Studies On The Anti-tumor Action And The Mechanism Of Active Components Of Menispermum Dauricum

Objective:To investigate the effect of active components of menispermum dauricum on inhibiting proliferation and inducing apoptosis of leukecythemia K562 cells and its antitumor action on S₁₈₀ sarcoma-bearing mice.Methods:The growth inhibition rates and cytotoxic effect of leukecythemia K562 cells were measured with MTT assay;Observing cells morphologic changes under inverted microscope,further testing the changed morphology by HE stain; Observing the ultrastructure changes of leukecythemia K562 cells with transmission electron microscope;The distribution of cells cycle and the apoptosis rate were observed by flow cytometry;The Caspase-9,Caspase-8 and Caspase-3 proteins were determined by immunohistochemical S-P staining technique.We explored the mechanism of inducing apoptosis on leukecythemia K562 cells from active components of menispermum dauricum.Korbor method was used to calculate mice acute oral LD₅₀ values(with their 95%confident limits).Active components of menispermum dauricum were administered orally to mice,and tumor inhibition action on S₁₈₀ sarcoma-bearing mice was observed.Anti-proliferation effect of active components of menispermum dauricum on leukecythemia K562 cells were observed by serum pharmacology method.We would adopt the prolongation of life span test to observe the tumor inhibition action on S₁₈₀ sarcoma-bearing mice.Results:(1)MTT assay showed active components of menispermum dauricum could significantly inhibit the growth of leukecythemia K562 cells.The effect of active components of menispermum dauricum was in a dose-dependent manner(P＜0.01);(2)Under inverted microscope the leukecythemia K562 cells showed the classical apoptosis changes as the cell membrane blurred and apoptotic bodies appeared by treated with 20μg/ml of active components of menispermum dauricum before 24 hours;after 24 hours cells showed necrosis;(3)HE stain showed that the observation was the same with in invert microscope,the apoptotic bodies were stained by the classical navy blue and pompadour color,the changes could be observed under the optical microscope;(4) Transmission electron microscope observed that chromatin condensed or ruptured,nucleolus disintegrated in leukecythemia K562 cells and vacuoles were found at cytoplasm and apoptotic bodies;(5) Flow cytometry showed active components of menispermum dauricum could induce the leukecythemia K562 cells apoptosis;some typical sub-dipioid peaks was observed,the percentage of cells in G_O/G₁ phase increased and the percentage of cells in S and G₂/M phase decreased after the cells treated by active components of menispermum dauricum;Active components of menispermum dauricum could arrest leukecythemia K562 cells in G_O/G₁ phase;(6)Immunocytochemistry showed that the expression of Caspases-9,Caspases-8 and Caspases-3 proteins increased after the leukecythemia K562 cells treated by active components of menispermum dauricum,there were different significantly compared with control.(7)Mice acute oral LD₅₀ values(with their 95% confident limits) of active components of menispermum dauricum were 52.72g/kg (38.12-63.87 g/kg).(8) In the group of low,middle and high dosage,the tumor inhibition rate on mice-bearing S₁₈₀ Were 17.19%,33.43%,42.29%respectively, active components of menispermum dauricum inhibited the growth of solid tumor of mice-bearing S₁₈₀ obviously(compared with model group P＜0.05 in middle dosage group,and P＜0.01 in high dosage group).(9) The growth inhibition rates of A549 ceils treated with active components of menispermum dauricum -containing rat serum with concentration of 10%,20%and 25%that were 29.03%,30.55%and 41.67% respectively(compared with model group P＜0.05);(10) The prolongation of life span test showed that the prolongation rates were 41.66%and 33.33%in the group of low and middle dosage(compared with model group P＜0.05).Conclusions:(1) Active components of menispermum dauricum have the effect of inhibiting proliferation and inducing apoptosis for leukecythemia K562 cells;(2) Before treated with the 20μg/ml active components of menispermum dauricum for24 hours,the effect of inhibitive proliferation mainly was completed by the induction of apoptosis;(3)Apoptosis of leukecythemia K562 cells induced by active components of menispermum dauricum may be related with arresting cell cycle in G_O/G₁ phase, The mechanism of inducing apoptosis for leukecythemia K562 cells might be related by up-regulation expression of Caspase-9,Caspase-8 and Caspase-3.It might be induce apoptosis in leukecythemia K562 cells by the Fas-dependent pathway and mitochondria pathway;(4)Active components of menispermum dauricum could prolong the life spans of mice bearing S₁₈₀ sarcoma.They have the effect of inhibiting the growth of S₁₈₀ sarcoma;(5) Active components of menispermum dauricum -containing serums have the effect of inhibition proliferation for A549 cells;(6)In the results of acute oral toxicity study test,active components of menispermum dauricum belong to low toxicity Chinese medicine.They have significantly anti-tumor effect and may be a new kind of antitumor drug.