Lappaconitine Hydrochloride Mediates the Cell Cycle Arrest of Human Cervical Cancer Hela Cell Through pRb Pathway

OBJECTIVE To explore the inhibitory effect of lappaconitine hydrochloride on proliferation of human cervical cancer Hela cells in vitro and its possible mechanism. METHODS Cell counting kit-8(CCK-8) and 5-ethynyl-2'-deoxyuridine(EdU) cell proliferation kit were used to detect the proliferation inhibition effect of lappaconitine hydrochloride on Hela cells. The effect of 4', 6-diamidino-2-phenylindole(DAPI) for nucleic acid staining on Hela nuclear morphology was studied. The technology of flow cytometry, propidium iodide(PI) single staining method and Annexin V-fluorescein isothiocyanate/propidium iodide(AnnexinV-FITC/PI) double staining method were employed to measure the cell cycle distribution and apoptosis of Hela cells after lappaconitine hydrochloride treatment. Western blotting was used to detect the effect of lappaconitine hydrochloride on the expression of p53, p21, p16, Cyclin D1, Rb and p-Rb. RESULTS Lappaconitine hydrochloride could inhibit the proliferation of human cervical cancer Hela cells in vitro, and the inhibition presented obvious dose-effect relationship(r=0.999 3, P<0.01). The IC₅₀ of lappaconitine hydrochloride on Hela cells was 0.772 6 mg·mL^-1. After dealing with lappaconitine hydrochloride, the nuclei of Hela cells were shrunken, deformed and fragmented. Hela cells showed obvious cell cycle arrest at G₀/G₁ phase, and the apoptotic ratio gradually increased with the increase of the concentration of lappaconitine hydrochloride. Lappaconitine hydrochloride could up-regulate the protein expression of p53, p16 and Rb in Hela cells, and down-regulate the protein expression of Cyclin D1 and p-Rb, but it had no significant effect on the expression of p21. CONCLUSION Lappaconitine hydrochloride might mediate G₀/G₁ phase arrest of Hela cells through pRb pathway, inhibit the proliferation of Hela cells and promote their apoptosis.