The diarylheptanoid hirsutenone sensitizes chemoresistant ovarian cancer cells to cisplatin via modulation of apoptosis-inducing factor and X-linked inhibitor of apoptosis

Abstract

Cisplatin (CDDP) and its derivatives are considered first-line treatments for ovarian cancer (OVCA). However, despite initial results that often appear promising, in most cases patients will return with recurrent disease that fails to respond to further chemotherapy. We assayed a number of food phytochemicals with reported PI3K inhibitory ability to identify candidates that can influence CDDP treatment outcomes in chemoresistant OVCA cell lines. A direct comparison revealed that the diarylheptanoid hirsutenone from the tree bark of Alnus hirsuta var. sibirica was superior at inducing CDDP sensitivity in a number of chemoresistant cancer cell lines. Whereas hirsutenone treatment activated p53, its modest efficacy in p53-mutant and -null cell lines suggested the existence of a p53-independent mode of action. Further investigation revealed that hirsutenone causes CDDP-dependent apoptosis in chemoresistant cells by ubiquitin-proteasome-dependent X-linked inhibitor of apoptosis degradation and by enhancing the translocation of apoptosis-inducing factor from the mitochondria to the nucleus. This was found to be, at least in part, under the influence of upstream Akt activity, linking hirsutenone-dependent PI3K inhibition with downstream effects on apoptosis-inducing factor, X-linked inhibitor of apoptosis, and apoptosis. Our findings provide rationale for further investigation of the effects of hirsutenone on chemoresistant OVCA in clinical studies.

Keywords: AIF; Akt; Chemoresistance; Hirsutenone; Ovarian Cancer; XIAP; p53.

FIGURE 1.

In vitro kinase assay data showing inhibitory properties of various food phytochemicals with reported PI3K inhibitory property. PI3K protein was preincubated with the indicated test compounds for 10 min at 30 °C and then incubated with phosphatidylinositol substrate and 10 μCi of [γ-³²P]ATP for an additional 10 min at 30 °C. The resulting ³²P-labeled phosphatidylinositol 3-phosphate was measured as described under “Experimental Procedures.” LY294002 was used as a positive control. BLK, blank without PI3K, includes substrate and ATP; CTL, control, PI3K without sample; Api, apigenin; Cya, cyanidin; Del, delphinidin; Lut, luteolin; Myr, myricetin; Pic, piceatannol; Que, quercetin; LY, LY294002; Iso, isorhamnetin; 734, 7,3,4′,4′-trihydroxyisoflavone; 674, 6,7,4′,4′-trihydroxyisoflavone; Aca, acacetin; Hir, hirsutenone.

FIGURE 2.

Comparison of the influence of food phytochemicals alone or in combination with CDDP on OVCA cell viability in vitro. Compounds with reported PI3K-inhibitory ability (10 μm) were assessed in the presence or absence of CDDP (10 μm; 24 h). Hirsutenone was more potent in sensitizing chemoresistant p53 wild-type OVCA (OVCAR-433, C13, and Hey) to CDDP. Hirsutenone decreased cell viability in p53-null/p53-mutant chemoresistant OVCA. DMSO was used as vehicle control; 24 h; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus respective DMSO control). +, p < 0.05; ++, p < 0.01; +++, p < 0.001 (versus respective CDDP-only treatment). ###, p < 0.05 (versus respective LY294002 plus CDDP treatment). Results are expressed as mean ± S.E. (error bars; n = 3 independent experiments).

FIGURE 3.

Hirsutenone (Hirs) sensitizes p53 wild-type chemoresistant OVCA cells to CDDP to a greater extent than null/mutant OVCA. Upper four panels, effects of CDDP (10 μm), hirsutenone (10 μm), and combined treatment on apoptosis in p53 wild-type chemoresistant OVCA. Hirsutenone treatment induces a left shift in concentration-response curves for CDDP-induced apoptosis in chemoresistant C13 and OVCAR-433. Lower four panels, hirsutenone-induced sensitivity in chemoresistant p53-mutant A2780cp* and p53-null SKOV3 cells at 10 μm concentration. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus respective DMSO control. Error bars, S.E. Apoptosis was assessed via the quantification of fragmented nuclear morphology, as described under “Experimental Procedures.”

FIGURE 4.

Reconstitution of wild-type p53 in p53-mutant and -null OVCA enhances hirsutenone-induced apoptosis. p53-mutant (A2780cp) and p53-null (SKOV3) OVCA cell lines were infected with adenoviral wild-type p53 or GFP control (multiplicity of infection = 10; 24 h). The presence of wild-type p53 induced sensitivity to CDDP, while enhancing apoptosis caused by hirsutenone alone (10 μm) and in combination with CDDP (10 μm). These events occurred with concomitant activation of p53 with serine 15 phosphorylation. Apoptosis was quantified using the Hoechst assay as described under “Experimental Procedures.” **, p < 0.01; ***, p < 0.001; error bars, S.E.

FIGURE 5.

Influence of hirsutenone (Hirs) on phospho-Akt, phospho-p53, caspase-3, and XIAP contents. A, hirsutenone down-regulates phospho-Akt contents (Ser⁴⁷³ and Thr³⁰⁸) and increases phospho-p53 levels in chemoresistant C13 cells. Cells were treated with CDDP (10 μm) and/or hirsutenone (10 μm) for 24 h. B, hirsutenone affected pro- and activated caspase-3 expression in chemoresistant C13 cells. Azacytidine (Aza; 5 μm) was used as a positive control for activated caspase-3. C, hirsutenone treatment (10 μm; 24 h) down-regulates XIAP in a concentration-dependent manner in chemoresistant (C13*) OVCA. D, hirsutenone-induced XIAP down-regulation in chemoresistant C13 cells is attenuated by the proteasome inhibitors epoxomicin (15 nm) and lactacystin (10 μm) but not by the pan-caspase inhibitor Z-VAD-fmk (20 μm). IP, immunoprecipitation; IB, immunoblotting. E, immunoprecipitation hirsutenone treatment effect on XIAP ubiquitination was analyzed. C13 cells were treated as indicated after transfection with ubiquitin-HA constructs (1 μg) in the presence of the proteasome inhibitor epoxomicin (15 nm). Native XIAP protein was immunoprecipitated using a monoclonal antibody conjugated to magnetic Dynabeads (Invitrogen).

FIGURE 6.

Effects of hirsutenone (Hirs) on CDDP-induced apoptosis are AIF-dependent and involve XIAP-AIF interaction as well as Akt inhibition. A, AIF silencing rescues C13 cells from apoptosis induced by hirsutenone and CDDP. Cells were transfected with AIF siRNA (100 nm, 48 h) or scrambled control siRNA prior to hirsutenone (10 μm) and CDDP (10 μm) treatment. Transfection with AIF siRNA significantly down-regulated AIF protein content while concomitantly attenuating apoptosis caused by hirsutenone treatment (10 μm) alone and in combination with CDDP (10 μm). B, immunoprecipitation (IP) of C13 whole cell lysate demonstrates XIAP-AIF interaction, which is enhanced by the presence of hirsutenone (10 μm). Proteins from C13 cell lysate were immunoprecipitated using anti-AIF or IgG (control antibodies) anchored to Invitrogen Dynabeads. IB, immunoblotting. C, Akt is an upstream modulator of hirsutenone action. Myristoylated AAkt1 (constitutively active Akt1) expression attenuates the cytotoxic action of CDDP (10 μm) and hirsutenone (10 μm) in C13 cells. Pc-Myr-Akt vectors were transfected into C13 cells prior to treatment as indicated. Error bars, S.E.

FIGURE 7.

Hirsutenone-facilitated CDDP-induced apoptosis in chemoresistant OVCA cells is mediated by AIF and by suppressed XIAP-AIF interaction. A, effects of hirsutenone (Hir) (10 μm) and CDDP (10 μm) treatment (12 h) on AIF nuclear translocation, in the presence and absence of XIAP overexpression. C13 cells were transfected with XIAP overexpression constructs (Pc-XIAP, 0.1 μg, 48 h) or GFP control constructs (Pc-GFP, 0.1 μg, 48 h), prior to treatment. Blue, DAPI; red, AIF; green, TOM20 (mitochondrial membrane marker). B, effects of XIAP overexpression on apoptosis induced by hirsutenone (10 μm) and CDDP (10 μm) treatment (24 h). C13 cells were transfected with XIAP overexpression constructs (Pc-XIAP, 1 μg, 48 h) or GFP control constructs (Pc-GFP, 1 μg, 48 h), prior to treatment. C, quantification of AIF nuclear localization data shown in A. Nuclear signal was quantified using ImageJ software. *, p < 0.05; **, p < 0.01 versus respective DMSO control. Error bars, S.E..

FIGURE 8.

Hypothetical model illustrating the actions of hirsutenone (HIR) and CDDP in chemoresistant OVCA. Although many further aspects remain to be determined, the PI3K/Akt pathway is known to be frequently overexpressed and XIAP stabilized at high levels in chemoresistant OVCA (29). A, Akt appears to inhibit apoptosis by stabilizing both XIAP and MDM2 at the protein level via direct phosphorylation of these proteins (12, 30). B, hirsutenone suppresses Akt function by inhibiting PI3K and induces degradation of XIAP and MDM2. C, in the presence of CDDP, hirsutenone further enhances p53 activation, down-regulates XIAP and MDM2 content, and facilitates AIF nuclear translocation, the latter possibly through direct action of p53 at the mitochondrial level (28).