The Effect Of Bcl-2Family In Apoptosis Of Salivary Adenoid Cystic Carcinoma Cell Line ACC-M Induced By Sulforaphane

Objective: Salivary adenoid cystic carcinoma(SACC) is one of the mostcommon salivary gland malignancy.It often occurings in the minor salivaryglands of the palate and the parotid gland, then the followed by submandibulargland posteriorly. This tumor has the important characteristics of highlyinvasive growth, infiltrating along facial nerve and high metastasis rate. Itsincidence ranks the second place of tumor in oral and maxillofacialmalignancies. Currently, the clinical treatment of adenoid cystic carcinoma issurgical excision. Local recurrence and even distant metastasis often occursafter surgery, and the metastasis rate of SACC is the highest among all oraland maxillofacial tumors. However, its mechanism of Morbidity andmetastasis is unclear now, it is very necessary to explore new therapies forSACC in order to improve the curative effect, decrease local recurrence anddistant metastasis, and improve the patients’ quality of life. Epidemiologicalstudies have suggested that increased dietary consumption of cruciferousvegetables may be protective against the risk for certain types of malignanciesAntineoplastic effects of cruciferous vegetables are credited to isothiocyanates(ITCs). Sulforaphane (SFN) attracted widespread attention because of its bestanti-tumor effects. Currently, with the continuous progress of the anti-tumoreffect of sulforaphane, the study of its anticancer mechanism is from thecellular level up to the molecular level.For example,in DU-145cells (prostatecancer), SFN induced cell apoptosis is associated with the increasing cleavageof caspase-3and reduced expression of Bcl-2. It can also cause the cleavage ofcaspase-3in pancreatic cancer cells.SFN can activate a variety of molecularmechanisms to induce cell apoptosis. Our previous study showed, SFN could inhibit the proliferation and induce apoptosis in ACC-M. And Caspases playimportant roles in the SFN-Induced apoptosis of ACC-M. However,in theprocess of SFN-Induced human adenoid cystic carcinoma ACC-M cellapoptosis, it has not been reported that whether Bcl-2family members play animportant role.The aim of present study is to investigate the molecularmechanism of cell apoptosis induced by Anti-apoptotic proteins Bcl-2,Bcl-xland Pro-apoptotic proteins Bax, Bak in human salivary adenoid cysticcarcinoma cell line ACC-M in vitro, which is affected by Sulforaphane atdifferent times, so as to providing the experimental evidence for theapplication of this new phytochemical in multimodal treatment of salivaryadenoid cystic carcinoma.Methods:1Materials:1.1Cell line ACC-M was purchased from the laboratory of oral andmaxillofacial surgery of Shanghai Jiao Tong University， which wasestablished in1995;1.2Sulforaphane(SFN) Provided by the Head and Neck Surgery ofPittsburgh University Medical Center,obtained from LKT lab(USA),purity is99%(HPLC).2Methods:2.1Preparation of physic liquor: SFN was dissolved with DMSO medium into100mmol/L and Stored in4℃which was diluted with RPMI1640before theassay.2.2Cell culture: Recover the cryopreserved ACC-M cells.Cell lines werecultured in RPMI1640medium containing10%(v/v) fetal bovine serum,penicillin(100U/ml)and streptomycin (100μg/ml),and then incubated at37℃in5%CO2gas incubator with saturation humidity. The medium waschanged the next day and About72-96hours passaged.2.3Cell seeding and Drug administration: when a new generation was formedand the cells in logarithmic phase were harvested with the mixed liquidcontaining0.25%trypsin and0.04%EDTA (1:1) to prepare a4.0×106/ml of cell suspension. According to the corresponding point in time,the cellsuspension was respectively seeded in five culture flasks which were markedas the groups of control,4hours,8hours,16hours and24hours. Everyculture flask contains2ml cell suspension. Drug treated cells after the cellsattached to the culture flasks by24hours incubation. Remove the adherentcells from the incubator, and discard supernatants, add a concentration of40μmol/L sulforaphane media3ml to four experimental groups. AddRPMI1640medium containing the same concentration of dimethylsulfoxide3ml to the control group.Then extract tissue protein after4,8,16, and24hours respectively.2.4Tissue protein extraction: Washed the control group and the experimentalgroup cells twice with PBS balanced salt solution,harvested the cells with themixed liquid containing0.25%trypsin and0.04%EDTA (1:1) to prepare cellsuspension, then transfer it into the corresponding EP tubes.4℃,3000rmp,centrifuged for15min. Supernatant was sucked away, leavingprecipitation,PBS washed again, then800rmp, centrifuged for5min,suckedaway the supernatant. In each of the control and experimental groups wereadded to cell lysates400μl, then placed them on ice to cleavage for about20-30min after shaked evenly. The lysis of the cells at4℃,13000rmp,centrifuged for15min, the supernatant is extracted cell protein.Stored theprotein at-80℃.2.5Protein quantification: determination of protein concentration byspectrophotometer. The principle of spectrophotometry is determined bymeasuring the test substance in the degree of light absorption in a specificwavelength or a certain wavelength range, which qualitative and quantitativeanalysis of the substance.This experiment measured absorbance values of eachsample at a wavelength of595nm. According to the formula (the concentrationof protein=absorbance value of measured protein/absorbance value ofstandard protein×the concentration of standard protein(0.563g/l)×10to derivethe concentration of sample protein.2.6Detect the expression of Bcl-2, Bax, Bcl-xl and Bak with Western blot： Through the process of SDS-PAGE (electrophoresis)；transmembrane；close；incubation of primary antibody, secondary antibody；washing；darkroomdeveloping, fixing etc.to obtain protein bands films, subsequently, with theelectrophoresis gel image analysis software to analysis the resultsquantificationally.3Statistical analyses:The above experiment was repeated three times. Make summary of theobtained experimental data variance analysis, correlation analysis wereperformed by SPSS13.0software.Results:By western blot analysis，we found that with the time extensionof SFN action the level of Bax and Bak proteins was up-regulated，whereas，the level of Bcl-2and Bcl-xl proteins was down-regulated．The ratio of Bax:Bcl-2increases. Treated with40μmol/L SFN for24hours, the expression levelof Bax protein was1.63folds of the control group (P<0.01), the expressionlevel of Bak protein was increased by44%in the control group(P<0.01), butthe expression level of Bcl-2and Bcl-xl proteins was reduced to43%and41%(P<0.01) of the control group, respectively. The ratio of Bax: Bcl-2increases. Compared with the control group, the groups of8hours,16hoursand24hours showed statistically significant comparing with control(P<0.01).Conclusion:This study suggested that the mechanism ofsulforaphane-induced salivary adenoid cystic carcinoma cell line ACC-Mapoptosis is likely related to the down-regulation of Bcl-2,Bcl-xl proteins andup-regulation of Bax,Bak proteins.