RESUMO
N6-adenosine methylation (m6A) is critical for controlling cancer cell growth and tumorigenesis. However, the function and detailed mechanism of how m6A methyltransferases modulate m6A levels on specific targets remain unknown. In the current study, we identified significantly elevated levels of RBM15, an m6A writer, in basal-like breast cancer (BC) patients compared to nonbasal-like BC patients and linked this increase to worse clinical outcomes. Gene expression profiling revealed correlations between RBM15 and serine and glycine metabolic genes, including PHGDH, PSAT1, PSPH, and SHMT2. RBM15 influences m6A levels and, specifically, the m6A levels of serine and glycine metabolic genes via direct binding to target RNA. The effects of RBM15 on cell growth were largely dependent on serine and glycine metabolism. Thus, RBM15 coordinates cancer cell growth through altered serine and glycine metabolism, suggesting that RBM15 is a new therapeutic target in BC.
Assuntos
Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glicina , Proteínas de Ligação a RNA , Serina , Neoplasias de Mama Triplo Negativas , Humanos , Serina/metabolismo , Glicina/metabolismo , Feminino , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Linhagem Celular Tumoral , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Metilação , Adenosina/metabolismo , Adenosina/análogos & derivadosRESUMO
Background: Ginseng has been used as a traditional medicine for treatment of many diseases and for general health maintenance. Previously, we showed that ginseng did not demonstrate estrogenic property in ovariectomized mouse model. However, it is still possible that disruption of steroidogenesis leading to indirect hormonal activity. Methods: The hormonal activities were examined in compliance with OECD guidelines for detecting endocrine disrupting chemicals: test guideline (TG) No. 456 (an in vitro assay method for detecting steroidogenesis property) and TG No. 440 (an in vivo short-term screening method for chemicals with uterotrophic property). Results: Korean Red Ginseng (KRG) and ginsenosides Rb1, Rg1, and Rg3 did not interfere with estrogen and testosterone hormone synthesis as examined in H295 cells according to TG 456. KRG treatment to ovariectomized mice did not show a significant change in uterine weight. In addition, serum estrogen and testosterone levels were not change by KRG intake. Conclusion: These results clearly demonstrate that there is no steroidogenic activity associated with KRG and no disruption of the hypothalamic-pituitary-gonadal axis by KRG. Additional tests will be performed in pursuit of cellular molecular targets of ginseng to manifest mode of action.
RESUMO
OBJECTIVE: Fenbendazole (FZ) has potential anti-cancer effects, but its poor water solubility limits its use for cancer therapy. In this study, we investigated the anti-cancer effect of FZ with different drug delivery methods on epithelial ovarian cancer (EOC) in both in vitro and in vivo models. METHODS: EOC cell lines were treated with FZ and cell proliferation was assessed. The effect of FZ on tumor growth in cell line xenograft mouse model of EOC was examined according to the delivery route, including oral and intraperitoneal administration. To improve the systemic delivery of FZ by converting fat-soluble drugs to hydrophilic, we prepared FZ-encapsulated poly(D,L-lactide-co-glycolide) acid (PLGA) nanoparticles (FZ-PLGA-NPs). We investigated the preclinical efficacy of FZ-PLGA-NPs by analyzing cell proliferation, apoptosis, and in vivo models including cell lines and patient-derived xenograft (PDX) of EOC. RESULTS: FZ significantly decreased cell proliferation of both chemosensitive and chemoresistant EOC cells. However, in cell line xenograft mouse models, there was no effect of oral FZ treatment on tumor reduction. When administered intraperitoneally, FZ was not absorbed but aggregated in the intraperitoneal space. We synthesized FZ-PLGA-NPs to obtain water solubility and enhance drug absorption. FZ-PLGA-NPs significantly decreased cell proliferation in EOC cell lines. Intravenous injection of FZ-PLGA-NP in xenograft mouse models with HeyA8 and HeyA8-MDR significantly reduced tumor weight compared to the control group. FZ-PLGA-NPs showed anti-cancer effects in PDX model as well. CONCLUSION: FZ-incorporated PLGA nanoparticles exerted significant anti-cancer effects in EOC cells and xenograft models including PDX. These results warrant further investigation in clinical trials.
Assuntos
Nanopartículas , Neoplasias Ovarianas , Humanos , Animais , Camundongos , Feminino , Fenbendazol/uso terapêutico , Neoplasias Ovarianas/patologia , Carcinoma Epitelial do Ovário/tratamento farmacológico , Linhagem Celular Tumoral , ÁguaRESUMO
Chemotherapy promotes phosphatidylserine (PS) externalization in tumors undergoing apoptosis, forms an immunosuppressive tumor microenvironment (TME), and inhibits dendritic cell (DC) maturation and antigen presentation by binding PS receptors expressed in DCs, thereby limiting naive T cell education and activation. In this study, we demonstrate a selective nanocarrier system composed of annexin A5-labeled poly (lactide-co-glycolide) nanoparticles (PLGA_NPs) encapsulating tumor specific antigen or neoantigen, to target apoptotic tumor cells expressing PS as an innate immune checkpoint inhibitor (ICI) that induces active cancer immunotherapy. Moreover, PLGA_NPs enhanced tumor-specific antigen-based cytotoxic T cell immunity via the original function of DCs by converting the tumor antigen-rich environment. Therefore, chemotherapy combined with an immunomodulatory nanocarrier system demonstrated an enhanced anticancer immune response by increasing survival rates, immune-activating cells, and pro-inflammatory cytokines in the spleen and TME. In contrast, the tumor mass, immune-suppressive cells, and anti-inflammatory cytokines were decreased. Furthermore, the combination of a nanocarrier system with other ICIs against large tumors showed therapeutic efficacy by immunosuppression in the TME and further amplified the anticancer immunity of interferon gamma+ (IFN-γ) CD8+ (cluster of differentiation 8) T cells. Taken together, our Annexin A5-labeled PLGA-NPs can be applied in various combination therapeutic techniques for cancer immunotherapy.
Assuntos
Agentes de Imunomodulação/farmacologia , Nanopartículas , Neoplasias , Anexina A5 , Apresentação de Antígeno , Antígenos de Neoplasias/metabolismo , Apoptose , Citocinas/metabolismo , Células Dendríticas , Humanos , Imunoterapia/métodos , Ácido Láctico , Neoplasias/tratamento farmacológico , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Microambiente TumoralRESUMO
Background: Di-(2-ethylhexyl) phthalate (DEHP) is the most common endocrine disrupting chemical used as a plasticizer. DEHP is associated with the development of endometrium-related diseases through the induction of inflammation. The major therapeutic approaches against endometrial cancer and endometriosis involve the suppression of inflammatory response. Korean Red Ginseng (KRG) is a natural product with anti-inflammatory and anti-carcinogenic properties. Thus, the purpose of this study is to investigate the effects of KRG on DEHP-induced inflammatory response in endometrial cancer Ishikawa cells and a mouse model of endometriosis. Methods: RNA-sequencing was performed and analyzed on DEHP-treated Ishikawa cells in the presence and absence of KRG. The effects of KRG on DEHP-induced cyclooxygenase-2 (COX-2) mRNA levels in Ishikawa cells were determined by RT-qPCR. Furthermore, the effects of KRG on the extracellular signal-regulated kinases (ERKs) pathway, COX-2, and nuclear factor-kappa B (NF-κB) p65 after DEHP treatment of Ishikawa cells were evaluated by western blotting. In the mouse model, the severity of endometriosis induced by DEHP and changes in immunohistochemistry were used to assess the protective effect of KRG. Results: According to the RNA-sequencing data, DEHP-induced inflammatory response-related gene expression was downregulated by KRG. Moreover, KRG significantly inhibited DEHP-induced ERK1/2/NF-κB/COX-2 levels in Ishikawa cells. In the mouse model, KRG administration significantly inhibited ectopic endometriosis growth after DEHP-induced endometriosis. Conclusions: Overall, these results suggest that KRG may be a promising lead for the treatment of endometrial cancer and endometriosis via suppression of the inflammatory response.
RESUMO
Various cancer therapies, such as surgery, radiotherapy, chemotherapy, and immunotherapy, have been used to treat cancer. Among cancer immunotherapies, stimulators of interferon genes (STING) activate various immune cells and induce them to attack cancer cells. However, the secretion of type I interferon (IFN α and ß) increases after stimulation of the immune cell as a side effect of STING agonist, thereby increasing the expression of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Therefore, it is necessary to reduce the side effects of STING agonists and maximize cancer treatment by administering combination therapy. Tumor-bearing mice were treated with cisplatin, tumor-specific peptide, neoantigen, DMXAA (STING agonist), and immune checkpoint inhibitor (ICI). The combination vaccine group showed a reduction in tumor mass, an increased survival rate, and IFN-γ+ (interferon gamma) CD8+ (cluster of differentiation 8) T cells in the spleen and TME. The distribution of immune cells in the spleen and TME was confirmed, and the number of active immune cells increased, whereas that of immunosuppressive cells decreased. When measuring cytokine levels in the tumor and serum, the levels of pro-inflammatory cytokines increased and anti-inflammatory cytokines decreased. This study demonstrated that when various cancer therapies are combined to treat cancer, it can lead to an anticancer immune synergistic effect by increasing the immune response and reducing side effects.
Assuntos
Interferon Tipo I , Neoplasias , Camundongos , Animais , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Antígeno B7-H1 , Interferon gama , Cisplatino , Imunoterapia , Neoplasias/terapia , Vacinas Combinadas , Microambiente TumoralRESUMO
BACKGROUND: Cytotoxic CD8+ T cell-based cancer immunotherapy has been extensively studied and applied, however, tumor cells are known to evade immune responses through the expression of immune checkpoints, such as programmed death ligand 1 (PD-L1). To overcome these issues, antibody-based immune checkpoint blockades (eg, antiprogrammed cell death 1 (anti-PD-1) and anti-PD-L1) have been revolutionized to improve immune responses. However, their therapeutic efficacy is limited to 15%-20% of the overall objective response rate. Moreover, PD-L1 is secreted from tumor cells, which can interrupt antibody-mediated immune reactions in the tumor microenvironment. METHODS: We developed poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) encapsulating PD-L1 small interfering RNA (siRNA) and PD-1 siRNA, as a delivery platform to silence immune checkpoints. This study used the TC-1 and EG7 tumor models to determine the potential therapeutic efficacy of the PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs, on administration twice per week for 4 weeks. Moreover, we observed combination effect of PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs using TC-1 and EG7 tumor-bearing mouse models. RESULTS: PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs boosted the host immune reaction by restoring CD8+ T cell function and promoting cytotoxic CD8+ T cell responses. We demonstrated that the combination of NP-based therapeutic vaccine and PLGA (siRNA)-NPs resulted in significant inhibition of tumor growth compared with the control and antibody-based treatments (p<0.001). The proposed system significantly inhibited tumor growth compared with the antibody-based approaches. CONCLUSION: Our findings suggest a potential combination approach for cancer immunotherapy using PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs as novel immune checkpoint silencing agents.
Assuntos
Antineoplásicos , Nanopartículas , Animais , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Receptor de Morte Celular Programada 1 , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêuticoRESUMO
The incidence of endometrial cancer is increasing worldwide. One of the main causes of this cancer is a hormone imbalance; progesterone derivatives have been used for treatment. However, reports have shown that hypoxia plays important and possibly beneficial roles in endometrial function. Here, we show the effect of hypoxia on the proliferation of human endometrial adenocarcinoma Ishikawa cells. Hypoxia induced caspase-dependent apoptosis in Ishikawa cells. Overexpression and siRNA-mediated knockdown of hypoxia-inducible factor-1α (HIF-1α) confirmed that HIF-1α accelerates hypoxia-induced cell death. Treatment with dimethyloxalglycine, which stabilizes HIF-1α, suppressed cell proliferation. Kaplan-Meier analysis showed that the expression level of HIF-1α has a significant positive effect on the survival rate of endometrial cancer patients. In our search for cellular targets involved in hypoxic apoptosis, we noticed that mammalian sterile 20-like kinase 2 (MST2), a member of the Hippo pathway, was positively correlated with HIF-1α expression in 176 endometrial cancer patients extracted from the TCGA database. Hypoxia induced caspase-dependent MST2 cleavage. In addition, a MST2 inhibitor suppressed HIF-1α-mediated reporter activity. These results suggest HIF-1α and the Hippo signaling pathway are involved in endometrial cancer.
Assuntos
Adenocarcinoma , Neoplasias do Endométrio , Animais , Apoptose , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias do Endométrio/patologia , Feminino , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mamíferos/metabolismoRESUMO
Drug-based chemotherapy is associated with serious side effects. We developed a chemotherapeutic system comprising a chitosan hydrogel (CH-HG) containing gold cluster-labeled liposomal doxorubicin (DOX) (CH-HG-GLDOX) as an injectable drug depot system. CH-HG-GLDOX can be directly injected into tumor tissue without a surgical procedure, allowing this system to act as a reservoir for liposomal DOX. CH-HG-GLDOX enhanced the retention time of DOX in tumor tissue and controlled its release in response to near-infrared (NIR) irradiation, resulting in significant inhibition of tumor growth and reduced DOX-related toxicity. The combined effect of CH-HG-GLDOX and poly (D,L-lactide-co-glycolic acid) nanoparticle-based vaccines increased cytotoxic CD8+ T cell immunity, leading to enhanced synergistic therapeutic efficacy. CH-HG-GLDOX provides an advanced therapeutic approach for local drug delivery and controlled release of DOX, resulting in reduced toxicity. Here, we suggest a combination strategy for chemo- and immunotherapies, as well as in nanomedicine applications. STATEMENT OF SIGNIFICANCE: We developed an injectable hydrogel containing gold cluster-labeled liposomes for sustained drug release at the tumor site. Moreover, we demonstrated the combined therapeutic efficacy of a hydrogel system and a nanoparticle-based immunotherapeutic vaccine for melanoma cancer. Thus, we show a potential combination approach for chemo- and immunotherapies for cancer treatment.
Assuntos
Lipossomos , Melanoma , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , HidrogéisRESUMO
Immune checkpoint inhibitors (ICIs), including programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 have shown promising cancer clinical outcomes. However, IC therapy has low patient response rates (10%-15%). Thus, ICIs and sufficient antigen combinations into the tumor microenvironment (TME) is important to produce strong tumor-specific adaptive immune responses. Mice were treated with cisplatin, and human cancer cells were exposed to inflammatory cytokines, to confirm increased PD-L1 and major histocompatibility complex (MHC) I expression by tumor cells or dendritic cells. TC-1, CT26, B16-F1, or B16-F10 tumor cells, and bone marrow-derived dendritic cells, were treated with interferon (IFN)-ß, IFN-γ, or tumor necrosis factor-α to identify the molecular mechanisms underlying tumor PD-L1 and MHC I upregulation, and to examine MHC I, CD40, CD80, CD86, or PD-L1 levels, respectively. For synergistic combination therapy, αPD-L1 monoclonal antibody (mAb) covalently linked to the long E7 peptide was generated. Chemotherapy shifted the TME to express high PD-L1 and MHC I, resulting in targeted ICI cargo delivery and enhanced generation and activation of tumor antigen-specific T cells. Synergistic effects of vaccination and IC blockade in the TME were demonstrated using an anti-PD-L1 mAb covalently conjugated to the E7 long peptide.
Assuntos
Antígenos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Imunoconjugados/farmacologia , Imunoterapia , Neoplasias Experimentais/prevenção & controle , Peptídeos/farmacologia , Animais , Antígeno B7-H1/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/imunologiaRESUMO
Cancer immunotherapy has fewer side effects and higher efficiency than conventional methods. Dendritic cell (DC)-based vaccine, a cancer immunotherapeutic, is prepared by processing mature DCs and pulsing with tumor antigen peptide ex vivo, to induce the activation of tumor-specific T lymphocytes followed by tumor clearance in vivo. Unfortunately, clinical trials of this method mostly failed due to low patient response, possibly due to the absence of novel adjuvants that induce DC maturation through Toll-like receptor (TLR) signals. Interestingly, immune checkpoint inhibitor (ICI) therapy has shown remarkable anti-tumor efficacy when combined with cancer vaccines. In this study, we identified 60S acidic ribosomal protein P2 (RPLP2) through pull-down assay using human cancer cells derived proteins that binds to Toll-like receptor 4 (TLR4). Recombinant RPLP2 induced maturation and activation of DCs in vitro. This DC-based vaccine, followed by pulsing with tumor-specific antigen, has shown to significantly increase tumor-specific CD8+IFN-γ+ T cells, and improved both tumor prevention and tumor treatment effects in vivo. The adjuvant effects of RPLP2 were shown to be dependent on TLR4 using TLR4 knockout mice. Moreover, ICIs that suppress the tumor evasion mechanism showed synergistic effects on tumor treatment when combined with these vaccines.
Assuntos
Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Proteínas Ribossômicas/metabolismo , Timoma/terapia , Receptor 4 Toll-Like/metabolismo , Neoplasias do Colo do Útero/terapia , Adjuvantes Imunológicos , Animais , Apoptose , Vacinas Anticâncer/imunologia , Proliferação de Células , Feminino , Humanos , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Timoma/imunologia , Timoma/patologia , Neoplasias do Timo/imunologia , Neoplasias do Timo/patologia , Neoplasias do Timo/terapia , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The roles of chromatin remodelers and their underlying mechanisms of action in cancer remain unclear. In this study, SMARCB1, known initially as a bona fide tumor suppressor gene, was investigated in liver cancer. SMARCB1 was highly upregulated in patients with liver cancer and was associated with poor prognosis. Loss- and gain-of-function studies in liver cells revealed that SMARCB1 loss led to reduced cell proliferation, wound healing capacity, and tumor growth in vivo. Although upregulated SMARCB1 appeared to contribute to switch/sucrose nonfermentable (SWI/SNF) complex stability and integrity, it did not act using its known pathways antagonism with EZH2 or association between TP53 or AMPK. SMARCB1 knockdown induced a mild reduction in global H3K27 acetylation, and chromatin immunoprecipitation sequencing of SMARCB1 and acetylated histone H3K27 antibodies before and after SMARCB1 loss identified Nucleoporin210 (NUP210) as a critical target of SMARCB1, which bound its enhancer and changed H3K27Ac enrichment and downstream gene expression, particularly cholesterol homeostasis and xenobiotic metabolism. Notably, NUP210 was not only a putative tumor supporter involved in liver cancer but also acted as a key scaffold for SMARCB1 and P300 to chromatin. Furthermore, SMARCB1 deficiency conferred sensitivity to doxorubicin and P300 inhibitor in liver cancer cells. These findings provide insights into mechanisms underlying dysregulation of chromatin remodelers and show novel associations between nucleoporins and chromatin remodelers in cancer. SIGNIFICANCE: This study reveals a novel protumorigenic role for SMARCB1 and describes valuable links between nucleoporins and chromatin remodelers in cancer by identifying NUP210 as a critical coregulator of SMARCB1 chromatin remodeling activity.
Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias Hepáticas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteína SMARCB1/genética , Acetilação , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Lisina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteína SMARCB1/metabolismo , Transdução de Sinais/genéticaRESUMO
Damage-associated molecular patterns (DAMPs) are danger signals (or alarmins) alerting immune cells through pattern recognition receptors (PRRs) to begin defense activity. Moreover, DAMPs are host biomolecules that can initiate a noninflammatory response to infection, and pathogen-associated molecular pattern (PAMPs) perpetuate the inflammatory response to infection. Many DAMPs are proteins that have defined intracellular functions and are released from dying cells after tissue injury or chemo-/radiotherapy. In the tumor microenvironment, DAMPs can be ligands for Toll-like receptors (TLRs) expressed on immune cells and induce cytokine production and T-cell activation. Moreover, DAMPs released from tumor cells can directly activate tumor-expressed TLRs that induce chemoresistance, migration, invasion, and metastasis. Furthermore, DAMP-induced chronic inflammation in the tumor microenvironment causes an increase in immunosuppressive populations, such as M2 macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). Therefore, regulation of DAMP proteins can reduce excessive inflammation to create an immunogenic tumor microenvironment. Here, we review tumor-derived DAMP proteins as ligands of TLRs and discuss their association with immune cells, tumors, and the composition of the tumor microenvironment.
Assuntos
Proteínas de Neoplasias/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Alarminas/genética , Alarminas/metabolismo , Animais , Biomarcadores Tumorais , Suscetibilidade a Doenças/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imunomodulação , Proteínas de Neoplasias/genética , Neoplasias/patologia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Ligação Proteica , Receptores Toll-Like/genética , Microambiente Tumoral/imunologiaRESUMO
Overcoming drug-resistance is a big challenge to improve the survival of patients with epithelial ovarian cancer (EOC). In this study, we investigated the effect of chloroquine (CQ) and its combination with cisplatin (CDDP) in drug-resistant EOC cells. We used the three EOC cell lines CDDP-resistant A2780-CP20, RMG-1 cells, and CDDP-sensitive A2780 cells. The CQ-CDDP combination significantly decreased cell proliferation and increased apoptosis in all cell lines. The combination induced expression of γH2AX, a DNA damage marker protein, and induced G2/M cell cycle arrest. Although the CQ-CDDP combination decreased protein expression of ATM and ATR, phosphorylation of ATM was increased and expression of p21WAF1/CIP1 was also increased in CQ-CDDP-treated cells. Knockdown of p21WAF1/CIP1 by shRNA reduced the expression of γH2AX and phosphorylated ATM and inhibited caspase-3 activity but induced ATM protein expression. Knockdown of p21WAF1/CIP1 partly inhibited CQ-CDDP-induced G2/M arrest, demonstrating that knockdown of p21WAF1/CIP1 overcame the cytotoxic effect of the CQ-CDDP combination. Ectopic expression of p21WAF1/CIP1 in CDDP-treated ATG5-shRNA/A2780-CP20 cells increased expression of γH2AX and caspase-3 activity, demonstrating increased DNA damage and cell death. The inhibition of autophagy by ATG5-shRNA demonstrated similar results upon CDDP treatment, except p21WAF1/CIP1 expression. In an in vivo efficacy study, the CQ-CDDP combination significantly decreased tumor weight and increased expression of γH2AX and p21WAF1/CIP1 in A2780-CP20 orthotopic xenografts and a drug-resistant patient-derived xenograft model of EOC compared with controls. These results demonstrated that CQ increases cytotoxicity in combination with CDDP by inducing lethal DNA damage by induction of p21WAF1/CIP1 expression and autophagy inhibition in CDDP-resistant EOC.
Assuntos
Autofagia/genética , Cloroquina/uso terapêutico , Inibidor de Quinase Dependente de Ciclina p21/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Regulação para Cima/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Autofagia/efeitos dos fármacos , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/genética , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Epithelial ovarian cancer (EOC) is a fatal gynecologic malignancy that is usually treated with chemotherapy after surgery. However, patients who receive chemotherapy experience severe side effects because of the inherent toxicity and high dose of chemotherapeutics. To overcome these issues, we suggest a combination therapeutic strategy using liposomes encapsulating linalool nanoemulsions (LN-NEs) and doxorubicin (DOX), a chemotherapeutic drug, to increase their synergistic antitumor efficacy and reduce the incidence of side effects from chemotherapeutics for EOC. METHODS: The physical properties of LN-NE-DOX-liposomes were characterized by light scattering with a particle size analyzer. Cell viability was determined by MTT assay. Therapeutic efficacy was evaluated in a mouse HeyA8 EOC tumor model of ovarian carcinoma. Additionally, biochemical toxicity was analyzed for levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) using BALB/c nude mice. RESULTS: The size of the liposomes encapsulating LN-NEs and DOX (LN-NE-DOX-liposomes) was 267.0 ± 4.6 nm, with a loading efficiency of 55.1 ± 3.1% and 27.2 ± 0.9% for linalool and DOX, respectively. Cell viability after treatment with LN-NE-DOX-liposomes was significantly decreased compared to that of cells treated with DOX liposomes, and apoptosis was significantly increased. Additionally, LN-NE-DOX-liposomes significantly inhibited HeyA8 EOC tumor growth compared to that of the control (p < 0.01) and DOX-liposome-treated groups (p < 0.05), while decreasing cell proliferation (Ki67) and microvessel density (CD31), and promoting apoptosis (caspase-3) compared to the control (p < 0.05). Moreover, the liposomal formulations induced no significant differences in biochemical toxicity (AST, ALT, and BUN) compared to healthy control mice, indicating that the liposomal formulations showed no overt toxicity in mice. CONCLUSION: This study demonstrates that the production of LN-NE-DOX-liposomes is a pivotal approach for EOC treatment, suggesting a novel combination therapeutic strategy.
Assuntos
Monoterpenos Acíclicos/uso terapêutico , Carcinoma Epitelial do Ovário/tratamento farmacológico , Doxorrubicina/análogos & derivados , Portadores de Fármacos/química , Monoterpenos Acíclicos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Doxorrubicina/uso terapêutico , Emulsões/química , Feminino , Humanos , Lipossomos/química , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Tamanho da Partícula , Polietilenoglicóis/uso terapêutico , Resultado do TratamentoRESUMO
Mammalian sterile 20-like kinase 1/2 (MST1/2) plays an important role in cell growth and apoptosis and functions as a tumor suppressor. Previously, we showed that MST2 overexpression activates Estrogen receptor alpha (ERα) in human breast cancer MCF-7 cells in the absence of a ligand. Here, we examined the role of MST2 in the growth of ER-positive MCF-7 cells. Cell cycle, apoptosis, and mammosphere formation assay method were implemented to detect the biological effects of MST2 ablation on the growth of MCF-7 cells in vitro. The effect of MST2-siRNA on MCF-7 cells tumor growth in vivo was studied in tumor-bearing mouse model. Kaplan-Meier plotter analysis was used to determine the effect of MST2 on overall survival in breast cancer patients. MST2 overexpression increased cell viability marginally. The ablation of MST2 using siRNA dramatically suppressed the viability of the MCF-7 cells, but not ER-negative MDA-MB-231 breast cancer cells. Furthermore, MST2 knockdown increased caspase-dependent apoptosis and led to decreased mammosphere formation. Treatment of MCF-7 tumor-bearing mice with MST2 siRNA significantly inhibited tumor growth. The tumor weight was reduced further when tamoxifen was added. Patients with ER-positive breast cancer with low MST2 expression had better overall survival than did those with high MST2 expression in Kaplan-Meier survival analyses using public datasets. Our results provide new insight into the role of MST2, a key component of the Hippo signaling pathway, in mediating breast cancer progression.