"Inhibitors and Mechanisms of Solanine and Curdione on HIF-1α in Renal Carcinoma Xenograft and Human Renal Carcinoma ACHN Cells"

The main purpose of this study was to observe the effect of Solanine and Curdione on HIF-1α in renal carcinoma transplanted tumor and human renal carcinoma ACHN cells, and to detect the upstream regulatory proteins of HIF-1α (PI3K/p-PI3K, Akt/pAkt, mTOR), and to explore the anti-renal cancer mechanism of Solanine and Curdione. Research is divided into two parts, the first part of the research in kidney transplantation tumor, the method is: take the subcutaneous injection method to establish nude mouse model of kidney transplantation tumor ACHN cells, divided into the Solanine, Curdione, Solanine&Curdione (1:1) group and the negative control group, the abdominal cavity injection drug delivery 1 times a day, for 28 days to death mice, tumors present organization, said the tumors had the quality, calculate the inhibitory rate. The expression of HIF-1α protein was detected by Western blotting. The content of ATP in tumor tissues was determined by biochemistry method. The expressions of p-PI3K, p-Akt, mTOR and other proteins were detected by Western Blot. The results showed that all nude mice were successfully molded, and no nude mice died during the medication period.

The results showed that the relative expression rate of HIF-1α protein in ACHN cells treated with Solanine and Curdione decreased compared with the control group (P < 0.05), and the combined administration of Solanine and Curdione had a more significant down-regulation effect on HIF-1α protein (P < 0.01). Solanine and Curdione significantly inhibited the proliferation of ACHN cells in vitro (P < 0.05), and the combination of Solanine and Curdione significantly inhibited the growth of ACHN cells (P < 0.01). The ATP production of ACHN cells after treatment with Solanine and Curdione was significantly decreased (P < 0.05), and the effect of combined treatment group was more significant than that of single treatment group (P < 0.01). The relative expression rates of p-PI3K, p-Akt and mTOR proteins in ACHN cells treated with Solanine and Curdione were lower than those in the control group (P < 0.05), and the combined administration had a significant down-regulation effect on p-PI3K, p-Akt and mTOR proteins (P < 0.01). Based on the above, we believe that the combination of Solanine and Curdione can significantly inhibit the energy metabolism of renal cancer, mainly by significantly down-regulating the expression of HIF-1α and inhibiting the PI3K/p-PI3K and Akt/p-Akt transformation process of the signal transduction pathway upstream of HIF-1α.

Introduction
Hypoxia-inducible factor-1 (HIF-1) is a protein complex composed of two similar subunits, HIF-1α and HIF-1β. HIF-1 has biological roles including mediating hypoxia and regulating metabolism. HIF-1α is a hypoxia-induced transcription factor that is present at high levels in malignant solid tumors, but not in normal tissues or slow-growing tumors. In fast-growing tumors, HIF-1α is and Curdione can inhibit the growth of kidney energy metabolism process of interference with, but the specific mechanism is unclear, thus, to explore the Solanine, Curdione, the function of kidney HIF -1 alpha and further study of related mechanisms, for the development and application of Solanine, Curdione has important value, and Solanine, Curdione will be safe and effective prevention and control of malignant tumor of cheap drugs [1][2][3].

Main Materials and Instruments
Human renal carcinoma ACHN cell line was preserved by the

Observation of Tumor Volume and Tumor Inhibition Rate:
At the end of administration, mice were killed, tumor mass was completely stripped, tumor mass was weighed, and tumor inhibition rate was calculated according to the following formula: tumor inhibition rate (%) = (1-average tumor weight in the treatment group/average tumor weight in the control group) ×100%.

ATP Level Detection in Tumor Mass Tissue: The transplanted
tumor was taken, weighed and prepared into 10% homogenate with normal saline, and the ATP content in the transplanted tumor tissue was detected according to the kit instructions.

Alpha HIF -1 Protein Expression in Tumor Tissue of
Detection: transplanted tumor, weighing 100 g after extraction of proteins, lines of sds-page electrophoresis (polyacrylamide gel electrophoresis), to set the conditions for 80 V, 25 min, 100 V, 80 min, the end will be transferred to the PVDF membrane protein, set conditions to 200 mA, 180 min, and in TBST containing 5% skimmed milk powder, enclosed 1 h at room temperature. Primary antibody HIF-1α (1∶1 000) and β-actin (1∶5 000) were incubated at 4 ℃ overnight. Wash 3 times with TBST. The second antibody was incubated at room temperature for 2 h. Wash 3 times with TBST.

In Vitro Experiment
Cell Culture: ACHN cells were cultured in DMEM containing 10% calf serum, 100 units /mL penicillin and 100μg/mL streptomycin. The cells were incubated at 37℃ and 5%CO2. The The lysates were centrifuged at 10000×g at 4℃ for 30min. The protein concentration of supernatant was measured by Bradford method. The protein was separated by 15% SDS-polyacrylamide gel electrophoresis. After electrophoresis will be transferred to nitrocellulose membrane protein, will use 5% skimmed milk powder solution 37 ℃ close after 2 h, respectively with p -PI3K, p -Akt, mTOR a react to 2 h, 37 ℃ PBST clean 2 times, each time 15 min, then 37 ℃ with horseradish enzyme labeled second antibody response 1 h, after washing, fully with PBST to DAB chromogenic, scan, use of actin antibody consistency on the sample amount is determined.

Statistical Treatment
SPSS20.0 was used for statistical analysis, and the experimental data were represented as the experimental data. Analysis of variance was used for comparison between multiple groups, and P <0.05 was considered statistically significant.

In Vivo Experiment Effect on the Growth of Transplanted Tumor in Nude Mice
with Tumor Bearing: After 28 days of treatment with Solanine and/or Curdione, the tumor weight was significantly lower than that of the control group (P<0.01), and the combination group was lower than that of Solanine and/or Curdione alone (P<0.05), as shown in Table 1.

Effect of ATP on Tumor Tissue in Nude Mice with Tumor:
After 28 days of treatment with Solanine or Curdione, ATP levels in the transplanted tumor tissues decreased, with statistical difference compared with the blank control group (P < 0.05), and the combined group was lower than the single drug group, as shown in Table 1.

Effect of HIF-1α Protein Expression in Tumor Tissues of
Nude Mice: Western blotting was used to detect the changes of HIF-1α protein expression in tumor block tissues of each group.
The results showed that the HIF-1α protein expression in tumor block tissues of the Solanine and or Curdione intervention group was significantly decreased compared with the blank control group (P < 0.05), and the decrease in the combination group was more significant than that in the single drug intervention group (P < 0.05), Table 1.

In Vitro Experiment
Effects on the Proliferation of ACHN Cells: After treatment with Solanine and Curdione for 24, 48 and 72h, CCK-8 kit was used to detect ACHN cells, and the results showed that the cell growth was inhibited to varying degrees in a time-dependent manner (P < 0.01), as shown in Table 2.

Effect on HIF-1α Protein Expression in ACHN Cells: Western
blot results showed that the relative expression rate of HIF-1α protein in ACHN cells treated with Solanine and Curdione was significantly lower than that in the control group (P < 0.05), and the combined administration of Solanine and Curdione significantly down-regulated HIF-1α protein (P < 0.01), as shown in Table 2. Effects of Solanine and Curdione on ATP Production in ACHN Cells: Results As shown in Table 2, after treatment with Solanine and Curdione, ATP production of ACHN cells decreased (P < 0.05), and the effect of the combined treatment group was more obvious than that of the single treatment group (P < 0.01).

Effects of Solanine and Curdione on p-PI3K, p-Akt and mTOR Protein Expression Levels in ACHN Cells: Western blot
results were shown in Table 2. The relative expression rates of

p-PI3K, p-Akt and mTOR proteins in ACHN cells treated with
Solanine and Curdione were significantly lower than those in the control group (P < 0.05), and the combined administration had a significant down-regulation effect on p-PI3K, p-Akt and mTOR proteins (P < 0.01).
In this study, it was confirmed that Solanine and Curdione had no effect on the protein expressions of PI3K and Akt but reduced the expressions of p-PI3K and p-Akt, suggesting that Solanine and Curdione down-regulated the PI3K/Akt pathway by inhibiting the phosphorylation of PI3K/ p-PI3K and Akt/p-Akt. Therefore,