calpain and Melanoma

There is increasing global incidence of highly metastatic melanoma and therapeutic strategies like those focusing on the downstream beta-catenin/MITF axis of invading melanoma cells are urgently needed. Targeting endoplasmic reticulum (ER) stress can promote cancer cell death and inhibit epithelial mesenchymal transition (EMT) in metastatic tumors. This study aimed to determine if Honokiol could promote ER stress-dependent apoptosis and regulate metastatic melanoma. The therapeutic efficacy of Honokiol was assessed using the highly metastatic melanoma xenograft mouse model for peritoneal metastasis and evaluated by computed tomography imaging. The ER stress marker, Calpain-10, delineated a novel proteolytic cleavage enzyme, while CHOP/GADD153-regulated apoptosis was used for gene silencing to determine the role of the β-catenin/MITF axis in melanoma cells. The results showed that Honokiol effectively decreased peritoneal dissemination and organ metastasis via ER stress activation and EMT marker inhibition. Knockdown Calpain-10 or CHOP/GADD153 blocked all of the biological effects in Honokiol-induced β-catenin/MITF cleavage, ERSE or TCF/LEF luciferase activity, and β-catenin kinase activity. These findings suggest that Honokiol can significantly thwart the progression of highly metastatic melanoma using the β-catenin/MITF axis via prompt Calpain-10 and CHOP/GADD153 regulated cascades.

Topics: Animals; Antineoplastic Agents, Phytogenic; beta Catenin; Biphenyl Compounds; Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 2; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Humans; Lignans; Male; Melanoma; Mice, Inbred BALB C; Mice, Nude; Microphthalmia-Associated Transcription Factor; Peritoneal Neoplasms; Skin Neoplasms; Transcription Factor CHOP; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Tyro3, a member of TAM receptor tyrosine kinase family, has been implicated in the regulation of melanoma progression and survival. In this study, we sought the molecular mechanism of Tyro3 effects avoiding endogenous background by overexpression of Tyro3 in fibroblasts that have negligible levels of Tyro3. This introduction triggers the tyrosyl-phosphorylation of ACTN4, a member of actin binding protein family involved in motility, a behavior critical for invasive progression, as shown by siRNA to Tyro3 limiting melanoma cell migration and invasion. Tyro3-mediated phosphorylation of ACTN4 required FAK activation at tyrosine 397 and the EGF receptor cascade, but not EGFR ligand binding. Using PCR-based mutagenesis, the sites of Tyro3-mediated ACTN4 phosphorylation were mapped to ACTN4 tyrosine 11 and 13, and this occurs in conjunction with EGF-mediated phosphorylation on Y4 and Y31. Interestingly, Tyro3-mediated phosphorylation only slightly decreases the actin binding activity of ACTN4. However, this rendered the phosphorylated ACTN4 resistant to the m-calpain cleavage between Y13 and G14, a limited proteolysis that prevents growth factor regulation of ACTN4 interaction with F-actin. Overexpression of both WT ACTN4 and ACTN4Y11/13E, a mimic of ACTN4 phosphorylated at tyrosine 11 and 13, in melanoma WM983b cells resulted in a likely mesenchymal to amoeboidal transition. ACTN4Y11/13E-expressing cells were more amoeboidal, less migratory on collagen I gel coated surface but more invasive through collagen networks. In parallel, expression of ACTN4Y11/13E, in ACTN4 knockdown melanoma WM1158 cells resulted in an increase of invasion compared to WT ACTN4. These findings suggest that Tyro3-mediated phosphorylation of ACTN4 is involved in invasion of melanoma cells.

Topics: Actinin; Amino Acid Substitution; Calpain; Cell Line; Cell Line, Tumor; Cell Movement; Enzyme Activation; Focal Adhesion Kinase 1; Gene Knockdown Techniques; Green Fluorescent Proteins; Humans; Melanoma; Mutation; Neoplasm Invasiveness; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein Stability; Proteolysis; Receptor Protein-Tyrosine Kinases; Recombinant Fusion Proteins; RNA Interference; Signal Transduction; Tyrosine

Melanoma, as one of the most highly metastatic types of cancer, is resistant to current treatment methods, including popular targeted molecular therapy. Consequently, it is essential to develop a deeper understanding of the mechanisms involved in melanoma progression so that alternative treatments may be identified. To date, accumulating evidence supports the use of calpains, including calpain small subunit 1 (also known as Capn4 or CAPNS1), which affect cancer progression through many pathways, such as epithelial‑mesenchymal transition (EMT), the Wnt/β-catenin (β-catenin) and the nuclear factor κB (NF-κB) signaling pathways. The EMT pathway is well known as one of the most important events in tumor metastasis. The present study observed cross-talk among the EMT, β-catenin and NF-κB pathways. To identify the underlying mechanisms of Capn4 activity in melanoma cells, we determined Capn4 expression by gene chip and immunohistochemistral analyses in melanoma tissues and cells in vitro. The extent of apoptosis as determined by TUNEL assay, DAPI staining, and cleaved-caspase-3 assay was increased in human melanoma cells in which Capn4 expression had been knocked down when compared with untreated cells. Transwell assays and xenograft tumorigenicity studies were also performed to assess the effects of Capn4 on migration and invasion in vitro and tumor growth in vivo, respectively. The levels of β-catenin, vimentin, E-cadherin and N-cadherin were altered in human melanoma cells as determined by western blot analysis assay. Our study demonstrated that Capn4 is an underlying target for melanoma treatment.

Topics: Animals; beta Catenin; Calpain; Caspase 3; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Humans; Melanoma; Mice, Inbred BALB C; NF-kappa B; RNA, Small Interfering; Signal Transduction; Transfection; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F2-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F2-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.

Topics: Benzothiazoles; Calpain; Cell Death; Cell Line, Tumor; Cell Proliferation; Humans; Melanoma; Muscle Proteins; Mutation; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Toluene; Tumor Suppressor Protein p53; Up-Regulation

Oncolytic virotherapy is a promising strategy for reducing tumor burden through selective virus replication in rapidly proliferating cells. However, the lysis of slowly replicating cancer stem cells (CSCs), which maintain neoplastic clonality, is relatively modest and the potential contribution of programmed cell death pathways to oncolytic activity is still poorly understood. We show that the oncolytic virus ΔPK lyses CSC-enriched breast cancer and melanoma 3D spheroid cultures at low titers (0.1 pfu/cell) without resistance development and it inhibits the 3D growth potential (spheroids and agarose colonies) of melanoma and breast cancer cells. ΔPK induces calpain activation in both melanoma and breast cancer 3D cultures as determined by the loss of the p28 regulatory subunit, and 3D growth is restored by treatment with the calpain inhibitor PD150606. In melanoma, ΔPK infection also induces light chain 3 (LC3)-II accumulation and p62/SQSTM1 clearance, both markers of autophagy, and 3D growth is restored by treatment with the autophagy inhibitor chloroquine (CQ). However, expression of the autophagy-required protein Atg5 is not altered and CQ does not restore p62/SQSTM1 expression, suggesting that the CQ effect may be autophagy-independent. PD150606 restores expression of p62/SQSTM1 in ΔPK-infected melanoma cultures, suggesting that calpain activation induces anti-tumor activity through p62/SQSTM1 clearance.

Topics: Acrylates; Adaptor Proteins, Signal Transducing; Apoptosis; Autophagy; Calpain; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Neoplastic Stem Cells; Oncolytic Virotherapy; Oncolytic Viruses; Sequestosome-1 Protein; Spheroids, Cellular; Virus Replication

Conventional calpains are ubiquitous cysteine proteases whose activity is promoted by calcium signaling and specifically limited by calpastatin. Calpain expression has been shown to be increased in human malignant cells, but the contribution of the calpain/calpastatin system in tumorigenesis remains unclear. It may play an important role in tumor cells themselves (cell growth, migration, and a contrario cell death) and/or in tumor niche (tissue infiltration by immune cells, neo-angiogenesis). In this study, we have used a mouse model of melanoma as a tool to gain further understanding of the role of calpains in tumor progression. To determine the respective importance of each target, we overexpressed calpastatin in tumor and/or host in isolation. Our data demonstrate that calpain inhibition in both tumor and host blunts tumor growth, while paradoxically increasing metastatic dissemination to regional lymph nodes. Specifically, calpain inhibition in melanoma cells limits tumor growth in vitro and in vivo but increases dissemination by amplifying cell resistance to apoptosis and accelerating migration process. Meanwhile, calpain inhibition restricted to host cells blunts tumor infiltration by immune cells and angiogenesis required for antitumor immunity, allowing tumor cells to escape tumor niche and disseminate. The development of highly specific calpain inhibitors with potential medical applications in cancer should take into account the opposing roles of the calpain/calpastatin system in initial tumor growth and subsequent metastatic dissemination.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line, Tumor; Disease Models, Animal; Male; Melanoma; Mice; Mice, Inbred C57BL; Mice, Transgenic

The molecular mechanisms responsible for the elevated metastatic potential of malignant melanoma are still not fully understood. In order to shed light on the molecules involved in the acquisition by melanoma of a highly aggressive phenotype, we compared the gene expression profiles of two cell clones derived from the human cutaneous metastatic melanoma cell line M14: a highly invasive clone (M14C2/MK18) and a clone (M14C2/C4) with low ability to invade the extracellular matrix (ECM). The highly invasive phenotype of M14C2/MK18 cells was correlated with overexpression of neuropilin-1, activation of a vascular endothelial growth factor (VEGF)-A/VEGFR-2 autocrine loop and secretion of matrix metalloprotease-2. Moreover, in an in vivo murine model, M14C2/MK18 cells displayed a higher growth rate as compared with M14C2/C4 cells, even though in vitro both clones possessed comparable proliferative potential. Microarray analysis in M14C2/MK18 cells showed a strong upregulation of platelet-derived growth factor (PDGF)-C, a cytokine that contributes to angiogenesis, and downregulation of calpain-3, a calcium-dependent thiol-protease that regulates specific signalling cascade components. Inhibition of PDGF-C with a specific antibody resulted in a significant decrease in ECM invasion by M14C2/MK18 cells, confirming the involvement of PDGF-C in melanoma cell invasiveness. Moreover, the PDGF-C transcript was found to be upregulated in a high percentage of human melanoma cell lines (17/20), whereas only low PDGF-C levels were detected in a few melanocytic cultures (2/6). By contrast, inhibition of calpain-3 activity in M14C2/C4 control cells, using a specific chemical inhibitor, markedly increased ECM invasion, strongly suggesting that downregulation of calpain-3 plays a role in the acquisition of a highly invasive phenotype. The results indicate that PDGF-C upregulation and calpain-3 downregulation are involved in the aggressiveness of malignant melanoma and suggest that modulators of these proteins or their downstream effectors may synergise with VEGF‑A therapies in combating tumour-associated angiogenesis and melanoma spread.

Topics: Animals; Calpain; Cell Line, Tumor; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Humans; Lymphokines; Matrix Metalloproteinase 2; Melanoma; Mice; Muscle Proteins; Neoplasm Invasiveness; Platelet-Derived Growth Factor; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

In this study we demonstrate that histone deacetylase (HDAC)-inhibitor mediated cell surface expression of the structural different NKG2D-ligands MICA/B and ULBP2 is calcium-dependent. Treatment with the calcium chelator EGTA inhibited constitutive as well as HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression on melanoma cells and Jurkat T-cells. A NKG2D-dependent cytolytic assay and staining with a recombinant NKG2D-Fc fusion protein showed that calcium chelation impaired the functional ability of NKG2D-ligands induced by HDAC-inhibitor treatment. The HDAC-inhibitor induced cell surface expression of ULBP2, but not MICA/B, was sensitive to treatment calmidazolium and trifluoperazine, two agents known to block calcium signaling. siRNA-mediated knock-down of the calcium-regulated proteins calmodulin or calpain did however not affect NKG2D-ligand cell surface expression on Jurkat T-cells. We further show that secretion and cell surface binding of the calcium-regulating protein galectin-1 is enhanced upon HDAC-inhibitor treatment of melanoma cells. However, binding of galectin-1 to cell surface glycoproteins was not critical for constitutive or HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression. We provide evidence that MICA/B and ULBP2 cell surface expression is controlled differently by calcium, which adds to the increasing perception that cell surface expression of MICA/B and ULBP2 is controlled by distinct signal transduction pathways.

Topics: Base Sequence; Calcium Signaling; Calmodulin; Calpain; Cell Line, Tumor; Cell Membrane; Depsipeptides; Galectin 1; Gene Knockdown Techniques; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Jurkat Cells; Ligands; Melanoma; RNA, Small Interfering; Trifluoperazine

Arginine deprivation is a promising strategy for treating ASS-negative malignant tumors including melanoma. However, autophagy can potentially counteract the effectiveness of this treatment by acting as a pro-survival pathway. By combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with arginine deprivation using ADI-PEG20 (pegylated arginine deiminase), we achieved enhanced apoptosis and accelerated cell death in melanoma cell lines. This implies a switch from autophagy to apoptosis. In our current investigation, we found that TRAIL could induce the cleavage of two key autophagic proteins, Beclin-1 and Atg5, in the combination treatment. Using specific inhibitors for individual caspases, we found that caspase-8 inhibitor could completely abolish the cleavage. Furthermore, caspase-8 inhibitor was able to fully reverse the enhanced cytotoxicity induced by TRAIL. Inhibitors for caspase-3, 6, 9, and 10 were able to block the cleavage of these two autophagic proteins to some extent and correspondingly rescue cells from the cytotoxicity of the combination of TRAIL and arginine deprivation. In contrast, calpain inhibitor could not prevent the cleavage of either Beclin-1 or Atg5, and was unable to prevent cell death. Overall, our data indicate that the cleavage of Beclin-1 and Atg5 by TRAIL-initiated caspase activation is one of the mechanisms that lead to the enhancement of the cytotoxicity in the combination treatment.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Arginine; Autophagy; Autophagy-Related Protein 5; Beclin-1; Calpain; Caspase 10; Caspase 3; Caspase 6; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Enzyme Activation; Humans; Hydrolases; Melanoma; Membrane Proteins; Microtubule-Associated Proteins; Polyethylene Glycols; TNF-Related Apoptosis-Inducing Ligand

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.. This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.. Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Brain Neoplasms; Calpain; Cell Line, Tumor; Dacarbazine; Glioma; Humans; Male; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Selenium; Skin Neoplasms; Temozolomide; Xenograft Model Antitumor Assays

Malignant melanoma is a highly aggressive and drug-resistant cancer. Virotherapy is a novel therapeutic strategy based on cancer cell lysis through selective virus replication. However, its clinical efficacy is modest, apparently related to poor virus replication within the tumors. We report that the growth compromised herpes simplex virus type 2 (HSV-2) mutant, DeltaPK, has strong oncolytic activity for melanoma largely caused by a mechanism other than replication-induced cell lysis. The ratio of dead cells (determined by trypan blue or ethidium homodimer staining) to cells that stain with antibody to the major capsid protein VP5 (indicative of productive infection) was 1.8-4.1 for different melanoma cultures at 24-72 h post-infection. Cell death was due to activation of calpain as well as caspases-7 and -3 and it was abolished by the combination of calpain (PD150606) and pancaspase (benzyloxycarbonyl-Val-Ala-Asp-fluormethyl ketone, z-VAD-fmk) inhibitors. Upregulation of the autopahgy protein Beclin-1 and the pro-apoptotic protein H11/HspB8 accompanied DeltaPK-induced melanoma oncolysis. Intratumoral DeltaPK injection (10(6)-10(7) plaque-forming unit (pfu)) significantly reduced melanoma tumor burden associated with calpain and caspases-7 and -3 activation, Beclin-1 and H11/HspB8 upregulation and activation of caspase-1-related inflammation. Complete remission was seen for 87.5% of the LM melanoma xenografts at 5 months after treatment termination. The data indicate that DeltaPK is a promising virotherapy for melanoma that functions through virus-induced programmed cell death pathways.

Topics: Animals; Apoptosis; Autophagy; Calpain; Capsid Proteins; Caspase 3; Caspase 7; Caspase Inhibitors; Cell Line, Tumor; Gene Deletion; Herpesvirus 2, Human; Humans; Melanoma; Mice; Mice, Nude; Oncolytic Virotherapy; Oncolytic Viruses; Protein Serine-Threonine Kinases; Ribonucleotide Reductases; Skin Neoplasms; Virus Replication; Xenograft Model Antitumor Assays

Resistance of melanoma cells to chemotherapeutics remains a major obstacle to successful treatment of melanoma once it has spread beyond locoregional sites. We report in this study that activation of the unfolded protein response (UPR) is involved in resistance of melanoma cells to two chemotherapeutic drugs, cisplatin (CDDP) and adriamycin, and this is associated with glucose-regulated protein 78 (GRP78)-mediated inhibition of activation of caspase-4 and -7. The UPR was constitutively activated in cultured melanoma cell lines and fresh melanoma isolates as evidenced by elevated expression levels of the GRP78 protein and the active form of x-box-binding protein 1 messenger RNA. Treatment with CDDP or adriamycin further increased the levels, indicative of induction of endoplasmic reticulum stress and activation of the UPR by the drugs. Inhibition of GRP78 by small-interference RNA (siRNA)-sensitized melanoma cells to CDDP- and adriamycin-induced apoptosis. This was associated with enhanced caspase-4 and -7 activation as siRNA knockdown of the caspases blocked induction of apoptosis. In contrast, overexpression of GRP78 attenuated activation of caspase-4 and -7 and induction of apoptosis by the drugs. CDDP- and adriamycin-induced activation of caspase-4 and -7 appeared to be mediated by calpain activity in that it was blocked by the calpain inhibitors calpeptin and PD150606 even when GRP78 was inhibited by siRNA. These results provide new insights into resistance mechanisms of melanoma cells to CDDP and adriamycin and identify GRP78 as a potential target for enhancing chemosensitivity in melanoma.

Topics: Antineoplastic Agents; Calpain; Caspase 7; Caspase 9; Caspases, Initiator; Cell Line, Tumor; Cisplatin; DNA-Binding Proteins; Doxorubicin; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Enzyme Activation; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Melanoma; Molecular Chaperones; Protein Folding; Regulatory Factor X Transcription Factors; RNA, Small Interfering; Signal Transduction; Transcription Factors

We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility.

Topics: Calpain; Cell Line, Tumor; Cell Movement; Chelating Agents; Contractile Proteins; Cytoskeleton; Egtazic Acid; Filamins; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Microfilament Proteins; Proto-Oncogene Proteins; Receptor Tyrosine Kinase-like Orphan Receptors; Receptors, Cell Surface; RNA, Small Interfering; Skin Neoplasms; Wnt Proteins; Wnt-5a Protein

Calpains are cysteine proteases comprising members ubiquitously expressed in human tissues and other tissue-specific isoforms. Alterations of calpain 3 (p94), the muscle-specific isoform that contains three peculiar sequences (NS, IS1 and IS2), are strictly associated to the limb-girdle muscular dystrophy type 2A, in which a myonuclear apoptosis has been documented. Our recent demonstration of a proapoptotic role of ubiquitous calpains in drug-induced apoptosis of melanoma cells prompted us to investigate the expression of calpain 3 in human melanoma cell lines undergoing apoptosis and in melanocytic lesions. In melanoma cell lines, we have identified two novel splicing variants of calpain 3 (hMp78 and hMp84): they have an atypical initiation exon and a putative nuclear localization signal, the shorter one lacks IS1 inset and both proteins are extremely unstable. Virtually, both isoforms (prevalently as cleavage forms) are localized in cytoplasm and in nucleoli. In cisplatin-treated preapoptotic cells, an increase of both transcription and autoproteolytic cleavage of the novel variants is observed; the latter event is prevented by the inhibitor of ubiquitous calpains, calpeptin, which is also able to protect from apoptosis. Interestingly, among melanocytic lesions, the expression of these novel variants is significantly downregulated, compared with benign nevi, in the most aggressive ones, i.e. in vertical growth phase melanoma and, even more, in metastatic melanoma cells, characterized by invasiveness properties and usually highly resistant to apoptosis. On the whole, our observations suggest that calpain 3 variants can play a proapoptotic role in melanoma cells and its downregulation, as observed in highly aggressive lesions, could contribute to melanoma progression.

Topics: Alternative Splicing; Antineoplastic Agents; Apoptosis; Biopsy; Calpain; Cell Line, Tumor; Cell Nucleolus; Cisplatin; Cytoplasm; Dipeptides; Dysplastic Nevus Syndrome; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Muscle Proteins; Neoplasm Metastasis; Nevus; RNA, Messenger; Skin Neoplasms

The contribution of different proteolytic systems, in particular calpains and effector caspases, in apoptotic cell death is still controversial. In this paper, we show that during cisplatin-induced apoptosis of human metastatic melanoma cells, calpain activation, as measured in intact cells by two different fluorescent substrates, is an early event, taking place well before caspase-3/-7 activation, and progressively increasing during 48 h of treatment. Such activation appears to be independent from any intracellular calcium imbalance; in fact, an increase of cytosolic calcium along with emptying of the reticular stores occur only at very late stages, uniquely in frankly apoptotic, detached cells. Calpain activation proves to be an early and crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples co-treated with the calpain inhibitors, MDL 28170, calpeptin and PD 150606, where a variable but significant reduction of both caspase-3/-7 activity and cell detachment is observed. Consistently, such a protective effect can be at least partially due to the impairment of cisplatin-induced p53 activation, occurring early in committed, preapoptotic cells. Furthermore, in late apoptotic cells, calpain activity is also responsible for the formation of a novel p53 proteolytic fragment (approximately 26 kDa), whose function is so far to be elucidated.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Calpain; Caspase 3; Caspase 7; Caspase Inhibitors; Cisplatin; Endoplasmic Reticulum; Enzyme Activation; Humans; Melanoma; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.

Topics: Acrylates; Butanes; Calpain; Cell Cycle; Cell Line, Tumor; Cisplatin; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Humans; I-kappa B Proteins; Melanoma; NF-kappa B; NF-KappaB Inhibitor alpha; Oligopeptides; Protease Inhibitors; Proteasome Inhibitors

In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.

Topics: Aged; Aged, 80 and over; Antigens, Differentiation, B-Lymphocyte; Calpain; Cell Line, Tumor; Computational Biology; Expressed Sequence Tags; Female; Gene Library; Histocompatibility Antigens Class II; Humans; Immunohistochemistry; Isoenzymes; Male; Melanoma; Middle Aged; Muscle Proteins; Neoplasm Staging; Polymerase Chain Reaction; Reproducibility of Results

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.

Topics: Calpain; Cell Cycle Proteins; Cell Division; Choroid Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Humans; In Vitro Techniques; MAP Kinase Signaling System; Melanocytes; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Substrate Specificity; Tumor Cells, Cultured; Tumor Suppressor Proteins

Topics: Apoptosis; Calcium; Calpain; Humans; Melanoma; Skin Neoplasms; Vitamin D