| Objective:Nephroblastoma is known as a Wilms' tumor (WT) after Dr Max Wilms who first described it in 1899. WT is the most common malignant renal cancer in children.As a result of the combination of surgery, chemotherapy, and radiotherapy, the outcome and long-term survival rate of WT has significantly improved over the past few decades. Although patients such as anaplasia, bilateral Wilms tumor and recrudescence received much more intensive treatment, including vincristine (VCR),actinomycin D, doxorubicin, and cyclophosphamide for a long period of time, the outcome was still poor. In some cases WT results in severe complications. Therefore,there is an urgent need for the development of a new agent or new combinations to increase the chemotherapy effect and decrease the chemotherapy complications.Andrographolide (AND) is the most active component that is isolated from the extract of Andrographis paniculata. Recently, it was found that AND possesses various biological activities, including anti-inflammatory, antiviral, immune modulatory,antitumor effects, and so on. It was found that AND has powerful antitumor effects on many tumor cells including live cancer, breast cancer, gastric cancer, lung cancer,oophoroma, and so on. AND inhibited tumor growth in cancer cells mainly through two ways. One way is apoptotic cell death through activation of proapoptotic JNK pathway, suppression of anti-apoptotic PI3K/AKT and ERK pathway, activation of ERK-p53 apoptotic pathway, or caspase-dependent apoptosis. The other way is cell cycle arrest. In addition, it was also found that AND enhanced the chemosensitivity of ovarian cancer cells to cisplatin. However, till date, no studies had been reported on the antitumor effects of AND on WT. Thus we carried out the relevant research in order to confirm that whether AND have antitumor effects on WT and whether AND can enhance the chemosensitivity of WT cells to VCR.The current study used many biochemical methods to investigate the antiproli-feration effect of AND and VCR on SK-NEP-1 cells in vitro. Meanwhile, SK-NEP-1 xenografts were used to detect the antitumor effect in vivo. The study also investigated the molecular mechanism of the antitumor effect of andrographolide combined with vincristine on Wlims tumor cells. This study represented a potential treatment for WT in the near future.Method:1. Cell culture and treatment: SK-NEP-1 human kidney (WT) cell line was kept in the Maccyo'5 medium supplented with 15% heat-inactivated fetal bovine serum in a humidified incubator with 5% CO 2 at 37 ?. Cells were treated according to the following methods, and treatment time was 24 hours, 48 hours and 72 hours for different purposes:(1) VCR or AND were dissolved in dimethyl sulfoxide (DMSO), then added to cell culture medium. The concentration of VCR reached 3,6,9,12,15nM respectively.The concentration of AND reached 5,10,15,20,25?M respectively.(2) VCR and AND were dissolved in DMSO together, then added to cell culture medium. The concentration of VCR is half maximal inhibitory concentration(IC 50),and the concentration of AND reached 10,20, 25p.M respectively.(3) SK-NEP-1 cells were pretreated with SC-79(an activator of AKT) for 6 hours.Then VCR were dissolved in DMSO and added to cell culture medium. The concentration of VCR is 10.8nM.(4) SK-NEP-1 cells were pretreated with SC-79 for 6 hours. Then VCR and AND were dissolved in DMSO together and added to cell culture medium. The concentration of VCR is 10.8nM, and the concentration of AND is 25?M.Control group: equal volume of DMSO were added to the cell culture medium.2. Detection of cell proliferation: cell proliferation was assessed by using the Cell Counting Kit-8(CCK-8). The IC 50 values of VCR solution or of VCR plus AND combination were calculated on the basis of a series of dose-response data using GraphPad Prism software, version 6. The CompuSyn software generates the combination index (CI) value for a particular combination of VCR with AND based on the data from the CCK-8 assay, whereby additivity, synergy, and antagonism were reflected by CI values of 0.9-1.1, <0.9, and >1.1, respectively.3. Apoptosis assay and Caspase-3 activity assay: apoptotic cells were stained by Annexin V/PI staining and analyzed by flow cytometry on FACScan. Caspase-3 activity was assayed in cellular extracts using the Caspase-3 Colorimetric Assay kit.4. Detection of autophagic vacuoles: monodansylcadaverine (MDC) staining was used to label autophagic vacuoles in vitro. These cells were analyzed using a fluorescence microscope.5. Western blotting: expression levels of ERK, p-ERK, p53,p-p53, AKT and p-AKT were determined using Western blotting assays.6. Tumor model: BALB/c-nude mice were used for the establishment of SK-NEP-1 xenografts model. The mice were randomized to four groups and received vehicle (normal saline, oral, once daily), 100 mg/kg AND (oral, once daily), 0.2 mg/kg VCR (intraperitoneal, once weekly), or AND plus VCR for 4 weeks. Tumor size was monitored once a week. The mice were sacrificed, and the tumors were weighted after 4 weeks of treatment. Body weight changes were monitored weekly during the process of efficacy study.7. Statistical analysis: all statistical analyses were performed using the Statistical Product and Service Solutions software (SPSS; Version 13.0). All data and results presented in this study are representative of, or calculated from, at least three independent experiments. Values are represented as the mean ± standard deviation.Data were analyzed by Student's t-test.P-values <0.05 were statistically significant.Results1. Combination treatment of VCR wtih AND had a synergistic antiproliferative effect on SK-NEP-1 cells: SK-NEP-1 cells were treated with empty vehicle,VCR(0-15 nM), or AND (0-25 ?M) for different time periods, and then, cell inhibition rate was determined by CCK-8 assay. The results shows that VCR decreased the SK-NEP-1 cell viability in a dose- and time-dependent manner, and the IC 50 value at 48 hours is 10.8 nM. However, the antiproliferative effect of AND was very weak. Cell inhibition rate of VCR+AND group is higher than VCR group. The IC50 values decreased when treated with both drugs than with either drug alone. The difference between the VCR+AND group and the VCR group was statistically significant(P<0.05). The Cl values at IC50 were <0.9 when AND was set at 10, 20,and 25?M. These results indicated that higher concentrations of AND had a synergistic interaction with VCR. Representative microscopy images captured by a Nikon phase contrast microscope showing the effect of cells treated with vehicle;10.8nM VCR; 10.8nM VCR+10?M AND; 10.8nM VCR+20?M AND; or 10.8nM VCR+25?M AND for 48 hours (magnification×100). The control group showed normal cellular morphology and density. Changed cellular morphology and a decreased cell number were observed upon drug treatment, especially in the VCR plus AND combination treatment group.2. AND promotes VCR-induced SK-NEP-1 tumor cell death, and Caspase-3-dependent apoptosis participates in the process: SK-NEP-1 cells were treated with vehicle; 10.8nM VCR; 10.8nM VCR+10?M AND; 10.8nM VCR+20?M AND; or 10.8nM VCR+25?M AND for 48 hours. Then, the cells were stained with V and PI for 15 minutes, and the typical images of apoptosis were analyzed by flow cytometry. Control group did not show any significant apoptosis, and the percentage of early apoptotic and late apoptotic cells was only 5%. After treatment with 10.8nM VCR for 48 hours, the corresponding quantity was 32%. Moreover, an addition of AND with 10.8nM VCR dramatically increased the apoptotic rate of SK-NEP-1 cells,and the corresponding values for 10, 20, and 25?M AND were 39%, 50%, and 56%,respectively. The difference between the 10.8nM VCR+20?M AND group and the VCR group was statistically significant(P < 0.05). AND the difference between the 10.8nM VCR+25?M AND group and the VCR group was statistically significant(P<0.05). The caspase-3 activity in the cells was tested by caspase-3 colorimetric assay kit. It was found that the combination of VCR with 20 or 25?M AND induced a significant increase of caspase-3 cleavage compared with VCR alone treatment(P <0.01).3. Autophagy was not involved in VCR- or/and AND-induced SK-NEP-1 cell death: cells were treated with vehicle; 10.8nM VCR; 10.8nM VCR+10?M AND;10.8nM VCR+20?M AND; or 10.8nM VCR+25?M AND for 48 hours.Representative fluorescence microscopy images of cells stained with MDC. The images under the fluorescence microscopy revealed that no matter whether it was treated with VCR alone or VCR plus AND combination, the percentage of autophagic vacuoles showed no difference compared with control group. In the normalized data,no more than 5% of cells were MDC positive.4. The combination treatment of VCR with AND upregulated p-AKT and p-p53 levels: cells were treated with vehicle; 10.8nM VCR; 10.8nM VCR+10?M AND;10.8nM VCR+20?M AND; or 10.8nM VCR+25?M AND for 48 hours. Protein expression changes were examined by using Western blotting. The results showed an increase in p-p53 level and a decrease in p-AKT level in the cells treated with VCR/AND combination compared with control or VCR alone, and p-ERK was not affected. In the normalized data, p-p53 protein reached its highest level at 20?M AND treatment.5. AKT activator partly reversed apoptosis induced by VCR plus AND combination treatment in SK-NEP-1 cells: SK-NEP-1 cells were pretreated with or without SC-79, an activator of AKT, for 6 hours and then treated with VCR, VCR+AND, VCR+ SC-79, or VCR+ AND + SC-79 for 48 hours. The results of CCK-8 indicated that cell growth inhibition rate caused by VCR plus AND combination treatment was partly reversed by SC-79 pretreatment(P < 0.05). Data from Western blotting indicated that SC-79 restored the protein expression change of p-AKT and p-p53 caused by VCR plus AND combination treatment.6. AND plus VCR significantly inhibited SK-NEP-1 xenograft tumor growth in vivo: the results showed tumor volume and tumor weight of VCR+AND group were lower than control group, and the difference between two groups was statistically significant(P< 0.05). Tumor volume and tumor weight of VCR group were also lower than control group, and the difference between two groups was statistically significant(P < 0.05). There are no difference between AND group and control group(P>0.05).Conclusion1. Combination treatment of VCR and AND had a synergistic antiproliferative effect on SK-NEP-1 cells in vitro and inhibited SK-NEP-1 xenograft tumor growth in vivo. AND enhanced the chemosensitivity of SK-NEP-1 cells to VCR.2. PI3K-AKT-p53 signal pathway was involved in VCR and AND-induced SK-NEP-1 apoptotic cell death.3. Caspase-3-dependent apoptosis was involved in VCR and AND-induced SK-NEP-1 cell death.4. Autophagy was not involved in VCR and AND-induced SK-NEP-1 cell death. |
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