RESUMO
Cholangiocarcinoma (CCA) is an aggressive malignant tumor of bile duct epithelia. Recent evidence suggests the impact of cancer stem cells (CSC) on the therapeutic resistance of CCA; however, the knowledge of CSC in CCA is limited due to the lack of a CSC model. In this study, we successfully established a stable sphere-forming CCA stem-like cell, KKU-055-CSC, from the original CCA cell line, KKU-055. The KKU-055-CSC exhibits CSC characteristics, including: (1) the ability to grow stably and withstand continuous passage for a long period of culture in the stem cell medium, (2) high expression of stem cell markers, (3) low responsiveness to standard chemotherapy drugs, (4) multilineage differentiation, and (5) faster and constant expansive tumor formation in xenograft mouse models. To identify the CCA-CSC-associated pathway, we have undertaken a global proteomics and functional cluster/network analysis. Proteomics identified the 5925 proteins in total, and the significantly upregulated proteins in CSC compared with FCS-induced differentiated CSC and its parental cells were extracted. Network analysis revealed that high mobility group A1 (HMGA1) and Aurora A signaling through the signal transducer and activator of transcription 3 pathways were enriched in KKU-055-CSC. Knockdown of HMGA1 in KKU-055-CSC suppressed the expression of stem cell markers, induced the differentiation followed by cell proliferation, and enhanced sensitivity to chemotherapy drugs including Aurora A inhibitors. In silico analysis indicated that the expression of HMGA1 was correlated with Aurora A expressions and poor survival of CCA patients. In conclusion, we have established a unique CCA stem-like cell model and identified the HMGA1-Aurora A signaling as an important pathway for CSC-CCA.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Camundongos , Animais , Proteína HMGA1a , Colangiocarcinoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Bradykinin has a wide variety of physiological functions, including vasodilation and blood pressure reduction. However, the physiological roles of bradykinin are not fully understood. We used the CRISPR/Cas9 method to generate BKdelK1 and BKdelK2 mutant mice, targeting the BK portion of mouse kininogen1 and kininogen2 genes, respectively. The BKdelK1 and BKdelK2 mutant mice had about 50% reductions in plasma low molecular weight kininogen and trypsin-released BK, compared to wild mice. Both BKdelK1 and BKdelK2 mice had significantly elevated systolic blood pressure compared to WT mice. These results suggest that plasma LKNG is a source of KNG in the vascular kallikrein-kinin system and contributes to maintaining lower systolic blood pressure.
Assuntos
Bradicinina , Hipertensão , Camundongos , Animais , Hipertensão/genética , Pressão Sanguínea , CalicreínasRESUMO
Primary effusion lymphoma (PEL) is a rare, aggressive B cell non-Hodgkin's lymphoma of the body cavities with malignant effusions. The prognosis is poor, and no optimal treatment has been established. CD38 is a type II transmembrane glycoprotein known to overexpress in multiple myeloma (MM). Daratumumab (DARA), a human CD38-targeting monoclonal antibody (mAb), is approved for MM treatment. In this study, we found expression of CD38 on PEL cells and assessed the anti-PEL activity of DARA. We found that both KHYG-1 and N6 (CD16-transfected KHYG-1) NK cell lines showed direct killing activity against PEL cells with induction of CD107a, and NK-mediated cytotoxicity by N6NK (CD16+) cells increased with DARA treatment. We confirmed direct NK activity and antibody-dependent cell cytotoxicity (ADCC) by expanded NK cells, indicating that DARA has high ADCC activity. We elucidated the antibody-dependent cell phagocytosis (ADCP) by using human monocyte-derived macrophages (MDMs) and mouse peritoneal macrophages. DARA also showed potent complement-dependent cytolysis (CDC) toward PEL. DARA also induced PEL cell death in the presence of a cross-linking antibody. Moreover, treatment with DARA inhibited tumor growth in a PEL xenograft mouse model. These results provide preclinical evidence that Ab targeting of CD38 could be an effective therapeutic strategy for the treatment of PEL.
Assuntos
Antineoplásicos , Linfoma de Efusão Primária , Mieloma Múltiplo , ADP-Ribosil Ciclase 1 , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/uso terapêutico , Humanos , Linfoma de Efusão Primária/tratamento farmacológico , CamundongosRESUMO
Primary effusion lymphoma (PEL) is a rare aggressive B-cell non-Hodgkin's lymphoma with no optimal treatment. Signaling lymphocytic activation molecule-F7 (SLAMF7, CD319), a type I transmembrane glycoprotein highly expressed in multiple myeloma (MM), represents a promising target for mAb-based immunotherapy. SLAMF7 also expresses on several hematopoietic lineages including NK cells. Elotuzumab (Elo), a humanized antibody targeting SLAMF7, is approved by FDA for MM treatment. In this study, we analyzed the expression of SLAMF7 on seven PEL cell lines. All PEL cells and NK cells showed high expression of SLAMF7. NK cells were enriched from PBMCs of healthy donors by MACS and expanded by co-culturing with MHC-class I negative K562 cells in the presence of IL-2 and IL-15. Expanded NK cells showed direct killing, and Elo demonstrated potent ADCC against PEL in an Effector:Target (E:T) dependent manner. Surface expression of CD107a on NK cells also increased in the process of ADCC. We also examined SLAMF7 expression of NK subpopulations and found that the CD56+CD16+ NK subpopulation demonstrated the highest SLAMF7 expression. Full-length-Elo but not F(ab')2-Elo exerts direct engagement to the expressing SLAMF7 on NK cells, promotes CD107a expression, and further augments NK cytotoxicity toward PEL. Elo enhanced survival of PEL-bearing immunodeficient mice with adoptive transfer of human NK cells. Taken together, our results show that NK cells play roles in PEL killing, and Elo causes ADCC/SLAMF7 ligation to boost NK cytotoxicity against PEL, offering promising preclinical evidence of Elo as a therapeutic monoclonal antibody treatment for PEL.
Assuntos
Antineoplásicos , Linfoma de Efusão Primária , Mieloma Múltiplo , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/uso terapêutico , Humanos , Células Matadoras Naturais , Linfoma de Efusão Primária/tratamento farmacológico , Linfoma de Efusão Primária/metabolismo , Camundongos , Mieloma Múltiplo/tratamento farmacológicoRESUMO
BACKGROUND: Tumor suppressor CYLD dysfunction by loss of its expression, triggers malignant transformation, especially drug resistance and tumor invasion/metastasis. Although loss of CYLD expression is significantly associated with poor prognosis in a large variety of tumors, no clinically-effective treatment for CYLD-negative cancer patients is available. METHODS: We focused on oral squamous cell carcinoma (OSCC), and sought to develop novel therapeutic agents for CYLD-negative cancer patients with poor prognosis. CYLD-knockdown OSCC cells by using CYLD-specific siRNA, were used to elucidate and determine the efficacy of novel drug candidates by evaluating cell viability and epithelial-mesenchymal transition (EMT)-like change. Therapeutic effects of candidate drug on cell line-derived xenograft (CDX) model and usefulness of CYLD as a novel biomarker using patient-derived xenograft (PDX) model were further investigated. RESULTS: CYLD-knockdown OSCC cells were resistant for all currently-available cytotoxic chemotherapeutic agents for OSCC, such as, cisplatin, 5-FU, carboplatin, docetaxel, and paclitaxel. By using comprehensive proteome analysis approach, we identified epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, played key roles in CYLD-knockdown OSCC cells. Indeed, cell survival rate in the cisplatin-resistant CYLD-knockdown OSCC cells was markedly inhibited by treatment with clinically available EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib. In addition, gefitinib was significantly effective for not only cell survival, but also EMT-like changes through inhibiting transforming growth factor-ß (TGF-ß) signaling in CYLD-knockdown OSCC cells. Thereby, overall survival of CYLD-knockdown CDX models was significantly prolonged by gefitinib treatment. Moreover, we found that CYLD expression was significantly associated with gefitinib response by using PDX models. CONCLUSIONS: Our results first revealed that EGFR-targeted molecular therapies, such as EGFR-TKIs, could have potential to be novel therapeutic agents for the CYLD-negative OSCC patients with poor prognosis.
RESUMO
CD99 is a surface molecule expressed on various cell types including cancer cells. Expression of CD99 on multiple myeloma is associated with CCND1-IGH fusion/t(11;14). This translocation has been reported to be a genetic hallmark of mantle cell lymphoma (MCL). MCL is characterized by overexpression of cyclin D1 and high tumor proliferation. In this study, high expression of CD99 on MCL cell lines was confirmed. Our generated anti-CD99 monoclonal antibody (mAb), termed MT99/3, exerted potent antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities against mantle B-cell lymphoma without direct cytotoxic effects. The anti-tumor activities of mAb MT99/3 were more effective in MCL than in other B-cell lymphomas. Moreover, in a mouse xenograft model using Z138 MCL cell line, treatment of mAb MT99/3 reduced tumor development and growth. Our study indicated that mAb MT99/3 is a promising immunotherapeutic candidate for mantle cell lymphoma therapy.
Assuntos
Antígeno 12E7/metabolismo , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/farmacologia , Ativação do Complemento , Linfoma de Célula do Manto/terapia , Antígeno 12E7/antagonistas & inibidores , Antígeno 12E7/imunologia , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acanthoic acid (AA) is an active substance that is extracted from Croton oblongifolius Roxb., a traditional plant in Thailand. The antiinflammatory effect of AA on NF-κB pathway has been exclusively reported, however, its anticancer effect is still lacking. PEL is a B cell lymphoma that is mostly found in HIV patients. The prognosis and progression of PEL patients are terribly poor with a median survival time less than 6 months, so the new effective treatment is urgently needed. In this study, we found that AA effectively inhibited PEL cell proliferation with IC50s at 120-130 µM in well-representative cells, while the IC50s of AA in PBMC were higher (>200 µM). AA increased percentages of Annexin V/PI positive cells, whereas adding of caspase inhibitor (Q-VD-OPh) prevented AA-induced cell death. The antiapoptotic protein, c-FLIP, was downregulated by AA which leading to the activation of caspase-8 and -3. Combination of AA and TRAIL dramatically enhanced apoptotic cell death. In PEL xenograft model, AA at the dose of 250 mg/kg effectively inhibited PEL tumor growth without detectable toxicities assessed by mice weight and appearance.
Assuntos
Diterpenos , Infecções por HIV , Linfoma de Efusão Primária , Animais , Apoptose , Linhagem Celular Tumoral , Humanos , Leucócitos Mononucleares , Linfoma de Efusão Primária/tratamento farmacológico , CamundongosRESUMO
Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte-monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.
Assuntos
Basigina/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Colangiocarcinoma/patologia , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Fosforilação/fisiologiaRESUMO
Long interspersed element-1 (LINE-1, L1) composes â¼17% of the human genome. However, genetic interactions between L1 and human immunodeficiency virus type 1 (HIV-1) remain poorly understood. In this study, we found that HIV-1 suppresses L1 retrotransposition. Notably, HIV-1 Vpr strongly inhibited retrotransposition without inhibiting L1 promoter activity. Since Vpr is known to regulate host cell cycle, we examined the possibility whether Vpr suppresses L1 retrotransposition in a cell cycle dependent manner. We showed that the inhibitory effect of a mutant Vpr (H71R), which is unable to arrest the cell cycle, was significantly relieved compared with that of wild-type Vpr, suggesting that Vpr suppresses L1 mobility in a cell cycle dependent manner. Furthermore, a host cell cycle regulator p21Waf1 strongly suppressed L1 retrotransposition. The N-terminal kinase inhibitory domain (KID) of p21 was required for this inhibitory effect. Another KID-containing host cell cycle regulator p27Kip1 also strongly suppressed L1 retrotransposition. We showed that Vpr and p21 coimmunoprecipitated with L1 ORF2p and they suppressed the L1 reverse transcriptase activity in LEAP assay, suggesting that Vpr and p21 inhibit ORF2p-mediated reverse transcription. Altogether, our results suggest that viral and host cell cycle regulatory machinery limit L1 mobility in cultured cells.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/fisiologia , HIV-1/fisiologia , Elementos Nucleotídeos Longos e Dispersos/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/fisiologia , Ciclo Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Endonucleases/metabolismo , Genes Reporter , Genes vpr , HIV-1/genética , Humanos , Domínios Proteicos , Proteínas/metabolismo , Interferência de RNA , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Gênica , Vírion/metabolismoRESUMO
Patient-derived xenograft (PDX) models can be created with the transplantation of cancerous cells or tissues from patients' primary tumors into immunodeficient mice. PDXs are now in the spotlight as more accurate human cancer models compared with mouse tumor and human cancer cell lines transplanted into mice. PDX technology leads to breakthroughs with the introduction of novel, highly immunodeficient mice such as NOG (NOD/Scid/IL2Rγnull), NSG (NOD/Scid/IL2Rγnull), and NOJ (NOD/Scid/Jak3null) mice. Xenograft efficiency differs by type of tumor, site of implantation, and tumor aggressiveness. Subcutaneous implantation is a standard method for PDX, and renal capsule or orthotropic implantation improves the efficiency. Despite positive test results in animal cancer models, significant numbers of novel drug candidates fail in clinical trials because conventional animal models such as murine tumor and human cancer cell line transplantation models do not always reflect the nature of human cancers. Since PDXs conserve the original tumor characteristics such as heterogeneous histology, clinical biomolecular signatures, malignant phenotypes and genotypes, tumor architecture, and tumor vasculature, they are currently believed to offer relevant predictive insights into clinical outcomes when evaluating the efficacy of novel cancer therapies. PDX banks with integrated genomic signatures are now established in many organizations including pharmaceutical companies. These PDX databases are becoming powerful tools for advancing precision cancer medicine.
Assuntos
Medicina de Precisão , Animais , Modelos Animais de Doenças , Humanos , Hospedeiro Imunocomprometido , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Autophagy plays a crucial role in cancer cell survival and the inhibition of autophagy is attracting attention as an emerging strategy for the treatment of cancer. Chloroquine (CQ) is an anti-malarial drug, and is also known as an inhibitor of autophagy. Recently, it has been found that CQ induces cancer cell death through the inhibition of autophagy; however, the underlying mechanism is not entirely understood. In this study, we identified the role of CQ-induced cancer cell death using Primary Effusion Lymphoma (PEL) cells. We found that a CQ treatment induced caspase-dependent apoptosis in vitro. CQ also suppressed PEL cell growth in a PEL xenograft mouse model. We showed that CQ activated endoplasmic reticulum (ER) stress signal pathways and induced CHOP, which is an inducer of apoptosis. CQ-induced cell death was significantly decreased by salbrinal, an ER stress inhibitor, indicating that CQ-induced apoptosis in PEL cells depended on ER stress. We show here for the first time that the inhibition of autophagy induces ER stress-mediated apoptosis in PEL cells. Thus, the inhibition of autophagy is a novel strategy for cancer chemotherapy.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cloroquina/administração & dosagem , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Linfoma de Efusão Primária/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/fisiologia , Feminino , Humanos , Linfoma de Efusão Primária/fisiopatologia , Camundongos , Camundongos NusRESUMO
UNLABELLED: The transcription factor NF-κB is important for HIV-1 transcription initiation in primary HIV-1 infection and reactivation in latently HIV-1-infected cells. However, comparative analysis of the regulation and function of NF-κB in latently HIV-1-infected cells has not been done. Here we show that the expression of IκB-α, an endogenous inhibitor of NF-κB, is enhanced by latent HIV-1 infection via induction of the host-derived factor COMMD1/Murr1 in myeloid cells but not in lymphoid cells by using four sets of latently HIV-1-infected cells and the respective parental cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during Toll-like receptor ligand and tumor necrosis factor alpha treatment and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the phosphoinositol 3-kinase (PI3K)-JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Our findings indicate that COMMD1 induction is the NF-κB inhibition mechanism in latently HIV-1-infected cells that contributes to innate immune deficiency and reinforces HIV-1 latency. Thus, COMMD1 might be a double-edged sword that is beneficial in primary infection but not beneficial in latent infection when HIV-1 eradication is considered. IMPORTANCE: HIV-1 latency is a major barrier to viral eradication in the era of combination antiretroviral therapy. In this study, we found that COMMD1/Murr1, previously identified as an HIV-1 restriction factor, inhibits the proteasomal degradation of IκB-α by increasing the interaction with IκB-α in latently HIV-1-infected myeloid cells. IκB-α protein was stabilized by COMMD1, which attenuated NF-κB signaling during the innate immune response and enhanced HIV-1 latency in latently HIV-1-infected cells. Activation of the PI3K-JAK pathway is involved in COMMD1 induction in latently HIV-1-infected cells. Thus, the host-derived factor COMMD1 is beneficial in suppressing primary infection but enhances latent infection, indicating that it may be a double-edged sword in HIV-1 eradication.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas I-kappa B/metabolismo , Latência Viral , Linhagem Celular , Humanos , Inibidor de NF-kappaB alfaRESUMO
Cholangiocarcinoma (CCA) is a unique liver cancer subtype with an increasing incidence globally. The lack of specific symptoms and definite diagnostic markers results in a delayed diagnosis and disease progression. Systemic chemotherapy is commonly selected for advanced CCA even though its advantages remain unknown. Targeted therapy, especially anti-vascular endothelial growth factor (VEGF) therapy, is promising for CCA; however, improvements in the therapeutic regimen are necessary to overcome subsequent resistance. We demonstrated VEGF expression was higher in CCA cell lines than in other liver cancer cells. Secreted VEGFs played roles in the induction of peri- and intra-tumoral vascularization. VEGF neutralization by bevacizumab effectively reduced tumor growth, mainly through the suppression of angiogenesis; however, increases in the expression of hypoxia-inducible factor 1α (HIF1α) and HIF1α-responsive genes (such as VEGF, VEGFR1, VEGFR2, carbonic anhydrase (CA) IX and CAXII) indicated the potential for subsequent therapeutic resistance. Supplementation with a carbonic anhydrase inhibitor, acetazolamide, enhanced the anti-CCA effects of bevacizumab. Anti-angiogenesis and anti-proliferation were observed with the combination treatment. These results suggested a novel treatment strategy to overcome anti-angiogenesis resistance and the importance of "induced essentiality" in the treatment of CCA.
Assuntos
Acetazolamida/farmacologia , Inibidores da Angiogênese/farmacologia , Bevacizumab/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Colangiocarcinoma/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Hep G2 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Natural killer cells, a critical component of the innate immune system, eradicate both virus-infected cells and tumor cells through cytotoxicity and secretion of cytokines. Human NK cell research has largely been based on in vitro studies because of the lack of appropriate animal models. In this study, a selective proliferation model of functional human NK cells was established in NOD/SCID/Jak3(null) (NOJ) mice transplanted with peripheral blood mononuclear cells (PBMC) and K562 cells. The antiviral effects of NK cells were evaluated by challenging this mouse model with HIV-1. The percentage of intracellular p24(+) T cells and the amount of plasma p24 was decreased compared with NOJ mice transplanted with PBMC. Our findings indicate that NK cells have an anti-HIV-1 effect through direct cytotoxicity against HIV-1-infected cells. These mice provide an important model for evaluating human NK function against human infectious diseases such as HIV-1 and malignancies.
Assuntos
Proliferação de Células , Modelos Animais de Doenças , Infecções por HIV/imunologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Citotoxicidade Imunológica , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Imunidade Inata , Imunoterapia Adotiva/métodos , Células K562 , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T/imunologiaRESUMO
Membrane fusion between host cells and HIV-1 is the initial step in HIV-1 infection, and plasma membrane fluidity strongly influences infectivity. In the present study, we demonstrated that GUT-70, a natural product derived from Calophyllum brasiliense, stabilized plasma membrane fluidity, inhibited HIV-1 entry, and down-regulated the expression of CD4, CCR5, and CXCR4. Since GUT-70 also had an inhibitory effect on viral replication through the inhibition of NF-κB, it is expected to be used as a dual functional and viral mutation resistant reagent. Thus, these unique properties of GUT-70 enable the development of novel therapeutic agents against HIV-1 infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Cumarínicos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Internalização do Vírus/efeitos dos fármacos , Antígenos CD4/genética , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Fluidez de Membrana/efeitos dos fármacos , Receptores CCR5/genética , Receptores CXCR4/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Linfócitos T/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti-apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF-α and IL-6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL-12 and anti-inflammatory cytokine IL-10. Interestingly, treatment with 5 µg/mL, but not 25 µg/mL, of APC significantly enhanced production of IL-10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co-stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti-inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF-α and/or IL-6 production.
Assuntos
Células Dendríticas/imunologia , Proteína C/imunologia , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Humanos , Interleucina-10/imunologia , Interleucina-12/imunologia , Interleucina-6/imunologia , Monócitos/citologia , Monócitos/imunologiaRESUMO
Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-ß-cyclodextrin (FA-M-ß-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-ß-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-ß-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-ß-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-ß-cyclodextrin, FA-M-ß-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-ß-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-ß-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-ß-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.
Assuntos
Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Transportadores de Ácido Fólico/metabolismo , Ácido Fólico/uso terapêutico , Melanoma/tratamento farmacológico , beta-Ciclodextrinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Ciclodextrinas/farmacologia , Ciclodextrinas/uso terapêutico , Combinação de Medicamentos , Endocitose , Ácido Fólico/metabolismo , Ácido Fólico/farmacologia , Humanos , Melanoma/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Fagossomos/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and ß2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.
Assuntos
Apoptose , Lactalbumina/farmacologia , Neoplasias/tratamento farmacológico , Ácidos Oleicos/farmacologia , Animais , Antineoplásicos/farmacologia , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Cabras , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Neoplasias/patologia , Ligação ProteicaRESUMO
Primary effusion lymphoma (PEL) is a subtype of aggressive and chemotherapy-resistant non-Hodgkin lymphoma that occurs predominantly in patients with advanced AIDS. In this study, we examined the antitumor activity of methyl-ß-cyclodextrin (M-ß-CyD) in vitro and in vivo. M-ß-CyD quickly induced caspase-dependent apoptosis in PEL cells via cholesterol depletion from the plasma membrane. In a PEL xenograft mouse model, M-ß-CyD significantly inhibited the growth and invasion of PEL cells without apparent adverse effects. These results strongly suggest that M-ß-CyD has the potential to be an effective antitumor agent against PEL.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Colesterol/química , Linfoma de Efusão Primária/tratamento farmacológico , Microdomínios da Membrana/química , beta-Ciclodextrinas/farmacologia , Animais , Antineoplásicos/química , Sobrevivência Celular , Meios de Cultura , Feminino , Hemólise , L-Lactato Desidrogenase/sangue , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Ensaios Antitumorais Modelo de Xenoenxerto , beta-Ciclodextrinas/químicaRESUMO
The anti-HIV-1 activity of cepharanthine (CEP), a natural product derived from Stephania cepharantha Hayata, was evaluated. CEP stabilized plasma membrane fluidity and inhibited HIV-1 envelope-dependent cell-to-cell fusion of HIV-1-infected cells as well as cell-free infection. It is suggested that CEP inhibited the HIV-1 entry process by reducing plasma membrane fluidity, and the plasma membrane is therefore an identical target to prevent viral infection.