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T-cell malignancies, including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL), present significant challenges to treatment due to their aggressive nature and chemoresistance. Chemotherapies remain a mainstay for their management, but the aggressiveness of these cancers and their associated toxicities pose limitations. Immunepotent CRP (ICRP), a bovine dialyzable leukocyte extract, has shown promise in inducing cytotoxicity against various cancer types, including hematological cancers. In this study, we investigated the combined effect of ICRP with a panel of chemotherapies on cell line models of T-ALL and T-LBL (CEM and L5178Y-R cells, respectively) and its impact on immune system cells (peripheral blood mononuclear cells, splenic and bone marrow cells). Our findings demonstrate that combining ICRP with chemotherapies enhances cytotoxicity against tumoral T-cell lymphoblasts. ICRP + Cyclophosphamide (CTX) cytotoxicity is induced through a caspase-, reactive oxygen species (ROS)-, and calcium-dependent mechanism involving the loss of mitochondrial membrane potential, an increase in ROS production, and caspase activation. Low doses of ICRP in combination with CTX spare non-tumoral immune cells, overcome the bone marrow-induced resistance to CTX cell death, and improves the CTX antitumor effect in vivo in syngeneic Balb/c mice challenged with L5178Y-R. This led to a reduction in tumor volume and a decrease in Ki-67 proliferation marker expression and the granulocyte/lymphocyte ratio. These results set the basis for further research into the clinical application of ICRP in combination with chemotherapeutic regimens for improving outcomes in T-cell malignancies.
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Ciclofosfamida , Espécies Reativas de Oxigênio , Animais , Camundongos , Humanos , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Ciclofosfamida/farmacologia , Sinergismo Farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Apoptose/efeitos dos fármacos , Bovinos , Morte Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Camundongos Endogâmicos BALB C , Proliferação de Células/efeitos dos fármacosRESUMO
Cancer immunotherapies include monoclonal antibodies, cytokines, oncolytic viruses, cellular therapies, and other biological and synthetic immunomodulators. These are traditionally studied for their effect on the immune system's role in eliminating cancer cells. However, some of these therapies have the unique ability to directly induce cytotoxicity in cancer cells by inducing immunogenic cell death (ICD). Unlike general immune stimulation, ICD triggers specific therapy-induced cell death pathways, based on the release of damage-associated molecular patterns (DAMPs) from dying tumour cells. These activate innate pattern recognition receptors (PRRs) and subsequent adaptive immune responses, offering the promise of sustained anticancer drug efficacy and durable antitumour immune memory. Exploring how onco-immunotherapies can trigger ICD, enhances our understanding of their mechanisms and potential for combination strategies. This review explores the complexities of these immunotherapeutic approaches that induce ICD, highlighting their implications for the innate immune system, addressing challenges in cancer treatment, and emphasising the pivotal role of ICD in contemporary cancer research.
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Antineoplásicos , Neoplasias , Humanos , Morte Celular Imunogênica , Neoplasias/patologia , Antineoplásicos/uso terapêutico , Sistema Imunitário/metabolismo , ImunoterapiaRESUMO
BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) represents one of the principal tumors of the head and neck. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are considered risk factors for the development and the clinical prognosis of LSCC. High levels of p16INK4a are suggested as a surrogate marker of HPV or EBV infection in some head and neck tumors but in LSCC is still controversial. Furthermore, pRb expression may be considered an additional biomarker but it has not been clearly defined. This work aimed to compare the expression of pRb and p16INK4a as possible biomarkers in tumor tissues with and without infection by EBV or different genotypes of HPV from patients with LSCC. METHODS: Tumor samples from 103 patients with LSCC were previously investigated for the presence and genotypes of HPV using the INNO-LiPA line probe assay and for the infection of EBV by qPCR. p16 INK4a and pRb expression was assessed by immunohistochemistry. RESULTS: Of the 103 tumor samples, expression of p16INK4a was positive in 55 (53.4%) and of this, 32 (56.1%) were positive for HPV whereas 11 (39.3%) were EBV positive but both without a significantly difference (p > 0.05). pRb expression was positive in 78 (75.7%) and a higher frequency of this expression was observed in HPV negative samples (87.0%) (p = 0.021) and in high-risk HPV negative samples (85.2%) (p = 0.010). No difference was observed when comparing pRb expression and EBV infection status (p > 0.05). CONCLUSION: Our results support the suggestion that p16INK4a is not a reliable surrogate marker for identifying HPV or EBV infection in LSCC. On the other hand, most of our samples had pRb expression, which was more frequent in tumors without HPV, suggesting that pRb could indicate HPV negativity. However, more studies with a larger number of cases are required, including controls without LSCC and evaluating other molecular markers to determine the real role of p16INK4a and pRb in LSCC.
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IMMUNEPOTENT CRP (ICRP) is an immunotherapy that induces cell death in cancer cell lines. However, the molecular mechanisms of death are not completely elucidated. Here, we evaluated the implication of intracellular Ca2+ augmentation in the cell death induced by ICRP on T-ALL and breast cancer cell lines. Cell death induction and the molecular characteristics of cell death were evaluated in T-ALL and breast cancer cell lines by assessing autophagosome formation, ROS production, loss of mitochondrial membrane potential, ER stress and intracellular Ca2+ levels. We assessed the involvement of extracellular Ca2+, and the implication of the ER-receptors, IP3R and RyR, in the cell death induced by ICRP, by using an extracellular calcium chelator and pharmacological inhibitors. Our results show that ICRP increases intracellular Ca2+ levels as the first step of the cell death mechanism that provokes ROS production and loss of mitochondrial membrane potential. In addition, blocking the IP3 and ryanodine receptors inhibited ER-Ca2+ release, ROS production and ICRP-induced cell death. Taken together our results demonstrate that ICRP triggers intracellular Ca2+-increase leading to different regulated cell death modalities in T-ALL and breast cancer cell lines. See also Figure 1(Fig. 1).
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Breast cancer (BC) is one of the leading causes of cancer death worldwide. Cyclophosphamide (CTX) remains a mainstay in cancer therapy despite harmful adverse effects and cell death-resistances. To face this, combinational therapy of chemotherapies and immunotherapies has been proposed. IMMUNEPOTENT CRP (ICRP) is an immunotherapy that has cytotoxic effects in several cancer cells without affecting peripheral blood mononuclear cells (PBMC) and CD3+ cells. The aim of this study was to evaluate cytotoxicity, the type of cytotoxic effect, and several features involved in cell death induced by the combination of CTX with ICRP (ICRP+CTX) in breast cancer cells as well as their effect on healthy cells. For this purpose, human and murine breast cancer cells, MCF-7, MDA-MB-231 and 4T1, or PBMC were treated for 24 hours with ICRP, CTX or ICRP+CTX in different combination ratios for the assessment of cell death. Flow cytometry and microscopy were used to determine biochemical and morphological characteristics of cell death. Assays showed that ICRP in combination with CTX induce potentiated cell death manifested with morphological changes, loss of mitochondrial membrane potential, reactive oxygen species (ROS) production, and caspase activation. In addition, it was determined that ICRP+CTX-cell death is caspase-independent in all the breast cancer cells assessed. On the other hand, ICRP did not affect CTX-cytotoxicity in PBMC. For all the above, we can propose that the combination of ICRP with CTX an effective combination therapy, promoting their use even in tumoral cells with defects on proteins implicated in the apoptotic pathway.
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Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide. Recent advances in the field of immuno-oncology demonstrate the beneficial immunostimulatory effects of the induction of immunogenic cell death (ICD). ICD increases tumor infiltration by T cells and is associated with improved prognosis in patients affected by triple negative breast cancer (TNBC) with residual disease. The aim of this study was to evaluate the antitumoral effect of PKHB1, a thrombospondin-1 peptide mimic, against breast cancer cells, and the immunogenicity of the cell death induced by PKHB1 in vitro, ex vivo, and in vivo. Our results showed that PKHB1 induces mitochondrial alterations, ROS production, intracellular Ca2+ accumulation, as well calcium-dependent cell death in breast cancer cells, including triple negative subtypes. PKHB1 has antitumor effect in vivo leading to a reduction of tumor volume and weight and promotes intratumoral CD8 + T cell infiltration. Furthermore, in vitro, PKHB1 induces calreticulin (CALR), HSP70, and HSP90 exposure and release of ATP and HMGB1. Additionally, the killed cells obtained after treatment with PKHB1 (PKHB1-KC) induced dendritic cell maturation, and T cell antitumor responses, ex vivo. Moreover, PKHB1-KC in vivo were able to induce an antitumor response against breast cancer cells in a prophylactic application, whereas in a therapeutic setting, PKHB1-KC induced tumor regression; both applications induced a long-term antitumor response. Altogether our data shows that PKHB1, a thrombospondin-1 peptide mimic, has in vivo antitumor effect and induce immune system activation through immunogenic cell death induction in breast cancer cells.
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Neoplasias da Mama , Morte Celular Imunogênica , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Morte Celular , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos/farmacologiaRESUMO
Chemotherapy Related Cognitive Impairment (CRCI), also called chemobrain, diminishes cancer patient's life quality. Breast cancer (BC) patients have been described to be importantly affected, however, the mechanism leading to CRCI has not been fully elucidated. Recent research proposes microglia as the main architect of CRCI, thus dysregulations in these cells could trigger CRCI. The aim of this research was to evaluate the effects of two drugs commonly used against breast cancer, cyclophosphamide (CTX) and epirubicin (EPI), on the microglia cell line SIM-A9, using the BC cell line, 4T1, as a control. Our results show that CTX and EPI decrease microglia-cell viability and increase cell death on a concentration-dependent manner, being 5 and 2 times more cytotoxic to microglia cell line than to breast cancer 4T1cells, respectively. Both chemotherapies induce cell cycle arrest and a significant increase in p53, p16 and γ-H2AX in breast cancer and microglia cells. Furthermore, mitochondrial membrane potential (ΔΨm) diminishes as cell death increases, and both chemotherapies induce reactive oxygen species (ROS) production on SIM-A9 and 4T1. Moreover, caspase activation increases with treatments and its pharmacological blockade inhibits CTX and EPI induced-cell death. Finally, low concentrations of CTX and EPI induce γ-H2AX, and EPI induces cytokine release, NO production and Iba-1 overexpression. These findings indicate that microglia cells are more sensitive to CTX and EPI than BC cells and undergo DNA damage and cell cycle arrest at very low concentrations, moreover EPI induces microglia activation and a pro-inflammatory profile.
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Cattle tick (Rhipicephalus microplus) represents a severe problem causing substantial economic losses, estimated in billions of dollars annually. Currently, chemical acaricides represent the most widely used control method. However, several problems such as resistance have been described. Phage-based vaccines represent a fast and low-cost tool for antigen delivery. In this regard, the objective of the present work was to develop a candidate phage-based vaccine displaying a cattle tick antigen (Bm86-derived Sbm7462 antigen) on the surface of bacteriophage M13. Phage ELISA and dot blotting analysis confirmed the display of the antigen. Vaccine immunogenicity was evaluated using a bovine monocyte-derived dendritic cell-based ex vivo assay and a murine in vivo assay. The ex vivo model showed the maturation of dendritic cells after being pulsed with the phage-based vaccine. The humoral response was confirmed in the in vivo assay. These results demonstrated the capacity of the phage-based vaccine to induce both humoral and cellular immune-specific responses. Importantly, this is the first report describing a control method for cattle ticks using a candidate phage-based vaccine. Further studies to evaluate the immunogenicity in a bovine model are needed. The current approach represents a promising alternative to control cattle tick infestations.
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(1) Background: Chitosan-coated gold nanoparticles (CH-AuNPs) have important theranostic applications in biomedical sciences, including cancer research. However, although cell cytotoxicity has been studied in cancerous cells, little is known about their effect in proliferating primary leukocytes. Here, we assessed the effect of CH-AuNPs and the implication of ROS on non-cancerous endothelial and fibroblast cell lines and in proliferative lymphoid cells. (2) Methods: The Turkevich method was used to synthetize gold nanoparticles. We tested cell viability, cell death, ROS production, and cell cycle in primary lymphoid cells, compared with non-cancer and cancer cell lines. Concanavalin A (ConA) or lipopolysaccharide (LPS) were used to induce proliferation on lymphoid cells. (3) Results: CH-AuNPs presented high cytotoxicity and ROS production against cancer cells compared to non-cancer cells; they also induced a different pattern of ROS production in peripheral blood mononuclear cells (PBMCs). No significant cell-death difference was found in PBMCs, splenic mononuclear cells, and bone marrow cells (BMC) with or without a proliferative stimuli. (4) Conclusions: Taken together, our results highlight the selectivity of CH-AuNPs to cancer cells, discarding a consistent cytotoxicity upon proliferative cells including endothelial, fibroblast, and lymphoid cells, and suggest their application in cancer treatment without affecting immune cells.
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BACKGROUND: Cancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. IMMUNEPOTENT CRP (ICRP) acts as an immunomodulator and can be cytotoxic to cancer cells. Thus, we evaluated if ICRP induces ICD in BC cells. METHODS: Immunogenicity of ICRP-induced cell death was evaluated in vitro, analysing the principal biochemical characteristics of ICD in MCF-7, MDA-MB-231 and 4T1 cells. Ex vivo, we assessed the ability of killed cancer cells (KCC) obtained from ICRP-treated 4T1 cells (ICRP-KCC) to induce DC maturation, T-cell priming and T-cell-mediated cancer cytotoxicity. In vivo, we evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice. RESULTS: ICRP induced caspase-independent, ROS-dependent cell death, autophagosome formation, P-eIF2α, chaperone protein exposure, CD47 loss, ATP and HMBG1 release in BC cells. Additionally, ICRP-KCC promoted DC maturation, which triggered T-cell priming and cancer cytotoxicity. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes. CONCLUSIONS: ICRP induces ICD in BC cells, leading to long-term antitumour memory.
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Neoplasias da Mama/prevenção & controle , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Recidiva Local de Neoplasia/prevenção & controle , Fator de Transferência/administração & dosagem , Animais , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Morte Celular Imunogênica , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Recidiva Local de Neoplasia/imunologia , Fator de Transferência/farmacologia , Vacinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: IMMUNEPOTENT CRP (ICRP) can be cytotoxic to cancer cell lines. However, its widespread use in cancer patients has been limited by the absence of conclusive data on the molecular mechanism of its action. Here, we evaluated the mechanism of cell death induced by ICRP in HeLa and MCF-7 cells. METHODS: Cell death, cell cycle, mitochondrial membrane potential and ROS production were evaluated in HeLa and MCF-7 cell lines after ICRP treatment. Caspase-dependence and ROS-dependence were evaluated using QVD.oph and NAC pre-treatment in cell death analysis. DAMPs release, ER stress (eIF2-α phosphorylation) and autophagosome formation were analyzed as well. Additionally, the role of autophagosomes in cell death induced by ICRP was evaluated using SP-1 pre-treatment in cell death in HeLa and MCF-7 cells. RESULTS: ICRP induces cell death, reaching CC50 at 1.25 U/mL and 1.5 U/mL in HeLa and MCF-7 cells, respectively. Loss of mitochondrial membrane potential, ROS production and cell cycle arrest were observed after ICRP CC50 treatment in both cell lines, inducing the same mechanism, a type of cell death independent of caspases, relying on ROS production. Additionally, ICRP-induced cell death involves features of immunogenic cell death such as P-eIF2α and CRT exposure, as well as, ATP and HMGB1 release. Furthermore, ICRP induces ROS-dependent autophagosome formation that acts as a pro-survival mechanism. CONCLUSIONS: ICRP induces a non-apoptotic cell death that requires an oxidative stress to take place, involving mitochondrial damage, ROS-dependent autophagosome formation, ER stress and DAMPs' release. These data indicate that ICRP could work together with classic apoptotic inductors to attack cancer cells from different mechanisms, and that ICRP-induced cell death might activate an immune response against cancer cells.
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Alarminas/metabolismo , Antineoplásicos/farmacologia , Autofagossomos , Neoplasias/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Fator de Transferência/administração & dosagem , Animais , Apoptose , Bovinos , Ciclo Celular , Proliferação de Células , Células HeLa , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/patologia , Estresse OxidativoRESUMO
Immunotherapies strengthen the immune system to fight multiple diseases such as infections, immunodeficiencies, and autoimmune diseases, and recently, they are being used as an adjuvant in cancer treatment. IMMUNEPOTENT-CRP (I-CRP) is an immunotherapy made of bovine dialyzable leukocyte extract (bDLE) that has chemoprotective and immunomodulatory effects in different cellular populations of the immune system and antitumor activity in different cancer cell lines. Our recent results suggest that the antineoplastic effect of I-CRP is due to the characteristics of cancer cells. To confirm, we evaluated whether the selectivity is due to cell lineage or characteristics of cancer cells, testing cytotoxicity in T-acute lymphoblastic leukemia cells and their cell death mechanism. Here, we assessed the effect of I-CRP on cell viability and cell death. To determine the mechanism of cell death, we tested cell cycle, mitochondrial and nuclear alterations, and caspases and reactive oxygen species (ROS) and their role in cell death mechanism. Our results show that I-CRP does not affect cell viability in noncancer cells and induces selective cytotoxicity in a dose-dependent manner in leukemic cell lines. I-CRP also induces mitochondrial damage through proapoptotic and antiapoptotic protein modulation (Bax and Bcl-2) and ROS production, nuclear alterations including DNA damage (γ-H2Ax), overexpression of p53, cell cycle arrest, and DNA degradation. I-CRP induced ROS-dependent apoptosis in leukemic cells. Overall, here, we show that I-CRP cytotoxicity is selective to leukemic cells, inducing ROS-dependent apoptosis. This research opens the door to further exploration of their role in the immune system and the cell death mechanism that could potentially work in conjunction with other therapies including hematological malignances.
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BACKGROUND: IMMUNEPOTENT-CRP® (I-CRP) is a bovine dialyzable leukocyte extract containing transfer factor. It is a cost-effective, unspecific active immunotherapy that has been used in patients with non-small cell lung cancer (NSCLC) as an adjuvant to reduce the side-effects of chemotherapy and radiotherapy, and has shown cytotoxic activity in vitro on different cancer cell lines. However, its mechanism of action against lung cancer cells has not been assessed. Therefore, the objective of this work was to assess the cytotoxic mechanism of I-CRP on lung cancer cell lines. METHODS: We assessed cell viability through MTT assay on the NSCLC cell lines A549, A427, Calu-1, and INER-51 after treatment with I-CRP. To further understand the mechanisms of cell viability diminution we used fluorescence-activated cell sorting to evaluate cell death (annexin-V and propidium iodide [PI] staining), cell cycle and DNA degradation (PI staining), mitochondrial alterations (TMRE staining), and reactive oxygen species (ROS) production (DCFDA staining). Additionally, we evaluated caspase and ROS dependence of cell death by pretreating the cells with the pan-caspase inhibitor Q-VD-OPH and the antioxidant N-acetylcysteine (NAC), respectively. RESULTS: Our data shows that I-CRP is cytotoxic to NSCLC cell lines in a dose and time dependent manner, without substantial differences between the four cell lines tested (A549, A427, Calu-1, and INER-51). Cytotoxicity is induced through regulated cell death and cell cycle arrest induction. I-CRP-induced cell death in NSCLC cell lines is characterized by DNA degradation, mitochondrial damage, and ROS production. Moreover, cell death is independent of caspases but relies on ROS production, as it is abrogated with NAC. CONCLUSION: Altogether, these results improve the knowledge about the cytotoxic activity of I-CRP on NSCLC cells, indicating that cell death, cell cycle arrest, DNA degradation and mitochondrial damage are important features, while ROS play the main role for I-CRP mediated cytotoxicity in lung cancer cells.
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BACKGROUND: Nanotechnology proposes the use of gold nanoparticles (AuNPs) for drug delivery, diagnosis, and treatment of cancer. Leukemia is a type of hematopoietic cancer that results from the malignant transformation of white blood cells. Chitosan-coated AuNPs (CH-AuNPs) are cell death inductors in HeLa and MCF-7 cancer cells without affecting peripheral blood mononuclear cells (PBMC). Considering the selectivity and versatile cytotoxicity of CH-AuNPs, we evaluated whether their selectivity is due to the cell lineage or the characteristics of the cancer cells, by assessing its cytotoxicity in leukemic cells. Moreover, we further examined the cell death mechanism and assessed the implication of nuclear damage, autophagosome formation, and the cell death mechanism induced in leukemic cells. MATERIALS AND METHODS: We synthesized CH-AuNPs by chemical methods and analyzed their cell death capacity in a T-acute lymphocytic leukemia cell line (CEM), in a chronic myeloid leukemia cell line (K562), and in healthy cells from the same lineage (PBMC and bone marrow, BM, cells). Then, we assessed ROS generation and mitochondrial and nuclear damage. Finally, we evaluated whether cell death occurred by autophagy, apoptosis, or necroptosis, and the role of ROS in this mechanism. RESULTS: We found that CH-AuNPs did not affect PBMC and BM cells, whereas they are cytotoxic in a dose-dependent manner in leukemic cells. ROS production leads to mitochondrial and nuclear damage, and cell death. We found that CH-AuNPs induce apoptosis in CEM and necroptosis in K562, both undergoing autophagy as a pro-survival mechanism. CONCLUSION: CH-AuNPs are selective cell death inductors in hematologic cancer cells, without affecting their healthy counterparts. Cell death induced by CH-AuNPs is independent of the cancer cell type; however, its mechanism is different depending on the type of leukemic cells.
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Apoptose , Quitosana/química , Ouro/química , Leucemia/patologia , Nanopartículas Metálicas/química , Espécies Reativas de Oxigênio/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , Ativação Enzimática , Humanos , Leucemia/enzimologia , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Necrose , Fosfatidilserinas/metabolismoRESUMO
In order to optimize the potency of the first serum-stable peptide agonist of CD47 (PKHB1) in triggering regulated cell death of cancer cells, we designed a maturation process aimed to mimic the trimeric structure of the thrombospondin-1/CD47 binding epitope. For that purpose, an N-methylation scan of the PKHB1 sequence was realized to prevent peptide aggregation. Structural and pharmacological analyses were conducted in order to assess the conformational impact of these chemical modifications on the backbone structure and the biological activity. This structure-activity relationship study led to the discovery of a highly soluble N-methylated peptide that we termed PKT16. Afterward, this monomer was used for the design of a homotrimeric peptide mimic that we termed [PKT16]3, which proved to be 10-fold more potent than its monomeric counterpart. A pharmacological evaluation of [PKT16]3 in inducing cell death of adherent (A549) and nonadherent (MEC-1) cancer cell lines was also performed.
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Desenho de Fármacos , Peptídeos/química , Peptídeos/farmacologia , Trombospondina 1/química , Células A549 , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Peptídeos/síntese química , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade , Trombospondina 1/farmacologiaRESUMO
Acute lymphocytic leukemia (ALL) is the most common pediatric cancer. Currently, treatment options for patients with relapsed and refractory ALL mostly rely on immunotherapies. However, hematological cancers are commonly associated with a low immunogenicity and immune tolerance, which may contribute to leukemia relapse and the difficulties associated with the development of effective immunotherapies against this disease. We recently demonstrated that PKHB1, a TSP1-derived CD47 agonist peptide, induces immunogenic cell death (ICD) in T cell ALL (T-ALL). Cell death induced by PKHB1 on T-ALL cell lines and their homologous murine, L5178Y-R (T-murine tumor lymphoblast cell line), induced damage-associated molecular patterns (DAMPs) exposure and release. Additionally, a prophylactic vaccination with PKHB1-treated L5178Y-R cells prevented tumor establishment in vivo in all the cases. Due to the immunogenic potential of PKHB1-treated cells, in this study we assessed their ability to induce antitumor immune responses ex vivo and in vivo in an established tumor. We first confirmed the selectivity of cell death induced by PKBH1 in tumor L5178Y-R cells and observed that calreticulin exposure increased when cell death increased. Then, we found that the tumor cell lysate (TCL) obtained from PKHB1-treated L5178YR tumor cells (PKHB1-TCL) was able to induce, ex vivo, dendritic cells maturation, cytokine production, and T cell antitumor responses. Finally, our results show that in vivo, PKHB1-TCL treatment induces tumor regression in syngeneic mice transplanted with L5178Y-R cells, increasing their overall survival and protecting them from further tumor establishment after tumor rechallenge. Altogether our results highlight the immunogenicity of the cell death induced by PKHB1 activation of CD47 as a potential therapeutic tool to overcome the low immunogenicity and immune tolerance in T-ALL.
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Cuphea aequipetala (C. aequipetala) has been used in Mexican traditional medicine since prehispanic times to treat tumors. In this paper, we evaluated the antiproliferative and apoptotic effect of the methanolic and aqueous extracts of C. aequipetala on several cancer cell lines including the B16F10 cell line of murine melanoma and carried a murine model assay. In vitro assay analyzed the effect in the cellular cycle and several indicators of apoptosis, such as the caspase-3 activity, DNA fragmentation, phosphatidylserine exposure (Annexin-V), and induction of cell membrane permeabilization (propidium iodide) in the B16F10 cells. In vivo, groups of C57BL/6 female mice were subcutaneously injected with 5x105 B16F10 cells and treated with 25 mg/mL of C. aequipetala extracts via oral. Aqueous and methanolic extracts showed a cytotoxic effect in MCF-7, HepG2, and B16F10 cell lines. The methanolic extract showed more antiproliferative effect with less concentration, and for this reason, the in vitro experiments were only continued with it. This extract was able to induce accumulation of cells on G1 phase of the cell cycle; moreover, it was able to induce DNA fragmentation and increase the activity of caspase-3 in B16F10 cells. On the other hand, in the murine model of melanoma, the aqueous extract showed a greater reduction of tumor size in comparison with the methanolic extract, showing an 80% reduction versus one of around 31%, both compared with the untreated control, indicating a better antitumor effect of C. aequipetala aqueous extract via oral administration. In conclusion, the in vitro data showed that both C. aequipetala extracts were able to induce cytotoxicity through the apoptosis pathway in B16F10 cells, and in vivo, the oral administration of aqueous extract reduces the melanoma tumoral mass, suggesting an important antitumoral effect and the perspective to search for effector molecules involved in it.
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Cuphea/química , Melanoma Experimental/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Feminino , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Melanoma Experimental/patologia , Metanol/química , Camundongos Endogâmicos C57BL , Extratos Vegetais/farmacologia , Água/químicaRESUMO
This work aimed to evaluate the mechanisms involved in the apoptosis induction of isorhamnetin-3-O-glucosyl-pentoside (IGP) in metastatic human colon cancer cells (HT-29). To achieve this, we assessed phosphatidylserine (PS) exposure, cell membrane disruption, chromatin condensation, cell cycle alterations, mitochondrial damage, ROS production, and caspase-dependence on cell death. Our results showed that IGP induced cell death on HT-29â¯cells through PS exposure (48%) and membrane permeabilization (30%) as well as nuclear condensation (54%) compared with control cells. Moreover, IGP treatment induced cell cycle arrest in G2/M phase. Bax/Bcl-2 ratio increased and the loss of mitochondrial membrane potential (63%) was observed in IGP-treated cells. Finally, as apoptosis is a caspase-dependent cell death mechanism, we used a pancaspase-inhibitor (Q-VD-OPh) to demonstrate that the cell death induced by IGP was caspase-dependent. Overall these results indicated that IGP induced apoptosis through caspase-dependent mitochondrial damage in HT-29 colon cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Glicosídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Opuntia/química , Quercetina/análogos & derivados , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Flavonóis , Glicosídeos/isolamento & purificação , Glicosídeos/uso terapêutico , Células HT29 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/patologia , Extratos Vegetais/farmacologia , Quercetina/isolamento & purificação , Quercetina/farmacologia , Quercetina/uso terapêuticoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin-1-derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first-described serum-stable CD47-agonist peptide) on CEM and MOLT-4 human cell lines (T-ALL) and on one T-murine tumor lymphoblast cell-line (L5178Y-R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non-tumor cells after CD47 activation. In vivo, we determined that PKHB1-treatment in mice bearing the L5178Y-R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage-associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high-mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase-independent and calcium-dependent cell death in leukemic cells while sparing non-tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.
Assuntos
Apoptose/efeitos dos fármacos , Antígeno CD47/agonistas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Antígeno CD47/metabolismo , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Estimativa de Kaplan-Meier , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Camundongos Endogâmicos BALB C , Peptídeos/química , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Trombospondina 1/químicaRESUMO
BACKGROUND: Human papillomavirus (HPV) is recognized as an important risk factor for laryngeal carcinogenesis. Although HPV-16 and 18 have been strongly implicated, the presence of other high-risk HPV (HR-HPV) genotypes or the coinfection with Epstein-Barr virus (EBV) or Merkel cell polyomavirus (MCPV) may increase the risk, but their etiological association has not been definitively established. METHODS: We characterized the genotype-specific HPV and the frequency of EBV and MCPV infections through the detection of their DNA in 195 laryngeal specimens of squamous cell carcinoma (SCC) histologically confirmed. RESULTS: HPV DNA was detected in 93 (47.7%) specimens. HPV-11 was the most frequent with 68 cases (73.1%), and HPV-52 was the most frequently HR-HPV found with 51 cases, which corresponds to 54.8% of all HPV-positive specimens. EBV DNA was detected in 54 (27.7%) tumor tissue specimens of which 25 (46.3%) were in coinfection with HPV. MCPV DNA was detected only in 11 (5.6%) cases of which 5 (45.4%) were in coinfection with an HR-HPV. No association between the presence of DNA of the three examined viruses and the patient smoking habits, alcohol consumption, age, the keratinization status, differentiation grade, or localization of the tumor in the larynx were found. DISCUSSION: HPV-52 was the most prevalent HR-HPV, which may suggest that this and other genotypes in addition to HPV-16 and 18 could be considered for prophylaxis. However, further studies including non-cancer larynx cases and the evaluation of other molecular markers and viral co-infection mechanisms are needed to determine the role of the different HR-HPV genotypes, EBV, and MCPV in the etiology of SCC of the larynx.