Disulfiram Inhibits Epithelial-Mesenchymal Transition In Oral Squamous Cell Carcinoma And Its Mechanism

Oral squamous cell carcinoma,the most common malignant tumor of the cranial and maxillofacial region,has become a major killer threatening human life and health due to its easy metastasis and recurrence.Numerous studies have shown that epithelial-mesenchymal transition(EMT)is closely related to tumor metastasis.Therefore,inhibiting tumor metastasis by regulating EMT may be a major direction for the treatment of oral squamous cell carcinoma.Heterogeneity caused by tumor mutation is one of the main reasons for tumor treatment failure.A large number of studies have confirmed that Smad4 mutations are widely present in oral squamous cell carcinoma.Therefore,selecting a broad-spectrum drug that has therapeutic effects on both Smad4 and non-mutated oral squamous cell carcinoma,has important clinical significance.As a traditional alcohol withdrawal drug,anticancer effect of disulfiram has been widely concerned by researchers in recent years.Disulfiram(DSF)exerts its anticancer effects through a variety of mechanisms,including induction of apoptosis,inhibition of proteasome activity,inhibition of angiogenesis,and so on.Studies have shown that disulfiram can inhibit EMT in breast cancer.The aim of this study was to investigate the effect of disulfiram on EMT of Smad4-mutation and Smad4-wild type oral squamous cell carcinoma cell line,to elucidate its specific molecular mechanism,and to explore the effect of disulfiram on the growth and metastasis of oral squamous cell carcinoma in vivo.In the second chapter,we investigated the effect of disulfiram on Smad4-mutation and Smad4-wild type oral squamous cell carcinoma cells EMT.We selected two oral squamous cell carcinoma cell lines-Smad4 mutant CAL 27 and Smad4 non-mutated SCC-25.The expression of Smad4 protein in these two cells was first examined by Western Blot,and the loss of Smad4 expression in CAL 27 cells was confirmed.The experimental concentration of disulfiram was then screened by CCK-8 method,and finally 5 μM,10 μM and 20 μM were selected as the subsequent experimental concentrations.Apoptosis assays and cell cycle analysis also confirmed the biosafety of selected concentrations.Then,EMT was induced by TGF-β1 in both cells,and the induced cells were pretreated by DSF were used to observe the effect of DSF.Morphology of the cells showed that disulfiram inhibited the morphological changes of the two cells induced by TGF-β1 from polygon to spindle shape;Western Blot detection of EMT markers revealed that disulfiram reversed TGF-β1-induced E-cadherin reduction and increased expression of Vimentin and Snail in both cells;immunofluorescence staining confirmed this result;cell migration and invasion experiments have shown that disulfiram can inhibit the increase of two cell migration and invasion caused by TGF-β1.The results of the first chapter demonstrate that disulfiram can inhibit TGF-β1-induced EMT in both Smad4-mutation and Smad4-wild type oral squamous cell carcinoma cells.In Chapter 3,we clarify the specific molecular mechanisms by which disulfiram inhibits EMT.Based on the experimental results in Chapter 2,disulfiram inhibits EMT in both Smad4-mutation and Smad4-wild type oral squamous cell carcinoma cells.We hypothesized that disulfiram may act by inhibiting non-Smad-dependent TGF-β signaling pathway.The MAPK signaling pathway is an important non-Smad-dependent TGF-β signaling pathway.We examined activation of three types of MAPK by Western Blot: ERK,p38 and JNK.The results showed that there was no significant change in p38 and JNK activation states after TGF-β1 and disulfiram treatment,and the phosphorylation level of ERK was increased rapidly after TGF-β1 treatment.The increase of ERK phosphorylation induced by TGF-β was significantly inhibited when pretreat with disulfiram,suggesting that disulfiram may play its role by inhibiting the ERK signaling pathway.To further confirm this result,we selected the ERK phosphorylation inhibitor U0126.Similar to the action of disulfiram,U0126 reversed the decreased E-cadherin expression and the increased Vimentin and Snail expression induced by TGF-β1,and also inhibited the increase of cell migration and invasion induced by TGF-β1.The results of the third chapter show that disulfiram exerts its inhibitory effect on EMT by inhibiting the ERK signaling pathway.In Chapter 4,we explored the effects of disulfiram on the growth and metastasis of oral squamous cell carcinoma in vivo.Since EMT is closely related to tumor metastasis,we first examined the effect of disulfiram on tumor metastasis.CAL 27 cells were treated with TGF-β1 and DSF,respectively,and a tumor metastasis model was constructed by injecting the treated cells into the tail vein of nude mice.General observation of lung tissue and HE staining showed that TGF-β1 caused significant lung metastasis,while disulfiram significantly inhibited lung metastasis caused by TGF-β1.In addition,the subcutaneous metastasis of the buttocks was unexpectedly found.The metastasis of the TGF-β1 treatment group was the largest,and the metastasis of the disulfiram and TGF-β1 co-treatment group was significantly smaller.Immunohistochemical staining of metastatic tumors showed that the expression of EMT markers Vimentin and Snail was significantly increased in the TGF-β1 treated group,while the expression in the disulfiram and TGF-β1 co-treated groups was significantly inhibited.Finally,the effects of disulfiram on tumor growth were investigated by intragastric administration of disulfiram in xenotransplant nude mice.Both the tumor growth curve and the general observation of the tumor indicate that disulfiram significantly inhibited tumor growth.Immunohistochemical staining of the tumor confirmed that disulfiram can inhibit the expression of the EMT markers Vimentin and Snail.The results of Chapter 4 experimental results indicate that disulfiram can inhibit tumor growth and metastasis in vivo,which may be related to the inhibition of EMT.This study demonstrates that disulfiram can inhibit EMT in both Smad4-mutation and Smad4-wild type oral squamous cell carcinoma cells.This effect is achieved by inhibiting the ERK signaling pathway,and disulfiram can also inhibit the growth and metastasis of oral squama squamous cell carcinoma in vivo,suggests that disulfiram has a broad spectrum of anticancer activity and has potential for clinical use in the treatment of oral squamous cell carcinoma.