The First Part Of Interferon ?-1a Induces Apoptosis Of Laryngeal Carcinoma Hep-2 Cells By Activating Endogenous Mitochondrial Apoptosis Pathway And Endoplasmic Reticulum Stress Apoptotic Pathway. The Second Part Of Microfluidic Western Blot Hybridization

Background and objectives:Traditionally,surgical therapy plus radiotherapy are the main popular treatments for patients with laryngeal carcinoma,while the potential voice loss and extensive rehabilitation after the oncologic larynx surgery may be detrimental to the quality of the patient's life.Thus,novel therapeutic approach to effectively restrict the neoplasm growth should be highly demanded.Interferon(IFN)is a well known cytokine with potent anti-viral,anti-proliferative and immuno-modulatory capacity.Interferon alpha(IFN a)exerts its anti proliferative effect on human laryngeal carcinoma cells.However,the mechanism underlining this IFN a mediated growth inhibition is still not completely explored.We used Hep-2 cell line as a model system to study the molecular mechanism of the IFN?-la mediated anti-proliferaion in laryngeal carcinoma cells.Materials and methods:Hep-2 cells were treated with IFN?-1a through either transient transfection or exogenous addition of the recombinant proteins.CCK8 and MTT tests were used to detect the anti-proliferative effect of IFN ?-1 a on Hep-2 cells,flow cytometric analysis showed that IFN?-1a promoted the apoptosis of Hep-2 cells.Western Blot analysis was employed to analyse the specific apoptosis pathways activated by IFN?-1a.To test the general effect of IFN?-1a on other cell lines,we tested the effect of the pro-apoptosis of IFN?-la to other cells,such as HEK-293T,HepG2 and A549 cells.Results:The results demonstrate that IFN?-1a markedly inhibited the proliferation and significantly promoted the apoptosis of Hep-2 cells.IFNa-la dramatically inhibited the expression of the anti apoptotic protein B cell lymphoma-XL(Bcl-XL),increased the cytoplasmic distribution of cytochrome c,promoted the cleavages of both caspase 3 and poly(ADP-Ribose)polymerase 1(PARP1)in a doses dependent manner,indicating that cell-intrinsic apoptotic pathway was activated by the over-expression of IFN?-1a gene products.IFN?-1a dramatically enhanced the expressions of GRP78 and CHOP and markedly promoted the activation of caspase 3 and caspase 4,indicating that IFN?-1a indeed promotes the ER stress mediated apoptosis.However,IFN?-1a failed to promote the cleavages of caspase 8 and 10,indicating that IFNo?-1a mediated apoptosis is independent of cell-extrinsic apoptotic pathway.In addtion,IFN?-1a did not induce the significant apoptosis of some other cell lines such as HEK293T,HepG2 and A549 cells.Conclusion:Mechanistic studies indicate that IFN ?-1 a mediated cell apoptosis was directly linked to the intrinsic as well as the ER-stress related apoptosis,but was independent of extrinsic apoptosis.There was no significant effect on HEK293T,HepG2 and A549 cells,indicating that IFN?-1a mediated apoptosis might have cell specific character.In conclusion,Current study unravels a detailed mechanism involved in IFNa-la mediated anti-proliferation in human laryngeal carcinoma cells.Background and instruction:Western Blotting,one of the popular application technologies used in the protein research in labs,can take a qualitative research and semi-quantitative research on the interest protein.Although Western Blotting has a huge application market,it actually do exist some disadvantages for researchers,such as,the large amount of required samples,the time and labor cost.Here,based on the principle of the reverse phase protein assay(RPPA),we develop a new Western Blotting-multiplex microfluidic paper-based immunoassay to take the qualitative and semi-quantitative research on the interest protein.Compared to the traditional Western Blotting,the new Western Blotting-multiplex microfluidic paper-based immunoassay has multiple advantages,the required sample as low as 1?L in each detecting channel,saving regents as low as 20mL,saving time as low as 1 hour,saving labors,high-throughput detection and so on.Especially,owing to the advantages of the microfludic paper chip of high specific surface area and capillary force,the paper chip Western Blotting can detect much smaller amounts of protein,as low as the pg level.Materials and Methods:In this paper,we chosen MGMT as our interest protein and compared the qualitative and semi-quantitative results of the MGMT expression in the MCF7 cells which was treated with MGMT inhibitor(Lomeguatrib)between the traditional Western Blotting and the paper chip immunoassay.Results:Microfluidic paper-based immunoassay can take the qualitative and relative quantitative detection.Compared to the 1-5ng of the traditional Western Blot,the Microfluidic paper-based immunoassay could detect as low as 10-25 pg in the protein detection limit.It improved 3 orders of magnitude.The entire operational process took only 1 hours,the reagent consume was less,the test cost was even low and it could make the high-throughput protein detection come true as the RPPA does.Conclusions:The experimental results revealed that the paper chip immunoassay could take the same qualitative and semi-quantitative detection for the MGMT as the traditional Western Blotting.Further,the microfludic paper chip could detect much smaller amounts of protein,detect high throughput samples as the reverse phase protein assay do,save more sample and reagent,save time,money and labor.