Konstantinos Alevizopoulos

Membrane androgen receptor activation in prostate and breast tumor cells: molecular signaling and clinical impact.

In recent years, membrane androgen receptors (mARs) have been identified in prostate and breast tumor cells, and their activation by specific mAR ligands was linked to the regulation of crucial cell responses, such as cell growth, motility, and apoptosis. Analysis of the molecular signals triggered by mAR in the presence of anti‐androgens has clearly differentiated mAR‐dependent biological actions from those induced by the activation of the classical intracellular androgen receptors (iARs). In this review, we summarize the specific cellular events attributed to mAR activation and the experimental results on distinct non‐genomic signaling cascades operating in various tumor cells independently of the iAR. Furthermore, we discuss the crucial role of actin cytoskeleton organization and signaling in mediating mAR responses. Finally, we assess the clinical impact of the reported mAR‐induced apoptotic regression of prostate cancer cells both in vitro and in vivo and discuss the potential role of mAR as a novel therapeutic target.

Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells

In this study we describe a novel Rho small GTPase dependent pathway that elicits apoptotic responses controlled by actin reorganization in hormone-sensitive LNCaP- and hormone insensitive DU145-prostate cancer cells stimulated with membrane androgen receptor selective agonists. Using an albumin-conjugated steroid, testosterone-BSA, we now show significant induction of actin polymerization and apoptosis that can be reversed by actin disrupting agents in both cell lines. Testosterone-BSA triggered RhoA/B and Cdc42 activation in DU145 cells followed by stimulation of downstream effectors ROCK, LIMK2 and ADF/destrin. Furthermore, dominant-negative RhoA, RhoB or Cdc42 mutants or pharmacological inhibitors of ROCK inhibited both actin organization and apoptosis in DU145 cells. Activation of RhoA/B and ROCK was also implicated in membrane androgen receptor-dependent actin polymerization and apoptosis in LNCaP cells. Our findings suggest that Rho small GTPases are major membrane androgen receptor effectors controlling actin reorganization and apoptosis in prostate cancer cells.

Membrane androgen receptor activation triggers down-regulation of PI-3K/Akt/NF-kappaB activity and induces apoptotic responses via Bad, FasL and caspase-3 in DU145 prostate cancer cells

BackgroundRecently we have reported membrane androgen receptors-induced apoptotic regression of prostate cancer cells regulated by Rho/ROCK/actin signaling. In the present study we explored the specificity of these receptors and we analyzed downstream effectors controlling survival and apoptosis in hormone refractory DU145-prostate cancer cells stimulated with membrane androgen receptor-selective agonists.ResultsUsing membrane impermeable conjugates of serum albumin covalently linked to testosterone, we show here down-regulation of the activity of pro-survival gene products, namely PI-3K/Akt and NF-κB, in DU145 cells. Testosterone-albumin conjugates further induced FasL expression. A FasL blocking peptide abrogated membrane androgen receptors-dependent apoptosis. In addition, testosterone-albumin conjugates increased caspase-3 and Bad protein activity. The actin cytoskeleton drug cytochalasin B and the ROCK inhibitor Y-27632 inhibited FasL induction and caspase-3 activation, indicating that the newly identified Rho/Rock/actin signaling may regulate the downstream pro-apoptotic effectors in DU145 cells. Finally, other steroids or steroid-albumin conjugates did not interfere with these receptors indicating testosterone specificity.ConclusionCollectively, our results provide novel mechanistic insights pointing to specific pro-apoptotic molecules controlling membrane androgen receptors-induced apoptotic regression of prostate cancer cells and corroborate previously published observations on the potential use of membrane androgen receptor-agonists as novel anti-tumor agents in prostate cancer.

Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo

BackgroundMembrane androgen receptors (mAR) have been implicated in the regulation of cell growth, motility and apoptosis in prostate and breast cancer. Here we analyzed mAR expression and function in colon cancer.ResultsUsing fluorescent mAR ligands we showed specific membrane staining in colon cell lines and mouse xenograft tumor tissues, while membrane staining was undetectable in healthy mouse colon tissues and non-transformed intestinal cells. Saturation/displacement assays revealed time- and concentration-dependent specific binding for testosterone with a KD of 2.9 nM. Stimulation of colon mAR by testosterone albumin conjugates induced rapid cytoskeleton reorganization and apoptotic responses, even in the presence of anti-androgens. The actin cytoskeleton drug cytochalasin B effectively inhibited the pro-apoptotic responses and caspase-3 activation. Interestingly, in vivo studies revealed that mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors.ConclusionOur results demonstrate for the first time that functional mARs are predominantly expressed in colon tumors and that their activation results in induction of anti-tumor responses in vitro and extensive reduction of tumor incidence in vivo.

Na+/K+ ATPase inhibitors in cancer.

Sodium potassium pump (Na(+)/K(+) ATPase) is a transmembrane protein complex found in all higher eukaryotes acting as a key energy-consuming pump maintaining ionic and osmotic balance in cells. Recently recognized as an important transducer and/or integrator of various signals as well as a protein-protein interaction scaffold forming receptor complexes with signaling properties, the most prominent pharmacological role of Na(+)/K(+) ATPase inhibitors is the increase of myocardial contractility in pathologic conditions such as congestive heart failure. Consequently, modulators of Na(+)/K(+) ATPase such as digoxin have been approved by regulatory authorities and are widely used in the treatment of cardiac failure since 1975. Initiating from early observations of reduction of cancer incidence in cardiac patients taking digoxin, recent epidemiological and other studies have consolidated the anti-cancer potential of Na(+)/K(+) ATPase inhibitors in indications such as prostate, breast, lung cancer or leukemia. More importantly, a new series of pharmacologically optimized Na(+)/K(+) ATPase inhibitors has recently shown strong anti-cancer activities in multiple preclinical assays and have reached early clinical trials. Altogether, these results suggest that Na(+)/K(+) ATPase is an emerging cancer target that merits further investigation. In this review, we summarize key functional properties of the enzyme that are highly relevant for cancer cell selectivity, review the most prominent chemical classes of Na(+)/K(+) ATPase inhibitors and analyze their downstream effectors. Moreover, we discuss overall development prospects of these candidate drugs on their way to becoming new effective treatments of cancer in patients.

Sclareol induces apoptosis in human HCT116 colon cancer cells in vitro and suppression of HCT116 tumor growth in immunodeficient mice.

Labd-14-ene-8, 13-diol (sclareol) is a labdane-type diterpene, which has demonstrated significant cytotoxic activity against human leukemic cell lines, but its effect on solid tumor-derived cells is uknown. Here, we demonstrate that addition of sclareol to cultures of human colon cancer HCT116 cells results in inhibition of DNA synthesis, arrest of cells at the G1 phase of the cell cycle, activation of caspases-8, -9, PARP degradation, and DNA fragmentation, events characteristic of induction of apoptosis. Intraperitoneal (ip) administration of sclareol alone, at the maximum tolerated dose, was unable to induce suppression of growth of HCT116 tumors established as xenografts in immunodeficient SCID mice. In contrast, ip administration of liposome-encapsulated sclareol, following a specific schedule, induced suppression of tumor growth by arresting tumor cell proliferation as assessed by detecting the presence of the cell proliferation-associated nuclear protein, Ki67, in thin tumor sections. These findings suggest that sclareol incorporated into liposomes may possess chemotherapeutic potential for the treatment of colorectal and other types of human cancer.

Serum- and glucocorticoid-dependent kinase-1-induced cell migration is dependent on vinculin and regulated by the membrane androgen receptor

The serum‐ and glucocorticoid‐dependent kinases 1–3 (SGK1–3) are downstream effectors of phosphatidylinositol 3‐kinases, implicated in various cell responses including colon cancer tumorigenesis in mice. Here, we investigated the role of SGK1 in the regulation of cell motility. Using Caco‐2 colon tumor and HEK293 embryonic kidney cells, we report that transfection with the constitutively active SGK1 mutant (SGK1‐SD) significantly enhanced cell motility. The cell‐adhesion protein vinculin was effectively dephosphorylated in SGK1‐SD‐transfected cells. Treatment of the cells with phosphatase inhibitors restored vinculin phosphorylation and inhibited cell migration, indicating a significant role for vinculin phosphorylation in SGK1‐induced motility. SGK1‐SD‐enhanced cell motility was inhibited by activation of membrane androgen‐binding sites (mAR) via testosterone‐conjugates in both cell lines, whereas intracellular androgen receptor (iAR)‐silencing and flutamide treatment revealed that these effects were clearly independent of the interaction of SGK1 with the classical androgen receptors (iAR). More importantly, mAR activation restored vinculin phosphorylation in SGK1‐SD‐transfected cells, whereas silencing of vinculin fully reversed the mAR‐induced inhibition of the migratory capacity, implying that this protein is directly involved in cell motility regulation by SGK1 and mAR. This study indicates for the first time that SGK1 regulates cell migration via vinculin dephosphorylation, a mechanism that is controlled by mAR function.

Membrane Androgen Receptor Down-Regulates c-Src-Activity and Beta-Catenin Transcription and Triggers GSK-3beta-Phosphorylation in Colon Tumor Cells

Background/Aims: Functional membrane androgen receptors (mARs) have recently been described in colon tumor cells and tissues. Their activation by specific testosterone albumin conjugates (TAC) down-regulates the PI-3K/Akt pro-survival signaling and triggers potent pro-apoptotic responses both, in vitro and in vivo. The present study explored the mAR-induced regulation of gene products implicated in the tumorigenic activity of Caco2 colon cancer cells. Methods: In Caco2 human colon cancer cells transcript levels were determined by RT-PCR, protein abundance and phosphorylation by Western blotting and confocal microscopy, as well as cytoskeletal architecture by confocal microscopy. Results: We report time dependent significant decrease in Tyr-416 phosphorylation of c-Src upon mAR activation. In line with the reported late down-regulation of the PI-3K/Akt pathway in testosterone-treated colon tumors, GSK-3beta was phosphorylated at Tyr-216 after long term stimulation of the cells with TAC, a finding supporting the role of this kinase to promote apoptosis. PCR analysis revealed significant decrease of beta-catenin and cyclin D1 transcript levels following TAC treatment. Moreover, confocal laser scanning microscopic analysis disclosed co-localization of beta-catenin with actin cytoskeleton. It is thus conceivable that beta-catenin may participate in the reported modulation of cytoskeletal dynamics in mAR stimulated Caco2 cells. Conclusions: Our results provide strong evidence that mAR activation regulates late expression and/or activity of the tumorigenic gene products c-Src, GSK-3beta, and beta-catenin thus facilitating the pro-apoptotic response in colon tumor cells.

Targeting membrane androgen receptors in tumors

Introduction: In the last decade androgen actions that are originated from non-genomic, rapid signaling have been described in a large number of cell models and tissues. These effects are initiated through the stimulation of membrane androgen-binding sites or receptors (mAR). Although the molecular identity of mARs remains elusive, their activation is known to trigger multiple non-genomic signaling cascades and to regulate numerous cell responses. In recent years specific interest is being paid to the role of mARs in tumors. Specifically, it was demonstrated that mAR activation by non-permeable testosterone conjugates induced potent anti-tumorigenic responses in prostate, breast, colon and glial tumors. In addition, in vivo animal studies further emphasized the potential clinical importance of these receptors. Areas covered: This review will summarize the current knowledge on the mAR-induced non-genomic, rapid androgen actions. It will focus on the molecular signaling pathways governed by mAR activation, discuss latest attempts to elucidate the molecular identity of mAR, address the plethora of cell responses initiated by mAR and evaluate the potential role of mAR and mAR-specific signaling as possible therapeutic targets in tumors. Expert opinion: mAR and mAR-induced specific signaling may represent novel therapeutic targets in tumors through the development of specific testosterone analogs.

The labdane diterpene sclareol (labd-14-ene-8, 13-diol) induces apoptosis in human tumor cell lines and suppression of tumor growth in vivo via a p53-independent mechanism of action.

The labdane diterpene sclareol has demonstrated significant cytotoxicity against human tumor cell lines and human colon cancer xenografts. Therefore, there is need to elucidate the mode of action of this compound as very little information is known for the anticancer activity of sclareol and other labdane diterpenes, in general. COMPARE analysis of GI(50) values for a number of human cancer cell lines was initially implicated in an effort to assign a putative mechanism of action to the compound. Sclareol-induced cell cycle arrest and apoptosis were assessed by flow cytometry and Western blot analyses. Finally, the anticancer ability of sclareol in vivo was assessed by using human colon cancer xenograft/mouse models. Sclareol arrested in vitro the growth of p53-deficient (HCT116(p53-/-)) human colon cancer cells and subsequently induced apoptosis by activating both caspases-8 and -9. Intraperitoneal administration of liposome-encapsulated sclareol at the maximum tolerated dose induced a marked growth suppression of HCT116(p53-/-) tumors established as xenografts in immunodeficient NOD/SCID mice. In conclusion, we demonstrate herein that sclareol kills human tumor cells by inducing arrest at the G(1)-phase of the cell cycle followed by apoptosis that involves activation of caspases-8, -9 and -3 via a p53-independent mechanism. These findings suggest that liposome-encapsulated sclareol possesses chemotherapeutic potential for the treatment of colorectal and other types of human cancer regardless of the p53-status.