Network analysis and basic experiments on the inhibition of renal cancer proliferation and migration by alpinetin through PI3K/AKT/mTOR pathway

doi:10.21203/rs.3.rs-1849984/v1

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Network analysis and basic experiments on the inhibition of renal cancer proliferation and migration by alpinetin through PI3K/AKT/mTOR pathway

https://doi.org/10.21203/rs.3.rs-1849984/v1

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To investigated the antitumor effect of on renal clear cell carcinoma (ccRCC). Network Pharmacology analysis of targets and molecular mechanisms of Alpinetin treating ccRCC. The Annexin V PE/7-AAD kit was used to detect apoptosis. Flow cytometry and Cell Counting Kit-8 (CCK-8) were used to detect cell proliferation and cycle. Cell migration analysis was performed by a 24-well transwell chamber and the ibidi scratch insert. The protein expression of the target molecule was detected by Western blotting. Nude mouse tumorigenesis assays were used to determine the in vivo antitumor effects of alpinetin. Network pharmacology revealed that GAPDH, HRAS, SRC, EGFR, and AKT1 are the main targets of alpinetin for the treatment of ccRCC, with the PIK/AKT signaling pathway being the main pathway of action. Alpinetin could significantly inhibit the proliferation and migration of ccRCC cells by inducing apoptosis. Alpinetin also inhibited the cycle progression of ccRCC cells by blocking them in the G1 phase. Furthermore, in vivo and in vitro, alpinetin could inhibit the activation of PI3K/Akt pathway involved in the proliferation and migration of ccRCC. Alpinetin can inhibit the growth of ccRCC cells by inhibiting the activation of PI3K/Akt pathway and can be a potential anti-cancer drug for ccRCC.

Proliferation

alpinetin

clear cell renal cell carcinoma

migration

PI3K/Akt/mTOR

Clear cell renal cell carcinoma (ccRCC), which mainly originates from the urinary tubule epithelial system of the renal parenchyma, is the most common renal malignancy in urology. The incidence of ccRCC has been gradually increasing worldwide over the past few years [1, 2]. In 2020, there were approximately 14,830 deaths in United States [3]. Surgery remains the mainstay of treatment for ccRCC. However, some patients have missed the opportunity of surgery when they are diagnosed [4]. Anti-angiogenic therapy and tumor immunotherapy once offered hope for patients without surgery, but side effects and resistance to treatment have made long-term outcomes poor [5]. Therefore, the development of new therapeutic drugs is of great importance for ccRCC.

Flavonoids, as a kind of ubiquitous natural polyphenolic plant extract, are of great interest due to their tremendous anticancer effects [6–8]. Alpinetin is one of the flavonoids, which arecommonly found in ginger plants such as turmeric, cardamom, and turmeric. Recent studies have shown that alpinetinis effective in treating ovarian, lung, liver, pancreatic, breast, colon, and gastric cancers as an antitumor agent [9–13]. However, it is unclear whether alpinetinhas the sameeffect onccRCC. Network pharmacology is an important method to study the relationship between drugs and diseases, and it is widely used in drug and disease research to comprehensively analyze drug components, targets, and mechanisms of action [14, 15]. In the present study, we used network pharmacology, cellular and animal experiments to illustrate the effects of alpine on ccRCC.

Network pharmacology analysis

The network pharmacology was analyzed in the same way as previously reported [14, 15]. Briefly, the relationship between alpinetin and ccRCC was analyzed using Swiss Target Prediction and PharmMapperdatabases to obtain the core targets of alpinetin forccRCC treatment. Based on R software, key target genes GO, and KEGG were analyzed for enrichment using Bioconductor bioinformatics package with P value < 0.05 and Q value < 0.05.

Cell culture and alpinetin preparation

786-O and OS-RC-2 were purchased from the American Type Culture Collection (ATCC, USA). OS-RC-2 and 786-O cells were cultured in RPMI-1640 medium (Gibco, USA) in a constant temperature incubator at 5% CO² and 37°C. Alpinetin (20 mmol/ml) was purchased from MedChemExpress (MCE, USA).

Cell viability and transwell assays

Cell viability and Transwell assays as previously reported [16]. Cell proliferation capacity was detected using CCK8 (Dojindo, Japan). Transwell assays were performed according to the instructions for transwellproducts (Corning, Cambridge, USA). Different concentrations (0, 50, 100 μmol/mL) of alpinetin were used for experiments CCK8 and Transwell assays.

Scratch wound healing assays

Scratch wound healing assays as previously reported [16]. 70μl of cells (OS-RC-2) treated with alpinetin (0, 50, 100 μmol/mL) were added to each well of the culture insert (ibidi, Germany) at a concentration of approximately 5 × 10⁵ cells/ml. The cells were then photographed under the microscope every 12 hours and analyzed using Image J software.

Cell cycle assays and apoptosis

Cell cycle and apoptosis analyses were as previously reported [16].After 48 hours of cell culture (at alpinetin concentrations of 0, 50, and 100 μmol/mL), cells were harvested and processed for cell cycle and apoptosis assays using flow cytometry.

Western blot assays

Western blot analysis as previously reported [7]. RIPA lysis buffer (Beyotime, Shanghai, China) was used for protein extraction and BCA Protein Assay Reagent (P0012, Beyotime, China) was used for protein concentration determination. 40 μg of protein was used for electrophoresis. PVDF membranes were incubated with primary antibodies against p-PI3K (1:5000, AP1208, Abclonal, China), PI3K (1:1000, 20584-1-AP, Proteintech, China), AKT (1:800, 60203-2-Ig, Proteintech, China), p-AKT (1:1000, 66444-1-Ig, Proteintech, China), Mtor (1:1000, 66888-1-Ig, Proteintech, China), GAPDH (1:1000, 60004, Proteintech, China), and p-mTOR (1:1000, 67778-1-Ig, Proteintech, China) in shake flasks at 4°C overnight. After the PVDF membranes were washed three times, the secondary antibody (goat anti-rabbit, 1:2000, SA00001-2, Proteintech, China and goat anti-mouse, 1:3000, SA00001-1, Proteintech, China) was incubated. Finally, we used enhanced chemiluminescent chromogenic substrates to observe the proteins.

Tumor xenograft experiment

Nude mice were purchased from the Chongqing Medical University. This study was approved by the Research Committee and Animal Care and Use Committee of Chongqing Medical University (approval NO. 2020-CY-187-21), and was performed in accordance with the Animal Welfare Guidelines and the Declaration of Helsinki. 6-week-old mice were used for the tumor xenograft experiment. 10⁶ OSRC-2 cells in 200 µL PBS were injected subcutaneously into mice (6 per group). As previously reported, one group was injected intraperitoneally with alpinetinat a dose of 100 mg/kg mouse body weight every three dayswith a caliper calculating tumor volume [13]. Four weeks after the injection of OSRC-2 cells, the mice were euthanized (inhaled with excessive amounts of the anesthetic isoflurane). Tumors were weighed and immunohistochemical staining was performed using paraformaldehyde.

Statistical analysis

Statistical analysis was performed using SPSS 20.0 software. Normally distributed measurements were expressed as mean ± standard deviation (M ± SD), and t-test was used for comparison between groups. Non-normal distributions were expressed as median. Multiple group comparisons of means were performed using one-way ANOVA or Kruskal-Wallis tests, depending on the variance distribution; post hoc comparisons were performed using the Student-Newman-Keuls test. Comparisons between non-normally distributed data were made using the Mann-Whitney U test, and rate tests were made using the X² test. p<0.05 indicates statistical significance.

Determination of the core target of alpinetin to treat ccRCC

As shown in Figure 1A, after using the database analysis, we obtained 987 ccRCC disease targets, 341 alpinetin drug targets, and 82 drug-disease common targets. In addition, we plotted the network diagram of "disease-target-component" interactions (Figure 1B) and the drug-disease common target protein interaction network (Figure 1C, the larger the node, the darker the color and the higher the degree parameter value. According to the PPI topology analysis (Figure 1D), we found that GAPDH, HRAS, SRC, EGFR, and AKT1 are the main targets of alpinetin.

GO and KEGG enrichment analysis of core targets

GO analysis of 82 common targets showed that the intersection gene set was enriched to 1721 biological processes, 52 cellular components and 111 related to molecular functions. As shown in Figure 2 A-C, we took the top 20 items of GO enrichment analysis and showed them in bar graphs. KEGG signaling pathway enrichment analysis was performed for 82 common targets, and 145 signaling pathways were obtained. As shown in Figure 2D, we selected the top 20 items of KEGG signaling pathway enrichment analysis and displayed them in the bar graph. KEGG enrichment analysis (Figure 2D) revealed that PI3K/AKT signaling pathway is the core signaling pathway for alpinetintreatment of ccRCC. Therefore, we selected the PI3K/AKT signaling pathway as the core pathway for cellular and animal experimental studies.

Alpinetin inhibited cell proliferation inccRCC

In this study, we found that alpinetin (0μm, 50μm, 100μm, and 200μm) inhibited the proliferation of ccRCC cells in a dose-dependent manner, and the higher the concentration, the stronger the inhibitory effect (Figure 3A, 3B and 3C). In addition, the results of the CCK-8 assay also showed that alpinetin inhibited the proliferation of ccRCC cells in a time- and dose-dependent manner (Figure 3D and 3E). Compared to the 0μm group, alpinetin at a concentration of 100 μm reduced the proliferation of 786-O and OS-RC-2 cells to about 20% on day 4. Thus, alpinetincan significantly inhibit the proliferative capacity of ccRCC cells.

Alpinetin inhibited cell migration Alpinetinin ccRCC

The migration ability of tumor cells is intently associated to the spread of malignant tumor cells [17]. To investigate whether alpinetin can affect the migratory ability of ccRCC cells, we performed transwell assay and wound healing (scratch) assay. As shown in Figures 4A, 4B and 4C, the transwell experiment showed that the number of cells penetrating the transwell membrane was significantly reduced in 786-O and OS-RC-2 cells after alpinetin treatment. Similarly, the results of the wound-healing (scratch) assay also showed that the migration ability of 786-O and OS-RC-2 cells was significantly decreased after alpinetin treatment (Figures 4D and 4E). Thus, all the above data suggest that alpinetincan inhibit the migratory ability of ccRCC cells.

Alpinetin promoted cell apoptosis and inhibited cell cycle progression in ccRCC

Disordered apoptosis and uncontrolled cell cycle are important features of ccRCC cells [18, 19]. We found that alpinetin treatment significantly promoted apoptosis in ccRCC cells, and the effect was more pronounced with higher alpinetin concentration (Figure 5A). Meanwhile, the results of cell cycle showed that ccRCC cell division could be blocked in G1 phase after treatment with alpinetin, thus inhibiting cell cycle progression and cell proliferation (Figure 5B). These results suggest that alpinetin can promote apoptosis and block the progression of the ccRCC cell cycle.

Alpinetin inhibited the PI3K/AKT/mTOR pathway activation in ccRCC

The PI3K/AKT/mTOR pathway is crucial in the development and progression of ccRCC [20, 21]. To investigate whether the effect of alpinetin on ccRCC is related to the PI3K/AKT/mTOR pathway, we adoptedwestern blot to detect the protein expression changes of PI3K/AKT/mTOR pathway in ccRCC cells after alpinetin treatment (Figure 6). The results showed that the expressions of p-PI3K, p-AKT and p-mTOR were significantly decreased in 786-O and OS-RC-2 cells after alpinetin treatment. The higher the concentration, the more obvious the decrease in protein expression (Figure 6).

Alpinetin inhibited RCC tumor growth in vivo

In animal experiments, we could clearly observe the difference between the alpinetinand NC groups (Figure 7A and B). The volume and weight of tumors were significantly reduced by 66.67% and 50% (Figure 7 C and D). On the molecular side, we selected two biomarkers, Ki-67 and P27, to indicate tumor growth by immunohistochemical analysis. As a proliferation marker, Ki-67 decreases after alpinetintreatment. Meanwhile, postulated biomarkersof RCC, P27 increased sharply (Figure 7E). Besides, we analyzed the PI3K/AKT/mTOR pathway, which is consistent with in vitro experiments (Figure 7F). These results suggest that alpinetinwas able ofinhibiting RCC growth in vivo. These results suggest that alpinetinmay act as a negative regulator of the PI3K/AKT/mTOR pathway, inhibiting its activation in ccRCC (Figure8).

In this study, we found through network pharmacology that GAPDH, HRAS, SRC, EGFR, and AKT1 are the main targets of alpinetin in the treatment of ccRCC, and alpinetin can exert anti-tumor effects through the PI3K/Akt signaling pathway. Cellular experiments further revealedthat alpinetin could inhibit the proliferation and migration of ccRCC cells. Moreover, alpinetin could induce cell cycle progression of ccRCC to be blocked by G1, thus also promoting apoptosis of ccRCC cells. In addition, alpinetin inhibited PI3K/Akt/mTOR activation in vivo and in vitro, which is an important pathogenic pathway of ccRCC.

Recent studies have led to important insights into the molecular biology of ccRCC, contributing significantly to the targeted treatment of ccRCC and prolonged the life expectancyof patients [22, 23]. Anti-angiogenic drugs targeting VEGFR, such as bevacizumab, sorafenib, axitinib, and pazopanib, have shown good efficacy in metastatic ccRCC [24, 25]. In addition, the mTOR pathway is another important target for ccRCCtreatment, and the mTOR inhibitors everolimus and temoximus can be used to treat advanced ccRCC [20, 26]. However, almost all patients, no matter how they are treated, eventually develop drug resistance [27]. Therefore, the development of new anti-ccRCC drugs has important clinical value.

In recent years, the good anti-cancer effects of compounds contained in natural plants have attracted the attention of an increasing number of researchers [28]. Alpinetin, a natural flavonoid, has shown its good antitumor effects in a variety of tumors. For example, Lin et al. confirmed that alpinetin inhibited the development of lung cancer and increased the sensitivity to cis-diammineddichloridoplatium [9]. Zhao et al. reported that alpinetin could inhibit the proliferation and migration of ovarian cancer cells and human hepatoma cells [10, 13]. Zhang et al. confirmed that alpinetin can inhibit breast cancer growth through the ROS/NF-κB/HIF-1α axis [13]. Wang et al. and Malami et al. demonstrated that alpinetin suppresses human gastric cancer cells and HT-29 cell growth [13–15]. Nevertheless, the role of alpinetin in ccRCC has been rarely reported. In this study, we explored the anticancer effects of alpinetin in ccRCC from the aspects of proliferation, migration, cycle, apoptosis and related pathways.

With the advanced understanding of the biological behavior of ccRCC cells, inhibiting the malignant proliferation and migration of ccRCC cells has become the focus of many researchers [29–31]. Therefore, we investigated the effects of alpinetin on the inhibition by CCK-8, transwell, and Scratch wound healing assays. Our results exhibited that alpinetin significantly inhibited the migration and proliferation of ccRCC. These results provide theoretical support for the treatment of ccRCC with alpinetin.

Currently, the restoration of tumor cell cycle control mechanisms as well as the sensitivity to apoptosis have been considered as two effective approaches for the treatment of tumors [32, 33]. Alpinetin has also been reported to show promising effects in promoting apoptosis and inhibiting cycle progression [13, 14]. In the present study, apoptotic ccRCC cells were significantly increased after treatment with alpinetin. In addition, the proportion of cells in G1 phase was also significantly increased. Therefore, alpinetin can promote ccRCC cell apoptosis and block cell cycle progression.

The PI3K/AKT/mTOR pathway is a key pathway in the development and progression of various human tumors [34–37]. In addition, inhibitors of the PI3K/AKT/mTOR signaling pathway are among the most commonly used targeted drugs for the treatment of ccRCC [37]. However, as previously mentioned, drug resistance almost always occurs in the final outcome. Therefore, there is a needto accelerate the development of new drugs against renal cancer. In this study, we found that alpinetin inhibited the phosphorylation of PI3K/AKT/mTOR pathway-related proteins in ccRCC, thereby suppressing the activation of PI3K/AKT/mTOR pathway and inhibiting the progression of ccRCC. In animal experiments, we found that the tumor volume and weight of nude mice were significantly reduced after using alpinetin. Immunohistochemical analysis showed a significant decrease in Ki-67 and a significant increase in p27 after alpinetin treatment. In addition, we further examined the protein expression of PI3K/Akt/mTOR pathway and found that the pathway was also blocked. These findings suggest that galangin has anti-tumor activity in vivo.

In conclusion, in the present study, we revealed that alpinetin can inhibit the proliferation and migration of ccRCC cells, induce apoptosis, block cell cycle progression, and inhibit the activation of PI3K/AKT/mTOR pathway. Thus, our findings offer new possibilities for the clinical treatment of ccRCC.

Funding

This work was supported by incentive plan for scientific institutions of Chongqing, Fund No. jxyn2021-1-20.

Data Availability

The datasets are available from the corresponding author on reasonable request.

Conflicts of Interest

None

Author contribution

Conceptualization: Guo Yu, Jiang Li, Luo Shengjun, Tang Wei; Methodology, formal analysis : Hu Daixing, Zhao Xin, Zhao Guozhi; validation: Guo Yu, Jiang Li, Luo Shengjun; writing and editing: Guo Yu, Jiang Li, Luo Shengjun, Tang Wei; supervision: Guo Yu, Jiang Li, Luo Shengjun, Tang Wei; funding acquisition, Guo Yu, Jiang Li, Luo Shengjun, Tang Wei.

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Network analysis and basic experiments on the inhibition of renal cancer proliferation and migration by alpinetin through PI3K/AKT/mTOR pathway

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