In the apoptotic process induced by the co-treatment of PL and Dox, caspase 8 is activated, which then initiates a caspase cascade, resulting in the release of cytochrome c from the mitochondria to the cytosol. The suppression of caspase activity by a generic caspase inhibitor z-VADfmk completely abolishes apoptosis induced by the co-treatment. Furthermore, the addition of PL alone is able to cause JNK activation, which subsequently downregulates the expression level of c-FLIPL in the cells. The addition of a JNK inhibitor or introduction of a dn-JNK suppresses the apoptotic process induced by the co-treatment. Therefore, our findings suggest that PL is a useful synergiser for augmenting Dox-mediated, apoptotic signalling. As such synergy on the induction of apoptosis only requires low doses of Dox, PL has potential for further developing a better therapeutic strategy to treat prostate cancer and avoid the side effects caused by high doses of Dox.
Because prostate cancer progression is partially affected by development of resistance of the cancer cells to apoptosis induced by anticancer drugs, the ability to sensitise apoptotic signalling pathways has important therapeutic implications. Genetic targeting or pharmacological manipulation of caspase family members and their regulators/modulators would provide better clinical strategies. Targeting the expression of caspase 7 has been demonstrated to be a useful approach for treating prostate cancer (Marcelli et al, 1998
). In this approach, overexpression of caspase 7 in LNCaP cells could effectively induce apoptosis and has been shown to be very promising clinically (Marcelli et al, 1998
). Therefore, the combination treatment of low doses of PL and Dox to activate caspases would have similar implications for treating prostate cancer patients.
Studies using specific caspase inhibitors have shown that Dox can activate caspases 8, 6 and 3 (Hannun, 1997
; Los et al, 1997
). The synergistic effect of Dox and TRAIL on caspase-induced apoptosis has been reported (Wu et al, 2002
). Here, we demonstrated that Dox is also able to synergise with PL at low doses for the induction of apoptosis. In this apoptotic process, caspases 3, 8 and BID are activated, leading to the release of cytochrome c
from the mitochondria to the cytosol. A generic caspase inhibitor blocked this synergistic, apoptotic effect on LNCaP cells, further suggesting the involvement of caspase activity in the cytotoxicity rendered by the combination treatment with PL and Dox. However, at low doses neither Dox nor PL elicited the activity of these caspase family members to induce apoptosis. It is possible that treatment with PL at low doses reduces the threshold of the sensitivity of LNCaP cells to Dox-induced apoptosis.
JNK is activated in response to various stresses. The exact mechanisms through which JNK participates in apoptosis remain unknown. JNK is not a direct apoptotic executor, however, instead, it often modulates the initiation of apoptosis through promoting processing of proapoptotic factors, such as the BH3-domain proteins or decaying of antiapoptotic factors (Luo et al, 1998
). In caspase-induced apoptosis, JNK has been suggested to act at a step upstream to caspase 8 (Deng et al, 2003
). Recently, experiments, in vitro
and in vivo
, have shown that JNK, during TNFα
-induced apoptotic processes, activates ubiquitin ligase Itch, which specifically interacts with c-FLIPL
to promote the degradation of this caspase inhibitor (Chang et al, 2006
). In our experimental settings, a low dose of PL was enough to elicit JNK phosphorylation, but not to induce apoptosis. However, this activation of JNK was responsible for the reduction of c-FLIPL
expression in LNCaP cells. Therefore, JNK, by promoting the degradation process, suppresses c-FLIPL
, and may sensitise the prostate cancer cells to apoptosis. Doxorubicin is known to be able to elicit cellular or DNA damage-induced responses at low-dose ranges and mobilise caspase family members to execute cell death programs at high-dose ranges. It is well possible that JNK, by lowering the threshold of LNCaP cells to apoptosis, synergises with Dox for the activation of caspases.
and Dox, at low doses, are G1
arrest inducers (Guo et al, 2006
). The data from colony formation assays demonstrated that LNCaP cells formed fewer colonies in soft agar medium in the presence of a low dose of Dox or PL alone. In contrast, the combination treatment with these two agents at low doses blocks LNCaP cells from growing and forming colonies in soft agar medium. The reduction of the numbers of colonies following treatment with Dox or PL alone at low concentrations is highly likely due to a negative effect of the treatment on cell proliferation, resulting in slow formation of colonies. In addition, the combination treatment of low doses of PL and Dox is nontoxic to normal prostate epithelial cells, causing only growth cessation. This suggests that PL can selectively sensitise cancer cells for apoptotic signalling.
The development of chemotherapeutic resistance to drugs is a major obstacle to the successful treatment of prostate cancer. A more effective anticancer therapy is required. Our present study demonstrates that PL acts as an enhancer to sensitise Dox-mediated, apoptotic signalling, and this sensitisation can be obtained at subtoxic concentrations of Dox. We previously have reported that PL, even at high doses, only causes normal fibroblasts or lung epithelial cells to arrest in the G1
phase of the cell cycle (Guo et al, 2006
). Altogether, our data suggest that PL is an apoptotic synergiser for conventional chemotherapeutics such as Dox, which can keep normal, surrounding cells unharmed. Therefore, PL, serving as a safe alternative medicine, has potential for more efficient therapies against drug-resistant prostate cancer.