AMTB,a TRPM8 antagonist,suppresses growth and metastasis of osteosarcoma through repressing the TGFβ signaling pathway

Since its first identification in prostate cancers and prostate tissues, transient receptor potential melastatin-subfamily member 8 (TRPM8) is subsequently found to be overexpressed in a wide range of cancers and is shown to be implicated in tumorigenesis and tumor progression. Here, we used N-(3-aminopropyl)-2-[(3-methylphenyl) methoxy] -N-(2-thienylmethyl) benzamide hydrochloride (AMTB), a specific TRPM8 antagonist, to explore its antitumoral effect on osteosarcoma. We find that AMTB suppresses osteosarcoma cell proliferation, metastasis and induces cellular apoptosis. Xenograft model in nude mice experiments also define that AMTB can increase the sensitivity of tumor cells to cisplatin, the cytotoxic chemotherapeutic regimens in treating osteosarcoma. Molecularly, AMTB specifically antagonizes TRPM8 which is upregulated in osteosarcoma and its expression level in osteosarcoma tissues is negatively related to patients’ prognosis. Finally, RNA sequencing analysis was performed to explore the mechanism underlying the antitumoral effect of AMTB on osteosarcoma cells and the results prove that AMTB suppresses the Transforming Growth Factor β (TGFβ) signaling pathway. Our study provides evidence that TRPM8 could be a potential therapeutic target and AMTB can suppress growth and metastasis of osteosarcoma cells through repressing the TGFβ signaling pathway and increase the sensitivity of tumor cells to cisplatin.Subject terms: Biochemistry, Bone cancer     

Transient receptor potential melastatin 2–antisense RNA is overexpresed in osteosarcoma and regulates cell proliferation and apoptosis

Accumulating evidence suggested that transient receptor potential melastatin 2–antisense RNA (TRPM2-AS) played crucial roles in the progression of human cancers. However, the role of TRPM2-AS was still unknown in osteosarcoma. The aim of this study was to explore the clinical significance of TRPM2-AS in osteosarcoma patients, and determine the role of TRPM2-AS on osteosarcoma cell proliferation and apoptosis. In our results, we identified a novel oncogenic long noncoding RNA TRPM2-AS, which was overexpressed in osteosarcoma tissues and cells, and correlated with advanced Enneking stage, large tumor size and high histological grade in osteosarcoma cases. Survival analysis indicated that osteosarcoma patients with high TRPM2-AS expression had an obviously shorter overall survival time than those with low TRPM2-AS expression. Loss-of-function studies suggested that suppression of TRPM2-AS expression inhibited osteosarcoma cell proliferation and induced cell apoptosis through upregulating cleaved caspase-3 and cleaved caspase-9 expression. In conclusion, TRPM2-AS acts as an oncogenic long noncoding RNA and predicts poor prognosis in osteosarcoma.     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

The LCK-14-3-3ζ-TRPM8 axis regulates TRPM8 function/assembly and promotes pancreatic cancer malignancy

Transient receptor potential melastatin 8 (TRPM8) functions as a Ca²⁺-permeable channel in the plasma membrane (PM). Dysfunction of TRPM8 is associated with human pancreatic cancer and several other diseases in clinical patients, but the underlying mechanisms are unclear. Here, we found that lymphocyte-specific protein tyrosine kinase (LCK) directly interacts with TRPM8 and potentiates TRPM8 phosphorylation at Y1022. LCK positively regulated channel function characterized by increased TRPM8 current densities by enhancing TRPM8 multimerization. Furthermore, 14-3-3ζ interacted with TRPM8 and positively modulated channel multimerization. LCK significantly enhanced the binding of 14-3-3ζ and TRPM8, whereas mutant TRPM8-Y1022F impaired TRPM8 multimerization and the binding of TRPM8 and 14-3-3ζ. Knockdown of 14-3-3ζ impaired the regulation of TRPM8 multimerization by LCK. In addition, TRPM8 phosphotyrosine at Y1022 feedback regulated LCK activity by inhibiting Tyr505 phosphorylation and modulating LCK ubiquitination. Finally, we revealed the importance of TRPM8 phosphorylation at Y1022 in the proliferation, migration, and tumorigenesis of pancreatic cancer cells. Our findings demonstrate that the LCK-14-3-3ζ-TRPM8 axis for regulates TRPM8 assembly, channel function, and LCK activity and maybe provide potential therapeutic targets for pancreatic cancer.Subject terms: Phosphorylation, Paediatric cancer     

Cytoskeletal Actin Structure in Osteosarcoma Cells Determines Metastatic Phenotype via Regulating Cell Stiffness,Migration, and Transmigration

Osteosarcoma is the most common primary malignant bone tumor. The cause of death due to osteosarcoma is typically a consequence of metastasis to the lung. Controlling metastasis leads to improved prognosis for osteosarcoma patients. The cell stiffness of several tumor types is involved in metastatic potential; however, it is unclear whether the metastatic potential of osteosarcoma depends on cell stiffness. In this study, we analyzed the cell stiffness of the low metastatic Dunn cell line and its highly metastatic LM8 subline, and compared actin organization, cell proliferation, and metastasis. Actin cytoskeleton, polymerization, stiffness, and other cellular properties were analyzed. The organization of the actin cytoskeleton was evaluated by staining F-actin with Alexa Fluor 488 phalloidin. Cell stiffness was measured using Atomic Force Microscopy (AFM). Cell proliferation, migration, invasion, and adhesion were also evaluated. All experiments were performed using mouse osteosarcoma cell lines cultured in the absence and presence of cytochalasin. In LM8 cells, actin polymerization was strongly suppressed and actin levels were significantly lower than in Dunn cells. Stiffness evaluation revealed that LM8 cells were significantly softer than Dunn. Young’s modulus images showed more rigid fibrillar structures were present in Dunn cells than in LM8 cells. LM8 cells also exhibited a significantly higher proliferation. The migration and invasion potential were also higher in LM8 cells, whereas the adhesion potential was higher in Dunn cells. The administration of cytochalasin resulted in actin filament fragmentation and decreased actin staining intensity and cell stiffness in both LM8 and Dunn cells. Cells with high metastatic potential exhibited lower actin levels and cell stiffness than cells with low metastatic potential. The metastatic phenotype is highly correlated to actin status and cell stiffness in osteosarcoma cells. These results suggest that evaluation of actin dynamics and cell stiffness is an important quantitative diagnostic parameter for predicting metastatic potential. We believe that these parameters represent new reliable quantitative indicators that can facilitate the development of new drugs against metastasis.     

Effects of luteolin on canine osteosarcoma: Suppression of cell proliferation and synergy with cisplatin

Canine osteosarcoma is characterized by aggressiveness, easy metastasis to the lungs, and high mortality after standard therapy. Luteolin is a flavonoid found in vegetables and fruits and has diverse functions. Elucidation of the biological mechanisms of luteolin on canine osteosarcoma will enhance the efficacy of chemotherapeutic agents in canine tumors. In this study, we examined the effects of luteolin in the canine osteosarcoma cell lines, D17 and DSN. The results of this study show that luteolin inhibited canine osteosarcoma cell proliferation and induced apoptosis by altering cell-cycle proportion, producing reactive oxygen species, increasing the loss of mitochondrial membrane potential, and reducing cytosolic Ca²⁺ concentration. In addition, luteolin activated ERK1/2 and inactivated phosphoinositide 3-kinase/AKT signaling in canine osteosarcoma cells. Moreover, luteolin showed synergistic effects with cisplatin to reduce cell proliferation. In summary, luteolin induced cell death by initiating mitochondrial dysfunction and regulating intracellular signal transduction in canine osteosarcoma cells.     

TRPM8 channel augments T-cell activation and proliferation

The transient receptor potential melastatin 8 (TRPM8) is an ion channel that has been widely studied as a cold-sensitive nociceptor. However, its importance in nonneuronal cells is mostly unexplored. Here, we describe the presence and functional significance of endogenous TRPM8, a nonselective Ca²⁺-channel in T cell functions. The major pool of TRPM8 resides at the T cell surface and its surface accumulation significantly increases in activated T cells. TRPM8 activation synergizes with T-cell receptor (TCR) stimulation to increase CD25, CD69 levels and enhances secretion of proinflammatory cytokine tumor necrosis factor. However, TRPM8 inhibition does not restrict TCR stimulation mediated activation of T cells, indicating that unlike the heat-sensitive TRPV1 and TRPV4 channels, the cold-sensitive TRPM8 channel may be dispensable during T-cell activation, at least in mice. In this study, we demonstrate that TRPM8 promotes TCR-induced intracellular calcium increase. TRPM8 activation is beneficial for T-cell activation and differentiation into effector cells. TRPM8 inhibition during the T-cell activation process may lead to altered phenotype and reduced proliferation, without affecting cell viability. These results collectively establish TRPM8 as a functional calcium channel whose activation may be utilized for mounting an effective immune response. The findings of this study will be relevant to the regulation and response of T cells during cell-mediated immunity. These results will likely further our understanding on the role of ion channels in T-cell activation.     

TRP channels in cancer

The progression of cells from a normal differentiated state in which rates of proliferation and apoptosis are balanced to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signalling proteins and the evolution and clonal selection of more aggressive cell phenotypes. These events are associated with changes in the expression of numerous other proteins. This process of tumorigenesis involves the altered expression of one or more TRP proteins, depending on the nature of the cancer. The most clearly described changes are those involving TRPM8, TRPV6 and TRPM1. Expression of TRPM8 is substantially increased in androgen-dependent prostate cancer cells, but is decreased in androgen independent and metastatic prostate cancer. TRPM8 expression is regulated, in part, by androgens, most likely through androgen response elements in the TRPM8 promoter region. TRPM8 channels are involved in the regulation of cell proliferation and apoptosis. Expression of TRPV6 is also increased in prostate cancer and in a number of other cancers. In contrast to TRPM8, expression of TRPV6 is not directly regulated by androgens. TRPM1 is highly expressed in early stage melanomas but its expression declines with increases in the degree of aggressiveness of the melanoma. The expression of TRPV1, TRPC1, TRPC6, TRPM4, and TRPM5 is also increased in some cancers. The level of expression of TRPM8 and TRPV6 in prostate cancer, and of TRPM1 in melanomas, potentially provides a good prognostic marker for predicting the course of the cancer in individuals. The Drosophila melanogaster, TRPL, and the TRPV1 and TRPM8 proteins, have been used to try to develop strategies to selectively kill cancer cells by activating Ca²⁺ and Na⁺ entry, producing a sustained increase in the cytoplasmic concentration of these ions, and subsequent cell death by apoptosis and necrosis. TRPV1 is expressed in neurones involved in sensing cancer pain, and is a potential target for pharmacological inhibition of cancer pain in bone metastases, pancreatic cancer and most likely in other cancers. Further studies are required to assess which other TRP proteins are associated with the development and progression of cancer, what roles TRP proteins play in this process, and to develop further knowledge of TRP proteins as targets for pharmaceutical intervention and targeting in cancer.     

LINC01140 regulates osteosarcoma proliferation and invasion by targeting the miR-139-5p/HOXA9 axis

Osteosarcoma is one of the commonest metastatic tumor in children and teenagers, and has a hopeless, prognosis. Long non-coding RNA (lncRNA) acts momentous roles as a regulator on the proliferation and migration of cancer. Here, we performed GEO database analysis and qPCR to identify differentially expressed lncRNAs in osteosarcoma cells. Knockdown of lncRNA LINC01140 was used to detect the effect of LINC01140 on the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. Bioinformatics analysis and qPCR identified the LINC01140/miR-139-5p/Homeobox A9 (HOXA9) regulatory axis. RNA immunoprecipitation assay, Dual-luciferase assay, and rescue experiments confirmed the interaction of LINC01140/miR-139-5p/HOXA9 in osteosarcoma. LINC01140 was overexpressed in osteosarcoma and knocking down LINC01140 restrained the proliferation and invasion of osteosarcoma cells and EMT. In Saos2 and MG63 cells, LINC01140 sponged miR-139-5p, and a miR-139-5p inhibitor overturned the suppression of LINC01140 knockdown on the proliferation and migration of osteosarcoma cells. Moreover, miR-139-5p depressed the invasion, proliferation, and EMT of osteosarcoma cells via targeting HOXA9. Our results indicate that LINC01140 downregulation inhibits the invasion, proliferation, and EMT in osteosarcoma cells through targeting the miR-139-5p/HOXA9 axis. Therefore, LINC01140 is a potential therapeutic target for osteosarcoma.     

Importance of melastatin-like transient receptor potential 7 and cations (magnesium, calcium) in human osteoblast-like cell proliferation

Abstract. Bone tissue in the adult is continuously being remodelled, and overall bone mass is maintained constant by the balance between osteoclastic bone resorption and osteoblastic bone formation. Adequate osteoblastic proliferation is essential for both appropriate formation and for regulation of resorption, and thereby the maintenance of bone remodelling equilibrium. Objectives: Here, we have investigated the roles of melastatin‐like transient receptor potential 6 and 7 (TRPM6, TRPM7), which are calcium (Ca²⁺) and magnesium (Mg²⁺) conducting channels, during proliferation of human osteoblasts. Results: Genetic expression of TRPM6 and TRPM7 was shown in human osteoblast‐like MG‐63, SaOS and U2‐OS cells, and reduction of extracellular Mg²⁺ or Ca²⁺ led to a decrease of cell proliferation. Concomitant reduction of both ions further accentuated reduction of cell proliferation. Expression of TRPM7 channels was increased under conditions of reduced extracellular Mg²⁺ and Ca²⁺ levels whereas expression of TRPM6 was not modified, suggesting compensatory mechanisms afforded by TRPM7 in order to maintain intracellular ion homeostasis. Pre‐incubation of cells in reduced extracellular Mg²⁺ conditions led to activation of Ca²⁺ and Mg²⁺ influx. Reduction of TRPM7 expression by specific siRNA prevented latter influx and inhibited cell proliferation. Conclusions: Our results indicate that extracellular Mg²⁺ and Ca²⁺ deficiency reduces the proliferation of human osteoblastic cells. Expression and activity of TRPM7 is modulated by extracellular Mg²⁺ and Ca²⁺ availability, indicating that TRPM7 channels are involved in intracellular ion homeostasis and proliferation of osteoblasts.     

TRPM2 Cation Channels Modulate T Cell Effector Functions and Contribute to Autoimmune CNS Inflammation

TRPM2, a highly Ca²⁺-permeable member of the transient receptor potential melastatin-related (TRPM) family of cation channels, is expressed in cells of the immune system. We demonstrate firstly that TRPM2 cation channels on T cells critically influence T cell proliferation and proinflammatory cytokine secretion following polyclonal T cell receptor stimulation. Consistently, trpm2-deficient mice exhibited an attenuated clincal phenotype of experimental autoimmune encephalomyelitis (EAE) with reduced inflammatory and demyelinating spinal cord lesions. Importantly, trmp2-deficient T cells were as susceptible as wildtype T cells to oxidative stress-induced cell death as it occurs in inflammatory CNS lesions. This supports the notion that the attenuated EAE phenotype is mainly due to reduced T cell effector functions but unaffected by potential modulation of T cell survival at the site of inflammation. Our findings suggest TRPM2 cation channels as a potential target for treating autoimmune CNS inflammation.     

TRPM7 ion channels are required for sustained phosphoinositide 3-kinase signaling in lymphocytes

Lymphocytes lacking the TRPM7 (transient receptor potential cation channel, subfamily M, member 7) dual function ion channel/protein kinase exhibit a unique phenotype: they are unable to proliferate in regular media, but proliferate normally in media supplemented with 10–15 mM extracellular Mg²⁺. Here, we have analyzed the molecular mechanisms underlying this phenotype. We find that upon transition from proliferation-supporting Mg²⁺-supplemented media to regular media, TRPM7-deficient cells rapidly downregulate their rate of growth, resulting in a secondary arrest in proliferation. The downregulated growth rate of transitioning cells is associated with a deactivation of signaling downstream from phosphoinositide 3-kinase, and expression of constitutively active p110 phosphoinositide 3-kinase is sufficient to support growth and proliferation of TRPM7-deficient cells in regular media. Together, these observations indicate that TRPM7 channels are required for sustained phosphoinositide 3-kinase-dependent growth signaling and therefore, that TRPM7 is positioned alongside phosphoinositide 3-kinases as a central regulator of lymphocyte growth and proliferation.     

Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells

Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients.     

Cholesterol-induced activation of TRPM7 regulates cell proliferation,migration, and viability of human prostate cells

Cholesterol has been shown to promote cell proliferation/migration in many cells; however the mechanism(s) have not yet been fully identified. Here we demonstrate that cholesterol increases Ca^2 + entry via the TRPM7 channel, which promoted proliferation of prostate cells by inducing the activation of the AKT and/or the ERK pathway. Additionally, cholesterol mediated Ca^2 + entry induced calpain activity that showed a decrease in E-cadherin expression, which together could lead to migration of prostate cancer cells. An overexpression of TRPM7 significantly facilitated cholesterol dependent Ca^2 + entry, cell proliferation and tumor growth. Whereas, TRPM7 silencing or inhibition of cholesterol synthesis by statin showed a significant decrease in cholesterol-mediated activation of TRPM7, cell proliferation, and migration of prostate cancer cells. Consistent with these results, statin intake was inversely correlated with prostate cancer patients and increase in TRPM7 expression was observed in samples obtained from prostate cancer patients. Altogether, we provide evidence that cholesterol-mediated activation of TRPM7 is important for prostate cancer and have identified that TRPM7 could be essential for initiation and/or progression of prostate cancer.     

Examination of the role of TRPM8 in human mast cell activation and its relevance to the etiology of cold-induced urticaria

Mast cells are considered the primary initiators of allergic diseases as a consequence of the release of multiple inflammatory mediators on activation. Although predominately activated through antigen-mediated aggregation of IgE-occupied-Fc?RI, they can also be induced to release mediators by other receptors and environmental stimuli. Based on studies conducted in the RBL 2H3 rodent mast cell line, the transient receptor potential melastatin 8 (TRPM8) cation channel has been implicated in the activation of mast cells in response to cold and, by inference, the development of urticaria. Here we investigated the expression and role of TRPM8 receptor, in both human and mouse non-transformed cells, with the aim of exploring the potential link between TRPM8 and the pathology of cold urticaria in humans. Although expressed in mouse mast cells, we found no evidence of TRPM8 expression in human mast cells or functional mutations in TRPM8 in cold urticaria patients. Furthermore, neither mouse nor human primary cultured mast cells degranulated in response to cold challenge or TRPM8 agonists and mast cell reactivity was unaffected in Trpm8^−/− mice. From these data, we conclude that TRPM8 is unlikely to directly regulate mast cell activation in cold urticaria. Thus, alternative mechanisms likely exist for the pathogenesis of this disease.     

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.     

Cantharidin inhibits osteosarcoma proliferation and metastasis by directly targeting miR-214-3p/DKK3 axis to inactivate β-catenin nuclear translocation and LEF1 translation

Background: As the leading primary bone cancer in adolescents and children, osteosarcoma patients with metastasis show a five-year-survival-rate of 20-30%, without improvement over the past 30 years. Wnt/β-catenin is important in promoting osteosarcoma development. DKK3 is a Wnt/β-catenin antagonist and predicted to have the specific binding site in 3′-UTR with miR-214-3p.Methods: miR-214-3p and DKK3 levels were investigated in human osteosarcoma tissues and cells by RT-qPCR; the prognostic importance of DKK3 level in osteosarcoma patients was determined with Log-rank test; direct binding between DKK3 with miR-214-3p was identified with targetscan; anti-osteosarcoma mechanism of cantharidin was investigated by miR-214-3p silence/over-expression with or without cantharidin treatment, and nuclear/cytoplasmic protein assay in osteosarcoma cells.Results: Down-regulated DKK3 indicated poor prognosis of osteosarcoma patients. Up-regulated miR-214-3p promoted proliferation and migration, while suppressed apoptosis of osteosarcoma cells by increasing β-catenin nuclear translocation and LEF1 translation via degradation of DKK3. Cantharidin suppressed viabilities, migration and invasion, while promoted cell cycle arrest and apoptosis in 143B and U-2 OS cells via down-regulating miR-214-3p to up-regulate DKK3, thus inhibited p-GSK-3β expression, β-catenin nuclear translocation and LEF1 translation. Meanwhile, cantharidin inhibited tumor growth in xenograft-bearing mice with 143B cell injection in tibia.Conclusion: miR-214-3p mediated Wnt/β-catenin/LEF1 signaling activation by targeting DKK3 to promote oncogenesis of osteosarcoma; cantharidin inhibited proliferation and metastasis of osteosarcoma cells via down-regulating miR-214-3p to up-regulate DKK3 and decrease β-catenin nuclear translocation, indicating that cantharidin may be a prospective candidate for osteosarcoma treatment by targeting miR-214-3p/DKK3/β-catenin signaling.     

The Role of TRP Channels in Oxidative Stress-induced Cell Death

The transient receptor potential (TRP) protein superfamily is a diverse group of voltage-independent calcium-permeable cation channels expressed in mammalian cells. These channels have been divided into six subfamilies, and two of them, TRPC and TRPM, have members that are widely expressed and activated by oxidative stress. TRPC3 and TRPC4 are activated by oxidants, which induce Na⁺ and Ca²⁺ entry into cells through mechanisms that are dependent on phospholipase C. TRPM2 is activated by oxidative stress or TNFα, and the mechanism involves production of ADP-ribose, which binds to an ADP-ribose binding cleft in the TRPM2 C-terminus. Treatment of HEK 293T cells expressing TRPM2 with H₂O₂ resulted in Ca²⁺ influx and increased susceptibility to cell death, whereas coexpression of the dominant negative isoform TRPM2-S suppressed H₂O₂-induced Ca²⁺ influx, the increase in [Ca²⁺]_i, and onset of apoptosis. U937-ecoR monocytic cells expressing increased levels of TRPM2 also exhibited significantly increased [Ca²⁺]_i and increased apoptosis after treatment with H₂O₂ or TNFα. A dramatic increase in caspase 8, 9, 3, 7, and PARP cleavage was observed in TRPM2-expressing cells, demonstrating a downstream mechanism through which cell death is mediated. Inhibition of endogenous TRPM2 function through three approaches, depletion of TRPM2 by RNA interference, blockade of the increase in [Ca²⁺]_i through TRPM2 by calcium chelation, or expression of the dominant negative splice variant TRPM2-S protected cell viability. H₂O₂ and amyloid β-peptide also induced cell death in primary cultures of rat striatal cells, which endogenously express TRPM2. TRPM7 is activated by reactive oxygen species/nitrogen species, resulting in cation conductance and anoxic neuronal cell death, which is rescued by suppression of TRPM7 expression. TRPM2 and TRPM7 channels are physiologically important in oxidative stress-induced cell death.