Study On The Mechanism Of Selective Killing Effect Of Hedyotis Diffusa Extract On Renal Carcinoma Cells

Renal cell carcinoma is a common tumor of urinary system,and the incidence of renal cancer in China has increased year by year.The main treatment for early kidney cancer is surgery that usually benefits patients with favorable prognosis.The patients with progressive and metastatic renal cell carcinoma,however,are mostly treated with comprehensive strategies based on internal medicine,since surgery is not practicable for these patients.Although targeted drug therapy can significantly improve their prognosis,some patients often fail to obtain clinical benefits after a long-term course of the therapy.Traditional Chinese medicine for the treatment of malignant tumors has multiple target effects and provides a systemic comprehensive conditioning that improves the quality of life of patients and prolong their survival.The pharmacological properties of certain traditional Chinese herbs are complex and may be associated with some side effects,appropriate application of Chinese medicine with the consideration of treatment individualization and comprehensive strategies for patients with advanced renal cancer,may be an effective way of decreasing the potential risk and preventing side effects in the treatments with Chinese medicine.Hedyotis diffusa Willd?HDW?has been documented as a Chinese herb that is effective in the clearance of heat and detoxification as well as in the improvement of other aspects,including blood circulation and diuresis,and dampness and swelling.The extract of the Hedyotis diffusa has strong and broad-spectrum anti-tumor activities and can inhibit growth of various types of tumors.Clinically,it has been used for anti-cancer and anti-infective treatment.The purpose of this study is to investigate whether and how the Hedyotis diffusa affects the proliferation,cycle,apoptosis,migration and invasion of human renal carcinoma cells,and to determine the function of the Hedyotis diffusa in selective killing of renal cancer cells.The data may support the potential for using the drug in the systematic treatment of renal cancer.In this study,human ACHN renal cell adenocarcinoma cells and human HK-2 renal proximal convoluted tubule cells were treated with HDW for 24 hours.CCK-8 assay and flow cytometry were used to measure cell proliferation,apoptosis and cell-cycle distribution.Scratch healing and transwell experiments were used to detect the migration and invasion ability of cells.Immunofluorescence staining was used to observe the morphologic changes of cells.Western blotting was used to detect the expression of some proteins related to epithelial and mesenchymal phenotypes and MAPK pathway-related proteins.The transcriptomes of ACHN cells were analyzed by RNA sequencing.Real-time fluorescence quantitative?RT-PCR?was used to detect gene expression in ACHN and HK-2 cells.The results showed that of HDW at a concentration of 12.5%could significantly inhibit the proliferation of ACHN cells.DNA staining with PI showed that HDW significantly inhibited cell cycle progression,showing a decrease in the proportion of cells in G₀/G₁ phase.The expression of CCRS2,CCNB1 and CCNB2 was significantly inhibited by HDW.AnnexinV/PI double staining detection found that HDW promotes early and late apoptosis of ACHN cells.Apoptosis-related gene detection showed that BIM,CASP10,and PUMA were up-regulated,and anti-apoptotic genes BCL-2 and BCL-XL were down-regulated.After treatment with 12.5%HDW for 24 hours,it was observed under light microscope that the cells were transformed from long spindle-shaped to round-shaped cell clusters.The staining of the intracellular microfilament protein with the labeled phalloidin-conjugates showed that the phenotype of cells was changed and the cell junction became tight.The tight junction marker CDH1 and the connexin CTNNB1 were detected by immunofluorescence,and the results showed that the cell side staining was enhanced.The expression of the mesenchymal surface markers,VIMENTIN and SNAIL1,were significantly reduced in cells with HDW treatment,indicating a significant inhibition of mesenchymal properties in cells examined;scratch healing and Transwell experiments showed that HDW significantly inhibited the migration and invasion of ACHN cells;HDW significantly suppressed the expression of RAP1 pathway-related genes and the levels of phosphorylated JNK protein in ACHN cells.These results suggest that HDW can selectively kill kidney cancer cells by inhibiting proliferation,promoting ACHN cell cycle arrest and apoptosis.HDW inhibites RAP1-JNK signaling pathway and selectively suppressed mesenchymal-like properties in ACHN renal cancer cells,indicating that HDW can induce MET transformation and inhibit migration and invasion of renal cancer cells.