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1.
Zhonghua Zhong Liu Za Zhi ; 43(9): 939-943, 2021 Sep 23.
Artigo em Chinês | MEDLINE | ID: mdl-34530576

RESUMO

Objective: Establishment of a new model of human primary colon cancer transplantation tumor in normal immune mice and to provide a reliable experimental animal model for studying the pathogenesis of colon cancer under normal immunity. Methods: Human colon cancer cells come from colon cancer patients who underwent surgery in the Affiliated Hospital of Jining Medical College in 2017. The mice in the cell control group were inoculated with phosphate buffered solution (PBS) containing colon cancer cells, the microcarrier control group was inoculated with PBS containing microcarrier 6, and the cell-microcarrier complex group was inoculated with the PBS containing colon cancer cell-microcarrier complex. The cells of each group were inoculated under the skin of the right axilla of mice by subcutaneous injection, and the time, size, tumor formation rate and pathological changes under microscope were recorded. The transplanted tumor tissue was immunohistochemically stained with the EnVisiion two-step method, and the tumor formation rate of the transplanted tumor was judged according to the proportion of positive cells in the visual field. The polymerase chain reaction (PCR) method was used to detect the expression of human-specific Alu sequence in mice tumor tissue. Results: After inoculation with tumor cells, the mice in the cell control group and the microcarrier control group did not die and did not form tumors; the mice in the cell-microcarrier complex group had palpable subcutaneous tumors in the right axillary subcutaneously on the 5th to 7th days after inoculation, and tumor formation rate is 67% (10/15), and the tumor volume can reach about 500 mm(3) 2 to 3 weeks after vaccination. The immunohistochemistry results showed that CK20, CDX-2 and carcinoembryonic antigen were all positively expressed. The PCR results showed that the expression of human-specific Alu sequence can be detected in the transplanted tumor tissue of tumor-bearing mice. Conclusion: Human primary colon cancer cells used microcarrier 6 as a carrier to form tumors in normal immunized mice, and successfully established a new model of human colon cancer transplantation tumor in normal immune mice.


Assuntos
Neoplasias do Colo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Carga Tumoral
2.
Anticancer Res ; 41(9): 4249-4258, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475044

RESUMO

BACKGROUND/AIM: Recent studies have indicated the clinical significance of tumor-associated macrophages (TAMs) in breast cancer; however, the detailed mechanisms of cell-cell interactions between TAMs and cancer cells remain unclear. MATERIALS AND METHODS: In vitro cell culture studies using human monocyte-derived macrophages and breast cancer cell lines were performed to test which cytokines would be involved in cell-cell interactions between cancer cells and macrophages. In addition, studies using human resected samples and animal breast cancer models were performed to examine the significance of TAMs in cancer development. RESULTS: Osteopontin, HB-EGF, and IL-6 were suggested to be macrophage-derived growth factors for breast cancer cells. FROUNT inhibitor significantly blocked TAM infiltration and subcutaneous tumor growth in an E0771 mouse breast cancer model. CONCLUSION: TAMs express growth factors, such as osteopontin, for cancer cells, and targeting of TAM infiltration might be a promising approach for anti-breast cancer therapy.


Assuntos
Neoplasias da Mama/patologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Interleucina-6/genética , Macrófagos/citologia , Osteopontina/genética , Macrófagos Associados a Tumor/citologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Interleucina-6/metabolismo , Células MCF-7 , Macrófagos/metabolismo , Camundongos , Transplante de Neoplasias , Osteopontina/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia
3.
J Immunol ; 207(5): 1456-1467, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34380650

RESUMO

Cancer immunotherapy has shown great promise as a new standard therapeutic strategy against cancer. However, the response rate and survival benefit remain unsatisfactory because most current approaches, such as the use of immune checkpoint inhibitors, depend on spontaneous antitumor immune responses. One possibility for improving the efficacy of immunotherapy is to promote antitumor immunity using adjuvants or specific cytokines actively. IL-33 has been a candidate for such cytokine therapies, but it remains unclear how and in which situations IL-33 exerts antitumor immune effects. In this study, we demonstrate the potent antitumor effects of IL-33 using syngeneic mouse models, which included marked inhibition of tumor growth and upregulation of IFN-γ production by tumor-infiltrating CD8+ T cells. Of note, IL-33 induced dendritic cells to express semaphorin 4A (Sema4A), and the absence of Sema4A abolished the antitumor activity of IL-33, indicating that Sema4A is intrinsically required for the antitumor effects of IL-33 in mice. Collectively, these results not only present IL-33 and Sema4A as potential therapeutic targets but also shed light on the potential use of Sema4A as a biomarker for dendritic cell activation status, which has great value in various fields of cancer research, including vaccine development.


Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Células Dendríticas/imunologia , Interleucina-33/metabolismo , Semaforinas/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Semaforinas/genética
4.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360757

RESUMO

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , MicroRNAs , RNA Neoplásico , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Xenoenxertos , Humanos , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Transplante de Neoplasias , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia
5.
J Immunol ; 207(5): 1298-1309, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34362833

RESUMO

Intralesional therapy is a promising approach for remodeling the immunosuppressive tumor microenvironment while minimizing systemic toxicities. A combinatorial in situ immunomodulation (ISIM) regimen with intratumoral administration of Fms-like tyrosine kinase 3 ligand (Flt3L), local irradiation, and TLR3/CD40 stimulation induces and activates conventional type 1 dendritic cells in the tumor microenvironment and elicits de novo adaptive T cell immunity in poorly T cell-inflamed tumors. However, the impact of ISIM on myeloid-derived suppressor cells (MDSCs), which may promote treatment resistance, remains unknown. In this study, we examined changes in the frequencies and heterogeneity of CD11b+Ly-6CloLy-6G+ polymorphonuclear (PMN)-MDSCs and CD11b+Ly-6ChiLy-6G- monocytic (M)-MDSCs in ISIM-treated tumors using mouse models of triple-negative breast cancer. We found that ISIM treatment decreased intratumoral PMN-MDSCs, but not M-MDSCs. Although the frequency of M-MDSCs remained unchanged, ISIM caused a substantial reduction of CX3CR1+ M-MDSCs that express F4/80. Importantly, these ISIM-induced changes in tumor-residing MDSCs were not observed in Batf3-/- mice. ISIM upregulated PD-L1 expression in both M-MDSCs and PMN-MDSCs and synergized with anti-PD-L1 therapy. Furthermore, ISIM increased the expression of IFN regulatory factor 8 (IRF8) in myeloid cells, a known negative regulator of MDSCs, indicating a potential mechanism by which ISIM decreases PMN-MDSC levels. Accordingly, ISIM-mediated reduction of PMN-MDSCs was not observed in mice with conditional deletion of IRF8 in myeloid cells. Altogether, these findings suggest that ISIM holds promise as a multimodal intralesional therapy to alter both lymphoid and myeloid compartments of highly aggressive poorly T cell-inflamed, myeloid-enriched tumors resistant to anti-PD-L1 therapy.


Assuntos
Células Dendríticas/imunologia , Imunoterapia/métodos , Fatores Reguladores de Interferon/metabolismo , Neoplasias Mamárias Animais/terapia , Proteínas de Membrana/uso terapêutico , Células Supressoras Mieloides/imunologia , Linfócitos T/imunologia , Animais , Antígeno B7-H1 , Fatores de Transcrição de Zíper de Leucina Básica/genética , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Terapia Combinada , Resistência a Medicamentos , Regulação da Expressão Gênica , Humanos , Injeções Intralesionais , Fatores Reguladores de Interferon/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Radioterapia , Proteínas Repressoras/genética , Receptor 3 Toll-Like/metabolismo , Microambiente Tumoral
6.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445503

RESUMO

Obesity is a major risk factor for developing cancer, with obesity-induced immune changes and inflammation in breast (BC) and colorectal cancer (CRC) providing a potential link between the two. This study investigates systemic effects of obesity on adaptive and innate immune cells in healthy and tumour-bearing mice. Immune cells from lean and obese mice were phenotyped prior to implantation of either BC (C57mg and EO771.LMB) or CRC (MC38) cells as tumour models. Tumour growth rate, tumour-infiltrating lymphocytes (TIL) and peripheral blood immune cell populations were compared between obese and lean mice. In vitro studies showed that naïve obese mice had higher levels of myeloid cells in the bone marrow and bone marrow-derived dendritic cells expressed lower levels of activation markers compared to cells from their lean counterparts. In the tumour setting, BC tumours grew faster in obese mice than in lean mice and lower numbers of TILs as well as higher frequency of exhausted T cells were observed. Data from peripheral blood showed lower levels of myeloid cells in tumour-bearing obese mice. This study highlights that systemic changes to the immune system are relevant for tumour burden and provides a potential mechanism behind the effects of obesity on cancer development and progression in patients.


Assuntos
Neoplasias da Mama/patologia , Neoplasias Colorretais/patologia , Linfócitos do Interstício Tumoral/metabolismo , Obesidade/imunologia , Imunidade Adaptativa , Animais , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Feminino , Humanos , Masculino , Camundongos , Células Mieloides/metabolismo , Transplante de Neoplasias , Microambiente Tumoral
7.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445387

RESUMO

Prostate cancer (PCa) is the leading cause of cancer-associated mortality in men, and new biomarkers are still needed. The expression pattern and protein tissue localization of proteoglycans of the syndecan family (SDC 1-4) and syntenin-1 (SDCBP) were determined in normal and prostatic tumor tissue from two genetically engineered mouse models and human prostate tumors. Studies were validated using SDC 1-4 and SDCBP mRNA levels and patient survival data from The Cancer Genome Atlas and CamCAP databases. RNAseq showed increased expression of Sdc1 in Pb-Cre4/Ptenf/f mouse Pca and upregulation of Sdc3 expression and downregulation of Sdc2 and Sdc4 when compared to the normal prostatic tissue in Pb-Cre4/Trp53f/f-;Rb1f/f mouse tumors. These changes were confirmed by immunohistochemistry. In human PCa, SDC 1-4 and SDCBP immunostaining showed variable localization. Furthermore, Kaplan-Meier analysis showed that patients expressing SDC3 had shorter prostate-specific survival than those without SDC3 expression (log-rank test, p = 0.0047). Analysis of the MSKCC-derived expression showed that SDC1 and SDC3 overexpression is predictive of decreased biochemical recurrence-free survival (p = 0.0099 and p = 0.045, respectively), and SDC4 overexpression is predictive of increased biochemical recurrence-free survival (p = 0.035). SDC4 overexpression was associated with a better prognosis, while SDC1 and SDC3 were associated with more aggressive tumors and a worse prognosis.


Assuntos
Perfilação da Expressão Gênica/métodos , Neoplasias da Próstata/patologia , Sindecana-1/genética , Sindecana-3/genética , Sindecana-4/genética , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Transplante de Neoplasias , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Análise Serial de Proteínas , Análise de Sequência de RNA , Análise de Sobrevida , Sindecana-1/metabolismo , Sindecana-3/metabolismo , Sindecana-4/metabolismo , Sinteninas/genética , Sinteninas/metabolismo
8.
J Cardiothorac Surg ; 16(1): 194, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233689

RESUMO

OBJECTIVE: C-erbB-2 has been confirmed to be an oncogene that participates in cell growth, differentiation and division of tumors. We are wondered if its silenced expression can exert an anti-tumor effect. Therefore, this study is conducted to investigate the mechanism of C-erbB-2 silencing and IGF-1 pathway on esophageal carcinoma (EC) cell biological behaviors. METHODS: The objects of study were 84 EC patients from Heping Hospital Affiliated to Changzhi Medical College, with the collection of EC tissue and adjacent normal tissue (> 5 cm away from cancer tissue). C-erbB-2 protein expression in EC tissues was detected by immunohistochemistry. Human EC cell line Eca-109 was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Based on different transfection protocols, EC cells with logarithmic growth phase of 3-5 passages were divided into blank control group, oe-C-erbB-2 NC group, siRNA C-erbB-2 NC group, oe-C-erbB-2 group, siRNA C-erbB-2 group, OSI-906 group, Rg5 group, Rg5 + siRNA C-erbB-2 NC group and Rg5 + siRNA C-erbB-2 group. Cell proliferation was detected by MTT assay; cell cycle distribution and apoptosis by flow cytometry; C-erbB-2, IGF-1, IGF-1R and Akt mRNA and protein expressions by qRT-PCR and western blot; and cell invasion and migration by Transwell assay and scratch test. Tumor growth was observed in male BALB/c nude mice (Shanghai Experimental Animal Center) based on Eca109 cell implantation, raising, and measurement. RESULTS: C-erbB-2, IGF-1, IGF-1R and Akt expression were higher in EC tissues than those in adjacent tissues (all P < 0.05). Compared with blank control group, both si-C-erbB-2 and OSI-906 groups had decreased IGF-1, IGF-1R and Akt mRNA and protein expressions, decreased cell proliferation, migration and invasion, prolonged G0/G1 phase, shortened S phase, increased cell apoptosis, and inhibited tumor growth (all P < 0.05); while opposite trends were detected in C-erbB-2 vector and Rg5 groups (all P < 0.05), without statistical differences in siRNA C-erbB-2 + Rg5 group (all P > 0.05). CONCLUSION: Silencing C-erbB-2 expression may inhibit EC cell proliferation, promote cell apoptosis and block cell cycle progression by inhibiting IGF-1 pathway activation. The beneficial effect of silencing C-erbB-2 expression can be reversed by promoting the activation of IGF-1 pathway. Findings in our study may provide potential reference for understanding the molecular mechanism of EC and supply possible axis for preventing the development of EC from the perspective of molecular biology.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Inativação Gênica/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Receptor ErbB-2/genética , Adulto , Idoso , Animais , Apoptose/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor IGF Tipo 1 , Transfecção
9.
Artigo em Chinês | MEDLINE | ID: mdl-34256488

RESUMO

Objective: To explore the role and mechanism of long non-coding RNA RP11-159K7.2 in the progression of sinonasal squamous cell carcinoma (SNSCC). Methods: Sixty-five cases of SNSCC tissues and adjacent tissues were selected from the Department of Otorhinolaryngology Head and Neck Surgery, the Second Affiliated Hospital of Harbin Medical University from 2009 to 2014. The expression of RP11-159K7.2 in SNSCC and adjacent tissues was detected by RNAscope in situ hybridization to observe its association with prognosis. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins 9 (CRISPR/Cas9) was used to knockout the expression of RP11-159K7.2 in RPMI-2650 cells (SNSCC cell line). Cell counting kit-8 (CCK-8), wound healing and Transwell were performed to observe the changes of proliferation, migration and invasion of SNSCC cells in vitro after down-regulation of RP11-159K7.2. Moreover, the growth of xenograft in nude mice after down-regulation of RP11-159K7.2 was examined in vivo. Mechanically, the protein chip, Western blot and RNA immunoprecipitation were performed to identify the proteins bound by RP11-159K7.2. SPSS 17.0 was used for statistical analysis. Results: The expression of RP11-159K7.2 in SNSCC tissue was significantly higher than that in adjacent tissues. RP11-159K7.2 expression was closely related with T grade, nodal metastasis and differentiation of SNSCC (χ2 value was 4.697, 4.235 and 10.753, respectively, all P<0.05). The five-year survival rate of RP11-159K7.2 high expression patients was significantly lower than that of RP11-159K7.2 low expression ones (P=0.013 7). After the down-regulation of RP11-159K7.2, the proliferation, migration and invasion ability of SNSCC cells decreased significantly, and the growth of SNSCC xenograft was significantly inhibited. There were 31 candidate proteins that may bind to RP11-159K7.2. RP11-159K7.2 directly bound to nuclear factor-κB (NF-κB) in SNSCC cells, and the regulation of RP11-159K7.2 on the proliferation and invasion of SNSCC cells depended on NF-κB. Conclusion: The increased expression of RP11-159K7.2 in SNSCC may serve as a potential molecular marker for SNSCC prognosis assessment. It is currently considered that the carcinogenic mechanism of RP11-159K7.2 in SNSCC is related to the regulation of NF-κB protein.


Assuntos
Carcinoma de Células Escamosas , RNA Longo não Codificante , Animais , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Prognóstico , RNA Longo não Codificante/genética
10.
Front Immunol ; 12: 636108, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34290694

RESUMO

Radiotherapy, the most frequent treatment of oral squamous cell carcinomas (OSCC) besides surgery is employed to kill tumor cells but, radiotherapy may also promote tumor relapse where the immune-suppressive tumor microenvironment (TME) could be instrumental. We established a novel syngeneic grafting model from a carcinogen-induced tongue tumor, OSCC13, to address the impact of radiotherapy on OSCC. This model revealed similarities with human OSCC, recapitulating carcinogen-induced mutations found in smoking associated human tongue tumors, abundant tumor infiltrating leukocytes (TIL) and, spontaneous tumor cell dissemination to the local lymph nodes. Cultured OSCC13 cells and OSCC13-derived tongue tumors were sensitive to irradiation. At the chosen dose of 2 Gy mimicking treatment of human OSCC patients not all tumor cells were killed allowing to investigate effects on the TME. By investigating expression of the extracellular matrix molecule tenascin-C (TNC), an indicator of an immune suppressive TME, we observed high local TNC expression and TIL infiltration in the irradiated tumors. In a TNC knockout host the TME appeared less immune suppressive with a tendency towards more tumor regression than in WT conditions. Altogether, our novel syngeneic tongue OSCC grafting model, sharing important features with the human OSCC disease could be relevant for future anti-cancer targeting of OSCC by radiotherapy and other therapeutic approaches.


Assuntos
Linfonodos/efeitos da radiação , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Tenascina/metabolismo , Neoplasias da Língua/radioterapia , Animais , Linhagem Celular Tumoral , Feminino , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Transplante de Neoplasias , Tolerância a Radiação , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/secundário , Tenascina/genética , Neoplasias da Língua/genética , Neoplasias da Língua/metabolismo , Neoplasias da Língua/patologia , Transplante Isogênico , Carga Tumoral/efeitos da radiação , Microambiente Tumoral
11.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298975

RESUMO

Previously, we showed that chemotherapy paradoxically exacerbated cancer cell colonization at the secondary site in a manner dependent on Atf3, a stress-inducible gene, in the non-cancer host cells. Here, we present evidence that this phenotype is established at an early stage of colonization within days of cancer cell arrival. Using mouse breast cancer models, we showed that, in the wild-type (WT) lung, cyclophosphamide (CTX) increased the ability of the lung to retain cancer cells in the vascular bed. Although CTX did not change the WT lung to affect cancer cell extravasation or proliferation, it changed the lung macrophage to be pro-cancer, protecting cancer cells from death. This, combined with the initial increase in cell retention, resulted in higher lung colonization in CTX-treated than control-treated mice. In the Atf3 knockout (KO) lung, CTX also increased the ability of lung to retain cancer cells. However, the CTX-treated KO macrophage was highly cytotoxic to cancer cells, resulting in no increase in lung colonization-despite the initial increase in cell retention. In summary, the status of Atf3 dictates the dichotomous activity of macrophage: pro-cancer for CTX-treated WT macrophage but anti-cancer for the KO counterpart. This dichotomy provides a mechanistic explanation for CTX to exacerbate lung colonization in the WT but not Atf3 KO lung.


Assuntos
Fator 3 Ativador da Transcrição/fisiologia , Ciclofosfamida/toxicidade , Neoplasias Pulmonares/secundário , Macrófagos/fisiologia , Neoplasias Mamárias Experimentais/genética , Metástase Neoplásica/fisiopatologia , Proteínas de Neoplasias/fisiologia , Estresse Fisiológico/genética , Macrófagos Associados a Tumor/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Ciclofosfamida/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genótipo , Humanos , Neoplasias Pulmonares/metabolismo , Ativação de Macrófagos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Terapia Neoadjuvante/efeitos adversos , Metástase Neoplásica/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias/métodos , Células-Tronco Neoplásicas/patologia , Migração Transendotelial e Transepitelial , Microambiente Tumoral , Macrófagos Associados a Tumor/efeitos dos fármacos
12.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208396

RESUMO

Non-small cell lung cancer (NSCLC) continues to be the leading cause of cancer death worldwide. Recently, targeting molecules whose functions are associated with tumorigenesis has become a game changing adjunct to standard anti-cancer therapy. As evidenced by the results of preclinical and clinical investigations, whole-body irradiations (WBI) with X-rays at less than 0.1-0.2 Gy per fraction can induce remissions of various neoplasms without inciting adverse side effects of conventional chemo- and radiotherapy. In the present study, a murine model of human NSCLC was employed to evaluate for the first time the anti-neoplastic efficacy of WBI combined with inactivation of CTLA-4, PD-1, and/or HSP90. The results indicate that WBI alone and in conjunction with the inhibition of the function of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the programmed death-1 (PD-1) receptor immune checkpoints (ICs) and/or heat shock protein 90 (HSP90) markedly reduced tumorigenesis in mice implanted by three different routes with the syngeneic Lewis lung cancer cells and suppressed clonogenic potential of Lewis lung carcinoma (LLC1) cells in vitro. These results were associated with the relevant changes in the profile of pro- and anti-neoplastic immune cells recruited to the growing tumors and the circulating anti- and pro-inflammatory cytokines. In contrast, inhibition of the tested molecular targets used either separately or in combination with each other did not exert notable anti-neoplastic effects. Moreover, no significant synergistic effects were detected when the inhibitors were applied concurrently with WBI. The obtained results supplemented with further mechanistic explanations provided by future investigations will help design the effective strategies of treatment of lung and other cancers based on inactivation of the immune checkpoint and/or heat shock molecules combined with low-dose radiotherapy.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Checkpoint Imunológico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Transplante de Neoplasias , Dosagem Radioterapêutica , Irradiação Corporal Total , Animais , Células Clonais , Pulmão/patologia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Ensaio Tumoral de Célula-Tronco
13.
Aging (Albany NY) ; 13(13): 17316-17327, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34238763

RESUMO

Anoctamin 5 (ANO5) is a member of the Anoctamin (ANO) family of calcium-activated chloride channels. Although ANO5 expression is upregulated in various cancers, its role in osteosarcoma remains largely unknown. In this study, bioinformatics analysis, western blot, and immunohistochemical staining revealed that ANO5 was upregulated in osteosarcoma cell lines and osteosarcoma tissues, and ANO5 expression was positively associated with tumor size, tumor grade, and metastasis. Functional experiments demonstrated that inhibition of ANO5 decreased, while ANO5 overexpression increased, osteosarcoma cell proliferation and mobility in vitro. Immunoprecipitation, western blot, and confocal microscopy experiments showed that ANO5 bound to and promoted the degradation of Nel-like proteins 1 (NELL1) and 2 (NELL2). Moreover, a subcutaneous tumor transplantation model revealed that ANO5 knockdown reduced osteosarcoma cell proliferation and increased NELL1 and NELL2 expression in vivo. Finally, rescue experiments showed that knockdown of NELL1 or NELL2 reversed the inhibitory effects of ANO5 knockdown on osteosarcoma cell proliferation and migration. These results demonstrated that upregulation of ANO5 promoted osteosarcoma development by decreasing the stability of the NELL1 and NELL2 proteins and that ANO5 may be an effective target for the treatment of osteosarcoma.


Assuntos
Anoctaminas/genética , Neoplasias Ósseas/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas do Tecido Nervoso/genética , Osteossarcoma/genética , Adolescente , Adulto , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Biologia Computacional , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/genética , Transplante de Neoplasias , Osteossarcoma/patologia , Regiões Promotoras Genéticas , Cicatrização , Adulto Jovem
14.
Nutrients ; 13(6)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199311

RESUMO

Despite multimodal treatment strategies, clinical outcomes of advanced stage colorectal cancer (CRC) patients remain poor. Neoadjuvant/adjuvant chemotherapy efficacy is limited due to chemoresistance, toxicity, and negative side effects. Since both melatonin and glycine have anti-cancer activities without relevant side effects, this study was designed to investigate their combined effects in experimental CRC liver metastases. CRC metastasis with CC531 cells were induced in male Wistar rats. Melatonin and glycine alone or their combination were supplemented for 14 days (n = 100). Blood parameters, a micro-computed tomography scan (tumor volume over time), and immunohistochemistry for Ki67 and CD31 expression in tumor tissue were compared between groups. Melatonin and glycine alone significantly reduced the tumor volume by 63.2% (p = 0.002) and 43% (p = 0.044) over time, respectively, while tumor volume increased by 8.7% in the controls. Moreover, treatment with melatonin and glycine alone reduced the tumor proliferation index. Most interestingly, the combination therapy did not have any influence on the above-mentioned tumor parameters. The leukocyte count was significantly increased with melatonin at the end of the experiment (p = 0.012) which was due to a high lymphocytes count. Tumor microvascular density was significantly reduced in all treatment groups. The results of this study suggest an inhibitory function for melatonin and glycine alone in the case of CRC liver metastasis growth by acting as natural antiangiogenic molecules, followed by angiogenesis-dependent cancer proliferation and immunomodulation.


Assuntos
Inibidores da Angiogênese/administração & dosagem , Neoplasias Colorretais/patologia , Glicina/administração & dosagem , Neoplasias Hepáticas/dietoterapia , Neoplasias Hepáticas/secundário , Melatonina/administração & dosagem , Animais , Linhagem Celular Tumoral , Dieta , Contagem de Leucócitos , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/patologia , Masculino , Microvasos , Transplante de Neoplasias , Ratos , Ratos Wistar , Carga Tumoral
15.
Methods Mol Biol ; 2329: 223-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085226

RESUMO

Among the methods for detecting cell cycle kinetics in tumor cells, fluorescent ubiquitination-based cell cycle indicator (Fucci) is innovative because it allows observation in live cells without losing spatiotemporal information. We succeeded in using the Fucci system to visualize radiation-induced G2 arrest in tumor cells with deficient p53 function. Here we describe protocols for establishing Fucci-expressing cell lines and analyzing radiation-induced G2 arrest kinetics in three different models: monolayer cell cultures, spheroids, and xenografted solid tumors in mice.


Assuntos
Técnicas de Cultura de Células/métodos , Ciclo Celular/efeitos da radiação , Neoplasias/metabolismo , Animais , Células HEK293 , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Transplante de Neoplasias , Ubiquitinação
16.
Cancer Sci ; 112(9): 3722-3731, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34115906

RESUMO

The rBC2LCN lectin, known as a stem cell marker probe that binds to an H type 3 fucosylated trisaccharide motif, was recently revealed to also bind to pancreatic ductal adenocarcinoma (PDAC) cells. A lectin-drug conjugate was generated by fusing rBC2LCN with a cytocidal toxin, and it showed a strong anticancer effect in in vitro and in vivo PDAC models. However, it is unclear which molecules are carrier proteins of rBC2LCN on PDAC cells. In this study, we identified a rBC2LCN-positive glycoprotein expressed in PDAC. Tumor lysates of PDAC patient-derived xenografts (PDXs) were coprecipitated with rBC2LCN lectin and analyzed by liquid chromatography-mass spectrometry. A total of 343 proteins were initially identified. We used a web-based database to select five glycoproteins and independently evaluated their expression in PDAC by immunohistochemistry (IHC). Among them, we focused on carcinoembryonic antigen 5 (CEA) as the most cancer-specific carrier protein in PDAC, as it showed the most prominent difference in expression rate between PDAC cells (74%) and normal pancreatic duct cells (0%, P > .0001). rBC2LCN lectin and CEA colocalization in PDAC samples was confirmed by double-staining analysis. Furthermore, rBC2LCN-precipitated fractions were blotted with an anti-CEA polyclonal antibody (pAb), and CEA pAb-precipitated fractions were blotted with rBC2LCN lectin. The results demonstrate that CEA is in fact a ligand of rBC2LCN lectin.


Assuntos
Antígeno Carcinoembrionário/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Transporte/metabolismo , Lectinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Anticorpos/imunologia , Antígeno Carcinoembrionário/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Xenoenxertos , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Ligantes , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Transplante de Neoplasias , Neoplasias Pancreáticas/patologia
17.
Cancer Sci ; 112(8): 3136-3149, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091990

RESUMO

Intratumoral heterogeneity, including in clear cell renal cell carcinoma, is a potential cause of drug resistance and metastatic cancer progression. We specified the heterogeneous population marked by endoglin (also known as CD105) in a preclinical model of clear cell renal cell carcinoma progression. Highly malignant derivatives of human clear cell renal cell carcinoma OS-RC-2 cells were established as OS5Ks by serial orthotopic inoculation in our previous study. Expression of both ENG (encoding endoglin) mRNA and protein were heterogeneously upregulated in OS5Ks, and the endoglin-positive (ENG+ ) population exhibited growth dependency on endoglin in anchorage-independent cultures. Despite the function of endoglin as a type III receptor, transforming growth factor ß and bone morphogenetic protein-9 signaling were unlikely to contribute to the proliferative phenotype. Although endoglin has been proposed as a marker for renal cancer-initiating cells, the OS5K-3 ENG+ population did not enrich other reported cancer-initiating cell markers or differentiate into the ENG- population. Mouse tumor inoculation models revealed that the tumor-forming capabilities of OS5K-3 ENG+ and ENG- cells in vivo were highly dependent on the microenvironment, with the renal microenvironment most preferable to ENG+ cells. In conclusion, the renal microenvironment, rather than the hypothesized ENG+ cell-centered hierarchy, maintains cellular heterogeneity in clear cell renal cell carcinoma. Therefore, the effect of the microenvironment should be considered when evaluating the proliferative capability of renal cancer cells in the experimental settings.


Assuntos
Carcinoma de Células Renais/patologia , Endoglina/genética , Endoglina/metabolismo , Neoplasias Renais/patologia , Regulação para Cima , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral
18.
Cancer Sci ; 112(8): 3278-3292, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091997

RESUMO

It is widely accepted that redox reprogramming participates in malignant transformation of lung adenocarcinoma (LUAD). However, the source of excessive reactive oxygen species (ROS) and the downstream signaling regulatory mechanism are complicated and unintelligible. In the current study, we newly identified the aquaporin 3 (AQP3) as a LUAD oncogenic factor with capacity to transport exogenous hydrogen peroxide (H2 O2 ) and increase intracellular ROS levels. Subsequently, we demonstrated that AQP3 was necessary for the facilitated diffusion of exogenous H2 O2 in LUAD cells and that the AQP3-dependent transport of H2 O2 accelerated cell growth and inhibited rapamycin-induced autophagy. Mechanistically, AQP3-mediated H2 O2 uptake increased intracellular ROS levels to inactivate PTEN and activate the AKT/mTOR pathway to subsequently inhibit autophagy and promote proliferation in LUAD cells. Finally, we suggested that AQP3 depletion retarded subcutaneous tumorigenesis in vivo and simultaneously decreased ROS levels and promoted autophagy. These findings underscore the importance of AQP3-induced oxidative stress in malignant transformation and suggest a therapeutic target for LUAD.


Assuntos
Adenocarcinoma de Pulmão/patologia , Aquaporina 3/genética , Aquaporina 3/metabolismo , Peróxido de Hidrogênio/metabolismo , Neoplasias Pulmonares/patologia , PTEN Fosfo-Hidrolase/metabolismo , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Autofagia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirolimo/farmacologia
19.
Cancer Sci ; 112(8): 3150-3162, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34097336

RESUMO

Glioma is one of the most commonly diagnosed intracranial malignancies. The molecular mechanism underlying the development of glioma is still largely unknown. In this study, we present the first report concerning the function and mechanism of cyclin-dependent kinase-like 3 (CDKL3) in the development and prognosis of glioma. It is shown that CDKL3 was upregulated in glioma tissues and could independently predict poor prognosis of patients. Silencing CDKL3 in glioma cells could inhibit cell proliferation and migration and induce cell apoptosis and cell cycle arrest, whereas the overexpression of CDKL3 promoted cell proliferation. The in vivo experiments also indicated that knockdown of CDKL3 significantly suppressed tumor growth of glioma. Gene expression profiling of CDKL3 knockdown U87 cells identified RRM2 as a potential target of CDKL3, which was proved to have direct interaction with CDKL3. Given similar effects on glioma development with CDKL3, knockdown of RRM2 could rescue the effects of CDKL3 overexpression on glioma cells. Moreover, knockdown of CDKL3 or RRM2 suppressed the activity of JNK signaling, whereas CDKL3 overexpression produced the opposite effect. In conclusion, our results identified CDKL3 as a promotor for glioma, probably through the regulation of RRM2 and activation of the JNK signalling pathway, highlighting the significance of CDKL3 as a promising therapeutic target of glioma.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleosídeo Difosfato Redutase/genética , Regulação para Cima , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Transplante de Neoplasias , Prognóstico , Ribonucleosídeo Difosfato Redutase/metabolismo , Análise de Sobrevida
20.
Cancer Sci ; 112(8): 3243-3254, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34097350

RESUMO

RNA N6 -methyladenosine (m6 A) is an emerging regulatory mechanism for tumor progression in several types of cancer. However, the underlying regulation mechanisms of m6 A methylation in colorectal cancer (CRC) remain unknown. Although the oncogenic function of methyl CpG binding protein 2 (MeCP2) has been reported, it is still unclear whether MeCP2 could alter RNA m6 A methylation state. Here, we systematically identified MeCP2 as a prometastasis gene to regulate m6 A methylation in CRC. Interestingly, MeCP2 could bind to methyltransferase-like 14 (METTL14) to coregulate tumor suppressor Kruppel-like factor 4 (KLF4) expression through changing m6 A methylation modification. Furthermore, insulin-like growth factor 2 mRNA-binding protein 2 recognized the unique modified m6 A methylation sites to enhance KLF4 mRNA stability. Taken together, these findings highlight the novel function of MeCP2 for regulating m6 A methylation and reveal the underlying molecular mechanism for the interaction between MeCP2 and METTL14, which offers a better understanding of CRC progression and metastasis.


Assuntos
Adenosina/análogos & derivados , Neoplasias Colorretais/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Metiltransferases/genética , Regulação para Cima , Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Camundongos , Transplante de Neoplasias , Estabilidade de RNA
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