Methylglyoxal-Derived Advanced Glycation End Products (AGE4) Promote Cell Proliferation and Survival in Renal Cell Carcinoma Cells through the RAGE/Akt/ERK Signaling Pathways

Abstract

Advanced glycation end products (AGEs) are the products formed through a non-enzymatic reaction of reducing sugars with proteins or lipids. There is a potential for toxicity in the case of AGEs produced through glycation with dicarbonyl compounds including methylglyoxal, glyoxal, and 3-deoxyglucosone. The AGEs bind the receptor for advanced glycation end products (RAGE) and stimulate the mitogen-activated protein (MAP) kinase signaling pathway that can increase the production of matrix metalloproteinases (MMPs). In addition, AGE-induced protein kinase B (Akt) signaling can promote cancer cell proliferation and contribute to many diseases such as kidney cancer. In light of the lack of extensive study of the relationship between methylglyoxal-induced AGEs (AGE4) and renal cancer, we studied the proliferous and anti-apoptotic effects of AGE4 on renal cell carcinoma (RCC) in this study. AGE4 treatment was involved in the proliferation and migration of RCC cells in vitro by upregulating proliferating cell nuclear antigen (PCNA) and MMPs while suppressing apoptotic markers such as Bax and caspase 3. Moreover, Akt and extracellular-signal-regulated kinase (ERK) were phosphorylated in RCC cells with AGE4 treatment. As a result, this study demonstrated that AGE4-RAGE axis can promote the growth ability of RCC by inducing PCNA, MMPs, and inhibiting apoptosis in RCC via the Akt and ERK signaling pathways.

INTRODUCTION

Advanced glycation end products (AGEs) are compounds developed through the non-enzymatic browning reaction between the amino group of protein and the carbonyl group or aldehyde group of reduced sugar.¹⁾ These AGEs can accumulate in the body and cause diseases such as atherosclerosis, diabetic retinopathy, neuropathy, and chronic kidney disease.^2–4) In addition, AGEs are related to changes in renal structure and contribute to chronic kidney diseases such as interstitial fibrosis and glomerulosclerosis.⁵⁾ Furthermore, accumulation of AGEs can cause DNA damage, resulting in apoptosis or cell proliferation.^6,7) These AGEs bind to their receptor (RAGE) and induce DNA damage, increase in metastasis and invasive properties which results in the development of cancer malignancy.^8–10) In addition, the interaction between AGEs and RAGE regulates several signaling pathways such as the extracellular-signal-regulated kinase (ERK) and protein kinase B (Akt) signaling pathways.^11,12) The Akt pathway modulates the growth, proliferation, and migration of tumor cells in breast cancer, colorectal cancer, kidney cancer, and ovarian cancer.^13,14) The ERK signaling pathway is known to be associated with cell survival and apoptosis. Also, the abnormal regulation of the ERK pathway leads to tumorigenesis.^15,16) In addition, both the Akt and the ERK signaling pathways play important roles in tumor growth, and inactivation of these pathways was studied as a cancer treatment.^17,18)

Methylglyoxal (MGO) is a dicarbonyl compound and a precursor capable of forming AGEs, and is highly reactive compared to glucose.¹⁹⁾ MGO can be ingested from foods with added sugars or can be endogenously formed via the glycolysis pathway.²⁰⁾ Increased levels of MGO in the body cause a corresponding increase in the production of MGO-derived AGEs (AGE4), which are renal toxic.¹⁹⁾ AGEs promote cell proliferation and migration by activating matrix metalloproteinases (MMPs) related to the progression of breast cancer.²¹⁾ Besides breast cancer, apoptosis resistance and tumor malignancy are increased by DNA damage and glycation-induced mutations of MGO.^22,23) Apoptosis is an important component of understanding cancer metastasis and progression.²⁴⁾ The p53 interacts with family Bcl-2 and stimulates the pro-apoptotic Bax protein directly.²⁵⁾ An increase in Bax negatively interacts with Bcl-2 and activates caspase3 to induce apoptosis.²⁶⁾ This apoptotic pathway cascade is a key part of renal cell carcinoma (RCC) development.²⁷⁾ Bcl-2, which prevents programmed cell death, promotes cancer cell proliferation and malignancy. However, p53 and Bax reduce cell viability and tumor progression.²⁸⁾

The most prevailing type of kidney cancer, accounting for more than 80% of malignant kidney cancers, is RCC.²⁹⁾ From 2013 to 2017, the incidence of RCC increased by approximately 15% of all new cancer cases, and RCC causes about 3.6% of total deaths by cancer.³⁰⁾ Also, in 2017, people with RCC in U.S.A. were estimated by 5.5 million.³⁰⁾ The causes of RCC are diverse such as smoking, obesity, hepatitis C, or dialysis. Also, RCC is more prevalent in diabetic patients with kidney disease.³¹⁾ Furthermore, metastasis and progression of kidney cancer can cause cancer in patients and eventually lead to death.³²⁾ According to aging, people generally have RCC, usually between ages 50 and 70.³³⁾ Although RCC is a severe disease, treating it at in early stage makes more likely to be cured.³⁴⁾ Previous study has shown that RAGE is expressed in RCC cells and is involved in cell growth.³⁵⁾ However, the significance of AGE4 in proliferative and anti-apoptotic effects is still unknown, particularly in RCC cells. Therefore, the purpose of the present study were, using RCC cell lines, to determine (1) whether AGE4 treatment has a proliferative effect on RCC and (2) the mechanism by which AGE4 treatment regulate cell proliferation, migration, and apoptosis in RCC cells.

MATERIALS AND METHODS

Reagents

Rosewell Park Memorial Institute (RPMI) 1640 medium and Minimum Essential Medium (MEM) were bought from Thermo Fisher Scientific (MA, U.S.A.). Penicillin–streptomycin solution, 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA), and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, U.S.A.), and MGO, LY294002, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and bovine serum albumin (BSA) were bought from Sigma-Aldrich (St. Louis, MO, U.S.A.) and PD98059 was bought from Cell Signaling Technology Inc. (MA, U.S.A.). RAGE antagonist, FPS-ZM1 (Lot. No. 553030) were purchased from Sigma-Aldrich.

Preparation of MGO-Derived AGEs (AGE4)

The preparation of AGE4 was slightly modified from previous methods.¹⁾ AGE4 were prepared by incubating BSA (50 mg/mL) in phosphate buffer (0.1 M, pH 7.4) with MGO (20 mM) at 37 °C for 7 d. The control was prepared using the same procedure in the presence of BSA but without MGO. The proteins were then dialyzed in phosphate buffer (0.1 M, pH 7.4) for 24 h at 4 °C. The protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (ThermoFisher Inc.), and the samples were diluted to 10 mg/mL and kept at −80 °C before use in the experiment.

Cell Culture

786-O and A498, the human renal cancer cell lines, were bought from ATC C (Manassas, VA, U.S.A.), and HK-2, the human proximal tubule epithelial cell line, was bought from the Korean cell line bank (Seoul, South Korea). A498 cells were incubated in MEM and 786-O and HK-2 cells were incubated in RPMI-1640 medium at 37 °C in a 5% CO₂ incubator. All media were supplemented with 100 units/mL streptomycin, 100 units/mL penicillin and 10% FBS.

Cell Viability Assay

The cell viability was measured by MTT assay. Briefly, cells (5 × 10⁴ cells/well) were seeded in 96-well plates. Next day, cells were treated with 100, 200, 400, and 800 µg/mL of BSA or AGE4 for 24 h. MTT solution was injected to each well. After 4 h cultivation, the MTT solution was discarded, and formazan crystals were lyzed in 100 µL of dimethyl sulfoxide (DMSO). The optical density was scanned at the 540 nm absorbance using a multi plate reader (ELx808, BioTek, VT, U.S.A.).

Wound Healing Migration Assay

786-O, A498, and HK-2 cells were seeded in 6-well plates (2 × 10⁵ cells/well) with FBS-free medium. After 24 h, cells were grown to >90% confluency and the monolayer of cells was scraped off using a 200 µL pipette tip and washed with phosphate buffered saline (PBS). Thereafter, cells were treated with either LY294002 (Akt inhibitor) or PD98059 (ERK inhibitor) in the presence of BSA or AGE4. Cells were cultured for 24 h and then photographed using light microscope (CKX41, Olympus, Tokyo, Japan). The wound width was quantified at 0 and 24 h using Image J software (National Institutes of Health, MD, U.S.A.).³⁶⁾

RNA Extraction and Quantitative Real Time PCR Analysis (qRT-PCR)

Cells were seeded in 6-well plates (2 × 10⁵ cells/well). After 24 h incubation, the media were changed with serum-free medium in the presence of 100 µg/mL BSA or AGE4 and cultured for 24 h. Next, total RNA was isolated using RNAiso Plus (TaKaRa, Kusatsu, Japan). Total mRNA was used for cDNA synthesis using the cDNA synthesis kit (the LeGene Premium Express first strand cDNA Synthesis System, LeGene Biosciences, CA, U.S.A.) manufacturer’s instructions. qRT-PCR was conducted with the PreMIX SYBR green (Enzynomics, Seoul, South Korea) kit using an iQ5 Thermal Cycler (Bio-Rad, CA, U.S.A.). The results were estimated to the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) and manifested as 2^−ΔΔCT values.

Western Blot Analysis

Cells were lysed by radio immunoprecipitation assay (RIPA) buffer at 4 °C and the supernatants were collected after centrifugation at 15000 × g for 20 min at 4 °C. The amount of protein was measured using the BCA protein assay kit. A consistent amount of protein was injected and then separated on either 7.5 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrotransferring to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, U.S.A.), the membrane were incubated with primary antibodies overnight at 4 °C, including anti-RAGE (sc-365154, 1 : 1000), anti-Bax (2772, 1: 1000), anti-Akt (9272, 1: 200), anti-phospho- Akt (4060, 1: 200, Cell Signaling Technology Inc.), anti-ERK1/2 (sc-514302, 1: 200), anti-phospho-ERK1/2 (sc-7383, 1: 200), anti-p53 (sc-126, 1: 200), anti-bcl-2 (sc-7382, 1: 200), anti-caspase 3(sc-56053, 1: 200), anti-proliferating cell nuclear antigen (PCNA) (sc-56, 1: 200), anti-GAPDH (sc-32233, 1: 200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A.), anti-cleaved caspase 3 (Asp175, 1: 1000, Cell Signaling Technology, Inc.), and secondary antibodies for 2 h at room temperature (r.t.), including anti-rabbit (sc-2357, 1 : 1,000), and anti-mouse (sc-2357, 1 : 1,000, Santa Cruz Biotechnology Inc.). Protein band images were acquired using the automatic X-ray film processor (JP-33, JPI Healthcare, Seoul, Republic of Korea).

Cell Cycle Analysis by Flow Cytometry Analysis

A498 cells were seeded in 60 mm petri dishes (1 × 10⁵ cells/well). After 24 h incubation, the cells were changed with serum-free medium in the presence of 100 µg/mL of BSA or AGE4 and cultured for 24 h. In the case of RAGE inhibitor treatment, it was incubated at 10 µM for 3 h, and washed on PBS. The cell was then transformed into a serum-free medium with 100 µg/mL of AGE4 for 24 h. The cells were collected after 24 h of treatment and fixed in 70% ethanol overnight at 4 °C. The propidium iodide (PI; Bioscience Co., San Diego, CA, U.S.A.) staining was performed with treatment of 50 µg/mL of ribonuclease (RNase) A (BIONEER CORPORATION, Daedeok-gu, Daejeon, Republic of Korea) to analyze cell cycle analysis for 30 min at r.t. A498 cells were divided into G1, and G2/M phases according to the PI fluorescence intensity. The percentage of cells distribution was measured with BD Accuri™plus flow cytometer (BD Bioscience Co., San Diego, CA, U.S.A.), and data were analyzed using BD Accuri™plus flow cytometer software system.

Statistical Analysis

All statistical analysis was conducted using GraphPad Prism software version 9 (GraphPad Prism Software, Inc., San Diego, CA, U.S.A.). All results are expressed as means ± standard deviations (S.D.), and all experiments were performed in triplicate. Significant differences between groups were confirmed using Tukey’s multiple comparison test. p Values of < 0.05< 0.01, 0.001, and < 0.001 were indicated statistically significant.

RESULTS

AGE4 Enhance Cell Proliferation of Renal Cell Carcinoma

RCC cells were treated with AGE4 and then subjected to qRT-PCR analysis to see if the treatment causes apoptosis. In Figs. 1A–C, cell viability of 786-O and A498 cells were increased after treatment of AGE4 for 24 h compared to BSA-treated cells. Interestingly, 786-O cells treated with AGE4 at the lowest dose of 100 µg/mL showed no significant difference in cell viability when compared to cells treated with BSA. However, HK-2 cells significantly (p < 0.01) reduced cell viability in a dose-dependent manner when exposed to more than 200 µg/mL of AGE4. We, therefore, decided on this concentration to use for the following study. On the basis of these results, we assumed that AGE4 reduce cell viability in normal kidney cells but increase in RCC cells. Using HPLC-electrospray ionization (ESI)-MS, the concentration of single AGE (CML, N^ε-carboxymethyl lysine; CEL, N^ε-carboxyethyllysine; MG-H1, methylglyoxal-derived hydroimidazolone; GOLD, glyoxal-derived lysine dimer; MOLD, methylglyoxal-derived lysine dimer; PYR, pyrimidine) was determined (Supplementary Table 1). In AGE4, the greatest concentration of MG-H1 (5410.16 ± 893.24 ng/mg protein) was found. CEL and PYR were not discovered in the BSA control, while other single AGEs were found at less than 100 ng/mg protein.

Fig. 1. Effects of Methylglyoxal-Derived Advanced Glycation End Products (AGE4) on the Cell Proliferation of Renal Cell Carcinoma (RCC) and Normal Kidney Cells

Cell viability and proliferation were measured by the MTT assay. (A) The RCC cell lines (A) 786-O and (B) A498, and the normal kidney cell line (C) HK-2, were treated with BSA or AGE4 (100–800 µg/mL) for 24 h. (D, E) qPCR was performed to determine the mRNA levels of MMP2 and MMP9 in 786-O and A498 cells. The cells were treated with AGE4 at the concentration of 100 µg/mL. (F, G) Total cell lysates of 786-O and A498 cells were probed for PCNA by Western blotting to measure protein levels. AGE4 was added to the cells at a dose of 100 g/mL. The values are normalized to BSA-treated cells. Densitometric analysis was quantified using ImageJ, and the level of the expression of the target protein to that of GAPDH was calculated. Data are expressed as mean ± S.D. of three independent experiments. NS, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (Color figure can be accessed in the online version.)

The expression of MMPs is associated with extracellular matrix (ECM) remodeling as well as cell migration and proliferation in tumor cells.³⁷⁾ To evaluate whether AGE4 treatment increases RCC cell proliferation, the cell lysate was analyzed by Western blotting for MMPs and PCNA. When AGE4 (100 µg/mL) was applied to A498 cells, the mRNA levels of MMP2 and MMP9 were significantly (p < 0.05) increased, whereas in 786-O cells, the mRNA level of MMP2 was significantly (p < 0.01) increased (Figs. 1D, E). Moreover, AGE4 treatment significantly (p < 0.05) increased the protein expression of PCNA in RCC in both RCC (Figs. 1F, G). Taken together, these results indicate that AGE4 treatment has the capability to promote the proliferation and migration of RCC.

AGE4 Promote Migration of RCC

Next, we determined whether AGE4 treatment promotes cell migration in RCC. The migration ability was determined by a wound-healing assay, which evaluates wound closure of a monolayer of cells. Using this assay, we observed that cell migration in 786-O and A498 cells was significantly (p < 0.05) increased, by approximately 26 and 45%, respectively, after 24 h of AGE4 treatment (Figs. 2A, B). Thus, AGE4 treatment increases the migration capabilities of tumor cells, which is linked to increased malignancy.

Fig. 2. AGE4 Promote Migration of RCC Cells

Images captured of cell migration and cell migratory distances of 786-O (A) and A498 (B) cells immediately (0 h), and 24 h after scratch generation. After a scratch was created, cells were treated with BSA or AGE4 (100 µg/mL) for 24 h. The ratio of migration was quantified using Image J. Cells treated with BSA were used as a control group. Data are expressed as mean ± S.D. of three independent experiments. ns, not significant, * p < 0.05.

AGE4 Suppress Apoptosis of RCC

To determine whether AGE4 treatment induce apoptosis in RCC, cells were treated to AGE4 and then subjected to qRT-PCR analyses. In 786-O cells, the mRNA levels of the pro-apoptotic markers p53, Bax, and caspase3 were significantly (p < 0.01) inhibited in the AGE4 treated cells compared with the control cells treated with BSA (Figs. 3A, B). The anti-apoptotic marker Bcl-2 was unchanged. Similarly, in A498 cells, p53 level was significantly (p < 0.0001) decreased after treatment with AGE4, whereas Bcl-2 were increased (Fig. 3B). Also, we determined the protein expression using Western blotting and the results showed that p53, Bax, and caspase3 expression were significantly (p < 0.05) lower and Bcl-2 was higher in the AGE4-treated cells in both RCC compared to BSA-treated cells. (Figs. 3C, D). These results suggest that AGE4 treatment downregulate pro-apoptotic and upregulate anti-apoptotic markers in RCC.

Fig. 3. AGE4 Inhibit Apoptosis of RCC Cells

786-O and A498 cells were treated with either BSA or AGE4 (100 µg/mL) for 2 h. (A, B) qPCR was performed to determine the mRNA levels of p53, Bax, Bcl-2, and caspase3 in 786-O and A498 cells. (C, D) The expression of p53, Bax, Bcl-2, caspase3 in 786-O and A498 cells were determined by Western blot analysis. Densitometric analysis was quantified using ImageJ, and the level of the expression of the target protein to that of GAPDH was calculated. Data are expressed as mean ± S.D. of three independent experiments. ns, not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001. (Color figure can be accessed in the online version.)

AGE4 Regulate Migration and Apoptosis via the Akt and ERK Signaling Pathways

Various signaling pathways are involved in cancer cell proliferation and apoptosis.^38,39) Akt and ERK signaling pathways play a part in AGEs-mediated cell proliferation and migration in previous studies.^40,41) Thus, cells were treated with AGE4, and Akt and ERK phosphorylation was determined. AGE4 treatment significantly (p < 0.01) activated Akt and ERK phosphorylation after 2 h compared to the BSA-treated cells (Fig. 4A, B). To investigate whether these AGE4-mediated Akt and ERK signaling play a role in cell migration of RCC, we used a specific inhibitor of either phosphatidylinositol 3-kinase (PI3K)/Akt (LY294002) or ERK (PD98059) signaling. After scratch was created, cells were treated with BSA or AGE4 along with the inhibitors (30 µM) for 24 h. As shown in Figs. 4C and D, the suppression of both Akt and ERK signaling strongly suppressed the cell migration in both RCC cell lines. BSA with LY (60.4%) and AGE4 with LY (35.7%) caused significant (p < 0.0001) changes in 786-O, but there was no significant difference in A498 cells. However, there were no significant differences between BSA with PD and AGE4 with PD in both RCC cell lines. Additionally, to evaluate whether RCC apoptosis is regulated by Akt or ERK signaling pathway, we assessed the expression of apoptosis markers on RCC cells treated with those inhibitors. As previously we have shown in Figs. 3C and D, the expression of Bax and caspase3 were decreased by treatment of AGE4 compared to BSA in both RCC cells (Figs. 4E, F). However, cells treated with 30 µM LY294002 or PD98059 for 4 h followed by AGE4 treatment for an additional 24 h, such decrease of Bax and caspase3 was alleviated in both RCC cell lines. Interestingly, LY294002 was more effective to block antiapoptosis than PD98059 in A498 cells (Fig. 4F). These results suggest that AGE4-mediated cell migration and anti-apoptosis are likely due to the phosphorylation of Akt and ERK signaling pathways.

Fig. 4. AGE4 Regulate Apoptosis and Migration via the Akt and ERK Signaling Pathways

(A, B) Western blot images show the effect of AGE4 on Akt and ERK activation in 786-O and A498 cells. Cells were treated with specific inhibitors, LY294002 (PI3K/Akt inhibitor, LY, 30 µM) or PD98059 (ERK inhibitor, PD, 30 µM) in the presence of either BSA or AGE4. (C, D) Scratch assay images were captured immediately (0 h), and 24 h after scratch generation. After a scratch was created, cells were treated with BSA or AGE4 (100 µg/mL) along with LY294002 or PD98059 for 24 h. (E, F) The expression of Bax and caspase3 in 786-O and A498 cells were determined by Western blot analysis. Densitometric analysis was quantified using ImageJ, and the level of the expression of the target protein to that of GAPDH was calculated. Data are expressed as mean ± S.D. of three independent experiments. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (Color figure can be accessed in the online version.)

Through AGE4-RAGE Axis, AGE4 Modulates Akt and ERK Activation, Apoptosis, and Cell Proliferation

The A498 cells were pretreated with 10 µM FPS-ZM1 (a selective RAGE inhibitor) for 3 h before being treated with 100 µg/mL of AGE4 for 24 h to see if the AGE4-RAGE axis affects RCC cell proliferation via Akt /ERK signaling pathways. In AGE4-treated RCC cells, RAGE inhibitor pretreatment significantly (p < 0.01) reduced phosphorylation of ERK and Akt protein compared to cells treated with AGE4 alone (Fig. 5A). Furthermore, inhibition of RAGE significantly (p < 0.01) increased the ratio of Bax/Bcl-2, and cleaved caspase 3 protein level, which were lowered by AGE4 treatment (Figs. 5B, C). While AGE4 treatment raised the expression of PCNA protein, a biomarker for cancer cell proliferation in A498 cells, FPS-ZM1 pretreatment significantly (p < 0.001) reduced the protein expression in the AGE4-treated cells (Fig. 5C). Figure 5D shows that AGE4 treatment increased the G1 phase of cell distribution in A498, compared with BSA control treatment (BSA:76.1 ± 5.1% vs. AGE4: 88.7 ± 5.7%), suggesting cell proliferation, while decreasing the G2/M phase cell cycle arrest (BSA:14.9 ± 3.7% vs. AGE4: 7.5 ± 2.7%), indicating inducing anti-apoptosis. In the cells treated with AGE4, however, when RAGE was inhibited, G1 phase significantly (p < 0.01) decreased (FPS-ZM1/AGE4: 80.9 ± 5.4%) and G2/M phase increased (FPS-ZM1/AGE4: 11.1 ± 1.9%).

Fig. 5. AGE4-RAGE Axis Regulates Apoptosis and Cancer Cell Proliferation via the Akt and ERK Signaling Pathways

The A498 cells were pre-treated with a specific RAGE inhibitor, FPS-ZM1 (10 µM) for 3 h, and treated with AGE4 (100 µg/mL, 24 h). (A) Western blot images show the effect of AGE4 on Akt and ERK activation in A498 cells. (B–D) The expression of Bax, Bcl-2, cleaved caspase3, and PCNA in A498 cells were determined by Western blot analysis. Densitometric analysis was quantified using ImageJ, and the level of the expression of the target protein to that of GAPDH was calculated. (E) The effect of AGE4 on the A498 cancer cell cycle was measured base on the G1, and G2/M phases by FACS analysis. Data are expressed as mean ± S.D. of three independent experiments. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (Color figure can be accessed in the online version.)

DISCUSSION

Numerous studies have shown that AGEs are known to be toxic in the kidney tissue and arise metastatic potential in several cancers including RCC, the most common form of kidney cancer.^22,42) ECM remodeling leads to the potential for tumor progression, and abnormal cell proliferation can initiate tumorigenesis.⁴³⁾ Normal (HK-2) cells had decreased cell vitality, but cancer (786-0 and A498) cells had no detrimental effect on proliferation with AGE4 treatment (Fig. 1A). One reason why HK-2 cells were more vulnerable to AGE4 treatment, but 786-0 and A498 cells proliferated more, might be differences in metabolic pathways between normal and malignant cells. Senavirathna et al.⁴⁴⁾ found that glyceraldehyde treatment of pancreatic ductal epithelial cells influenced the formation of intracellular AGEs differently in normal (HPDE) and cancer (PANC-1 and MIA PaCa-2) cells, with HPDE cells being more sensitive to glyceraldehyde-derived AGE toxicity. They also suggested that PANC-1 cells accumulate less AGEs than normal HPDE cells, probably due to increased expression of glyoxalase 1 which detoxifies the methylglyoxal precursor of AGEs. We observed that treatment of AGE4, methylglyoxal-derived AGEs increased the wound healing process and upregulate PCNA and MMP2/9 expression, which are linked to proliferation and invasion⁴⁵⁾ in RCC cells (Figs. 1, 2) suggesting that AGE4 are likely to promote tumor progression and cancer metastasis.

Normal cells undergo apoptosis due to intercellular stimuli, including oxidative stress, DNA damage, and growth factor deprivation, while the apoptosis is often dysregulated in tumor cells, leading to abnormal growth.⁴⁶⁾ We observed that AGE4 significantly downregulated the tumor suppressor p53, Bax, and caspase3, and upregulated an anti-apoptotic marker, Bcl-2 (Fig. 3). These results suggest that AGE4 accelerate RCC cell proliferation by negatively regulate apoptosis. The Akt and ERK signaling pathways are involved in different cancer processes, especially cell death and proliferation, and regulating these signaling pathways can be key for cancer treatment.^47,48) Previous studies have shown that the Akt pathway regulates cell survival and plays an important role in human cancer.⁴⁷⁾ Furthermore, the ERK pathway is essential for intrinsic or extrinsic cell functions and regulates apoptosis and proliferation.⁴⁸⁾ We found that AGE4-mediated anti-apoptosis and wound healing are due to the activation of Akt and ERK signaling pathways (Fig. 4). Furthermore, the ERK and PI3K)/Akt pathways are well known for their involvement in regulating RCC invasion and migration.⁴⁹⁾ ERK inhibitor PD98059 and PI3K/Akt inhibitor LY294002 suppressed the migration of 786-O and A498 cells stimulated by AGE4 (Figs. 4C, D) confirming the mechanism by which AGE4 induced migration and anti-apoptosis in RCC through the Akt and EKR signaling pathways. A previous study supported that a combination of MEK inhibitor AZD6244 and a low dose of antitumor reagent induced apoptosis in primary RCC cells resulting in cell proliferation reduction and tumor growth suppression.⁵⁰⁾ As a result, we confirmed that AGE4 treatment promote cell proliferation, migration, and anti-apoptosis likely due to the activation of Akt and ERK pathways in RCC. Nonetheless, further study is required to determine whether AGE4 enhance RCC cell growth in vivo and the specific inhibitors of RAGE, Akt, or ERK signaling suppress such tumor growth.

The impact of AGE4 on activating ERK and Akt, inhibiting apoptosis, and increasing cell proliferation in RCC can be regulated via the AGE4-RAGE axis, as demonstrated in Fig. 5. To the best of our knowledge, no research has been conducted comparing the expression of RAGE in cancer patients and healthy individuals. However, Wu et al.⁵¹⁾ revealed for the first time that RAGE expression was elevated in RCC tumor tissue relative to normal kidney tissue in paired samples of RCC and peritumoral normal kidney tissue collected from 30 patients. Because RAGE is a multi-ligand receptor known to bind to AGEs, S100 proteins, high mobility group box 1 (HMGB1), and amyloid β-peptide, resulting in cellular effects through the AGEs-RAGE axis,⁵²⁾ it is also possible that the increased proliferation and migration in RCC cells with AGE4 treatment in the current study was also due to increased generations of HMB1 and S100. The reactive oxygen species (ROS) generated by AGEs and their interaction with RAGE can enhance the production of HMB1 and S100.⁵³⁾ It has been reported that the treatment of 50 µg/mL of AGE4 significantly increased ROS content in macrophages compared to BSA.⁵⁴⁾ Wu et al.⁵¹⁾ have shown that the HMGB1 protein interacts to the RAGE receptor and functions as a crucial regulator in the development and angiogenesis of RCC cancer. It should be noted, therefore, that activation of RAGE by various ligands, including HMGB1 and S100 proteins, as well as AGE4, may lead to enhanced proliferation and migration in RCC cells.

In conclusion, our study found that AGE4 interaction with RAGE enhances RCC cell proliferation and migration while inhibiting RCC cell death via activation of the ERK and Akt signaling pathways and up-regulation of MMP2/9. More research is needed to determine the involvement of AGE4 in the pathological mechanisms of RCC as well as its clinical significance in the disease.

Acknowledgments

This research was supported by the Main Research Program (E0164400-04) of the Korea Food Research Institute (KFRI) funded by the Ministry of Science, the National Research Foundation of Korea (NRF) Grant funded by the Korea government (MEST) (No. 2017R1A2 B4012182), Korea University Grant (K1901241), and the School of Life Science and Biotechnology for BK21 PLUS, Korea University.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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