The major findings in our current study are that high doses of PL elicit two major apoptotic pathways in prostate cancer cells: a caspase 8-induced cascade and the unfolded protein response. The activation of caspase 8 and its downstream effectors may be a general effect of PL on tumours. The unfolded protein response in the ER appears to be prostate cancer specific, and is regulated by the AR and caspase 2. Our study suggests a molecular target of PL, which provides a better understanding of the potential of PL to treat prostate cancer.
It is known that PL is not toxic in general (Song et al, 1995
). We have demonstrated that PL, at high doses, causes lung epithelial cells to arrest in the G1
phase of the cell cycle by blocking the expression of cyclin D1
and its further interaction with cell cycle-dependent kinases 4 and 6 (Guo et al, 2006
). It is possible that treatment with PL elicits G1
checkpoint control in normal prostate epithelial cells, such as PrEC, resulting in the cessation of cell cycle progress. In tumour cells, cell cycle checkpoints are often impaired. Under such conditions, high doses of PL become apoptotic and activate the cell death programme in prostate cancer cells.
It has been reported that caspase 2 expression is regulated by the AR. As an unusual member of the caspase family, caspase 2 possesses features that can both initiate and execute programmes of cell death (Harvey et al, 1997
; Droin et al, 2001
). Activation of caspase 2 results in a complex that consists of the death-domain-containing protein and the adapter protein. It has also been shown that activated caspase 2 can directly cause the cleavage of BID (Harvey et al, 1997
; Droin et al, 2001
). In addition, studies have demonstrated that caspase 2, upon different apoptotic stimulations, re-distributes to the nuclei or other subcellular membrane compartments to participate in the execution of the cell death programme (Mancini et al, 2000
; Chae et al, 2004
; Rokhlin et al, 2005
). Our results here demonstrate the interconnection between AR and caspase 2 in the regulation of PL-induced apoptosis in prostate cancer cells. Studies to identify molecular targets in the cooperation between AR and caspase 2 during PL-induced apoptosis are under way.
In the progression of prostate cancer, AR is often mutated or its expression is lost, which plays an important role in the development of the resistance of cancer cells to treatment. Therefore, the discovery of molecular targets 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. It has been shown that overexpression of caspase 7 in LNCaP cells could effectively induce apoptosis, and this has been very promising clinically (Marcelli et al, 1999
). Our present study indicates that caspase 2 is an intracellular target for upregulating the susceptibility of PC3 cells or prostate cancer cells containing a mutated AR to PL-induced apoptosis.
Activation of caspase family members is at the core of apoptosis, representing a point of intersection of various apoptotic pathways. Tumour necrosis factor or Fas/CD95 receptors, mitochondrial proteins (such as cytochrome c
) and granzyme are able to induce cell death through activation of the caspase cascade (Schmitz et al, 2000
; Shi, 2002
). Damage to or stress in the ER or Golgi has been shown to be able to trigger apoptosis (Herr and Debatin, 2001
; Breckenridge et al, 2003
). Studies have shown that the unfolded protein response or lack of calcium is responsible for ER-mediated apoptosis (Breckenridge et al, 2003
). ER stress has been demonstrated to cause the translocation of certain caspases to the ER or Golgi and the execution of apoptosis there. Calcium released from the ER during times of ATP deficiency is an important element in apoptosis induced by ischaemia–reperfusion injury. It is possible that PL treatment upregulates and activates caspase 2 that subsequently translocates to the ER and causes the unfolded protein response, resulting in apoptosis.
Caspases 8, 6 and 3 are major players in caspase-induced apoptosis (Nicholson and Thomberry, 1997
). We demonstrated that PL, at low doses, is able to synergise with anticancer drugs (such as doxorubicin) for the induction of apoptosis in LNCaP cells (Collins et al, 2006
). We also demonstrated that high doses of PL elicit a caspase cascade in human and murine lung cancer cells, but not in normal lung epithelial cells, in which caspase 3, caspase 8 and BID are activated (Guo et al, 2006
). In this study, we further demonstrate that treatment with high doses of PL can activate caspase 8-initiated apoptotic signalling in both AR-expressing (LNCaP) and AR-null (PC3) prostate cancer cells. It seems that the high doses of PL, by activating the caspase 8-regulated signalling pathway, are generally toxic to various types of tumours and have no effect on normal cells.
The successful treatment of prostate cancer requires identification of specific intracellular targets for sensitising the tumour to apoptosis. Our previous study demonstrated that PL, at low doses, acts as an enhancer to sensitise anticancer drug-mediated, apoptotic signalling, and this sensitization can be obtained at subtoxic concentrations of the drug. In this study, we showed that high doses of PL can mobilize multiple apoptotic signalling pathways to cause different degrees of cytotoxic effects on prostate cancer cells, depending on the expression of AR. We also conclude that caspase 2, in an AR-dependent fashion, may be a specific intracellular switch for the regulation of the susceptibility of tumour cells. Our data suggest that PL, by modulating caspase activity, can be developed for more efficient therapies against refractory prostate cancer.