Mechanisms Of Imiquimod On Human Skin Squamous Cell Carcinoma SCL-1 Cell Line

Skin suqamous cell carcinoma (SCC), a kind of skin tumor origined fromkeratinocyte or skin appendant organ suqmaous cell, is one of the most commonnon-melanoma skin cancers (about 20%). With the using and spreading of Mohs′operation, a good treatment effect can be achieved toward most of SCC cases. Buttoward some special SCC cases which are not suitable to adopte skin surgical therapy,it is important to choose effective and appropriate topical medicins to progress localtreatment. Imiquimod, an imidazoquinoline member, is an immune response modifierwith potent antitumor activity. The Food and Drug Administration of USA hasapproved the use of this topical medicine for treatment of different malignant andpremalignant tumors such as actinic keratoses and basal cell carcinoma. Althoughimiquimod's exact mechanisms of action in neoplastic diseases has not yet been fullyelucidated, animal and human studies have shown its ability to enhance the productionof cytokines such as interferon-[alpha], interleukin (IL)-12, and tumor necrosis factor(TNF)-[alpha] though conjugations with Toll like receptors on the surface of dendriticcells of the monocyte-macrophage lineage. These cytokines will activate the innateimmune response and indirectly will also stimulate adaptive (acquired) immunity andshow its antitumor activity, Further studies also indicate that apoptosis and restrain cellproliferation might play important role in imiquimod's mechanisms of treatingneoplastic diseases. In this study, we evaluated wether or not imiquimod can induce theinhibition and apoptosis of SCL-1 cell line, and the period,dosage and effect ofimiquimod on SCL-1 cell line were to be observed. To study the mechanisms ofimiquimod induce SCL-1 cell line apoptosis and down regulate SCL-1 cell line 1. Cell cultureSCL-1 cells were cultured in RPMI 1640 medium containing 10% fetal calf serum(FCS), 1mM pyruvate, 2mM glutamine, penicillin(100μg/mL), and streptomycin(100μg/mL), in 5% CO², at 37℃saturation humidity box. When the cells grew to forma monolayer, they were stimulated with Imiquimod for 12, 24, and 48 hours and thenbe collected.2. MTTSCL-1 cells were incubated in the 96-well culture board. Culture fluid containswith25μg/mL,50μg/mL,75μg/mL,100μg/mL,125μg/mL,150μg/mL,175μg/mL,200μg/mL and 225μg/mL imiquimod, which contrast to the culture medium cellswithout imiquimod. Each well add MTT solution (5μg/mL) reagent 20μL, to culture 4h,at 37℃, and stop culture. Each well add DMSO 150μL, concusing 10min, using ELISAreader to detect the OD at wavelength 570nM of each well. Restrain rate (%)=[1-OD(the disposal team) / OD (the contrast team)×100%]3. Cell morphology observationWith the culture fluid in which the concentration of imiquimod is 0μg/mL and150μg/mL, obsering the cell morphology and taking photoes with inverted phasecontrast microscrope after 48h culture.4. Detection the early stage apoptosis cells by flow cytornetryIncubating SCL-1 cells in 6-well culture medium with 1×10⁶/well, after 12～48hculture with the concentration of imiquimod 0μg/mL and 150μg/mL, gathering cellsgrowing along the wall and making monocellular suspension, washing in PBS for 2times, and then add 5μL Annexin V/FITC and 10μL PI solutions, reaction for 15min avoiding light, detection the early stage apoptosis cells by flow cytometry. Analyse theresult with software (Cell Quest).5. Detection cell apoptosis rate with Cell Death Detection KitSCL-1 cells were incubated in 96-well culture board with 1×10⁶/ well. Theculture fluid contains 50μg/mL,75μg/mL,100μg/mL,125μg/mL,150μg/mLimiquimod. After cultured for 12～48h, Samples were centrifuged at 200×g for 10minand the supernatant was removed, add 200μL lysis buffer in each well, cultured for30min at room temperature, centifuging again and gathering 20μL samples at thebottome of the wells, add samples and 80μL immunoreagent (Anti-histone-biotin,Anti-DNA-POD) into the wells of streptavidin coated MP, 300rpm×2h, after washingwith culture buffer for 3 times, add 100μL ABTS solution into each well and shake250rpm×15min. Detection the OD with ELISA reader at wavelength 490nM.6. Observation cell later period apoptosis with AO stainSCL-1 cells were incubated with 0μg/mL and 150μg/mL imiquimod for 48h,washing with PBS for 5min, add 5μL AO into 95μL cell suspension (1×10⁷/mL). Take1 drop sample on the surface of a clean glass slide and observe with fluorescencemicroscope at 490nM.7. Detection oftelomerase activitySCL-1 cells were incubated with 0μg/mL,50μg/mL,75μg/mL,100μg/mL,125^μ/mL,150μg/mL imiquimod for 48h, preparing telomerase extraction solution,RT-PCR amplification according to the manufacturers procedures. Add 5μL amplifiedproduction and 20μL denaturalization into 225μtL hybridisation solution, shaking for10min, add 100μL samples into the wells of streptavidin coated MP, hybridisating for2h at 37℃. Add 100μL of anti-DNP antibody-HRP conjμgation into each well,incubating at room temperature for 30min, after add 100μL of TMB solution andincubate 10min, add stop solution into each well, using a microtiter plate reader,measure the OD of the samples at 450nM.8. Immunocytochemistry and immunocytofluorescence SCL-1 cells were harvested at 48h after being stimulated with 150μg/mLimiquimod for 48h, and the cells were washed twice with PBS. The cells were thencollected onto glass slides by cytospin centrifμgating at 1000rpm for 5min, and thenincubated in acetone at 4℃for 10min. The preparations were incubated in a 0.3%H₂O₂ solution for 30min in order to quench endogeneous peroxidase activity. Mouseantibody against human Fas, bcl-2, c-Myc and goat antibody against human hTERTwere applied as the first antibody and incubated at 4℃for whole night. After washingfor 2 times with PBS, horseradish peroxidase-labelled and FITC-labelled IgG wereused as the second antibody. DAB was applied as coloring substrate and oberservationwith fluorescence microscope.9. RT-PCRAfter SCL-1 cells were stimulated with 0μg/mL,50μg/mL and 150μg/mLimiquimod for 48h, harvesting them with cellscraper. To detect the changes of Fas,bcl-2, hTERT, c-Myc mRNA level, total RNA was isolated from 3×10⁶ cells by Trizolreagent, RT-PCR amplification is operated according to the instructions of the AccessRT-PCR kit. PCR products were electrophoresed on 1.5% agarose gels stained withethidium bromide. The ethidium bromide-stained PCR products were quantified bydensitometry using Perkin-Elmer Imaging System. The results were expressed as theratio between the amount of the Fas, bcl-2, hTERT, c-Myc amplication product overthe internal control. These ratios were called target genes relative transcript or mRNAlevels.Experimental Results1. MTTThe inhibition of imiquimod to SCL-1 cell is dose-depent and IC30=47μg/mL,IC50=152μg/mL.2. Cell morphology observationThe shape of SCL-1 cell has not regular and the clearance has gradually became wider, volume smaller by imiquimod along with the time prolonging and theconsistence increasing.3. Detection the early stage apoptosis cells using flow cytometryIncubating with 150μg/mL imiquimod for 24h, the SCL-1 cells early stageapoptosis is extrmely obvious. The early stage apoptosis rate is 47.23%.4. Detection cell apoptosis rate with Cell Death Detection KitThe ELISA test result shows that the apoptosis rate of SCL-1 cells to imiquimodis dose-dependentandtime-dependent. F=15.96, P＜0.01; F=15.78, P＜0.01.5. Observation cell later period apoptosis with AO stainObservation with fluorescence microscope at 490nM, the later stage apoptosis ofSCL-1 cell treated with 150μg/mL imiquimod for 48h is obvious.6. Detection of telomerase activityThe telomerase activity of SCL-1 cell treated with imiquimod is dose-dependent,F=615.90,P＜0.01.7. Immunocytochemistry and immunocytofluorescenceImmunocytochemistry shows that Fas protein expression upgrade and bcl-2,c-Myc protein expression decrease aider treated with 150μg/mL imiquimod for 48h. Atthe same time, immunocytofluorescence shows hTERT protein expression decrease.8. RT-PCRAlong with the increase of the imiquimod reacting concentration, the expressionof mRNA of bcl-2,c-Myc and hTERT are decreasing, however, the expression of Fasis on the contrary-upgrade. The difference is significant P＜0.01.Conclusions1. Imiquimod can inhibit SCL-1 cell proliferation and induce SCL-1 cellapoptosis, and the fashion is dose-dependent and time-dependent in a given range.2. Imiquimod can upgrade Fas mRNA and protein expression level of SCL-1 cellline, and at the same time downgrade bcl-1 mRNA and protein expression level. 3. Imiquimod can upgrade hTERT mRNA and protein level of SCL-1 cell, andthe apoptosis rate of SCL-1 cell is closely inverse correlation with hTERT expression.4. Imiquimod can downgrade SCL-1 cell telomerase activity in a dose-dependentway.5. Imiquimod can downgrade c-Myc mRNA and protein level of SCL-1 cell linein a dose-dependent fashion.