CD133 expression was evaluated after the administration of 10 μM lupeol for 72 hours. Western blot analysis revealed that CD133 expression decreased in a time-dependent manner learn more in both Huh-7 and PLC-8024 cells (Fig. 2C). Moreover, using flow cytometry analysis, CD133 expression was found to decrease by 86% and 82% in Huh-7 and PLC-8024 cells, respectively, following lupeol administration (Fig. 2D). Accompanied with decrease of CD133 expression upon lupeol treatment, stemness genes including Sox2, Oct4, Nanog, Nestin, and β-catenin were down-regulated (Fig. 2E). CD133+ cells demonstrated resistance to chemotherapeutic agents compared with CD133− cells,28 and expansion of the CD133+ population was observed
in PTEN knockout mice.29 Based on the results shown in Fig. 2B, we hypothesized that lupeol chemosensitized HCC cells to chemotherapeutic agents through modulation of the PTEN pathway. Using MTT assay, the IC10 and IC30 of Huh-7 and PLC-8024 cells in response to cisplatin and doxorubicin were determined. The IC10 and IC30 values, defined as 10% and 30% growth inhibition of Huh-7 and PLC-8024 cells in response to cisplatin or doxorubicin, were 0.5 and 0.9 μg/mL (Huh-7, cisplatin), 0.3 and 0.6 μg/mL (PLC-8024, cisplatin), 0.7 and 1.45 μg/mL (Huh-7, doxorubicin), and 0.25 and AZD3965 in vivo 0.66 μg/mL (PLC-8024,
doxorubicin) (Fig. 3A). When lupeol (10 μM) was combined with cisplatin at the IC10 and IC30 doses, growth was inhibited by 32.3% and 58.2%, respectively, in Huh-7 cells and 30.1% and 55.2%, respectively, in PLC-8024 cells (Fig. 3A). Similarly, when lupeol (10 μM) was combined with doxorubicin at the IC10 and IC30 doses, growth was inhibited by 40.2% and 69.2%, respectively, in Huh-7 cells and 37.2% and 61.3%, respectively, in PLC-8024 cells (Fig. 3A). To determine whether this suppressive growth effect
of lupeol was mediated through the PTEN pathway, we evaluated PTEN and Akt protein expression when Huh-7 and PLC-8024 were treated with 10 μM lupeol. Western blot analysis revealed that increased PTEN protein levels were accompanied by decreased expression levels of phosphorylated AktSer473 (Fig. 3B). Akt has been shown to regulate ABCG2 expression in stem-like cells in glioma,30 which is important in drug efflux response to chemotherapeutic agents. Consistent with these findings, we observed selleck screening library a decrease in ABCG2 protein expression and a decrease in AktSer473 phosphorylation (Fig. 3B). We evaluated the role of PTEN in chemoresistance and formation of hepatospheres by knocking down PTEN expression in HCC cells using short hairpin RNA knockdown approach. Upon PTEN knockdown in Huh-7 and PLC-8024 cells, AktSer473 expression was up-regulated, whereas CD133 and ABCG2 protein expression was consistently decreased in the two PTEN knockdown clones (#2130 and #31001) each of Huh-7 and PLC-8024 (Fig. 4A). Knockdown of PTEN also decreased hepatosphere formation and the ability to form secondary hepatospheres (Fig. 4B).