Study On The Toxin Gene Therapy For Oral Tongue Squamous Cell Carcinoma With A PE38KDEL Toxin Expression System Regulated By CEACAM6 Promoter

Human oral tongue squamous cell carcinoma(hOTSCC)is a malignant neoplasm with the highest morbidity and mortality of oral cancers and is characterized by a high relapse rate and poor prognosis.At present,surgery,radiotherapy,and chemotherapy are still the mainstay treatments for hOTSCC.However,due to a lack of targeting,these therapeutic approaches have high toxic side effects and have great impacts on the quality of life of patients.Therefore,the development of genomically targeted agents has become a research interest in recent years.In toxin gene therapy,the expression of the toxin is regulated by a tumor-specific promoter,so that the toxin gene is only expressed in tumor cells,but not normal cells,which makes it safer and more effective during the treatment course and receive a growing body of attention.To date,there are still few reports on hOTSCC toxin gene therapy.Carcinoembryonic antigen-related cell adhesion molecule6(CEACAM6)is a cell-surface adhesion molecule and is overexpressed in numerous cancers.Glycosylated CEACAM6 is even a tumor marker associated with recurrence in early-stage OSCC patients.In this research,we used the CEACAM6 promoter to regulate the expression of Pseudomonas exotoxin A and explore its therapeutic effects on OTSCC.Firstly,the expression levels of CEACAM6 in different cells were detected by qRT-PCR.The results showed that CEACAM6 was highly expressed in hOTSCC cells but expressed at low levels in normal cells,which suggested its robust OTSCC cell-specific expression.Then,by gene recombination method,the toxin expression vector(p CEACAM6-PE38KDEL)and the firefly luciferase expression vector(p CEACAM6-Basic)were constructed.Luciferase assay was performed to detect the activity of the CEACAM6 promoter,and we found that the CEACAM6 promoter exhibited strong OTSCC cell-specificity.For the PE38 KDEL toxin can inactivate eukaryotic elongation factor 2(e EF2),we used a co-transfection method to determine the specific inhibitory effects of the toxin expression plasmid on protein synthesis of OTSCC cell.Meanwhile,the cell proliferation inhibition assay,flow cytometry,TUNEL,western blotting,JC-1 fluorescent dye,transwell,and wound healing assays were conducted to evaluate the influence of the plasmid on cell proliferation,invasion,migration,apoptosis,and cycle distribution.With the regulation of the CEACAM6 promoter,the PE38 KDEL toxin strongly inhibited the protein synthesis,proliferation,invasion,and migration of TCA8113 cells and also induced its apoptosis and cell cycle arrest.However,for normal cells and CEACAM6-negative cells,there were no obvious inhibitory effects.With a nude mouse xenograft tumor model of hOTSCC cells,we had further determined the therapeutic effect of the toxin expression plasmid on OTSCC in vivo and evaluated its systemic toxicity by H&E and immunohistochemistry.It was showed that p CEACAM6-PE38 KDEL performed strong inhibition on the formation and growth of the tumor without causing any damage to normal tissues.The results of this research indicated that PE38 KDEL toxin regulated by CEACAM6 can effectively inhibit and kill hOTSCC cells with favorable targeting ability and biosafety.This strategy is expected to be a candidate for the clinically targeted therapy of hOTSCC.