Overexpression of Bax induces down-regulation of store-operated calcium entry in prostate cancer cells

Store-operated Ca2+ channels control homeostasis between extracellular Ca2+ reservoir and intracellular Ca2+ storage and play important roles in apoptosis in a wide variety of cells, including prostate epithelia. Recent studies have shown that the acquired apoptosis-resistant nature of androgen-independent prostate cancer is associated with reduced function of store-operated Ca2+ entry (SOCE). This study investigates the functional interaction between Bax and SOCE in the apoptosis signaling cascade in prostate cancer. Our previous findings show that NRP-154, an androgen-independent prostate cancer cell line, could sustain overexpression of exogenous Bax without undergoing apoptosis. Here we show that sustained overexpression of Bax in NRP-154 cells leads to down-regulation of SOCE and reduced Ca2+ storage inside the endoplasmic reticulum. While reduced SOCE may represent an adaptive mechanism for cell survival, increased levels of Bax in the latent state enhances the sensitivity of NRP-154 cells to TGF-beta and thapsigargin-induced apoptosis. This enhanced apoptosis can be reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of SOCE, or reversed under conditions where SOCE is only partially activated. Our results demonstrate a functional interaction between Bax and SOCE in apoptosis of prostate cancer, and support the concept that improving this interaction has therapeutic implications for prostate cancer.     

Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins

Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.     

Ca2+ homeostasis and apoptotic resistance of neuroendocrine-differentiated prostate cancer cells

Neuroendocrine (NE) differentiation is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. NE tumor cells are nonproliferating and escape apoptotic cell death; therefore, an understanding of the apoptotic status of the NE phenotype is imperative for the development of new therapies for prostate cancer. Here, we report for the first time on alterations in intracellular Ca(2+) homeostasis, which is a key factor in apoptosis, caused by NE differentiation of androgen-dependent prostate cancer epithelial cells. NE-differentiating regimens, either cAMP elevation or androgen deprivation, resulted in a reduced endoplasmic reticulum Ca(2+)-store content due to both SERCA 2b Ca(2+) ATPase and luminal Ca(2+) binding/storage chaperone calreticulin underexpression, and to a downregulated store-operated Ca(2+) current. NE-differentiated cells showed enhanced resistance to thapsigargin- and TNF-alpha-induced apoptosis, unrelated to antiapoptotic Bcl-2 protein overexpression. Our results suggest that targeting the key players determining Ca(2+) homeostasis in an attempt to enhance the proapoptotic potential of malignant cells may prove to be a useful strategy in the treatment of advanced prostate cancer.     

Association of plasminogen with dipeptidyl peptidase IV and Na+/H+ exchanger isoform NHE3 regulates invasion of human 1-LN prostate tumor cells

Binding of plasminogen type II (Pg 2) to dipeptidyl peptidase IV (DPP IV) on the surface of the highly invasive 1-LN human prostate tumor cell line induces an intracellular Ca2+ ([Ca2+]i) signaling cascade accompanied by a rise in intracellular pH (pHi). In endothelial cells, Pg 2 regulates intracellular pH via Na+/H+ exchange (NHE) antiporters; however, this mechanism has not been demonstrated in any other cell type including prostate cancer cells. Because the Pg 2 receptor DPP IV is associated with NHE3 in kidney cell plasma membranes, we investigated a similar association in 1-LN human prostate cancer cells and a mechanistic explanation for changes in [Ca2+]i or pHi induced by Pg 2 in these cells. Our results suggest that the signaling cascade initiated by Pg 2 and its receptor proceeds via activation of phospholipase C, which promotes formation of inositol 3,4,5-trisphosphate, an inducer of Ca2+ release from endoplasmic reticulum stores. Furthermore, our results suggest that Pg 2 may regulate pHi via an association with NHE3 linked to DPP IV in these cells. These associations suggest that Pg has the potential to simultaneously regulate calcium signaling pathways and Na+/H+ exchanges necessary for tumor cell proliferation and invasiveness.     

Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84--> Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via beta1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-alpha-induced apoptosis. Evidence is provided that expression of beta1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-alpha-induced apoptosis. In contrast, the beta1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-alpha in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis.     

Requirement for store-operated calcium entry in sodium butyrate-induced apoptosis in human colon cancer cells

The SOCE (store-operated Ca2+ entry) pathway plays a key role in both normal cells and cancerous cells. However, its molecular mechanism remains a long-lasting puzzle of Ca2+ signalling. In this paper, we provide evidence that butyric acid, a dietary fibre-derived short-chain fatty acid, induces apoptosis of colon cancer cells via SOCE signalling networks. We found that sodium butyrate (NaB) induces Ca2+ release from endoplasmic reticulum, which in turn causes extracellular Ca2+ influx in HCT-116 cells. The Ca2+ release and influx are important, because the addition of chelators, EGTA or BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] respectively blocked NaB-induced apoptosis. Furthermore, down-regulation of STIM1 (stromal interaction molecule 1) by RNA interference or pharmacological blockade of the SOCC (store-operated Ca2+ channel) by 2-APB (2-aminoethoxydiphenyl borate) or SKF-96365 inhibited NaB-induced extracellular Ca2+ influx and apoptosis in HCT-116 cells. Thus we conclude that NaB triggers colon cancer cell apoptosis in an SOCE-dependent manner. This finding provides new insights into how butyric acid suppresses colon carcinogenesis.     

Store-operated Ca2+ channels in prostate cancer epithelial cells: function,regulation, and role in carcinogenesis

Ca2+ homeostasis mechanisms, in which the Ca2+ entry pathways play a key role, are critically involved in both normal function and cancerous transformation of prostate epithelial cells. Here, using the lymph node carcinoma of the prostate (LNCaP) cell line as a major experimental model, we characterize prostate-specific store-operated Ca2+ channels (SOCs)--a primary Ca2+ entry pathway for non-excitable cells--for the first time. We show that prostate-specific SOCs share major store-dependent, kinetic, permeation, inwardly rectifying, and pharmacological (including dual, potentiation/inhibition concentration-dependent sensitivity to 2-APB) properties with "classical" Ca2+ release-activated Ca2+ channels (CRAC), but have a higher single channel conductance (3.2 and 12pS in Ca2+- and Na+-permeable modes, respectively). They are subject to feedback inhibition via Ca2+-dependent PKC, CaMK-II and CaM regulatory pathways and are functionally dependent on caveolae integrity. Caveolae also provide a scaffold for spatial co-localization of SOCs with volume-regulated anion channels (VRAC) and their Ca2+-mediated interaction. The TRPC1 and TRPV6 members of the transient receptor potential (TRP) channel family are the most likely molecular candidates for the formation of prostate-specific endogenous SOCs. Differentiation of LNCaP cells to an androgen-insensitive, apoptotic-resistant neuroendocrine phenotype downregulates SOC current. We conclude that prostate-specific SOCs are important determinants in the transition to androgen-independent prostate cancer.     

Clusterin inhibits apoptosis by interacting with activated Bax

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.     

TCTP Is an Androgen-Regulated Gene Implicated in Prostate Cancer

TCTP has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. In addition to these intracellular functions, TCTP has extracellular functions and plays an important role in immune cells. TCTP expression was previously shown to be deregulated in prostate cancer, but its function in prostate cancer cells is largely unknown. Here we show that TCTP expression is regulated by androgens in LNCaP prostate cancer cells in vitro as well as human prostate cancer xenografts in vivo. Knockdown of TCTP reduced colony formation and increased apoptosis in LNCaP cells, implicating it as an important factor for prostate cancer cell growth. Global gene expression profiling in TCTP knockdown LNCaP cells showed that several interferon regulated genes are regulated by TCTP, suggesting that it may have a role in regulating immune function in prostate cancer. In addition, recombinant TCTP treatment increased colony formation in LNCaP cells suggesting that secreted TCTP may function as a proliferative factor in prostate cancer. These results suggest that TCTP may have a role in prostate cancer development.     

Assessment and validation of a microinjection method for kinetic analysis of [Ca2+]i in individual cells undergoing apoptosis

Normal and malignant epithelial cells are induced to undergo apoptosis by a large variety of mechanistically diverse agents. Regardless of inducing agent, apoptosis characteristically occurs asynchronously within a population of epithelial cells over a period of 12-96 h and is associated with permeability and enzymatic perturbations. Pre-loading of cells with acetoxymethyl esters (AM) derivatives of fluorescent Ca2+ indicators (i.e. fura-2, indo-1, fluo-3) by passive diffusion allows longitudinal kinetic analysis of acute [Ca2+] changes subsequent to exposure to apoptosis inducing agents. Using prostate cancer cell lines, however, it is demonstrated that dye leakage and compartmentalization into organelles limit such passive loading to longitudinal [Ca2+]i measurements of < 2 h. Post-loading of cells exposed to the apoptosis inducing agent for several hours is also inaccurate owing to decreased loading efficiency and de-esterification of the probes resulting in increased production of fluorescent Ca(2+)-insensitive dye species. To accurately measure kinetics of [Ca2+]i changes longitudinally in individual cells undergoing apoptosis, cells were microinjected with fura dextran and maintained in a physiologic environment. [Ca2+] and morphological changes characteristic of apoptosis were then followed simultaneously in individual cells over several days following exposure to the apoptosis inducing agent.     

Phosphorylation of Bcl-2 and activation of caspase-3 via the c-Jun N-terminal kinase pathway in ursolic acid-induced DU145 cells apoptosis

There is currently no successful therapy for androgen-independent prostate cancer. Ursolic acid (UA), a pentacyclic triterpenoid compound, has been shown to have an anti-proliferative effect on various tumors. We investigated the effect of UA on cell viability in the human hormone-refractory prostate cancer cell line DU145, as well as the molecular mechanisms underlying its growth inhibiting effect. We demonstrated that UA induces apoptosis and the activation of caspase-3 in DU145 cells. UA also causes the activation of c-Jun N-terminal kinase (JNK), but has no effect on extracellular signal-regulated protein kinases (ERK1/2) and p38 MAP kinases (p38). UA-induced JNK activation could result in Bcl-2 phosphorylation (Ser70) and degradation in DU145 cells, which may be one of the molecular mechanisms by which it induces apoptosis. Although further evaluation, such as in vivo testing, is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer.     

NPC-14686 (Fmoc-l-homophenylalanine)-induced CaCa2+ increases and death in human prostate cancer cells

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca2+ levels ([Ca2+]i) and growth in PC3 human prostate cancer cells was examined by using fura-2 as a fluorescent Ca2+ indicator and WST-1 as a fluorescent growth dye. NPC-14686 at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 100 microM. NPC-14686-induced Ca2+ influx was confirmed by Mn2+ quench of fura-2 fluorescence. The Ca2+ signal was also reduced by removing extracellular Ca2+. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ nearly abolished 200 microM NPC-14686-induced Ca2+ release; and conversely pretreatment with NPC-14686 completely inhibited thapsigargin-induced Ca2+ release. The Ca2+ release induced by 200 microM NPC-14686 was not affected by inhibiting phospholipase C with 2 microM U73122. Overnight treatment with 1-500 microM NPC-14686 decreased cell viability in a concentration-dependent manner. These findings suggest that in human PC3 prostate cancer cells, NPC-14686 increases [Ca2+]i by evoking extracellular Ca2+ influx and releasing intracellular Ca2+ from the endoplasmic reticulum via a phospholiase C-independent manner. NPC-14686 may be cytotoxic to prostate cancer cells.     

Comparative studies of intracellular Ca2+ in strongly and weakly metastatic rat prostate cancer cell lines

The metastatic ability of prostate cancer cells involves differential expression of ionic mechanisms. In the present study, using electrophysiological recordings and intracellular Ca2+ measurements, we investigated Ca2+ related signalling in two rat prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic potential. Whole-cell voltage clamp experiments indicated the absence of an inward current carried through voltage-dependent Ca2+ channels in either cell line. A Ca2+-dependent component was also absent in the voltage-activated outward K+ currents. Indo-1 microfluorimetry confirmed these results and also revealed marked differences in the resting level of intracellular Ca2+ and the ability of the two cell lines to regulate intracellular Ca2+. The weakly metastatic AT-2 cells displayed a significantly higher resting intracellular Ca2+ than the related but strongly metastatic MAT-LyLu cell line. Increasing extracellular K+ decreased intracellular Ca2+ in the AT-2 but had no effect on intracellular Ca2+ levels in the MAT-LyLu cells. Furthermore, increasing extracellular Ca2+ increased intracellular Ca2+ in AT-2 but, again, had no effect on MAT-LyLu cells. These results suggested the presence of a tonic, voltage-independent Ca2+ permeation mechanism operating specifically in the AT-2 cells. The influx of Ca2+ into the AT-2 cells was suppressed by both CdCl2 (100-300 microM) and SKF-96365 (10-30 microM). It is concluded that the strongly metastatic MAT-LyLu cell line lacks a voltage-independent basal Ca2+ influx mechanism that is present in the weakly metastatic AT-2 cells.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I(cold/menthol)) that shows inward rectification and high Ca(2+) selectivity, which are dramatically different properties from "classical" TRPM8-mediated I(cold/menthol). Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I(cold/menthol) and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca(2+) release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca(2+) release channel, potentially involved in a number of Ca(2+)- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.     

Genistein induces Ca2+ -mediated, calpain/caspase-12-dependent apoptosis in breast cancer cells

Genistein, a soy-derived isoflavone, has been suggested for breast cancer prevention; however, use of soy products for this purpose remains controversial. Genistein has been reported to regulate growth of tumor cells, although the involved molecular mechanisms are not defined. Here we report that genistein induces apoptosis in breast cancer cells via activation of the Ca2+ -dependent proapoptotic proteases, mu-calpain, and caspase-12. The treatment of MCF-7 breast cancer cells with genistein induced a sustained increase in concentration of intracellular Ca2+ resulting from depletion of the endoplasmic reticulum Ca2+ stores. This increase in Ca2+ was associated with activation of mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 substrates and antibodies to active (cleaved) forms of the enzymes. Selective inhibition of Ca2+ binding sites of mu-calpain, forced increase of the cytosolic Ca2+ buffering capacity, and caspase inhibition decreased apoptotic indices in the genistein-treated cells. Our results suggest that Ca2+ -dependent proteases are potential targets for genistein in breast cancer cells and that the cellular Ca2+ regulatory activity of genistein underlies its apoptotic mechanism.     

Cytoskeleton Reorganization as an Alternative Mechanism of Store-Operated Calcium Entry Control in Neuroendocrine-Differentiated Cells

Neuroendocrine differentiation (NED) is a hallmark of advanced androgen-independent prostate cancer, for which no successful therapy exists. NED tumour cells escape apoptotic cell death by alterations of Ca²⁺ homeostasis where the store-operated Ca²⁺ entry (SOCE) is known to be a key event. We have previously shown that the downregulation of Orai1 protein representing the major molecular component of endogenous SOCE in human prostate cancer cells, and constituting the principal source of Ca²⁺ influx used by the cell to trigger apoptosis, contributes to the establishment of an apoptosis-resistant phenotype (Cell Death Dis. 2010 Sep 16;1:e75.). Here, we report for the first time that the decrease of SOCE during NED may be caused by alternative NED-induced mechanism involving cytoskeleton reorganisation. NED induced by androgen deprivation resulted in a decrease of SOCE due to cortical F-actin over-polymerization which inhibits thapsigargin-induced SOCE. The disruption of F-actin polymerization by Cytochalasin D in NED cells restored SOCE, while the induction of F-actin polymerization by jasplakinolide or calyculin A diminished SOCE without changing the expression of key SOCE players: Orai1, STIM1, and TRPC1. Our data suggest that targeting cytoskeleton-induced pathways of malignant cells together with SOCE-involved channels may prove a useful strategy in the treatment of advanced prostate cancer.