Dihydroartemisinin induces apoptosis and sensitizes human ovarian cancer cells to carboplatin therapy

Abstract

The present study was designed to determine the effects of artemisinin (ARS) and its derivatives on human ovarian cancer cells, to evaluate their potential as novel chemotherapeutic agents used alone or in combination with a conventional cancer chemotherapeutic agent, and to investigate their underlying mechanisms of action. Human ovarian cancer cells (A2780 and OVCAR-3), and immortalized non-tumourigenic human ovarian surface epithelial cells (IOSE144), were exposed to four ARS compounds for cytotoxicity testing. The in vitro and in vivo antitumour effects and possible underlying mechanisms of action of dihydroartemisinin (DHA), the most effective compound, were further determined in ovarian cancer cells. ARS compounds exerted potent cytotoxicity to human ovarian carcinoma cells, with minimal effects on non-tumourigenic ovarian surface epithelial (OSE) cells. DHA inhibited ovarian cancer cell growth when administered alone or in combination with carboplatin, presumably through the death receptor- and, mitochondrion-mediated caspase-dependent apoptotic pathway. These effects were also observed in in vivo ovarian A2780 and OVCAR-3 xenograft tumour models. In conclusion, ARS derivatives, particularly DHA, exhibit significant anticancer activity against ovarian cancer cells in vitro and in vivo, with minimal toxicity to non-tumourigenic human OSE cells, indicating that they may be promising therapeutic agents for ovarian cancer, either used alone or in combination with conventional chemotherapy.

Figure 1

DHA selectively decreases cell viability and inhibits the growth of human ovarian carcinoma cells, but not non-tumourigenic ovarian surface epithe-lial cells. (A) Chemical structures of the four artemisinin (ARS) compounds; (B) Viability of human ovarian carcinoma cells (ovarian carcinoma A2780 and OVCAR-3) and non-tumourigenic OSE cells (IOSE144) after 48 hrs exposure to the ARS compounds as determined by MTT assay; C, Cell growth inhibition after 0, 24, 48 and 72 hrs exposure of A2780 and OVCAR-3 cells to DHA. Values are representative of at least three independent experiments with similar results, and are presented as the percentage of cell inhibition where vehicle-treated cells were regarded as 100% viable/0% growth inhibition.

Figure 2

DHA induces dose-dependent apoptosis in human ovarian carcinoma cells. (A) Apoptosis in A2780 and OVCAR-3 cells treated with DHA (0, 5, 10, 25, 50 μM) for 24 hrs; (B) Data summary and analysis (*, P < 0.001 versus the control, respectively). Data are representative of values from at least three independent experiments with similar results. The percentage of cellular apoptosis in control cells was regarded as 100%.

Figure 3

Western blot analysis of protein expression levels indicating the effects of DHA on A2780 and OVCAR-3 ovarian cancer cell lines after 24 hrs exposure to specific dosesof DHA.

Figure 4

DHA causes disruption of the mitochondrial membrane potential and cytochrome c release. (A) and (B) Fluorescence (red and green) intensity values emitted by JC-1 fluorescent dye at specific excitation wave-lengths (detailed information is described in the Materials and methods) and the corresponding ratio of red/green (% of the control) after exposure to different concentrations of DHA for 24 hrs. Values are representative of at least three independent experiments with similar results (*, P < 0.001 versus the control). (C) Western-blot analysis of the effects of 24 hrs DHA exposure (0, 5, 10, 25, 50 μM) on cytochrome c release from the mitochondria to the cytosol (mito-, mitochondrial; cyto-, cytoplasmic).

Figure 5

DHA significantly decreases cell viability and inhibits cell growth in human ovarian carcinoma cells by increasing apoptosis, both alone and in combination with carboplatin (CBP). (A) Viability of ovarian epithelial cells after 48 hrs exposure to CBP (0, 1, 10, 50,100, 500,1000 μM) in the presence or absence of 1 μM DHA; (B) Cell growth inhibition following exposure to CBP (10 μM) with 1 μM DHA for 0, 24, 48 or 72 hrs; (C) Apoptosis of ovarian cancer cells after exposure to 500 μM CBP with or without 1 μM DHA for 24 hrs, and the corresponding data summary and analysis (*, P < 0.001 versus the control, respectively).

Figure 6

DHA significantly inhibits tumour growth and induces apoptosis alone or in combination with carboplatin (CBP) in mice bearing A2780 and OVCAR-3 xenograft tumours. (A) and (B) Inhibition of tumour growth in mice bearing A2780 or OVCAR-3 xenograft tumours, and the corresponding body weight changes during the treatments; (C) Western-blot analysis of proteins involved in the apoptotic pathway.

Figure 7

Cartoons of the proposed mechanisms of action of DHA alone and in combination with CBP: (A) The ‘death receptor- and mitochondrion-mediated caspase-dependent apoptotic pathway’ demonstrates how DHA may exert anticancer effects in ovarian cancer cells; (B) Mechanism by which DHA enhances the therapeutic effects of carboplatin.