RESUMEN
The study's aim was to investigate the S100A4-mediated mechanisms of the regulation of tumor cell proliferation and migration in the human triple-positive breast carcinoma cell line MCF-7 (TPBC) and triple-negative breast carcinoma cell line MDA-MB-231 (TNBC). The proliferative activity of TNBC more than doubled during the incubation in the conditioned medium of TPBC. Extracellular S100A4 dose-dependently decreased the proliferative response of TPBC. TPBC negatively impacted the growth of TNBCs during their co-culturing. TPBC significantly decreased the migration activity of the TNBC cells while the S100A4 intracellular level in the TNBC was also decreasing. The decrease in the S100A4 intracellular level occurred due to the protein's monomeric form while the contribution of the dimeric form into the overall S100A4 concentration in TNBC cells increased 1.5-2-fold. The S100A4 pathway in the intercellular communication between TNBC and TPBCs also included the dexamethasone-sensitive mechanisms of S100A4 intra- and extracellular pools regulation.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Medios de Cultivo Condicionados/farmacología , Humanos , Proteína de Unión al Calcio S100A4 , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
S100A4, a small intra- and extracellular Ca(2+)-binding protein, is involved in tumor progression and metastasis with S100A4 level shown to be correlated with tumor cells metastatic potential. Simultaneously, Octamer transcription factor 1 (Oct-1) regulates a wide range of genes and participates in tumor cell progression with high Oct-1 level associated with a poor prognosis for different tumors. In this study, following the establishment of Oct-1 binding site, we used Burkit lymphoma B cells (Namalwa cells) which express different isoforms of Oct-1 (Oct-1A, Oct-1L and Oct-1X) to investigate the role of Oct-1 in S100A4 expression and sustaining intra- and extra-cellular S100A4 levels. As antitumor agents, we used dexamethasone which effect is mediated by the activation of intracellular glucocorticoid receptors and camptothecin which molecular target is nuclear DNA topoisomerase I (TOP1). We established that, firstly, the most significant increase in S100A4 gene expression has been demonstrated in the cells transfected with Oct-1A. Secondly, we have established that high level of Oct-1 and decreased intracellular S100A4 level decline the survival of Namalwa cells under dexamethasone treatment. Thirdly, we have shown that the tumor cells transformation by different Oct-1 isoforms retained those cells' sensitivity to the antitumor effect of combined dexamethasone and camptothecin. In contrast, in the non-transformed Namalwa cells, dexamethasone decreased the camptothecin effect on the cells survivorship, thus, emphasizing Oct-1 role in the regulation of cell response to different antitumor agents. The results identify a necessity to consider Oct-1 level for combined chemotherapeutic drug treatment.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/farmacología , Dexametasona/farmacología , Regulación Neoplásica de la Expresión Génica , Factor 1 de Transcripción de Unión a Octámeros/genética , Proteína de Unión al Calcio S100A4/genética , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Combinación de Medicamentos , Humanos , Concentración 50 Inhibidora , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Plásmidos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteína de Unión al Calcio S100A4/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Tag7 (also known as peptidoglycan recognition protein PGRP-S, PGLYRP1), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. In this study, we have analyzed the programmed cell death mechanisms that are induced when cells interact with the Tag7-Hsp70 complex, which was previously shown to be released by human lymphocytes and is cytotoxic to cancer cells. We show that this complex induces both apoptotic and necroptotic processes in the cells. Apoptosis follows the classic caspase-8 and caspase-3 activation pathway. Inhibition of apoptosis leads to a switch to the RIP1-dependent necroptosis. Both of these cytotoxic processes are initiated by the involvement of TNFR1, a receptor for TNF-α. Our results suggest that the Tag7-Hsp70 complex is a novel ligand for this receptor. One of its components, the innate immunity protein Tag7, can bind to the TNFR1 receptor, thereby inhibiting the cytotoxic actions of the Tag7-Hsp70 complex and TNF-α, an acquired immunity cytokine.
Asunto(s)
Apoptosis , Citocinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Animales , Caspasas/metabolismo , Línea Celular , Células Clonales , Células HEK293 , Humanos , Ratones , Necrosis , Unión Proteica , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Tag7 (PGRP-S) was described as an innate immunity protein. Earlier we have shown that Tag7 forms with Hsp70 a stable complex with cytotoxic and antitumor activity. The same complex is formed in and secreted by cytotoxic T-lymphocytes. We have also found that Hsp-binding protein HspBP1 incapacitates the Tag7-Hsp70 complex. Here we have studied the interaction of extracellular Tag7 and HspBP1. We have shown that HspBP1 binds Tag7 in the conditioned medium of tumor CSML0 cells, thereby preventing formation of the cytotoxic Tag7-Hsp70 complex. We have also found that Tag7, if present in serum (in every third donor on average), is always in complex with HspBP1. This may be a protective measure against indiscriminate attack of the cytotoxic complex on normal cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Citocinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Línea Celular Tumoral , Medios de Cultivo Condicionados , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas In VitroRESUMEN
Heat shock-binding protein HspBP1 is a member of the Hsp70 co-chaperone family. The interaction between HspBP1 and the ATPase domain of the major heat shock protein Hsp70 up-regulates nucleotide exchange and reduces the affinity between Hsp70 and the peptide in its peptide-binding site. Previously we have shown that Tag7 (also known as peptidoglycan recognition protein PGRP-S), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. This complex can be produced in cytotoxic lymphocytes and released during interaction with tumor cells. Here the effect of HspBP1 on the cytotoxic activity of the Tag7-Hsp70 complex was examined. HspBP1 could bind not only to Hsp70, but also to Tag7. This interaction eliminated the cytotoxic activity of Tag7-Hsp70 complex and decreased the ATP concentration required to dissociate Tag7 from the peptide-binding site of Hsp70. Moreover, HspBP1 inhibited the cytotoxic activity of the Tag7-Hsp70 complex secreted by lymphocytes. HspBP1 was detected in cytotoxic CD8+ lymphocytes. This protein was released simultaneously with Tag7-Hsp70 during interaction of these lymphocytes with tumor cells. The simultaneous secretion of the cytotoxic complex with its inhibitor could be a mechanism protecting normal cells from the cytotoxic effect of this complex.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Complejos Multiproteicos/metabolismo , Neoplasias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Adenosina Trifosfato/genética , Adenosina Trifosfato/inmunología , Adenosina Trifosfato/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Citocinas/genética , Citocinas/inmunología , Citotoxinas/genética , Citotoxinas/inmunología , Citotoxinas/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/inmunología , Humanos , Células K562 , Ratones , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Neoplasias/genética , Neoplasias/inmunología , Unión ProteicaRESUMEN
We consider the novel means of attack and defense in the host versus cancer combat that involve interactions between widespread multifunctional proteins, focusing on the aspects that may seem paradoxical in the framework of established notions. Particularly, we show that a protein broadly known for its protective functions such as Hsp70 can make a tumoricidal "binary weapon" with another nontoxic protein Tag7 (PGRP-S); that the same Hsp70, a ubiquitous intracellular chaperone, when expressed on the MHC-negative tumor cell surface, can itself be the hallmark of immune evasion rather than a primordial MHC substitute; that a device functionally equivalent to the T-cell receptor (Tag7-Centered Recognizer) can be assembled of components in no way related to the classical pathways of T-cell-mediated immunity, and operate where the orthodox immunosurveillance fails; and that one and the same protein Mts1 (S100A4) under different circumstances may work as "reactive armor" of a tumor cell against humoral agents and as a vital part of the T-cell machinery aimed against immunoevasive cells, i.e., perform both prometastatic and antimetastatic functions.
Asunto(s)
Citocinas/uso terapéutico , Proteínas HSP70 de Choque Térmico/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas S100/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Humanos , Células K562 , Ratones , Ratones Endogámicos DBA , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína de Unión al Calcio S100A4 , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
We compare the physical and functional interactions between three widespread multifunctional proteins [metastasin (Mts1/S100A4), innate immunity-related Tag7/PGRP-S, and Hsp70] in two experimental models relevant to host-tumor relationships on humoral and cellular levels. (i) Tag7 and Hsp70 in solution or in a lymphocyte make a stable binary complex that is highly cytotoxic for some tumor cells. Here, we show that Mts1 prevents Tag7.Hsp70 assembly in solution, and an excess of Mts1 disrupts the existing Tag7.Hsp70 complex; accordingly, Tag7.Hsp70 cytotoxicity (exemplified with L929 cells) is diminished in the presence of excess Mts1. (ii) Tag7 exposed on a specialized subset of lymphokine-activated killer cells makes specific contact with Hsp70 exposed on some HLA-negative tumor cells, thus enabling FasL/Fas-mediated induction of apoptosis. Here, we show that some CD4(+)CD25(+) cells coexpose Mts1 with Tag7 and FasL, that Mts1 and Tag7 closely contact the same Hsp70 molecule on the target K562 cell (as evidenced by cross-linking), and that killing of such targets is abolished by Mts1-specific antibodies (or selective removal of Mts1-exposing lymphocytes). Thus, this phenotype active against immunoevasive cancerous cells is defined as CD4(+)CD25(+), FasL(+), Tag7(+)Mts1(+) (approximately 0.5% of total lymphocytes in culture). Remarkably, similar effectors with at least the same activity are often found in fresh donor blood samples (approximately 10(4) effectors/mL). Thus, our models suggest that interactions between the three proteins in different situations may have opposite functional outcomes as regards antitumor defense, immune escape, and metastasis.
Asunto(s)
Citocinas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas S100/fisiología , Animales , Biotinilación , Linfocitos T CD4-Positivos/metabolismo , Membrana Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Células K562 , Leucocitos Mononucleares/metabolismo , Ratones , Proteína de Unión al Calcio S100A4 , Proteínas S100/biosíntesisRESUMEN
Within the broad problem of host immune surveillance versus tumor immune evasion, a most intriguing question is how the cellular immunity can cope with cancerous cells that have gotten rid of the classical antigen-presenting machinery. One such option stems from (1) the fact that HLA loss is often attended with expression of Hsp70 on the tumor cell surface, and (2) our findings that human lymphocytes express a protein Tag7 (also known as PGRP-S) capable of tight and specific interaction with cognate Hsp70. Here we show that a subpopulation of human CD4(+)CD25(+) lymphocytes, obtained either in culture as lymphokine-activated killers or directly from healthy donors, carry Tag7 and FasL on their surface and can indeed kill the HLA-negative tumor-derived cells K562 and MOLT-4 that expose Hsp70 and Fas. The primary binding of lymphocyte Tag7 to target-cell Hsp70 is very specific (eg, it is blocked by preincubating either cell with minimal peptides from the "partner" protein), and secures cell contact indispensable for subsequent FasL/Fas-triggered apoptosis. Unrelated to natural killer cell action or the putative role of Hsp as an antigen-presenting substitute, this novel mechanism is rather a backup analog of orthodox (CD8(+)) target recognition (Tag7 acting as built-in T-cell receptor and Hsp70 itself as ligand).
Asunto(s)
Apoptosis , Proteína Ligando Fas/inmunología , Antígenos HLA/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Linfocitos T Citotóxicos/inmunología , Receptor fas/inmunología , Biotinilación , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Citotoxicidad Inmunológica , Proteína Ligando Fas/metabolismo , Citometría de Flujo , Humanos , Inmunoprecipitación , Células K562/inmunología , Células K562/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Linfocitos T Citotóxicos/metabolismo , Receptor fas/metabolismoRESUMEN
The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumor-derived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450-463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis. Thus, we demonstrate how a heat shock protein may perform functions clearly distinct from chaperoning or cell rescue and how peptidoglycan recognition proteins may be involved in innate immunity and anti-cancer defense.