Ⅰ Chemoresistance Of Human Laryngeal Cancer Stem Cells And The Chemosensitivity Of Targeted Notch Signaling Pathway Ⅱ The Killing Effect Of GP73 Regulated-oncolytic Adenovirus GD55 On Prostate Cancer Stem Cells

Ⅰ.Purpose Head and neck squamous cell carcinoma（HNSCC）is one of the most common malignant cancers with high morbidity and mortality,which seriously threats to human health.As the development of molecular medicine,proteomics and bioinformaticset al,many molecular targets that are associated with HNSCC tumorigenesis and development and involved signaling pathways are identified,including two most important TP53 and Notch.Cisplatin,doxorubicin hydrochloride and others is essential in reducing laryngeal cancer metastasis,improve patient survival,protection of organ function.Targeting or selectively killing cancer stem cells is expected to improve the sensitivity of chemoradiation to reduce cancer metastasis and recurrence.Methods In this study,the suspension culture was used to enrich cancer stem cells in vitro.p CK-Notch1 and p LK0.1-sh RNA vectors were constructed by molecular cloning technique..Laryngeal carcinoma cells were infected with lentiviruses which can silence Notch1 signal or make Notch1 signal overexpression.Hep2^Notch1+ cells and Hep2^Notch1-cells were screened out.The biological characteristics of cell-associated factor and cell cycle were detected by cell counting,QRT-PCR,Weastern blot and other methods.Moreover,the killing effect of DDP for Hep2 WT,Hep2^Notch1+,Hep2^Notch1-cells were measured by MTT assay and flow cytometric analysis.Results Using cloning formation experiment and cell counting method to detect type of laryngeal stem cell found its proliferation ability is weak,many of Hep2 sphere cells in the resting state G0-G1 phase.q RT-PCR and western blot data revels that Hep2 sphere cells over expressed many laryngeal CSCs-assaciated genes（e.g.CD44,ALDH1）and downregulated Notch1,Bax,AKT genes when compared to Hep2 cells.The proliferation of Hep2^Notch1-cells was accelerated.The protein expression of AKT and p21 were downregulated,and the proapoptotic protein Bax was also decreased expression.After treatment with cisplatin,the cell viability was higher than that of wild-type laryngeal carcinoma cells.In contrast,cell proliferation rate of Hep2^Notch1+ cells were decreased.The expression of pro-apoptotic protein Bax were increased.Evenmore,p AKT protein and tumor suppressor protein P21 were also overexpression.The survival rate of Hep2^Notch1+ was significantly lower than Hep2 WT and Hep2^Notch1-cells after treatment with the same concentration of DDP.Conclusion The results show that Aspirin and adenovirus ZD55-TRAIL has strong killing effect to Hep2 sphere cells.DDP,DOX and 5-Fu are on the contrary.Noch1 signal in laryngeal cancer cells inhibite proliferation of laryngeal cancer cell and promote apoptosis of Hep2 cells,and induce the expression of p21 signal which can start the tumor suppressor mechanism and make more cells stay in the G1 period.At the same time,overexpression of Notch1 signaling enhanced the cytotoxicity of chemotherapeutic drug cisplatin to laryngeal carcinoma cells.Blocking the Notch1 signaling pathway can promote the cell proliferation of Hep2^Notch1-,reduce apoptosis,and enhance resistance of Hep2^Notch1-to DDP.Thus,Notch1 signal could be a tumor suppressor in laryngeal cancer.Ⅱ.An oncolytic virus is able to specifically infect and lyse tumor cells,while without causing darnage to nonnal cells.In the last few years,with the significant progress in the fields of cancer biology,virology and modern biotechnology,some oncolytic virus have been developed as anticancer drugs by genetic engineering,and these oncolytic virus can specifically target tumor cell.However,the major limitation for oncolytic virus to acting as anticancer drugs is its ability to infecting tumor cell specifically and efficiently.Currently,a novel GP73-regulated oncolytic adenovirus GD55 was developed to target liver cancer and exhibited obvious cytotoxicity effect.Prostate cancer is one of the major problems of men’s health.It has more obvious cancer heterogeneity,affecting the diagnosis,treatment and prognosis monitoring when comparing with other cancer.Thus,cancer related biomarkers are urgently needed to guide biopsy,treatment selection and follow-up.This research utilized the suspension culture to enrich the prostate CSCs-like cells.The properties of GD55 in the self-renewal,differentiation,cell cycle,drug resistance and tumorigenicity of prostate CSCs-like cells were detected by MTT,Q-PCR and Western blot.The results showed that GD55 could significantly induce apoptosis of prostate CSCs-like cells compared with normal oncolytic virus ZD55,and showed stronger oncolytic effect.Additionally,GD55 possessed the greater efficacy in suppressing the growth of implanted tumors derived from prostate CSC-like cells than ZD55.Furthermore,GD55 could remarkedly induce the apoptosis of prostate CSC-like cells in vitro and in vivo,and inhibited the propogation of cells and angiogenesis in xenograft tumor tissues.Thus,GD55 may actually represent an attractive therapeutic agent for targeting prostate CSCs-like cells in order to achieve better clinical outcomes in prostate cancer.