| Objective:Head and neck squamous cell carcinoma(HNSCC)accounts for more than 90%of head and neck malignant tumors,with an annual increase of about 600,000 cases worldwide,ranking the sixth among new malignant tumor patients.Tongue squamous cell carcinoma(TSCC)is a common type of HNSCC.In the early stage,surgery was the main treatment,and in the late stage,cisplatin-based chemotherapy was mainly used.Because some patients are insensitive to chemotherapy or resistant to chemotherapy,the overall 5-year survival rate is limited.Therefore,it is urgent to find new drugs with high efficiency and low toxicity to improve the survival rate of patients.More and more studies have shown that the drugs that induce ferroptosis have great application prospects in tumor treatment,and are expected to become a new means to treat tumors.Erastin is one of the earliest and most widely used ferroptosis inducers.It has been proved that Erastin can be combined with a variety of anti-tumor drugs to increase the sensitivity of tumor cells and overcome drug resistance.In recent years,the development of chemical drug libraries has made it possible to screen synergistic lethal agents.Therefore,based on the clinical problems,this study selected the kinase inhibitor library as the screening object to screen the synergistic lethal drugs with Erastin in tongue squamous cell carcinoma cell lines,and explored the corresponding targets and related mechanisms in vivo and in vitro,,aiming to provide new treatment options for TSCC patients who are not sensitive to or resistant to chemotherapy.Methods:1.High throughput screening of synergistic lethal drugs with Erastin in the kinase inhibitor libraryWe selected the tongue squamous cell carcinoma cell line CAL27 for screening,with Erastin as the background drug,and screened it in the kinase inhibitor library containing 644 compounds.In the first round,we screened drugs with a combined inhibition rate greater than 50%,and in the second round,we screened drugs with a dose dependence.In the third round,we selected two tongue squamous cell carcinoma cell lines CAL27 and SCC25 as screening objects,and the kinase inhibitor with the lowest drug concentration in the two cell lines was selected as the final research object when the two drugs reached the same inhibitory level.2.Verify that the combination of two drugs has synergistic lethal effect in tongue squamous cell carcinoma cellsFirst,the drug concentrations were determined by calculating IC50 in two tongue squamous cell carcinoma cell lines CAL27 and SCC25.Then,the CCK8 assay was used to verify the effect of DMSO,Erastin,kinase inhibitor and the combination of the two drugs on the viability of tongue squamous cell carcinoma cells,and also verified in other ferroptosis inducers.The cytotoxicity was detected by LDH release assay,The clone formation ability of each group was compared by plate cloning assay,and the combined index of the two drugs was calculated by Calcusyn software.3.Verify that the combination of the two drugs causes ferroptosis of tongue squamous cell carcinoma cellsThere are many ways of cell death.To verify the way of cell death caused by the combination of the two drugs,tongue squamous cell carcinoma cells were treated with ferroptosis inhibitors and apoptosis inhibitors,and their cell viability was detected by CCK8 assay,their morphological characteristics were observed by optical microscopy.The expression of ferroptosis marker PTGS2 was detected by q PCR assay.Lipid peroxidation ROS and intracellular total ROS levels were determined by flow cytometry.4.Explore the synergistic mechanism of kinase inhibitor combined with ferroptosis inducersAfter CAL27 and SCC25 were treated with DMSO,Erastin,kinase inhibitor and the combination of the two drugs,transcriptome sequencing was performed.Bioinformatics analysis of the common differential genes between the combination group and other groups was conducted.It was verified whether they affected the combined effect of the two drugs by interfering with the expression of differential genes,and the downstream targets of the differential genes were found.Western blot and q PCR assays were used to detect the expression levels of related genes.5.Effects of drug combination and target knockout on the growth of xenograft in nude miceThe nude mice were subcutaneously injected with tongue squamous cell carcinoma cell line CAL27 to construct the xenograft model,and were divided into four groups,which were respectively treated with DMSO,RSL3,kinase inhibitor and the combination of the two drugs.In addition,to verify the effectiveness of the synergistic target of two drugs,we used a bilateral tumor formation model.The right side was inoculated with vector group,and the left side was inoculated with target knockdown group.After tumor formation,they were randomly divided into two groups,one group was given solvent control treatment,and the other group was given drug treatment.After death,the tumor was dissected and its size and volume were observed.Results:1.High-throughput screening of synergistic lethal drugs with Erastin in the kinase inhibitor libraryIn the first round of screening,23 kinase inhibitors with a combined inhibition rate of more than 50% were identified.In the second round,5 kinase inhibitors were screened in a dose-dependent manner.Later,these 5 kinase inhibitors were simultaneously verified in two tongue squamous cell carcinoma cell lines CAL27 and SCC25.The results showed that when Erastin combined with different kinase inhibitors reached the same inhibition level on cell activity,the concentration of ALK inhibitor Lorlatinib is the lowest.Therefore,we selected Lorlatinib for subsequent study.2.Ferroptosis inducers combined with Lorlatinib has a synergistic lethal effect in tongue squamous cell carcinoma cellsWe determined the concentration of the two drugs in CAL27 and SCC25,and treated them with DMSO,Erastin,Lorlatinib and the combination of the two drugs,respectively.CCK8 results showed that the combination of the two drugs substantially promoted cell death compared with the single drug group.In addition,other ferroptosis inducers also have combined effects with Lorlatinib.The cell function assays showed that the LDH release in the combination group was significantly increased,and the clone formation ability was significantly weakened.The CI analysis showed that there was a synergistic effect between ferroptosis inducers and Lorlatinib.3.Ferroptosis inducers combined with Lorlatinib induces lipid peroxidation and ferroptosis in tongue squamous cell carcinoma cellsFerroptosis inducer combined with Lorlatinib can induce significant ferroptosis in tongue squamous cell carcinoma cells CAL27 and SCC25,which can be reversed by a variety of ferroptosis inhibitors.In addition,the specific morphology of ferroptosis can be observed under the optical microscope.The expression level of ferroptosis molecular marker PTGS2 is significantly increased.The levels of lipid peroxidation ROS and intracellular total ROS were significantly increased.4.SAT1 is an important target for ferroptosis inducers combined with Lorlatinib to synergistically induce ferroptosis of tongue squamous cell carcinoma cells.Inhibiting SAT1 can reduce the level of lipid peroxidation and the occurrence of ferroptosisFirst,we verified other ALK inhibitors,none of which showed significant synergies with Erastin.Moreover,CRISPR/Cas9 system knockout or overexpression of ALK also cannot change the effect of Erastin.Next,transcriptome sequencing was performed,and bioinformatics analysis showed that the expression of SAT1 was significantly increased in the combination group.After SAT1 knockdown,cell proliferation activity increased significantly,LDH release decreased significantly,clone formation ability increased significantly,the levels of lipid peroxidation ROS and intracellular total ROS decreased significantly compared with the control group.The results indicated that SAT1 was one of the synergistic targets of the two drugs.5.SAT1/ALOX15 pathway mediates ferroptosis of tongue squamous cell carcinoma cells induced by ferroptosis inducers combined with LorlatinibThrough literature review,we knowed that ALOX15 may be the downstream regulatory gene of SAT1.q PCR results showed that its expression level is down-regulated with the knockdown of SAT1,and the specific inhibitors of ALOX15 could substantially reverse the ferroptosis caused by the combination of the two drugs.After ALOX15 knockdown,cell proliferation activity increased significantly,the levels of lipid peroxidation ROS and intracellular total ROS decreased significantly compared with the control group.These results indicated that ferroptosis inducers combined with Lorlatinib further regulated the expression of ALOX15 by up-regulation of SAT1,and then induced lipid peroxidation of tongue squamous cell carcinoma cells,eventually leading to ferroptosis.6.Ferroptosis inducers combined with Lorlatinib has obvious combined anti-tumor effect in vivo,and the combined effect is significantly weakened after SAT1knockdownBecause of the poor solubility and unstable metabolism of Erastin,we chose RSL3 to conduct xenograft experiment in nude mice.Tongue squamous cell line CAL27 was injected subcutaneously into nude mice to construct a xenograft model,and the combined effect of RSL3 and Lorlatinib was verified in vivo.The results showed that the combination of the two drugs could substantially inhibit tumor growth.In addition,we verified the effectiveness of the synergistic target of the two drugs by knockdown SAT1,and found that compared with the control group,the SAT1 knockdown group could significantly resistant to the ferroptosis induced by the combination of the two drugs,which was consistent with the results in the cell experiments.Conclusions:1.Screening synergistic lethal drugs from the kinase inhibitor library is an efficient choice for exploring new drug combinations.2.Ferroptosis inducers combined with Lorlatinib has a synergistic lethal effect in tongue squamous cell carcinoma cells.3.Ferroptosis inducers combined with Lorlatinib induces lipid peroxidation and ferroptosis in tongue squamous cell carcinoma cells.4.SAT1 is an important target for ferroptosis inducers combined with Lorlatinib to synergistically induce ferroptosis of tongue squamous cell carcinoma cells.Inhibiting SAT1 can reduce the level of lipid peroxidation and the occurrence of ferroptosis.5.SAT1/ALOX15 pathway mediates ferroptosis of tongue squamous cell carcinoma cells induced by ferroptosis inducers combined with Lorlatinib.6.Ferroptosis inducers combined with Lorlatinib has obvious combined anti-tumor effect in vivo,and the combined effect is significantly weakened after SAT1 knockdown. |
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