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OBJECTIVES: Chemoprevention can be a treatment for potentially malignant lesions (PMLs). We aimed to evaluate whether artemisinin (ART) and cisplatin (CSP) are associated with apoptosis and immunogenic cell death (ICD) in vitro, using oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) cell lines, and whether these compounds prevent OL progression in vivo. METHODS: Normal keratinocytes (HaCat), Dysplastic oral cells (DOK), and oral squamous cell carcinoma (SCC-180) cell lines were treated with ART, CSP, and ART + CSP to analyze cytotoxicity, genotoxicity, cell migration, and increased expression of proteins related to apoptosis and ICD. Additionally, 41 mice were induced with OL using 4NQO, treated with ART and CSP, and their tongues were histologically analyzed. RESULTS: In vitro, CSP and CSP + ART showed dose-dependent cytotoxicity and reduced SCC-180 migration. No treatment was genotoxic, and none induced expression of proteins related to apoptosis and ICD; CSP considerably reduced High-mobility group box-1 (HMGB-1) protein expression in SCC-180. In vivo, there was a delay in OL progression with ART and CSP treatment; however, by the 16th week, only CSP prevented progression to OSCC. CONCLUSION: Expression of proteins related to ICD and apoptosis did not increase with treatments, and CSP was shown to reduce immunogenic pathways in SCC-180, while reducing cell migration. ART did not prevent the malignant progression of OL in vivo; CSP did despite significant adverse effects.
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Apoptose , Artemisininas , Movimento Celular , Cisplatino , Progressão da Doença , Leucoplasia Oral , Neoplasias Bucais , Artemisininas/farmacologia , Animais , Leucoplasia Oral/patologia , Leucoplasia Oral/tratamento farmacológico , Humanos , Cisplatino/farmacologia , Camundongos , Neoplasias Bucais/patologia , Neoplasias Bucais/tratamento farmacológico , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proteína HMGB1/metabolismo , Antineoplásicos/farmacologiaRESUMO
Photodynamic therapy (PDT) uses a photosensitizer to generate reactive oxygen species (ROS) that kill target cells. In cancer treatments, PDT can potentially induce immunogenic cell death (ICD), which is characterized by a well-controlled exposure of damage-associated molecular patterns (DAMPs) that activate dendritic cells (DCs) and consequently modulate the immune response in the tumor microenvironment. However, PDT still has limitations, such as the activity of photosensitizers in aqueous media and poor bioavailability. Therefore, a new photosensitizer system, SLN-AlPc, has been developed to improve the therapeutic efficacy of PDT. In vitro experiments showed that the light-excited nanocarrier increased ROS production in murine melanoma B16-F10 cells and modulated the profile of DCs. PDT induced cell death accompanied by the exposure of DAMPs and the formation of autophagosomes. In addition, the DCs exposed to PDT-treated B16-F10 cells exhibited morphological changes, increased expression of MHCII, CD86, CD80, and production of IL-12 and IFN-γ, suggesting immune activation towards an antitumor profile. These results indicate that the SLNs-AlPc protocol has the potential to improve PDT efficacy by inducing ICD and activating DCs.
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The BRAFV600E mutation, found in approximately 50% of melanoma cases, plays a crucial role in the activation of the MAPK/ERK signaling pathway, which promotes tumor cell proliferation. This study aimed to evaluate its impact on the melanoma immune microenvironment and therapeutic responses, particularly focusing on immunogenic cell death (ICD), a pivotal cytotoxic process triggering anti-tumor immune responses. Through comprehensive in silico analysis of the Cancer Genome Atlas data, we explored the association between the BRAFV600E mutation, immune subtype dynamics, and tumor mutation burden (TMB). Our findings revealed that the mutation correlated with a lower TMB, indicating a reduced generation of immunogenic neoantigens. Investigation into immune subtypes reveals an exacerbation of immunosuppression mechanisms in BRAFV600E-mutated tumors. To assess the response to ICD inducers, including doxorubicin and Me-ALA-based photodynamic therapy (PDT), compared to the non-ICD inducer cisplatin, we used distinct melanoma cell lines with wild-type BRAF (SK-MEL-2) and BRAFV600E mutation (SK-MEL-28, A375). We demonstrated a differential response to PDT between the WT and BRAFV600E cell lines. Further transcriptomic analysis revealed upregulation of IFNAR1, IFNAR2, and CXCL10 genes associated with the BRAFV600E mutation, suggesting their involvement in ICD. Using a gene reporter assay, we showed that PDT robustly activated the IFN-1 pathway through cGAS-STING signaling. Collectively, our results underscore the complex interplay between the BRAFV600E mutation and immune responses, suggesting a putative correlation between tumors carrying the mutation and their responsiveness to therapies inducing the IFN-1 pathway, such as the ICD inducer PDT, possibly mediated by the elevated expression of IFNAR1/2 receptors.
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INTRODUCTION: Cancer is an individual disease and its formation and development are specific to each host. Conventional treatments are ineffective in complex cases, such as metastasis, and have severe adverse side effects. New strategies are needed to address the problem, and the use of immunogenic cell death (ICD) as a trigger or booster of the immune system through the exposure of damage-associated molecular patterns, along with tumor antigens, by cancerous cells is presented as an immunization approach in this work. METHODS: For this purpose, 4T1 cells were exposed to doxorubicin (DOX) for 24 hours and then, these cells undergoing ICD were subcutaneously administered to mice. The ICD induction by DOX on 4T1 was assessed by flow cytometry and image analysis. This immunization process was performed three times and after the last administration, the immunized mice were challenged with a subcutaneous xenograft of live cancer cells. RESULTS: The results demonstrate that the mice immunized with cells undergoing ICD after exposure to DOX presented no primary tumor or indications of distant metastatic lesion development. CONCLUSION: In summary, our findings indicate that the immunization process utilizing ICD is indeed efficacious in managing this aggressive form of pre-clinical breast cancer.
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BACKGROUND: Mesenchymal stromal cells (MSCs) tropism for tumours allows their use as carriers of antitumoural factors and in vitro transcribed mRNA (IVT mRNA) is a promising tool for effective transient expression without insertional mutagenesis risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with antitumor properties by stimulating the specific immune response. The aim of this work was to generate modified MSCs by IVT mRNA transfection to overexpress GM-CSF and determine their therapeutic effect alone or in combination with doxorubicin (Dox) in a murine model of hepatocellular carcinoma (HCC). METHODS: DsRed or GM-CSF IVT mRNAs were generated from a cDNA template designed with specific primers followed by reverse transcription. Lipofectamine was used to transfect MSCs with DsRed (MSC/DsRed) or GM-CSF IVT mRNA (MSC/GM-CSF). Gene expression and cell surface markers were determined by flow cytometry. GM-CSF secretion was determined by ELISA. For in vitro experiments, the J774 macrophage line and bone marrow monocytes from mice were used to test GM-CSF function. An HCC model was developed by subcutaneous inoculation (s.c.) of Hepa129 cells into C3H/HeN mice. After s.c. injection of MSC/GM-CSF, Dox, or their combination, tumour size and mouse survival were evaluated. Tumour samples were collected for mRNA analysis and flow cytometry. RESULTS: DsRed expression by MSCs was observed from 2 h to 15 days after IVT mRNA transfection. Tumour growth remained unaltered after the administration of DsRed-expressing MSCs in a murine model of HCC and MSCs expressing GM-CSF maintained their phenotypic characteristic and migration capability. GM-CSF secreted by modified MSCs induced the differentiation of murine monocytes to dendritic cells and promoted a proinflammatory phenotype in the J774 macrophage cell line. In vivo, MSC/GM-CSF in combination with Dox strongly reduced HCC tumour growth in C3H/HeN mice and extended mouse survival in comparison with individual treatments. In addition, the tumours in the MSC/GM-CSF + Dox treated group exhibited elevated expression of proinflammatory genes and increased infiltration of CD8 + T cells and macrophages. CONCLUSIONS: Our results showed that IVT mRNA transfection is a suitable strategy for obtaining modified MSCs for therapeutic purposes. MSC/GM-CSF in combination with low doses of Dox led to a synergistic effect by increasing the proinflammatory tumour microenvironment, enhancing the antitumoural response in HCC.
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Carcinoma Hepatocelular , Doxorrubicina , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Neoplasias Hepáticas , Células-Tronco Mesenquimais , RNA Mensageiro , Animais , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Linhagem Celular Tumoral , Transplante de Células-Tronco Mesenquimais/métodos , Humanos , Camundongos Endogâmicos C3H , TransfecçãoRESUMO
Novos fármacos, como a artemisinina (ART), podem ser promissores no tratamento de lesões potencialmente malignas (LPM) e podem ser úteis quando usados em associação com outros quimioterápicos, especialmente na redução dos seus efeitos colaterais. A leucoplasia oral (LO) é a LPM mais comum da cavidade bucal e, pode evoluir para um carcinoma de células escamosas oral (CCEO). Não há terapia para evitar a sua transformação maligna, a quimioprevenção pode iniciar a morte celular imunogênica (MCI) que ativa o sistema imunológico para que reconheça e elimine as células malignas ou pré-malignas, sendo um potencial tratamento para as LPM. O presente estudo objetivou avaliar se a ART e a cisplatina (CSP) associadas ou não seriam capazes de induzir a MCI em linhagens celulares de LO (DOK) e CCEO SCC180). Material e métodos: As linhagens celulares HaCat (controle), DOK e SCC-180 foram tratadas por ART e CSP de forma combinada ou isolada, a fim de analisar a citotoxicidade e a genotoxicidade destes fármacos, além da capacidade destes em reduzir a migração celular e, se os compostos seriam capazes de induzir a expressão da proteína box de alta mobilidade (HGMB-1), caspase 3, 8, 9, e Calreticulina (CALR). Resultados: Em todas as linhagens celulares a CSP e CSP+ART causaram uma resposta dose dependente, apresentando maior citotoxicidade com doses mais altas, o que não foi observado com a ART. A formação de micronúcleos não foi observada no teste de genotoxicidade. A taxa de migração foi reduzida com as concentrações de IC50 de CSP e ART+CSP para as células de CCEO. Não foram encontradas expressões significativas de proteínas relacionadas à MCI ou apoptose nas linhagens de LO e CCEO, tratadas com ART, CSP ou ART+CSP, indicando que outro tipo de morte celular possa ter ocorrido. Conclusão: A MCI e a apoptose não foram evidenciadas como forma de morte celular após as linhagens de LO e CCEO receberem tratamentos com ART, CSP e a associação de ambas. Efeitos genotóxicos não foram observados nas doses testadas. O tratamento de CSP e ART+CSP foi capaz de reduzir a migração de células de CCEO. Também concluímos que novos estudos são necessários para elucidar se a ART e CSP podem ocasionar a MCI ou apoptose em linhagens celulares de LO e CCEO (AU)
New drugs, such as artemisinin (ART), may be promising in the treatment of potentially malignant lesions (PML) and may be useful when used in combination with other chemotherapy drugs, especially in reducing their side effects. Oral leukoplakia (OL) is the most common LPM of the oral cavity and can progress to oral squamous cell carcinoma (OSCC). There is no therapy to prevent its malignant transformation, chemoprevention can initiate immunogenic cell death (ICM) that activates the immune system to recognize and eliminate malignant or pre-malignant cells, being a potential treatment for LPM. The present study aimed to evaluate whether or not ART and cisplatin (CSP) combined would be capable of inducing MCI in LO (DOK) and CCEO SCC-180) cell lines. Material and methods: HaCat (control), DOK and SCC-180 cell lines were treated by ART and CSP in combination or alone, in order to analyze the cytotoxicity and genotoxicity of these drugs, in addition to their ability to reduce cell and, whether the compounds would be able to induce the expression of high mobility box protein (HGMB-1), caspase 3, 8, 9, and Calreticulin (CALR). Results: In cytotoxicity at higher doses, which was not observed with ART. The formation of micronuclei was not observed in the genotoxicity test. The migration rate was reduced with the IC50 concentrations of CSP and ART+CSP for OSCC cells. No significant expressions of proteins related to MCI or apoptosis were found in the LO and CCEO lines, treated with ART, CSP or ART+CSP, indicating that another type of cell death may have occurred. Conclusion: MCI and apoptosis were not evidenced as a form of cell death after the LO and CCEO lines received treatments with ART, CSP and the combination of both. Genotoxic effects were not observed at the doses tested. CSP and ART+CSP treatment was able to reduce OSCC cell migration. We also conclude that new studies are necessary to elucidate whether ART and CSP can cause MCI or apoptosis in LO and CCEO cell lines.(AU)
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Leucoplasia Oral , Imunomodulação , Carcinoma de Células Escamosas de Cabeça e Pescoço , Morte Celular Imunogênica , Células Apresentadoras de AntígenosRESUMO
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
Capsaicinoids are a unique chemical species resulting from a particular biosynthesis pathway of hot chilies (Capsicum spp.) that gives rise to 22 analogous compounds, all of which are TRPV1 agonists and, therefore, responsible for the pungency of Capsicum fruits. In addition to their human consumption, numerous ethnopharmacological uses of chili have emerged throughout history. Today, more than 25 years of basic research accredit a multifaceted bioactivity mainly to capsaicin, highlighting its antitumor properties mediated by cytotoxicity and immunological adjuvancy against at least 74 varieties of cancer, while non-cancer cells tend to have greater tolerance. However, despite the progress regarding the understanding of its mechanisms of action, the benefit and safety of capsaicinoids' pharmacological use remain subjects of discussion, since CAP also promotes epithelial-mesenchymal transition, in an ambivalence that has been referred to as "the double-edge sword". Here, we update the comparative discussion of relevant reports about capsaicinoids' bioactivity in a plethora of experimental models of cancer in terms of selectivity, efficacy, and safety. Through an integration of the underlying mechanisms, as well as inherent aspects of cancer biology, we propose mechanistic models regarding the dichotomy of their effects. Finally, we discuss a selection of in vivo evidence concerning capsaicinoids' immunomodulatory properties against cancer.
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Capsicum , Neoplasias , Humanos , Capsaicina/farmacologia , Frutas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , BiologiaRESUMO
Immunogenic cell death (ICD) is a type of cell death capable of stimulating immunity against cancer through danger signals that lead to an adaptive immune response. Silver nanoparticles (AgNPs) have been shown to have a cytotoxic effect on cancer cells; however, their mechanism of action is not fully understood. The present study synthesized, characterized, and evaluated the cytotoxic effect of beta-D-glucose-reduced AgNPs (AgNPs-G) against breast cancer (BC) cells in vitro; and assess the immunogenicity of cell death in vitro and in vivo. The results showed that AgNPs-G induce cell death in a dose-dependent manner on BC cell lines. In addition, AgNPs show antiproliferative effects by interfering with the cell cycle. Regarding the detection of damage-associated molecular patterns (DAMPs), it was found that treatment with AgNPs-G induces calreticulin exposure and the release of HSP70, HSP90, HMGB1, and ATP. In vivo, prophylactic vaccination did not prevent tumor establishment; however, tumor weight was significantly lower in AgNPs-G vaccinated mice, while the survival rate increased. In conclusion, we have developed a new method for the synthesis of AgNPs-G, with in vitro antitumor cytotoxic activity on BC cells, accompanied by the release of DAMPs. In vivo, immunization with AgNPs-G failed to induce a complete immune response in mice. Consequently, additional studies are needed to elucidate the mechanism of cell death that leads to the design of strategies and combinations with clinical efficacy.