| BackgroundRenal cell carcinoma is the malignant tumor originated from the tubules of urinary system, takes up 80 to 90 percent of kidney cancer, is one of the most common malignant tumors in urinary system. With the means of checking progressions, the incidence of renal cancer increase about 2% per year in China. The incidence in men is about twice in women, the most high period of being diagnosed is 50-70. Although China’s incidence rate is lower than the global average, but in 2012 alone,66,466 cases was newly diagnosed,about twice cases in the United States, this difference may be due to genetic predisposition and environmental exposure. The incidence of kidney cancer in men ranks seventh in tumors incidence,and it imposes great economic pressure to the family and the whole society.With the detection means of nowadays,25～40 percent of the renal cancer was spotted accidentally.25-30 percent of the patients was first diagnosed with remote metastasis,some of them subject to sub-clinical metastasis. Laparoscopic Radical nephrectomy is the main curable choice to the early-diagnosed patient. The patients diagnosed with remote metastasis at the first time have a poor prognosis with a median-survive time less than a year.Renal caner is resistent to both radiotherapy and chemotherapy,so the measures were limited for the patients who had local or remote metastasis.Though the cytokine therapy was implied to the advanced in the last two decades,it only has a remission rate less than 30 percent.In western countries,Target therapy is the main stream measure for the advanced renal cancer.There are mainly two kinds of target agents used in the therapy of the advanced.One kind is tyrosinase inhibitors (TKIs) target tumor blood vessels and the other is mammals rapamycin target protein (mTOR) inhibitors. Sorafenib is the first TKIs that used in the treatment of stage IV kidney cancer and recommended as first-line or second-line.Others including sunitinib, everolimus and Axitinib, each of them are expensive or lacking sufficient domestic clinical experimental results.For a long time,searching for agents sensitive to kidney cancer interests the scientific researchers and medical staff. Lycorine, a natural alkaloid extracted from the Amaryllidaceae plant family, has been reported to exhibit a wide range of physiological effects, including the potential effect against cancer.Activity study showed that Lycorine and its derivatives have anti-inflammatory, anti-viral, anti-malarial, anti-parasite, inhibiting acetylcholinesterase, cardiovascular protection and anti-tumor effect, and recently discovered to inhibit osteoclast formation and other biological role.In recent years, many domestic and foreign synthesized Lycorine derivatives, such as hydrochloric acid Lycorine (Lycorine hydrochloride), Lycoris alkali oxide (Lycobetaine), pseudo Lycorine (Pseudolycorine) and some other structural modification done yet unnamed compounds. And Lycorine in effect and mechanism of various tumors do more research. Currently no Lycorine renal cancer studied systematically.ObjectiveTo explore the effect of lycorine on 786-0 and ACHN cells. To observe the biological behavior changes and investigate the possible mechanism caused by lycorine. So as to seek a new approach for the chemotherapy of Renal cell carcinoma.Method1. Biological behavior changes caused by Lycorine1.1 Apoptosis inducing and cell cycle analysisCells In logarithmic phase were used to cultivate for flow cytometry to apoptosis detecting or cell cycle analysis.Cells made into single cell suspension, adjust the density to 3 x 105 ml, and seed into the 6-well plate,37℃ and 5% CO2 for the night, to grow to 50% confluence take out 6-well plate,discard the culture medium, and add in the pre-made 0,1,2,5,10,25 μmol/L of lycorine solution 2 ml in the prepared cells, cultivate for 24 h, collected the cells and the supernatant, Then send it to flow cytometry room of Sun yat-sen Memorial Hospital, apoptosis and cell cycle analysis was detected at the same time.1.2 MTS assay was used to detect the proliferation of 786-0 and ACHN cellsMark the 96-well plates with normal or control group,set five parallel holes at each concentration. Cells in logarithmic phase were seeded into 96-well plate,adjust the density to 2×103 per well.Then cultivated in the atmosphere of 37 ℃ and 5% CO2 for indicated time.Then add 20ul MTS to the well and incubate for an hour before reading from the Microplate System at 490nm.1.3 Wound-healing and Transwell assaysMark the 96-well plates with normal or control group,cultivate the cells to almost full confluence.the"wounds" were created by a sterile pipette tip. Fresh medium containing 10% FBS and indicated concentrations of Lycorine was added. Take photos of each time point when terminate the culture.Set three parallel holes.Use the Imge-Pro plus 6.0 software to measure the migration distance.Cells In logarithmic phase were used.After culturing 12h in the pre-made medium,then made it into single cell suspension.Add the cells to the Transwell chamber,then add the 15% FBS to the 24-well plate. Cultured for 48h and the stain it with crystal violet solution.Then dry the chamber in the room temperature and snapshot it under the inverted microscope. At last,count the number of cells manually.1.4 Colony forming assayCells In logarithmic phase were used.Dilute the single cell suspension to 200 cells per well.Add the 5uM/L Lycorine solution to the experimental group,then cultured for ten day.Fix the colonies and then stain it with crystal violet.Then count the colonies and calculate the formation rate.2. Study of the mechanism of the effect of Lycorine on 786-O cells2.1 Detecting the ROS Level in 786-O cells by DCF-DASeed the cells in 96-well plate 1.5×104 per well.Culture the cells to almost full confluence and change the medium and incubate with 25uM DCFH-DA for 30min.Then get rid of the medium and wash it before snapshoting.Choose the 525nm (green) laser to excitation the fluorescence stain.2.2 Real-time PCRSearched the primers from the Primerbank (https://pga.mgh.harvard.edu/ primerbank/) and synthesized it by the help of Songon Biotech company(Shanghai).Havested the cells and extracted the total RNA.Then,used the Reverse transcription kit to synthesis related gene cDNA and performed the real time PCR. Analyze the result by 2 formula.2.3 Western-blotHarvested the 786-O cells of two groups at indicated time; then lysed in RIPA buffer. Protein concentration was determined using a Bicinchoninca acid assay (Thermo Scientific). Protein samples were run on 8% to 12% SDS-PAGE gels and transferred to nitrocellulose filter membrane. The membranes were incubated overnight at 4℃ using specific antibodies. The signals were visualized via the Bio-RAD Chemidox XRS+Western blotting detection system.Analyze the grey value of the pictures.Result1. Effects of Lycorine on 786-0 and ACHN renal carcinoma cell lines.1.1 As to 786-O cells,lycorine has a IC5O of 11.3umol/L,while 24.3μumol/L as to ACHN cells.1.2786-0 cells was significant arrested in S phase(p<0.05);ACHN cells were arrested in G0/G1 phase.1.3 Compared with the normal control group,Lycorine induced apoptosis in 786-O(p<0.01) and ACHN cells (p<0.01) significantly. Lycorine inhibited the proliferation of 786-O(p<0.01) and ACHN(p<0.01) cells significantly.Lycorine inhibited the migration and invasion ability of 786-O(p<0.01) and ACHN(p<0.01) cells significantly.1.4 Lycorine inhibited the colony formation ability of 786-O(p<0.01) and ACHN(p<0.01) cells significantly.2. The pilot study of the mechanism of Lycorine on 786-0 cells2.1 The mechanism of Lycorine on renal cell carcinoma cell line 786-0We treat the 786-0 cells with 5uM Lycorine for 24h before observing it under the inverted microscope.As a result,both group cells emit green fluorescence.The difference is that the experimental group cells have a brighter fluorescence and the shape of the cells became round while the control group cells with a dim fluorescence and have little changes in its shape.2.2 Treated with 5uM Lycorine for 24h,our real-time PCR showed that lycorine downregulated the expression of CDK4,CyclinD1,STAT3,JNK,,ERK1/2,IKBA 90RSK and while upregulated AKT1,MSK1,STAT3,P38 and RELA.2.3 Treated with 5uM Lycorine for 24h,The western-blot assay show that the protein level of Bax、p-AKT、p-Bad、p-p38、p-ERK were upregulated,while the CDK4、 CyclinD1、Bcl-2、Bax、survivim、p-P65、IKBa were downregulated.The expression level of p21,JNK、p38、IKKα、p65 have little change.Conclusion1.Lycorine can inhibits the proliferation of 786-0 and ACHN cell line. The inhibition effect on 786-0 is more significant.2.Lycorine could induce both 786-0 and ACHN cells apoptosis.3.Lycorine has a inhibition effect on the migration and invasion ability of 786-0 cells and ACHN cells.4.Lycorine could inhibit the colony formation ability of 786-0 and ACHN cells.5. Lycorine might affect 786-0 cells on accumulating reactive oxygen species and activating apoptosis pathway and NF-κB pathway.In a word, Lycorine has a significant anti-tumor effect on renal cancer lines.Comparatively speaking,Lycorine has a stronger effect on 786-0 cells than on ACHN,which may attributes to the different malignant degrees of the cell lines.This agent may provides new approach to the chemotherapy of the renal cell carcinoma or its adjuvant therapy. |
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