The Inhibitory Effect Of Itraconazole On Cutaneous Squamous Cell Carcinoma And Its Mechanism

Cutaneous squamous cell carcinoma(cSCC)is a cutaneous malignant tumor originating from epidermal keratinocytes with complex pathogenesis.The incidence of cSCC is high and still shows a rising trend worldwide.High-grade and metastatic cSCC have a poor prognosis and relatively high mortality.Early detection and rational treatment of cSCC are of great significance.Surgical resection is the main treatment option,but this approach is only effective for early and well-defined cSCC.External medication,freezing,photodynamic therapy and some other therapeutic methods are associated with adverse reactions such as pain,local irritation,and irreversible pigmentation,with low compliance and high treatment failure rates.Therefore,there is an imperative need to find more effective treatment strategies for cSCC.Itraconazole is a broad-spectrum antifungal drug with a recognized safety profile.A large number of basic and clinical studies have shown that itraconazole has inhibitory effects on basal cell carcinoma,non-small cell lung cancer,prostate cancer,breast cancer,medulloblastoma,esophageal squamous cell carcinoma,oral squamous cell carcinoma,glioblastoma,etc.However,the role and mechanism of itraconazole in cSCC remain unclear.Therefore,our study investigated the effects of itraconazole on the biological behaviors of cSCC in vitro and in vivo,and explored mechanisms of action.Part 1 Itraconazole inhibits cell proliferation of cutaneous squamous cell carcinomaObjective:To evaluate whether itraconazole affects the proliferation of cSCC cells in vitro and in vivo.Methods:A431 and Colo 16 cells were treated with itraconazole for 36 h,48 h and 72 h respectively,and the inhibition rate was detected by CCK8 assay.After treated with itraconazole for 48 h and then incubated for 2 weeks,the proliferation of A431 and Colo 16 cells were assessed by colony formation assay.We used flow cytometry to discover the cell cycle distribution of A431 and Colo 16 cells after treatment with itraconazole.Annexin V/PI staining assay and Tunel assay were utilized to investigate whether itraconazole activating apoptosis mechanisms in A431 and Colo 16 cells.A xenografted tumor model derived from A431 cells was established,and the inhibitory efect of itraconazole on the growth of xenograft tumor was evaluated.We used immunohistochemistry to detect the expression of Ki-67 and cleaved caspase-3.HE staining was performed on liver tissues.Results:Itraconazole inhibited the proliferation of cSCC cells in a time and concentration-dependent manner.Itraconazole increased the percentage of cSCC cells in the G2/M phase.Itraconazole increased the numbers of apoptotic cSCC cells in a dose-dependent manner compared with the controls.Itraconazole treatment could inhibit the growth of A431 cells xenograft tumors and reduce tumor weight.Itraconazole increased the expression of cleaved caspase-3,and decreased the expression of Ki-67 in xenograft tumor tissue.Liver tissue was not significantly affected.Conclusions:Itraconazole inhibits the proliferation of cSCC cells in vitro and in vivo,and induces cycle arrest and promoted cell apoptosis.Part 2 Transcriptomic and proteomic profiling of cSCC cells treated with itraconazoleObjective:To explore the mechanism of itraconazole in inhibiting cSCC cells proliferation and promoting cell apoptosis by using transcriptome and proteome sequencing technology.Methods:After A431 cells were treated with itraconazole(1 μM,48 h),transcriptome and proteome sequencing were performed respectively.Then,the transcriptome and proteome sequencing data were combined for bioinformatics analysis.Results:A total of 1309 up-regulated genes and 1467 down-regulated genes were found by transcriptome sequencing.Differentially expressed genes were related to various cellular metabolic processes.Up-regulated differentially expressed genes are enriched with IL-17,NF-kappaB,MAPK,steroid synthesis,TNF,AMPK,P53,PI3K/Akt and some other pathways.7362 differential splicing events were found,including 4732 exon skipping splicing events.63 up-regulated proteins and 61 down-regulated proteins were identified by proteome sequencing analysis.Differentially expressed proteins were related to biological processes such as cholesterol anabolism and steroid metabolism,and were enriched with pathways such as cholesterol synthesis.HMGCS1 was found to interact with various proteins.Combined analysis of transcriptome and proteome obtained 34 up-up regulated genes(up-regulated in transcriptome and up-regulated in proteome)and 14 down-down regulated genes(down-regulated in transcriptome and down-regulated in proteome).Differentially expressed genes were enriched with cholesterol synthesis processes.Conclusions:Itraconazole changes expression levels of various genes in cSCC cells,and affects cholesterol synthesis,NF-kappaB,MAPK and some other pathways.Itraconazole causes numerous mRNA differential splicing events dominated by exon skipping in cSCC cells.Itraconazole exerts its anti-cSCC mechanism in various ways.Part 3 Itraconazole inhibits the proliferation of cutaneous squamous cell carcinoma cells by transcriptionally upregulating HMGCS1 expressionObjective:To explore the role of HMGCS1 in the inhibition of cSCC cell proliferation by itraconazole.Methods:RT-qPCR and western blot were used to detect the expression level of HMGCS1 in A431 cells treated with itraconazole.We used immunohistochemistry to detect the expression of HMGCS1 in xenograft tumor.HMGCS1 stable knockdown A431 cell line was constructed by using lentiviral vector.The effect of itraconazole on the HMGCS1 promoter in A431 cells was tested using dual-luciferase reporter gene assay.Results:Itraconazole significantly up-regulated the expression of HMGCS1 in A431 cells.Itraconazole increased the expression of HMGCS1 in xenograft tumor tissue.Knockdown of HMGCS1 could partially reverse the antiproliferative effect of itraconazole on A431 cells.Itraconazole dramatically increased the HMGCS1 promoter-luciferase reporter activity compared with the control.Conclusions:Itraconazole inhibits the proliferation of cSCC cells by transcriptionally upregulating the expression of HMGCS1.Part 4 Itraconazole induces ferroptosis in cutaneous squamous cell carcinoma cells via the HMGCS1/ACSL4 axisObjective:To explore the effect of itraconazole on ferroptosis in cSCC cells.Methods:RT-qPCR and western blot were used to verify the effect of itraconazole on ACSL4 mRNA and protein expression levels in A431 cells.We used immunohistochemistry to detect the expression of ACSL4 in xenograft tumor.MDA detection kit and iron colorimetric kit were used to detect the effects of itraconazole on malondialdehyde and iron in A431 cells.Then,the expression of HMGCS1 and ACSL4 in A431 sh-Con cells and A431 sh-HMGCS1#2 cells were detected.The effects of itraconazole on malondialdehyde and iron levels in A431 sh-Con cells and A431 shHMGCS 1#2 cells were examined.Results:Itraconazole significantly up-regulated the expression of ACSL4 in A431 cells.Itraconazole increased the expression of ACSL4 in xenograft tumor tissue.Itraconazole could increase the levels of malondialdehyde and iron levels in A431 cells.shRNA-mediated silencing of HMGCS1 reduced the protein expression of ACSL4 in A431 cells.Silencing of HMGCS1 weakened the promoting effects of itraconazole on malondialdehyde and iron accumulation in A431 cells.Conclusions:Itraconazole induces ferroptosis in cutaneous squamous cell carcinoma cells via the HMGCS1/ACSL4 axis.