Inhibition On Proliferation And Induction Of Apoptosis In Human Tongue Cancer Cells And Antiangiogenic Potential Of Camptothecin

Objective: Camptothecin (CPT) is a new anticancer agent because of its inhibition on DNA topoisomerase I . Recent researches show that CPT and its analogs can inhibit proliferation, induce apoptosis and promote differentiation in cells. The growth, aggression and metastasis of tumors depend on the development of an adequate blood supply via angiogenesis. Therefore, an anti-angiogenesis strategy offers a new target for therapies on tumors, psoriasis and other benign proliferation diseases. CPT and its analogs can inhibit angiogenesis in vitro and the effect may result from inhibition on the proliferation and reduction of endothelial cell apoptosis. In this study, human tongue cancer Tca8113 cells and human umbilical venular endothelial cell ECV304 are treated with CPT and the cell proliferation and apoptosis are detected in order to investigate the effect of CPT on Tca8113 and the mechanism of antiangiogenesis.Methods: Tca8113 and ECV304 are incubated with CPT at different concentrations for 24,48 and 72 hours, respectively. Inhibition on cells proliferation and effective concentrations of CPT are measured by MTT assay. Cells apoptosis is evaluated by inverted microscope, electron microscopy, flow cytometry and agarose gel electrophoresis.Results: (1) CPT can inhibit the proliferation of Tca8113 cells and ECV304 cells. The maximal inhibition ratio on Tca8113 is 43%, 63% and 82.8% for 24h, 48h and 72h, respectively, with the best inhibition concentration as 8ug/ml. The maximal inhibition ratio on ECV304 is 67.9%, 96.7% and 97.2% for 24h, 48h and 72h, respectively, with the best inhibition concentration as 0.25ug/ml. The inhibition effect of CPT shows time and dose dependence relationship. The effective concentration range for Tca8113 and ECV304 is from 0.016ug/ml to 8ug/ml, and from 0.004ug/ml to 0.25ug/ml, respectively. The inhibition doesn't increase with the concentration of CPT afterexceeding this effective range. The inhibition concentration of CPT to ECV304 is significantly lower than that to Tca8113.(2) After exposure to CPT, Tca8113 and ECV304 cells present typical morphologic features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, formation of apoptotic bodies.(3) Flow cytometry test showed that CPT with a concentration higher than 1ug/ml can induce apoptosis in Tca8113 cells after 48h treatment. And the Tca8113 cell cycle is arrested in S phase. Apoptosis can be induced in ECV304 cells by CPT with a concentration higher than 0.125ug/ml, and the cell cycle is arrested in G1 phase.(4) Agarose gel electrophoresis shows characteristic DNA ladder after ECV304 and Tca8113 is treated with CPT.Conclusions: (1) The inhibition of CPT on the proliferation of Tca8113 and ECV304 shows time and dose dependence relationship. CPT may have a clinical therapeutic effect on squamous carcinoma.(2) CPT can induce Tca8113 apoptosis and arrest the cell in S phase by the inhibition of DNA replication. Its anticancer mechanism may be relative to apoptosis induction.(3) CPT can interference ECV304 cell cycle, arrest the cell in Gl phase and induce ECV304 apoptosis.(4) The inhibition of CPT to Tca8113 and ECV304 is within an effective concentration range with the best inhibiting concentration as 8ug/ml and 0.25ug/ml, respectively. And the inhibition doesn't increase with the concentration beyond the range.(5) The concentrations of proliferation inhibition and apoptosis induction of CPT to Tca8113 were higher than that to ECV304 notably. It is demonstrated that CPT is an anti-tumor agent with double effect due to its inhibition effect both on tumor cell colony and endothelial cell colony.