Diallyl Trisulfide And Simvastatin Induce Apoptosis And Inhibits Proliferation Of A549Cells

Lung cancer has been the leading cause of cancer-related mortality all over the world. It happens not only in China, where a peak is still expected in its incidence, but also around the world for decades. Unfortunately, the majority of lung cancer patients are not diagnosed until the disease has progressed to an advanced stage. Squamous-cell carcinoma (SCC), adenocarcinoma, small cell carcinoma and large cell carcinoma are the four most common histological subtypes of lung cancer. The incidence rates for three major types of female lung cancer (SCC, adenocarcinoma and small cell carcinoma) have increased, with an especially rapid increase in adenocarcinoma, which is now the predominant type in women while SCC prevails in men. This is probably due to changes in smoking habits and tobacco products (e.g. lower tar contents and use of a filter or inhalation). In recent years, pulmonary adenocarcinoma, surpassing squamous cell carcinoma in frequency, has been the predominant form of lung cancer among males in many countries. As women have an increased susceptibility to adenocarcinoma, more studies are needed to throw light upon the molecular mechanism and eventually find the ideal therapeutic method.Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate for many cancers. A number of epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate for malignant tumor of digestive tract, especially the mortality of gastric cancer in China. A wealth of laboratory evidence for possible lung cancer-preventive biological mechanisms of a series of garlic derived compounds (also known as:organsulfer compounds OSC), including water-soluble sulfur compounds such as S-allylcysteine (SAC) and S-allylmercaptocysteine (SMAC) and oil-soluble sulfur compounds, namely diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS) and ajoene, has been published. Diallyl trisulfide (DATS) is one of garlic derived compounds (also known as:organsulfer compounds OSC). DATS could induce apoptosis and inhibit the growth of many cancer cells lines. Although, whether biochemistry or toxicology of individual allyl sulfides are different in the number of sulfur atoms in molecule have not been sufficiently concluded yet, DATS, cantaining sulfane sulfur in its structure, has stronger biological activity and greater toxicity than SAC, SMAC and DAS, DADS. Increasing evidence suggests that DATS shows a potential ability to inhibit proliferation and induce apoptosis of hepatocellular carcinoma and colon, prostate cancer. It has be shown that DATS induced apoptosis by inducing proapoptotic proteins and decreasing the expression of antipoptotic proteins in human lung cancer cell lines H358and H460, without knockdown of Bax and Bak proteins, but not in normal human bronchial epithelial cell line BEAS-2B. However, the exact pathway by which DATS induces apoptosis and inhibits the growth of lung cancer cells lines while the normal cells can survival is still not clear and conflicting results came out occasionally even with same cell line. Moreover, systematical research data of DATS both in vitro and in vivo are relatively rare. Above all these correlative studies were followed up by only one intervention trial about gastric cancer which examined the chemopreventive effects of DATS and selenium in Chinese population of natural villages. As the number of females in this trial was too small, more studies are needed. Lower cytotoxicity in nonneoplastic cells compared to cancer cells is prerequisite for any chemopreventive agent. All the efforts of our research to make clear the molecular events contribute to DATS-induced apoptosis of lung cancer cell and how nonneoplastic cells evade apoptotic death is to validate the feasibility of DATS being an ideal cancer chemopreventive agent. Our study demonstrated that apoptotic incidents induced by DATS were mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic bcl-2that result in up-regulation of the ratio of Bax/Bcl-2and the activity of caspase-3,-8. And DATS induced the apoptosis and inhibited the proliferation in a concentration-and time-dependent manner. Furthermore, by establishing an animal model of female BALB/c nude mice with A549xenografts, we found that oral gavage of DATS significantly retarded growth of A549xenografts in nude mice without causing weight loss or any other side effects compared with the control group. All the evidences above both in vitro and in vivo suggested that DATS could be an ideal anticancer drug.Statins, as hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are a class of drugs that potently decrease the plasma cholesterol levels. Statins have a well-established role in reducing cardiovascular morbidity and mortality, and has seen widespread use for primary and secondary prevention of heart disease. In addition to their lipid-lowering property, statins exhibit anticancer effect in vitro and in vivo models. Statins mediated proteasome activity, which were related to cell cycle arrest and apoptosis. Recently, it has been reported that statins altered Akt signaling and has a role inreduced proliferation rate in vitro. In addition, it has been reported that statins enhance the sensitiveness of a variety of tumor cells to conventional chemotherapeutic drugs. Indeed, statins have been found to have anticancer properties in many tumor cell types, such as colorectal and prostate. However, to date, use of statins against cancer development remains controversial. Some reports showed that statins did not decrease the incidence of cancer in experimental models, and even conflicting results occasionally came out in same cell line. To our best knowledge, few associations between lung cancer and statins have been detected. Moreover, the molecular mechanisms underlying the anticancer effects of statins are still unclear. So, further studies about anticancer and cancer chemopreventive effects of statins on lung cancer are needed. In this study, we hypothesized that statins therapy may reduce the progress of lung cancer by inhibiting cell proliferation, influencing the cell cycle, down-regulating cyclin D1and CDKs protein expression, inducing apoptosis and decreasing MMP-9levels. Experiments in vitro were designed using A549cells and performed to test this hypothesis. We hope to make clear wheather simvastatin could be an ideal anticancer drug by the second part of our study.This study was divided into two parts: PART IDiallyl trisulfide induces apoptosis and inhibits proliferation of A549cells in vitro and in vivoAimsTo observe apoptotic incidents induced by DATS were mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic bcl-2that result in up-regulation of the ratio of Bax/Bcl-2and the activity of caspase-3,-8. And DATS induced the apoptosis and inhibited the proliferation in a concentration-and time-dependent manner. By establishing an animal model of female BALB/c nude mice with A549xenografts, we found that oral gavage of DATS significantly retarded growth of A549xenografts in nude mice without causing weight loss or any other side effects compared with the control group. Try to validate the feasibility of DATS being an ideal cancer chemopreventive agent.Methods1.Cell cultureHuman lung cancer cell line (A549, SIBCB, China) were cultured in RPMI1640containing10%FBS,1%penicillin-streptomycin at37℃in5%CO2,95%air humidified cell culture incubator.2.MTT assayMTT assay were carried to determined the cytotoxicity of to A549cells. The cells were plated into96-well (5×105cells/ml) and treated at three concentrations (25μm,50μm,100μm) of DATS (Sigma, USA) for1h. Then the mediums of per well were replaced and incubated for48h in new RPMI1640medium. All cells were determined by MTT assayas previous described. Every group had5wells and average values were calculated.3. RNA isolation and Real time PCR for caspase-3and-8The cells among groups was harvested and total RNA was extracted using TRIzol Kit. Real time PCR were performed for caspase-3and-8. The primer for GAPDH, caspase-3and-8as follows: GAPDH, forward,5’ AGGTCGGTGTGAACGGATTTG-3’, reverse,5’TGTAGACCATGTAGTT GAGGTCA-3’; caspase-3, forward,5’-TGCATACTCCACAGCACCTGGTTA-3’, reverse,5’-CATGGCACAAAGCGACTGGATGAA-3’; caspase-8, forward:5’-TTTCACTGTGTTAGCCAGGGTGGTA-3’, reverse,5’-CCTGTAATCCCAGC A CTTTGGGAG-3’. The experiments were repeated five times and GAPDH was used as a reference transcript.4. Caspase-3and-8activityCaspase-3and-8activity in cells among the groups were assayed using colorimetric assay kits (Beyotime Institute of Biotechnology, China) as previous described. A549were cultured alone or100μm DATS for72h and the activities of Caspase-3and-8were deternmined. The experiment was performed in triplicate.5. Western blot analysisA549were cultured alone or with DATS and the protein was extracted from all groups of cells and applied to a12%SDS-polyacrylamide gel. Then the proteins were transferred on a PVDF membrane. After a block in5%not-fat milk for1hour, the membrane was inbucated with primary antibodies overnight, including mouse anti-BcI-2Ab (1:500, Abeam, USA), rabbit anti-Bax antibody (1:1000, Abeam, USA). Followed a incubation with a horseradish peroxidase-conjugated antibody and washing three times with TBST, the immunoactive proteins were detected with chemiluminescence.6. Animals experimentsForty female BALB/c nude mice (Six-week-old) were obtained from the Institute of Zoology, Chinese Academy of Sciences. The mice were housed under specific pathogen-free conditions and food and water ad libitum throughout the experimental periods. Animal protocols according to the permission and rules of the animal ethics committee of Shandong University.A549cells were used to established the mouse tumor models. Briefly, a single cell suspension of A549cells was prepared. Then, the mice were injected subcutaneously with A549cells (5×106/mouse) which suspended in100μl PBS. At the same time, the mice were divided into two groups randomly. One group received by oral with6μm DATS in100μl PBS every other day (DATS group, n=20); The other group was provided100μl PBS (control group, n=20). The general state of the animals were observed. At30days, the mice were killed and the incidence and volume of tumor were assayed.7. Statistical Analysis:The datas were shown as mean±D. Statistical significance was established by One-way ANOVA and t-test. Statistical analyses were carried out using SPSS13.0software. P<0.05was considered significant.Result1.DATS treatment inhibited the survival of A549cells in vitroWe assayed the effect of DATS on cancer cells survival by MTT and we found that A549cells were significantly inhibited after incubation with DATS.(P<0.05). Furthermore, this effect on tumor cells of DATS was dose-dependently.(Figure1)2. DATS treatment increased the expression caspase-3and-8in vitroThe mRNA expression of caspase-3and-8was marked increased in DATS-treated A549cells than those in control group (P<0.05). Also, we assayed the actiivity of caspase-3and-8in vitro. After incubation with100μm DATS for48h, the actiivity of caspase-3and-8significantly enhanced in comparision with control group (Figure2, P<0.05).3. DATS treatment induced the expression of Bcl-2in vitroBcl-2played a central role in apoptosis, proliferation and invasion of tumor cells. In this study,we examined the protein expression of Bcl-2to explored the inherent mechanism of DATS on A549cells. The result showed that DATS treatment lead to a marked downregulation of Bcl-2protein expression in A549cells (P<0.05).As shown in figure3, we established3concentration and time in this experiment, respectively. We found that100μm DATS achieved the larger effect compared other2groups (P<0.05), so it documented that the effect of DATS on A549cells was dose-dependently. Compared to control group and24h group, Bcl-2expression was significant decreased at48h than that in24h group (P<0.05). However, the expression of Bcl-2did not further decreased at72h (P>0.05).4. DATS treatment inhibited the growth of lung cancer cells in vivoWe assessed the antitumor effect of DATS used BALB/c nude mice models. As shown in figure4, a single injection of DATS induced a marked reduction in the incidence and volume of tumor in the mice conpared with those of in control group (P <0.05).ConclusionOur study demonstrated that apoptotic incidents induced by DATS were mitochondria-dependent caspase cascade through a significant decrease of the anti-apoptotic bcl-2that result in up-regulation of the ratio of Bax/Bcl-2and the activity of caspase-3,-8. And DATS induced the apoptosis and inhibited the proliferation in a concentration-and time-dependent manner. Furthermore, by establishing an animal model of female BALB/c nude mice with A549xenografts, we found that oral gavage of DATS significantly retarded growth of A549xenografts in nude mice without causing weight loss or any other side effects compared with the control group. All the evidences above both in vitro and in vivo suggested that DATS could be an ideal anticancer drug. PART ⅡSimvastatin induces apoptosis and inhibits proliferation of A549cellsAimsLung cancer is the one of the most frequent causes of malignant tumors. In recent years, it has been documented that statins have anticancer and cancer chemopreventive properties. However, the mechanism of simvastatin on lung cancer is still unclear. Methods1. Cell cultureThe human lung cancer cell line A549(SIBCB, China) was grown in RPMI1640supplemented with10%FBS,1%penicillin/streptomycin at37℃in a5%CO2,95%air humidified cell culture incubator.2. MTT assayMTT assay was carried out to determine the effect of simvastatin on the survival of A549cells. The cells were seeded into96-well plates (5×104cells/well in200μl) and incubated overnight. Then, the cells were treated with or without simvastatin (Sigma, USA) at different concentrations (1μm,10μm, and50μm) for48h. After incubation, MTT (Amresco,Solon, OH, USA) solution was added to each well to determine the cytotoxicity to A549cells. The cells in each well were examined by MTT assay as previously described. Every group had5wells and the average values were calculated.3. Cell cycle analysisThe cells in each well were incubated alone (control group) or with50μm simvastatin (simvastatin group) for48h. Then cell cycle analysis was performed following the methods as previously described.4. RNA isolation and Real time PCRThe cells in all groups were harvested. Then, the total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and cDNA was prepared (Invitrogen). The gene levels were quantified using the SYBR Premix Ex Tag quantitative PCR kit (Takara, Ossu, Japan) according to the manufacturer’s instructions. Real time PCR were performed and the primer sequences for caspase-3, caspase-8, caspase-9, Xiap, and GAPDH, were as follows:caspase-3, forward:5’-TGCATACTCCACAGCACCT GGTTA-3’, reverse,5’-CATGGCACAAAGCGACTGGATGAA-3’; caspase-8, forward:5’-TTTCACTGTGTTAGCCAGGGTGGTA-3’, reverse,5’-CCTGTAATCC CAGCACTTTGGGAG-3’; caspase-9, forward:5’-GCTTCGTTTCTGCGAACTAAC A-3’, reverse,5’-GTTGGCTTCGACAACTTTGCT-3’; Xiap, forward,5’-CACTTGA GGTTCTGGTTGCAG-3’, reverse,5’-TGCAAAGCTT CTCCTCTTGC-3’; GAPDH, forward,5’AGGTCGGTGTGAACGGATTTG-3’, reverse,5’TGTAGACCATGTA GTTGAGGTCA-3’. GAPDH expression was used as a reference transcript. The samples were repeated five times and the relative expression of the target gene to GAPDH was analyzed.5. Western blot analysisAfter the incubation of A549cells with or without simvastatin, the cells in all groups were washed with PBS and lysed, and then the total protein was extracted and the protein concentration of each sample was measured using the BCA protein assay. In brief, the samples were subjected to a10%acrylamide gel by sodium dodecul sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and thereafter transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated in5%fat-skimmed milk for2hours at room temperature, followed by the primary antibodies overnight, including mouse Bcl-2antibody (1:500, Abeam, USA), rabbit Bax antibody (1:1000, Abeam), rabbit Cyclin D1antibody (1:2000, Abeam), rabbit cyclin-dependent kinase4antibody (CDK4,1:1000, Abeam), rabbit caspase-3antibody (1:2500, Abeam), rabbit caspase-8antibody (1:500, Abeam), rabbit caspase-9antibody (1:1000, Abeam), rabbit X-linked inhibitor of the apoptosis protein antibody (Xiap,1:1000, Abeam), rabbit matrix metalloproteinase-9antibody (MMP-9,1:1000, Abcam),rabbit nuclear factor κB antibody (NF-κB p65,1:1000, Cell Signaling, USA) and rabbit β actin antibody (1:1000, Cell Signaling). After incubation with a horseradish peroxidase-conjugated secondary antibody for2h at room temperature, the membrane was washed with TBST three times and the immunoactive proteins were detected by chemiluminescence. The levels of β-actin protein were used to verify equal loading of samples in each lane.6. Statistical Analysis:All data were presented as mean±SD. One-way ANOVA and t-test were performed to determine the the differences between control and treatment groups. Statistical analysis was carried out using SPSS version13.0(SPSS Inc., Chicago, IL). A P-value<0.05was considered significant..Results1. Simvastatin treatment inhibited survival of A549cellsTo evaluate whether simvastatin affects the survival of A549cells, the MTT assay was performed. A549cells were incubated with various concentration of simvastatin (1μM,10μM, and50uM) for48h. Compared to the control group, cell survival was significantly reduced in10u M and50u M simvastatin groups (P<0.05). Though1μM simvastatin also slightly reduced the value of cell survival, the difference was not significant (P>0.05). Simvastatin treatment inhibited the survival of A549cells in a dose-dependent manner.2. Simvastatin treatment down-regulated Bcl-2expression and up-regulated Bax expression in A549cellsBcl-2and the Bax family play an important role in regulation of apoptosis, proliferation and invasion of tumor cells. In this study, the protein expression of Bcl-2and Bax were determined by Western blot. It is noteworthy, in order to assess the time-and dose-response, A549cells were cultured with different concentrations of simvastatin for different time. For time-response, simvastatin achieved the largest effect in48h group (P<0.05). In addition,50μm simvastatin significant down-regulated Bcl-2protein expression and up-regulated Bax protein expression in A549cells compared other groups (P<0.05). The result showed that the effect of simvastatin on A549cells was time-and dose-dependently. So,50u m simvastatin for48h was applied for further experiment.3. The effects of simvastatin on cell cycle regulationWe assayed the cell cycle distribution of tumor cells to explore the mechanism of statins induced growth inhibition. In comparison with the control group, the percentage of G1phase cells was markedly increased after simvastatin treatment (P<0.05). These data showed that simvastatin blocked cells in the G1phase of the cell cycle, and this may be a mechanism of its antitumor effect. In addition, we examined the cell cycle checkpoint proteins, Cyclin Dl and CDK4, which are associated with distributional change. It was found that cyclin Dl and CDKs protein expression markedly decreased after pretreatment with simvastatin for48h in a dose-dependent manner (P<0.05).4. Simvastatin induced the apoptosis in A549cellsTo determine whether simvastatin induced the apoptosis in A549cells, several assays were performed. After the cells were cultured with50μm simvastatin for48h, caspase-3,-8and-9mRNA and protein expression were assayed to evaluate the effect of caspase in simvastatin-induced apoptosis. Notably, caspase-3, caspase-8and caspase-9mRNA and protein expressions were more significantly increased in simvastatin-treated A549cells than those in the control group (P<0.05). These results showed that simvastatin activate the caspase cascade reaction, and thereby plays a central role in apoptosis in A549cells.Xiap play a central role in regulation of apoptosis of tumor cells. In this study, we measured the mRNA and protein expression of Xiap in A549cells and found that simvastatin significant down-regulated Xiap the expression of mRNA and protein (P <0.05). So, the result showed that simvastatin induced the apoptosis of A549cells.5. Simvastatin suppressed MMP-9expression and NF-κB activation in A549cellsMMP-9protein expression were assayed by western blot, and we found that simvastatin treatment decreased the expression of MMP-9protein in a dose-dependent manner (P<0.05).p65is a major component of NF-κB, and the levels of NF-κB p65was examined. After coculture with50u M simvastatin for48h, the levels of NF-κB p65was significantly suppressed in simvastatin group (P<0.05). The results showed that simvastatin suppressed MMP-9expression and NF-κB activation in A549cellsConclusionStatins contribute to lung cancer therapy and may be an ideal anticancer and cancer chemopreventive agent for lung cancer.