Ginsenoside Rg3Inhibit The Proliferation Of NCI-H446Small Cell Lung Cancer Cell Line And Down Regulate The Expression Of Caveolin-1

Objective: Ginsenoside Rg3, which is a triterpenoid saponin extracted fromPanax ginseng, had been demonstrated of many favorable effects on anti-proliferation,anti-infiltration, angiogenesis, induce apoptosis and reverse of the multidrug resistance.Caveolin-1(Cav-1) anchored into the bilayer of cell membrane with the assistantof cholesterol making the bilayer to form a stable sort of lipid raft which is calledcaveolae and it is the most important one of the three family members. Its majorfunction is to limit various signal proteins to caveolae. Cav-1’s expression is abnormalin numerous tumors such as gastric cancer, prostatic cacinoma and lung cancer. It hadbeen demonstrated that both the protein and the cave are related with cell proliferation,cell signal and substance exchange.Small cell lung cancer (SCLC) is a highly malignant type of lung cancer whichhas shorter doub and progress time and often accompany cryptorrhea and cavinoidsyndrome. The NCI-H446cell line was derived by D. Carney, A.F. Gazdar andassociates in1982from the pleural fluid of a patient with small cell cancer of the lung.The morphology of the original tumor was not characteristic of SCLC and it is abiochemical and morphological variant of SCLC. It had been demonstrated that Cav-1influenced the cell proliferation and inhibit the apoptosis of NCI-H446. As numerousstudies had been performed on antitumor effect of Rg3, there is no report about therelationships between Rg3with SCLC and Cav-1. On the other hand, mechannismrelated to Rg3actting upon Cav-1needs to be further expounded.In this experiment, Rg3was added to NCI-H446to observe the inhibition effect ofcell proliferation and the changes of caveolae and Cav-1to further explore themechanisms of Rg3’s inhibition effect. Materials and Methods: NCI-H446cells were selected for the study,(1) NCI-H446cells were treated in culture medium with50μmol/l Rg3forconsecutive seven days, and cell counting and optical microscope image were takeneach day. RT-PCR and Western Blot were used to detect the differences about the levelof the PCNA mRNA and the translated protein between dosing groups and controlgroup on the fifth and the seventh day.(2) Adding different concentrations of Rg3(0.4μmol/l、2μmol/l、10μmol/l、50μmol/l) in the cell culture medium, the different inhibition efficiency of drugconcentrations to Cav-1expression was measured by Western Blot. NCI-H446cellswere treated in culture medium with50μmol/l Rg3for24h, Transmission electronmicroscope was used to observe the different amount of caveolae; Sucrose densitygradient centrifugation was used to observe the different distribution of Cav-1(3) Indirect immunofluorescence was used to observe the different distribution ofCav-1which was treated with pure medium、50μmol/l Rg3、25mmol/l cholesterol andcombined50μmol/l Rg3with25mmol/l cholesterol separately. Western Blot was usedto detect the differences of Cav-1expression.Results:(1) Seven days consecutive culture pictures shown that Rg3groupmorphological changes occured and the cell number decreased significantly comparedwith control group. PCNA synthesis of Rg3treated NCI-H446cells was decreased.(2) Rg3decreased the expression of Cav-1in dose dependent way; the amount ofcaveolae was significantly decreased when treated with50μmol/l Rg3, typical flask-likestructures were hardly observed. Cav-1expression of Rg3treated cells was decreased inlipidraft area and increased in non-lipidraft area.(3) Rg3not only downregulated the expression of Cav-1but also made Cav-1shiftout of lipid raft and internalized into cell cytoplasm and these effects could be weakenedby cholesterol.Conclusion:(1) Ginsenoside Rg3inhibits NCI-H446proliferation.(2) Ginsenoside Rg3further destruct the presence of caveolea in bilayer; Rg3shiftthe distribution and decrease the expression of Cav-1.(3) The effect that Ginsenoside Rg3towards Cav-1could be weakened by denseconcentration of cholesterol.