Study On Anti-tumor Activities And Mechanism Of A Novel Cyclophosphamide Derivate9b In Vivo And Vitro

AimTo investigate the anti-tumor activity of a novel cyclophosphamidederivate9b in vivo and in vitro，and its possible mechanism of action.Methods1. The inhibitory effects of9b on human hepatoma cell lineHepG2, human breast carcinoma cell line MCF-7, human myeloidleukemia cell line K562, human ovarian cancer cell line SK-OV3andhuman osteosarcoma cell line143B cells proliferation were analyzedby MTT assay in vitro.2. The effect of9b on apoptotic rate and cell cycle distributionwere evaluated by flow cytometry (FCM).3. To evaluate the anti-tumor effect of9b in vivo, mouse modelbearing inoculated H22, S180and B16tumor were established.4. The mRNA expression levels of cell cycle-related genescyclinD1and p21was treated by RT-PCR; the protein expression ofcyclinD1and p21was assessed by western blot. Results1. MTT assay revealed that9b could inhibit the proliferation ofHepG2, MCF-7, K562, SK-OV3and143B cells in a dose and timedependent manner. The IC50value of9b was32.34μmol·L-1toHepG2cells,87.07μmol·L-1to MCF-7cells,49.10μmol·L-1to K562cells,71.27μmol·L-1to SK-OV3cells and98.50μmol·L-1to143Bcells respectively when incubation for48h.2. The results of flow cytometry indicated that after treated for48h with different concentration of9b, the ratios of HepG2, MCF-7,143Bcells in the G0/G1phase and K562, SK-OV3cells in the G0/G1phaseand G2/M phase were significantly increased compared with controlgroup, and the apoptotic rate increased with the increase of theconcentration of9b.3.9b (3,10and30mg·kg-1·d-1) could significantly reducetumor weight in H22, S180and B16solid tumor mouse model in vivo.4. RT-PCR and western blot showed that9b decreased theexpression of mRNA and protein of cyclinD1, while increased theexpression of mRNA and protein of p21.Conclusion9b showed significantly anti-tumor activity in vivo and in vitro, ofwhich the mechanism might be associated with the change of cell cycledistribution and induction of tumor cell apoptosis. The role of human hepatoma cell line HepG2on G1phase arresting might be related to theexpression of mRNA and protein of cycle-related gene cyclinD1and p21.