while the relative expressions of PI3 K, FasL mRNA and protein and the relative expression of pAkt protein were lower in the intervention groups(P<0.05). As compared with the cinobufotalin low dose group, the apoptotic rate of the tumors and relative expression of Fas and protein were higher in the cinobufotalin high dose group, cisplatin group and cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower(P<0.05). As compared with the cinobufotalin high dose group and the cisplatin group, apoptotic rate of the tumors and the relative expression of Fas mRNA and protein were higher in the cisplatin+cinobufotalin group, while the relative expressions of PI3 K, FasL mRNA and protein and pAkt protein were lower in the cisplatin+cinobufotalin group(P<0.05). In summary, cinobufotalin has significant anti-tumor effect on H22 liver cancer mice, and can enhance the immune function of mice and synergistically enhance the effect of chemotherapy. Its mechanism may be associated with regulating PI3 K/Akt/Fas/FasL signaling pathway related genes and protein expression.

Podophyllotoxin derivative LN-13 induced multidrug resistant cell K562/A02 apoptosis and its molecular mechanism / 中国药理学通报

Aim To study the mechanism of action of the new derivative of podophyllotoxin(LN-13)in indu-cing the apoptosis of K562/A02 cells.Methods The MTT method was taken to detect the inhibition of LN-13 and VP-16 on K562/A02 proliferation and inhibi-tion rate and IC50 values were obtained 48 hours later. The K562/A02 cell morphological change induced by LN-13 were observed through Hochest33342 and PI staining after 48 hours later.Flow cytometry was taken to detect the apoptosis of K562/A02 cells induced by LN-13.The reverse transcription-polymerase chain re-action was taken to detect the Bcl-2,Bax,caspase-3 and mdr-1 mRNA expression.The expression of P-gp was detected by Western blot.Results The growth of K562 /A02 cells was obviously inhibited by LN-13 when IC50 value was 3.32 μmol · L-1 .LN-13 could obviously induced cell apoptosis observed by Ho-chest33342 and PI staining.Flow cytometry detection showed that LN-13(2,4,8 μmol·L-1 )could induce cell apoptosis and apoptosis ratio reached 15.0%, 48.0%,68.96%,respectively.The reverse transcrip-tion-polymerase chain reaction showed that LN-13 in-creased the Bax and Caspase-3 mRNA expression,and meanwhile the expression of mdr-1 mRNA decreased. Western blot showed that P-gp expression was de-creased as the LN-13 dose increased.The data were significantly different from those of control group.Con-clusion Podophyllotoxin derivative LN-13 can induce the apoptosis of K562 /A02 cells,which may be close-ly-related to regulating P-gp expression and apoptosis related gene mRNA expression.

Effects of electro-acupuncture on functional performance and the expression of apoptosis-related proteins Bcl-2 and Bax in the hippocampal CA1 region of rats with cerebral infarction / 中华物理医学与康复杂志

Objective To observe effects of electro-acupuncture (EA) on the expression of apoptosis related protein B-cell lymphoma/leukemia gene 2 (Bcl-2) and Bcl-associated protein X (Bax) in the hippoeampal CA1 region of rats with cerebral infarction,and on their behavior. MethodsAn animal model of cerebral ischemia was established by right side middle cerebral artery occlusion using thread in 48 male,adult Wistar rats,who were then randomly divided equally into an EA group and a control group.Both groups were sub-divided into 1 week,2 weeks and 3 weeks subgroups.The EA group began receiving EA 24h after the occlusion,applied at the Baihui (DU20) and Dazhui (BU14) points,once daily,for one,two or three weeks.The control group was reared conventionally and was not given any special treatment.The rats'learning and memory,motion and neural function were evaluated.The expression of Bcl-2 and Bax in the CA1 region of the hippocampus on the infarcted side and changes of apoptosis indexes were detected using immunohistochemical techniques.ResultsThe learning,memory,motion and neural function of the EA group rats were better on average than those of the control group at all observation time points.The expression of Bcl-2 protein increased along with reduced expression of Bax protein in the EA group significantly more than among the controls.TUNEL positive cells in the CA1 region of the hippocampus were reduced significantly in the EA group compared with the control group.ConclusionsBehavioral ability after cerebral infarction can be improved by EA at the Baihui (DU20) and Dazhui (BU14) points.EA can up-regulate the expression of Bcl-2 and down-regulate the expression of Bax and reduce TUNEL positive cells in the CA1 region of the hippocampus after cerebral infarction,which might be the mechanism of its neuroprotection.

Expression of Apoptosis Related Genes from HaCaT Cell after UVB Irradiation / 대한피부과학회지

BACKGROUND: Ultraviolet B (UVB) radiation is a major harmful environmental factor causing cutaneous changes such as sunburn, skin aging and skin cancer. Excessive UVB irradiation induces apoptosis of keratinocytes through several molecular pathways. However, the precise molecular mechanisms have been underinvestigated. OBJECTIVE: The purpose of this study was to investigate expression levels of apoptosis-related genes in UVB- irradiated HaCaT ketratinocyte cell lines. METHODS: Cells were irradiated by UVB at various doses (0, 100, 200 and 400 mJ/cm2). Expression levels of caspases, Bax, Bcl2, and Bcl-XL were confirmed by RT-PCR analysis and western blotting. RESULTS: Expression of cytochrome C was increased followed by activation of caspase-3, 8 and 9 in UVB-irradiated HaCaT cells. Furthermore, the expression of Bcl-XL was decreased from UVB 200 mJ/cm2 and Bcl2 was decreased weakly from UVB 400 mJ/cm2, implying that the expression of Bcl-XL is more sensitive to UVB. Interestingly, the down-regulation of Bcl-XL may be mediated by proteasome dependent pathways. CONCLUSION: Excessive UVB-irradiated HaCaT cells undergo apoptotic cell death following activation of caspases; degradation of Bcl-XL is particularly sensitive to UVB.

Expression of Apoptosis Related Genes from HaCaT Cell after UVB Irradiation / 대한피부과학회지

BACKGROUND: Ultraviolet B (UVB) radiation is a major harmful environmental factor causing cutaneous changes such as sunburn, skin aging and skin cancer. Excessive UVB irradiation induces apoptosis of keratinocytes through several molecular pathways. However, the precise molecular mechanisms have been underinvestigated. OBJECTIVE: The purpose of this study was to investigate expression levels of apoptosis-related genes in UVB- irradiated HaCaT ketratinocyte cell lines. METHODS: Cells were irradiated by UVB at various doses (0, 100, 200 and 400 mJ/cm2). Expression levels of caspases, Bax, Bcl2, and Bcl-XL were confirmed by RT-PCR analysis and western blotting. RESULTS: Expression of cytochrome C was increased followed by activation of caspase-3, 8 and 9 in UVB-irradiated HaCaT cells. Furthermore, the expression of Bcl-XL was decreased from UVB 200 mJ/cm2 and Bcl2 was decreased weakly from UVB 400 mJ/cm2, implying that the expression of Bcl-XL is more sensitive to UVB. Interestingly, the down-regulation of Bcl-XL may be mediated by proteasome dependent pathways. CONCLUSION: Excessive UVB-irradiated HaCaT cells undergo apoptotic cell death following activation of caspases; degradation of Bcl-XL is particularly sensitive to UVB.

RELATION OF ANTIRADIATION EFFECT OF LAMINARIA JAPONICA POLYSACCHARIDES AND LYMPHOCYTE APOPTOSIS / 营养学报

Objective:To investigate the relation of antiradiation effect of LJP and lymphocyte apoptosis. Methods:36 Wistar rats were randomly divided into 6 groups (n=6): control, model,and LJP given i.g.at 4 doses(100,200,300,400 mg/ kg bw) for 10d before whole-body irradiation with 9.0 Gy Co?-ray. 18h later,the effects of LJP on the indices of immune function of the irradiated rats 60 were measured. TUNEL and flow cytometry were used to study the effects of LJP on splenic lymphocyte apoptosis and immunohistochemical technique was used to detect the expression of Bcl-2 and the Bax protein. Results:LJP significantly modulated immune function in irradiated rats. The apoptosis ratio of splenic lymphocyte of the model group was higher than those of other groups. LJP could markedly inhibit the effects of irradiation on apoptosis and increase the ratio of bcl-2/bax protein in dose-effect manner. Conclusion:LJP could inhibit splenic lymphocyte apoptosis induced by irradiation, and its mechanism is associated with regulating the expression of bcl-2 and bax protein of splenic lymphocyte. Key word:laminaria japonica polysaccharides; irradiation; lymphocyte; apoptosis apoptosis-related genes

Effects of Iron Chelators on Labile lron Pool and Apoptosis Related Genes Ex pression in K562 Cells / 实用儿科临床杂志

Objective To explore the effect of iron chelators on labile iron pool and expression of apoptosis associated genes in cells of K562, an erythroleukemia cell line.Methods K562 cells were incubated at 37 ℃ in RPMI 1640 containing 10% heat-inactived fetal bovine serum in an saturated humidity and 5% CO_2 incubator. K562 cells were incubated with different concentrations of desferro-(xamine(DFO)). The study groups were divided as following: DFO group, iron+DFO group and the control group. Following indices were detected which included apoptosis by flow cytometry (FCM) assay, expression of Rb, c-myc, bax mRNA by RT-PCR. The intracellular LIP was measured with a fluorimetric assay using the metalsensitive probe calcein-AM.Results 1. The viability of K562 cells incubated with different concentrations of DFO was lower than that of control group at 12 h,24 h and 48 h (P

Effects of apoptosis-related protein on apoptosis of gastric cancer cells / 中华消化杂志

Objective To investigate the effects of nu cleus transcription factor (NF)-?B, survivin, Bcl-2 and Caspase-3 on apoptos is of gastric cancer cells induced by tumor necrosis factor related apoptosis in ducing ligand(TRAIL). Methods Gastric cancer cells were cultured in RPMI-1640 and the proportions of apoptosis were analyzed by flow cytometry. The expressions of N F-?B, survivin, Bcl-2 and Caspase-3 in gastric cancer cells were evaluated b y Western blot. Results After gastric cancer cells were exposed to 50 ng/ml TRAIL f or 24 hours, the proportions of apoptosis were 24.05% in line MKN28, 7.83% in MK N45, 8.05% in AGS and 3.17% in SGC-7901 respectively; after 300 ng/ml TRAIL for 24 ho urs, the proportions of apoptosis were 36.05% in MKN28, 20.27% in MKN45, 16.50% in AGS and 11.80% in SGC-7901 respectively. The expressions of NF-?B and surv ivin under Western blot were lower in MKN28 than that in MKN45, AGS and SGC-79 0 1. In contrast, the expression of Caspase-3 was higher in MKN28 than that in MK N45, AGS a nd SGC-7901. The expression of Bcl-2 had no difference among the 4 lines of ga stric cancer cells. Conclusions The anti-tumor sensitivity of TRAIL to gastric cancer c ell may be associated with both increased expressions of NF-?B and survivin an d decreased expression of Caspase-3.

A study on the mechanisms of apoptosis of gastric cancer line induced by Nimesulide / 中华消化杂志

Objective Non steroidal anti inflammatory drugs(NSAIDs)can induce apoptosis in gastric cancer cell and the mechanism is not clear. We aimed to study the mechanism of selective COX 2 inhibitor Nimesulide induced apoptosis of human gastric cancer line SGC 7901 by detecting the expressions of COX 2 at mRNA level, c myc, Bcl 2 and caspase 3 at protein level. Methods Apoptosis was determined by electronic microscopy, Annexin V FITC staining and flow cytometry. The mRNA of COX 2 was detected by RT PCR. The protein expressions of c myc, Bcl 2 and caspase 3 were examined by immunohistochemistry. Results Nimesulide of 50 ?mol/L at 48 and 72 h, and of 100 ?mol/L and 200 ?mol/L at 24, 48 and 72 h induced apoptosis of gastric cancer cells in a dose and time dependent manner.Their apoptotic rates were 7.51%, 9.86% and 11.58%, 12.45%, 16.66% and 12.21%, 15.38%, 20.28% respectively. It increased c myc and caspase 3 expression and decreased Bcl 2 expression and COX 2 mRNA expression. The positive protein expression rates of Bcl 2, c myc and capase 3 were (20.2?7.6)%,(49.2?15.1)% and (34.6?12.9)% respectively with Nimesulide of 200 ?mol/L at 72 h,while the controls being (44.6?12.1)%, (24.7 ?9.5)% and (14.8?6.4)% the three comparative P

The mechanisms of apoptosis induced by hydroxycamptothecin in gastric cancer cells / 中华消化杂志

Objective To study the mechanisms of Hydroxycamptothecin (HCPT) induced apoptosis of gastric cancer cells by detecting the expressions of p53, c myc, bcl 2, bcl xl, bcl xs genes. Methods Apoptosis of gastric cancer cells (SGC 7901,MKN 45) was measured by TUNEL and flow cytometry. The levels of mRNA and protein of p53, c myc, bcl 2, bcl xl,bcl xs genes were determined by reverse transcriptase polymerase chain reaction analysis and immunohistochemistry, respectively. Results Gastric cancer cells SGC 7901, MKN 45 presented some morphologic features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, formation of apoptotic bodies, after 12 hours expourse to HCPT. Some typical subdiploid peaks before G0/G1 phase were observed. The apoptotic rates of SGC 7901, MKN 45 were 21.88% and 12.34%, respectively. The levels of p53 and bcl 2 mRNA and protein were downregulated after treated with HCPT in SGC 7901 cells. The levels proteins of c myc, bcl xl, bcl xs were unchanged after expourse to HCPT in SGC 7901 cells. The levels of p53 mRNA and protein were increased after treated with HCPT in MKN 45 cells. While the expressions of protein of bcl 2, c myc, bcl xl, bcl xs genes were not affected by HCPT in MKN 45 cells. Conclusion The results have showed HCPT induced apoptosis in gastric cancer cells might be regulated through influencing the expressions of p53 and bcl 2 genes.

Studies on the relationship of apoptosis and multidrug resistance in 5-fluorouracil resistant human hepatocellular carcinoma cell line Bel7402/5-FU / 中国药理学通报

AIM To elucidate whether apoptosis is involved in the development of multidrug resistance in 5-fluorouracil resistant cells Bel7402/5-FU. METHODS Multidrug resistance of Bel7402/5-FU cells was determined by SRB assay, apoptosis was measured by Annexin V-FITC/PI double labeled assay, cell cycle phases were analysed by flow cytometry, and expression of genes related to apoptosis bcl-2, bcl-xl, bcl-xs, bax, fas, p53, and cpp32 was detected by RT-PCR assay. RESULTS Bel7402/5-FU cells were resistant not only to 5-FU but also to multiple anticancer drugs. When treated with the same concentration of 5-FU (30 ?mol?L -1 or 150 ?mol?L -1 ), the proportion of apoptosis was significantly increased and G_0/G_1 phase was increased and S and G_2/M phases were reduced in Bel7402 cells, but the proportion of apoptosis and cell cycle was not changed in Bel7402/5-FU cells. The expression of bcl-xl, bcl-xs, and bax mRNA were significantly increased and the expression of p53 and cpp32 mRNA were significantly decreased in resistant Bel7402/5-FU cells, as compared with Bel7402 cells. CONCLUSION Decrease of apoptosis may be related to multidrug resistance of human hepatocellular carcinoma cells Bel7402/5-FU. The up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes are involved in the development of multidrug resistance in Bel7402/5-FU cells.