Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells

Abstract

Artemisinin is a plant-derived anti-malarial drug that has relatively low toxicity in humans and is activated by heme and/or intracellular iron leading to intracellular free radical formation. Interestingly, artemisinin has displayed anti-cancer activity, with artemisinin dimers being more potent than monomeric artemisinin. Intracellular iron uptake is regulated by the transferrin receptor (TfR), and the activity of artemisinin depends on the availability of iron. We examined the level of TfR in prostate cancer (PCa) tumor cells, synthesized two new artemisinin dimers, and evaluated the effect of dihydroartemisinin and artemisinin dimers, ON-2Py and 2Py, on proliferation and apoptosis in PCa cells. TfR was expressed in the majority of PCa bone and soft tissue metastases, all 24 LuCaP PCa xenografts, and PCa cell lines. After treatment with dihydroartemisinin, ON-2Py, or 2Py all PCa cell lines displayed dose-dependent decrease in cell number. 2Py was most effective in decreasing cell number. An increase in apoptotic events and growth arrest was observed in the C4-2 and LNCaP cell lines. Growth arrest was observed in PC-3 cells, but no significant change was observed in DU 145 cells. Treatment with 2Py resulted in a loss of the anti-apoptotic protein survivin in all four cell lines. 2Py treatment also decreased androgen receptor and prostate-specific antigen expression in C4-2 and LNCaP cells, with a concomitant loss of cell cycle regulatory proteins cyclin D1 and c-Myc. This study shows the potential use of artemisinin derivatives as therapeutic candidates for PCa and warrants the initiation of preclinical studies.

Figure 1. Structures of dihydroartemisinin (DHA), ON-2Py and 2Py

Figure 2. Immunohistochemical analysis of transferrin receptor (TfR) expression

in human PCa bone (Panel A) and lymph node (Panel B) metastases from 22 patients (200-fold magnification). There was no evidence that TfR staining intensity varies between bone and soft tissue metastases. Specific immunostaining was assessed on a four-point scale: 3 = intense, 2 = defined, 1 = faint, and 0 = absent.

Figure 3. Cell number as assessed by crystal violet assay in LNCaP, C4-2 and PC-3 cells

Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO, 10, or 25 µM DHA, ON-2Py or 2Py for 24, 48, and 72 h. Relative cell number was assessed by crystal violet assay (n = 4). Results are expressed as the mean ± SD. * indicates significant difference from control (p < 0.05).

Figure 4. Effect of 2Py on apoptosis and cell cycle in LNCaP, C4-2, PC-3 and DU 145 cells

Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h. The cells were trypsinized and stained with propidium iodide and analyzed on a flow cytometer. The percentage of apoptotic events (Panel A), cell viability (Panel B), and the percentage of cells in G0 and G2M and S phase of the cell cycle (Panels C, D and E) were determined (n=3). Histograms of cell cycle distribution in control and 2Py treated cells (Panel F).

Figure 5. Effect of 2Py on survivin, Bcl-2, and Bax in LNCaP, C4-2, PC-3 and DU 145 cells

Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and western blotting analysis performed using antibodies to survivin (n=4), Bax (n=3), and Bcl-2 (n=3), with β-actin as control.

Figure 6. Effect of 2Py on C-Myc, Cyclin D1, AR, PSA, and β-catenin expression in LNCaP and C4-2 cells

(A) Cells were brought to 50% confluence and then cultured in RPMI 1640 with 10% FBS medium with DMSO or 15 µM 2Py for 48 h, cell lysates were isolated and Western blotting analysis performed using antibodies to Cyclin D1 (n=3), c-Myc (n=3), AR (n=3), PSA (n=3), and β-catenin (n=2) with β-actin as control (n=3). (B) Immunocytochemical analysis of β-catenin in LNCaP and C4-2 cells cultured in 10% FBS medium with DMSO or 15 µM 2Py for 24 h. Arrows indicate cytoplasmic relocalization of β-catenin after 2Py treatment.