Rosmarinic acid rich fraction from Orthosiphon aristatus (Blume) Miq. leaves induces apoptosis by inhibiting the rapamycin signalling pathway

Abstract

The mammalian target of the rapamycin (mTOR) pathway is critical for cell survival, metabolism, growth, and protein synthesis. Nevertheless, its hyperactivation can lead to the tumour development and progression. Consequently, inhibiting the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and mTOR signalling routes is an effective approach to initiate apoptosis in cancer cells. Orthosiphon aristatus (Blume) Miq.has been reported to exhibit anticancer properties against many cancer cell lines. Notably, the rosmarinic acid-rich fraction of Orthosiphon aristatus (Blume) Miq. (RA-OA) have been reported to significantly contribute to the antiproliferative activities on DU-145 cell line. In this study, the antiproliferative mechanism of RA-OA was explored via PI3K/Akt/mTOR pathway using in vitro experiments and in silico molecular docking. For in vitro study, annexin V staining and flow cytometry were utilized to determine apoptosis and cell cycle distribution, respectively. A quantitative reverse transcription-polymerase chain reaction was conducted to detect the changes in PI3K, Akt, mTOR and PTEN gene expression after treatment with RA-OA while molecular docking was performed to evaluate the binding affinity of rosmarinic acid towards the key proteins in the PI3K, Akt and mTOR pathway. Apoptosis was significantly induced in cells treated with the RA-OA, where the cell cycle progression of DU-145 cell line was arrested in the S phase. The RA-OA treatment has lowered PI3K, Akt and mTOR while enhancing PTEN gene expression. RA molecule exhibited strong binding (inhibition) affinity towards PI3K, Akt, mTOR proteins with docking energies of -9.10–7.58 kcal/mol. In conclusion, antiproliferative of RA-OA occurs by inhibiting the gene expression of PI3K/Akt/mTOR pathway and its protein phosphorylation.