Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells.

We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome ß5 and ß1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF(2α)), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF(2α) and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as a potential agent in cancer chemotherapy.

Vitamin Bs, one carbon metabolism and prostate cancer.

Somatic genetic and epigenetic alterations have been suggested to be crucially involved in development and progression of cancers including prostate cancer (PCa). Epigenetic alterations such as chemical modification of chromatin associated proteins and DNA methylation can largely affect gene expression that may be important for early normal organ development, and also produces favorable conditions for cancer cell formation, growth, and survival. Aberrant DNA methylation (hyper- or hypo-methylation) may lead to chromosomal instability, and transcriptional gene silencing for tumor suppressors or overexpression for oncogenes. Vitamin Bs play important roles in one carbon metabolism that provides critical metabolites for DNA methylation, DNA repair and nucleic acid synthesis. Nutrition uptake and circulating levels of these vitamin Bs as well as genetic polymorphisms of related key enzymes in the one carbon metabolism pathway may govern bioavailability of the metabolites, and therefore to affect the phenotypic changes (e.g., cellular malignancy) via genetic and epigenetic alterations. This article will summarize recent new findings from laboratory, epidemiological or clinical trial studies regarding influence of vitamin B and one carbon metabolism on PCa development or progression.

Mutant epidermal growth factor receptor vIII increases cell motility and clonogenecity in a prostate cell line RWPE1.

Epidermal growth faxtor receptor (EGFR)-vIII mutant has been demonstrated to over-express as prostatic neoplasms progressed from intraepithelial changes to metastatic disease. In this study, we transfected the EGFRvIII expression vector into an immortalized normal prostate epithelium cell line RWPE-1 and established stable transfectants. The cell growth, glandular morphogenesis, cell motility, and soft-agar colony formation efficiency were then studied. The results showed that EGFR-vIII mutation increased the RWPE1 cell motility and clone formation efficiency, while it had no significant effect on the cell growth when compared to non-transfected as well as mock transfected RWPE-1 cells. Moreover, EGFR-vIII changed the RWPE1 acinar morphogenesis. Further study showed that these effects of EGFR-vIII mutation may be related to down-regulation of E-cadherin and up-regulation of beta-catenin.

Carotenoids and prostate cancer risk.

Chemoprevention is presumably one of most effective means to combat prostate cancer (PCa). Patients usually require more than a decade to develop a clinically significant Pca, therefore, an ideal target for chemoprevention. This review will focus on recent findings of a group of naturally occurring chemicals, carotenoids, for potential use in reducing PCa risk.

ZNF185, an actin-cytoskeleton-associated growth inhibitory LIM protein in prostate cancer.

We have recently identified ZNF185 as a gene that is downregulated in prostate cancer (PCa), in part via epigenetic alteration, and maybe associated with disease progression. In this study, we cloned the ZNF185 cDNA from normal human prostate tissues and investigated its biological function. We show that ZNF185 is a novel actin-cytoskeleton-associated Lin-l 1, Isl-1 and Mec-3 (LIM) domain-containing protein that localizes to F-actin structures, and is enriched at focal adhesions. We find that the NH(2)-terminal region, which we designate the actin-targeting domain, facilitates ZNF185 binding to actin in vitro and is both necessary and sufficient to mediate actin-cytoskeleton targeting of ZNF185, whereas the LIM domain, which is localized in the COOH-terminus is dispensable for this phenomenon. Interestingly, ectopic expression of full-length ZNF185, but not a mutant lacking the actin-targeting domain, could suppress proliferation and anchorage-independent growth of PCa cells. Together, our data suggest that ZNF185 may function as a tumor-suppressor protein by associating with the actin-cytoskeleton.

Forced expression of heat-shock protein 70 increases the secretion of Hsp70 and provides protection against tumour growth.

Although heat-shock protein 70 (Hsp70) has been considered an intracellular protein, we report that Hsp70 is secreted under normal cell culture conditions by human prostate cell lines, LAPC-4, PC-3, CWR-22, RWPE-1 and -2, LNCaP, and TRAMP (transgenic adenocarcinoma mouse prostate)-C2. We found that the secretion can be enhanced by transfection with cDNA encoding for Hsp70. To verify that the Hsp70 detected in the supernatant was not secondary to cell leakage, C2 cells were cotransfected with cytoplasmic Renilla luciferase as a reporter. High levels of activities were noted in the cell extracts, while no enzyme activities were detected in the supernatants. To verify that forced oversecretion of Hsp70 could protect against tumour growth, mice were injected with C2 cells transfected with an Hsp70 DNA construct and challenged with live tumour cells. Mice injected with cells transfected with the Hsp70 DNA construct demonstrated a significantly decreased rate of tumour growth compared to those injected with empty vector. In addition, a difference in survival rate as defined by a surrogate end point was noted between the two groups. In a second experiment, we developed a cell line that stably overexpressed Hsp70. Mice injected with these cells also demonstrated a significant decrease in tumour growth and significantly increased survival.

Retinoic acid-induced stimulation of sodium iodide symporter expression and cytotoxicity of radioiodine in prostate cancer cells.

We reported recently the induction of androgen-dependent iodide uptake activity in the human prostatic adenocarcinoma cell line LNCaP using a prostate-specific antigen (PSA) promoter-directed expression of the sodium iodide symporter (NIS) gene. This offers the potential to treat prostate cancer with radioiodine. In the current study, we examined the regulation of PSA promoter-directed NIS expression and therapeutic effectiveness of (131)I in LNCaP cells by all-trans-retinoic acid (atRA). For this purpose, NIS mRNA and protein expression levels in the NIS-transfected LNCaP cell line NP-1 were examined by Northern and Western blot analysis following incubation with atRA (10 (-9) to 10(-6) M) in the presence of 10(-9) M mibolerone (mib). In addition, NIS functional activity was measured by iodide uptake assay, and in vitro cytotoxicity of (131)I was examined by in vitro clonogenic assay. Following incubation with atRA, NIS mRNA levels in NP-1 cells were stimulated 3-fold in a concentration-dependent manner, whereas NIS protein levels increased 2.3-fold and iodide accumulation was stimulated 1.45-fold. This stimulatory effect of atRA, which has been shown to be retinoic acid receptor mediated, was completely blocked by the pure androgen receptor antagonist casodex (10(-6) M), indicating that it is androgen receptor dependent. The selective killing effect of (131)I in NP-1 cells was 50% in NP-1 cells incubated with 10(-9) M mib. This was increased to 90% in NP-1 cells treated with atRA (10(-7) M) plus 10(-9) M mib. In conclusion, treatment with atRA increases NIS expression levels and selective killing effect of (131)I in prostate cancer cells stably expressing NIS under the control of the PSA promoter. Therefore atRA may be used to enhance the therapeutic response to radioiodine in prostate cancer cells following PSA promoter-directed NIS gene delivery.