Cancer Associated Fibroblasts-induced Chemoresistance And Natural Compound In The Treatment Of Esophageal Squamous Cell Carcinoma

Malignant tumors are common diseases worldwide.Among them,the incidence of esophageal cancer ranked eighth in all malignant tumors,and the mortality ranked sixth.Esophageal cancer can be divided into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC)according to tissue type.Among them,ESCC is the most common type of esophageal cancer in the world,especially in high-risk East Asia and Southeastern Africa,including some high-risk areas.In China,about 90%of patients are esophageal squamous cell carcinoma.According to the latest global data,the total death number of esophageal cancer in the world is 402,000,of which in China is 197,500,accounting for 49.35%of the global total number.At present,the mortality rate of esophageal cancer ranks fourth in malignant tumors in China.Although the understanding of esophageal cancer has gradually deepened in recent years and there have been considerable advances in prevention,diagnosis,and treatment,the overall condition of esophageal cancer is late detection,delayed treatment,and poor prognosis.The five-year survival rate has remained between 15%to 25%.The main treatment for ESCC is endoscopy,surgery,radiotherapy and chemotherapy,among which preoperative and postoperative adjuvant chemotherapy are important means for the treatment of ESCC.The development of resistance to chemotherapy is a major problem affecting the efficacy of treatment and the prognosis of patients.We urgently need to develop new and effective drugs for the treatment of ESCC.At the same time,we need to further study the resistance mechanism of currently used chemotherapy drugs such as cisplatin in order to overcome it,to improve prognosis,and ultimately to be good for patients.The previous studies on chemotherapy resistance focused on the tumor cells themselves,and the possible mechanisms include gene mutations of tumor cells,changes in drug transporters on the surface of the cell membrane,changes in cellular metabolism pathways and so on.In recent years,studies have found that the tumor microenvironment played an important role in drug resistance.The tumor microenvironment includes cancer-associated fibroblasts(CAF),tumor-associated macrophages,endothelial cells,and immune cells.The CAF is one of the main components of the tumor microenvironment,which plays a key role in tumorigenesis,development,and chemoresistance and can be considered a potential therapeutic target for cancer.The CAF mainly regulates the tumor microenvironment through cytokines,cell adhesion and immune responses.In our study,CAF was first isolated and cultured from the tissues of ESCC patients and immunofluorescence was used to identify the specific markers of CAF.Next,we used a co-culture system to find that cisplatin-pretreated CAF can cause ESCC cell proliferation and drug resistance compared to untreated CAF.Then,by using a protein array,a secreted cytokine plasminogen activator inhibitor 1(PAI-1)was found to be upregulated from cisplatin-pretreated CAF.When we used the cytokine PAI-1 alone,it could promote the growth of ESCC cells in vitro and in vivo by paracrine and reduce the killing effect of cisplatin on ESCC cells.It was also found that PAI-1 could play a role by activating AKT and ERK1/2 signaling pathways and inhibiting caspase-3 activity and ROS accumulation.In summary,our results confirm that CAF plays an important role in the development and resistance of ESCC.Cisplatin stimulates secretion of PAI-1 by CAF after stimulation.The cytokine PAI-1 promotes proliferation of ESCC and induces drug resistance through paracrine,which may become a new target for the treatment of ESCC.The PAI-1 inhibitor can play a synergistic role in vivo and is worthy of further study.The incidence and mortality of esophageal cancer rank 8th and 6th respectively in all malignancies worldwidely.The common pathological types of esophageal cancer are divided into squamous cell carcinoma and adenocarcinoma,of which China is dominated by esophageal squamous cell carcinoma,accounting for more than 90%of the total cases.Esophageal squamous cell carcinoma(ESCC)lacks specific symptoms.Despite considerable progress in diagnosis,treatment,and other aspects in recent years,there is still lack of effective treatments for ESCC.The main treatment includes endoscopic therapy,surgery and chemotherapy,while the overall survival rate of ESCC has not improved significantly,ranging from 15%to 25%.Therefore,there is an urgent need to look for effective drugs for the treatment of ESCC.Plant extracts are one of the important sources of anticancer drugs.The Isodon plant is a well-known biologically active diterpene which has a variety of skeletal structures,especially the enantio-kaurene.Longikaurin A(LK-A)is an ent-kaurane diterpenoid isolated from Isodon ternifolius,and its anticancer effect has been studied in other tumors including nasopharyngeal carcinoma and hepatocellular carcinoma.It can exert anti-cancer effects by causing DNA damage in tumor cells,inducing cell cycle arrest,promoting apoptosis,and increasing the level of oxidative stress in cells.We are trying to explore whether LK-A has the same anti-cancer effects in ESCC,the possible mechanisms and future clinical applications of LK-A.After preliminary screening,we found that LK-A has a significant inhibitory effect on ESCC cells.We found that LK-A could significantly inhibit the proliferation of ESCC cells(KYSE30 and KYSE450)by CCK8 assay.At the same time,the inhibition ability of non-tumor normal cells(NIH3T3 and HEK293)significantly weakened.The 24 h IC50 values of LK-A in ESCC were 1.259 μM and 1.370 μM,respectively,while the IC50 values in non-tumor normal cells were 3.941 μM and 5.744μM,respectively.With the increase of LK-A dose,the colony formation of ESCC could be significantly inhibited.Also,LK-A could cause G2/M phase arrest of ESCC cells and reduce the expression of Cyclin B1 and cdc2.LK-A could induce apoptosis of ESCC cells in a time-and dose-dependent manner.Western blot results showed that LK-A may induce apoptosis of ESCC cells through caspase-dependent mitochondrial apoptosis pathway,while using caspase inhibitors,Z-VAD(OMe)-FMK,pretreatment partially attenuated LK-A-induced apoptosis.LK-A could cause a significant increase in reactive oxygen species(ROS)in ESCC cells.The use of the antioxidant N-acetylcysteine(NAC)could attenuate LK-A-induced apoptosis.LK-A could activate JNK and p38 MAPK signaling pathways.JNK and p38 MAPK inhibitors could attenuate LK-A-induced apoptosis and NAC could also reduce the phosphorylation of JNK and p38 MAPK.Finally,in vivo study found that LK-A could significantly inhibit tumor growth in nude mice compared with the control group.At the same time,LK-A had no significant effect on the general condition and body weight of nude mice.H&E staining of liver and kidney tissue showed no obvious toxic side effects.These results confirmed that LK-A was a relatively safe and effective anti-cancer drug.In conclusion,our study confirmed that LK-A could inhibit the proliferation of ESCC cells in vivo and in vitro,promote its apoptosis and exert new anti-tumor effects in ESCC cells,and at the same time had a relative safety.This indicated that the compound might have the potential as a clinical therapeutic drug and deserved further study.