The glycosylation characteristic of hepatoma-associated antigen HAb18G/CD147 in human hepatoma cells

HAb18G/CD147 is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. Our previous studies have demonstrated that overexpressing HAb18G/CD147 enhances the metastatic potentials of human hepatoma cells. In the present study, to investigate the glycosylation characteristic of HAb18G/CD147 in human hepatoma cells, HAb18G/CD147 was first purified from human FHCC-98 hepatoma cells by immunoaffinity chromatography, and then introduced into human fibroblasts culture system for matrix metalloproteinases induction. As a result, the elevated levels of matrix metalloproteinases secreted by fibroblasts were detected by gelatin zymography. The lysates of human hepatoma FHCC-98 cell revealed two major forms of HAb18G/CD147 (43-66 and 35 kDa) by western blot assay. To elucidate whether the variation of molecule size were caused by different glycosylation, two different approaches were employed to accomplish this goal: deglycosylation with N-glycosylation inhibitor tunicamycin or endoglycosidases. A single deglycosylated core protein with molecular weight approximately 27 kDa was obtained from both methods. Furthermore, the results of endoglycosidases treatment also showed that two forms of HAb18G/CD147 contain different types of oligosaccharide chains, thus sensitive to different endoglycosidase. In conclusion, the present study demonstrated that purified native HAb18G/CD147 has the bioactivity of stimulating human fibroblasts to produce elevated levels of matrix metalloproteinases, and that the two different forms of HAb18G/CD147 are derived from the single core protein but differ in their degree and types of glycosylation.     

BetaIg-h3 is involved in the HAb18G/CD147-mediated metastasis process in human hepatoma cells

HAb18G/CD147, a new hepatoma-associated antigen cloned and screened from human hepatocellular carcinoma cDNA library, is closely correlated with metastasis process in human hepatoma cells. In the present study we aimed to identify the pivotal molecules of the HAb18G/CD147 signal transduction pathway. The investigation showed that betaig-h3, a secretory extracellular matrix (ECM) protein, was upregulated in HAb18G/CD147-expressing human hepatoma T7721 cells and was downregulated by depressing HAb18G/CD147 expression. The expression of betaig-h3, upregulated in human hepatoma cells, was positively relative to the expression of HAb18G/CD147 in different human hepatoma cell lines. By overexpressing betaig-h3 in human SMMC-7721 hepatoma cells, we discovered that betaig-h3 promoted cell adhesion, invasion, and matrix metalloproteinase (MMP) secretion potential. HAb18G/CD147-induced invasion and metastasis potential of human hepatoma cells can be attenuated by antibodies specific for betaig-h3, and no significant differences on inhibitory effects were observed among T7721 cells incubated with antibodies for betaig-h3 or HAb18G/CD147 or both types together. Taken together, our study suggests that betaig-h3, regulated by the expression of HAb18G/CD147, is involved in the HAb18G/CD147 signal transduction pathway and mediates the HAb18G/CD147-induced invasion and metastasis process of human hepatoma cells.     

HAb18G/CD147 functions in invasion and metastasis of hepatocellular carcinoma

CD147 molecule is reported to be correlated with the malignancy of some cancers; however, it remains unclear whether it is involved in the progression of hepatocellular carcinoma (HCC). Here, we investigated the function of HAb18G/CD147, a member of CD147 family, and its antibodies, HAb18 and LICARTIN, in HCC invasion and metastasis. We observed that HAb18G/CD147 gene silence in HCC cells significantly decreased the secretion of matrix metalloproteinase (MMP) and the invasive potential of HCC cells (P < 0.001). MMP silence in HCC cells also significantly suppressed the invasion of the cells when cocultured with fibroblasts; however, its inhibitory effect was significantly weaker than that of both HAb18G/CD147 silence in HCC cells and that of MMP silence in fibroblasts (P < 0.001). Blocking theHAb18G/CD147 molecule on HCC cells with HAb18 monoclonal antibody resulted in a similar suppressive effect on MMP secretion and cell invasion, but with no significant effects on the cell growth. (131)I-labeled HAb18 F(ab')(2) (LICARTIN), however, significantly inhibited the in vitro growth of HCC cells (P < 0.001). In an orthotopic model of HCC in nude mice, HAb18 and LICARTIN treatment effectively reduced the tumor growth and metastasis as well as the expression of three major factors in the HCC microenviroment (MMPs, vascular endothelial growth factor, and fibroblast surface protein) in the paracancer tissues. Overall, these results suggest that HAb18G/CD147 plays an important role in HCC invasion and metastasis mainly via modulating fibroblasts, as well as HCC cells themselves to disrupt the HCC microenviroment. LICARTIN can be used as a drug targeting to HAb18G/CD147 in antimetastasis and recurrence therapy of HCC.     

cGMP-regulated store-operated calcium entry in human hepatoma cells

This study aimed to investigate cGMP-regulated store-operated Ca(2+)entry in human 7721 hepatoma cells. [Ca(2+)](i)was measured using Fura2/AM. After incubation of the cells with 4 microm thapsigargin, Ca(2+)entry was evoked by application of 1 mMm Ca(2+)to extracellular solution and was blocked by 3 m m Ni(2+), indicating the presence of store-operated Ca(2+)entry in human 7721 hepatoma cell line. Application of 8-Br-cGMP reduced the [Ca(2+)](i)in hepatoma 7721 cells by 80%. These data demonstrated for the first time that store-operated Ca(2+)entry pathway is present in human hepatoma cells, which is regulated by cGMP.     

Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

HAb18G/CD147, a glycoprotein of the immunoglobulin super‐family (IgSF), is a T cell activation‐associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4⁺ and CD8⁺ T cells was up‐regulated. In vitro cross‐linking of T cells with an anti‐HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co‐stimulation inhibited T cell proliferation by down‐regulating the expression of CD25 and interleukin‐2 (IL‐2), decreased production of IL‐4 but not interferon‐γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti‐HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody–antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N‐terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co‐stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.     

Crystal structure of HAb18G/CD147: implications for immunoglobulin superfamily homophilic adhesion

CD147, a member of the immunoglobulin superfamily (IgSF), plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. Importantly, our previous studies have demonstrated that HAb18G/CD147 is a novel hepatocellular carcinoma (HCC)-associated antigen, and HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several matrix metalloproteinases, facilitating invasion and metastasis of HCC cells. In addition, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers. However, the structural basis underlying the multifunctional character of CD147 remains unresolved. We report here the crystal structure of the extracellular portion of HAb18G/CD147 at 2.8A resolution. The structure comprises an N-terminal IgC2 domain and a C-terminal IgI domain, which are connected by a 5-residue flexible linker. This unique C2-I domain organization is distinct from those of other IgSF members. Four homophilic dimers exist in the crystal and adopt C2-C2 and C2-I dimerization rather than V-V dimerization commonly found in other IgSF members. This type of homophilic association thus presents a novel model for homophilic interaction between C2 domains of IgSF members. Moreover, the crystal structure of HAb18G/CD147 provides a good structural explanation for the established multifunction of CD147 mediated by homo/hetero-oligomerizations and should represent a general architecture of other CD147 family members.     

Dimerization is essential for HAb18G/CD147 promoting tumor invasion via MAPK pathway

HAb18G/CD147 is a transmembrane glycoprotein of the immunoglobulin superfamily (IgSF) and is reported to be correlated with invasion and metastasis of many cancers. The crystal structure of HAb18G/CD147 ectodomain has shown that it can form homodimers in crystal. However, the functional significance of HAb18G/CD147 dimerization remains unclear. In the present study, guided by the crystal structure, we performed extensive mutational and functional studies to identify residues critical for dimerization and molecular function of HAb18G/CD147. Fourteen mutants were purified and evaluated for their ability to form dimers in solution and in living cells. Subsequent functional validation revealed that K63E and S193A mutants, which disrupted CD147 dimerization both in solution and in living cells, showed clearly dominant-negative effects on MAPK activation, MMP2 induction and invasiveness in tumor cells. Taken together, the present study provides mutational and functional evidences demonstrating for the first time the functional importance of CD147 dimerization and its direct correlation with invasion and metastasis of tumor cells.     

HAb18G/CD147 Regulates Vinculin-Mediated Focal Adhesion and Cytoskeleton Organization in Cultured Human Hepatocellular Carcinoma Cells

Focal adhesions (FAs), integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147) is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC) cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001), length/width ratios (P<0.0001), and mean intensities (P<0.0001). CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t _1/2) recovery times (22.7±3.3 s) of vinculin-mediated focal adhesions (P<0.0001). In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2). Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.     

HAb18G/CD147 enhances the secretion of matrix metalloproteinases (MMP) via cGMP/NO-sensitive capacitative calcium entry (CCE) and accordingly attenuates adhesion ability of fibroblasts

The present study examined the effect of hepatoma-associated antigen HAb18G (homologous to CD147) expression on the NO/cGMP-regulated Ca2+ mobilization to induce matrix metalloproteinases (MMP) production and attenuate adhesion ability of mouse fibroblast NIH/3T3 cells. HAb18G/CD147 cDNA was transfected into fibroblast 3T3 cells to obtain a cell line stably expressing HAb18G/CD147, t3T3, as demonstrated by immunofluorescence staining and flow cytometry assays. 8-Bromo-cGMP inhibited the thapsigargin-induced Ca2+ entry in 3T3 cells, whereas an inhibitor of protein kinase G, KT5823 (1 microM), led to an increase in Ca2+ entry. Expression of HAb18G/CD147 in t3T3 cells decreased the inhibitory response to cGMP. A similar effect on the Ca2+ entry was observed in 3T3 cells in response to an NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca2+ entry was also reduced in HAb18G/CD147-expressing t3T3 cells, indicating a role for HAb18G/CD 147 in NO/cGMP-regulated Ca2+ entry. Results of gelatin zymography assays showed that addition of extracellular Ca2+ induced MMP (MMP-2, MMP-9) release and activation in a dose-dependent manner, and expression of HAb18G/CD147 enhanced the secretion of MMP-2 and MMP-9 in 3T3 cells. 8-Bromo-cGMP and SNAP reduced the production of MMP in 3T3 cells but not in t3T3 with HAb18G/CD147 expression. RT-PCR experiments substantiated that the expression of MMP-2 and MMP-9 mRNA in HAb18G/CD 147-expressing t3T3 cell was significantly greater than that in 3T3 cells. Experiments investigating adhesion potentials demonstrated that HAb18G/CD147-expressing t3T3 cells pretreated with Ca2+ attached to Matrigel-coated culture plates significantly less efficiently than 3T3 cells. The proportion of attached cells could be increased by treatment with 8-bromo-cGMP and SNAP in 3T3 cells, but not in t3T3. These results suggest that HAb18G/CD147 attenuates adhesion potentials in fibroblasts by enhancing the secretion of MMP through NO/cGMP-sensitive capacitative Ca2+ entry.     

HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner

Background  

Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory.     

The heterogeneity of store-operated calcium entry in melanoma

Calcium is a key regulator of many physiological processes that are perturbed in cancer, such as migration, proliferation and apoptosis. The proteins STIM and Orai mediate store-operated calcium entry (SOCE), the main pathway for calcium entry in non-excitable cells. Changes in the expression and function of STIM and Orai have been found in a range of cancer types and thus implicated in disease progression. Here we discuss the role of STIM, Orai and the SOCE pathway in the progression of melanoma and explore how the heterogeneous nature of melanoma may explain the lack of consensus in the field regarding the role of SOCE in the progression of this disease.     

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.     

Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets

Two mechanisms for store-operated Ca(2+) entry (SOCE) regulated by two independent Ca(2+) stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP(3) receptor (IP(3)RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca(2+) entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca(2+)-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca(2+) stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca(2+) stores and the DTS, the major releasable Ca(2+) store in platelets. These findings were confirmed by using Sr(2+) as a surrogate for Ca(2+) entry. Colchicine attenuated the coupling between IP(3)RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP(3)RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca(2+). These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca(2+) stores.     

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.     

High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells

Diabetes mellitus causes multiple cardiovascular complications. Previous studies have shown that prolonged exposure (96 h) of human umbilical vein endothelial cells (HUVECs) to hyperglycemia causes a significant increase in apoptosis. We report here that this increase in apoptosis is associated with an increase in Ca(2+) current (whole cell patch-clamp recorded) resulting from Ca(2+) entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high glucose (HG, 30 mmol/L) exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La(3+). A marked increase (approximately 80%) in Ca(2+)-dependent calcineurin (CN-A) phosphatase activity also occurred after prolonged HG exposure. Prolonged HG exposure-induced increase in CN-A activity was prevented by 2-APB, and selective CN-A phosphatase inhibition by FK506 or calmodulin inhibition by calmidazolium decreased HG-induced apoptosis. Blocking hydrogen peroxide production using catalase or inhibiting the tyrosine kinase pp60(src) during prolonged exposure to HG, resulted in a marked decrease in apoptosis and was further associated with a significant reduction in CN-A phosphatase activity. The results demonstrate a significant role for Ca(2+) entry in HG-induced apoptosis in HUVECs, and suggest that this role is mediated via H(2)O(2) generation and the action of the Ca(2+)-activated protein phosphatase calcineurin.     

The cellular and molecular basis of store-operated calcium entry

The impact of calcium signalling on so many areas of cell biology reflects the crucial role of calcium signals in the control of diverse cellular functions. Despite the precision with which spatial and temporal details of calcium signals have been resolved, a fundamental aspect of the generation of calcium signals -- the activation of 'store-operated channels' (SOCs) -- remains a molecular and mechanistic mystery. Here we review new insights into the exchange of signals between the endoplasmic reticulum (ER) and plasma membrane that result in activation of calcium entry channels mediating crucial long-term calcium signals.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

Expression of CD20 reveals a new store-operated calcium entry modulator in skeletal muscle

Among the scarce available data about the biological role of the membrane protein CD20, there is some evidence that this protein functions as a store-operated Ca²⁺ channel and/or regulates transmembrane Ca²⁺ trafficking. Recent findings indicate that store-operated Ca²⁺ entry (SOCE) plays a central role in skeletal muscle function and development, but there remain a number of unresolved issues relating to SOCE modulation in this tissue. Here we describe CD20 expression in skeletal muscle, verifying its membrane localization in myoblasts and adult muscle fibers. Additionally, we show that inhibition of CD20 through antibody binding or gene silencing resulted in specific impairment of SOCE in C2C12 myoblasts. Our results provide novel insights into the CD20 expression pattern, and suggest that functional CD20 is required for SOCE to consistently occur in C2C12 myoblasts. These findings may contribute to future identification of mechanisms and molecules involved in the fine regulation of store-operated Ca²⁺ entry in skeletal muscle.     

MURC/CAVIN-4 facilitates store-operated calcium entry in neonatal cardiomyocytes

Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca²⁺ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca²⁺ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies.     

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.