The Effect Of The Inhibitor Of Apoptosis Protein Family In The Growth Of Human Salivary Gland Adenoid Cystic Carcinoma ACC-M Xenografts Inhibited By Sulforaphane In Vivo

Background:Salivary adenoid cystic carcinoma?SACC?,is a kind of malignant tumor,mainly derived from salivary gland epithelium,charactered by perineural vascular growth,local invasive and blood metastasis.The local recurrence rate was high and the distant metastasis was high too after surgical resection.The rate of distant metastasis of SACC takes the first rank in oral and maxillofacial region.The mechanism of onset and metastasis of SACC is not very clear yet,that we need to seek new treatment methods to improve the curative effect and reduce the rate of local recurrence and distant metastasis.Sulforaphane?SFN?,a natural-occurring isothiocyanate?ITCS?from cruciferous vegetables,is an effective cancer chemoprotective agent in cell culture and animal studies.Our previous data showed SFN inhibited proliferation of cultured ACC-M cells by inducing apoptosis in vitra.SFN-induced apoptosis was associated with activation of caspases-3,-9 and-8 and reversed by Caspase inhibitor Z-VAD-FMK.Moreover,modulation of Bcl-2 family was involved in SFN-induced apoptosis.Oral administration of SFN can effectively reduce the volume and weight of ACC-M xenografts in nude mice,but not cause body weight loss.Growth inhibition of nude race xenografts was due to SFN-induced apoptosis.Inhibitor of apoptosis protein?IAP?is a family which can inhibit Caspase,regulate the cell cycle and cell differenation,etc.Members of IAP family include c-IAP1,c-IAP2,Survivin,Livin,X-IAP,NAIP,Bruce and ILP-2.c-IAP1,c-IAP2,X-IAP can directly inhibit the Caspase 3,7,9,and Survivin can not only inhibit Caspase 3,7,but also enhance X-IAP and other members of the family of IAP antiapoptotic effect.Livin can inhibit the Caspase activity-3,7,9 too.ILP leads to increase in the expression of Bcl-2,to down-regulate the activation of Caspase-9,thus inhibiting apoptosis.Research on NAIP mainly involve in the nervous system.Bruce inhibits the activity of Caspase-9 and Smac.However,it is not clear whether IAP family members played a role in inhibiting ACC-M proliferation by SFN.Objective:In the present study,we subcutaneously vaccinated salivary gland adenoid cystic cancer cells ACC-M into nude mice to establish nude race xenografts model,and observe the expression of IAP family in xenografts after oral administration of SFN in vivo,in order to explore the possible molecular mechanism and provide theoretical evidence for the clinical application of SFN.Methods: 1 Materials 1.1 Human salivary adenoid cystic carcinoma cell line?ACC-M?It was purchased from Shanghai Jiaotong University,laboratory of maxillofacial surgery.The cell line was established in 1995.1.2 Sulforaphane?SFN?Sulforaphane?purity is no less than 99%?was provided from LKT laboratories?USA?.1.3 Nude mouseThe nude mice were purchased from Military Medical Sciences Academy of PLA,Beijing,10 males,aged 4⁶ weeks,weight 16²0 grams.1.4 AntibodyX-IAP polyclonal antibody,c-IAP2 polyclonal antibody,c-IAP1 polyclonal antibody,Livin polyclonal antibody are provided from the British Abcam company.Survivin polyclonal antibody is provided from American AR company.Rabbit IgG polymer was purchased from dongyuan biological technology company.2 Method 2.1 Culture of cellsWe recovered the frozen human salivary adenoid cystic carcinoma cells?ACC-M?and cultured in the constant temperature incubator under 37?,5% CO₂.When the cell density reaches a certain concentration,we harvested the cells.2.2 Establish xenografts modelThe prepared cell suspension were injected into nude mice left front and right rear back,and grouped?the experimental group and control group?after oral administration of SFN.The experimental group were treated orally with SFN?6 mmol SFN in 0.1 ml PBS?,and the control group received an equal volume of PBS,give medicine three times a week.2.3 Dissect the tumorsWe euthanized the nude mice with the method of breaking neck after three weeks.Then the tumors was dissected with the surgical method,and the necrotic tissue and the blood vessels on the surface of the tumors was removed.The tumor tissue was embedded with paraffin,and the immunohistochemical experiment was performed at last.2.4 Immunohistochemical experimentIn this study,DAB staining was performed by immunohistochemical method.To observe the expression of five proteins in IAP family,including Survivin,Livin,c-IAP1,c-IAP2 and X-IAP.Then,the numbers of positive cells in five high power fields per tumor were counted.2.5 Data of statistical analysisIn this experiment,t-test was used for statistical analysis,then draw the conclusions.Results:1 in the experiment group,the numbers of c-IAP1 positive cells in five high power fields were 1027.2±107.88,there is significant difference comparing with control?1210.6±108.31??P<0.05?;2 in the experiment group,the numbers of c-IAP2 positive cells in five high power fields were 995.4±113.35,there is significant difference comparing with control?1140.30±122.35??P<0.05?;3 in the experiment group,the numbers of Livin positive cells in five high power fields were 1038±156.10,there is significant difference comparing with control?1163.5±85.21??P<0.05?;4 in the experiment group,the numbers of Survivin positive cells in five high power fields were 547.80±189.77,there is significant difference comparing with control?959.30±179.77??P<0.01?;5 in the experiment group,the numbers of X-IAP positive cells in five high power fields were 1072±86.95,there is significant difference comparing with control?1183.2±127.49??P<0.05?.Conclusions:The IAP family were inhibited in xenografts by sulforaphane in vivo,which may be one of the mechanisms in the apoptosis induced by sulforaphane.