| Objective: Salivary adenoid cystic carcinoma (SACC), is one of the mostcommon salivary gland cancers originated from epithelial tissues in oral andmaxillofacial region. This tumor has the important characteristics of highlyinvasive growth, infiltrating along facial nerve and high metastasis rate. Theincidence of local recurrence and distant metastasis of SACC is very high afterthe surgical removal of the tumor for its high malignancy and lower radiationsensitivity. The metastasis rate of SACC is the highest among all oral andmaxillofacial tumors. However, its mechanism of morbidity 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 patients’ quality of life. Sulforaphane (SFN) is a substancenaturally occurring in cruciferous vegetables belonging to isothiocyanates(ITCS)with strong anti-cancer effects. Recently, Sulforaphane (SFN) attractedwidespread attention because of its strong anti-tumor effects. Our previous invitro experiments showed that sulforaphane can inhibit proliferation ofadenoid cystic carcinoma cell line ACC-M, triggered by the Caspase-mediatedapoptosis and Bcl-2family members played an important role in apoptosis.However, to our best knowledge, it has not been reported if sulforaphane caninhibit SACC cell growth in vivo. In this experiment, we established a nudemouse xenograft model by subcutaneous injecting human lung highly meta-static salivary cystic carcinoma cell line ACC-M and give certain concent-ration sulforaphane by oral administration to observe whether sulforaphanehave inhibition of proliferation and induction of apoptosis for human salivaryadenoid cystic carcinoma xenograft and explore its possible molecularmechanism, in order to develop new ways for plant chemotherapy treatment for SACC and provide related experimental data.Methods:1Material1.1Human salivary adenoid cystic carcinoma cell line ACC-M: Cell lineACC-M was provided by the laboratory of oral and maxillofacial surgery ofShanghai Jiao Tong University，which was established in1995.1.2Sulforaphane(SFN): Sulforaphane was purchased from LKTlaboratories(USA), gifted by Pittsburgh University Medical Center, Head andNeck Surgery, purity is no less than99%(HPLC).1.3Nude mouse: The nude mice is provided from Beijing ExperimentalAnimal Center of Military Medical Sciences,10male nude mice,4~6weeksold, weight16~20grams.1.4In situ apoptosis detection kit: Purchased from the ROCHE Company.1.5DAB detection kit: Purchased from Hebei Bohai BiologicalEngineering Co,Ltd.2Method2.1Preparation of components: Sulforaphane was diluted with phosphatebuffer solution (PBS)100mmol/L concentration of stock solution, frozen at-20℃refrigerator.2.2Cell culture: Recovered the cryopreservation of ACC-M cells. Celllines were cultured in RPMI1640medium containing10%(v/v) fetal bovineserum, penicillin(100U/ml) and streptomycin (100μg/ml), and then preparedaccording to the proportion of cell incubated at37℃in5%CO2gas incubatorwith saturation humidity. The media was changed every other day and cellswere generated about every72-96hours.2.3Establish animal models and cell inoculation: Cells were harvested inlogarithmic growth phase and adjusted to cell suspension with density of2×107/ml. and a0.2ml suspension containing2×106cells was injectedsubcutaneously on both left and right flank of each mouse.2.4Treatment: The nude mice were randomly divided into experimentaland control groups of five mice/group (2tumors/mouse). Experimental animals were treated orally with SFN (6mmol SFN in0.1ml PBS)3times/week (Monday, Wednesday and Friday) beginning the day of tumor cellimplantation. Control mice received an equal volume of the PBS. Physicalstate, appetite, activities, body weight, tumor weight and volume of nude micewere observed and measured.2.5Measurement and calculation of tumor volume inhibition rate oftumor growth: The diameter (a), short axis (b) and high (c) of tumors weremeasured with a vernier caliper after treatment. Tumor volume was calculatedwith the formula:(tumor volume V=π×a×b×c/6). And tumor growth curvewere made. The inhibition rate of tumor growth was calculated with theformula: inhibition rate=the average volume of the control group-the averagevolume of experimental group/the average volume of control group×100%.Mice were killed by cervical dislocation, povidone-iodine disinfection, withophthalmic forceps, scalpels quickly peel the tumor tissue. Removal ofnecrotic tissue on the surface of the tumors, as well as the small blood vesselsand so on. Tumor diameter and weight of each group were measured.2.6Detected apoptosis by TUNEL assay: Tumor tissue specimens werefixed with4%paraformaldehyde for24hours then were embedded withparaffin. Two4-5uM Biopsy was cut. After deparaffinied with xylene andgraded ethanol dehydration, biopsies were stained with TUNEL. BiotinylateddUTP labeled DNA was broken to form3`-OH ends mediated by the terminaldeoxynucleotidyl transferase. Peroxidase was binded to residual DNA bymeans of biotin and avidin binding specificity. Finally, the substrate was addedto stain by3,3-diamino-benzidine(DAB).2.7Judgment of results: Brown apoptotic cells were observed in thenuclei under light microscope. Ten400times (10×40eyepiece lens) highpower field in each biopsy were shoot randomly with Motic Med6.0imageanalysis system. Tumor cells nuclei were blue, positive apoptotic cells werebrown. The positive cells were counted with immunohistochemical score(IHS). For data statistics and analysis, the negative and weakly positive wereclassified as negative group. Moderate positive and strong positive were classified as positive group.2.8Statistical analysis: The tumor volume and weight were analyzed witht-test using SPSS13.0statistical software. Immunohistochemical score forTUNEL were analyzed with χ2test and Fisher exact probability. P<0.05wasconsidered to indicate a statistically significant result.Result:The result show: After60mmol/L SFN treatment, SFN inhibitedsignificantly growth of human adenoid cystic carcinoma xenografts in nudemice. There are significant differences between two groups in tumor size andweight. The average tumor volume in the experimental group was signify-cantly smaller than the control group. No significant difference in body weightof nude mice in the experimental group compared with the control group.Positive rate of TUNEL staining in experimental group was significantlyhigher than the control group.Conclusion:1Sulforaphane could inhibit ACC-M adenoid cystic carcinoma xenografttumor proliferation and growth.2Sulforaphane can induce apoptosis of adenoid cystic carcinomaACC-M nude mouse xenograft in vivo. |
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