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1.
J Vis Exp ; (168)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616115

RESUMO

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

2.
Dermatol Ther ; : e14751, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33406278

RESUMO

Aberrant expression of long non-coding RNA (lncRNA) zinc finger protein, FOG family member 2 antisense RNA 1 (ZFPM2-AS1) has been identified in many tumors, but its role in cutaneous malignant melanoma remains largely obscure. Our present study was intended to unveil the role and potential mechanism of ZFPM2-AS1 in cutaneous malignant melanoma. RT-qPCR was utilized to analyze ZFPM2-AS1 expression in cutaneous malignant melanoma cells. Cell counting kit-8 (CCK-8), colony formation, flow cytometry, and transwell analyses were utilized to assess ZFPM2-AS1 function on cell proliferation, apoptosis, and migration. Luciferase reporter, RNA immunoprecipitation, and RNA-pull down assays were applied to probe the regulatory mechanism of ZFPM2-AS1 in cutaneous malignant melanoma cells. Up-regulation of ZFPM2-AS1 was discovered in cutaneous malignant melanoma cells. ZFPM2-AS1 deletion restrained cell proliferation, migration, and elevated cell apoptosis in cutaneous malignant melanoma. ZFPM2-AS1 regulated notch receptor 1 (NOTCH1) to activate the NOTCH pathway. ZFPM2-AS1 acted as a competing endogenous RNA (ceRNA) to affect NOTCH1 expression via sponging miR-650. Collectively, ZFPM2-AS1 exerted an oncogenic role in cutaneous malignant melanoma progression via targeting miR-650/NOTCH1 signaling. Our study might offer a novel sight for cutaneous malignant melanoma treatment.

3.
Commun Biol ; 4(1): 72, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452400

RESUMO

Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited tumor growth and metastasis in IBC xenograft mouse models. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology.

4.
Acta Neuropathol ; 141(2): 303-321, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33394124

RESUMO

The deadly complication of brain metastasis (BM) is largely confined to a relatively narrow cross-section of systemic malignancies, suggesting a fundamental role for biological mechanisms shared across commonly brain metastatic tumor types. To identify and characterize such mechanisms, we performed genomic, transcriptional, and proteomic profiling using whole-exome sequencing, mRNA-seq, and reverse-phase protein array analysis in a cohort of the lung, breast, and renal cell carcinomas consisting of BM and patient-matched primary or extracranial metastatic tissues. While no specific genomic alterations were associated with BM, correlations with impaired cellular immunity, upregulated oxidative phosphorylation (OXPHOS), and canonical oncogenic signaling pathways including phosphoinositide 3-kinase (PI3K) signaling, were apparent across multiple tumor histologies. Multiplexed immunofluorescence analysis confirmed significant T cell depletion in BM, indicative of a fundamentally altered immune microenvironment. Moreover, functional studies using in vitro and in vivo modeling demonstrated heightened oxidative metabolism in BM along with sensitivity to OXPHOS inhibition in murine BM models and brain metastatic derivatives relative to isogenic parentals. These findings demonstrate that pathophysiological rewiring of oncogenic signaling, cellular metabolism, and immune microenvironment broadly characterizes BM. Further clarification of this biology will likely reveal promising targets for therapeutic development against BM arising from a broad variety of systemic cancers.

5.
Oncogene ; 37(22): 2921-2935, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29515234

RESUMO

ADP-ribosylation, including poly-ADP-ribosylation (PARylation) and mono-ADP-ribosylation (MARylation), is a multifunctional post-translational modification catalyzed by intracellular ADP-ribosyltransferases (ARTDs or PARPs). Although PARylation has been investigated most thoroughly, the function of MARylation is currently largely undefined. Here, we provide evidences that deficiency of PARP10, a mono-ADP-ribosyltransferase, markedly increased the migration and invasion of tumor cells through regulation of epithelial-mesenchymal transition (EMT), and PARP10 inhibited tumor metastasis in vivo, which was dependent on its enzyme activity. Mechanistically, we found that PARP10 interacted with and mono-ADP-ribosylated Aurora A, and inhibited its kinase activity, thereby regulating its downstream signaling. Moreover, the expression level of PARP10 was downregulated in intrahepatic metastatic hepatocellular carcinoma (HCC) compared with its corresponding primary HCC and adjacent non-tumorous tissues. Taken together, our results indicated that PARP10 has an important role in tumor metastasis suppression via negatively regulation of Aurora A activity.


Assuntos
Aurora Quinase A/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/secundário , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Poli(ADP-Ribose) Polimerases/deficiência , Proteínas Proto-Oncogênicas/deficiência , Animais , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Transplante de Neoplasias , Transdução de Sinais
6.
Tumour Biol ; 39(10): 1010428317713390, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29064327

RESUMO

Triple-negative breast cancer is a kind of breast cancer with poor prognosis and special biological behavior, which lacked endocrine therapy and targeted therapy. We investigate the effect of human APE1 (apurinic/apyrimidyl endonuclease 1), a rate-limiting enzyme of base excision repair, on the prognosis in triple-negative breast cancer and drug sensitivity of olaparib. The expression of APE1 was detected by immunohistochemistry in the triple-negative breast cancer tissues and its effect on survival of triple-negative breast cancer patients was followed. To find whether APE1 effect the drug sensitivity in triple-negative breast cancer cells, the APE1-knockout HCC1937 cell line (triple-negative breast cancer cell line) was established by CRISPR/Cas9 system. Then, we use the wild-type and knockout one to test the drug sensitivity of olaparib. The expression of APE1 in triple-negative breast cancer tissues was significantly higher than that in the adjacent tissues (85.6% vs 14.4%) and its expression was related to tumor size (p < 0.05). We also found that it is an independent prognostic factor in patients with triple-negative breast cancer (overall survival, p = 0.01). In vitro assay, the half maximal inhibitory concentration of olaparib in HCC1937-APE1-KO was significantly increased (17.22 vs 91.85 µM) compared to the wild type. The growth curve showed that olaparib had a stronger lethality on HCC1937 compared to HCC1937- APE1-KO (p < 0.05 on day 3). HCC1937 resulted in more mitotic G2/M arrest and increased apoptosis rate after treatment with 40 µM of olaparib, while HCC1937-APE1-KO did not change significantly. When HCC1937 was treated with different concentrations of olaparib, it was found that APE1 expression decreased more significantly at 15 µM of olaparib was. In HCC1937-APE1-KO, the expression of endogenous poly (ADP-ribose) polymerase 1 was also less than that of HCC1937. These results suggested that the expression of APE1 was an important basis for the maintenance of poly (ADP-ribose) polymerase 1, and the deletion of APE1 may be related to the resistance of olaparib.


Assuntos
Antineoplásicos/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Técnicas de Inativação de Genes , Humanos , Imuno-Histoquímica , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Análise Serial de Tecidos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/mortalidade
7.
Neoplasia ; 19(7): 583-593, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28628795

RESUMO

Human hepatocellular carcinoma (HCC) is a malignant cancer. It is a challenge to develop anti-HCC drugs due to HCC's extreme aggressiveness and with the sensitivity of the liver to show severe adverse effects. More importantly, the precise mechanisms causing HCC pathogenicity are not known. Our previous study disclosed Nogo-B as a reticulon 4 (Rtn4) family member. In the present study, we first identified that Nogo-B played a critical role in HCC progression. We found, via in vitro and in vivo assays, that Nogo-B was expressed aberrantly in primary HCC tumor tissues and immortal HCC cells but was relatively scarce in the normal liver tissues or cells. Nogo-B knockout, via the CRISPR-Cas9 technique, resulted in significant suppression of HCC cell proliferation and tumor growth. Next-generation sequencing analysis showed that Nogo-B knockout have effects on interleukin-6 (IL-6) signaling pathway. Furthermore, we observed that IL-6 induced phosphorylation of STAT3 (pSTAT3) in wild-type HCC cells, but Nogo-B knockout could reduce IL-6-induced increase of pSTAT3, supporting that Nogo-B affects HCC tumor progression possibly via regulating the IL-6/STAT3 signaling pathway. In conclusion, Nogo-B is expressed aberrantly in HCCs and plays an oncogenic role. These findings support that Nogo-B may be a novel anti-HCC therapeutic target.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Nogo/genética , Adulto , Idoso , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Feminino , Expressão Gênica , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Proteínas Nogo/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo , Carga Tumoral
8.
Biochem Biophys Res Commun ; 488(1): 211-217, 2017 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-28487110

RESUMO

Poly (ADP-ribose) polymerase 1 (PARP1) is an ADP- ribosylation enzyme and plays important roles in a variety of cellular processes, including DNA damage response and tumor development. However, the post-transcriptional regulation of PARP1 remains largely unknown. In this study, we identified that the mRNA of PARP1 is associated with nuclear factor 90 (NF90) by RNA immunoprecipitation plus sequencing (RIP-seq) assay. The mRNA and protein levels of PARP1 are dramatically decreased in NF90-depleted cells, and NF90 stabilizes PARP1's mRNA through its 3'UTR. Moreover, the expression levels of PARP1 and NF90 are positively correlated in hepatocellular carcinoma (HCC). Finally, we demonstrated that NF90-depleted cells are sensitive to PARP inhibitor Olaparib (AZD2281) and DNA damage agents. Taken together, these results suggest that NF90 regulates PARP1 mRNA stability in hepatocellular carcinoma cells, and NF90 is a potential target to inhibit PARP1 activity.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas do Fator Nuclear 90/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas do Fator Nuclear 90/isolamento & purificação , RNA Mensageiro/genética
9.
Oncol Lett ; 11(2): 1173-1178, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26893714

RESUMO

Liver cancer is one of the most common types of cancer, and hepatoma demonstrates a poor long-term prognosis. The present study reports that glaucocalyxin A (GLA), a natural product isolated from Rabdosia umbrosa, inhibits the growth of the liver cancer Focus and SMMC-7721 cell lines in a dose- and time-dependent manner. The present study revealed that GLA arrested the liver cancer cells at the G2/M stage of the cell cycle and led to decreased expression of caspase 3 and the cleavage of poly(adenosine diphosphate-ribose) polymerase. Overall, the present study demonstrated that GLA inhibits the growth of liver cancer cells by G2/M stage cell-cycle arrest and cell apoptosis.

10.
PLoS One ; 8(4): e60414, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23577108

RESUMO

OBJECTIVE: To down-regulate expression of mRNA for the platelet-derived growth factor receptor (PDGFR)-α, block the signalling pathway of PDGF and its receptor, and study their influence on fibroblast transdifferentiation to myofibroblasts in systemic sclerosis (SSc). METHODS: Fibroblasts from skin lesions of SSc patients and health adult controls were cultured in vitro, and α-smooth muscle actin (α-SMA) expression was determined by immunocytochemistry. Both groups of fibroblasts were stimulated with PDGF-AA, transforming growth factor ß1 (TGF-ß1), and costimulated with PDGF-AA and TGF-ß1, then PDGFR-α and α-SMA mRNA and protein expression were detected with RT-PCR and WB respectively. Three pairs of siRNAs targeting different PDGFR-α mRNA sequences were synthesized for RNAi. SSc and control fibroblasts were transfected with PDGFR-α siRNA; stimulated with PDGF-AA; and assessed for PDGFR-α and α-SMA mRNA and protein expression. RESULTS: Although the fibroblasts from both groups had similar morphology, the SSc skin lesions had significantly more myofibroblasts than control skin lesions. PDGF-AA stimulation, TGF-ß1 stimulation, and costimulation significantly up-regulated PDGFR-α and α-SMA mRNA and protein expression in SSc fibroblasts compared to control (P<0.05), and costimulation had the strongest effects (P<0.05). All three pairs of siRNAs suppressed PDGFR-α mRNA and protein expression (P<0.05), but siRNA1495 had the highest gene-silencing efficiency (P<0.05). PDGFR-α siRNA attenuated the effects of PDGF-AA through up-regulating PDGFR-α and α-SMA mRNA and protein expression and inhibiting fibroblast transdifferentiation to myofibroblasts in SSc (P<0.05). CONCLUSIONS: PDGFR-α over-expression in SSc fibroblasts bound PDGF-AA more efficiently and promoted fibroblast transdifferentiation, which was enhanced by TGF-ß1. PDGFR-α siRNA down-regulated PDGFR-α expression, blocked binding to PDGF-AA, and inhibited fibroblast transdifferentiation to myofibroblasts.


Assuntos
Transdiferenciação Celular/genética , Fibroblastos/patologia , Interferência de RNA , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/deficiência , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Actinas/genética , Adulto , Transdiferenciação Celular/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/farmacologia
11.
Nan Fang Yi Ke Da Xue Xue Bao ; 31(11): 1840-5, 2011 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-22126761

RESUMO

OBJECTIVE: To explore the role of the fibroblast transdifferentiation into myofibroblasts (MFBs) in the pathogenesis of systemic sclerosis (SSc) and investigate the influence of transforming growth factor ß(1) (TGF-ß(1)) and blocking of its signal transduction on fibroblast transdifferentiation. METHODS: Fibroblasts derived from the skin lesions of SSc patients and normal skin tissue were cultured in vitro. The proportion of MFBs in the fibroblast culture was analyzed qualitatively using immunocytochemistry and quantitatively with ELISA for α-smooth muscle actin (α-SMA). The changes in fibroblast transdifferentiation were observed after addition of TGF-ß(1) in the cell culture and after blocking the signal transduction of TGF-ß(1). RESULTS: The fibroblasts isolated from SSc patients and control subjects showed a similar morphology. The mean number of cells positive for α-SMA in SSc group was significantly higher than that in the control group (P<0.01). As culture time extended, α-SMA levels of the two groups both increased gradually (P<0.01), but the increments were significantly greater in SSc group than in the control group at 24, 48, and 72 h (P<0.05 all). Addition of TGF-ß(1) resulted in significantly increased α-SMA levels in both groups (P<0.05), and SSc group showed significantly higher α-SMA levels than the control group at 24, 48, and 72 h (P<0.01). In the presence of TGF-ß(1), blocking of Smads, ERK/MAPK, and p38MAPK pathways, but not JNK/MAPK pathway, caused an obvious decrease in α-SMA levels in the fibroblasts in both groups. CONCLUSION: The fibroblasts in the skin lesion of SSc patients have strong potential of transdifferentiation into MFBs, and TGF-ß(1) can promote this transdifferentiation process possibly involving Smads, and ERK/MAPK, and p38MAPK signalling pathways.


Assuntos
Transdiferenciação Celular/fisiologia , Fibroblastos/patologia , Miofibroblastos/patologia , Escleroderma Sistêmico/patologia , Fator de Crescimento Transformador beta1/metabolismo , Actinas/metabolismo , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Transdução de Sinais , Pele/patologia
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 27(12): 1312-4, 1318, 2011 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-22152813

RESUMO

AIM: The study was to explore the effects of the fibroblast transdifferentiation for myofibroblast (MFB) in the pathogenesis of systemic sclerosis (SSc) and to research the effect mechanism of H2 Relaxin (H2-RLX) against fibrosis in SSc. METHODS: The fibroblasts derived from the skin lesion of the SSc patients and normal skin tissue were respectively cultured in vitro and demonstrated. The MFB proportion in fibroblast was known by α-smooth muscle actin (α-SMA) in fibroblast culture detected with immunohistochemistry for qualitative study and ELISA for quantitative analysis. The influence on fibroblast proliferation and transdifferentiation for MFB was observed by adding H2-RLX. RESULTS: There were no apparent differences for cell morphology between the fibroblasts from SSc patients and controls. The means of positive α-SMA in SSc group were higher than those in controls (P<0.01). With culture time extended, α-SMA levels of the two groups all increased gradually (P<0.01 all), but there were higher α-SMA levels in SSc group than those in controls separately at H24, H48, H72 in culture (P<0.05 all). Fibroblast proliferation and α-SMA levels were not influenced after adding 1 µg/L of H2-RLX, but 10 µg/L and 100 µg/L of it could completely inhibit the fibroblast proliferation and α-SMA levels (P<0.05 all), with the strongest repressing effect after adding 100 µg/L of it. CONCLUSION: There is a specific property of fibroblasts transdifferentiation for MFB strongly from the skin lesion of the SSc patients. H2-RLX could give play to the antifibrotic effects by repressing the fibroblast proliferation and transdifferentiation for MFB in SSc.


Assuntos
Proliferação de Células , Transdiferenciação Celular , Miofibroblastos/patologia , Relaxina/fisiologia , Escleroderma Sistêmico/patologia , Pele/patologia , Actinas/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Escleroderma Sistêmico/metabolismo
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