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New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is aberrantly expressed in multiple myeloma (MM) patients, however, its role remains largely unknown. The present study aimed to investigate the effect of NY-ESO-1 knockdown on MM impact and provide evidence for targeting treatment of MM. Human MM U266 cells were infected with lentivirus-based small hairpin RNA-targeting NY-ESO-1 (LV-shNY-ESO-1). Cellular proliferation, colony-forming, migration, and invasion assays were employed. The expressions of cell cycle and epithelial-mesenchymal transition (EMT)-related molecules, MM growth, and mouse osteolytic lesions were evaluated. The results showed that the LV-shNY-ESO-1-U266 cells had a lower expression of NY-ESO-1 and a higher expressions of p21 and E-cadherin, and a weaker abilities of colony formation, drug-resistant to adriamycin, migration, and invasion than those of the control cells. Importantly, the knockdown of NY-ESO-1 inhibited significantly the U266 cell-induced MM growth and osteolytic lesions along with increasing the expressions of E-cadherin, p21, and p53 in mice challenged with LV-shNY-ESO-1-U266 cells. Collectively, our findings demonstrate that knockdown of NY-ESO-1 suppressed the U266 cell-induced MM growth and osteolytic lesions by inhibition of the MMs cell cycle and EMT. The NY-ESO-1 knockdown may be considered for future clinical trials in MM.
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Proliferação de Células/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago/genética , Mieloma Múltiplo/genética , Animais , Biomarcadores Tumorais/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , New YorkRESUMO
Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44+ CD24- and CD44+ CD24- ESA+ BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44+ CD24- and CD44+ CD24- ESA+ BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3'-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44+ CD24- ESA+ BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.
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Aldeído Oxirredutases/genética , Neoplasias da Mama/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Aldeído Desidrogenase/metabolismo , Animais , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
BACKGROUND: Breast cancer stem cells (BCSCs) are typically seed cells of breast tumor that initiate and maintain tumor growth. MiR-7, as a cancer inhibitor, decreases the BCSC subset and inhibits tumor progression through mechanisms that remain unknown. METHODS: We examined miR-7 expression in breast cancer and developed a BCSC-driven xenograft mouse model, to evaluate the effects of miR-7 overexpression on the decrease of the BCSC subset in vitro and in vivo. In addition, we determined how miR-7 decreased the BCSC subset by using the ALDEFLUOR, lentivirus infection, dual-luciferase reporter, and chromatin immunoprecipitation-PCR assays. RESULTS: MiR-7 was expressed at low levels in breast cancer tissues compared with normal tissues, and overexpression of miR-7 directly inhibited lncRNA XIST, which mediates the transcriptional silencing of genes on the X chromosome, and reduced epithelium-specific antigen (ESA) expression by increasing miR-92b and inhibiting slug. Moreover, miR-7 suppressed CD44 and ESA by directly inhibiting the NF-κB subunit RELA and slug in breast cancer cell lines and in BCSC-driven xenografts, which confirmed the antitumor activity in mice injected with miR-7 agomir or stably infected with lenti-miR-7. CONCLUSIONS: The findings from this study uncover the molecular mechanisms by which miR-7 inhibits XIST, modulates the miR-92b/Slug/ESA axis, and decreases the RELA and CD44 expression, resulting in a reduced BCSC subset and breast cancer growth inhibition. These findings suggest a potentially targeted treatment approach to breast cancer.
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Neoplasias da Mama/prevenção & controle , Molécula de Adesão da Célula Epitelial/metabolismo , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Fatores de Transcrição da Família Snail/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Molécula de Adesão da Célula Epitelial/genética , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/patologia , RNA Longo não Codificante/genética , Fatores de Transcrição da Família Snail/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Genetically modified cells have been shown to be one of the most effective tumor vaccine strategies. However, in many cases, such as in melanoma, induction of a potent immune responses against the disease still remains a major challenge. Thus, novel strategies to reinforce tumor vaccine efficacy are needed. Using microRNA (miR) and Zinc-finger E-box binding homeobox (ZEB) have received much attention for potentially regulating tumor progression. To elicit a potent antitumor efficacy against melanoma, we used tumor vaccine in combination with miR200c overexpression or ZEB1 knockdown to assess the efficacy of treatment of murine melanoma. METHODS: B16F10 cell vaccine expressing interleukin 21 (IL-21) in the glycosylpho- sphatidylinositol (GPI)-anchored form (B16F10/GPI-IL-21) were developed. The vaccine was immunized into mice challenged by B16F10 cells or B16F10 cells stably transduced with lentiviral-miR200c (B16F10/miR200c) or transfected with the ZEB1-shRNA recombinant (B16F10/shZEB1) or the B16F10/GPI-IL-21 vaccine. The immune responses, tumorigenicity and lung metastasis in mice were evaluated, respectively. RESULTS: The vaccination with B16F10/GPI-IL-21 markedly increased the serum cytokine levels of IFN-γ, TNF-α, IL-4 and decreased TGF-ß level as well as augmented the cytotoxicity of splenocytes in immunized mice compared with control mice. In addition, the tumor vaccine B16F10/GPI-IL-21 significantly inhibited the tumor growth and reduced counts of lung metastases in mice challenged by B16F10/GPI-IL-21, B16F10/shZEB1 and B16F10/miR200c respectively compared with the control mice challenged by B16F10 cells. The efficacy mechanisms may involve in reinforcing immune responses, increasing expression of miR200c, E-cadherin and SMAD-7 and decreasing expression of TGF-ß, ZEB1, Vimentin and N-cadherin in tumor tissues from the immunized mice. CONCLUSIONS: These results indicate that the tumor vaccine B16F10/GPI-IL-21 in combination with miR200c overexpression or ZEB1 knockdown effectively inhibited melanoma growth and metastasis a murine model. Such a strategy may, therefore, be used for the clinical trials.
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Vacinas Anticâncer/imunologia , Regulação Neoplásica da Expressão Gênica , Glicosilfosfatidilinositóis/imunologia , Proteínas de Homeodomínio/genética , Interleucinas/imunologia , Fatores de Transcrição Kruppel-Like/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , MicroRNAs/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/sangue , Citotoxicidade Imunológica/genética , Transição Epitelial-Mesenquimal/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Imunização , Fatores de Transcrição Kruppel-Like/metabolismo , Melanoma Experimental/sangue , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Metástase Neoplásica/genética , Metástase Neoplásica/imunologia , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Baço/metabolismo , Baço/patologia , Transdução Genética , Transfecção , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Zinc-finger E-box binding homeobox 1 (ZEB1) is a master regulator of epithelial-mesenchymal transition (EMT) and has been implicated in primary epithelial cancer biological processes, such as invasion and metastasis. However, the role of ZEB1 in progression of melanoma and cancer stem cells (CSCs) remains obscure. In this study, the recombinant plasmids of t3 shRNAs targeting mouse ZEB1 were constructed and transfected into melanoma B16F10 cells. The stable transfected cells were selected and the characteristics of ZEB1 downregulated B16F10 cells was assessed. The tumourigenicity of CD44(+) CD133(+) CSCs isolated from B16F10 cells stably transfected with the ZEB1-shRNA2 recombinant was also assessed. ZEB1-shRNAs B16F10 showed a lower expression of ZEB1 and vimentin, weaker migration, invasiveness, colony forming, and proliferation, and a lower tumourigenicity than the control cells. The tumourigenicity of the ZEB1-shRNA2 CD44(+) CD133(+) CSCs was also inhibited. In conclusion, ZEB1-shRNA2-mediated downregulation of ZEB1 expression in B16F10 cells and CSCs is involved in the inhibition of the EMT process. ZEB1 may be a potential target in melanoma targeted.
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Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Células-Tronco Neoplásicas/metabolismo , Vimentina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Transformação Celular Neoplásica , Regulação para Baixo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/citologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
OBJECTIVE: To investigate the effect and mechanism of B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine on pulmonary metastasis in mouse model of melanoma. METHODS: Twelve 8-week old female C57BL/6 mice were used in this study. The mice were injected with wild-type B16F10 cells through tail vein after immunization with B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine, and the pulmonary metastasis was observed. The CD4(+) and CD8(+) T cells were isolated by magnetic activated cell sorting, and then used for the detection of CFSE/7-AAD cytotoxicity by flow cytometry. Serum from the mice immunized with tumor-cell vaccine was used to detect IFN-γ expression by ELISA. The expression of TGF-ß2, ZEB1, E-cadherin, and N-cadherin of tumor tissues was detected by RT-PCR and immunofluorescence, respectively. RESULTS: The mice vaccinated with B16F10-ESAT-6-gpi/IL-21 had significantly fewer nodules in the lung and lower lung weight [(285.8 ± 19.01) mg vs. (406.3 ± 27.12) mg], with lower levels of TGF-ß2, ZEB1 and N-cadherin proteins but higher level of E-cadherin protein within the tumor tissue, as compared with the control mice. Meanwhile, the immunized mice had significantly increased CD8(+) T cell killing activity [(42.62 ± 3.465)% vs. (22.29 ± 1.804)%] and IFN-γ expression level [(55.200 ± 7.173) pg/ml vs. (6.435 ± 1.339) pg/ml] over the control mice. CONCLUSIONS: The B16F10-ESAT-6-gpi/IL-21 vaccine can inhibit the metastasis of melanoma in the lung in vaccinated melanoma-bearing mice. This inhibitory effect is associated with CD8(+) T cell immune response and a higher level of IFN-γ, which may influence on the mesenchymal-epithelial transition of tumor cells.
Assuntos
Vacinas Anticâncer/imunologia , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/secundário , Melanoma/patologia , Animais , Linfócitos T CD8-Positivos/imunologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Interferon gama/metabolismo , Interleucinas/imunologia , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Tamanho do Órgão , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Advanced ovarian cancer (OC) patients have limited benefit from current relevant cytotoxic and targeted therapies following debulking surgery. Therefore, new therapeutic strategies are in urgent need. Immunotherapy has shown great potential in tumor treatment, especially in tumor vaccine development. The study objective was to evaluate the immune effects of cancer stem cells (CSCs) vaccines on OC. The CD44+CD117+CSCs were isolated from human OC HO8910 and SKOV3 cells using the magnetic cell sorting system; the cancer stem-like cells were selected from murine OC ID8 cell by no-serum formed sphere culture. The CSC vaccines were prepared by freezing and thawing these CSCs, which were then injected into mice followed by challenging the different OC cells. The in vivo antitumor efficacy of CSC immunization revealed the vaccines were capable of significantly provoking immune responses to autologous tumor antigens in vaccinated mice as the mice were found to have markedly inhibited tumor growth, prolonged survival, and decreased CSC counts in OC tissues when compared to mice without the CSC vaccination. The in vitro cytotoxicities of immunocytes toward SKOV3, HO8910 and ID8 cells indicated a significant killing efficacy compared with the controls. However, the antitumor efficacy was remarkably reduced whilst the mucin-1 expression in CSC vaccines was down-regulated by small interfering RNA. Overall, findings from this study provided the evidence that has deepened our understanding of CSC vaccine immunogenicity and anti-OC efficacy, particularly for the role of dominant antigen mucin-1. It is possible to turn the CSC vaccine into an immunotherapeutic approach against ovarian cancer.
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Vacinas Anticâncer , Neoplasias Ovarianas , Humanos , Camundongos , Animais , Feminino , Mucina-1/metabolismo , Neoplasias Ovarianas/metabolismo , Vacinação , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular TumoralRESUMO
Background: Immunotherapy-based approaches are important breakthroughs with potential treatment benefits for melanoma patients. Mucin 1 (MUC1) is significantly upregulated in melanoma relative to normal cells. It has been reported that MUC1 influences cancer cell proliferation, apoptosis, invasion, and metastasis.The study aimed to explore the effect of MUC1 knockdown on the biological characteristics of the melanoma cell line B16F10 and evaluate whether MUC1 is an effective candidate target antigen for melanoma vaccine development. Methods: First, lentiviral vector-mediated short hairpin RNA (shRNA) was used to knockdown MUC1 in B16F10 cells (shMUC1-B16F10 cells). Next, we examined epithelial-mesenchymal transition (EMT), migration, proliferative capacity, clone formation, and distribution of cell cycle in shMUC1-B16F10 cells. Finally, the vaccine was prepared by repeated freeze-thawing of the shMUC1-B16F10 cells and used to subcutaneously immunize C57BL/6 mice, which were then challenged using B16F10 cells 10 days after the final vaccination. Results: It was revealed that shMUC1 suppressed B16F10 proliferative and colony formation capacity, induced the arrest of cell cycle in the G0/G1 phase, and adjusted the expression of EMT-associated factors. MUC1 downregulation markedly suppressed the effect of B16F10 vaccine against melanoma in a mouse model. As compared with B16F10-vaccinated mice, B16F10-vaccinated mice in which MUC1 was silenced had reduced natural killer (NK) cytotoxicity, lower production of interferon-γ (IFN-γ), anti-MUC1 antibodies, perforin, granzyme B, and elevated tumor growth factor-ß (TGF-ß) level. Conclusions: MUC1 has strong melanoma vaccine immunogenicity, and induces the host's anti-tumor reaction. MUC1 knockdown inhibits the immune activity of B16F10 cell vaccine and anti-melanoma effect, suggesting the MUC1 is an important candidate target antigen of the melanoma vaccine.
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CSCs (cancer stem cells) are a small subset of cells within a tumour that possesses the characteristics of stem cells and are considered to be responsible for resistance to chemoradiation. Identification of CSCs through stem cell characteristics might have relevant clinical implications. In this study, SP (side population ) cells were sorted from a human ovarian cancer cell line by FACS to determine whether cancer stem cell-like SP cells were present. A very small fraction of SP cells (2.6%) was detected in A2780 cells. SP cells possessed the following characteristics: highly proliferative activity, marked ability for self-renewal in soft agar and culture medium, high expression of ABCG2, drug resistance to vinblastine in vitro, and strong tumourigenic potential in Balb/c nude mice. It is concluded that there exists in the A2780 cell line a small number of SP cells with high expression of ABCG2. The cells have the characteristics of cancer stem-like cells, and identification and cloning of such human SP cells can help in improving therapeutic approaches to ovarian cancer in patients.
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Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Células da Side Population/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/citologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Células da Side Population/citologia , Vincristina/uso terapêuticoRESUMO
The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. In this study, we developed a DNA vaccine expressing HSV-1 glycoprotein D (gD) and mouse interleukin-21(IL-21) and intramuscularly inoculated mice 3 times at 2-week intervals with a total of 300 ?g/mouse. Two weeks after the last immunization the specific antibody, splenocyte proliferative response to gD, IFN-? and IL-4 as well as the cytotoxic activities of splenocytes and natural killer (NK) cells were assayed. Immune protection against herpes keratitis was concurrently evaluated in the immunized mice after HSV-1 challenge of the mouse cornea. The results showed that the DNA vaccine pRSC-gD-IL-21 generated higher levels of antibody, IFN-? and IL-4, and enhanced the splenocyte proliferative response to gD as well as the cytotoxic activity of splenocytes and NK cells to target cells compared with the response in either the pRSC-gD or mock plasmid pRSC immunized mice. Importantly, the pRSC-gD-IL-21 ameliorated herpes keratitis severity and time course after corneal infection with HSV-1. The findings suggest that the DNA vaccine pRSC-gD-IL-21 may induce an immune response that can limit HSV-1 infection and development of herpes keratitis in the immunized mice.
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Interleucinas/imunologia , Ceratite Herpética/prevenção & controle , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Citocinas/sangue , Feminino , Imunidade Celular/imunologia , Interleucinas/genética , Ceratite Herpética/patologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas de DNA/genética , Células Vero , Proteínas do Envelope Viral/genética , Vacinas Virais/genéticaRESUMO
Inhibition of aldehyde dehydrogenase 1 family member A3 (ALDH1A3) has been revealed to lead to significant increase of microRNA (miR)-7 expression and decrease of CD44 expression in breast cancer stem cells (BCSCs), however the mechanism is not clear. The aim of the present study was to investigate the regulatory relationship between ALDH1A3, miR-7, and CD44 in BCSCs. The expression of ALDH1A3 was inhibited by small interfering RNA (siRNA or si), and the expression of miR-7 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Then, the ratio of CD44+ cells was analyzed by flow cytometry in MDA-MB-231 cells. The dual-luciferase reporter system was used to demonstrate that miR-7 binds to transforming growth factor-ß receptor 2 (TGFBR2) 3'UTR, and ChIP-PCR determined whether the transcription factor Smad3 binds to the upstream regulatory region of the CD44 promoter. The results revealed that siALDH1A3 downregulated ALDH1A3 and promoted miR-7 expression, which resulted in downregulation of CD44 expression. siALDH1A3 also downregulated the CD44 expression on the surface of MDA-MB-231 cells and inhibited the G2/M phase in BCSCs as analyzed by flow cytometry. In addition, lenti-miR-7 cells transfected with TGF-ß1 + SB431542 revealed that lenti-miR-7 inhibited the TGF-ß1 pathway by inhibiting Smad2/3/4 expression and, thus, downregulated CD44 expression. miR-7 was revealed to directly bind to the TGFBR2 3'UTR through dual-luciferase reporter assay, and Smad3, a transcription factor, through ChIP-PCR was demonstrated to bind to the upstream region of the CD44 promoter. These results demonstrated the existence of the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 axis in MDA-MB-231 cells. RT-qPCR results of 12 breast cancer surgical specimens and SK-BR-3, MCF-7, and LD cell lines further confirmed the presence of the regulatory axis. In conclusion the findings from the present study demonstrated that the ALDH1A3-miR-7-TGFBR2-Smad3-CD44 regulatory axis was highly efficient in the inhibition of CD44 expression in BCSCs, and that the regulatory expression of ALDH1A3 and miR-7 may provide a strategy in the therapy of breast cancer.
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BACKGROUND AND AIM: Acute myeloid leukemia (AML), initiated and maintained by leukemia stem cells (LSCs), is often relapsed or refractory to therapy. The present study aimed at assessing the effects of nanozyme-like Fe3O4 nanoparticles (IONPs) combined with cytosine arabinoside (Ara-C) on LSCs in vitro and in vivo. METHODS: The CD34+CD38-LSCs, isolated from human AML cell line KG1a by a magnetic activated cell sorting method, were treated with Ara-C, IONPs, and Ara-C+ IONPs respectively in vitro. The cellular proliferation, apoptosis, reactive oxygen species (ROS), and the related molecular expression levels in LSCs were analyzed using flow cytometry, RT-qPCR, and Western blot. The nonobese diabetic/severe combined immune deficiency mice were transplanted with LSCs or non-LSCs via tail vein, and then the mice were treated with Ara-C, IONPs and IONPs plus Ara-C, respectively. The therapeutic effects on the AML bearing mice were further evaluated. RESULTS: LSCs indicated stronger cellular proliferation, more clone formation, and more robust resistance to Ara-C than non-LSCs. Compared with LSCs treated with Ara-C alone, LSCs treated with IONPs plus Ara-C showed a significant increase in apoptosis and ROS levels that might be regulated by nanozyme-like IONPs via improving the expression of pro-oxidation molecule gp91-phox but decreasing the expression of antioxidation molecule superoxide dismutase 1. The in vivo results suggested that, compared with the AML bearing mice treated with Ara-C alone, the mice treated with IONPs plus Ara-C markedly reduced the abnormal leukocyte numbers in peripheral blood and bone marrow and significantly extended the survival of AML bearing mice. CONCLUSION: IONPs combined with Ara-C showed the effectiveness on reducing AML burden in the mice engrafted with LSCs and extending mouse survival by increasing LSC's ROS level to induce LSC apoptosis. Our findings suggest that targeting LSCs could control the AML relapse by using IONPs plus Ara-C.
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Citarabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Nanopartículas Magnéticas de Óxido de Ferro/química , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Separação Celular , Citarabina/farmacologia , Hemoglobinas/metabolismo , Humanos , Leucemia Mieloide Aguda/sangue , Contagem de Leucócitos , Camundongos Endogâmicos NOD , Camundongos SCID , NADPH Oxidase 2/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The microtubule actin cross-linking factor 1 (MACF1) is involved in cellular migration, adhesion, and invasion processes. Its abnormal expression initiates tumor cell proliferation and metastasis in numerous cancer types. METHODS: In this study, we utilized short hair-pin RNA interference of MACF1 to assess the inhibitory effects on the metastatic potential of B16F10 melanoma cells both in vitro and in vivo a mouse model. RESULTS: The MACF1 expression was increased in B16F10 cells-induced tumor tissues; while the down-regulation of MACF1 impacted the B16F10 melanoma cell metastatic behavior by decreasing the ability of colony formation and invasion in vitro as well as inhibiting B16F10 cells-induced tumor growth and lung metastasis in vivo. The results of Western blot and immunohistochemistry indicated that the expression of E-cadherin and Smad-7 was significantly increased whereas the expression of N-cadherin and TGF-ß1 was significantly decreased in tumor tissue of mice challenged with the B16F10/MACF1-RNAi cells when compared with the B16F10 cells challenged mice. CONCLUSION: The data presented in this study demonstrated that down-regulated MACF1 expression decreased B16F10 melanoma metastasis in mice by inhibiting the epithelial to mesenchymal transition program. Thus, MACF1 may be a novel target for melanoma therapy.
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Increasing knowledge of colorectal cancer stem cells (CCSCs) and tumor microenvironment improves our understanding of cellular mechanisms involved in the immunity against colorectal cancer (CRC). Tumor associated antigens were evaluated via RNA-seq and bioinformatics analysis, evoking promising targets for tumor immunotherapy. MUC1 has been demonstrated to participate in the maintenance, tumorigenicity, glycosylation and metastasis of CCSCs, which may provide a new priority for CSC vaccination. In the present study, the vaccination with CCSCs with high expression of MUC1 was evaluated in a murine model for the vaccine's immunogenicity and protective efficacy against CRC. CD133+ CCSCs were isolated from SW620 cell line using a magnetic-activated cell sorting system, and shMUC1 was further used to knock down the expression of MUC1 in CD133+ CCSCs. Mice were subcutaneously immunized with the cell lysates of CCSCs and shMUC1 CCSCs, followed by a challenge with SW620 cells at ten days after final vaccination. The results indicated CCSC vaccine significantly reduced the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+ cells and ALDH+ cells in tumors. Moreover, CCSC vaccine resulted in the elevated NK cytotoxicity, production of perforin, granzyme B, IFN-γ, memory B cells, and anti-MUC1 antibodies. Of note, MUC1 knockdown partly impaired the anti-tumor efficacy of CCSC vaccine. Importantly, the CCSC vaccine has no toxic damage to organs. Overall, CCSC vaccine could serve as a potent and safe vaccine for CRC treatment, and MUC1 might play an essential role in CCSC vaccine.
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Vacinas Anticâncer , Neoplasias Colorretais/imunologia , Mucina-1/imunologia , Células-Tronco Neoplásicas/imunologia , Animais , Linfócitos B/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Colorretais/terapia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Mucina-1/genéticaRESUMO
Targeted clearance of colorectal cancer stem cells (CCSCs) has become a novel strategy for tumor immunotherapy. Molecule mucin1 (MUC1) is one of targetable cell surface antigens in CCSCs. However, the critical role of MUC1 in anti-tumor effects of CCSC vaccine remains unclear. In the present study, we showed that MUC1 may be required for CCSC vaccine to exert tumor immunity. CD133+CCSCs were isolated from CT26 cell line using a magnetic-activated cell sorting system, and MUC1 shRNA or recombinant plasmid was further used to decrease or increase the expression of MUC1 in CD133+CCSCs. Mice were subcutaneously immunized with the CCSC lysates, MUC1 knockin CCSCs, and MUC1 knockdown CCSCs respectively, followed by a challenge with CT26 cells. We found that CCSC vaccine significantly reduced the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+ cells and ALDH+ cells in tumors. Moreover, CCSC vaccine markedly increased the cytotoxicity of NK cells and the splenocytes, and promoted the release of IFN-γ, Perforin, and Granzyme B, and also reduced the TGF-ß1 expression. Additionally, CCSC vaccination enhanced the antibody production and decreased the myeloid derived suppressor cells and Treg subsets. More importantly, MUC1 knockdown partly impaired the anti-tumor efficacy of CCSC vaccine, whereas MUC1 overexpression dramatically enhanced the CCSC vaccine immunity. Overall, these results reveal a novel role and molecular mechanisms of MUC1 in CCSC vaccine against colorectal cancer.
Assuntos
Vacinas Anticâncer/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/prevenção & controle , Mucina-1/biossíntese , Mucina-1/genética , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Antígeno AC133/metabolismo , Aldeído Desidrogenase/metabolismo , Animais , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Feminino , Granzimas/metabolismo , Imunoterapia/métodos , Interferon gama/sangue , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Crescimento Transformador beta1/sangue , Carga Tumoral/efeitos dos fármacosRESUMO
This study aimed to present evidence that miR-7 inhibited the metastasis of breast cancer stem cells (BCSCs) and elucidated the mechanisms that have remained unknown. The samples collected from miR-7 agomir-treated, BCSC-driven tumors were subjected to a protein array to analyze the protein expression profiles. A dual-luciferase reporter and chromatin immunoprecipitation-PCR were used to validate and evaluate the molecular expressions of interest in the collected breast cancer tissues and cell lines. miR-7 overexpression affecting metastasis of BCSCs was further evaluated in mice. The endothelial cell-selective adhesion molecule (ESAM) was highly expressed in breast cancer tissues and in BCSC-driven xenografts. Results of the dual-luciferase reporter and chromatin immunoprecipitation-PCR indicated that the miR-7 mimic reduced RELA expression by directly targeting the 3' UTR of RELA to inhibit ESAM expression in MDA-MB-231 cells. Moreover, the expression levels of RELA, CD44, and ESAM were significantly decreased in lentivirus (Lenti)-miR-7-BCSC-driven xenografts compared with the control xenografts, accompanied with an increase in E-cadherin and a decrease in vimentin expression, as well as reduction in tumor growth and metastasis to lungs. Our data demonstrated that miR-7 overexpression reduced the metastasis of BCSCs via inhibiting ESAM, suggesting that ESAM could be a potential target for breast cancer therapy.
RESUMO
Colorectal cancer (CRC), initiated and maintained by colorectal cancer stem cells (CCSCs), ranks the third most common cancers and has drawn wide attentions worldwide. Therefore, targeting clearance of CCSCs has become an important strategy of CRC immunotherapy. Mucin1 (MUC1) is a tumor-associated cell surface antigen of CRC, but its role in CCSC vaccine remains unclear. In the study, we demonstrated that MUC1 may be a dominant antigen to exert antitumor immunity in CCSC vaccine. First, CCSCs were enriched from CT26 cell line via a serum-free sphere formation approach, and were identified by detecting expression of CD133, ALDH, and ALCAM. Then, the isolated CCSCs were frozen for 30 min and thawed for 30 min to prepare the cell lysate. The specific anti-MUC1 antibody was added to the cell lysate to neutralize the dominant antigen MUC1. Finally, mice were subcutaneously immunized with the cell lysate, followed by a challenge with CT26 cells at one week after final vaccination. Attractively, CCSC vaccine significantly activated the NK cells, T cells, and B cells, resulting in inhibiting the tumor growth via a target killing of CCSCs as evidenced by a decrease of CD133+cells in tumor compared to CCSC vaccine with specific anti-MUC1 antibody. In addition, CCSC vaccine reduced expression of inflammatory factors in vaccinated mice. As expected, neutralizing antibody against MUC1 significantly impaired the antitumor efficacy of CCSC vaccine. Overall, CCSC vaccine could serve as a potent vaccine for CRC immunotherapy. The surface dominant antigen MUC1 may play a key role in regulating immunogenicity of CCSCs.
Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias Colorretais/prevenção & controle , Mucina-1/imunologia , Células-Tronco Neoplásicas/imunologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Feminino , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , VacinaçãoRESUMO
Increasingly more evidence shows that TSCs possess the characteristics of stem-like cells. However, a link between stem cells and TSCs remains to be shown. We have sorted SP cells and non-SP cells from the B16F10 cell lines by FACS, and then studied their cellular biological characteristics by using a SFCM culture method, proliferative assay in vitro, clone formation assays in soft agar and normal media, tumorigenic assays in C57BL/6 mice, and resistance to chemotherapy assay in vitro, the quantitative detecting expression of ABCG2 and their CD phenotype analysis by a FCM. We detected 0.96% SP cells in the B16F10 cells and found that they had obvious differences in characteristics from non-SP cells. They possessed a marked capacity for self-renewal in soft agar and culture medium, strong tumorigenic potential in C57BL/6 mice, apparent resistance to vinblastin in vitro, upregulated ABCG2 expression, and a high expression of CD44+CD133+CD24+ phenotypes. We conclude that there were a few of SP cells that had the characteristics of tumor stem-like cells which may provide a useful tool and a readily accessible source for further study when specific TSCs markers are unknown.
Assuntos
Melanoma Experimental/metabolismo , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antígenos CD/metabolismo , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Citometria de Fluxo , Glicoproteínas/metabolismo , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Peptídeos/metabolismo , Vimblastina/farmacologiaRESUMO
Tumor vaccines offer a number of advantages for cancer treatment. In the study, the vaccination with cancer stem cells (CSCs) with high expression of the type I receptor tyrosine kinase-like orphan receptor (ROR1) was evaluated in a murine model for the vaccine's immunogenicity and protective efficacy against epithelial ovarian carcinoma (EOC). CD117+CD44+ CSCs were isolated from human EOC HO8910 cell line using a magnetic-activated cell sorting system; murine ID8 EOC suspension sphere cells, which are collectively known as cancer stem-like cells, were acquired from serum-free suspension sphere-forming culture. Mice were subcutaneously immunized with the repeat cycles of freezing and thawing whole HO8910 CD117+CD44+ CSCs and ID8 cancer stem-like cells, respectively, followed by a challenge with HO8910 or ID8 cells at one week after final vaccination. The results showed that the CSC vaccination significantly induced immunity against EOC growth and markedly prolonged the survival of EOC-bearing mice in the prophylactic setting compared with non-CSC vaccination. Flow cytometry showed significantly increased immunocyte cytotoxicities and remarkably reduced CSC counts in the CSC-vaccinated mice. Moreover, the protective efficacy against EOC was decreased when the ROR1 expression was downregulated by shRNA in CSC vaccines. The findings from the study suggest that CSC vaccines with high ROR1 expression were highly effective in triggering immunity against EOC in vaccinated mice and may serve as an effective vaccine for EOC immunoprophylaxis.
Assuntos
Vacinas Anticâncer/imunologia , Carcinoma Epitelial do Ovário/prevenção & controle , Células-Tronco Neoplásicas/imunologia , Neoplasias Ovarianas/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Animais , Vacinas Anticâncer/administração & dosagem , Carcinoma Epitelial do Ovário/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias Ovarianas/prevenção & controle , VacinaçãoRESUMO
In this paper, we addressed the immune adjuvant effects of interleukin(IL)-21 on DNA vaccine constructs expressing mycobacterium tuberculosis (TB) Ag85A and compared immune responses induced in mice inoculated DNA vaccine constructs expressing Ag85A and IL-21 with mice inoculated DNA vaccine constructs expressing Ag85A alone or Bacillus Galmette-Guérin(BCG.). In this experiment, the gene of IL-21 was firstly amplified from plasmid pcDNA3.1-mIL21 by PCR and cloned into the plasmid pRSC, forming recombinant plasmid pRSC-IL21. Then, the gene of Ag85A was amplified from the plasmid pIRES-Ag85A by PCR and cloned into the recombinant pRSC-IL21 again, finally forming co-expression DNA vaccine constructs pRSC-IL21-Ag85A. It was identified by the analysis of endonuclease digestion, DNA sequencing, the IL-21 and Ag85A expression in SP2/0 cells. Mice were i.m. immunized with BCG, DNA vaccine constructs pRSC-Ag85A or pRSC-IL21-Ag85A respectively, and the immune responses induced in mice was compared with other vaccines. The results showed that the DNA vaccine constructs pRSC-IL21-Ag85A was successfully constructed since the Ag85A and IL-21 was correctly expressed in SP2/0 cells respectively, and it elicited stronger immune responses in Balb/c mice than that of mice immunized with pRSC-Ag85A and the efficiency was as BCG did. We concluded that the IL-21 was a promising immune adjunctive modality to enhance immunigenicity of DNA vaccine containing Ag85A and the study provided the possibility of further development of immune accessory effect of IL-21 on DNA vaccine against TB.