Can remdesivir and its parent nucleoside GS-441524 be potential oral drugs? An

Remdesivir (RDV) is the only US Food and Drug Administration (FDA)-approved drug for treating COVID-19. However, RDV can only be given by intravenous route, and there is a pressing medical need for oral antivirals. Significant evidence suggests that the role of the parent nucleoside GS-441524 in the clinical outcomes of RDV could be largely underestimated. We performed an

Effect of adenosine kinase small interfering RNA modified corneal endothelial cells on the proliferation and secretion of regulatory T cells / 中华实验眼科杂志

Objective To investigate the role of adenosine kinase (ADK) small interfering RNA (siRNA) modified corneal endothelial cells on the proliferation and secretion of regulatory T cells (Treg cells).Methods The experiment was divided into transfected group and non-transfected group,FAM-ADK siRNA was transfected into corneal endothelial cells with Lipofectamine 3000 in transfected group,and the average mass concentration of adenosine was detected by high performance liquid chromatography in both groups.The experiment was divided into control group (transfected with control siRNA),rapamycin group (added 10 ng/ml rapamycin to culture supernatant after transfected with control siRNA) and ADK siRNA group (added 10 ng/ml rapamycin to culture supernatant after transfected with ADK siRNA).TUNEL staining was used to detect the proportion of apoptosis.Flow cytometry was used to detect the expressions of intercellular adhesion molecule-1 (ICAM-1),vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in corneal endothelial cells.The cells were divided into single cultured group (lymphocytes were cultured separately),co-culture group (lymphocytes were co-cultured with corneal endothelial cells) and ADK siRNA group (lymphocytes were co-cultured with ADK siRNA transfected corneal endothelial cells),and the proportion of CD4+ CD25+ Foxp3 + Treg cells was detected by flow cytometry,and the content of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the supernatant was detected by ELISA in the three groups.The access and use of clinical specimens was approved by Ethic Committee of Tianjing Taida Hospital (TD201705140901).Results Flow cytometry showed that 95.1％ of the endothelial cells expressed siRNA.The average mass concentration of adenosine in the supernatant of the transfection group was significantly higher than that in the control group ([38.020±6.658] ng/ml vs.[1.663 ±0.581] ng/ml) (t =5.437,P =0.006).TUNEL staining showed that the average apoptotic cell proportion of corneal endothelial cells in the rapamycin group was significantly higher than that in the control group,and the average apoptotic cell proportion of corneal endothelial cells in the ADK siRNA group was significantly lower than that in the rapamycin group,with significant differences between them (t =3.763,P =0.020;t =4.405,P =0.012).Flow cytometry showed that the average fluorescence intensities of ICAM-1,VCAM-1 and E-selectin in the ADK siRNA group was weaker than that in the control group (4.060± 1.179 vs.11.600±2.427,3.600 ± 1.234 vs.11.030 ± 2.291,5.223 ± 1.734 vs.24.270 ± 4.332),with significant differences between them (t =2.794,P =0.049;t =2.857,P =0.046;t =4.081,P =0.015).The proportions of Treg cells in the single cultured group,co-culture group and ADK siRNA group was significantly different (F =12.890,P =0.007),and the proportion of Treg cells in the ADK siRNA group was significantly lower than that in the co-culture group (t =3.650,P =0.022).ELISA assay showed that,the contents of IL-10 and TGF-β in the supernatant in the single cultured group,co-culture group and ADK siRNA group were significantly different (F =20.960,P =0.003;F =27.320,P=0.001),and the content of IL-10 and TGF-β in the supernatant in the ADK siRNA group was significantly higher than that in the co-culture group,respectively (t =4.492,P =0.011;t =5.280,P =0.006).Conclusions ADK siRNA modified corneal endothelial cells can induce Treg cells to proliferate and secrete IL-10 and TGF-β,which provides a new method for induction of corneal allograft immune tolerance.

Expression levels of serum osteopontin, adenosine kinase 1 and Dikkopf 1 in patients with lung cancer and their clinical significances / 肿瘤研究与临床

Objective To observe the expression levels of serum osteopontin (OPN),adenosine kinase 1 (TK1) and secretory protein Dikkopf 1 (DKK1) in patients with lung cancer and their clinical significances.Methods Lung cancer patients treated in the First Affiliated Hospital of Xi'an Medical University from February 2017 to April 2018 were selected as the lung cancer group (60 cases),and 60 healthy adults who received physical examination in the same period were selected as the control group.The differences of serum OPN,TK1 and DKK1 levels between the lung cancer group and the control group and lung cancer patients with different characteristics were compared.Measurement data were compared by using t test.Results The levels of serum OPN,TK1 and DKK1 in lung cancer patients were (38.56±3.18) μg/L,(4.69±1.03) pmol/L and (3.76±0.89) ng/ml,respectively,which were higher than those in the control group [(15.98±2.06) μg/L,(1.01±0.22)pmol/L,(1.21±0.24) ng/ml;t =-46.162,-27.064,-21.428,all P ＜ 0.01].The levels of serum OPN,TK1 and DKK1 in lung cancer patients of different ages and gender had no statistical differences (all P ＞ 0.05).The levels of serum OPN,TK 1 and DKK1 in stage Ⅲ-Ⅳ lung cancer patients were (57.18 ±3.12) μg/L,(6.26±1.28) pmol/L and (4.98±1.03) ng/ml,respectively,which were higher than those in stage Ⅰ-Ⅱ lung cancer patients [(30.35±2.96) μg/L,(3.49±0.67) pmol/L,(3.01±0.96) ng/ml;t =-34.156,-10.690,-7.665,all P ＜ 0.01].The levels of serum OPN,TK 1 and DKK1 in non-small cell lung cancer (NSCLC) patients were (55.13±5.02) μg/L,(5.96±1.11) pmol/L and (5.02±1.32) ng/ml,respectively,which were higher than those in small cell lung cancer (SCLC) patients [(29.68±3.16) μg/L,(3.13±0.98) pmol/L,(2.86±0.56) ng/ml;t =-22.353,-10.213,-7.688,all P ＜ 0.01].Conclusion The levels of serum OPN,TK1 and DKK1 in patients with lung cancer are higher,which are related to the type and stage of lung cancer.

Efeitos da 5-iodotubercidina em linhagens de melanoma humano parentais e resistentes ao inibidor de BRAF / Effects of 5-iodotubercidin on human melanoma lines of parental and resistant to the BRAF inhibitor

O melanoma é responsável por menos de 5% dos cânceres de pele, porém, 95% das mortes ocorrem devido a ocorrência de metástases. O melanoma metastático é refratário às terapias convencionais e rapidamente adquire resistência às terapias como as oncogene-dirigidas, como o inibidor de BRAF, da via de MAPK. Estudos prévios de screening in silico do nosso grupo, onde se utilizou as bases de dados TCGA e GEO, identificaram o gene adenosina quinase (ADK) como sendo diferencialmente expresso entre o melanoma invasivo e os nevus. A 5-iodotubercidina (5-ITu) é um potente inibidor farmacológico da ADK que dentre os diversos efeitos relatados na literatura destaca-se pelo potencial genotóxico. Os danos no DNA são os principais ativadores de checkpoint do ciclo celular, que levam a parada do ciclo celular transitória ou permanente, além de induzir morte celular, levando a hipótese de que ADK possa ser potencial agente anti-melanoma. Este trabalho objetivou avaliar a expressão do gene ADK em melanomas humanos e quimiorresistentes ao inibidor de BRAF (iBRAF), avaliou os impactos de 5-ITu sobre a proliferação, progressão do ciclo celular e morte celular e por fim avaliamos sua capacidade de aumentar a sensibilidade das células. Foi realizado PCR em tempo real para avaliar os níveis de expressão de mRNA de ADK em linhagens de melanoma e na cultura primária de melanócitos; a fim de avaliar a citotoxicidade de 5-ITu foram realizados os ensaios de exclusão por azul de tripan e de apoptose - Anexina V e PI e em modelo de esferoide, usando live/dead; também foi avaliada a influência de 5-ITu sobre a capacidade clonogênica e seus efeitos sobre a proliferação celular, a partir dos ensaios de ciclo celular e avaliação de marcadores de proliferação por imunofluorescência; as linhagens foram submetidas a diferentes regimes de tratamento com 5-ITu e o iBRAF, a fim de avaliar a curva de crescimento e a sensibilidade ao iBRAF por MTT níveis de expressão de mRNA de ADK maiores nas linhagens tumorais em relação aos melanócitos. 5-ITu mostrou-se capaz de inibir a proliferação (IC50) das linhagens de melanoma em concentrações de 1,9 a 3,5 µM. 5-ITu não foi capaz de induzir inviabilidade celular, apesar de reduzir a quantidade de células viáveis em todas as condições de tratamento, também não foi capaz de induzir aumento significativo de células apoptóticas, nem mesmo necróticas. No entanto, o tratamento com 5-ITu reduziu a capacidade clonogênica de linhagens de melanoma e promoveu parada de ciclo celular nas fases G1 e G2/M, levou ao aumento da população subG1. O tratamento com 5-ITu promoveu a redução da expressão de marcadores de proliferação, como ki67, e a combinação de tratamentos 5-ITu e iBraf foi capaz de aumentar o tempo de dobramento das linhagens de melanoma, embora tenha se mostrado incapaz de sensibilizar as células de melanoma ao tratamento com iBRAF. Desse modo, pode-se concluir que 5-ITu induz o efeito citostático e se mostra um potente agente antiproliferativo para melanoma parental e resistente

Melanoma accounts for less than 5% of skin cancers, but 95% of deaths occur due to metastases. Metastatic melanoma is refractory to conventional therapies and rapidly acquires resistance to therapies such as oncogene-directed, such as the BRAF inhibitor, of the MAPK pathway. Previous studies of screening in silico of our group, using the databases TCGA and GEO, identified the adenosine kinase gene (ADK) as differentially expressed between invasive melanoma and nevus. 5-iodotubercidin (5-ITu) is a potent pharmacological inhibitor of ADK that among the several effects reported in the literature stands out for the genotoxic potential. DNA damage is the main activator of the cell cycle checkpoint, which leads to transient or permanent cell cycle arrest, in addition to inducing cell death, leading to the hypothesis that ADK may be a potential anti-melanoma agent. This work aimed to evaluate the expression of the ADK gene in human melanomas and chemoresistants to the BRAF inhibitor (iBRAF), evaluated the impacts of 5-ITu on proliferation, cell cycle progression and cell death and finally we evaluated its ability to increase the sensitivity of cells. Real-time PCR was performed to assess the levels of ADK mRNA expression in melanoma lines and primary melanocyte culture; in order to evaluate the cytotoxicity of 5-ITu, the trypan blue and apoptosis - Annexin V and PI exclusion and blue spheroid models were performed using live / dead; the influence of 5-ITu on the clonogenic capacity and its effects on cell proliferation, from the cell cycle assays and the evaluation of proliferation markers by immunofluorescence; the cell lines were submitted to different treatment regimens with 5-ITu and iBRAF in order to evaluate the growth curve and the sensitivity to iBRAF by MTT levels of mRNA expression of ADK higher in the tumor lines in relation to the melanocytes. 5-ITu was able to inhibit the proliferation (IC 50) of melanoma lines at concentrations of 1.9 to 3.5 181;M. 5-ITu was not able to induce cell non-viability, although it reduced the amount of viable cells in all treatment conditions, nor was it able to induce a significant increase in apoptotic or even necrotic cells. However, treatment with 5-ITu reduced the clonogenic capacity of melanoma cells and promoted cell cycle arrest in the G1 and G2 / M phases, leading to an increase in the subG1 population. Treatment with 5-ITu promotes the reduction of expression of proliferation markers, such as ki67, and the combination of 5-ITu and iBRAF treatments was able to increase the doubling time of melanoma cells, although it has been shown to be unable to sensitize melanoma cells to treatment with iBRAF. Thus, it can be concluded that 5-ITu induces the cytostatic effect and shows a potent antiproliferative agent for parental and resistant melanoma

Effect of morphine on gene expression of enzymes related to purine nucleotides metabolism in PC12 cells / 吉林大学学报(医学版)

Objective To study the effect of morphine on gene expression of enzymes involved in salvage and catabolism pathways of purine nucleotides metabolism in nerve cells.Methods PC12 cells were cultivated and divided into morphine treated groups which were treated with morphine(10 mg?L~(-1) culture) for 12,24,48, 72 h,and control groups which were treated with normal saline for 12,24,48 and 72 h.Total RNA of PC12 cells was isolated.HGPRT,AK,ADK mRNA levels were determined by using the reverse transcription polymerase chain reaction(RT-PCR); the ?-actin mRNA expression was used as an internal control.Results As compared with corresponding control groups,HGPRT mRNA levels in PC12 cells were increased significantly after 12 and 24 h treatment with morphine(P