In our previous study, we demonstrated that isochaihulactone was efficacious against various models of human solid tumors but not prostate cancer . We also have shown recently that isochaihulactone triggers an apoptotic pathway in human A549 lung cancer cells that occurs via the ERK1/2 and NAG-1 pathway . To clarify the mechanisms of isochaihulactone-induced tumor apoptosis between different types of cancer cells, we further investigated the antitumor potential and mechanisms of isochaihulactone action in human prostate cancer cells. Three human prostate cell lines were used to test the cytotoxicity of isochaihulactone, only the LNCaP prostate cancer cells showed sensitivity to isochaihulactone treatment. This phenomenon might be important to the antitumor potential of isochaihulactone and is discussed later.
In this study, we demonstrated that isochaihulactone apparently induced G2/M cell cycle arrest and cell death in LNCaP cells. The tumor suppressor protein p53 plays a role in the molecular response to DNA damage and cell cycle arrest. The cyclin-dependent kinase inhibitor p21 also helps to maintain G2/M cell cycle arrest by inactivating the cyclin B1/cdc2 complex, disrupting the interaction between proliferating cell nuclear antigen and cdc25c . Our result showed that increased levels of p53 and p21 proteins were expressed in LNCaP cells in response to treatment with isochaihulactone (Figure 2B). The transition from G2 phase to mitosis is triggered by the cdc25c-mediated activation of the cyclin B1/cdc2 complex. Cyclin B1/cdc2 activation is triggered when cdc25c dephosphorylates Thr15 [12, 13]. In our study, isochaihulactone-mediated LNCaP cell cycle arrest at G2/M phase (Figure 2B) was accompanied by decreased expression of cyclin B1 and cdc2 kinase. The decrease in the levels of cdc2 may be due to the decrease in cdc25 activation by phosphorylation, leading to subsequent G2 arrest (Figure 2B).
Activation of aspartate-specific cysteine protease (caspase) represents a crucial step in the induction of drug-induced apoptosis, and cleavage of PARP by caspase-3 is considered to be one of the hallmarks of apoptosis . Isochaihulactone-induced caspase 3 cleavage was observed by immunocytochemistry (Figure 3B), and late-stage apoptosis was revealed by TUNEL staining (Figure 3D). Furthermore, isochaihulactone inhibited Bcl-2 expression, induced caspase-9 and caspase-3 cleavage, and induced PARP activation were also observed (Figure 3E). It is interesting to note that isochaihulactone-induced Bcl-2 phosphorylation, caspase-9 cleavage, and PARP cleavage were observed at nearly the same time point, suggesting that the isochaihulactone-induced Bcl-2 phosphorylation is related apoptosis (Figure 3E). Recent reports have revealed the involvement of JNK-mediated Bcl-2 phosphorylation and degradation, and also the activation of caspase-9 in the apoptosis of both the androgen-dependent and -independent human prostate cancer cells . Bcl-2 and Bcl-XL inhibit apoptosis by regulating the mitochondrial membrane potential, whereas cytochrome c release is required for activation of caspase-9 and subsequent activation of caspase-3 . Thus, increased levels of Bcl-2 phosphorylation, caspase-9 and -3 activation appeared to correlate with mitochondrial apoptosis in isochaihulactone-induced LNCaP cell death.
Many microtubule-destabilizing agents are activators of caspase-9, a major key player in mitochondrial apoptotic pathway [17, 18]. Microtubule depolymerization agents arrest the cell cycle in G2/M phase by acting through several types of kinases, which lead to phosphorylation cascades, activation of the cyclin B1/cdc2 complex, and the phosphorylation of Bcl-2 . The MAPK inhibitor PD98059 has been shown to partially inhibit isochaihulactone-induced cdc2 phosphorylation, causing G2/M arrest in A549 cells. The activation of NAG-1 expression via ERK1/2 pathway is involved in isochaihulactone-induced G2/M arrest in A549 cells [7, 9]. To determine which MAPK family member is involved in the major signaling pathway for isochaihulactone-mediated cell growth inhibition, MAPK inhibitors were used to study the growth inhibition induced by isochaihulactone in LNCaP cells. Only JNK1/2 inhibitor SP600125 significantly decreased the growth inhibition induced by isochaihulactone (Figure 4A), and neither the p38 inhibitor SB203580 nor the ERK1/2 inhibitor PD98059 reversed isochaihulactone-induced growth inhibition. Phosphorylation of JNK kinase was also observed with western blot analysis after isochaihulactone treatment (Figure 4B). In cell cycle analysis, pre-treatment of JNK1/2 inhibitor SP600125 significantly reduces sub-G1 population (Figure 4C). These data suggest that JNK1/2 signaling pathway is involved in isochaihulactone-induce cell death.
Increased NAG-1 expression results in the induction of apoptosis in several cancer cell lines [20, 21]. NAG-1 is induced not only by NSAIDs but also by several anti-tumorigenic compounds including dietary compounds, peroxisome proliferator-activated receptor-γ ligands, phytochemicals [16–18], as well as resveratrol, genistein, diallyldisulfide, 5F203, and retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) [22–24]. NAG-1 appears to be a key downstream target of EGR-1.
In our previously studies, we confirmed the antitumor effect of isochaihulactone , and the inhibition of tumor growth that was attributable to NAG-1 protein expression in a nude mice xenograft model . Thus, NAG-1 is an essential factor in the antitumor activity of isochaihulactone. Our current results show that isochaihulactone induced EGR-1 and NAG-1 protein expression in LNCaP cells in a time-dependent manner (Figure 5A). Furthermore, only the JNK1/2 inhibitor SP600125 reduced isochaihulactone-induced NAG-1 protein expression (Figure 5B). These data support that isochaihulactone-induced JNK1/2 activity is critical in regulating NAG-1 expression. In addition, we further confirmed by using siRNA approach that NAG-1 expression has an apoptosis-promoting effect (Figure 5D).
In summary, we found that isochaihulactone increased NAG-1 expression, suggesting that the antitumor effect of isochaihulactone is mediated via this tumor suppressor protein. NAG-1 mRNA is highly expressed in the human prostate epithelium , suggesting its role in prostate homeostasis. Despite this, NAG-1 negatively affects LNCaP cell survival , and is overexpressed in many tumors including prostate cancer [27, 28]. NAG-1 may be like other members of the TGF-β superfamily, acting as a tumor suppressor in the early stages but becoming pro-tumorigenic during the later stages of tumor progression. The effects of NAG-1 appear to be ambiguous, and under different conditions, NAG-1 exhibits either tumorigenic or anti-tumorigenic activity . Epidemiological studies have shown that patients who use NSAIDs for 10-15 years have a reduced risk of developing cancer . NSAIDs inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Several studies have suggested that the tumorigenic or anti-tumorigenic activity of NAG-1 may be due to the interaction of NAG-1 and cyclooxygenase [21, 30, 31].
Recent study has revealed a new pathway that Retinoblastoma (RB; encoded by RB1) depletion induced unchecked androgen receptor (AR) activity that underpinned therapeutic bypass and tumor progression . The hypo-phosphorylation form of RB suppresses E2F1-mediated transcriptional activation and induces cell cycle arrest. Loss of RB1 was observed in most of the castrate-resistant prostate cancer (CRPC), and AR as a gene under the control of E2F1, which in turn is stringently regulated by RB. Since hypo-phosphorylation of RB was observed after isochaihulactone treatment in LNCaP cells (data not shown), this might explain why LNCaP is more sensitive to isochaihulactone than the other two androgen-independent prostate cancer cell lines. However, the exact mechanism of these differences needs to be extensively investigated.