Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

AIM： To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin （ADFMChR） on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved.METHODS： HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry （FCM） using propidium iodide （PI） fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ （PPARγ）, NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting.RESULTS： MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose- dependent manner, with little effect on growth of L-02 cells, and when ICs0 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil （5-FU, ICso was 9.27 μmol/L）. The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 （191.55/8.45）, higher than 5-FU （SI was 7.05 （65.37/9.27）. FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR （16.0%） （P 〈 0.05） and were similar to those obtained with 30.0 μmol/L 5-FU（41.0%）. DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 pmol/1 GW9662, a blocker of PPARy. Western blotting analysis revealed that aEer 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFMChR, PPARy and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARy and NF-KB protein expression in HepG2 cells.CONCLUSION： ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.     

Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating peroxisome proliferator-activated receptor γ

AIM:To examine whether and how rosiglitazone enhances apoptosis induced by fluorouracil in human colon cancer(HT-29)cells.METHODS:Human colon cancer HT-29 cells were cultured in vitro and treated with fluorouracil and/or rosiglitazone.Proliferation and growth of HT-29 cells were evaluated by MTT assay and trypan blue exclusion methods,respectively.The apoptosis of HT-29 cells was determined by acridine orange/ethidium bromide staining and flow cytometry using PI fluorescence staining.The expressions of peroxisome proliferator-activated receptor γ(PPARγ),Bcl-2 and Bax in HT-29 cells were analyzed by Western blot.RESULTS:Although rosiglitazone at the concentration below 30 μmol/L for 72 h exerted almost no inhibitory effect on proliferation and growth of HT-29 cells,it could significantly enhance fluorouracil-induced HT-29 cell proliferation and growth inhibition.Furthermore,10 μmol/L rosilitazone did not induce apoptosis of HT-29 cells but dramatically enhanced fluorouracil-induced apoptosis of HT-29 cells.However,rosiglitazone did not improve apoptosis induced by fluorouracil in HT-29 cells pretreated with GW9662,a PPARγ antagonist.Meanwhile,the expression of Bax and PPARγ was up-regulated,while the expression of Bcl-2 was down regulated in HT-29 cells treated with rosiglitazone in a time-dependent manner.However,the effect of rosiglitazone on Bcl-2 and Bax was blocked or diminished in the presence of GW9662.CONCLUSION:Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating PPARγ.     

Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

AIM: To evaluate the effects of tributyrin, a pro-drug of natural butyrate and a neutral short-chain fatty acid triglyceride, on the growth inhibition of human gastric cancer SGC-7901 cell. METHODS: Human gastric cancer SGC-7901 cells were exposed to tributyrin at 0.5, 1, 2, 5, 10 and 50 mmol/L(-1) for 24-72 h. MTT assay was applied to detect the cell proliferation. [(3)H]-TdR uptake was measured to determine DNA synthesis. Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect tributyrin-triggered apoptosis. The expressions of PARP, Bcl-2 and Bax were examined by Western blot assay. RESULTS: Tributyrin could initiate growth inhibition of SGC-7901 cell in a dose- and time-dependent manner. [(3)H]-TdR uptake by SGC-7901 cells was reduced to 33.6 % after 48 h treatment with 2 mmol/L(-1) tributyrin, compared with the control (P<0.05). Apoptotic morphology was detected by TUNEL assay. Flow cytometry revealed that tributyrin could induce apoptosis of SGC-7901 cells in dose-dependent manner. After 48 hours incubation with tributyrin at 2 mmol/L(-1), the level of Bcl-2 protein was lowered, and the level of Bax protein was increased in SGC-7901, accompanied by PARP cleavage. CONCLUSION: Tributyrin could inhibit the growth of gastric cancer cells effectively in vitro by inhibiting DNA synthesis and inducing apoptosis, which was associated with the down-regulated Bcl-2 expression and the up-regulated Bax expression. Therefore, tributyrin might be a promising chemopreventive and chemotherapeutic agent against human gastric carcinogenesis.     

Apoptosis of human gastric adenocarcinoma cells induced by β-ionone

AIM:To investigate the effect of β-ionone on the growth and apoptosis of gastric adenocarcinoma cell line SGC-7901.METHODS: Using M-IT, fluorescence dye (Hoechst-33258),transmission electron microscopy and the TUNEL assay,we examined growth and apoptosis of SGC-7901 cells treated with β-ionone at various concentrations (i.e. 25, 50, 100 and 200μmol/L) for 24h,48h.RESULTS:The growth of SGC-7901 cells was inhibited by β-ionone. Seven days after treatment with β-ionone at four concentrations, the inhibition rates were 12.04%, 30.59%,78.25% and 94.15%, respectively. The IC50 value of β-ionone for SGC-7901 cells was estimated to be 89μmol/L.The apoptotic morphology was demonstrated in SGC-7901 cells treated with β-ionone by Hoechst-33258 staining and electron microscopy. Apoptosis was also shown in β-iononetreated SGC-7901 cells by the TUNEL assay.CONCLUSION:β-ionone can inhibit cell proliferation and induce apoptosis of SGC-7901 cells.However, the mechanism needs to be further investigated.     

Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

AIM: To evaluate the effects of tributyrin, a pro-drug of natural butyrate and a neutral short-chain fatty acid triglyceride, on the growth inhibition of human gastric cancer SGC-7901 cell.METHODS: Human gastric cancer SGC-7901 cells were 24-72 h. MTT assay was applied to detect the cell proliferation.[3H]-TdR uptake was measured to determine DNA synthesis.Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect tributyrin-triggered apoptosis. The expressions of PARP, Bcl-2 and Bax were examined by Western blot assay.RESULTS: Tributyrin could initiate growth inhibition of SGC7901 cell in a dose- and time-dependent manner. [3H]-TdR uptake by SGC-7901 cells was reduced to 33.6% after 48 h control (P＜0.05). Apoptotic morphology was detected by TUNEL assay. Flow cytometry revealed that tributyrin could induce apoptosis of SGC-7901 cells in dose-dependent manner. After 48 hours incubation with tributyrin at 2 mmol.L-1, the level of Bcl-2 protein was lowered, and the level of Bax protein was increased in SGC-7901, accompanied by PARP cleavage.CONCLUSION: Tributyrin could inhibit the growth of gastric cancer cells effectively in vitro by inhibiting DNA synthesis and inducing apoptosis, which was associated with the downregulated Bcl-2 expression and the up-regulated Bax expression. Therefore, tributyrin might be a promising chemopreventive and chemotherapeutic agent against human gastric carcinogenesis.     

Inhibitory effects of apigenin on the growth of gastric carcinoma SGC-7901 cells

AIM: To explore the growth inhibition and apoptosis-inducing effect of apigenin on human gastric carcinoma SGC-7901 cells. METHODS: The effects of apigenin on the growth, clone formation and proliferation of human gastric carcinoma SGC-7901 cells were observed by MTT, clone-forming assay, and morphological observation. Fluorescent staining and flow cytometry analysis were used to detect apoptosis of cells. RESULTS: Apigenin obviously inhibited the growth, clone formation and proliferation of SGC-7901 cells in a dosedependent manner. Inhibition of growth was observed on d 1 at the concentration of 80 μmol/L, while after 4 d, the inhibition rate (IR) was 90%. The growth IRs at the concentration of 20, 40, and 80 μmol/L were 38%, 71%, and 99% respectively on the 7~(th) d. After the cells were treated with apigenin for 48 h, the number of clone-forming in control, 20, 40, and 80 μmol/L groups was 217±16.9, 170±11.1 (P<0.05), 98±11.1(P<0.05), and 25±3.5 (P<0.05) respectively. Typical morphological changes of apoptosis was found by fluorescent staining. The cell nuclei had lost its smooth boundaries, chromatin was condensed, and cell nuclei were broken. Flow cytometry detected typical apoptosis peak. After the cells were treated with apigenin for 48 h, the apoptosis rates were 5.76%, 19.17%, and 29.30% respectively in 20, 40, and 80 μmol/L groups. CONCLUSION: Apigenin shows obvious inhibition on the growth and clone formation of SGC-7901 cells by inducing apoptosis.     

Paclitaxel induces apoptosis in human gastric carcinoma cells

AIM: To investigate the apoptosis in gastric cancer cells induced by paclitaxel, and the relation between this apoptosis and expression of Bcl-2 and Bax. METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitively detect the apoptosis status of gastric cancer cell line SGC-7901 before and after the paclitaxel treatment. Immunohistochemical staining was used to detect the expression of apoptosis-regulated gene Bcl-2 and Bax. RESULTS: Paclitaxel inhibited the growth of gastric cancer cell line SGC-7901 in a dose-and time-dependent manner. Paclitaxel induced SGC-7901 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. Paclitaxel could reduce the expression of apoptosis-regulated gene Bcl-2, and improve the expression of apoptosis-regulated gene Bax. CONCLUSION: Paclitaxel is able to induce the apoptosis in gastric cancer. This apoptosis may be mediated by down-expression of apoptosis-regulated gene Bcl-2 and up-expression of apoptosis-regulated gene Bax.     

Effects of c9,t11-conjugated linoleic acid on adhesion of human gastric carcinoma cell line SGC-7901

AIM: To investigate the effect of c9, t11-conjugated linoleic acid (c9,t11-CLA) on the adhesion of human gastric carcinoma cell line (SGC-7901).METHODS: SGC-7901 cells were at first treated with different concentrations (25, 50, 100, 200 μmol/L) of c9,t111-CLA and 1 mL/L ethanol (as a negative control) for 24 h.Using adhesion assay and Western blot, we investigated the ability of SGC-7901 cells to adhere to intracellular matrix and examined the expression of E-cadherin (ECD), α-catenin,intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in these cells.RESULTS: The attachment rate to laminin of SGC-7901 cells treated with different concentrations of c9,t11-CLA (0,25, 50, 100, and 200 μmol/L) was 100.0&#177;3.3, 95.7&#177;4.0,89.2&#177;4.6, 87.9&#177;6.1, and 65.9&#177;5.8, respectively. The attachment rate to fibronectin was 100.0&#177;4.7, 96.8&#177;3.8,94.5&#177;4.1, 76.5&#177;4.3, and 61.8&#177;4.8, respectively. The attachment rate to Matrigel was 99.9&#177;6.6, 91.4&#177;6.8,85.5&#177;7.4, 79.3&#177;5.6, and 69.6&#177;5.1, respectively. Besides,c9,t11-CLA could increase the level of ECD and α-catenin,and decrease the level of ICAM-1 and VCAM-1 in SGC-7901 cells.CONCLUSION: c9, t11-CLA can reduce the adhesion of human gastric carcinoma cells to laminin, fibronectin and Matrigel. c9,t11-CLA can increase the level of ECD and α-catenin, and decrease the level of ICAM-1 and VCAM-1 in human gastric carcinoma cells.     

Inhibitory effects of c9, t11-conjugated linoleic acid on invasion of human gastric carcinoma cell line SGC-7901

AIM: To investigate the effect of c9, t1l-conjugated linoleic acid (c9, t11-CLA) on the invasion of human gastric carcinoma cell line and its possible mechanism of preventing metastasis.METHODS: Using reconstituted basement membrane invasion, chemotaxis, adhesion, PAGE substrate zymography and RT-PCR assays, we analyzed the abilities of invasion,direct migration, adhesion of intracellular matrix, as well as the activity of type IV collagenase and expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 mRNA in SGC-7901 cells which were treated with gradually increased concentrations (25, 50, 100 and 200μmol/L) of c9,c11-CLA for 24 h.RESULTS: At the concentrations of 200μmol/L, 100μmol/L and 50μmol/L, c9,tll-CLA suppressed the invasion of SGC-7901 cells into the reconstituted basement membrane by 53.7 %, 40.9 % and 29.3 %, respectively, in comparison with the negative control. Only in the 200 μmol/L c9,tll-CLA group, the chemotaxis of SGC-7901 cells was inhibited by 16.0 % in comparision with the negative control. C9,tll-CLA also could inhibit the adhesion of SGC-7901 cells to laminin, fibronectin and Matrigel, increase the expression of TIMP-1 and TIMP-2 mRNA, and reduce type IV collegenase activities in the serum-free medium supernatant of SGC-7901 cells.CONCLUSION: c9,t11-C:LA can inhibit the invasion of SGC-7901 cells at multiple procedures in tumor metastasis cascade, which may be associated with the induction of TIMP-1 and TIMP-2 mRNA expression.     

Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating peroxisome proliferator-activated receptor γ

AIM: To examine whether and how rosiglitazone enhances apoptosis induced by fluorouracil in human colon cancer (HT-29) cells.METHODS: Human colon cancer HT-29 cells were cultured in vitro and treated with fluorouracil and/or rosiglitazone. Proliferation and growth of HT-29 cells were evaluated by MTT assay and trypan blue exclusion methods, respectively. The apoptosis of HT-29 cells was determined by acridine orange/ethidium bromide staining and flow cytometry using PI fluorescence staining. The expressions of peroxisome proliferator-activated receptor y (PPARy), Bcl-2 and Bax in HT-29 cells were analyzed by Western blot.RESULTS: Although rosiglitazone at the concentration below 30 umol/L for 72 h exerted almost no inhibitory effect on proliferation and growth of HT-29 cells, it could significantly enhance fluorouracil-induced HT-29 cell proliferation and growth inhibition. Furthermore, 10 umol/L rosilitazone did not induce apoptosis of HT-29 cells but dramatically enhanced fluorouracil-induced apoptosis of HT-29 cells. However, rosiglitazone did not improve apoptosis induced by fluorouracil in HT-29 cells pretreated with GW9662, a PPARy antagonist. Meanwhile, the expression of Bax and PPARy was up-regulated, while the expression of Bcl-2 was down regulated in HT-29 cells treated with rosiglitazone in a time-dependent manner. However, the effect of rosiglitazone on Bcl-2 and Bax was blocked or diminished in the presence of GW9662.CONCLUSION: Rosiglitazone enhances fluorouracil-induced apoptosis of HT-29 cells by activating PPARγ.     

Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer. METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting. RESULTS: The number of viable cells decreased in a dose-and time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10μmol/L for 48 h or 8μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G_1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h. CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.     

Apoptosis of human gastric adenocarcinoma cells induced by β-ionone

AIM: To investigate the effect of β-ionone on the growthMETHODS: Using MTT, fluorescence dye (Hoechst-33258),transmission electron microscopy and the TUNEL assay, we examined growth and apoptosis of SGC-7901 cells treated with β-ionone at various concentrations (i.e. 25, 50, 100 and 200 μmol/L) for 24 h, 48 h.RESULTS: The growth of SGC-7901 cells was inhibited by β-ionone, Seven days after treatment with β-ionone at four concentrations, the inhibition rates were 12.04%, 30.59%,78.25% and 94.15%, respectively. The IC50 value of β-ionone for SGC-7901 cells was estimated to be 89 μmol/L. The apoptotic morphology was demonstrated in SGC-7901 cells treated with β-ionone by Hoechst-33258 staining and electron microscopy. Apoptosis was also shown in β-iononetreated SGC-7901 cells by the TUNEL assay.CONCLUSION: β-ionone can inhibit cell proliferation and induce apoptosis of SGC-7901 cells. However, the mechanism needs to be further investigated.     

Reversal of multidrug resistance in vincristine-resistant human gastric cancer cell line SGC7901/VCR by LY980503

AIM： To investigate the reversal effect of LY980503, a benflumetol derivative, on multidrug resistance in vincristine （VCR） -resistant human gastric carcinoma cell line SGC7901/VCR.
METHODS： Cells of a human gastric cancer cell line, SGC7901, and its VCR-resistant variant, SGC7901/VCR, were cultivated with LY980503 and/or doxorubicin （DOX）. The cytotoxicity of drugs in vitro was assayed by M-IF method. Based on the flow cytometric technology, the uptake of DOX was detected in these cells by measuring DOX -associated mean fluorescence intensity （MFI）.
RESULTS： SGC7901/VCR cells were 23.5 times more resistant to DOX in comparison with 5GC7901 cells. LY980503 at the concentrations of 2.0 μmol/L -10 μmol/ L had no obvious cytotoxicity to SGC7901 and SGC7901/ VCR cells. After simultaneous treatment with LY980503 at the concentrations of 2.0, 4.0 and 10 μmol/L, the ICso of DOX to SGC7901/VCR cells decreased from 1.6 ± 0.12 μmol/L to 0.55 ± 0.024, 0.25 ± 0.032 and 0.11 ± 0.015 μmol/L, respectively, thus, increasing the DOX sensitivity by 2.9-fold （P 〈 0. 05）, 6.4-fold （P 〈 0. 01） and 14.5-fold （P 〈 0. 01）, respectively. In the uptake study of DOX, simultaneous incubation of SGC7901/VCR cells with LY980503 significantly increased the DOX -associated MFI in SGC7901/VCR cells. No such results were found in parental SGC7901 cells.
CONCLUSION： LY980503 at non-cytotoxic concentrations can effectively circumvent resistance of SGC7901/VCR cells to DOX by increasing intracellular DOX accumulation.     

Inhibition of DNA primase and induction of apoptosis by 3,3′-diethyl-9-methylthia-carbocyanine iodide in hepatocellular carcinoma BEL-7402 cells

AIM:To evaluate the effects of 3,3′-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells.METHODS: DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit.RESULTS:DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC50 value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC50 value of 2.09μmol/L. Furthermore,DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53,Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI.CONCLUSION: The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.     

Effect of cis—9,trans—11—conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line（SGC—7901）

AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P<0.05 and P<0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P<0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).     

熊果酸对肝星状细胞增殖与凋亡的影响

目的 体外观察熊果酸对肝星状细胞增殖与凋亡的影响,探讨熊果酸诱导肝星状细胞凋亡的可能作用机制. 方法 将不同浓度熊果酸作用于肝星状细胞HSC-T6及肝细胞L02,分别在药物作用24、48、72 h后用四甲基偶氮唑盐法检测熊果酸对HSC-T6及L02细胞增殖的影响;流式细胞仪检测熊果酸对HSC-T6凋亡的影响;光学显微镜观察熊果酸作用后细胞形态学变化情况;免疫细胞化学法检测HSC-T6中Bcl-2、Bax和Caspase-3蛋白的表达情况. 结果 各种浓度的熊果酸均可抑制HSC-T6细胞的增殖,且呈剂量-时间依赖性;当熊果酸浓度为25、50、75μmol/L时可促进L02细胞增殖,浓度＞75μmol/L则表现为抑制L02细胞增殖.在病理形态学方面,熊果酸作用HSC-T6细胞48 h后,光学显微镜下可见细胞缩小变圆、核浓缩等.25、50、75 μmol/L熊果酸作用HSC-T6细胞48 h后,流式细胞仪检测显示细胞凋亡率分别为10.30%±3.85%、21.87%±4.46%、31.33%±6.18%,比对照组(2.93%±1.60%)明显升高(P＜0.01).免疫细胞化学显示Bax及Caspase-3蛋白表达较对照组升高(P＜0.05),且呈剂量依赖性,而Bcl-2蛋白表达水平与对照组无明显差异(P＞0.05).结论 在体外熊果酸可较明显地抑制HSC-T6细胞增殖,诱导其凋亡;对L02细胞的生长具有双向调节作用.熊果酸诱导HSC-T6细胞凋亡可能与降低Bcl-2/Bax比值、激活Caspase-3蛋白有关.