Effect Of Sulforaphane On Proliferation And Apoptosis In Salivary Adenoid Cystic Carcinoma Cell Line ACC-M In Vitro

Objective: Salivary adenoid cystic carcinoma (SACC) is one of the mostcommon malignant tumors in human salivary glands ranking at the secondplace in oral and maxillofacial tumors. Highly invasive and easy to distantmetastasis is an important feature of adenoid cystic carcinoma. Even aftersurgical resection, local recurrence and distant metastasis often befall and themetastasis rate of SACC is in the first place of all oral and maxillofacialtumors. However, its mechanism of the tumor's incidence and metastasis isunclear now. It is very necessary to explore new therapies for SACC in orderto improve the curative effect, decrease local recurrence and distant metastasis,and improve the quality of life of the patients. Sulforaphane (SFN) is a kind ofmedicine, which is mainly isolated from various cruciferous vegetables. Andmore and more studies showed that Sulforaphane and its major activeingredients had the effect of anti-tumor. Nevertheless, whether the SFN caninhibit the proliferation and induce the apoptosis and necrosis of SACC hasnot been reported yet. The aim of this study is to observe the effect ofSulforaphane on proliferation and apoptosis in salivary adenoid cysticcarcinoma cell line ACC-M in vitro at different times and concentrations invitro and investigate the molecular mechanism for providing the experimentalbasis for the application of this new phytochemical in Multimodal treatment ofSalivary adenoid 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) was obtained from LKT lab(USA),purity is 98%(HPLC).2Methods:2.1Preparation of physic liquor: SFN was dissolved with DMSO mediuminto100μmol/L and Stored in4°C, which was diluted with RPMI1640beforeeach assay2.2Cell culture: ACC-M cells were cultured in RPMI1640mediumcontaining10%(v/v) fetal bovine serum, penicillin(100U/ml)and streptomycin(100μg/ml),and then incubated at37℃i n5%CO2gas incubator withsaturation humidity. After24~48hours,a new generation was formed and thecells were harvested with the mixed liquid containing0.25%trypsin and0.04%EDTA (1:1).2.3MTT assay:5×104/ml cell suspension in logarithmic phase wereseeded into96-well plates at100μl culture medium every well. After24hincubating,200μl SFN solution was added into each well, the finalconcentrations were5μM,10μM,20μM,30μM,40μM,60μM,80μM,100μM, respectively. And blank and control were set.Sextuplicate were donein each experimental group. After cultured for24,48and72h,20μl MTTsolution (5mg/ml in PBS) was added to each well. Then additional4hincubation was carried on, supernatant was sucked out and200μl DMSO wasadded to solubilize crystallize with concussion sufficiently. The absorbancewas measured with Microplate reader, the inhibition rate of growth wascalculated.2.4Trypan blue exclusion assay:5×104/ml cell suspension in logarithmicphase were seeded into6-well plates at2ml tissue culture medium every well.After24h incubating,3ml SFN solution was added into each well, the finalconcentrations were20μM,40μM, respectively. And negative control was set.After cultured for24,48and72h, all of the groups were stained with Trypanblue solution and the number of both stained cells(dead cells) and unstainedcells(the living cells)would be recorded for calculating.2.5Observation under the inversion phase contrast microscope: Cellswere seeded into50ml glass culture flask, the growth status of cells in control group and different concentration groups (5μM,10μM,20μM,30μM,40μM,60μM,80μM,100μM) were observed at different times(24,48,72h).2.6Observation under the light microscope: Cells were inoculated into6-well plate, and grown on coverslips placed in every well.After the cellsattached to the coverslips by overnight incubation the culture medium withand without SFN were added and cultured for different time (24,48,72h).Then after Giemsa staining, cells were observed under the microscope.2.7Observation under the transmission electronmicroscopy: Cells wereinoculated into50ml culture flask.After scheduled processing and preparationof electron microscope specimens,the growth status of cells in control groupand40μM SFN treatment group were observed at48h.Flow cytometric analyses (FCM) of apoptotic ACC-M: After cells wereTreated with and without SFN at different concentrations (0μM,20μM,40μMfor different time (24,48,72h). Cells were determined with double staining ofAnnexin-V-FITC/PI as analyzed by FCM.3Statistical analyses:Make summary of the obtained experimental dataVariance analysis, correlation analysis were performed by SPSS13.0software.Results:1MTT assay: SFN could markedly inhibit the growth of ACC-M atdifferent times (24h,48h and72h) respectively. The the maximum growthinhibition rate could reach81.05%. By statistics analysis, the inhibition rate ofgrowth is significantly different among groups at different times at the sameconcentration (P <0.01). The inhibitory effect of SFN on ACC-M is positivecorrelated with the concentration and time.2Trypan blue exclusion assay：The ratio of living cell in the treatedgroup showed a clear downward trend with the growth of drug concentrationand time And significant difference (P <0.01) between each time group andeach concentration group were found in statistical analysis.3Observation under the inversion phase contrast microscope: the cells ofcontrol groups grew quickly and attached in the wall like paving stones, mostof which were flat and polymorphic,full of cytoplasm,and the rounded nucleus was found at the center of cells. In the contrary, the growth of SFN-treatedcells got slower markedly.Also a few treated cells were found desquamatedfrom the glass wall to the medium, the shape of the cells became round andsmaller than the control and the nucleus were into shade while the cell's indexof refractive got enhancement.4Observation under the light microscope:Compared to the controlgroup, the proportion of nucleus to cytoplasm of the treated cells decreasedand multiple nucleoli were found in the cells of control group. The cytoplasmcondensed and the nucleolus reduced or disappeared with treated cells, thechromatins clustered under the nuclear membrane and nuclear fragmentationwas observed.5Observation under the transmission electronmicroscopy:The proportionof nucleus to cytoplasm of the treated cells decreased in comparison with thecontrol group, and there were multiple nucleoli in control group And theorganelles hyperthyroidism of the treated cells showed obvious mitotic figures.Nucleoli of treated cells, were reduce or even disappear And the phenomenoncalled chromosome set of edges, topical features of apoptotic cells, andapoptotic bodies were obviously observed6FCM assay: In the control cells,the rate of apoptosis was too low toshow statistically significance.But the apoptosis rate of the treated groupswere markedly observed,which was depending on the dose of the drug andprocessing time. The difference between groups was significant by statisticalanalysis (P <0.01).Conclusion: This study suggested that Sulforaphane could inhibit theproliferation of the salivary adenoid cystic carcinoma ACC-M cells and themain mechanism of this inhibition of proliferation is due to induction ofapoptosis, and inhibition was dependent association of the drug concentrationand processing time.