Identification of a novel autophagic inhibitor cepharanthine to enhance the anti-cancer property of dacomitinib in non-small cell lung cancer

OBJECTIVE Identification of novel autophagy inhibitors for the combinational treatment of non-small cell lung cancer(NSCLC).METHODS MTT assay and annexin V/PI staining assay were used to evaluate the cell proliferation and apoptosis,respectively.Immunofluorescence staining and cathepsin activity assay were used to detect autophagy.Small interfering RNA was performed to silence the genes and Western blot assay was used to evaluate the protein express levels.Xenograft experiments were applied for in vivo evaluation.RESULTS Cepharanthine,a natural compound,increased LC3-II expression and GFP-LC3 puncta formation in NSCLC NCI-H1975 cells.Numerous yellow puncta were observed in cepharanthine-treated cells with m RFP-EGFP-LC3 transfection.Co-staining of GFP-LC3 with LysoT racker red or LAMP1 antibody suggested that cepharanthine inhibits autophagosomes-lysosomes fusion.Moreover,cepharanthine attenuated the lysosomal cathepsins maturation.We also confirmed that dacomitinib induced cytoprotective autophagy.Combined treatment with cepharanthine increased the anti-cancer effects of dacomitinib in vitro and in vivo.Besides,cepharanthine could not enhance the anti-cancer effect of dacomitinib in autophagy deficient cells.CONCLUSION Cepharanthine might be further developed as a promising autophagic inhibitor,and combined treatment cepharanthine with dacomitinib could pose as an effective strategy for NSCLC treatment.

Antiviral activity of cepharanthine against severe acute respiratory syndrome coronavirus in vitro

Severe acute respiratory syndrome(SARS) is the first severe viral epidemic we encountered his century,which once spread in more than thirty countriesin2003.1 The etiological agent of SARS has beenc onfirmed to be a novel coronavirus,namely SARS coronavirus(SARS-CoV),2,3 and the first outbreak of SARS has been successfully controlled world wide,but the identification of SARS-CoV isolated from wildanimals,the emergence of some sporadic SARS cases laterafter that outbreak,all suggest that the recurrence of such an epidemic is not unlikely in the future.In this case,development of SARS vaccines and specific drugs is undoubtedlyessential to the control and prevention from the possible outbreak.4,5

N-glycoproteomic profiling revealing novel coronavirus therapeutic targets potentially involved in Cepharanthine's intervention

The Coronavirus disease 2019(COVID-19)has posed a serious threat to global health and the world economy.Antiviral therapies targeting coronavirus are urgently required.The Cepharanthine(CEP)is a traditional Chinese herbal extract.Our previous research revealed that CEP has a very potent anti-coronavirus effect,but its mechanism of action was not fully understood.To investigate the effect of novel coronavirus on protein glycosylation in infected cells and to further investigate the mechanism of action of CEP against coronavirus,a cellular model using coronavirus GX_P2V infection of Vero E6 cells was established.The effect of coronavirus GX_P2V on host cell protein glycosylation was investigated by N-glycoproteomic analysis,and the antagonistic effect of CEP on the abnormal protein glycosylation caused by coronavirus was analyzed.The results showed that GX_P2V could cause abnormal changes in protein glycosylation levels in host cells,while CEP could partially antagonize the abnormal protein glycosylation caused by GX_P2V.In addition,we also found that CEP could regulate the glycosylation level of coronavirus S protein.In conclusion,this article provides important ideas about the infection mechanism of novel coronaviruses,providing evidence for CEP as a promising therapeutic option for coronavirus infection.

Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model

Background:Medicines for the treatment of 2019-novel coronavirus(2019-nCoV)infections are urgently needed.However,drug screening using live 2019-nCoV requires high-level biosafety facilities,which imposes an obstacle for those institutions without such facilities or 2019-nCoV.This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019(COVID-19)in a 2019-nCoV-related coronavirus model.Methods:A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described.Whether GX_P2V uses angiotensin-converting enzyme 2(ACE2)as the cell receptor was investigated by using small interfering RNA(siRNA)-mediated silencing of ACE2.The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection.Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection.The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated.Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction(qRT-PCR)and plaque assay,respectively.Results:The spike protein of coronavirus GX_P2V shares 92.2%amino acid identity with that of 2019-nCoV isolate Wuhanhu-1,and uses ACE2 as the receptor for infection just like 2019-nCoV.Three drugs,including cepharanthine(CEP),selamectin,and mefloquine hydrochloride,exhibited complete inhibition of cytopathic effects in cell culture at 10μmol/L.CEP demonstrated the most potent inhibition of GX_P2V infection,with a concentration for 50%of maximal effect[EC50]of 0.98μmol/L.The viral RNA yield in cells treated with 10μmol/L CEP was 15,393-fold lower than in cells without CEP treatment([6.48±0.02]×10-4vs.1.00±0.12,t=150.38,P<0.001)at 72 h post-infection(p.i.).Plaque assays found no production of live viruses in media containing 10μmol/L CEP at 48 h p.i.Furthermore,we found CEP had potent anti-viral activities against both viral entry(0.46±0.12,vs.1.00±0.37,t=2.42,P<0.05)and viral replication([6.18±0.95]×10-4vs.1.00±0.43,t=3.98,P<0.05).Conclusions:Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research.CEP,selamectin,and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection.Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus,and further study of CEP for treatment of 2019-nCoV infection is warranted.