Chemopreventive effect of PSP through targeting of prostate cancer stem cell-like population

Abstract

Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer.

Figure 1. PSP down-regulates prostate CSC markers in PC-3 cells.

A) Western blotting of prostate CSC markers CD44 and CD133 in PC-3 cells after PSP treatment. Note that PSP significantly down-regulates both stem cell markers in a dose- and time-dependent manner. B) Viability of PC-3 cells after treatment with 5, 25, 125, 250 and 500 µg/ml of PSP for 48 or 72 hr was measured with MTT assay. Results are presented as mean ± s.d. C) Flow cytometry analysis of PC-3 cells after treatment with 250 µg/ml of PSP for 72 hr. Note that no significant difference in cell cycle distribution was observed. D) Western blotting results for apoptotic markers (left panel) and stem cell maintenance proteins (right panel) in PC-3 cells after PSP treatment. Note that no changes in Bax and Bcl-2 or cleavage of PARP were detected.

Figure 2. Effects of PSP on CSC properties.

A) Spheroid formation assay was performed with PC-3 and Du145 cells. Two hundred of cells were seeded onto polyHEMA pre-coated plates and treated with either 500 µg/mL of PSP or vehicle for 14 days. The number of prostaspheres formed was counted, and the result was presented as the mean ± s.d. Note that γ-T3 treatment efficiently suppresses the spheroid formation ability of PC-3 cells. Image of the prostaspheres was captured under microscope. Note that no prostaspheres can be found in cells treated with 500 µg/mL of PSP. (B) PSP inhibited the formation of secondary prostaspheres. Primary prostaspheres were dissociated and re-seeded into polyHEMA pre-coated plate. PSP was added 24 hr after the plating. Note that prostasphere formation was inhibited by more than 70% and 90% in the presence of 250 µg/mL and 500 µg/mL of PSP respectively. * P<0.001, ** P<0.0001, t test.

Figure 3. PSP inhibits tumorigenicity of PC-3 cells in vivo.

A) Bioluminescent images of SCID mice orthotopically injected with PC-3-luc cells for two weeks. SCID mice in the upper row were injected with vehicle-treated PC-3-luc cells, whereas mice in the bottom row were injected with PSP-treated PC-3-luc cells. B) Table summarizes the percentages of mice developing detectable tumors at week 2. Approximately 40% of mice in the PSP pretreated group did not form detectable tumors, whereas 100% tumor formation was found in the control group (p = 0.07). C) Selected ex vivo images of the prostate from both groups. Note that in PSP-treated mice with negative luciferase signal, no visible tumor were found in the prostate tissue.

Figure 4. Effect of PSP on PIN development in the TgMAP transgenic mouse model.

A) Time frame of the PIN and PCa development in TgMAP mice and the schedule of the PSP treatment. Fourteen-week old TgMAP mice were treated with 200 mg/kg of PSP by oral gavage feeding for 4 weeks and sacrificed at the time when PIN has developed (20 weeks old). The table summarizes the results of the histology examination of the prostate from the vehicle- and PSP-treated TgMAP mice. C) Representative photos of the Hematoxylin & Eosin staining of the prostatic tissues from the TgMAP mice. Note that both control- and PSP-treated TgMAP mice developed prostatic intraepithelial neoplasia (PIN), as indicated by the arrows.

Figure 5. Effect of PSP on prostate tumor development of the TgMAP transgenic mouse model.

A) Outline of the schedule for PSP treatment. Eight-week old TgMAP mice were treated with 300 mg/kg of PSP by oral gavage feeding for 20 weeks and sacrificed at age of 28 weeks. B & C) Representative photos of the Hematoxylin & Eosin staining of the prostate tissues from the vehicle and PSP-treated TgMAP mice. Note that tumors were found in all of the mice that were treated with vehicle only but were absent in all the PSP-treated mice. D) The table summarizes the results of the histology examination of the prostate tissues from the vehicle and PSP-treated TgMAP mice. *P<0.05 compared to control treatment by Fisher's exact test. E) Average body weight of the mice during the PSP treatment.