Effect Of Caspase-3,8,9on Apoptosis Induced By SFM 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 salivary glands, and ranks the second place inoral and maxillofacial tumors. Its characteristics are infiltrating growth andhigh metastasis rate. However, its pathogenesis is unclear now. It is verynecessary to explore new therapies for the tumor in order to improve thecurative effect and the patients' quality of life from starting at the point ofcontrolling cell cycle and inducing apoptosis. Caspase with the full name ofcysteinyl aspartate specific proteinase plays an important role in apoptosis.Sulforaphane(SFN) which is one kind of isothiocyanates extracting fromisothiocyanates has been focused on because of the best anti-tumor effect ontumortherapy. Recent studies indicated that caspases participated in apoptosisin tumor, ischemia reperfusion, inflammation, damage of tissue. Caspase is akey point on apoptosis. Recent research showed that SFN can inhibittumorigenesis and induce apoptosis in adenoid cystic carcinoma cell lineACC-M. Nevetheless, the effect of caspase-3,8,9on apoptosis in salivaryadenoid cystic carcinoma cell line ACC-M in vitro induced by SFN has notbeen reported yet. In present study, we detect the activities of caspase-3,8,9byspectrophotometry at different times in salivary adenoid cystic carcinoma cellline ACC-M in vitro induced by40μM SFN and the apoptosis in the cellspretreated by50μM Caspase Inhibitor Z-VAD-FMK by flow cytometric toexplore the possible mechanism and to provide theoretical basis for clinicalapplication in treating SACC.Methods:1Materials(1) Cell line ACC-M was purchased from the laboratory of oral and maxillofacial surgery of Shanghai Jiao Tong University,which was established in 1995;(2) Caspase-3,8,9activity assay kit and Caspase Inhibitor Z-VAD-FMK werepurchased from Beyotime Institute of Biotechnology；(3) SFN was purchased from LKT Laboratory (USA), its purity≥98%.2Methods2.1Preparation of reagent:(1) SFN was dissolved with DMSO to100μM liquid, stored under-20℃anddiluted with RPMI1640before used.(2) Caspase inhibitor Z-VAD-FMK was dissolved with4mL DMSO anddivided evenly into eppendoff tubes.2.2Cell culture: ACC-M cells were cultured in RPMI1640mediumcontaining10%(v/v) fetal calf serum, streptomycin (100μg/mL) andpenicillin (100U/mL), then incubated at37℃in5%CO2incubator withsaturation humidity. The cells were harvested with the mixed liquid containing0.25%trypsin and0.04%EDTA (1:1) when cells grown to about80%confluence in the flask.2.3Analyses the activities of caspase-3,8,9with spectrophotometry: To madethe standard curve of A405with the standard substance of pNA. Cellsuspensions in logarithmic phase were seeded into96well plates in theconcentration of2.0×105/mL. After24h incubating, media containing40μMSFN was added into each well. The cells were collected and washed aftercultured for0,4,8,6,24h. After centrifugalization, discard, the supernatantliquid were absorbed carefully, the lysis buffer was added into each wellaccording to20×105cells with100μL lysis buffer. And then the cells wereprecipitated and resuspended, and cracked in low temperature. The samplewas collected by centrifuging.10μL sample,80μL buffer solution, and10μLAc-DEVD-pNA(or Ac-IETD-pNA or Ac-LEHD-pNA)were added into96well plates with blank control. The absorbances were measured on ELIASA.According to the standard curve of A405, the quantities of pNA werecalculated, and the activities of caspase-3,8,9were described by graphics.2.4Detect the apoptosis of ACC-M with Flow cytometric analyses (FCM): Cells were divided into three groups. Caspase Inhibitor Z-VAD-FMK wasadded in the group of SFN+Caspase inhibitor2h before treatment at a finalconcentration of50μM. And then cells were incubated with0.04%DMSO,40μM SFN, or40μM SFN+50μM Caspase inhibitor for24h. The apoptosis ofthe cells was detected with Annexin-V-FITC and PI double labeling by flowcytometry.3Statistical analyses:The values of absorbance, activities of caspase-3,8,9and apoptosis rates wereanalyzed with One-way ANOVA by SPSS13.0sofeware.Results:1Analyses the activities of caspase-3with Spectrophotometry：The activitiesof caspase-3on apoptosis induced by SFN in salivary adenoid cysticcarcinoma cell line ACC-M could obviously increase and reach the peak valueat16h. By statistics analysis, there was significantly different between groupsat different time and blank control (0h vs4h: P<0.01;0h vs8h: P<0.01;0h vs16h: P<0.001;0h vs24h: P<0.01). There was no significantly different among4,8and24h by multiple comparison (P>0.05). There was significantlydifferent between16h and4,8,24h (P<0.05).2Analyses the activities of caspase-8with Spectrophotometry：The activitiesof caspase-8on apoptosis induced by SFN in salivary adenoid cysticcarcinoma cell line ACC-M could obviously increase and reach the peak valueat16h. By statistics analysis, there was significantly different between groupsat different time and black control (0h vs4h: P<0.05;0h vs8h: P<0.01;0h vs16h: P<0.001;0h vs24h: P<0.01. There was no significantly different among4,8and24h by multiple comparison (P>0.05). There was significantlydifferent between16h and4,8,24h (P<0.001).3Analyses the activities of caspase-9with Spectrophotometry：The activitiesof caspase-9on apoptosis induced by SFN in salivary adenoid cysticcarcinoma cell line ACC-M could obviously increase and reach the peak valueat16h. By statistics analysis, there was significantly different between groupsat different time and black control (0h vs4h: P<0.05;0h vs8h: P<0.001;0h vs 16h: P<0.001;0h vs24h: P<0.001). There was significantly different amongeach groups by multiple comparison (4h vs8h: P<0.001;4h vs16h: P<0.001;4h vs24h: P<0.001;8h vs16h: P<0.001;8h vs24h: P<0.001;16h vs24h:P<0.05).4FCM assay: Although apoptosis could be found in the DMSO andCaspase+Inhibitor cells, the rates of apoptosis were very low. The apoptosiswere obviously observed in the cells induced by SFN. There was significantlydifferent between SFN and Caspase+Inhibitor (P<0.01). There was nosignificantly different between DMSO and Caspase+Inhibitor by statisticalanalysis (P＞0.05).Conclusion:1This study suggested that the activities of caspase-3,8,9on apoptosisinduced by SFN in salivary adenoid cystic carcinoma cell line ACC-Mobviously increased from4h to24h and reach the peak value at16h.2The apoptosis in salivary adenoid cystic carcinoma cell line ACC-M added50μM Caspase inhibitor Z-VAD-FMK for2h before SFN was significantlyinhibited. These results identified caspase-3,8,9played a role in apoptosisinduced by SFN in salivary adenoid cystic carcinoma cell line ACC-M.