| Background:Colorectal cancer is the fourth most common cancer in the world.About 639,000 people die from colorectal cancer every year.In the past few years,the morbidity and mortality of colorectal cancer patients in China have been increasing significantly,and many patients have been diagnosed at the advanced stage,the treatment effect is poor.To find an effective and reliable treatment for colorectal cancer has become an urgent problem in modern medicine.At present,there are many methods for the treatment of colorectal cancer,including surgery,radiotherapy,chemotherapy and biotherapy.At this stage,it is believed that surgery and chemotherapy are one of the most effective treatments for colorectal cancer.Although some early patients can be radically cured by surgical treatment,many patients with colorectal cancer are in the middle and advanced stage when they are clinically diagnosed.Some patients will have recurrence metastasis after surgical resection of the tumor,so chemotherapy becomes one of the necessary treatments for colorectal cancer patients after surgery.Chemotherapy after surgery can prevent tumor recurrence and metastasis to a great extent;when some patients with advanced colorectal cancer can not be completely removed by surgery,chemotherapy can kill or inhibit the residual tumor cells to continue to grow;for some patients with advanced colorectal cancer at the time of diagnosis,There is a wide range of metastasis and can not be operated on,through chemotherapy can reduce the size of the tumor,so that some symptoms of patients alleviate,sometimes even to the possibility of surgery.However,in the process of chemotherapy for colorectal cancer patients,in addition to some of the primary drug resistance,there are still some cancer patients may be in the course of treatment with the increase of chemotherapy course,and eventually appear to be resistant to chemotherapy drugs,and even to a variety of chemotherapy drugs,which lead to chemotherapy failure,tumor recurrence and metastasis.Some studies have shown that due to the emergence of multidrug resistance of tumor,about one third of patients will have recurrence and metastasis of tumor.Therefore,the emergence of multidrug resistance is one of the major obstacles to successful chemotherapy for colorectal cancer.Among the chemotherapeutic agents for colorectal cancer,5-fluorouracil(5-Fu),as one of the most effective clinical drugs,has been widely used in clinical treatment.However,the resistance of multidrug-resistant colorectal cancer cells to 5-Fu is also one of the common reasons for the failure of colorectal cancer patients in clinical chemotherapy.Therefore,to explore the mechanism of multidrug resistance of colorectal cancer cells and to find a new and effective method to reverse multidrug resistance of colorectal cancer is one of the hot issues in colorectal cancer research.For this reason,we have established a pentafluorouracil-resistant colorectal cancer LOVO/5-Fu resistant cell line,and used this multidrug-resistant cell line to study the molecular mechanism of drug resistance in colorectal cancer,reversing multidrug-resistant colorectal cancer LOVO/5-Fu resistant cells to chemotherapy-sensitive tumor cells.P-glycoprotein(P-gp)is an important transporter protein on the cell membrane.It belongs to the superfamily of ABC transporters(ATP-dependent transporters)and plays an important role in inducing multidrug resistance(MDR)in tumor cells.One of the important reasons for MDR in tumor cells is the increase of P-gp,which is encoded by MDR1 gene in tumor cells.In humans,the expression of MDR1 mRNA is low in adrenal glands,pancreas,large intestine,small intestine and other tissues,it participates in the growth of normal human tissue cells;MDR1/P-gp overexpression in MDR phenotypic tumor cells.In vitro studies have shown that the higher the expression level of MDR1 gene and P-gp,the lower the concentration of anti-tumor drugs in MDR cells,the stronger the drug resistance.Studies have shown that P-gp has the function of a drug pump,which can actively pump chemotherapeutic drugs into tumor cells out of the cell,so as to reduce the accumulation of antitumor drugs in tumor cells,thereby causing MDR in tumor cells.C-Jun N-terminal kinase(JNK)is also known as stress activated protein kinase(SAPK).It is a kind of serine/threonine kinase and one of the important members of mitogen activated protein kinase(MAPK)family in mammalian cells.JNK signaling pathway can be activated by osmotic pressure,ultraviolet,oxidative stress,ionizing radiation,heat shock and other environmental stress factors,as well as growth factors,cytokines(such as TNF,IL21),resulting in the activation of JNK/SAPK signaling pathway by threonine and serine diphosphorylation,and ultimately JNK/c-Jun signaling pathway is activated.JNK/SAPK signaling pathway plays an important role in cell growth,stress response,oncogene transformation,cell differentiation and apoptosis.In recent years,it has been found that JNK signaling pathway is closely related to the occurrence,development and drug resistance mechanism of colorectal cancer.Tetrandrine(Tet)is a double benzyl isoquinoline alkaloid extracted from the root of Tetrandrine powder.It has a wide range of pharmacological effects,such as anti-inflammation,lowering blood pressure,blocking calcium channel and so on.It has been reported that it can reverse multidrug resistance of leukemia cells in vitro and in vivo.The mechanism of reversing multidrug resistance is to down-regulate the expression of MDR1 mRNA/P-gp,to accumulate antineoplastic drugs in cells,and to increase the sensitivity of antineoplastic drugs,leading to apoptosis of tumor cells.Based on this,we speculate that Tet may regulate the expression of MDR1 mRNA/P-gp and MDR in colorectal cancer during chemotherapy.In this study,Tet was applied to LOVO/5-Fu resistant colorectal cancer cell lines in vitro to study the growth inhibition and apoptosis-inducing effect of Tet on LOVO/5-Fu resistant colorectal cancer cell lines,and to understand its reversal effect on multidrug resistant colorectal cancer LOVO/5-Fu cells.The results showed that Tet could reverse the resistance of LOVO/5-Fu to 5-Fu by inhibiting the expression of MDR1 gene and decreasing the expression of P-gp.At the same time,through the study of JNK signaling pathway,we found that tet can regulate the phosphorylation level of JNK and c-jun in cells,and then regulate the drug resistance of cells.Tet may be an important reversal drug for reversing the multidrug resistance of colorectal cancer.Methods:1.Establishment of LOVO/5-Fu multidrug-resistant cell lineIn this study,a multidrug resistance LOVO/5-Fu cell line was screened by increasing the concentration of 5-Fu gradually and continuously acting on LOVO cell line,which was sensitive to colorectal cancer.MTT assay was used to detect the multidrug resistance,which confirmed that our multidrug resistance cell line LOVO/5-Fu had good multidrug resistance and stability.2.The expression of MDR and P-gp protein in colorectal cancer cell LOVO and LOVO/5-Fu were analyzed.Real-time fluorescence quantitative PCR(Realtime-PCR)was used to detect the mRNA expression of multidrug resistance genes in LOVO and LOVO/5-Fu cells.Western blot was used to detect the expression of P-glycoprotein in LOVO and LOVO/5-Fu cells.The expression of MDR and P-gp protein in colorectal cancer LOVO and LOVO/5-Fu cells was revealed.3.The concentration of reversal agent was determined by MTT method.Different concentrations of Tet were used to treat LOVO cells and LOVO/5-Fu cells respectively.MTT assay was used to detect the effect of Tet on the inhibition rate of LOVO/5-Fu cells.4.Drug resistance reversal test.After Tet was used to treat LOVO/5-Fu cells,MTT assay was used to detect the inhibition rate of LOVO/5-Fu cells,and IC50 and drug resistance index were further calculated.5.Cell cycle distribution was detected by flow cytometry.After Tet was applied to LOVO and LOVO/5-Fu cells,the cell cycle distribution of LOVO and LOVO/5-Fu cells was detected by flow cytometry.6.Apoptosis was detected by flow cytometry.Flow cytometry was used to detect the apoptosis rate of LOVO and LOVO/5-Fu cells treated with Tet respectively.7.The expression of P-gp was detected by flow cytometry.After Tet was used to treat LOVO and LOVO/5-Fu cells,the expression of P-gp in LOVO and LOVO/5-Fu cells was detected by flow cytometry.8.Detection of MDR1 gene expression by PCR.After Tet was applied to LOVO and LOVO/5-Fu cells,the expression of MDR1 gene was detected by PCR.9.Detection of P-gp,JNK,p-JNK,c-jun and p-c-jun protein by Western blot.After Tet was applied to LOVO and LOVO/5-Fu cells,the expression of P-gp,JNK,p-JNK,c-jun and p-c-jun were detected by Western blot.Results:In this study,we found that MDR mRNA expression was significantly up-regulated and P-gp protein content was significantly increased in pentafluorouracil-resistant LOVO/5-Fu cells compared with the control group.We also found that Tet could enhance the sensitivity of LOVO/5-Fu cells to pentafluorouracil.The apoptosis rate of LOVO/5-Fu cells was increased and the number of cells blocked in G1 phase was increased,but the number of cells entering division phase was decreased significantly after Tet treatment on LOVO and LOVO/5-Fu cells.The expression of MDR1 and P-gp protein in LOVO/5-Fu cells were significantly lower than those before Tet treatment.When tet was applied to LoVo/5-FU cells,p-JNK was significantly higher than that of LoVo/5-FU cells without Tet,and the expression of p-c-jun downstream was also significantly increased;when sp600125 was added,Western blot showed that the expression of p-JNK and p-c-jun in tet group was significantly lower than that before sp600125.There was no significant change in the expression of JNK and c-jun in each group.Conclusion:1.The LOVO/5-Fu resistant cell line established by our experimental method has good drug resistance and stability.2.The MDR mRNA expression and the P-gp protein content in LOVO/5-Fu group were significantly higher than those in LOVO group.3.Tet can reverse the MDR of human colorectal cancer multidrug-resistant cell line LOVO/5-Fu in vitro.4.Tet can change the multidrug resistance of human colorectal cancer cell line LOVO/5-Fu by regulating the apoptosis rate and cell cycle.5.The mechanism of Tet reversing MDR in LoVo/5-FU cells may be realized by regulating JNK signal pathway and down regulating the expression of MDR mRNA and P-gp protein. |
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