Experimental Study For Inhibitory Efficacy Of Gossypol Acetic Acid On Human Tongue Squamous Cell Carcinoma Cal-27 Transplanted In Nude Mice

[Objective] To study the therapeutic effect of gossypol acetic acid (GAA) on human tongue squamous cell carcinoma Cal-27 transplanted in nude mice, and also preliminarily discuss the inhibitory efficacy and its mechanism of gossypol acetic acid on head and neck squamous cell carcinoma in order to discover new ways and provide scientific theory basis for clinical relating tumor treatment.[Materials and Methods] 1. Animal model establishment. The selected 4 to 6 week-old BALB/C-nu nude mice were subjected to subcutaneous inoculation with cultured tongue squamous cell carcinoma Cal-27 cells in right armpits. Each mouse was inoculated with a dose of 0.1ml, and with cell density 3×107/ml. Then, the condition of tumor growth and tumor formation rate were checked and recorded. 2. Experiment grouping and drug intervention. Two weeks after inoculation, the nude mice were randomly divided into 3 groups (8 mice for each group), which are control group, DMSO group and GAA group. All groups were subjected to abdomen injection with once two days, lasting for 6 times. The inoculums for control group were normal saline, and the dosage was 0.2ml for one mouse per injection. For DMSO group, DMSO with concentration of 0.2% and dosage of 0.2ml was injected into one mouse per injection. For GAA group, the injection was GAA with dosage of 15mg for one kilogram mouse weight per injection, and the solvent for GAA was DMSO with concentration of 0.2%.3. Observation of therapeutic effect. The mouse weight, condition of tumor growth and tumor growth curve were checked and recorded during the injection period. The mice were put to death 2 days after the last injection. After peeling off the tumor tissues and main organs of mice, the tumor weight was weighed and inhibition rate of tumor was calculated. 4. Paraffin section making. The pathology of tumor tissues and tumor cell metastasis of main organs were observed by hematoxylin and eosin staining (HE) for all groups. Apoptosis rate of tumor cell was detected for all groups by terminal transferase-mediated dUTP nick end-labeling (TUNEL).5. Immunohistochemistry for CD31 detection. Microvessel density (MVD) counts expressions for tumor of each group were collected.[Results] 1. Animal models were successfully established for human tongue squamous cell carcinoma Cal-27 transplanted in nude mice and tumor formation rate was 96%.2. The variation of mouse weight and tumor volume in each group. The average mouse weight of control group was heaviest, while GAA group was lightest, and DMSO group was medium. There was no significant difference between control group and DMSO group weight change (P>0.05), significant difference between the GAA group and the other two groups were between weight change (P<0.05). Drug intervention had an obvious influence on tumor growth in GAA group, which tumor growth slowing down. The tumor volume of GAA group was obvious less than that of control group and DMSO group, which showing statistical significance.3. Inhibition rate of tumor in each group. The average mice weight for control group, DMSO group and GAA group were (1.835±0.293)g, (1.764±0.184)g and (0.839±0.091)g respectively, while the inhibition rate of tumor were 0%,(3.87±3.7)% and (54.28±6.9)% respectively. Compared with control group and DMSO group, the tumor inhibition rate of GAA group was higher obviously, showing statistical significance (P<0.05).4. HE staining results. For control group, there were the highest arranged cells density with big, condensed nuclei and the most number of vessels compared with the other two groups. Loosely-arranged cells, less vessels, more intercellular substance, with hyperchromatic nuclei were seen for DMSO group. For GAA group, there was unobvious nuclear polymorphism, the least number of tumor vessels, and the cells were in nests, strip arrangement. Apoptotic cells could be observed for GAA group. No tumor cell metastases of main organs were observed for all the groups.5. Apoptotic index (AI) detecting by TUNEL for each group. The apoptotic index for control group, DMSO group and GAA group were (21.14±10.01)%,(23.80±4.45)% and (96.16±2.80)% respectively. But there was no statistical significance of AI between blank group and that of DMSO group (P>0.05).While it showed a statistical significance of AI between GAA group and the other two groups (P<0.01).6. MVD value for tumor of each group. The MVD for control group and DMSO group was higher than that of GAA group, and it showed a significant difference between GAA group and the other two groups (P<0.01) by statistical analysis. But there was no statistical significance of MVD between control group and DMSO group (P>0.05).[Conclusions] 1. Animal models were successfully established for human tongue squamous cell carcinoma Cal-27 transplanted in nude mice and tumor formation rate was 96%.2. Gossypol acetic acid had an obvious inhibitory effect on the growth of human tongue squamous cell carcinoma Cal-27 transplanted in nude mice. And no distant metastases of human tongue squamous cell carcinoma Cal-27 transplanted in nude mice.3. Gossypol acetic acid could induce the cell apoptosis of human tongue squamous cell carcinoma Cal-27 transplanted in nude mice.4. The inhibition of tumor growth which was human tongue squamous cell carcinoma Cal-27 transplanted in nude mice, maybe due to the inhibition of angiogenesis and decreased MVD value by GAA.