A Meta-analysis On Cefixime And Adenovirus-mediated IL-24 Gene Induces Apoptosis Of Cutaneous Squamous Cell Carcinoma Cells

IL-24 (Interleukin-24), formerly known as melanoma differentiation associated antigen 7 (melanoma differentiat ion-associated antigen, MDA-7). And it is mainly secreted by CD3+ T lymphocytes and monocytes express, as a secreted protein. IL-24 is the members of IL-10 family. Current study Shows that, IL-24 is not only inhibit the growth of a tumor cell, but also hinder the formation of blood vessels, at the same time, and almost no effect on normal cells.What’s more,IL-24 also can enhence the tumor cells’ sensitivity to radio and the characteristics. A large number of experiments have shown, IL-24 gene can effectively inhibit the growth of tumor cells, such as stomach cancer, liver cancer, but the research on skin squamous cell carcinoma has not been reported.CSCC (cutaneous squamous cell carcinoma) is the most common form of skin cancer, which accounts for non-melanocytic skin tumors of 20%, rather than the no-melanin skin cancer accounts for all skin tumors’ 96%. In addition to using sunscreen as the prevention and surgical resection of the tumor lesions, gene therapy may become a important measures and ways for the treatment of skin cancer. This topic has been successfully constructed utilizing adenovirus-mediated vector --Ad-IL-24, make IL-24 gene expression, skin squamous cell COLO-16, and SCL-1 cell for the purpose cells, IL-24 on whether it has the growth inhibition and induction of apoptosis, thereby discuss IL-24 effects on the skin squamous cell carcinoma, and squamous cell carcinoma pathogenesis of skin, squamous cell carcinoma of the skin gene therapy provide laboratory evidence.The experiment is divided into four parts.Part 1 Establish IL-24 overexpression of skin squamous cell modelObjective Establish IL-24 overexpression of skin squamous cell modelMethods Using an adenovirus vector-mediated (Ad-IL-24) infection of squamous cell carcinoma of the skin, and assay IL-24 gene expression in squamous cell carcinoma cell line COLO-16 cells, and SCL-1 Cells by QPCR.1. Construction of adenovirus(1) Preparation of adenovirus(2) adenovirus packaging, collecting(3) Purification of the virus2. infect and culture the target cellsThe target cells used in this experiment is skin squamous cell COLO-16 cells, and SCL-1 cells.3. To choice the best MOI in infected cellsIn COLO-16 cells were used as a screening cell, select the best MOI in infected cells4.The expression of IL-24 gene in skin squamous cell carcinoma COLO-16, and SCL-1 by QPCRThe experiment was divided into 3 groups:Ad-IL-24 group, Ad-GFP group and CONTROL. With 37 ℃,5% CO2 in the incubator for 72 h, collected cells(COLO-16, SCL-1), total RNA was extracted, then dothe real-time PCR reaction.Results COLO-16 cells and SCL-1 cells:The expression of IL-24 gene significantly increased in Ad-IL-24 group cells compared with the control group and Ad-GFP group (P<0.05).Conclusion:cutaneous squamous cell of IL-24 overexpression model is successfully establishedPart 2 Ad-IL-24 effects the proliferation on COLO-16 cells and SCL-1 cellsObjective Research SCC proliferation by IL-24, and provide evidences for the anti-tumor mechanism of IL-24Methods Cell viability was determined by MTT assayThe cells were seeded in 96-well plates and was divided into 3 groups:Ad-IL-24 group, Ad-GFP group and CONTROL. At its 0,1,2,3,4,5,6 day MTT solution was added (already formulated,5mg/ml), 10ul/hole, incubate in an incubator 4h, then added 150ul DMSO, in the micro-shock device the moderate concussion 8min, measured by immunoassay microplate OD value (490), according to data obtained growth curve.Results COLO-16 cells:The proliferation assay(MTT)showed that the proliferation of cells from Ad-IL-24 Group was inhibited in a time-depending manner from day 4 (P<0.05),and day 6 is the greatest difference.SCL-1 cells:The proliferation assay(MTT)showed that the proliferation of cells from Ad-IL-24 Group was inhibited from day 4 (P<0.05),and day 6 is the greatest difference.Conclusion:IL-24 inhibit the growth of COLO-16 cells and SCL-1 cells.Part 3 The effect of apoptosis in COLO-16, SCL-1 cells by Ad-IL-24 adenoviral transfectionObjective Study the effection on the skin squamous cell carcinoma, provide the basis for the anti-squamous cell carcinoma of IL-24Methods(1)The morphological change of COLO-16 cells and SCL-1 cells was observed by laser scanning confocal microscopyThe experiment was divided into 3 groups:Ad-IL-24 group, Ad-GFP group and CONTROL. With 37 ℃,5% CO2 in the incubator for 72 h, and then dyeing by PI staining and DAPI staining. Then assy the morphological change of cells by laser scanning confocal microscopy.(2)cell apoptosis rate were analyzed by flow cytometerWith 37℃,5% CO2 in the incubator for 72 h, collected the cell,and washed with PB 3 times, fixation cells with 70% cold ethanol, add reagents, analys the apoptosis by flow cytometry.ResultsCOLO-16 cells:(1)Confocal laser scanning microscopy confirmed that IL-24 can accelerate apoptosis of COLO-16 cells.(2)The apoptosis rate was identified by flow cytometry.And there is a significant difference between the Ad-IL-24 group[(13.1±O.92)%] and control group[(3.69± 0.36)%](P<0.05),and there is no significant difference between Ad-GFP group[(3.39± 1.06)%] and control group (P<0.05)SCL-1 cells:(1)Confocal laser scanning microscopy confirmed that IL-24 can accelerate apoptosis of SCL-1 cells.(2)The apoptosis rate was identified by flow cytometry.And there is a significant difference between the Ad-IL-24 group[(18.6± 1.25)%] and control group[(3.81± 0.46)%](P<0.05),and there is no significant difference between Ad-GFP group[(6.07± 0.97)%] and control group (P<0.05).Conclusion:IL-24 can induce the apoptosis of COLO-16 cells and SCL-1 cells.Part 4 Effect of expression of IL-24 on apoptosis marker in COLO-16, SCL-1 cell:bax, bcl-2,caspase-3Objective Detect the expression of IL-24 on COLO-16, SCL-1 cell apoptosis marker:bcl-2, bax, caspase-3 and discover the mechanism.Methods Detect apoptosis marker in skin squamous cell (COLO-16,SCL-1), bcl-2、bax and cleaved caspase-3 by Western blotting、qPCR and immunofluorescence.1. TO detect the expression of apoptotic marker in COLO-16 and SCL-1:Bax、 Bcl-2 and cleaved caspase-3 by Western blotting.2. Assay the expression ofapoptosis marker (Bax, Bcl-2) in COLO-16 and SCL-1 cells by QPCR’.The experiment was divided Ad-IL-24 group, Ad-GFP group and control group. COLO-16 and SCL-1 cells were incubated at 37 ℃,5% CO2 condition for 72 h, then, collected the cells, PBS washed 3 times, total RNA was extracted and reverse transcription to generate cDNA, conduct real-time PCR reactions.3. Immunofluorescence detect expression of apoptosis marker (Bax, Bcl-2) in COLO-16 and SCL-1 cells after IL-24 overexpressionThe experiment was divided Ad-IL-24 group, Ad-GFP group and control group. cells were incubated at 37 ℃,5% CO2 condition for 72 h, washed with PBS for 3 times, with 4% paraformaldehyde for lOmins, then through the permeable fluid treatment for 30mins, added rabbit anti-human Bax, Bcl-2. Incubated overnight under 4℃ condition, washed with PBS; coupled with goat anti-rabbit IgG secondary antibody, incubated at room temperature for 1 h, then, observe cell under the microscope.Results (1)COLO-16 cells:The result shows that the expression of BAX is increased and the expression of Bcl-2 is decreased in Ad-IL-24 group by immunofluorescence staining、Western Blot and qPCR,and the expression of cleaved caspase-3 is up regulated by Western Blot.(2) SCL-1 cells:The result shows that the expression of BAX is increased and the expression of Bcl-2 is decreased in Ad-IL-24 group by immunofluorescence staining、Western Blot and qPCR,and the expression of cleaved caspase-3 is up regulated by Western Blot.Conclusion:(1)Ad-IL-24 can induce apoptosis of COLO-16 cells,probably through up-regulating Bax expression, down-regulating Bcl-2 expression, and inducing caspase-3 activation.(2) Ad-IL-24 can induce apoptosis of SCL-1 cells,probably through up-regulating Bax expression, down-regulating Bcl-2 expression, and inducing caspase-3 activation.Summary1. This study were infected skin squamous cell line COLO-16、and SCL-1 cells with adenovirus, and the IL-24 were over expressed. To provide a model for the the experiment.2. IL-24 inhibits the proliferation of COLO-16 cells, and start from day 4, the inhibition is time-dependent. Similarly, SCL-1 cells can also be significantly inhibited from day 4.3. IL-24 can induce the apoptosis of COLO-16 cells and SCL-1 cells. And the COLO-16 cells’ late apoptosis was more pronounced by IL-24.4. The reaction mechanisms of IL-24 induce apoptosis of COLO-16 cells and SCL-1 cells, may be related with increase the Bax/Bcl-2 ratio, and activate the ceaspase-3.