| Objective: To observe the effects of resolving heat-phlegm-masses formula(RHPMF)and its separate prescription—clearing heat formula(CHF),dispersing phlegm formula(DPF)and resolving masses formula(RMF)on the proliferation inhibition and PI3K-Akt-mTOR signaling transduction pathway in human lung squamous carcinoma cell line SK-MES and small cell lung carcinoma cell line NCI-H69,study the mechanism of RHPMF and its separate prescription—CHF,DPF and RMF on lung carcinoma treatment,seek effective formula and Chinese medicine,explore the mechanism of lung carcinoma treated by traditional Chinese medicine from the level of molecular.Methods: Human lung squamous carcinoma cell line SK-MES and small cell lung carcinoma cell line NCI-H69 were selected as the experimental objects,cells were detected by inverted microscope,and calculated the inhibition rate with MTT method for screening effective herbs wich can inhibit both the SK-MES cells and the NCI-H69 cells effectively.According to the efficacy of herbs,orthogonal design and supervisor's experience,select the best curative effect prescription;cell growth state was observed under inverted microscope;flow cytometry method was used to measure the cell cycle of SK-MES cells and NCI-H69 cells after effected by RHPMF and its separate prescription—CHF,DPF and RMF;western blot method was used to check PI3K,Akt,mTOR protein expression in PI3K-Akt-mTOR signal transduction pathway.Results:(1)Five herbs of inhibiting both the SK-MES cells and the NCI-H69 cells effectively were screened;(2)Selected out RHPMF and its separate prescription—CHF,DPF and RMF;(3)RHPMF and its separate prescription—CHF,DPF and RMF all could obviously inhibit SK-MES cells and NCI-H69 cells proliferation,their effects on cellular morphology were obviously different compared with the control group;Squamous lung carcinomacell line SK-MES: There were dead cells in all treatmet groups,cell in the CHF group cellular space increased,cells became shrinking;cell in the DPF group cellular morphology obviously shrank,cellular space increased;cell in the RMF group was visualized,smaller than the control group,some cells shrank,some cells presented vacuous-liking construction;cells in the RHPMF group was less than the control group,cellular space increased,some cells shrank.Small cell lung carcinoma cell line NCI-H69: RHPMF and its separate prescription—CHF,DPF and RMF all could significantly inhibit the growth of NCI-H69 cell,cell quantity was less than the control group,more single cell,cell in the RMF group was smaller.(4)RHPMF and its separate prescription—CHF,DPF and RMF all could significantly inhibit the proliferation of SK-MES cell and NCI-H69 cell,and effects of RHPMF group were the strongest,and presented dosage-effects and time-effects dependent relationship with the increasing of medicine concentration and time.(5)RHPMF and its separate prescription—CHF,DPF and RMF all had obviously effects on SK-MES cell and NCI-H69 cell cycle.Squamous lung carcinoma cell SK-MES cell and the small cell lung carcinoma NCI-H69 cell :Cellular percent of S stage increased in all treatment group of RHPMF and its separate prescription—CHF,DPF and RMF,the DPF group increased most obviously,the effects intensity of SK-MES cell was followed by DPF group,CHF group,RHPMF group,RMF group,and RMF could also effect the G2 stage.Small cell lung carcinoma NCI-H69 cells: the effects intensity of NCI-H69 cell was followed by DPF group,CHF group,RMF group,RHPMF group,and RHPMF group can also effect the G2 stage.(6)PI3K,Akt,mTOR were expressed in human lung squamous carcinoma cell line SK-MES cell and small cell lung carcinoma cell line NCI-H69,after the cells were treated with IC50 dosage,the protein expression level of PI3K,Akt,mTOR in the cells decreased,the protein expression level of PI3K and mTOR in the two cells is highest in RHPMF group,then RMF group,CHF group,DPF group;the protein expression level of Akt in the two cells is highest in RHPMF group,then CHF group,RMF group,DPF group.Conclusion:(1)RHPMF and its separate prescription — CHF,DPF and RMF all can inhibit the lung squamous carcinoma cell line SK-MES and small cell lung cancer cell line NCI-H69.(2)RHPMF and its separate prescription— CHF,DPF and RMF all inhibit the lung squamous carcinoma cell line SK-MES and small cell lung cancer cell line NCI-H69 mainly in the S stage.(3)RHPMF and its separate prescription—CHF,DPF and RMF all can inhibit the expression of Akt,mTOR and PI3K proteins,so that inhibit the growth of lung carcinoma cells.(4)In the pathological process of lung carcinoma,phlegm,heat and masses are closely related to the protein expression of Akt-mTOR-PI3K signaling pathways. |
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