Susceptibility Of Prostate Cancer Cells Research Articles

TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

BackgroundAs a novel necrosis manner, ferroptosis has been increasingly reported to play a role in tumor progression and treatment, however, the specific mechanisms underlying its development in prostate cancer remain unclear. Growing evidence showed that peroxisome plays a key role in ferroptosis. Herein, we identified a novel mechanism for the involvement of ferroptosis in prostate cancer progression, which may provide a new strategy for clinical treatment of prostate cancer.MethodsLabel-Free Mass spectrometry was used to screen and identify candidate proteins after ferroptosis inducer-ML210 treatment. Immunohistochemistry was undertaken to explore the protein expression of AGPS in prostate cancer tissues compared with normal tissues. Co-immunoprecipitation and GST pull-down were used to identify the directly binding of AGPS to MDM2 in vivo and in vitro. CCK8 assay and colony formation assay were used to illustrate the key role of AGPS in the progression of prostate cancer in vitro. The xenograft model was established to verify the key role of AGPS in the progression of prostate cancer in vivo.ResultsAGPS protein expression was downregulated in prostate cancer tissues compared with normal tissues from the first affiliated hospital of Zhengzhou University dataset. Lower expression was correlated with poorer overall survival of patients compared to those with high expression of AGPS. In addition, AGPS can promote ferroptosis by modulating the function of peroxisome-resulting in the lower survival of prostate cancer cells. Furthermore, it was shown that AGPS can be ubiquitinated and degraded by the E3 ligase-MDM2 through the proteasomal pathway. Meanwhile, kinase TrkA can promote the combination of AGPS and MDM2 by phosphorylating AGPS at Y451 site. It was verified that kinase TrkA inhibitor—Larotrectinib can increase the susceptibility of prostate cancer cells to ferroptosis, which leads to the inhibition of prostate cancer proliferation to a great extent in vitro and in vivo.ConclusionBased on these findings, we proposed the combination of ferroptosis inducer and TrkA inhibitor to synergistically exert anti-tumor effects, which may provide a new strategy for the clinical treatment of prostate cancer.

Lipocalin-2 regulates the expression of interferon-stimulated genes and the susceptibility of prostate cancer cells to oncolytic virus infection

Lipocalin-2 (LCN2) performs pleiotropic and tumor context-dependent functions in cancers of diverse etiologies. In prostate cancer (PCa) cells, LCN2 regulates distinct phenotypic features, including cytoskeleton organization and expression of inflammation mediators. Oncolytic virotherapy uses oncolytic viruses (OVs) to kill cancer cells and induce anti-tumor immunity. A main source of specificity of OVs towards tumor cells stems from cancer-induced defects in interferon (IFN)-based cell autonomous immune responses. However, the molecular underpinnings of such defects in PCa cells are only partially understood. Moreover, LCN2 effects on IFN responses of PCa cells and their susceptibility to OVs are unknown. To examine these issues, we queried gene expression databases for genes coexpressed with LCN2, revealing co-expression of IFN-stimulated genes (ISGs) and LCN2. Analysis of human PCa cells revealed correlated expression of LCN2 and subsets of IFNs and ISGs. CRISPR/Cas9-mediated stable knockout of LCN2 in PC3 cells or transient overexpression of LCN2 in LNCaP cells revealed LCN2-mediated regulation of IFNE (and IFNL1) expression, activation of JAK/STAT pathway, and expression of selected ISGs. Accordingly, and dependent on a functional JAK/STAT pathway, LCN2 reduced the susceptibility of PCa cells to infection with the IFN-sensitive OV, EHDV-TAU. In PC3 cells, LCN2 knockout increased phosphorylation of eukaryotic initiation factor 2α (p-eIF2α). Inhibition of PKR-like ER kinase (PERK) in PC3-LCN2-KO cells reduced p-eIF2α while increasing constitutive IFNE expression, phosphorylation of STAT1, and ISG expression; and decreasing EHDV-TAU infection. Together, these data propose that LCN2 regulates PCa susceptibility to OVs through attenuation of PERK activity and increased IFN and ISG expression.

Abstract B022: CHD1 promotes sensitivity to aurora kinase inhibitors by modulating the interaction of AURKA with TPX2

Abstract Clinical studies have shown that subsets of cancer patients achieve a significant benefit from Aurora kinase inhibitors, suggesting an urgent need to identify biomarkers for predicting drug response. Chromodomain-helicase-DNA-binding protein 1 (CHD1) is involved in chromatin remodeling, DNA repair, and transcriptional plasticity. Prior studies have demonstrated that CHD1 has distinct expression patterns in cancers with different molecular features, but its impact on drug responsiveness remains understudied. Here, we show that CHD1 promotes the susceptibility of prostate cancer cells to inhibitors targeting Aurora kinases, while depletion of CHD1 impairs their efficacy in vitro and in vivo. Pan-cancer drug sensitivity analyses revealed that high expression of CHD1 was associated with increased sensitivity to Aurora kinase A (AURKA) inhibitors. Mechanistically, KPNA2 served as a direct target of CHD1 and suppressed the interaction of AURKA with the co-activator TPX2, thereby rendering cancer cells more vulnerable to AURKA inhibitors. Consistent with previous research reporting that loss of PTEN elevates CHD1 levels, studies in a genetically engineered mouse model, patient-derived organoids, and patient samples showed that PTEN defects are associated with a better response to AURKA inhibition in advanced prostate cancer. These observations demonstrate that CHD1 plays an important role in modulating Aurora kinases and drug sensitivities, providing new insights into biomarker-driven therapies targeting Aurora kinases for future clinical studies. Citation Format: Haoyan Li, Yin Wang, Kevin Lin, Varadha Balaji Venkadakrishnan, Martin Bakht, Kaitlyn Tremble, Yue Lu, Himisha Beltran, Di Zhao. CHD1 promotes sensitivity to aurora kinase inhibitors by modulating the interaction of AURKA with TPX2. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B022.

Acquired drug resistance interferes with the susceptibility of prostate cancer cells to metabolic stress

BackgroundMetformin is an inhibitor of oxidative phosphorylation that displays an array of anticancer activities. The interference of metformin with the activity of multi-drug resistance systems in cancer cells has been reported. However, the consequences of the acquired chemoresistance for the adaptative responses of cancer cells to metformin-induced stress and for their phenotypic evolution remain unaddressed.MethodsUsing a range of phenotypic and metabolic assays, we assessed the sensitivity of human prostate cancer PC-3 and DU145 cells, and their drug-resistant lineages (PC-3_DCX20 and DU145_DCX20), to combined docetaxel/metformin stress. Their adaptation responses have been assessed, in particular the shifts in their metabolic profile and invasiveness.ResultsMetformin increased the sensitivity of PC-3 wild-type (WT) cells to docetaxel, as illustrated by the attenuation of their motility, proliferation, and viability after the combined drug application. These effects correlated with the accumulation of energy carriers (NAD(P)H and ATP) and with the inactivation of ABC drug transporters in docetaxel/metformin-treated PC-3 WT cells. Both PC-3 WT and PC-3_DCX20 reacted to metformin with the Warburg effect; however, PC-3_DCX20 cells were considerably less susceptible to the cytostatic/misbalancing effects of metformin. Concomitantly, an epithelial–mesenchymal transition and Cx43 upregulation was seen in these cells, but not in other more docetaxel/metformin-sensitive DU145_DCX20 populations. Stronger cytostatic effects of the combined fenofibrate/docetaxel treatment confirmed that the fine-tuning of the balance between energy supply and expenditure determines cellular welfare under metabolic stress.ConclusionsCollectively, our data identify the mechanisms that underlie the limited potential of metformin for the chemotherapy of drug-resistant tumors. Metformin can enhance the sensitivity of cancer cells to chemotherapy by inducing their metabolic decoupling/imbalance. However, the acquired chemoresistance of cancer cells impairs this effect, facilitates cellular adaptation to metabolic stress, and prompts the invasive front formation.

Pleiotrophin affects the susceptibility of prostate cancer cells to cisplatin.

Drug resistance is a major problem in cancer therapy with cisplatin. It has not been reported that pleiotrophin, which is anti-apoptotic in some cancer cells, is associated with cisplatin resistance. Pleiotrophin was exogenously expressed in 293 cells. Viability and apoptosis of PC3 cells treated with different concentrations of cisplatin in the presence or absence of purified pleiotrophin were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. PC3 cells transfected with shRNAs were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting 24 h after transfection. MTT assay data indicated that the EC50 value of cisplatin for PC3 cells was significantly increased in the presence of pleiotrophin. Flow cytometry data demonstrated the pleiotrophin dose-dependent anti-apoptosis in PC3 cells treated with cisplatin. Knockdown of pleiotrophin with sh-RNA, as justified by RT-PCR and western blotting analysis, led to increased cisplatin induced-apoptosis in PC3 cells with an increased level of the cleaved poly ADP-ribose polymerase protein. Pleiotrophin may be a potential antiapoptotic protein associated with cisplatin susceptibility, which warrants further study on the role of pleiotrophin in cisplatin resistance.

Combined genetic and epigenetic interferences with interferon signaling expose prostate cancer cells to viral infection.

Interferons (IFNs) induce anti-viral programs, regulate immune responses, and exert anti-proliferative effects. To escape anti-tumorigenic effects of IFNs, malignant cells attenuate JAK/STAT signaling and expression of IFN stimulated genes (ISGs). Such attenuation may enhance the susceptibility of tumor cells to oncolytic virotherapy. Here we studied genetic and epigenetic mechanisms of interference with JAK/STAT signaling and their contribution to susceptibility of prostate cancer cells to viral infection. Bioinformatics analysis of gene-expression in cohorts of prostate cancer patients revealed genetic and epigenetic interference with the IFN program. To correlate lack of IFN signaling and susceptibility to viral infection and oncolysis; we employed LNCaP prostate cancer cells as cellular model, and the human metapneumovirus and the epizootic hemorrhagic disease virus as infectious agents. In LNCaP cells, JAK1 is silenced by bi-allelic inactivating mutations and epigenetic silencing, which also silences ISGs. Chemical inhibition of epigenetic silencing partially restored IFN-sensitivity, induced low levels of expression of selected ISGs and attenuated, but failed to block, viral infection and oncolysis. Since viral infection was not blocked by epigenetic modifiers, and these compounds may independently-induce anti-tumor effects, we propose that epigenetic modifiers and virotherapy are compatible in treatment of prostate tumors defective in JAK1 expression and IFN signaling.

The first experience in using abiraterone acetate in patients with castration-refractory prostate cancer

Even in the mid-twentieth century, Huggins and Hodges proved the susceptibility of prostate cancer cells to hormonal manipulations, by using surgical castration as an example. An average of 18–36 months after initiation of first-line hormonal therapy, patients develop the castration resistance in prostate cancer, one of the causes of which was hyperproduction of the tumor receptors of prostate cancer cells and their hypersusceptibility to the castration levels of testosterone. Long-term treatment in patients with castration-refractory prostate cancer was extremely symptomatic and quality of life and overall survival were low. In the 2000s, investigations aimed at designing drugs to treat this category of patients were underway, which have culminated in the advent of three drugs (two of which belong to chemotherapy) that are now used in the Russian Federation. The second-line hormonal agent abiraterone acetate (Zytiga) is one of these drugs, which was officially registered in 2011. Its mechanism of action is due to inhibition of the enzyme CYP17, leading to the blocked synthesis of testosterone at all levels, including at the intracrine level, and achieving testosterone levels below the postcastration ones. The paper reviews the literature regarding abiraterone acetate and the first experience in using second-line hormonal therapy in three patients.

Abstract 5054: The interplay between Disabled-2 and protein kinase C epsilon in the regulation of prostate cancer cell survival

Abstract Prostate cancer is the leading cause of cancer-related death in men in the world. Previous studies have demonstrated that Disabled-2 (DAB2) is a tumor suppressor and down-regulation of DAB2 expression is accompanied with prostate cancer progression. On the other hands, protein kinase C epsilon (PKCε) functions as an oncogene and promotes human prostate cancer cell survival upon treatment of methyseleninic acid (MSA), an anti-cancer agent that induces prostate cancer cell apoptosis. However, little is known whether there is a crosstalk between DAB2 and PKCε. In this study, we first analyzed DAB2 and PKCε protein expression in three prostate cancer cell lines DU145, PC3 and C4-2. Western blot analysis revealed that DAB2 was relatively abundant in PC3 when compared with the DU145 and C4-2 cells. In contrast, the expression of PKCε was higher in DU145 and C4-2 cells when compared with the PC3 cells. Consistent with these findings, ectopic expression of DAB2 resulted in down-regulation of PKCε. These observations implicate a reverse association of DAB2 and PKCε and suggest that DAB2 is a regulator of PKCε expression. To further explore the interplay between DAB2 and PKCε, we determined the effect of DAB2 overexpression on DU145 cell susceptibility to MSA-induced cell death. Consistent with a previous report, overexpression of PKCε increased DU145 cell survival and knockdown of PKCε by small interfering RNA augmented cell death upon treatment of MSA but not Ibupofen. Accordingly, expression of DAB2 down-regulated PKCε and increased in cell susceptibility to MSA that resulted in a 40% decrease of cell survival. These findings suggest that DAB2 modulates PKCε expression and affects prostate cancer cell susceptibility to selective anti-cancer agents. This study represents the first report to unveil the interplay between DAB2, PKCε and prostate cancer cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5054.

Fibronectin confers survival against chemotherapeutic agents but not against radiotherapy in DU145 prostate cancer cells: Involvement of the insulin like growth factor‐1 receptor

Tumor growth is influenced by an increase in cell proliferation and a reduction in apoptosis; both of which are affected by alterations in extracellular matrix (ECM). Our aim was to assess if the susceptibility of prostate cancer cells to apoptosis induced by either chemotherapeutics or radiotherapy was altered by changes in the ECM. Prostate cancer cell lines LNCaP and DU145 (androgen independent) cells were treated with chemotherapeutics (ceramide and docetaxel) or radiotherapy in the presence or absence of fibronectin, laminin, or vitronectin. Cell death was assessed using Trypan blue cell counting and apoptosis was confirmed by measuring PARP cleavage by Western immunoblotting (WIB). To identify a mechanism of action, changes in the abundance (WIB) or association (immunoprecipitation followed by WIB) of key proteins was also assessed. We found that fibronectin, but not laminin or vitronectin activated a survival pathway that protected DU145 but not LNCaP prostate cancer cells against ceramide and docetaxel-induced apoptosis but not that induced by radiotherapy. This survival effect involved the insulin-like growth factor (IGF-I) and beta1 integrin receptors and was associated with an increase in the recruitment of the beta1 integrin to a complex containing the IGF-IR and protein receptor for activated C kinase (RACK-1) and an increase in the abundance of a MAPK-phosphatase-1 (MKP-1). Changes in the ECM associated with disease progression may contribute to resistance to chemotherapeutic drugs but not to radiation therapy. The susceptibility to chemotherapy may be improved by targeting either the IGF-I or beta1 integrin receptors.

Combination of ginsenoside Rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of NF-κB

Ginsenoside Rg3 has been a subject of interest for use as a cancer preventive or therapeutic agent. Nuclear factor-kappa (NF-kappaB) is constitutively activated in prostate cancer, and gives cancer cells resistance to chemotherapeutic agents. To investigate whether Rg3 can suppress the activation of NF-kappaB, and thus increase susceptibility of prostate (LNCaP and PC-3, DU145) cells against chemotherapeutics, prostate cancer cell growth as well as activation of NF-kappaB was examined. We found that a combination treatment of Rg3 (50 microM) with a conventional agent docetaxel (5 nM) was more effective in the inhibition of prostate cancer cell growth and induction of apoptosis as well as G(0)/G(1) arrest accompanied with the significant inhibition of NF-kappaB activity than those by treatment of Rg3 or docetaxel alone. It was also found that NF-kappaB target gene expression of Bax, caspase-3, and caspase-9 was much more significantly enhanced, but the expression of Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP), and the expression of cell cycle regulatory proteins cyclin B, D1 and E, and cyclin dependent kinases 2 and 4 was also much more significantly inhibited by the combination treatment. The combination of Rg3 (50 microM) with cisplatin (10 microM) and doxorubicin (2 microM) was also more effective in the inhibition of prostate cancer cell growth and NF-kappaB activity than those by the treatment of Rg3 or chemotherapeutics alone. These results indicate that ginsenoside Rg3 inhibits NF-kappaB, and enhances the susceptibility of prostate cancer cells to docetaxel and other chemotherapeutics. Thus, ginsenoside Rg3 could be useful as an anti-cancer agent.

Anti-Cancer Effect of the Combination of Thiacremonone and Docetaxel by Inactivation of NF-κB in Human Cancer Cells

Abstract － Thiacremonone, the main component isolated from heated garlic ( Allium sativum L.), is inter-ested for using as a cancer preventive or therapeutic agent since garlic has been known to be useful plant in the treatment of cancers. Nuclear factor kappaB (NF-κB) is constitutively activated in the prostate cancer and activation of NF-κB is implicated in drug resistance in cancer cells. Docetaxel, a semisynthetic analog of pa-clitaxel, is an antineoplastic drug widely used for advanced various cancer. In previous studies, we found that thiacremonone inhibited activation of NF-κB in cancer cells and marcrophages. In the present study, we in-vestigated whether thiacremonone could increase susceptibility of prostate cancer cells (PC-3 and DU145) to docetaxel via inactivation of NF-κB. We found that the combination treatment of thiacremonone (50 μg/ml) with docetaxel (5 nM) was more effective in the inhibition of prostate cancer cell growth and induction of apoptosis accompanied with the significant inhibition of NF-κB activity than those by the treatment of thiacre-monone or docetaxel alone. It was also found that NF-κB target gene expression of Bax, caspase-3 and cas-pase-9 was much more significantly enhanced, but the expression of Bcl-2 was also much more significantly inhibited by the combination treatment. These results indicate that thiacremonone inhibits NF-κB, and enhan-ces the susceptibility of prostate cancer cells to docetaxel. Thus, thiacremonone could be useful as an ad-juvant anti-cancer agent.Keywords: Thiacremonone, Docetaxel, NF-κB, Apoptotic cell death, Prostate cancer

Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation

BackgroundManipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP) Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis.MethodscIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation.ResultsPC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression.ConclusionSimultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

307 POSTER Effects of triple knockdown of cIAP-1, c-IAP-2 and XIAP on prostate cancer cell susceptibility to apoptosis

307 POSTER Effects of triple knockdown of cIAP-1, c-IAP-2 and XIAP on prostate cancer cell susceptibility to apoptosis

493 POSTER Manipulating prostate cancer cell susceptibility to docetaxel and novel titanocene analogues induced apoptosis

The overexpression of AXL receptor tyrosine kinase is a frequent finding that has been associated with poor prognosis in esophageal adenocarcinoma (EAC). As the majority of EAC are intrinsically resistant to DNA-damaging therapies, an alternative therapeutic approach based on the activation of death receptors may be warranted. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been evaluated in clinical trials and found promising as anticancer agent with mild side effects; unfortunately, resistance to TRAIL remains a major clinical problem. Herein, we explored the role of AXL in TRAIL resistance and elucidated the underlying mechanism. Overexpression of AXL in OE33 and OE19 cells promoted cell survival and attenuated TRAIL-induced cellular and molecular markers of apoptosis. In contrast, knockdown of endogenous AXL sensitized FLO-1 cells to TRAIL. The mechanism by which AXL regulates TRAIL resistance was examined. Protein and mRNA expression of DR4 and DR5 death receptors was not downregulated by AXL. In addition, the possible involvement of FLICE-inhibitory protein (FLIP) in regulating the interaction of caspase-8 with Fas-associated death domain protein (FADD) was excluded, as AXL did not enhance FLIP expression or FLIP/FADD association. Alternatively, protein association of AXL with DR5, independent of TRAIL, was confirmed, suggesting that AXL could regulate DR5 receptor activity. The AXL/DR5 association had no negative effect on TRAIL-induced interaction with FADD. However, the AXL/DR5 interaction blocked the recruitment of caspase-8 to the death-inducing signal complex (DISC). Collectively, our findings uncover a novel mechanism of TRAIL resistance mediated by AXL through regulation of the DISC and provide strong evidence that AXL could be exploited as a therapeutic target to circumvent TRAIL resistance.

O-Glycosylation Regulates LNCaP Prostate Cancer Cell Susceptibility to Apoptosis Induced by Galectin-1

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.

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

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.

Targeting apoptosis in prostate cancer: focus on caspases and inhibitors of apoptosis proteins

Androgens are critical to the growth and differentiation of prostate epithelial cells. Removal of androgen normally results in apoptosis, but androgen-independent tumours have developed mechanisms that allow cells to survive the loss of androgen. The caspases are central mediators of cell death. An important area for research involves manipulating caspases by novel mechanisms to induce apoptosis. However, such mechanisms as diethylmaleate priming are limited by an inability to selectively target tumour cells. Inhibitors of apoptosis proteins (IAPs) are recently identified anti-apoptotic caspase regulators. Each IAP homologue has a different mechanism of action. Because more than one member of the IAP family may be overexpressed in prostate cancer, successful treatment strategies will be defined by the ability to block all of the IAP expressed. Anti-sense oligonucleotide strategies have been shown to decrease IAP expression and increase prostate cancer cell susceptibility to apoptotic induction, although not by mitochondrial-mediated pathways. Fully understanding the basic apoptotic pathway and its regulation in prostate cancer will lead to more targets for manipulation, which can be translated into novel therapies. This article focuses on the role of the caspases and IAP in developing a rational approach to using apoptosis as a therapeutic target.

Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which has been implicated in inhibition of apoptosis and control of mitotic progression. The finding that survivin is overexpressed in most human tumors but absent in normal adult tissues has led to the proposal of survivin as a promising therapeutic target for anticancer therapies. We decided to evaluate the effects of a ribozyme-based strategy for survivin inhibition in androgen-independent human prostate cancer cells. We constructed a Moloney-based retroviral vector expressing a ribozyme targeting the 3' end of the CUA(110) triplet in survivin mRNA, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations obtained by infection with the retroviral vector of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were selected for the study. Ribozyme-expressing prostate cancer cells were characterized by a significant reduction of survivin expression compared to parental cells transduced with a control ribozyme; the cells became polyploid, underwent caspase-9-dependent apoptosis and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of prostate cancer cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted in athymic nude mice. These findings suggest that manipulation of the antiapoptotic survivin pathway may provide a novel approach for the treatment of androgen-independent prostate cancer.

Protein kinase Cepsilon interacts with Bax and promotes survival of human prostate cancer cells.

Prostatic glandular epithelial cells express protein kinase Cepsilon (PKCepsilon ), an oncoprotein that coordinately disrupts the reactivation of the tumor suppressor Rb, derepressess transcriptional elongation of the c-myc oncogene, and propagates survival signals in LNCaP cells. Since the activation of such a program may contribute to the progression of human prostate cancer, a proteomic analysis was performed to gain a more global perspective on the signaling network that PKCepsilon might be capable of engaging in prostate cancer cells. Using CWR22 xenografts, we identified at least 18 different structural, signaling, and stress-related proteins that associated with PKCepsilon, including an interaction with the proapoptotic protein Bax that was novel to recurrent CWR22 tumors. An investigation into the biological significance of the PKCepsilon association with Bax provided the first evidence of an inverse relationship between endogenous levels of PKCepsilon and susceptibility of prostate cancer cells to the apoptotic effects of phorbol esters. Western blot and antisense experiments demonstrated that CWR-R1 cells expressed moderate levels of PKCepsilon and relied on this protein to survive in the presence of phorbol esters, while the apoptosis normally induced by phorbol esters in PKCepsilon -deficient LNCaP cells was dependent on the presence of Bax. Forced expression of PKCepsilon in LNCaP cells was sufficient to confer a significant resistance to phorbol esters and this resistance was associated with an inhibition of phorbol ester-induced Bax conformational rearrangements that are important for Bax oligomerization, mitochondrial integration, and cytochrome c release. Considered in their entirety, our data suggest that an association of PKCepsilon with Bax may neutralize apoptotic signals propagated through a mitochondrial death-signaling pathway.