The Curative Effect Of Survivin Antisense Oligonucleotide On Salivary Adenoid Cystic Carcinoma

Purpose: Salivary adenoid cystic carcinoma (SACC) is a common malignancy of salivary gland. Recurrence or/and early metastasis is its biological properties. It is difficult to be entirely resected surgically and is invalid to radiotherapy and chemotherapy. It is imperative to find a perfect auxiliary therapeutic treatment. The gene encoding the inhibitor of aoptosis proteins (IAP) survivin was cloned recently, and the protein was characterized. Unlike other IAP family members, survivin expression is found during embryonic and fetal development, but is undetectable in terminally differentiated adult tissues. It subsequently becomes re-expressed in most common human tumors and a variety of human cancer cell lines. So gene therapy target survivin has perfect pertinency and security. In this study, adenoid cystic carcinoma cell line with high tendency of lung metastasis (Acc-M) cell line was used to investigate the effect of survivin antisense oligonucleotide (ASODN) on expression of survivin and apoptosis in this cancer cell line. SACC nude mice model was used to observe the curative effect of survivin ASODN on this tumor.Materials and Methods: 1 Materials: (1) Cell line:a human adenoid cystic carcinoma cell line with high tendency of lung metastasis(Acc-M); (2) Antisense oligonucleotide of specific target survivin; (3) Animal: Fifteen SPF grade, 4-5 week-old female Balb/c nude mice were used in this study. 2 Methods: (1) Acc-M cells were cultured in RPMI-1640 supplemented with born calf serum and streptomycin (0.1mg/ml) and penicillin (100IU/ml) in an incubator at 37℃; (2) A phosphorothioated antisense oligonucleotide of specific target survivin was designed, synthesized and then transferred to Acc-M cell line by lipfectin. Simultaneously blank control group, sense oligonucleotide (SODN) group were set up for comparison. MTT assay was used to detect cell growth. Apoptosis was observed by flow cytometry. Survivin expression was determined by RT-PCR and Western-Blotting; (3) Acc-M cell line was implanted subcutaneously into nude mice. Twelve days after implantation, the tumor diameter grew to about 1.0 centimeter. Then the mice were divide into 3 groups: NS group (the control group), group ASODN and group SODN . Thirteen days after treatment, the tumor volum and weight were measured. The percentage of apoptotic and cell cycle distribution were detected by the flow cytometer. The tumors were observed by light microscope.Results: 1 (1) Compared with the control group and group SOND, in group ASODN, 48 hours after transplantation, the expression of survivin mRNA and protein level were viability decreased as 43.03% and 36.03%, the difference was significant (P﹤0.05); (2) Apoptosis rate apparently increased, the results of flow cytometer showed that the percentage of apoptotic cells was 25.9% in group ASODN, 1.98% in group SODN, 2.08% in group lipfectin, and 1.93% in the control group. There was significantly difference between ASODN group and other groups (P<0.05), and there was no obvious difference between either two groups among group SODN, group lipfectin and the control group (P>0.05). (3) Cell growth was inhibited in MTT assay. The IR value of ASODN group was increased as 37.61% and 46.42% in 800nmol/L and 1000nmol/L ASODN groups, the increase was significant (P<0.05). There was no obvious difference in group SODN and the blank control group. 2 (1) After union treated with survivin ASODN and lipfectin, the growth of transplanted tumor in nude mice was obviously inhibited. The tumor volumes of group NS, group SODN, group ASODN (cm3) were 3.09±0.06,2.92±0.13,1.309±0.27, and the weights(g) were 4.97±0.54, 4.40±0.51,2.32±0.87. There were significantly difference between group ASODN and other two groups(P<0.05). (2) Morphologic observations:①Gross: The transplanted tumor was ellipsoidal shape with intact membrane and smooth surface. Occasionally, the surface of tumor was broken. The cross section of tumor was solid with gray-white color. Some yellowish-white materials like bean dregs were seen in a few sections, which look like necrotic tissue.②Light microscope (LM): The result revealed that the transplanted tumor was arranged in mass. The tumor cells were round or multisided. The cytoplasm of cells was stained red. The proportion of the nuclei and cytoplasm was imbalance. Nuclear division could be easily observed. There were many apoptotic cells with their cytoplasm shrank in group ASODN,and the chromatin condensed into block , and dispersed unevenly. Apoptotic bodies were formed. Few apoptotic cells were seen in group SODN and group NS. (3) After treated with ASODN the percentage of apoptotic cells were increased to 32.9% by the flow cytometry, and the value of group SODN was 6.22%, the control group was 5.60%.Conclusions: 1 Survivin antisense oligonucleotide can down-regulated the expression of survivin gene in Acc-M cell line specifically. 2 Survivin antisense oligonucleotide plays an important role in inducing tumor apoptosis and suppressing cell proliferation. Survivin antisense oligonucleotide can suppress the growth of Acc-M transplanted tumors in nude mice. 3 Survivin antisense oligonucleotide can be an effective method in the future gene therapy of SACC.