RESUMEN

BACKGROUND: Chemoresistance remains a major treatment obstacle in multiple myeloma (MM). Novel new therapies are thus in need. Transient Receptor Potential Vanilloid type 1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. Calcium channels have been shown to be involved in the regulation of cell proliferation, chemoresistance, migration and invasion. The aim of the current study was to evaluate its possible role in MM. METHODS: Pharmacological inhibitor was used to evaluate the role of TRPV1 in MM cell lines and primary MM cells. Flow cytometry, molecular analysis, fluorescent microscopy, proteomic analysis and xenograft in vivo model of MM with BM involvement were employed to assess the effect of TRPV1 inhibition and decipher its unique mechanism of action in MM. RESULTS: TRPV1 was found to be expressed by MM cell lines and primary MM cells. TRPV1 inhibition using the antagonist AMG9810-induced MM cell apoptosis and synergized with bortezomib, overcoming both CXCR4-dependent stroma-mediated and acquired resistance. In accordance, AMG9810 suppressed the expression and activation of CXCR4 in MM cells. TRPV1 inhibition increased mitochondrial calcium levels with subsequent mitochondrial ROS accumulation and depolarization. These effects were reversed by calcium chelation, suggesting the role of calcium perturbations in oxidative stress and mitochondrial destabilization. Furthermore, AMG9810 abolished bortezomib-induced accumulation of mitochondrial HSP70 and suppressed protective mitochondrial unfolded protein response. Proteomics revealed unique molecular signature related to the modification of ubiquitin signaling pathway. Consequently, 38 proteins related to the ubiquitylation machinery were downregulated upon combined bortezomib/AMG9810 treatment. Concomitantly, AMG9810 abolished bortezomib-induced ubiquitination of cytosolic and mitochondrial proteins. Furthermore, bortezomib/AMG9810 treatment induced mitochondrial accumulation of PINK1, significantly reduced the mitochondrial mass and promoted mitochondrial-lysosomal fusion, indicating massive mitophagy. Finally, in a recently developed xenograft model of systemic MM with BM involvement, bortezomib/AMG9810 treatment effectively reduced tumor burden in the BM of MM-bearing mice. CONCLUSIONS: Altogether, our results unravel the mechanism mediating the strong synergistic anti-MM activity of bortezomib in combination with TRPV1 inhibition which may be translated into the clinic.

Asunto(s)

Acrilamidas/farmacología , Antineoplásicos/farmacología , Bortezomib/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Mitofagia/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Acrilamidas/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Bortezomib/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Calcio/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Mieloma Múltiple/metabolismo , Canales Catiónicos TRPV/metabolismo , Ubiquitina/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos

RESUMEN

Polyclonal anti-human thymocyte globulins (ATG) have been recently shown to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells has been suggested as one of the possible mechanisms. The aim of current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance. Phenotypic characterization revealed a significant increase in the expression of FoxP3, GITR, CD95, PD-1 and ICOS as well as the complement inhibitory molecules CD55, CD58 and CD59 on CD4+CD25+ T cells upon ATG treatment. Addition of ATG-treated cells to autologous and allogeneic peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/anti-CD28 antibodies resulted in significant inhibition of proliferation. Moreover, T-cell activation and IFNγ secretion were reduced in the presence of ATG-induced Treg cells. The CD4+CD25+CD127-low Treg fraction sorted from ATG-treated culture demonstrated greater suppressive potency than negative fraction. Conditioned medium produced by ATG-treated but not IgG-treated cells contained TGFß and suppressed T cell proliferation and activation in a TGFß receptor-dependent manner. TGFß receptor kinase inhibitor SB431542 interfered with the suppressive activity of ATG-primed cells, enabling partial rescue of proliferation and IFNγ secretion. Moreover, SB431542 prevented Treg phenotype induction upon ATG treatment. Altogether, our data reveal the role of TGFß signaling in ATG-mediated immunosuppression and further support the use of ATG, a potent inducer of regulatory T cells, for prevention of GVHD post HSCT and potentially other therapeutic applications.