| Objective:Based on the previous studies, to study the effect of tretinoin injection on the growth of hepatocellular carcinoma cells SMMC-7721in vitro and nude mice tumor xenograft in vivo, to detect tumor tissue cell apoptosis and caspase-3expression, to aims to clarify tretinoin injection action and possible mechanism of anti-liver tumor, and to provide a scientific experiment basis for its clinical application on the treatment of liver cancer. Methods:(1) Take SMMC-7721cells in the growing period of logarithm to be intervented by different concentration tretinoin injection (final concentration was100μg/mL,50μg/mL,25μg/mL,12.5μg/mL,6.2μg/mL and3.125μg/mL) for48hours. The negative control was culture medium for equal volume. The positive control was anti-cancer drug cisplatin (DDP), then its final concentration was respective10μg/mL,1μg/mL and0.1μg/mL. The CCK-8Kit (Cell Counting Kit-8) was used to test the influence for human liver cells SMMC-7721proliferation;(2) To establish the transplant tumor model in nude mice for human liver cells SMMC-7721:After15days, the nude mice, with developed tumors≥50mm3were chosen and the nude mice were removed with transplant tumor no growth or transplant tumor slow growth or transplant tumor fast growth. Based on the experiment scheme, nude mice were randomly divided into5groups:negative control group(saline), positive control group (cisplatin,1mg/kg weight) and low-dosage, medium-dosage and high-dosage for tretinoin injection(7.5mg/kg weight,15mg/kg weight and30mg/kg weight), by intraperitoneal injection. For a total of four weeks to medicine in five days given drug withdrawal2days, tumor size and nude mice weight were measured every week for three times. Tumor size was calculated for tumor volume(TV), relative tumor size(RTV) and relative tumor proliferation rate [T/C (%)] at each time for detecting the influence of growth of transplant tumor in nude mice for human liver cells SMMC-7721.(3) Nude mice with tumor-burdened were given drugs for4weeks, with drug withdrawal1day, and killed by taking off the neck. The subcutaneous transplant tumors were removed, fixed by4%neutral formaldehyde, dehydrated conventionally, put into paraffin, cut into4-μm thick slices and dewaxed hydration. The apoptosis of tumor tissue cell was detected by TUNEL method, and the caspase-3expression in the tumor tissue was detected by immunohistochemical method. Results:(1) With the increase of concentration, the inhibition of tretinoin injection to human liver cell lines SMMC-7721proliferation enhanced. It presented a good concentration-response relationship, and the IC50value was (15.14±0.46) μg/mL.(2) Tretinoin injection treatment group and positive control group(DDP) could inhibit transplant tumor growth in the nude mice. After intraperitoneal injection, TV, RTV and T/C (%) could decline obviously. And T/C (%) was less than60%obviously, compared with the negative control group(saline)(P<0.05). DDP could reduce the weight of nude mice, but tretinoin injection did not. It revealed that tretinoin injection may have lower toxicity.(3) The TUNEL method showed that positive cells nucleus were brown. Some cells performanced pyknotic or fractured nucleus, in which chromatin was divided into block or apoptotic body. The positive rate of apoptotic cells in the tretinoin injection treatment group was higher than that in the negative control group, and the difference was statistically significant(P<0.05). Cisplatin in the positive control group had no significant apoptotic effect on the cells, and the positive rate of apoptotic cells in the tretinoin injection treatment group was lower. There were no obvious difference(P>0.05); The Immunohistochemical method showed that tumor cells presented caspase-3expression in the tretinoin injection treatment group, and there were brown markings in the cytoplasm. But those did not presented in the positive control group. Caspase-3expression in the tretinoin injection treatment group was significantly higher than those in the positive and negative control group, and the difference was statistically significant(P<0.05). Conclusion:Tretinoin injection could obviously inhibit human liver cells SMMC-7721proliferation and the growth of transplant tumor in nude mice. It presented a good concentration-response relationship and revealed strong anti-tumor activity in vitro and in vivo. The mechanisms of anti-tumor activity of tretinoin may be that it could promote caspase-3activation, and induce tumor cell apoptosis. |
