Phellinus linteus sensitises apoptosis induced by doxorubicin in prostate cancer

Abstract

It has been demonstrated that the Phellinus linteus (PL) mushroom, which mainly consists of polysaccharides, possesses antitumour activity. The mechanisms of PL against malignant growth remain unknown. The anticancer drug doxorubicin (Dox) has been shown to induce apoptosis via initiating a caspase cascade. In this investigation, we tested the effect of PL on Dox-induced apoptosis in prostate cancer LNCaP cells. We showed that PL or Dox, at relatively low doses, does not induce apoptosis in the cells. However, combination treatment with low doses of PL and Dox results in a synergistic effect on the induction of apoptosis. In this apoptotic process, caspases 8, 3 and BID are cleaved, and the addition of caspase inhibitor z-VADfmk completely blocks apoptosis. In addition, JNK is activated in response to PL or the combination treatment in LNCaP cells. The suppression of JNK partially inhibits the induction of apoptosis elicited by the co-treatment. These findings indicate that PL has a synergistic effect with Dox to activate caspases in prostate cancer LNCaP cells. Our study also suggests that PL has therapeutic potential to augment the magnitude of apoptosis induced by antiprostate cancer drugs.

Figure 1

DNA fragmentation and [³H]thymidine incorporation in response to the treatment of PL, Dox and PL plus Dox. (A) and (B) LNCaP or PrEC cells were cultured in the growth medium in the presence of different concentrations of PL, Dox, or PL plus Dox. After treatment for 48 h, the percentage of cells with fragmented DNA was analysed by a flow cytometer. Error bars represent the s.d. over five independent experiments. (C) Following serum-starvation (0.5% serum) for 48 h, the cells were refed with growth medium containing 10% serum and [³H]thymidine (2 μCi ml⁻¹) in the presence or absence of PL, Dox or both for 24 h. Subsequently, trichloroacetic acid-insoluble radioactivity was determined. Error bars represent the s.d. from five independent experiments.

Figure 2

Activation of caspase 3, 8 or BID following the treatmet with PL, Dox or PL plus Dox. (A) After treating with PL, Dox, or PL plus Dox, LNCaP cells were harvested and lysates were prepared. The existence of activated forms of caspases 3, 8 and BID was determined by Western blot. Equal loading of total proteins was verified by β-Actin expression. (B) Following the treatments, lysates were prepared to measure the activity of these caspase family members using a colorimetric assay. Error bars represent the s.d. over five independent experiments.

Figure 3

Releasing of cytochrome c to the cytosol following the treatment with PL, Dox, or PL plus Dox. The mitochondrial or cytosolic fractions from untreated or treated cells were isolated and analysed for the expression of cytochrome c by Western blot. Equal loading of proteins in the mitochondrial or cytosolic fraction was determined by reprobing the blot with antitubulin or Bcl-2 Ab.

Figure 4

JNK activation and c-FLIP_L expression. (A) After treatment with PL, Dox or PL plus Dox, cells were harvested and lysates were prepared. The protein expression level of JNK or the presence of the phosphorylated form of JNK was determined by Western blot using the corresponding antibodies. Equal loading of total proteins in each sample was verified by β-actin. (B) c-FLIP_L expression upon treatment with PL, Dox, or PL plus Dox. The cells with or without addition of a dn-JNK were treated with PL, Dox, or PL plus Dox. Subsequently, lysates were prepared to analyse the expression level of c-FLIP_L. The percentages of the cells with fragmented DNA were determined by flow cytometry. The error bars represent the s.d. over five independent experiments.

Figure 5

Effect of JNK inhibition on apoptosis induced by the combination treatment. After introducing a dn-JNK or treatment with a JNK inhibitor, the percentages of DNA fragmentation in LNCaP cells with or without treatment with PL plus Dox were analysed by a flow cytometer. Error bars represent the s.d. over five independent experiments.