Rhus verniciflua Stokes (RVS) and butein induce apoptosis of paclitaxel-resistant SKOV-3/PAX ovarian cancer cells through inhibition of AKT phosphorylation

Abstract

Background: Rhus verniciflua Stokes (RVS) belongs to the Anacardiaceae family and traditionally used for cancer treatment. RVS and butein, a major compound of RVS, were known to induce apoptosis via AKT inhibition in cancer cells. Thus, in this study, we investigated the effect of RVS and its derivative compounds (fisetin, quercetin, butein) on cell death in SKOV-3/PAX cells.

Methods: The 80 % ethanol extract of RVS and its derivative compounds (fisetin, quercetin, butein) were prepared. The cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Apoptotic cells were detected by staining with propidium iodide (PI) and Annexin V-fluorescein isothiocyanate/7-aminoactinomycin D (Annexin V-FITC/7-AAD). The expression level of intracellular signaling related-proteins in apoptosis and growth were measured by western blot assay.

Results: We found that RVS and butein suppressed the growth of SKOV-3/PAX cells in a dose-dependent manner. We also found that RVS and butein produced the cleavage of caspase-9, -8, -3, and PARP. Similarly, sub-G1 phase and Annexin V-FITC positive cells were increased by RVS and butein. Moreover, RVS and butein significantly reduced AKT phosphorylation in SKOV-3/PAX cells. PI3K inhibitor LY294002 caused PARP cleavage supporting our finding.

Conclusion: Our data clearly indicate that RVS and butein induce apoptosis of SKOV-3/PAX cells through inhibition of AKT activation. RVS and butein could be useful compounds for the treatment for paclitaxel resistant-ovarian cancer.

Keywords: AKT; Apoptosis; Butein; Paclitaxel-resistant ovarian cancer; Rhus verniciflua Stokes.

Fig. 1

Proliferation of parental and paclitaxel resistant ovarian cancer cells by paclitaxel or RVS treatment for 72 hours. a The effect of paclitaxel on the viability in SKOV-3 or SKOV-3/PAX cells was determined by MTT assay. b The effect of RVS on the viability in SKOV-3/PAX cells was determined by MTT assay (mean ± SD; n = 6). *, p < 0.05 versus control in parental. **, p < 0.05 versus control in resistant

Fig. 2

RVS induces apoptotic-cell death in paclitaxel resistant ovarian cancer cells for 24 hours. a Left panel, the cytotoxic effect of RVS in SKOV-3/PAX cells was determined by PI staining assay. Right panel, data represent quantitative results for left panel (mean ± SD; n = 3). *, p < 0.05. b The dead cells were differentiated between apoptosis and necrosis by Annexin V-FITC and 7-AAD dual staining. c The expression of procaspase-9, cleaved caspase-9, cleaved caspase-8, procaspase-3, caspase-3, and PARP in cell lysates from SKOV-3/PAX cells treated with RVS was determined by Western blot assay

Fig. 3

The effect of apoptosis in butein-treated SKOV-3/PAX cells. (a) The effect of butein, fisetin, and quercetin on the viability in SKOV-3/PAX cells for 72 hours was determined by MTT assay. (b) The cytotoxic effect of butein in SKOV-3/PAX cells for 24 hours was determined by PI staining assay. Bottom panel, data represent quantitative results for top panel (mean ± SD; n = 3). *, p < 0.05 versus control. (c) The dead cells were differentiated between apoptosis and necrosis by Annexin V- FITC and 7-AAD dual staining. (d) The expression of procaspase-9, cleaved caspased-9, cleaved caspase-8, procaspase-3, caspase-3, and PARP in cell lysates from SKOV-3/PAX cells treated with butein were determined by Western blot assay

Fig. 4

RVS and butein inhibit AKT signaling in SKOV-3/PAX cells for 2 hours. (a and b) The expression of AKT, Raf-1, ERK, p38, and JNK in cell lysates from SKOV-3/PAX cells treated with RVS or butein were determined by Western blot assay. (c) SKOV-3/PAX cells were pretreated with AKT inhibitor, LY294002 (50 μM) for 1 hour and then added with RVS or butein for another 24 hours. The expression levels of AKT phosphorylation and PARP cleavage were determined by Western blot assay. (d) A schematic model for the apoptosis via inhibition of AKT activity by RVS or butein