Effect of Diallyl Trisulfide on Human Ovarian Cancer SKOV-3 / DDP Cell Apoptosis

Ovarian cancer is one of the most common malignant tumors of the female productive tract. Its mortality is the highest in gynecological tumors (Chauhan et al., 2009; Rouzier et al., 2010). At present, chemotherapy is one of the most important methods to treat ovarian cancer. However, drug resistance is a major reason to cause reoccurrence of ovarian cancer and treatment failure. Thus, it is necessary to seek anti-drug resistance, nontoxic and effective drugs to treat ovarian cancer. Diallyl trisulfide (DT) is the main bioactive compound in garlic. Some studies have revealed that, DT can induce the cell apoptosis of many tumors such as lung cancer, breast cancer, colon cancer and skin cancer (Fleischauer and Arab, 2001; Mao et al., 2010; Altonsy and Andrews, 2011; Jeong et al., 2011; Tsubura et al., 2011; Li et al., 2012). In this study, cisplatin (DDP)-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP) were treated with DT or combined DT and DDP. The effects of DT on apoptosis of SKOV-3/DDP cells and expressions of p53 upregulated modulator of apoptosis (PUMA), Bax and Bcl-2 were investigated. The role of PUMA in cell


Introduction
Ovarian cancer is one of the most common malignant tumors of the female productive tract.Its mortality is the highest in gynecological tumors (Chauhan et al., 2009;Rouzier et al., 2010).At present, chemotherapy is one of the most important methods to treat ovarian cancer.However, drug resistance is a major reason to cause reoccurrence of ovarian cancer and treatment failure.Thus, it is necessary to seek anti-drug resistance, nontoxic and effective drugs to treat ovarian cancer.Diallyl trisulfide (DT) is the main bioactive compound in garlic.Some studies have revealed that, DT can induce the cell apoptosis of many tumors such as lung cancer, breast cancer, colon cancer and skin cancer (Fleischauer and Arab, 2001;Mao et al., 2010;Altonsy and Andrews, 2011;Jeong et al., 2011;Tsubura et al., 2011;Li et al., 2012).In this study, cisplatin (DDP)-resistant human epithelial ovarian cancer SKOV-3 cells (SKOV-3/DDP) were treated with DT or combined DT and DDP.The effects of DT on apoptosis of SKOV-3/DDP cells and expressions of p53 upregulated modulator of apoptosis (PUMA), Bax and Bcl-2 were investigated.The role of PUMA in cell

Hoechst33342 fluorescent staining assay
Cultured cells were seeded into 6-well plates and treated with drugs for 48 h, and then 10 μg/ml Hoechst33342 (Shanghai Shize Biological Technology Co., Ltd., Shanghai, China) was added, followed by culture for 0.5 h.After the used medium containing Hoechst33342 was removed, plates were washed with PBS or medium in triplicate.Finally, cells were observed and photographed under fluorescence microscope.

Flow cytometry
The apoptosis rate of SKOV-3/DDP cells was detected using Annexin V-FITC and PI combined staining in flow cytometry, which could distinguish different stages of cell apoptosis.After treatment with trypsinase solution without EDTA, cells were washed with PBS twice and centrifugated (2000 rp/min) for 5 min, then cells were collected and put into different tubes (1-5×10 5 cells/tube)

RT-PCR
mRNA expressions of PUMA, Bax and Bcl-2 in SKOV-3/DDP cells were detected by RT-PCR method.Total RNA was extracted from peripheral blood mononuclear cells with TRIzol reagent according to manufacturer's protocol, followed by synthesis of cDNA and amplification.The amplified products were detected by 1.5% agarose gel electrophoresis.Compared with band of β-actin (Santa Cruz Inc., California, USA), relative gene expressions of each group were calculated by Launch SensiAnsys Image Acquisition and Analysis Software.Sequences of gene primers were shown in Table 1.

Western blot assay
Expressions of PUMA, Bax and Bcl-2 protein in SKOV-3/DDP cells were detected by western blot assay.The cells were collected and total protein was extracted and quantitated by Bradford method.The lysates (30 μg of protein for each well) were electrophoresed on SDS-PAGE gel for 4 h before blotting onto PVDF membranes.The blots were incubated with primary antibodies of PUMA, Bax and Bcl-2 (1: 200 dilution, ABcam Inc., Massachusetts, USA) overnight at 4 oC, followed by washing and incubation for 1.5 h with horseradish peroxidase-conjugated secondary antibody (1: 2000 dilution, Beijing ZSGB-BIO Company, Beijing, China).After washing, the membranes were developed in ECL system.Protein expression levels were determined by analyzing the signals captured on membranes using Image-Pro Plus6.0 analyzer.β-actin was used as internal control.

Specific PUMA siRNA experiment
According to cDNA sequences of PUMA, specific PUMA siRNA primers (5'-GGGUCCUGUACAAUCUCA UTT-3', 5'-AUGAGAUUGUACAGGACCCTT-3') were designed and synthesized by Shanghai GenePharma Co., Ltd., Shanghai, China.PUMA siRNA was transfected into SKOV-3/DDP cells using LipofectamineTM2000 (Invitrogen Corp., California, USA).The effects of DT on SKOV-3/DDP cell apoptosis and protein expression were detected.There were four groups to be performed, including control group (cells were cultured with noserum medium for 6 h, and then cultured with normal medium for 48 h), negative control (NC) group (the protocols were the same with control group, expect adding transfected siRNA products into medium), siRNA group (the protocols were the same with control group, expect adding transfected si-PUMA fragment I into medium) and siRNA+DT group (after transfection with si-PUMA fragments for 6 h, medium containing 20 µg/ml DT was used for culture for 48 h).

Statistical analysis
All results were presented as mean±SD.Statistical analysis was performed using SPSS 17.0 software.Oneway analysis of variance was used for comparison among different groups.P < 0.05 was considered as statistically significant.

Effect of DT on apoptosis of SKOV-3/DDP cells
Effect of DT on apoptosis of SKOV-3/DDP cells in different groups were shown in Figure 1A and B. After treatment with DT and DDP for 48 h, compared with control group, the apoptosis rates in three DT groups, DDP group and DDP+DT group were significantly increased (P < 0.05 or P < 0.01).Compared with DDP group and DT groups, the early apoptosis rate of DDP+DT group was obviously increased (P < 0.01).This indicated that, the treatment effect of combined DDP and DT was better than that of DT or DDP alone.

Effects of specific PUMA siRNA on SKOV-3/DDP cell apoptosis induced by DT
After knockdown of PUMA expression with specific siRNA, SKOV-3/DDP cells were treated with 20 μg/ ml DT for 48 h. Figure 2 showed that, compared with control group, both the apoptosis rates at early and late stages in si-PUMA group were significantly reduced (P < 0.05).Furthermore, compared with si-PUMA group, the apoptosis rate at late stage in si-PUMA+DT group was increased (P < 0.05).This result implied that, PUMA could promote the apoptosis of SKOV-3/DDP cells.

Changes of morphology
The morphology of SKOV-3/DDP cells was observed under fluorescence microscope.Figure 3 showed that, morphological changes of cells were obvious in DT groups after treatment for 48 h, presenting karyopyknosis, chromatin condensation and crescent shape around karyotheca.In 40 μg/ml DT group, with increase of DT dose, the nuclei were darker gradually, with obvious karyorrhexis.This indicated that, DT could induce SKOV-3/DDP cell apoptosis.However, there was not obvious morphological change in DDP group.In DT+DDP group, karyorrhexis was more obvious, and nuclei presented different sizes and irregular morphology.These results further demonstrated that, combined DT and DDP could significantly promote the apoptosis of SKOV-3/DDP cells.

Expressions of PUMA, Bax and Bcl-2 mRNA in SKOV-3/ DDP cells
Figure 4 showed that, compared with control group, mRNA expressions of PUMA and Bax were up-regulated in DDP group and DT group, with down-regulation of Bcl-2 mRNA expression (P < 0.05 or P < 0.01).
Moreover, compared with DDP group or DT group, the mRNA expressions of PUMA and Bax in DDP+DT group were increased, and the mRNA expression of Bcl-2 was decreased.This indicated that, DT could regulate the mRNA expressions of PUMA and Bax.Furthermore, combined DT and DDP had more obvious effects on genes expression.

Expressions of PUMA, Bax and Bcl-2 protein in SKOV-3/ DDP cells
As shown in Figure 5, compared with control group, the protein expressions of PUMA and Bax were upregulated and that of Bcl-2 was down-regulated in DDP group and DT group (P < 0.05 or P < 0.01).Compared with DDP group or DT group, the protein expressions of PUMA and Bax in DDP+DT group were up-regulated, and that of Bcl-2 was down-regulated.These results indicated that DT could regulate the protein expressions of PUMA and Bax, and combination of DT and DDP had a stronger effect.

Expressions of PUMA, Bax and Bcl-2 protein in SKOV-3/ DDP cells after knockdown of PUMA
Figure 6 showed that, the levels of PUMA and Bax protein in si-PUMA group were significantly lower than control group (P < 0.05), and that of Bcl-2 protein was significantly increased in si-PUMA group (P <0.05).The levels of PUMA and Bax protein in si-PUMA+DT group were significantly lower than control group (P < 0.05), but significantly higher than si-PUMA group (P < 0.05).Expressions of Bcl-2 protein in si-PUMA+DT group were down-regulated significantly (P < 0.05), compared with si-PUMA group (P < 0.05).Additionally, there was no significant difference of PUMA, Bax and Bcl-2 protein level between control group and NC group.This indicated that, knockdown of PUMA could lead to decrease of Bax protein level and increase of Bcl-2 protein level, which could be inhibited by treatment with DT.

Discussion
Chemotherapeutic drugs exert anti-cancer effect mainly through inducing cell apoptosis.There are some reasons to severely obstruct drugs clinical application, like tumor cells resistance to drugs and toxic side effect.Now, it's generally considered that classic pathways of cell apoptosis include mitochondrial pathway, endoplasmic reticulum pathway and death signal receptor pathway.Mitochondrial apoptotic pathway is a close correlation with Bcl-2 gene family (Xiao and Singh, 2006;Gong et al., 2012;Tu et al., 2012).Mechanisms of DT antitumor effect are related to antioxidation, inducing apoptosis and so on (Oommen et al., 2004;Kim et al., 2011;Na et al., 2012;Wang et al., 2012;Yu et al., 2012;Chandra-Kuntal et al., 2013;Yi and Su, 2013).Hassan (2004) has found that allicin could enhance the apoptotic effect of chemotherapeutic drugs through up-regulating the expression of Bcl-2 and Caspase-3 in human leukemia CD4 positive cells.Zhang et al. (2007) have reported that combined treatment of allicin with paclitaxel has a synergistic inhibiting effect on growth of gastric cancer cell lines, and induction of apoptosis through enhancing Bax expression and decreasing bcl-2 expression, interestingly, there was the same effect of allicin in prostatic cancer (Chen et al., 2012).Our results indicated that allicin inhibited the proliferation of SKOV-3/DDP cells via mediating the expressions of PUMA, Bax and Bcl-2, moreover, combined allicin with DDP had a synergistic inhibiting effect on proliferation of SKOV-3/DDP cells.Furthermore, we explored the molecular mechanism of tumor cell apoptosis induced by DT through mitochondria initiated apoptotic pathway.Results suggested that DT induced cell apoptosis through up-regulating the expression of PUMA, which could increase Bax expression and reduce Bcl-2 expression.
Many studies have revealed that PUMA played an important role in chemotherapeutic drugs by inducing tumor cells apoptosis (Wang et al., 2006;Jiang et al., 2006;Zhang et al., 2008), for example, treatment of DDP caused renal tubular epithelial cells apoptosis and necrosis, even renal injury, and meanwhile, there was an increase of PUMA expression (Jiang et al., 2006) in a time-dose dependence after treated with DDP.Liu and Wan also reported that allicin up-regulated the expression of PUMA to induce LoVo cells apoptosis (Liu and Wan, 2012).Once PUMA expression was inhibited, allicin had little effect on inducing cell apoptosis.Wang et al. (2008) found the same phenomenon in LoVo cells after treatment of green tea phlyphenols.Some studies demonstrated that after treatment of prostate cancer cells with wogonin, PUMA protein is increased, which release cytochrome C from mitochondria so as to activate caspases cascade reaction (Lee et al., 2008).We used RNAi technology to knockdown PUMA and find that apoptosis rate of SKOV-3/DDP cells was obviously decreased in si-PUMA transfection group, compared with treatment of DT after si-PUMA transfection.Moreover, si-PUMA significantly down-regulated the protein expression of PUMA and Bax more than control group, but up-regulated Bcl-2 expression more than control group.Compared with