Phellinus linteus activates different pathways to induce apoptosis in prostate cancer cells

Abstract

It is known that polysaccharides extracted from the Phellinus linteus (PL) mushroom possess antitumour activity. We previously have demonstrated that high doses of PL render murine or human lung cancer cells susceptible to apoptosis. However, the molecular mechanisms of PL-mediated apoptosis have not been fully explored. In this study, we demonstrate that LNCaP cells expressing the androgen receptor (AR) are highly susceptible to apoptosis in response to treatment with high doses of PL. In this process, caspase 8 and its downstream effectors (such as BID), as well as ER stress-related, apoptotic signalling, are activated. In contrast, a moderate amount of apoptosis occurs in PC3 cells (that lack AR) after the same treatment, which does not activate ER-mediated apoptotic signalling. We also show that, in the process of PL-induced apoptosis, caspase 2 is induced in LNCaP cells, but not in PC3 cells. However, LNCaP cells that express a mutated AR or LNCaP cells treated with a caspase 2 inhibitor blocked ER stress-induced apoptotic signals. The magnitudes of the induction of apoptosis in these cells are comparable with what occurred in the PC3 cells. The data demonstrate that high doses of PL activate the AR-dependent and independent apoptotic pathways. Our study also suggests that caspase 2 is a key target in the determination of the susceptibility of prostate cancer cells to PL-induced apoptosis.

Figure 1

AR function and PL-mediated DNA fragmentation. (A) PrEC, LNCaP and PC3 cells were cotransiently transfected with an AR-responsive element-luciferase and β-gal (internal control) constructs. Forty-eight hours post-transfection, cells were cultured in the medium without growth factors for 6 h and stimulated with DHT for 24 h. Subsequently, luciferase activity was analysed. Error bars represent the standard deviation over five independent experiments. (B) The cells were treated with different doses of PL for 48 h, and the percentage of the cells with fragmented DNA was analysed by a flow cytometer. Error bars represent the standard deviation over five independent experiments. (C) After treatment with PL, the percentage of the cells stained with Annexin V was measured.

Figure 2

Activation of caspase 8, caspase 3 and BID in response to treatment with PL. (A) After treating with different concentrations of PL, cell lysates from PrEC, LNCaP and PC3 cells were prepared. Expression of the activated form of caspase 8 was determined by Western blot. Equal loading of total proteins was verified by β-actin expression. (B) After PL treatment, cell lysates were prepared to analyse caspase 8 activity (B–D). Following the treatments, lysates were prepared to analyse the presence of the cleaved, active forms of caspase 3 or BID. Equal loading of total proteins was verified by β-actin expression.

Figure 3

Caspase 2 activation in response to different doses of PL treatment in prostate cancer cells. (A) After treating LNCaP and PC3 cells, cell lysates were prepared and subsequently analysed for the expression of caspase 2. Equal loading of total proteins was verified by β-actin expression. (B) After PL treatment, cell lysates were prepared to analyse caspase 2 activity.

Figure 4

Expression of the ER proteins BiP and ATF-4 in PL-treated prostate cancer cells. LNCaP and PC3 cells were untreated or treated with 1 mg ml⁻¹ of PL for 24 h and lysates were prepared. Subsequently, immunoblotting was conducted to detect BiP (A) and ATF-4 (B) expression. Equal loading of total proteins was verified by β-actin expression.

Figure 5

Effect of mutant AR or caspase 2 inhibitor on PL-induced apoptosis. (A) After treating LNCaP or LNCaP cells expressing a mutated AR with different doses of PL, the percentage of DNA fragmentation was measured by a flow cytometer (upper panel). The lysates from untreated or treated cells were also prepared, and immunoblotting was conducted to detect the expression of BiP and ATF-4 (lower panel). Equal loading of total proteins was verified by β-actin expression. (B) LNCaP, mutant LNCaP and PC3 cells were treated with a caspase 2 inhibitor before PL treatment. Subsequently, the percentage of DNA fragmentation was measured by a flow cytometer. (C) The lysates from untreated or treated LNCaP or mutant LNCaP cells were also prepared for immunoblotting to detect the expression of BiP and ATF-4. Equal loading of total proteins was verified by β-actin expression.