20(S)-Protopanaxadiol (PPD) is one of the major active metabolites of ginseng. tensin homologue erased on chromosome 10 (p-PTEN) manifestation and downregulate PI3K/AKT/mTOR-pathway protein manifestation. Moreover, G0/G1 cell cycle arrest in MCF-7 cells could be induced by 20(S)-PPD treatment at high concentrations. Furthermore, overexpression or knockdown of mTOR could inhibit or promote the apoptotic effects of 20(S)-PPD. In addition, tumor volumes were partially reduced by 20(S)-PPD at 100 mg/kg inside a MCF-7 xenograft model. Immunohistochemical staining indicated a detailed relationship between the inhibition of tumor growth and the PI3K/AKT/mTOR transmission pathway. PI3K/AKT/mTOR pathway-mediated apoptosis Fst may be one of the potential mechanisms of 20(S)-PPD treatment. 0.05 compared to 0 M. 2.2. 20(S)-PPD-Induced Cell Cycle Arrest of G1/G0 Phase in MCF-7 Cells In order to examine whether cell cycle arrest was related to 20(S)-PPD-inhibited MCF-7 cell growth, PI staining was used to perform cell cycle analysis. As demonstrated in Number 3A, G0/G1 cell cycle arrest could be induced by treatment with 20(S)-PPD in MCF-7 cells at 60 M ( 0.05), and the percentage of cells in G0/G1 phase were 53.89 8.55%, 56.62 7.62%, 61.31 8.12%, and 74.61 4.67%, respectively (Figure 3B). To investigate the underlying mechanism of G0/G1 cell arrest induced by 20(S)-PPD, European blot was used to analyze cell cycle regulatory proteins. After 20(S)-PPD treatment, the level of p53 was markedly elevated and the expression of p27kip1, c-myc, CDK 4, and cyclin D1 were dramatically decreased by varying degrees at high doses (30C60 M) (Figure 3C,D). These data suggest that 20(S)-PPD exhibited cell viability inhibition of MCF-7 cells via inducing G0/G1 phase cell arrest. Open in a separate window Figure 3 Effects of 20(S)-PPD on cell cycle arrest and the arrest-related proteins in MCF-7 cells. (A,B) Flow cytometry was used to detect cell cycle distribution. After 24 h treatment with 20(S)-PPD (0, 15, 30, and 60 M), a propidium iodide (PI) staining assay was performed on MCF-7 cells. (C,D) In MCF-7 cells treated with 20(S)-PPD, the expression of cell cycle arrest-related proteins p53, p27kip1, c-myc, CDK 4, and cyclin D1 was detected by Western blot. G0 phase is a resting phase where the cell has left the cycle and has stopped dividing. G1 Phase is the first phase within interphase, from the end ACY-1215 small molecule kinase inhibitor of the previous M phase until the beginning of DNA synthesis. S phase starts when DNA synthesis commences, when it is complete, all of the chromosomes have been replicated. G2 phase occurs after DNA replication and is a period of protein synthesis and rapid cell growth to prepare the cell for mitosis. M phase is called chromosome separation phase. All data were represented as suggest ACY-1215 small molecule kinase inhibitor S.D. * 0.05 in comparison to 0 M. 2.3. 20(S)-PPD-Induced Apoptosis Was Reversed by Transfection with mTOR Plasmid To determine if the PI3K/AKT/mTOR signaling pathway performed a leading part of 20(S)-PPD-induced MCF-7 cell apoptosis, mTOR plasmid was transiently transfected in to the cells which were consequently incubated with 20(S)-PPD (30 M) for 24 h. After transfection with mTOR plasmid, the manifestation of mTOR considerably was upregulated, as noticed by Traditional western blot evaluation, and cell viability was also improved weighed against treatment with 20(S)-PPD (30 M) just (Shape 4A). As demonstrated in Shape 4B, transfection with mTOR plasmid could weaken the result of 20(S)-PPD-induced apoptosis. Furthermore, Western blot evaluation indicated how the protein manifestation of Bax, Bcl-2, and p-mTOR (Ser2448) had been controlled by 20(S)-PPD, and these results mediated by 20(S)-PPD had been partially reversed by transfection with mTOR plasmid (Figure 4C,D). Open in a separate window Figure 4 20(S)-PPD-induced apoptosis was reversed by transfection with mTOR plasmid. (A) Expression of mTOR after transfection of mTOR plasmid ACY-1215 small molecule kinase inhibitor was viewed by Western blot (upper line). After 20(S)-PPD (30 M) treatment in MCF-7 cells for 24 h, the MTT assay was used to determine the cell viability (lower line). (B) Flow cytometry was used.