20(S)-25-methoxyl-dammarane-3beta, 12beta, 20-triol, a novel natural product for prostate cancer therapy: activity in vitro and in vivo and mechanisms of action

Abstract

We recently isolated 20(S)-25-methoxyl-dammarane-3beta, 12beta, 20-triol (25-OCH3-PPD), a natural product from Panax notoginseng, and demonstrated its cytotoxicity against a variety of cancer cells. Here we report the effects of this compound in vitro and in vivo on human prostate cancer cells, LNCaP (androgen-dependent) and PC3 (androgen-independent), in comparison with three structurally related ginsenosides, ginsenoside Rh2, ginsenoside Rg3, and 20(S)-protopanaxadiol. Of the four test compounds, 25-OCH3-PPD was most potent. It decreased survival, inhibited proliferation, induced apoptosis, and led to G1 cell cycle arrest in both cell lines. It also decreased the levels of proteins associated with cell proliferation (MDM2, E2F1, cyclin D1, and cdks 2 and 4) and increased or activated pro-apoptotic proteins (cleaved PARP, cleaved caspase-3, -8, and -9). In LNCaP cells, 25-OCH3-PPD inhibited the expression of the androgen receptor and prostate-specific antigen. Moreover, 25-OCH3-PPD inhibited the growth of prostate cancer xenograft tumours. Combining 25-OCH3-PPD with conventional chemotherapeutic agents or with radiation led to potent antitumour effects; tumour regression was almost complete following administration of 25-OCH3-PPD and either taxotere or gemcitabine. 25-OCH3-PPD also demonstrated low toxicity to noncancer cells and no observable toxicity in animals. In conclusion, our preclinical data indicate that 25-OCH3-PPD is a potential therapeutic agent against both androgen-dependent and androgen-independent prostate cancer.

Figure 1

Chemical structures and anticancer activities in vitro. (A) Structures of 25-OCH₃-PPD, PPD, Rg3, and Rh2. (B) Antiproliferative effects of the compounds on prostate cancer cells. LNCaP (B1) and PC3 (B2) cells were exposed to various concentrations of the four compounds for 24 h followed by assessment of cell proliferation by BrdUrd. All assays were performed in triplicate. (C) Induction of apoptosis in LNCaP (C1) and PC3 (C2) cells. These cells were exposed to various concentrations of ginsenosides for 48 h followed by assessment of apoptosis. All assays were performed in triplicate.

Figure 2

Effects of ginsenosides on cell cycle progression of prostate cancer cells. LNCaP (A) and PC3 (B) cells were exposed to various concentrations of the compounds for 24 h followed by determination of cell cycle distribution. All assays were performed in triplicate.

Figure 3

Effects of ginsenosides on expression of various cell cycle-related (A and B) and apoptosis-related (C and D) proteins in human prostate cancer cells. LNCaP (A and C) and PC3 (B and D) cells were exposed to various concentrations of the compounds for 24 h, and the target proteins were detected by immunoblotting with specific antibodies.

Figure 4

Effects on AR and PSA. (A) Comparison of the effects of 72-h exposure of LNCaP cells to increasing concentrations of 25-OCH₃-PPD, Rg3, Rh2, or PPD with resveratrol on secreted PSA levels in conditioned medium. (B) Effects of exposure for 24 h to 50 μM of 25-OCH₃-PPD (lane 2), resveratrol (lane 3), Rg3 (lane 4), Rh2 (lane 5), and PPD (lane 6) on cellular PSA and AR protein expression, as determined by western blots. (C) Comparison of the effects of 25-OCH₃-PPD with resveratrol on the steady-state level of mRNA transcripts of PSA, AR, and NSE after 24 h of exposure, as determined by reverse transcriptase–polymerase chain reaction. Neuron-specific enolase mRNA served as a molecular marker for neuroendocrine differentiation.

Figure 5

Antitumour activity and effects on body weight of 25-OCH₃-PPD administered alone or in combination with taxotere, gemcitabine, or radiation to nude mice bearing PC3 xenograft tumours. 25-OCH₃-PPD was given by intraperitoneal injection at doses of 5, 10, or 20 mg kg⁻¹ day⁻¹, 3 days per week for 4 weeks (A1 and A2), or 5 or 10 mg kg⁻¹ day⁻¹, 5 days per week for 4 weeks (B1 and B2). Taxotere (15 mg kg⁻¹) (C1 and C2) or gemcitabine (160 mg kg⁻¹) (D1 and D2) was given on days 5 and 12 by intraperitoneal injection, radiation (3 Gy) (E1 and E2) was administered on days 5, 12, and 19. The dose of 25-OCH₃-PPD was 10 mg kg⁻¹, 5 days per week for studies combining ginsenoside with conventional therapy.

Figure 6

Proposed mechanisms of action. The cartoon, showing the effects of 25-OCH₃-PPD on various proteins, demonstrates how the compound could exert its anticancer effects.