# Inhibitory Effect of Ursolic Acid on Proliferation and Migration of Renal Carcinoma Cells and Its Mechanism

Authors: Xiao Lyu; Xuhui Zhang; Libin Sun; Jingqi Wang; Dongwen Wang
Journal: Computational Intelligence and Neuroscience
 (2022)

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## Abstract

Background. Renal carcinoma is one of the most common malignant tumors in the urinary system. Autophagy can be both activated and inhibited in renal carcinoma, and it plays a double-edged role in the development of renal carcinoma. In the early stage of cancer, autophagy can suppress tumors. In the late stage, autophagy contributes to the survival of tumor cells in an unfavorable environment, and some autophagy-related proteins P62, LC3B, and beclin-1 have become indicators of the prognosis of patients with renal carcinoma. Aim. To demonstrate that ursolic acid activates autophagy in renal carcinoma 786-O cells by inhibiting the hedgehog signaling pathway. Methods. The effect of ursolic acid on the viability of 786-O cells was determined by the MTT method; the effect of ursolic acid on the proliferation and migration of 786-O cells was examined by crystalline violet staining and scratch assay, respectively. For the study of autophagy, we firstly screened the time points. Western blot assay was used to detect the expression level of autophagic protein P62 at different time points of ursolic acid on 786-O. Then, the Cell MeterTM Autophagy Assay Kit was used to detect the effect of different doses of ursolic acid on the autophagic fluorescence intensity of 786-O cells; the Western blot method was used to detect the effect of different doses of ursolic acid on the expression levels of LC3II and P62 proteins in 786-O cells. Further, AdPlus-mCherry-GFP-LC3B adenovirus transfection was used to detect the effect of ursolic acid on the autophagic flow of 786-O cells; ursolic acid was combined with the autophagy inhibitor chloroquine (CQ) to detect the expression level of autophagy protein LC3II by Western blot. In terms of mechanism, the effect of ursolic acid on hedgehog signaling pathway-related proteins in 786-O cells was detected by Western blot.Results. Ursolic acid inhibited the activity, proliferation, and migration of 786-O cells, enhanced the fluorescence intensity of autophagosomes in 786-O cells, increased the expression level of autophagy marker protein LC3II, and inhibited the expression level of P62 in a time and dose-dependent manner; ursolic acid activated the autophagic flow in 786-O cells, which showed that ursolic acid caused the accumulation of autophagic fluorescent spots and enhanced the fluorescence intensity of autophagosomes. Ursolic acid activated the autophagic flow in 786-O cells, as evidenced by the accumulation of autophagic fluorescent spots and enhanced fluorescence intensity of autophagosomes, and the combined use of the autophagy inhibitor CQ increased the expression level of LC3II compared to ursolic acid alone; ursolic acid decreased the expression levels of PTCH1, GLI1, SMO, SHH, and c-Myc and increased the expression level of Sufu in the hedgehog signaling pathway. Conclusion. Ursolic acid activates autophagy in renal carcinoma 786-O cells, probably by inhibiting hedgehog signaling pathway activity.


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## Body

## 1. Introduction

Renal carcinoma is one of the most common cancers is one of the most common malignant tumors in the urinary system [1]. By 2030, the number of new cases is expected to exceed 2.2 million and the number of deaths will exceed 1.1 million [2]. Hedgehog signaling pathway plays an important role in the growth and differentiation of human tissues and is critical for the body to maintain homeostasis in a physiological state [3]. Activation of the hedgehog signaling pathway involves multiple molecular components [4]. When the Hh ligand binds to PTCH1, a typical receptor for SMO inhibition, it leads to SMO aggregation and phosphorylation, which induces the segregation of downstream GLI1 proteins from Sufu and then into the nucleus to induce transcription of a range of oncogenes [5]. Most studies have shown that aberrant activation of the hedgehog signaling pathway is associated with the development, progression, and recurrence of renal carcinoma [6]. Autophagy is a highly evolutionarily conserved process that occurs when cellular contents, including damaged proteins, organelles, etc., are transferred to lysosomes and digested and degraded by enzymes for recycling [7]. When cells are exposed to unfavorable environments, such as nutrient deficiency, the mammalian target of rapamycin (mTOR) will be inhibited, followed by activation of the ULK complex (FIP200, Atg13, ULK) and then the downstream PI3KIII complex (hvps34, beclin-1, p150, UVRAG) to induce autophagy. Atg12-Atg5 and Atg7-Atg3, two interconnected systems, play an important role in the extension of autophagic membranes to form autophagosomes and their fusion with lysosomes to form autophagic lysosomes to disassemble their contents. Recent studies have shown that autophagy is closely associated with aberrant activation of the hedgehog signaling pathway. In this study, we investigated whether ursolic acid could regulate the proliferation, migration, and autophagy of renal carcinoma 786-O cells. For the study of autophagy, we firstly screened the time points. Western blot assay was used to detect the expression level of autophagic protein P62 at different time points of ursolic acid on 786-O. Then, the Cell MeterTM Autophagy Assay Kit was used to detect the effect of different doses of ursolic acid on the autophagic fluorescence intensity of 786-O cells; the Western blot method was used to detect the effect of different doses of ursolic acid on the expression levels of LC3II and P62 proteins in 786-O cells. Also, we further explored whether its mechanism was related to its inhibition of hedgehog signaling pathway activity to provide an experimental basis for the search for new anti-renal carcinoma active ingredients from traditional Chinese medicine.


## 2. Materials and Methods

### 2.1. Materials

Renal carcinoma cell line 786-O was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The sources of reagent instruments used in this study are as follows: Ursolic acid (Chengdu Manstead Biotechnology Co., Ltd., Lot No. MUST-17070501); GANT61 (Sigma-Aldrich, Lot No. 21798); MyCoy5A medium (Beijing Wokawei Biotechnology Co., Ltd., Lot No. AF29530969); Fetal bovine serum (Beijing Sai Aomei Cell Technology Co. 20180524); RIPA lysate (Hubei Dr.D. Biological Co., Ltd., Lot No. 15E28C05); Protease inhibitor (Lot No. P1010-1), AdPlusmCherry-GFP-LC3B adenovirus (Lot No. C3012), 4% paraformaldehyde (Lot No. 1808573), crystalline violet (Lot No. C0121) (Shanghai Ltd.); GLI1 (Beijing Biosun Biotechnology Co., Ltd., Lot No. A103296468); SHH (Lot No. 20697-1-AP), Sufu (Lot No. 26759-2-AP) (Wuhan Sanying Biotechnology Co., Ltd.); PTCH1 (Immunoway Inc., Lot No. YT3598); c Myc (Chengdu Zhengneng Biotechnology Co., Ltd., Lot No. 20190313); SMO (Hangzhou Hua'an Biotechnology Co., Ltd., Lot No. HN0716); GAPDH primary antibody (Lot No. A00131), HRP-labeled sheep anti-rabbit IgG secondary antibody (Lot No. YR202912) (Hangzhou Unitech Biotechnology Co., Ltd.); P62 (Lot No. 39749), LC3 (lot 12741) (CST, USA); Cell MeterTM Autophagy Assay Kit (AAT, USA, lot 23000);CO2 incubator (Thermo, USA, model Forma3111); Ultra-clean bench (Suzhou Antai Air Technology Co., Ltd, model SW-CJ-2FD); Confocal microscope (Leica GmbH, Germany, model SP8SR); Bio-Rad gel imaging system (Burroughs Corporation, USA, model 721BR11389).


### 2.2. Methodology

#### 2.2.1. Effect of Ursolic Acid on the Viability of 786-O Cells Measured by MTT Method

786-O cells in the logarithmic growth phase were inoculated in 6-well plates at 5 000 cells per well, and the cell-free wells were set as zeroing wells. After about 8 h, the original medium was discarded and a series of drug-containing medium containing different concentrations of ursolic acid (90, 60, 45, 30, 15, and 7.5μmol−L−1) was added. After 24 h of incubation, 10 μL of MTT working solution was added to each well and incubated at 37°C with 5% CO2 for 4 h. The liquid in the wells was discarded and DMSO (150 μL/well) was added. After incubation, the absorbance (A) at 570 nm was measured by ELISA, and the cell survival rate and IC50 were calculated. Cell survival rate = (Aadministered wells − Azeroed wells)/(Anormal wells − A zeroed wells) × 100%.


#### 2.2.2. Effect of Ursolic Acid on the Proliferation of 786-O as Detected by Crystalline Violet Staining

786-O cells in good growth condition were digested and inoculated with 1 000 cells per well in 6-well plates, and the culture was continued for 7 d. The medium was changed every 2 d. The medium was discarded, and a medium containing different doses of ursolic acid (20, 10, 5μmol-L-1) and GANT61 (20 μmol-L-1) was added. The medium was discarded, and a medium containing different doses of ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) were added. After 72 h of action, the original medium was discarded, 4% paraformaldehyde was fixed for 15 min, and the crystalline violet staining solution was added for 10 min and then washed three times with PBS to observe the effect of drugs on the proliferation of 786-O cells.


#### 2.2.3. Scratch Test for the Effect of Ursolic Acid on the Migration of 786-O

After trypsin digestion, 786-O cells were collected and inoculated in 24-well plates (1 ×105 cells/well), and when the monolayer of cells spread across the bottom of the plate, a cross was crossed with a 10-μL gun, washed with PBS, and drug-containing medium containing different doses of ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) was added immediately. Then, photographs were taken under an inverted microscope at 0, 12, and 24 h. The healing area was measured by ImageJ, and the percentage of wound healing was calculated.


#### 2.2.4. Selection of Autophagy Time Points

(1)
CellMeterTM Autophagy Assay Kit was used to detect the number of autophagosomes at different time points. 786-O cells at logarithmic growth stage were inoculated in 96-well plates (5 000 cells/well) at 5 time points (0, 4, 8, 12, and 24 h). After about 8 h, the old medium was discarded and 200 μL of ursolic acid at a concentration of 20 μmol−L−1 was added at different time points, and then the old medium was discarded. The autophagy fluorescence intensity was observed by inverted fluorescence microscopy.(2)
Western blot method is to detect the expression level of autophagy protein P62 at different time points. 786-O cells were inoculated in 6-well plates at 6×105 cells/well, and after cell apposition, 2 mL of ursolic acid at a concentration of 20 μmol-L-1 was administered at 4, 8, 12, and 24 h, respectively. Cells were collected, total cell protein was lysed, and protein concentration was determined by the BCA method. The protein concentration was determined by the BCA method. A certain amount of loading buffer was added according sample volume, denatured at high temperature and then electrophoresed, blocked by 5% BSA for 90 min, and the primary antibody was washed three times for 5 min each time in TBST.


#### 2.2.5. CellMeterTM Autophagy Assay Kit Detects the Effect of Different Doses of Ursolic Acid on the Autophagosome of 786-O Cells

786-O cell suspensions in logarithmic growth phase were collected and inoculated in 30 mm confocal dishes at1×104 cells/well. After about 8 h, the old medium was discarded, 1 mL of medium was given to the blank group, and ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) were given to each group with corresponding doses of drug-containing medium, and the old medium was discarded after 24 h. Each well was washed once with 500 μL of PBS, and 100 μL of LCell MeterTM Autophagy Assay Kit was added. Then, the cell was incubated for 30 min at 37 °C and observed with a Leica confocal microscope. Fluorescence images were taken with a 630x oil microscope, and the fluorescence intensity of each image was analyzed using ImageJ software.


#### 2.2.6. Effect of Ursolic Acid on Autophagic Flow in 786-O Cells

(1)
AdPlus-mCherry-GFP-LC3B adenovirus transfection assay to detect the effect of ursolic acid on autophagic flow in 786-O cells. 30 mm laser copolymer dishes were submerged in filtered 100μg-mL−1 poly-D-lysine (PLL) solution for 5 min and then placed in an ultra-clean table with open air blowing and UV sterilization. 786-O cells in logarithmic growth phase were inoculated with 2×104 cells per well in 30 mm laser copolymer dishes. The cells were incubated in a CO2 incubator (5% CO2, 37°C), and adenovirus transfection was performed when the fusion level of the cells was 50% under the microscope in the laser confocal dish. The cells in each dish were infected with mCherry-GFP-LC3B adenovirus solution (MOI = 40) for 24 h. The adenovirus solution was discarded, and the cells were continued to be cultured for 24 h. The successful transfection was observed under an inverted fluorescence microscope. After the adenovirus solution was discarded, the normal group was given 1 mL (0.1% DMSO) of complete medium, and ursolic acid (20, 10, 5 μmol−L−1) was given 1 mL of drug-containing medium in each group.(2)
Combination of ursolic acid and the autophagy inhibitor chloroquine (CQ) to detect the expression level of the autophagy protein LC3II by Western blot.786-O cells were inoculated in6×105 cells/well in 6-well plates, and after the cells were fused to about 40%, ursolic acid high-dose group (20 μmol−L−1), GANT61 group (20 μmol−L−1), ursolic acid high-dose (20 μmol−L−1) + CQ (25 μmol−L−1) group, GANT61 (20 μmol−L−1) + CQ (25μmol−L−1) group, and blank control group were set up. 1) +CQ (25 μmol−L−1) group, CQ group (25 μmol−L−1), and blank control group. The drug-containing medium was washed once with PBS, and the wells were incubated with the corresponding drug-containing medium for 24 h. Western blot was then performed as described above, and the protein bands were analyzed by Image Lab after completion.


#### 2.2.7. Western Blot Assay to Detect the Effect of Ursolic Acid on the Expression Levels of Hedgehog Signaling Pathway and Autophagy Proteins in 786-O Cells

786-O cells were inoculated at6×105 cells/well in a 6-well plate, and the cells were fused to about 40% and given medium containing ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) at different concentrations, respectively. The protein bands were analyzed by Image Lab.


### 2.3. Statistical Analysis

The data were analyzed by SPSS 19.0 statistical software, and the experimental data were expressed as mean ± standard deviation (x¯±s). t-Test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. <0.05 indicates that the difference is statistically significant.


## 2.1. Materials

Renal carcinoma cell line 786-O was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The sources of reagent instruments used in this study are as follows: Ursolic acid (Chengdu Manstead Biotechnology Co., Ltd., Lot No. MUST-17070501); GANT61 (Sigma-Aldrich, Lot No. 21798); MyCoy5A medium (Beijing Wokawei Biotechnology Co., Ltd., Lot No. AF29530969); Fetal bovine serum (Beijing Sai Aomei Cell Technology Co. 20180524); RIPA lysate (Hubei Dr.D. Biological Co., Ltd., Lot No. 15E28C05); Protease inhibitor (Lot No. P1010-1), AdPlusmCherry-GFP-LC3B adenovirus (Lot No. C3012), 4% paraformaldehyde (Lot No. 1808573), crystalline violet (Lot No. C0121) (Shanghai Ltd.); GLI1 (Beijing Biosun Biotechnology Co., Ltd., Lot No. A103296468); SHH (Lot No. 20697-1-AP), Sufu (Lot No. 26759-2-AP) (Wuhan Sanying Biotechnology Co., Ltd.); PTCH1 (Immunoway Inc., Lot No. YT3598); c Myc (Chengdu Zhengneng Biotechnology Co., Ltd., Lot No. 20190313); SMO (Hangzhou Hua'an Biotechnology Co., Ltd., Lot No. HN0716); GAPDH primary antibody (Lot No. A00131), HRP-labeled sheep anti-rabbit IgG secondary antibody (Lot No. YR202912) (Hangzhou Unitech Biotechnology Co., Ltd.); P62 (Lot No. 39749), LC3 (lot 12741) (CST, USA); Cell MeterTM Autophagy Assay Kit (AAT, USA, lot 23000);CO2 incubator (Thermo, USA, model Forma3111); Ultra-clean bench (Suzhou Antai Air Technology Co., Ltd, model SW-CJ-2FD); Confocal microscope (Leica GmbH, Germany, model SP8SR); Bio-Rad gel imaging system (Burroughs Corporation, USA, model 721BR11389).


## 2.2. Methodology

### 2.2.1. Effect of Ursolic Acid on the Viability of 786-O Cells Measured by MTT Method

786-O cells in the logarithmic growth phase were inoculated in 6-well plates at 5 000 cells per well, and the cell-free wells were set as zeroing wells. After about 8 h, the original medium was discarded and a series of drug-containing medium containing different concentrations of ursolic acid (90, 60, 45, 30, 15, and 7.5μmol−L−1) was added. After 24 h of incubation, 10 μL of MTT working solution was added to each well and incubated at 37°C with 5% CO2 for 4 h. The liquid in the wells was discarded and DMSO (150 μL/well) was added. After incubation, the absorbance (A) at 570 nm was measured by ELISA, and the cell survival rate and IC50 were calculated. Cell survival rate = (Aadministered wells − Azeroed wells)/(Anormal wells − A zeroed wells) × 100%.


### 2.2.2. Effect of Ursolic Acid on the Proliferation of 786-O as Detected by Crystalline Violet Staining

786-O cells in good growth condition were digested and inoculated with 1 000 cells per well in 6-well plates, and the culture was continued for 7 d. The medium was changed every 2 d. The medium was discarded, and a medium containing different doses of ursolic acid (20, 10, 5μmol-L-1) and GANT61 (20 μmol-L-1) was added. The medium was discarded, and a medium containing different doses of ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) were added. After 72 h of action, the original medium was discarded, 4% paraformaldehyde was fixed for 15 min, and the crystalline violet staining solution was added for 10 min and then washed three times with PBS to observe the effect of drugs on the proliferation of 786-O cells.


### 2.2.3. Scratch Test for the Effect of Ursolic Acid on the Migration of 786-O

After trypsin digestion, 786-O cells were collected and inoculated in 24-well plates (1 ×105 cells/well), and when the monolayer of cells spread across the bottom of the plate, a cross was crossed with a 10-μL gun, washed with PBS, and drug-containing medium containing different doses of ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) was added immediately. Then, photographs were taken under an inverted microscope at 0, 12, and 24 h. The healing area was measured by ImageJ, and the percentage of wound healing was calculated.


### 2.2.4. Selection of Autophagy Time Points

(1)
CellMeterTM Autophagy Assay Kit was used to detect the number of autophagosomes at different time points. 786-O cells at logarithmic growth stage were inoculated in 96-well plates (5 000 cells/well) at 5 time points (0, 4, 8, 12, and 24 h). After about 8 h, the old medium was discarded and 200 μL of ursolic acid at a concentration of 20 μmol−L−1 was added at different time points, and then the old medium was discarded. The autophagy fluorescence intensity was observed by inverted fluorescence microscopy.(2)
Western blot method is to detect the expression level of autophagy protein P62 at different time points. 786-O cells were inoculated in 6-well plates at 6×105 cells/well, and after cell apposition, 2 mL of ursolic acid at a concentration of 20 μmol-L-1 was administered at 4, 8, 12, and 24 h, respectively. Cells were collected, total cell protein was lysed, and protein concentration was determined by the BCA method. The protein concentration was determined by the BCA method. A certain amount of loading buffer was added according sample volume, denatured at high temperature and then electrophoresed, blocked by 5% BSA for 90 min, and the primary antibody was washed three times for 5 min each time in TBST.


### 2.2.5. CellMeterTM Autophagy Assay Kit Detects the Effect of Different Doses of Ursolic Acid on the Autophagosome of 786-O Cells

786-O cell suspensions in logarithmic growth phase were collected and inoculated in 30 mm confocal dishes at1×104 cells/well. After about 8 h, the old medium was discarded, 1 mL of medium was given to the blank group, and ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) were given to each group with corresponding doses of drug-containing medium, and the old medium was discarded after 24 h. Each well was washed once with 500 μL of PBS, and 100 μL of LCell MeterTM Autophagy Assay Kit was added. Then, the cell was incubated for 30 min at 37 °C and observed with a Leica confocal microscope. Fluorescence images were taken with a 630x oil microscope, and the fluorescence intensity of each image was analyzed using ImageJ software.


### 2.2.6. Effect of Ursolic Acid on Autophagic Flow in 786-O Cells

(1)
AdPlus-mCherry-GFP-LC3B adenovirus transfection assay to detect the effect of ursolic acid on autophagic flow in 786-O cells. 30 mm laser copolymer dishes were submerged in filtered 100μg-mL−1 poly-D-lysine (PLL) solution for 5 min and then placed in an ultra-clean table with open air blowing and UV sterilization. 786-O cells in logarithmic growth phase were inoculated with 2×104 cells per well in 30 mm laser copolymer dishes. The cells were incubated in a CO2 incubator (5% CO2, 37°C), and adenovirus transfection was performed when the fusion level of the cells was 50% under the microscope in the laser confocal dish. The cells in each dish were infected with mCherry-GFP-LC3B adenovirus solution (MOI = 40) for 24 h. The adenovirus solution was discarded, and the cells were continued to be cultured for 24 h. The successful transfection was observed under an inverted fluorescence microscope. After the adenovirus solution was discarded, the normal group was given 1 mL (0.1% DMSO) of complete medium, and ursolic acid (20, 10, 5 μmol−L−1) was given 1 mL of drug-containing medium in each group.(2)
Combination of ursolic acid and the autophagy inhibitor chloroquine (CQ) to detect the expression level of the autophagy protein LC3II by Western blot.786-O cells were inoculated in6×105 cells/well in 6-well plates, and after the cells were fused to about 40%, ursolic acid high-dose group (20 μmol−L−1), GANT61 group (20 μmol−L−1), ursolic acid high-dose (20 μmol−L−1) + CQ (25 μmol−L−1) group, GANT61 (20 μmol−L−1) + CQ (25μmol−L−1) group, and blank control group were set up. 1) +CQ (25 μmol−L−1) group, CQ group (25 μmol−L−1), and blank control group. The drug-containing medium was washed once with PBS, and the wells were incubated with the corresponding drug-containing medium for 24 h. Western blot was then performed as described above, and the protein bands were analyzed by Image Lab after completion.


### 2.2.7. Western Blot Assay to Detect the Effect of Ursolic Acid on the Expression Levels of Hedgehog Signaling Pathway and Autophagy Proteins in 786-O Cells

786-O cells were inoculated at6×105 cells/well in a 6-well plate, and the cells were fused to about 40% and given medium containing ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) at different concentrations, respectively. The protein bands were analyzed by Image Lab.


## 2.2.1. Effect of Ursolic Acid on the Viability of 786-O Cells Measured by MTT Method

786-O cells in the logarithmic growth phase were inoculated in 6-well plates at 5 000 cells per well, and the cell-free wells were set as zeroing wells. After about 8 h, the original medium was discarded and a series of drug-containing medium containing different concentrations of ursolic acid (90, 60, 45, 30, 15, and 7.5μmol−L−1) was added. After 24 h of incubation, 10 μL of MTT working solution was added to each well and incubated at 37°C with 5% CO2 for 4 h. The liquid in the wells was discarded and DMSO (150 μL/well) was added. After incubation, the absorbance (A) at 570 nm was measured by ELISA, and the cell survival rate and IC50 were calculated. Cell survival rate = (Aadministered wells − Azeroed wells)/(Anormal wells − A zeroed wells) × 100%.


## 2.2.2. Effect of Ursolic Acid on the Proliferation of 786-O as Detected by Crystalline Violet Staining

786-O cells in good growth condition were digested and inoculated with 1 000 cells per well in 6-well plates, and the culture was continued for 7 d. The medium was changed every 2 d. The medium was discarded, and a medium containing different doses of ursolic acid (20, 10, 5μmol-L-1) and GANT61 (20 μmol-L-1) was added. The medium was discarded, and a medium containing different doses of ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) were added. After 72 h of action, the original medium was discarded, 4% paraformaldehyde was fixed for 15 min, and the crystalline violet staining solution was added for 10 min and then washed three times with PBS to observe the effect of drugs on the proliferation of 786-O cells.


## 2.2.3. Scratch Test for the Effect of Ursolic Acid on the Migration of 786-O

After trypsin digestion, 786-O cells were collected and inoculated in 24-well plates (1 ×105 cells/well), and when the monolayer of cells spread across the bottom of the plate, a cross was crossed with a 10-μL gun, washed with PBS, and drug-containing medium containing different doses of ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) was added immediately. Then, photographs were taken under an inverted microscope at 0, 12, and 24 h. The healing area was measured by ImageJ, and the percentage of wound healing was calculated.


## 2.2.4. Selection of Autophagy Time Points

(1)
CellMeterTM Autophagy Assay Kit was used to detect the number of autophagosomes at different time points. 786-O cells at logarithmic growth stage were inoculated in 96-well plates (5 000 cells/well) at 5 time points (0, 4, 8, 12, and 24 h). After about 8 h, the old medium was discarded and 200 μL of ursolic acid at a concentration of 20 μmol−L−1 was added at different time points, and then the old medium was discarded. The autophagy fluorescence intensity was observed by inverted fluorescence microscopy.(2)
Western blot method is to detect the expression level of autophagy protein P62 at different time points. 786-O cells were inoculated in 6-well plates at 6×105 cells/well, and after cell apposition, 2 mL of ursolic acid at a concentration of 20 μmol-L-1 was administered at 4, 8, 12, and 24 h, respectively. Cells were collected, total cell protein was lysed, and protein concentration was determined by the BCA method. The protein concentration was determined by the BCA method. A certain amount of loading buffer was added according sample volume, denatured at high temperature and then electrophoresed, blocked by 5% BSA for 90 min, and the primary antibody was washed three times for 5 min each time in TBST.


## 2.2.5. CellMeterTM Autophagy Assay Kit Detects the Effect of Different Doses of Ursolic Acid on the Autophagosome of 786-O Cells

786-O cell suspensions in logarithmic growth phase were collected and inoculated in 30 mm confocal dishes at1×104 cells/well. After about 8 h, the old medium was discarded, 1 mL of medium was given to the blank group, and ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) were given to each group with corresponding doses of drug-containing medium, and the old medium was discarded after 24 h. Each well was washed once with 500 μL of PBS, and 100 μL of LCell MeterTM Autophagy Assay Kit was added. Then, the cell was incubated for 30 min at 37 °C and observed with a Leica confocal microscope. Fluorescence images were taken with a 630x oil microscope, and the fluorescence intensity of each image was analyzed using ImageJ software.


## 2.2.6. Effect of Ursolic Acid on Autophagic Flow in 786-O Cells

(1)
AdPlus-mCherry-GFP-LC3B adenovirus transfection assay to detect the effect of ursolic acid on autophagic flow in 786-O cells. 30 mm laser copolymer dishes were submerged in filtered 100μg-mL−1 poly-D-lysine (PLL) solution for 5 min and then placed in an ultra-clean table with open air blowing and UV sterilization. 786-O cells in logarithmic growth phase were inoculated with 2×104 cells per well in 30 mm laser copolymer dishes. The cells were incubated in a CO2 incubator (5% CO2, 37°C), and adenovirus transfection was performed when the fusion level of the cells was 50% under the microscope in the laser confocal dish. The cells in each dish were infected with mCherry-GFP-LC3B adenovirus solution (MOI = 40) for 24 h. The adenovirus solution was discarded, and the cells were continued to be cultured for 24 h. The successful transfection was observed under an inverted fluorescence microscope. After the adenovirus solution was discarded, the normal group was given 1 mL (0.1% DMSO) of complete medium, and ursolic acid (20, 10, 5 μmol−L−1) was given 1 mL of drug-containing medium in each group.(2)
Combination of ursolic acid and the autophagy inhibitor chloroquine (CQ) to detect the expression level of the autophagy protein LC3II by Western blot.786-O cells were inoculated in6×105 cells/well in 6-well plates, and after the cells were fused to about 40%, ursolic acid high-dose group (20 μmol−L−1), GANT61 group (20 μmol−L−1), ursolic acid high-dose (20 μmol−L−1) + CQ (25 μmol−L−1) group, GANT61 (20 μmol−L−1) + CQ (25μmol−L−1) group, and blank control group were set up. 1) +CQ (25 μmol−L−1) group, CQ group (25 μmol−L−1), and blank control group. The drug-containing medium was washed once with PBS, and the wells were incubated with the corresponding drug-containing medium for 24 h. Western blot was then performed as described above, and the protein bands were analyzed by Image Lab after completion.


## 2.2.7. Western Blot Assay to Detect the Effect of Ursolic Acid on the Expression Levels of Hedgehog Signaling Pathway and Autophagy Proteins in 786-O Cells

786-O cells were inoculated at6×105 cells/well in a 6-well plate, and the cells were fused to about 40% and given medium containing ursolic acid (20, 10, 5 μmol−L−1) and GANT61 (20 μmol−L−1) at different concentrations, respectively. The protein bands were analyzed by Image Lab.


## 2.3. Statistical Analysis

The data were analyzed by SPSS 19.0 statistical software, and the experimental data were expressed as mean ± standard deviation (x¯±s). t-Test was used for comparison between two groups, and one-way ANOVA was used for comparison between multiple groups. <0.05 indicates that the difference is statistically significant.


## 3. Results

### 3.1. Inhibition of 786-O Activity by Ursolic Acid In Vitro

Compared with the solvent control, different doses of ursolic acid (90, 60, 45, 30, and 15μmol−L−1) significantly inhibited 786-O cell activity (p < 0.01), and the inhibitory effect increased with higher doses of ursolic acid (Table 1).Table 1
Effect of ursolic acid on the activity of 786-O cells (x¯±s, n = 6).
Ursolic acid (μmol/L)A570 nm00.82 ± 0.12900.13 ± 0.021)600.16 ± 0.031)450.20 ± 0.051)300.31 ± 0.101)150.48 ± 0.081)7.50.68 ± 0.13Note:1)refers to compared to solvent control, P < 0.01.


### 3.2. Ursolic Acid Inhibits the Proliferation of 786-O Cells

The high dose of ursolic acid (20μmol/L) and the GANT61 (20 μmol/L) group almost completely inhibited the clonal growth of 786-O cells, followed by the medium dose of ursolic acid (10 μmol/L) and to a lesser extent the low dose (5 μmol/L) group (Figure 1).Figure 1
Effect of ursolic acid on the proliferation of 786-O cells.C, solvent control group; P, GANT61 20μmol−L−1 group; L, ursolic acid 5 μmol−L−1 group; M, ursolic acid 10 μmol−L−1 group; H, ursolic acid 20 μmol−L−1 group (notations are similar for Figures 2–5).Figure 2
Effect of ursolic acid on the migration of 786-O cells.p < 0.05 compared with solvent control; ∗p < 0;01; ∗∗p < 0.01; ##p < 0.01 compared with 12 h.Figure 3
Effect of ursolic acid on autophagy in 786-O cells.Figure 4
Effect of ursolic acid on autophagic flow in786-O cells. Notes: CQ, chloroquine 25μmol·L−1 group; P+CQ. GANT61 20 μmol·L−1 + chloroquine 25 μmol·L−1 group; H+CQ. ursolic acid 20 μmol·L−1 + chloroquine 25 μmol·L−1 group; 2 groups compared, #P < 0.05.Figure 5
Effect of ursolic acid on the expression of hedgehog signaling pathway proteins and autophagy-related proteins.


### 3.3. Ursolic Acid Inhibits Migration of 786-O Cells

Compared with the solvent control group, the GANT61 group and ursolic acid high (20μmol/L) and medium (10 μmol/L) dose groups could inhibit the migration of 786-O cells at 12 and 24 h (p < 0.01 or p < 0.05); compared with 12 h, the GANT61 (20 μmol/L) group and ursolic acid at different doses (20, 10, 5) could inhibit the migration of 786-O cells at 24 h (p < 0.01). The inhibition of migration of 786-O cells was more obvious in the GANT61 (20 μmol-L-1) and ursolic acid groups (20, 10, 5 μmol−L−1) at 24 h. The difference was statistically significant (p < 0.01) (Figure 2).


### 3.4. Autophagy Induction by Ursolic Acid in 786-O Cells

Compared with the solvent control group, the ursolic acid high-dose group was able to inhibit the expression of the autophagy-related protein P62 at the time point of 24 h (Figure6) (p < 0.05) (Figure 3). Therefore, the autophagy time point was set at 24 h for the subsequent experiments.Figure 6
Effect of ursolic acid on autophagy of 786-O cells at different time points.∗p  < 0.05 compared with 0 h.


### 3.5. Enhanced Autophagosome Fluorescence of 786-O Cells by Ursolic Acid

Compared with the solvent control group, the GANT61 (20μmol/L) group and ursolic acid high (20 μmol/L) and medium (10 μmol/L) dose groups enhanced the fluorescence intensity of autophagosomes in 786-O cells (p < 0.01 or P < 0.05), and unlike the solvent control group, which showed diffuse and weak fluorescence, the above groups could be seen as aggregates with enhanced fluorescence. The fluorescence of autophagosomes was enhanced in the above groups, suggesting that autophagy was activated (Figure 3).


### 3.6. Activation of 786-O Cellular Autophagic Flow by Ursolic Acid

Compared with the solvent control group, yellow fluorescent spots were clustered in the GANT61 (20μmol/L) group and ursolic acid high (20 μmol/L), medium (10 μmol/L), and low (5 μmol/L) dose groups, and red fluorescent spots increased with increasing ursolic acid dose, suggesting that autophagic processes were not blocked and autophagic flow was activated; the autophagy late inhibitor CQ inhibited the degradation of LC3II (p < 0.05), and the expression level of LC3II was elevated in the ursolic acid high dose (20 μmol/L) and GANT61 (20 μmol/L) groups (p < 0.05); after CQ was combined with ursolic acid and GANT61, the expression of LC3II was increased compared with that before the combination (p < 0.05), indicating that ursolic acid and GANT61 activated autophagosome formation rather than inhibiting autophagosome degradation (Figure 4).


### 3.7. Ursolic Acid Inhibited Hedgehog Signaling Pathway Activity to Activate Autophagy Compared with the Solvent Control Group

Medium (10μmol/L) and low (5 μmol/L) dose groups significantly inhibited the expression of the pathway protein PTCH1 (P < 0.01 or P < 0.05); the GANT61 (20 μmol/L) group and the ursolic acid high (20 μmol/L) and medium (10 μmol/L) dose groups increased the expression of LC3II, a marker protein of autophag (P < 0.01 or P < 0.05), while the expression level of P62 was significantly decreased (P < 0.01) (Figure 6).


## 3.1. Inhibition of 786-O Activity by Ursolic Acid In Vitro

Compared with the solvent control, different doses of ursolic acid (90, 60, 45, 30, and 15μmol−L−1) significantly inhibited 786-O cell activity (p < 0.01), and the inhibitory effect increased with higher doses of ursolic acid (Table 1).Table 1
Effect of ursolic acid on the activity of 786-O cells (x¯±s, n = 6).
Ursolic acid (μmol/L)A570 nm00.82 ± 0.12900.13 ± 0.021)600.16 ± 0.031)450.20 ± 0.051)300.31 ± 0.101)150.48 ± 0.081)7.50.68 ± 0.13Note:1)refers to compared to solvent control, P < 0.01.


## 3.2. Ursolic Acid Inhibits the Proliferation of 786-O Cells

The high dose of ursolic acid (20μmol/L) and the GANT61 (20 μmol/L) group almost completely inhibited the clonal growth of 786-O cells, followed by the medium dose of ursolic acid (10 μmol/L) and to a lesser extent the low dose (5 μmol/L) group (Figure 1).Figure 1
Effect of ursolic acid on the proliferation of 786-O cells.C, solvent control group; P, GANT61 20μmol−L−1 group; L, ursolic acid 5 μmol−L−1 group; M, ursolic acid 10 μmol−L−1 group; H, ursolic acid 20 μmol−L−1 group (notations are similar for Figures 2–5).Figure 2
Effect of ursolic acid on the migration of 786-O cells.p < 0.05 compared with solvent control; ∗p < 0;01; ∗∗p < 0.01; ##p < 0.01 compared with 12 h.Figure 3
Effect of ursolic acid on autophagy in 786-O cells.Figure 4
Effect of ursolic acid on autophagic flow in786-O cells. Notes: CQ, chloroquine 25μmol·L−1 group; P+CQ. GANT61 20 μmol·L−1 + chloroquine 25 μmol·L−1 group; H+CQ. ursolic acid 20 μmol·L−1 + chloroquine 25 μmol·L−1 group; 2 groups compared, #P < 0.05.Figure 5
Effect of ursolic acid on the expression of hedgehog signaling pathway proteins and autophagy-related proteins.


## 3.3. Ursolic Acid Inhibits Migration of 786-O Cells

Compared with the solvent control group, the GANT61 group and ursolic acid high (20μmol/L) and medium (10 μmol/L) dose groups could inhibit the migration of 786-O cells at 12 and 24 h (p < 0.01 or p < 0.05); compared with 12 h, the GANT61 (20 μmol/L) group and ursolic acid at different doses (20, 10, 5) could inhibit the migration of 786-O cells at 24 h (p < 0.01). The inhibition of migration of 786-O cells was more obvious in the GANT61 (20 μmol-L-1) and ursolic acid groups (20, 10, 5 μmol−L−1) at 24 h. The difference was statistically significant (p < 0.01) (Figure 2).


## 3.4. Autophagy Induction by Ursolic Acid in 786-O Cells

Compared with the solvent control group, the ursolic acid high-dose group was able to inhibit the expression of the autophagy-related protein P62 at the time point of 24 h (Figure6) (p < 0.05) (Figure 3). Therefore, the autophagy time point was set at 24 h for the subsequent experiments.Figure 6
Effect of ursolic acid on autophagy of 786-O cells at different time points.∗p  < 0.05 compared with 0 h.


## 3.5. Enhanced Autophagosome Fluorescence of 786-O Cells by Ursolic Acid

Compared with the solvent control group, the GANT61 (20μmol/L) group and ursolic acid high (20 μmol/L) and medium (10 μmol/L) dose groups enhanced the fluorescence intensity of autophagosomes in 786-O cells (p < 0.01 or P < 0.05), and unlike the solvent control group, which showed diffuse and weak fluorescence, the above groups could be seen as aggregates with enhanced fluorescence. The fluorescence of autophagosomes was enhanced in the above groups, suggesting that autophagy was activated (Figure 3).


## 3.6. Activation of 786-O Cellular Autophagic Flow by Ursolic Acid

Compared with the solvent control group, yellow fluorescent spots were clustered in the GANT61 (20μmol/L) group and ursolic acid high (20 μmol/L), medium (10 μmol/L), and low (5 μmol/L) dose groups, and red fluorescent spots increased with increasing ursolic acid dose, suggesting that autophagic processes were not blocked and autophagic flow was activated; the autophagy late inhibitor CQ inhibited the degradation of LC3II (p < 0.05), and the expression level of LC3II was elevated in the ursolic acid high dose (20 μmol/L) and GANT61 (20 μmol/L) groups (p < 0.05); after CQ was combined with ursolic acid and GANT61, the expression of LC3II was increased compared with that before the combination (p < 0.05), indicating that ursolic acid and GANT61 activated autophagosome formation rather than inhibiting autophagosome degradation (Figure 4).


## 3.7. Ursolic Acid Inhibited Hedgehog Signaling Pathway Activity to Activate Autophagy Compared with the Solvent Control Group

Medium (10μmol/L) and low (5 μmol/L) dose groups significantly inhibited the expression of the pathway protein PTCH1 (P < 0.01 or P < 0.05); the GANT61 (20 μmol/L) group and the ursolic acid high (20 μmol/L) and medium (10 μmol/L) dose groups increased the expression of LC3II, a marker protein of autophag (P < 0.01 or P < 0.05), while the expression level of P62 was significantly decreased (P < 0.01) (Figure 6).


## 4. Discussion

Renal carcinoma is one of the most common malignant tumors in the urinary system. Recent studies have shown that autophagy is closely associated with aberrant activation of the hedgehog signaling pathway. Autophagy can be both activated and inhibited in renal carcinoma, and it plays a double-edged role in the development of renal carcinoma [8, 9]; in the early stage of cancer, autophagy can suppress tumors, while in the late stage, autophagy contributes to the survival of tumor cells in an unfavorable environment, and some autophagy-related proteins P62, LC3B, and beclin-1 have become indicators of the prognosis of patients with renal carcinoma [10, 11]. Autophagy is a dynamically changing process [12, 13], and this experiment was conducted to map the autophagy time point, and the results showed that the activation of autophagy was more obvious when ursolic acid acted on 786-O cells at 24 h. Therefore, the time point of the subsequent experiment was set to 24 h. It was found that ursolic acid could increase the expression level of LC3II protein in 786-O cells, which is considered as autophagy. However, the reason for this may be the increased autophagosome production and also the blocked degradation phase of autophagosomes, so the degradation phase was inhibited using the autophagic tool drug CQ in combination to observe whether the expression level of LC3II was changed [14]. AdPlus-mCherry-GFP-LC3B adenovirus was also used to transfect cells, based on the principle that in the case of non-autophagy, mCherry-GFP-LC3B is present in the cytoplasm as diffuse yellow fluorescence under fluorescence microscopy, In the case of autophagy, mCherry-GFP-LC3B aggregated on the autophagosome membrane under fluorescence microscope, presenting as yellow spots; When the autophagosome and lysosome fuse, they appear as red spots due to partial quenching of GFP fluorescence. The above experimental results suggest that ursolic acid and GANT61 activate the autophagosome formation phase, but do not inhibit the lysosome fusion degradation phase [15, 16]. Currently, hedgehog pathway inhibitors have been widely used as a treatment for various types of cancers caused by pathway overactivation.


## 5. Conclusion

The present experimental study showed that ursolic acid, a novel inhibitor of hedgehog signaling pathway, could induce autophagy in renal carcinoma cells 786-O (Figure7), but since the role of autophagy in renal carcinoma remains controversial, whether it will promote or prevent renal carcinoma progression remains to be further investigated. Our future research direction is to analyze the effect of autophagy cells on carcinoma.Figure 7
Ursolic acid activates 786-O cell autophagy by inhibiting the hedgehog signaling pathway.



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*Source: 1529132-2022-05-04.xml*