| BackgroundTongue squamous cell carcinoma(TSCC)is a common malignant tumor,which can lead to speech,chewing and swallowing disorders.The main treatment method for tongue squamous cell carcinoma is multidisciplinary cooperation and comprehensive sequencing based on surgical treatment.However,surgical operations often cause dysfunctions such as speech,eating,breathing and maxillofacial deformities,with a high recurrence rate,and prone to lymph node metastasis or distant organ metastasis.The molecular mechanism of tongue squamous cell carcinoma is not fully understood,and there is no specific targeted therapy drug.Therefore,the research on the molecular mechanism of the biological characteristics of tongue squamous cell carcinoma and the exploration of targeted therapy will provide new ideas for the treatment of tongue squamous cell carcinoma.Carbon monoxide releasing molecule-3(CORM-3)is a water-soluble carbon monoxide(CO)releasing molecule whose actual effect is to exert a biological effect through CO.CORM-3 has important biological functions in the body.In cardiovascular diseases,CORM-3 can show obvious vasodilation effects by regulating vascular tension and blood pressure;in the inflammatory response,the CO produced by CORM-3 has anti-apoptotic function and can resist oxidative damage.Promote endothelial healing.Currently known studies show that CORM-3 has therapeutic effects on transplantation,myocardial infarction and rheumatoid arthritis.At present,more and more studies have shown that CO-releasing molecules(CORM)that release CO in a controlled manner under physiological conditions can increase heme oxygenase-1(HO-1)in various animal models and cell types.HO-1 has powerful antioxidant and anti-apoptotic effects and has an important regulatory effect on cancer cell growth and treatment resistance.Nuclear factor E2-related factor 2(Nrf2)is an important upstream regulator of HO-1.It responds to the redox reaction in the cell and then participates in the regulation of normal healthy cell structure and normal physiological functions.Nrf2/HO-1 anti-oxidative stress signaling pathway may have multiple roles in tumors and play different biological roles in different tumor types or different tumor microenvironments.This study will focus on exploring the biological effects of CORM-3 on tongue squamous cell earcinoma and study the therapeutic effects of CORM-3 on tongue squamous cell carcinoma from in vivo and in vitro experimental levels.In addition,we will also discuss the molecular mechanism of CORM-3 inhibiting tongue squamous cell carcinoma and study the regulation of CORM-3 on the Keap1/Nrf2/HO-l anti-oxidative stress signaling pathway.This study will provide scientific theory and basis for the role of CORM-3 in the treatment of tongue squamous cell carcinoma.The research plan is as follows:MethodsPart Ⅰ Effects of CORM-3 on biological behavior of TSCCCORM-3 with different concentrations(0 μM,50 μM,100μM,200 μM,400μM,800 μM)and treatment time(0,6 h,12 h,24 h,48 h,and 72 h)were used to treat CAL27 and SCC4 cells of TSCC.CCK-8 and clonogenesis assay was used to detect the proliferation of CAL27 and SCC4 cells.The invasion of CAL27 and SCC4 cells were detected by Transwell assay.CAL27 and SCC4 cell migration were detected by cell scratch test.The apoptosis of CAL27 and SCC4 cells was detected by flow cytometry.Part Ⅱ Study on the mechanism of CORM-3 against TSCCExperiment 1 Expression of HO-1 in human TSCCImmunohistochemistry was used to detect the expression and distribution of HO-1 in human TSCC.The correlation between HO-1 and Ki67 was analyzed,and the role of HO-1 in the occurrence and development of TSCC was preliminarily analyzed.Experiment 2 Effects of CORM-3 on the expression of HO-1 in TSCCCAL27 and SCC4 cells were treated with different concentrations of CORM-3(0μM,100 μM,200 μM,400 μM),and total proteins of CAL27 and SCC4 cells were extracted 12 h later.Western Blot was used to detect HO-1 expression in TSCC cells.CAL27 and SCC4 cells were treated with CORM-3 at different treatment times(0,0.25 h,0.5 h,1 h,3 h,6 h,12 h).The expression of HO-1 protein and mRNA in CAL27 and SCC4 was detected by Western Blot and qRT-PCR.TSCC cells were treated with HO-1 inhibitor(HO-1-in-1)and HO-1 gene silencing.The inhibitory effect of HO-1 and shHO-1 on CORM-3 was determined by CCK-8,clonal formation assay,cell scratched assay,Transwell assay and Annexinv-FITC/PI double staining assay.Experiment 3 Effects of CORM-3 on Keapl/Nrf2/HO-1 signaling pathway in TSCCCAL27 and SCC4 cells were treated with CORM-3 at different treatment times(0,0.25 h,0.5 h,1 h,3 h,6 h,12 h).Western Blot was used to detect the expression of Keapl and Nrf2 proteins in CAL27 and SCC4.The expressions of Keapl and Nrf2 mRNA in CAL27 and SCC4 cells were detected by qRT-PCR.After CAL27 and SCC4 cells with stable Nrf2 interference were constructed,Western Blot was used to detect the effect of CORM-3 on HO-1 protein expression level in CAL27 and SCC4 cells with Nrf2 interference.CAL27 and SCC4 cells were treated with 400 μM CORM-3 for 12 h,and Nrf2 expression was detected by immunofluorescence.Cytoplasmic extraction kit was used to extract proteins from cytoplasm and nucleus,and Western Blot was used to detect Nrf2 protein levels.Part Ⅲ Effects of CORM-3 on the growth of transplanted human TSCC in vivoTwelve 4-week-old BALB/C-NUDE male Nude mice were subcutaneously inoculated with human tongue squamous cell carcinoma CAL27 cells in their axils.After tumor formation,they were randomly divided into blank control group and CORM-3 experimental group,with 6 mice in each group.Nude mice in CORM-3 treatment group were intraperitoneally injected with CORM-3 solution every other day,with an injection volume of 30mg/kg.The Control group were injected with the same amount of PBS for 4 weeks.The mental,dietary and activity of nude mice were observed every day.The size of subcutaneous tumors in nude mice was measured every 3 days or so,and the maximum and minimum diameters of subcutaneous tumors were recorded.At the end of the treatment,the nude mice were sacrificed,and the tumor was stripped and weighed.The effect of CORM-3 on tumor was determined by measuring animal body weight,tumor size and immunohistochemical staining.ResultsPart I Effects of CORM-3 on biological behavior of TSCC1.CCK8 showed that CORM-3 had inhibitory effects on CAL27 and SCC4 cells at 400 μM.CORM-3 group and blank control group had no inhibitory effect on cell proliferation.CAL27 and SCC4 cells were treated with 400 μM and 800 μM CORM-3 for 24 h,The cell proliferation was significantly inhibited under the treatment of 400 μM and 800 μm of CORM-3.The inhibitory effect of CORM-3 was reduced.Since 400 μM was the lowest inhibitory concentration for CORM-3 to inhibit cell proliferation,400 μM was selected as the working concentration of CORM-3 in subsequent experiments.The results of cell clone formation assay showed that the clone formation ability of CAL27 and SCC4 cells was significantly inhibited under the treatment of 400 μM CORM-3.2.Transwell and scratch test showed that CORM-3 could significantly inhibit the invasion and migration of CAL27 and SCC4 cells of TSCC.After treating CAL27 and SCC4 cells with 400 μM CORM-3 for 24h,the number of cells crossing the polycarbonate membrane was significantly decreased in CAL27 and SCC4 cells,45%and 67%of control group.After being treated with 400 μM CORM-3 for 6 h,12 h and 24 h,the cell migration rates of CAL27 cells were 44%,47%and 83%of the control group,respectively.When treated with SCC4 cells,the cell migration rates of SCC4 cells were 53%,48%and 78%of the control group,respectively.No significant difference can be seen between iCORM-3 group and control group,indicating that CORM-3 inhibits tongue squamous cell carcinoma invasion by releasing CO.Western Blot was used to detect the expression of invasion and migration related proteins in tongue squamous cell carcinoma cells.The results showed that with the increase of CORM-3 treatment time,the expression of E-cadherin,a key protein molecule in epithelial mesenchymal transition,was up-regulated,and N-cadherin was down-regulated.3.Annexin V-FITC/PI double staining showed that CORM-3 could significantly promote the apoptosis of tongue cell carcinoma.After treated with 400 μM CORM-3 for 24 h,the apoptosis rate of CAL27 and SCC4 cells increased by nearly 50%compared with the negative blank control group.Part Ⅱ Study on the mechanism of CORM-3 against TSCCExperiment 1 Expression of HO-1 in human TSCCImmunohistochemical results showed that the expression of HO-1 in paracancer tissues was higher than that in tongue carcinoma tissues.HO-1 positive reactants were deposited as brownish yellow mainly in the nucleus.It was negatively correlated with the proliferation index Ki67.Immunohistochemistry and statistical analysis of clinical data showed that HO-1 expression was not correlated with age(P=0.0926),gender(P=0.5593),tumor size(P=0.2970),pathological grade(P=0.0686),but with lymph node metastasis(P<0.05).HO-1 was more strongly expressed in TSCC without lymph node metastasis.Experiment 2 Effects of CORM-3 on the expression of HO-1 in TSCC1.After CAL27 and SCC4 cells were treated with CORM-3 at 0 μM,100 μM,200 μM,400 μM concentrations for 12 h.The results showed that when 0 μM was used as blank control group,HO-1 protein expression levels were increased to varying degrees under the action of CORM-3 at 100 μM,200 μM and 400 μM,and the highest expression level was observed at 400 μM,with statistically significant differences.2.After CAL27 and SCC4 cells were treated with CORM-3 at different treatment times(0 h,0.25 h,0.5 h,1 h,3 h,6 h,12 h).The results showed that HO-1 protein expression in CAL27 and SCC4 cells increased gradually at 0.25 h,0.5 h,1 h,3 h,6 h and 12 h with 0 h as blank control group,and reached the highest expression at 12 h.The expression of HO-1 mRNA in CAL27 and SCC4 cells of TSCC cells was detected by qRT-PCR after 400 μM CORM-3 treatments for 12 h.The results showed that the expression of HO-1 mRNA was increased compared with 0 h.3.CAL27 and SCC4 cells were treated with HO-1 inhibitor(CORM-3)to inhibit HO-1 activity and silencing HO-1 gene.The results of CCK8 and clonogenesis showed that CORM-3 and shHO-1 could partially restore the inhibitory effect of CORM-3 on the proliferation of TSCC cells.Transwell assay showed that CORM-3 and shHO-1 could partially restore the inhibitory effect of CORM-3 on TSCC.Scratch test showed that CORM-3 and shHO-1 could partially recover the inhibitory effect of CORM-3 on the migration of TSCC.Annexin V-FITC/PI double staining assay showed that CORM-3 and shHO-1 could partially recover the apoptotic effect of CORM-3 on TSCC cells.Experiment 3 Effects of CORM-3 on Keapl/Nrf2/HO-1 signaling pathway in TSCC1.Western Blot showed that the expression of Keapl protein was down-regulated with the increase of corm-3 treatment time after CAL27 and SCC4 cells were treated with 400 μM CORM-3.Contrary to the change trend of Keapl protein expression,Nrf2 protein expression levels were up-regulated with the increase of CORM-3 treatment time.qRT-PCR results showed that Keap1 mRNA expression was down-regulated and Nrf2 mRNA expression was up-regulated in CAL27 and SCC4 cells treated with 400 μM CORM-3 for 12h.2.After the successful construction of CAL27 and SCC4 TSCC cell lines with stable Nrf2 interference,Western Blot was used to detect the effect of CORM-3 on HO-1 protein expression level in CAL27 and SCC4 cells with Nrf2 interference.The results showed that the protein expression levels of HO-1 and Nrf2 in shNrf2 group were lower than those in NC group.After 400 μM CORM-3 was added to CAL27 and SCC4 cells that interfered with Nrf2 for 12 h,the protein expression levels of HO-1 and Nrf2 increased compared with that without CORM-3.The protein expression level of HO-1 decreased after down-regulation of Nrf2 and increased slightly after addition of CORM-3.3.The expression of Nrf2 in human TSCC cells was detected by immunofluorescence after CAL27 and SCC4 cells were treated with 400 μM CORM-3 for 12 h.The results showed that Nrf2 was mainly located in the cytoplasm of cells.After 400 μM CORM-3 was added,the expression of Nrf2 nucleus increased gradually,indicating that CORM-3 can significantly promote Nrf2 entry into the nucleus.Western Blot was used to detect the expression of Nrf2 protein in cytoplasm and nucleus after extracting proteins from cytoplasm and nucleus with nuclear cytoplasm extraction kit.The results showed that the expression of Nrf2 protein in cytoplasm was down-regulated after 12 h of CORM-3 treatment.The expression of Nrf2 protein in the nucleus was up regulated.Part Ⅲ Effects of CORM-3 on the growth of transplanted human TSCC in vivoAfter subcutaneous injection of CAL27 cell suspension,all nude mice survived,the cell suspension was completely absorbed 24 hours later,and tumor appeared 5-6 days later,and the tumor formation rate was 100%.14 days after tumor formation,nude mice were given drugs in groups.No obvious discomfort was found in mental,activity and diet during the administration.Before death,the weight of nude mice in each group was 21.50 ± 0.70 g in blank control group.The body weight of nude mice in CORM-3 group was 23.22 ± 1.88g.The average tumor volume of the blank control group was543.74±82.78 mm3,and that of the CORM-3 group was 383.87±138.70 mm3.HE staining results showed that the tumor tissues in the blank group and CORM-3 group were consistent with the pathological characteristics of cancer cells.Cytoskeleton structure disorder,nuclear size,nuclear hyperchromia and nuclear malformation,nuclear membrane thickening,irregular cells.Immunohistochemical results showed that Ki67,E-Cadherin and HO-1 were expressed in blank control group and CORM-3 group.The expression of Ki67 was in the nucleus and nucleoli and presented as brownish yellow particles.The expression level of the blank group was higher than that of the CORM-3 group.Conclusions:1.CORM-3 has a certain anti-tumor effect in TSCC.CORM-3 can inhibit the proliferation,migration and invasion of TSCC cells and promote the apoptosis of TSCC cells in vitro.2.CORM-3 can regulate the Keapl/Nrf2/HO-1 antioxidant stress signaling pathways in TSCC cells,thereby regulating the proliferation,apoptosis,migration and invasion of TSCC cells.3.CORM-3 inhibits the growth of TSCC in vivo,inhibits the tumor proliferation and increases the expression of HO-1,suggesting that CORM-3 has certain anti-tumor effects. |
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