Simultaneous mitochondrial Ca(2+) overload and proteasomal inhibition are responsible for the induction of paraptosis in malignant breast cancer cells

In this study, we investigated the role of Ca(2+) in curcumin-induced paraptosis, a cell death mode that is accompanied by dilation of mitochondria and the endoplasmic reticulum (ER). Curcumin induced mitochondrial Ca(2+) overload selectively in the malignant breast cancer cells, but not in the normal breast cell, contributing to the dilation of mitochondria/ER and subsequent paraptotic cell death. In addition, we found that simultaneous inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (mNCX) and proteasomes can trigger a sustained mitochondrial Ca(2+) overload and effectively induce paraptosis in malignant breast cancer cells.     

Ca2+ influx-mediated dilation of the endoplasmic reticulum and c-FLIPL downregulation trigger CDDO-Me–induced apoptosis in breast cancer cells

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me–induced increases in intracellular Ca²⁺ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca²⁺ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca²⁺ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIP_L (cellular FLICE-inhibitory protein) and overexpression of c-FLIP_L blocked CDDO-Me–induced cell death, but not vacuolation. These results suggest that c-FLIP_L downregulation is a key contributor to CDDO-Me–induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca²⁺ influx and ROS generation as well as caspase activation via c-FLIP_L downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.     

Decreased expression of 17β-hydroxysteroid dehydrogenase type 1 is associated with DNA hypermethylation in colorectal cancer located in the proximal colon

Background

Celastrol is an active ingredient of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo; however, the lack of information on the target and mechanism of action of this compound have impeded its clinical application. In this study, we sought to determine the mode of action of celastrol by focusing on the processes that mediate its anticancer activity.

Methods

The downregulation of heat shock protein 90 (HSP90) client proteins, phosphorylation of c-Jun NH2-terminal kinase (JNK), and cleavage of PARP, caspase 9 and caspase 3 were detected by western blotting. The accumulation of reactive oxygen species (ROS) was analyzed by flow cytometry and fluorescence microscopy. Cell cycle progression, mitochondrial membrane potential (MMP) and apoptosis were determined by flow cytometry. Absorption spectroscopy was used to determine the activity of mitochondrial respiratory chain (MRC) complexes.

Results

Celastrol induced ROS accumulation, G2-M phase blockage, apoptosis and necrosis in H1299 and HepG2 cells in a dose-dependent manner. N-acetylcysteine (NAC), an antioxidative agent, inhibited celastrol-induced ROS accumulation and cytotoxicity. JNK phosphorylation induced by celastrol was suppressed by NAC and JNK inhibitor SP600125 (SP). Moreover, SP significantly inhibited celastrol-induced loss of MMP, cleavage of PARP, caspase 9 and caspase 3, mitochondrial translocation of Bad, cytoplasmic release of cytochrome c, and cell death. However, SP did not inhibit celastrol-induced ROS accumulation. Celastrol downregulated HSP90 client proteins but did not disrupt the interaction between HSP90 and cdc37. NAC completely inhibited celastrol-induced decrease of HSP90 client proteins, catalase and thioredoxin. The activity of MRC complex I was completely inhibited in H1299 cells treated with 6 μM celastrol in the absence and presence of NAC. Moreover, the inhibition of MRC complex I activity preceded ROS accumulation in H1299 cells after celastrol treatment.

Conclusion

We identified ROS as the key intermediate for celastrol-induced cytotoxicity. JNK was activated by celastrol-induced ROS accumulation and then initiated mitochondrial-mediated apoptosis. Celastrol induced the downregulation of HSP90 client proteins through ROS accumulation and facilitated ROS accumulation by inhibiting MRC complex I activity. These results identify a novel target for celastrol-induced anticancer activity and define its mode of action.     

Involvement of Ca2+ and ROS in α‐tocopheryl succinate‐induced mitochondrial permeabilization

Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox‐silent analog of vitamin E, α‐tocopheryl succinate (α‐TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax‐mediated permeabilization of the outer mitochondrial membrane. Here we show that α‐TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T‐lymphocytes. In particular, in addition to ROS production, α‐TOS stimulates rapid Ca²⁺ entry into the cells with subsequent accumulation of Ca²⁺ in mitochondria—a prerequisite step for MPT induction. Alteration of mitochondrial Ca²⁺ buffering capacity was observed as early as 8 hr after incubation with α‐TOS, when no activation of Bax was yet detected. Ca²⁺ accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca²⁺ uptake significantly mitigated the apoptotic response. Importantly, Ca²⁺‐induced mitochondrial destabilization might cooperate with Bax‐mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria.     

High endoplasmic reticulum activity renders multiple myeloma cells hypersensitive to mitochondrial inhibitors

Multiple myeloma (MM) cells continuously secrete large amounts of immunoglobulins that are folded in the endoplasmic reticulum (ER) whose function depend on the Ca²⁺ concentration inside its lumen. Recently, it was shown that the ER membrane leaks Ca²⁺ that is captured and delivered back by mitochondria in order to prevent its loss. Thus, we hypothesized that the highly active and abundant ER in MM cells results in greater Ca²⁺-regulation by mitochondria which would render them sensitive to mitochondrial inhibitors. Here, we indeed find that Ca²⁺ leak is greater in 3 MM, when compared to 2 B-cell leukemia cell lines. Moreover, this greater leak in MM cells is associated with hypersensitivity to various mitochondrial inhibitors, including CCCP. Consistent with our hypothesis, CCCP is more potent in inducing the unfolded protein response marker, CHOP/GADD153 in MM versus B-cell leukemia lines. Additionally, MM cells are found to be significantly more sensitive to clinically used fenofibrate and troglitazone, both of which were recently shown to have inhibitory effects on mitochondrial function. Overall, our results demonstrate that the unusually high ER activity in MM cells may be exploited for therapeutic benefit through the use of mitochondrial inhibitors including troglitazone and fenofibrate.     

Molecular interaction and functional coupling between type 3 inositol 1,4,5-trisphosphate receptor and BKCa channel stimulate breast cancer cell proliferation

BackgroundThe implication of ion channels and inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ signalling (IICS) in the carcinogenesis processes, including deregulation of cell proliferation, migration and invasion, is increasingly studied. Studies from our laboratory have shown that type 3 IP₃ receptor (IP₃R3) and voltage- and Ca²⁺-dependent K⁺ channels BK_Ca channels are involved in human breast cancer cell proliferation. In this context, we investigated the probable interaction between these two proteins (IP₃R3 and BK_Ca channel) in normal and in breast cancer cells.MethodsMCF-7 and MCF-10A cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay in the presence or absence of adenosine triphosphate (ATP). Furthermore, cell-cycle analysis was carried out and cell cycle protein expression was examined by Western blotting. Immunocytochemistry and co-immunoprecipitation assays were used to check co-localisation of BK_Ca and IP₃R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca²⁺ imaging were performed to assess the functional interaction.ResultsOur results are in favour of a functional and a molecular coupling between IP₃R3 and BK_Ca channel that is involved in MCF-7 proliferation. Indeed, ATP increased MCF-7 cell proliferation and this effect was impaired when the expression of BK_Ca and/or IP₃R3 has been reduced by specific small interfering RNAs (siRNAs). Flow cytometry experiments showed that both siRNAs led to cell cycle arrest in the G0/G1 phase and these results were confirmed by the analysis of cell cycle protein expression. Specifically, BK_Ca and IP₃R3 silencing decreased both cyclin-D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Furthermore, ATP elicited a phospholipase C (PLC)-dependent elevation of internal Ca²⁺ that triggered in turn an iberiotoxin (IbTx)- and a tetra-ethyl-ammonium (TEA)-sensitive membrane hyperpolarisation that was strongly reduced in the cells with silenced IP₃R3 or BK_Ca. In the same way, intracellular application of Ins(2,4,5)P₃ triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca²⁺ chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) prevented ATP-induced BK_Ca activation. BK_Ca and IP₃R3 also co-immunoprecipitated and this interaction seemed to occur in cholesterol-enriched microdomains. Conversely, in the normal breast cell line MCF-10A, neither ATP application nor BK_Ca silencing affected cell proliferation. Furthermore, IP₃R3 and BK_Ca did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BK_Ca and IP₃R3 in the MCF-10A normal cell line.ConclusionAltogether, our results suggest a molecular and functional link between BK_Ca channel and IP₃R3 in cancer cells. Our findings led us to propose this coupling between BK_Ca and IP₃R3 as an important mechanism for tumour cell proliferation.     

Suppression of STIM1 inhibits human glioblastoma cell proliferation and induces G0/G1 phase arrest

Background

Depletion of calcium (Ca²⁺) from the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca²⁺ entry (SOCE) pathway which sustains long-term Ca²⁺ signals and is critical for cellular functions. Stromal interacting molecule 1 (STIM1) serves a dual role as an ER Ca²⁺ sensor and activator of SOCE. Aberrant expression of STIM1 could be observed in several human cancer cells. However, the role of STIM1 in regulating tumorigenesis of human glioblastoma still remains unclear.

Methods

Expression of STIM1 protein in a panel of human glioblastoma cell lines (U251, U87 and U373) in different transformation level were evaluated by Western blot method. STIM1 loss of function was performed on U251 cells, derived from grade IV astrocytomas-glioblastoma multiforme with a lentvirus-mediated short harpin RNA (shRNA) method. The biological impacts after knock down of STIM1 on glioblastoma cells were investigated in vitro and in vivo.

Results

We discovered that STIM1 protein was expressed in U251, U87 and U373 cells, and especially higher in U251 cells. RNA interference efficiently downregulated the expression of STIM1 in U251 cells at both mRNA and protein levels. Specific downregulation of STIM1 inhibited U251 cell proliferation by inducing cell cycle arrest in G0/G1 phase through regulation of cell cycle-related genes, such as p21^Waf1/Cip1_, cyclin D1 and cyclin-dependent kinase 4 (CDK4), and the antiproliferative effect of STIM1 silencing was also observed in U251 glioma xenograft tumor model.

Conclusion

Our findings confirm STIM1 as a rational therapeutic target in human glioblastoma, and also indicate that lentivirus-mediated STIM1 silencing is a promising therapeutic strategy for human glioblastoma.     

EFHD1, a novel mitochondrial regulator of tumor metastasis in clear cell renal cell carcinoma

The biological function of many mitochondrial proteins in mechanistic detail has not been well investigated in clear cell renal cell carcinoma (ccRCC). A seven-mitochondrial-gene signature was generated by Lasso regression analysis to improve the prediction of prognosis of patients with ccRCC, using The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium cohort. Among those seven genes, EFHD1 is less studied and its role in the progression of ccRCC remains unknown. The decreased expression of EFHD1 was validated in clinical samples and was correlated with unfavorable outcome. Overexpression of EFHD1 in ccRCC cells resulted in the reduction of mitochondrial Ca²⁺, and the inhibition of cell migration and invasion in vitro and tumor metastasis in vivo. Mechanistically, EFHD1 physically bound to the core mitochondrial calcium transporter (mitochondrial calcium uniporter, MCU) through its N-terminal domain. The interaction between EFHD1 and MCU suppressed the uptake of Ca²⁺ into mitochondria, and deactivated the Hippo/YAP signaling pathway. Further data revealed that the ectopic expression of EFHD1 upregulated STARD13 to enhance the phosphorylation of YAP protein at Ser-127. The knockdown of STARD13 or the overexpression of MCU partly abrogated the EFHD1-mediated induction of phosphorylation of YAP at Ser-127 and suppression of cell migration. Taken together, the newly identified EFHD1–MCU–STARD13 axis participates in the modulation of the Hippo/YAP pathway and serves as a novel regulator in the progression of ccRCC.     

Increased intracellular Ca2+ signaling caused by the antitumor agent helenalin and its analogues

The antitumor sesquiterpene lactone helenalin, which is found in species of the plant genusHelenium, cause a marked potentiation of the increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) produced by mitogens such as vasopressin, bradykinin, and platelet-derived growth factor in Swiss mouse 3T3 fibroblasts. Removing external Ca²⁺ partly attenuated the increased [Ca²⁺]_i response. caused by helenalin. The increased [Ca²⁺]_i responses occurred at concentrations of helenalin that inhibited cell proliferation. At higher concentrations, helenalin inhibited the [Ca²⁺]_i responses. No change in resting [Ca²⁺]_i was caused by helenalin even at high concentrations. Other helenalin analogues also increased the [Ca²⁺]_i response. Helenalin did not inhibit protein kinase C (PKC) and PKC appeared to play a minor role in the effects of helenalin on [Ca²⁺]_i responses in intact cells. Studies with saponin-permeabilized HT-29 human colon carcinosarcoma cells indicated that helenalin caused an increased accumulation of Ca²⁺ into nonmitochondrial stores and that the potentiating effect of helenalin on mitogen-stimulated [Ca²⁺]_i responses was due in part to an increase in the inositol-(1,4,5)-trisphosphate-mediated release of Ca²⁺ from these stores.     

Diosgenin induces cell cycle arrest and apoptosis in human leukemia K562 cells with the disruption of Ca2+ homeostasis

Purpose Diosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells.Methods Cell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca²⁺ concentration and reactive oxygen species (ROS) generation.Results Cell cycle analysis showed that diosgenin caused G₂/M arrest independently of p53. The levels of cyclin B1 and p21^Cip1/Waf1 were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca²⁺ concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-x_L proteins were downregulated, whereas the proapoptotic Bax was upregulated.Conclusions Diosgenin inhibits K562 cell proliferation via cell cycle G₂/M arrest and apoptosis, with disruption of Ca²⁺ homeostasis and mitochondrial dysfunction playing vital roles.     

A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2

Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP₃Rs), inhibiting pro-apoptotic Ca²⁺ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP₃R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP₃ Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP₃R interaction and thus inducing Ca²⁺-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton’s tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP₃R interaction.     

Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway

Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G₂/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G₁ population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-X_L and an increase in Bax expression. Ectopic expression of Bcl-X_L but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca⁺⁺ content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.     

The marine sponge toxin agelasine B increases the intracellular Ca2+ concentration and induces apoptosis in human breast cancer cells (MCF-7)

Purpose

In search for new drugs derived from natural products for the possible treatment of cancer, we studied the action of agelasine B, a compound purified from a marine sponge Agelas clathrodes.

Methods

Agelasine B was purified from a marine sponge Agelas clathrodes and assayed for cytotoxicity by MTT on two human breast cancer cells (MCF-7 and SKBr3), on a prostate cancer cells (PC-3) and on human fibroblasts. Changes in the intracellular Ca²⁺ concentrations were assessed with FURA 2 and by confocal microscopy. Determination of Ca²⁺-ATPase activity was followed by Pi measurements. Changes in the mitochondria electrochemical potential was followed with Rhodamine 123. Apoptosis and DNA fragmentation were determined by TUNEL experiments.

Results

Upon agelasine B treatment, cell viability of both human breast cancer cell lines was one order of magnitude lower as compared with fibroblasts (IC₅₀ for MCF-7?=?2.99???M; SKBr3: IC₅₀?=?3.22???M vs. fibroblasts: IC₅₀?=?32.91???M), while the IC₅₀ for PC-3 IC₅₀?=?6.86???M. Agelasine B induced a large increase in the intracellular Ca²⁺ concentration in MCF-7, SKBr3, and PC-3 cells. By the use of confocal microscopy coupled to a perfusion system, we could observe that this toxin releases Ca²⁺ from the endoplasmic reticulum (ER). We also demonstrated that agelasine B produces a potent inhibition of the ER Ca²⁺-ATPase (SERCA), and that this compound induced the fragmentation of DNA. Accordingly, agelasine B reduced the expression of the anti-apoptotic protein Bcl-2 and was able to activate caspase 8, without affecting the activity of caspase 7.

Conclusions

Agelasine B in MCF-7 cells induce the activation of apoptosis in response to a sustained increase in the [Ca²⁺] _i after blocking the SERCA activity. The reproduction of the effects of agelasine B on cell viability and on the [Ca²⁺] _I obtained on SKBr3 and PC-3 cancer cells strongly suggests the generality of the mechanism of action of this toxin.     

Inositol 1,4,5-trisphosphate-induced Ca<Superscript>2+</Superscript> signalling is involved in estradiol-induced breast cancer epithelial cell growth

Background  

Ca²⁺ is a ubiquitous messenger that has been shown to be responsible for controlling numerous cellular processes including cell growth and cell death. Whereas the involvement of IP₃-induced Ca²⁺ signalling (IICS) in the physiological activity of numerous cell types is well documented, the role of IICS in cancer cells is still largely unknown. Our purpose was to characterize the role of IICS in the control of growth of the estrogen-dependent human breast cancer epithelial cell line MCF-7 and its potential regulation by 17β-estradiol (E₂).     

Epi-reevesioside F inhibits Na+/K+-ATPase,causing cytosolic acidification,Bak activation and apoptosis in glioblastoma

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC₅₀ values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na⁺/K⁺-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na⁺ but not Ca²⁺, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na⁺/K⁺-ATPase, leading to overload of intracellular Na⁺ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.     

A novel interaction between calcium-modulating cyclophilin ligand and Basigin regulates calcium signaling and matrix metalloproteinase activities in human melanoma cells

Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca²⁺]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca²⁺]i and the [Ca²⁺]i increase induced by the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca²⁺]i signaling. Meanwhile upregulating the [Ca²⁺]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca²⁺]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment.