Tao HUANG,Liyan LI,Xiaona GUO,Zhigang GUO,Yalin Zhang

Medical School,Huanghe Science&Technology College,Zhengzhou 450063,China

Changes of NF-κB,Bax and Caspase 3 in Apoptosis Induced by Ligustrazine Combined with Cis-dichlorodiamine Platinum in Human Gastric Carcinoma SGC-7901 Cell Lines

Tao HUANG,Liyan LI*,Xiaona GUO,Zhigang GUO,Yalin Zhang

Medical School,Huanghe Science&Technology College,Zhengzhou 450063,China

[Objective] This study aimed to investigate the mechanism of apoptosis induced by ligustrazine(TMP)and cis-dichlorodiamine platinum(DDP)in SGC-7901 cell lines in vitro.[Methods]SGC-7901 cell lines were treated with ligustrazine and DDP alone or combined for 48 h for Western blot analysis,respectively.Western blot analysis was used to determine the expression of proteins involved in apoptosis including NF-κB p65,bax and caspase-3.[Results]The viability of SGC-7901 cells was inhibited after treated with ligustrazine and/or combined with DDP.The expression of NF-κB P65 protein decreased after treated with drugs,in which the protein decreased significantly in 1.2 mg/ml of TMP combined with 2 μg/ml of DDP group. Meanwhile,we investigated the protein expression of bax and caspase-3.The results showed that the expression of the two proteins increased following with the increasing concentration of TMP.[Conclusion]All the results indicated that ligustrazine combined with DDP could induce the apoptosis of SGC-7901 cell lines,and NF-κB maybe the possible way to induce the cell apoptosis.

Ligustrazine;Cis-dichlorodiamine pliatinum(DDP);Human gastric carcinoma;Apoptosis

N uclear factor-κB is an ubiquitously expressed family of Relrelated transcription factors[1]. Typically,in unstimulated cells,NF-κB is sequestered in the cytoplasm by binding to inhibitory κB protein(IκB).In response to a variety of stimuli,such as imflammatory cytokins,oncogenes, and viruses,the proteasome-dependent degradation of IκB allows the translocation of NF-κB to the nucleus, where it binds to the promoter region of target genes involved in the control of different cellular responses,including apoptosis[2-4].It has been reported that NF-κB is activated in cancer cells by several chemotherapies and by radiation,and in many cases this response inhibits the radiotherapy-and chemotherapy-induced cell death[5-6].It was predicted that activation of autophagy by blocking NF-κB may contribute to the anti-tumor actions of NF-κB inhibitors.

Ligustrazine(tetramethylpyrazine, TMP),a bioactive component contained in Chuanxiong(Ligusticum chianxiong Hort),has been reported the antitumor effect through inhibiting tumor cells proliferation directly,inducing cancer cells apoptosis,immune-regulating,enhancing the sensitivity of cancer cells to chemotherapeutic drug or decreasing the toxicity[7]. Our research has been confirmed that TMP could induce apoptosis of human gastric carcinoma SGC-7901 cell lines and the apoptosis rate was high significantly when combined with DDP[8]. In this study,we will investigate preliminarily the mechanism of apoptosis induced by TMP combined DDP in gastric cancer cell line SGC-7901.

Materials and Methods

Chemicals

Ligustrazine hydrochloride was purchased from Zhengzhou Zhuofeng Pharmaceutical Co.Ltd(Zhengzhou, China).HyClone RPMI 1640 medium and serum were purchased from Thermo Scientific Company(Logan, Utah,USA).Antibodies used in this study included NF-κB p65,bax,caspase 3 and β-action were obtained from Sangon Biotech(Shanghai)Co., Ltd.HRP-conjugated Affinipure Goat anti-rabbit IgG(H+L)was purchased from Sangon Biotech(Shanghai)Co., Ltd.DAB kit and RIPA I lysate were purchased from Sangon Biotech (Shanghai)Co.,Ltd.PMSF was obtained from Sigma company.BCA protein concentration assay kit was purchased from Beyotime Biotech. Co.,Ltd(Shanghai,China).

Cell culture

Human gastric carcinoma SGC-7901 cell lines presented as a gift by professor Xiaoping Le at Zhengzhou University were maintained at 37℃in a humidified incubator with 5% CO2/95%air and propagated in RPMI 1640 medium supplement with 10% fetal bovine serum,100 U/ml penicillin and 100 U/ml streptomycin.

Apoptosis-inducing and Western blot analysis

SGC-7901 cells were harvested in a 6-well plate(1×105cells per well)after being treated with DDP(2 μg/ml), DDP(2 μg/ml)combined with different concentrations of ligustrazine(0.3,0.6 and 1.2 mg/ml)for 48 h.Cells were washes twice in PBS and the supernatant was discarded.The cells were ruptured and extracted with lysozymeand were harvested for total proteins. Protein concentration of cells was measured by BCA protein assay kit and was unified with PBS.

The protein samples were heated with 100℃for 3 min to denature the protein,and then were separated with SDS-PAGE electrophoresis.The separated protein was transferred to a piece of PVDF(Millipore Company, USA)membrane with semi-dry transfer technique at 100 V for 2 hours.The membrane was stained with ponceau-S to confirm whether the protein transfer was successful.Five percent of defat milk powder-PBS solution was used to block the unspecific antibody binding site for one hour.Then the membrane was washed 3 times in PBS-T solution,15 min each time.The membrane was incubated with polyclonal antibody solution(1∶1 000)of rabbit antihuman NF-κB p65,Bax, Caspase 3 overnight at 4℃,respectively.After washing 3 times in PBS-T solution,the membranes were then incubated with HRP-conjugated secondary antibody goat antirabbit IgG solution(1∶1 000)for one hour at room temperature and subsequently analyzed by DAB kit.Protein β-action (1∶1 000)was used as loading control.Statistical analysis

Optical density was analyzed by Quantity One v4.62.All data were presented as mean±SD.Statistical analysis was carried out by ANOVA followed by a Dennett’s test and P＜0.05 was considered statistically significant.

Results

Effects of ligustrazine and DDP on the expression of NF-κB p65

NF-κB is heterodimer consisting of p50 and p65.P65 antibody was used to detect the expression of NF-κB by western blot.The expression of NF-κB(p65)in SGC-7901 cells was shown in Fig.1.NF-κB expression decreased after treated with TMP and DDP,and the expression of TMP combined with DDP groups were lower than that of DDP group,and decreased followed with the increasing concentration of TMP.The decreasing of expression of DDP combined with 1.2 mg/ml of TMP for 48 h was much more significant.It showed a dose-dependent negative correlation between NF-κB expression and the dose of TMP.

Our results showed that TMP combined with DDP could induce SGC-7901 cells to apoptosis[8],and the expression of NF-κB decreased following with the concentration of TMP. It indicated that TMP combined with DDP inhibited tumor cells proliferation and may induce apoptosis through inhibiting NF-κB activity.

Effects of ligustrazine and DDP on the expression of bax

Meanwhile,the expression of bax was investigated and shown in Fig.2. Opposite to expression of NF-κB,bax protein increased after treated with DDP and TMP in 48 h,and the increasing of bax protein was the most significant in 2 μg/ml of DDP combined with 1.2 mg/ml of TMP group.It showed a dose-dependent correlation between bax expression and the dose of TMP.

Effects of ligustrazine and DDP on the expression of Caspase-3

Caspase-3 is a kind of key effect enzyme in caspase family which mediates apoptosis,so it was also investigated in present study and shown in Fig.3.Caspase-3 protein increased after treated with DDP and TMP in 48h. It also showed a dose-dependent negative correlation between caspase-3 protein expression and the dose of TMP.

Discussion

NF-κB signaling pathways play critical roles in a variety of physiological and pathological processes.One function of NF-κB is to promote cell survival through induction of target genes,whose products inhibit components of the apoptotic machinery in normal and cancerous cells.Regardless of mechanism,many cancer cells, of either epithelial or hematopoietic origin,use NF-κB to achieve resistance to anticancer drugs and radiation. Hence,inhibition of NF-κB activation offers a strategy for treatment of dif-ferent malignancies and can induce apoptosis in gastric cancer SGC-7901 cells.

Till now,PDTC as selective inhibitor of NF-κB,was reported that it could induce apoptosis of SGC-7901 cells,which were human gastric carcinoma cells[9].Resveratrol could downregulate the expression of bc 1-2 gene of HepG2 cell,increasing the expression of bax gene,and inhibit the activity of NF-κB.It was possible pathway of resveratrol inducing apoptosis of HepG2 cells[10].Dejardin reported that non-steroidal anti-inflammatory drug Sulindac could prevent I-κB from phosphorylation,and inhibit the activation of NF-κB[11].Our research also confirms that the treatment of TMP combined with DDP could induce the SGC-7901 cells apoptosis by inhibiting the activity of NF-κB.It maybe a NF-κB inhibitor and offer a new strategy for clinic treatment of gastric cancer and DDP resistance.Bax(Bcl-2 associated X protein)is a promoting gene apoptosis in bcl-2 gene family.Overexpression of bcl-2 could antagonize the protective effect of bcl-2 and make the cell apoptosis.The expression of bcl-2 may be an early behavior in the gastric carcinogenesis.Bax had antagonistic effect on bcl-2.The expression of bax and bcl-2 in gastric carcinoma showed a reverse expression,it indicated that the two proteins inhibited each other[12-13].Therefore,expression of bax was investigated basing on above report in this study and the results showed that the expression of bax increased after treated by TMP combined with DDP for 48h.It was similar with the report of HepG2 treated with resveratrol.Resveratrol downregulated the expression of bc1-2 gene of HepG2 cell,increased the expression of bax gene,and inhibited the activity of NF-κB[10].Caspase-3 is a kind of key effect enzyme in caspase family which mediates apoptosis. Caspase-3 could be activated when cells appeared apoptosis.Hasegaw et al.found that caspase-3 showed high activity in tumor tissue,and played a major role in process of apoptosis mediated by Fas in Human Hepatic Carcinoma cell lines[14].Meanwhile,NF-κB, as a kind of transcript factor inhibiting apoptosis,could up-regulate bcl-2 expression,and bcl-2 inhibit the activity of caspase-3 or caspase-8 directly or indirectly,also may inhibit the synthesis of caspase-3,and inhibit cancer cells apoptosis finally,so the expression of caspase-3 is negatively correlated with NF-kB p65[15-16].It has been confirmed that PDTC up-regulated caspase-3 expression and induced apoptosis of SGC-7901 cells[9].TMP combined with DDP also up-regulated the expression of caspase-3 when NF-κB was inhibited and down-regulated in our research.In a word,SGC-7901 cells apoptosis is a complex process,and TMP combined with DDP treatment may induce cells apoptosis through inhibiting NF-κB activity and up-regulating the expression of bax and caspase-3 proteins.

Conclusion

All the results indicated that ligustrazine combined with DDP or treated alone could induce the apoptosis of SGC-7901 cell lines,and NF-κB maybe the possible way to induce the cell apoptosis.

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Responsible editor:Yong XU

Responsible proofreader:Xiaoyan WU

Supported by the Fund for Excellent Young Teachers by Education Department of Henan(2010GGJS-224).

*Corresponding author.E-mail:liyanli0921@163.com

Received:March 10,2015 Accepted:June 16,2015