Baicalein inhibits renal cancer cell growth and metastasis via ERK and p38 pathway

doi:10.21203/rs.3.rs-1847253/v1

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Baicalein inhibits renal cancer cell growth and metastasis via ERK and p38 pathway

https://doi.org/10.21203/rs.3.rs-1847253/v1

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Background

Baicalein has exhibited anti-tumor effects against several cells of different origins. However, the potential effects of baicalein on renal cancer has not been investigated.

Methods

Human A498 and 786-O cell lines were used to investigate the antirenal acitivity of baicalein. CCK-8 assay was used to detect cell viability. Cell migration was assessed by scratch assay and transwell migration assay in vitro. Transwell invasion assay was carried out to evaluate the effect of baicalein on cell invasion. Apoptosis assay was performed to detect cell apoptosis using the Annexin V-FITC/PI apoptosis detection kit. The protein expression of ERK, p-ERK, p38, p-p38, p53, bax, bcl-2 and survivin were measured by WB assay.

Results

Baicalein significantly inhibited the proliferation of A498 and 786-O cells in a time- and dose-dependent manner. The similar effect of baicalein on cell migration, invasion and cell apoptosis were observed. Mechanismlly, ERK and p38 MAPK pathways account for the anti-tumor effects of baicalein.

Conclusions

Baicalein can induce apoptosis and inhibit metastasis of renal caner cells through inhibition of the ERK and p38R pathway.

Renal cancer

Baicalein

Metastasis

Apopotosis

ERK1/2

p38

Renal cell carcinoma (RCC) is the most common type of renal cancer, and its incidence is increasing worldwide over recent years [1]. About 20–30% of patients have metastases as soon as they are found, and their median survival is 8 months [2]. At the moment, only the surgical intervention has limited benefit for patients. In addition, RCC is very insensitive to chemotherapy and radiotherapy, and the efficacy of cytokine therapy is less than 20% [3]. Thus, new treatments are needed to treat RCC. In recent years, many people have begun to try to use new bioactive compounds from plants and other natural sources as new anticancer drugs. It is reported that several traditional Chinese medicines (TCMs) can be used to prevent and treat various cancers [4, 5].

In China, Scutellaria baicalensis (SB), known as Huang-Qin, is used to treat a variety of diseases, including inflammation, high blood pressure, cardiovascular disease, bacterial and viral infections, with very few side effects [6]. Baicalein (Ba) is a flavonoid isolated from the root of scutellaria baicalensis and has significant biological activity. Both alone and in combination with other drugs have shown anti-tumor effects in a number of malignancies, including breast cancer, hepatocellular carcinoma, leukemia and colon cancer [7–10]. However, there are few studies on the exact effect and molecular mechanism of baicalein on RCC cells.

In this study, we found that baicalein can inhibit the growth of RCC cells and promote their apoptosis in vitro. At the same time, we found that baicalein could significantly inhibit the migration and invasion of RCC cells. We investigated the underlying molecular mechanisms and found that the ERK1/2 and p38 MAPK pathways play a key role. Together, these new findings provide new ideas for the treatment of renal cell carcinoma.

Cell lines and reagents

This study was approved by the Qilu Hospital Committee of Shandong University. Human A498 and 786-O cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were grown in RPMI-1640 medium (Hyclone, Utah, USA) containing 10% fetal calf serum (Gibco, California, USA) at 37°C in a 5% CO2 incubator. Baicalein was purchased from Selleck Chemicals (Houston, TX, USA), and was dissolved according to the manufacturer’ instructions. Antibodies against phospho-p38 (Ser473), p38, phospho-ERK1/2, ERK1/2, GAPDH, and HRP-conjugated goat anti-rabbit and anti-mouse IgG were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p53, Bax, Bcl-2, survivin were obtained from abcam (Abcam, Cambridge, UK).

Cell viability assay

CCK-8 assay was used to detect cell viability. Briefly, A498 and 786-O (3×10³ cells/well) cells in 200μl of medium were seeded in 96-well plates. After 12h for adherence, the medium were replaced with the medium containing different concentrations of baicalein and the plate was incubated for 24, 48 and 72h. Subsequently, the cultured cells were treated with 20μl of Cell Counting kit-8 (Dojindo, Japan), and incubated at 37°C for additional 4h according to the instructions. The absorbance values were determined at 450nm with the Microplate Reader (Bio-Rad, Hercules, CA, USA).

In vitro scratch assay

Cell migration was assessed by scratch assay. After incubation with baicalein (10 and 20μM respectively) for 12h, each well was manually scratched with a 200ul pipette tip, washed with PBS three times and then incubated with baicalein (10 and 20μM respectively) at 37°C. Images were taken again after 24h. The distance between two cell edges were analyzed by ImageJ software.

Transwell invasion and migration assay

Transwell migration assay was carried out to further evaluate the effect of baicalein on cell migration. It was done as described before [11]. Briefly, a total of 5×10⁴ cells were seeded into the upper chamber using serum-free medium. Meanwhile, RPMI 1640 containing 20% FBS and baicalein (10 and 20μM) or PD98059 (50μM) or SB203580 (5μM) were added to the lower chamber. After 24h, the non-invaded cells in the upper chamber were gently removed with a cotton swab whereas the cells attaching to the lower surface were fixed with precooled methanol and stained with 0.1% crystal violet. At least five fields of each chamber were randomly selected and the cell numbers were counted under the microscope.

For invasion assay, the cells were seeded in upper chambers coated with 2mg/ml Matrigel (BD Biosciences, MA, USA). The rest of assay was performed as the migration assay. After 24h, the cells on lower surface were also counted in at least five randomly fields, then the cell number was analyzed statistically.

Apoptosis assay

This assay was performed to detect cell apoptosis using the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA). In brief, 1×10⁵ treated with baicalein cells were harvested following trypsinization and centrifugation. Cells were washed with PBS and resuspended in 500μl binding buffer. Then, 5μl Annexin V-FITC and 5μl PI (propidium iodide) were added, followed by 15-min incubation in the dark. The data was analyzed by WinMDI V2.9 software (The Scripps Research Institute, San Diego, CA, USA).

Western blotting

Cell lysates were prepared by using radioimmune precipitation assay (RIPA) lysis buffer in the presence of protease inhibitors. Total protein concentration was determined by BCA reagent following the manufacturer's instruction (Thermo Scientific, Rockford, IL). Equal amounts of proteins from each sample were separated by SDS-PAGE and transferred onto a PVDF membrane using a wet transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk for 2h at room temperature and incubated with the primary antibodies overnight at 4°C, followed by incubation with the horseradish peroxidase-labeled secondary antibodies for 1h at room temperature. Protein bands were detected by enhanced chemiluminescence detection system (Amersham, Pittsburg, PA). Protein levels were analyzed by ImageJ software.

Statistical analysis

All Data were presented as the mean ± SD of three independent experiments. The student two-tailed t-test or one-way analysis of variance was used to analyze the statistical differences between treatment and control values, as appropriate. Differences were considered statistical significant when p<0.05.

Baicalein inhibits cell proliferation of renal cancer cells

CCK-8 assay was used to detect the proliferation of 5 to 80 μM baicalein for 48 and 72h. Baicalein significantly inhibited the proliferation of A498 and 786-O cells in a time and dose dependent manner (*P<0.05, **P<0.01) (Fig. 1a and 1b). After treatment with baicalein (20μM) for 72h, the viability of A498 and 786-O cells decreased to 61.33% and 58.67%, respectively. The IC50 value of baicalein in A498 and 786-O cells ranged from 10 μmol/ L to 20μmol/ L, and was selected as the IC50 value in subsequent experiments.

Migration and invasion are inhibited by baicalein in renal cancer cells

The effect of baicalein on the migration of renal carcinoma was evaluated by scratch assay. Our results showed that baicalein inhibited the migration of A498 cells in a dose-dependent manner (Figs. 2a and 2b). Baicalein also had the same effect in 786-O cells (FIG. 2c and 2d). To further study the effects of baicalein on cell migration and invasion, A498 cells were used for transwell migration and Matrigel-underprinning transwell invasion assays. As shown in Fig. 3a and 3b, baicalein significantly inhibited the migration and invasion of renal carcinoma cells (*P<0.05, **P<0.01) in a dose-dependent manner. The similar effect of baicalein was also observed in 786-O cells (*P<0.05, **P<0.01)(Figure 3c and 3d).

Baicalein induces apoptosis of renal cancer cells

To investigate whether baicalein induced growth inhibition was related to apoptosis, apoptosis assay was used. Annexin V-conjugated Alexa Fluor 488 and propidium iodide staining were used to analyze the percentage of baicalein-induced apoptotic cells. The lower right quadrant (LR) and upper right quadrant (UR) of the histograms showed the percentages of early and late apoptotic cells, respectively. The percentage of total apoptosis (UR+LR) of A498 cells increased from 6.9% in non-baicalein treatment to 13.63% and 26.5% in baicalein treatment (10 μM and 20μM) for 48h (Fig. 4a and 4c) (*P<0.05, **P<0.01). A similar phenomenon was also found in 786-O cells, and the percentage of total apoptotic cells increased from 7.32% to 15.4% and 21.9% (*P<0.05 and **P<0.01), respectively (Fig. 4b and 4d). After treatment with baicalein 10 μM and 20μM for 48h, apoptotic cells of both cell lines increased in a dose-dependent manner. Baicalein can induce apoptosis of renal carcinoma cells, which indicates that baicalein has certain anticancer effect on renal carcinoma cells.

Baicalein induces the expression of apoptosis-related proteins in renal carcinoma cells

P53 is associated with cell cycle arrest, DNA repair or apoptosis [12]. Therefore, we evaluated p53 expression in two cell lines that responded to baicalein. As shown in Figure 5a, baicalein treatment resulted in a dose-dependent accumulation of P53. The pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 are members of the Bcl-2 family and are central regulators of apoptosis. The expression levels of Bcl-2, Bax and Survivin proteins were detected by western blotting. A498 and 786-O cells were treated with baicalein (10 and 20uM) for 48h. The expression of Bax increased gradually in a dose-dependent manner after baicalein treatment, while the expression of Bcl-2 decreased significantly (Figure. 5a). In addition, Survivin expression was slightly down-regulated. The ratio of Bax/Bcl-2 protein level is a decisive factor in apoptosis signal transduction. By comparing the intensities of their bands, we found a dose-dependent increase in the ratio of Bax/Bcl-2 (*P<0.05, **P<0.01) (Fia. 5b).

The ERK1/2 and p38 MAPK signaling pathway is involved in the anti-tumor mechanism of baicalein

Mitogen-activated protein kinases (MAPKs) regulate a variety of cellular programs [13], and some evidence shows that ERK1/2 and p38 kinase signaling pathway regulate metastasis and apoptosis of renal carcinoma cells [14-16]. To investigate whether baicalein induced apoptosis of A498 and 786-O cells was related to the regulation of MAPKs pathway, western blotting was used to detect the activation of ERK1/2 and p38. The results showed that baicalein significantly inhibited ERK1/2 phosphorylation in a dose-dependent manner (Fig. 5c and 5d). In addition, baicalein could inhibit p38 activation (Fig. 5e and 5f).

To further clarify the roles of erk1/2 and p38 signaling pathway in the anti-tumor activity of baicalein, A498 cells were cultured in the presence of baicalein (20uM) and/or PD98059 (50μM) or SB203580 (5μM) for 24h. As shown in Figure 6a, inhibition of ERK activity with PD98059 enhanced baicalein-induced up-regulation of Bax expression and down-regulation of Bcl-2 expression. Similar result was also observed in A498 cells treated with SB203580 (Fig. 6b). Next, we evaluated the growth inhibition and anti-metastasis activities of baicalein and/or PD98059 or SB203580 on A498 cells. The results showed that inhibition of ERK1/2 or p38 significantly inhibited the proliferation and invasion of RCC cells (Fig. 6c, 6d, 6e, 6f), suggesting that ERK1/2 and p38 MAPK pathway played important roles in the proliferation and invasion of RCC cells. In addition, baicalein also inhibited the proliferation and motility of ERK1/2 or p38 knockdown cells, suggesting that inhibition of ERK1/2 or p38 signaling pathway could enhance the anti-tumor effect of baicalein.

Baicalein, one of the main flavonoids in scutellaria baicalensis, can inhibit the proliferation, migration and induce apoptosis of cancer cells [17, 18]. Our previous studies have shown that baicalein could inhibit the metastasis and induce apoptosis of prostate cancer cells. However, the anti-metastasis effect of baicalein in renal carcinoma cells and its related mechanism remain unclear. In this study, we demonstrated for the first time that baicalein could effectively inhibit the proliferation and migration of renal carcinoma cells in vitro. Accordingly, we also observed the pro-apoptotic effect of baicalein. Induction of tumor cell apoptosis is a key strategy for anti-tumor therapy.

In addition, we also studied the effect of baicalein concentration on apoptosis markers of renal carcinoma cells. The results showed that baicalein up-regulated the expression of anti-apoptotic proteins Bcl-2 and p53, and down-regulated the expression of pro-apoptotic proteins Bax and Survivin. P53, as a transcription factor, played a role in regulating downstream genes in cell apoptosis and has been reported to induce p53-dependent cell apoptosis in renal carcinoma cells [12]. In this study, the level of p53 was upregulated after baicalein treatment of renal carcinoma cells, suggesting that p53 was involved in baicalein-induced apoptosis. The Bcl-2 family is closely related to cancer cell apoptosis. It has been reported that Bax expression was associated with increased apoptosis, while Bcl-2 expression was associated with inhibition of apoptosis of target cells [19, 20]. Baicalein could induce apoptosis of lung cancer cells [21] by activating Bax and inhibiting Bcl-2. We also found similar phenomena in this study, suggesting that baicalein-induced up-regulation of Bax and down-regulation of Bcl-2 could induce apoptosis of renal carcinoma cells.

ERK and p38 are often abnormally activated in cancer cells and are thought to play important roles in many signaling pathways [22, 23]. ERK pathway is involved in tumor initiation and progression by promoting cell proliferation and metastasis [24]. As for the effect of ERK on apoptosis, it has been reported that in many kinds of cancer cells, cell survival and apoptosis are regulated by the ERK/MAPK pathway [25]. Therefore, ERK was considered as a potential pharmacological target for tumor therapy [26]. Our study is consistent with these results. We found that ERK activity was significantly decreased in the presence of baicalein. In addition, inhibition of ERK activity by PD98059 significantly increased the sensitivity of renal carcinoma cells to baicalein-induced apoptosis and metastasis inhibition. P38 signaling pathway is widely expressed in various tissues, and its physiological functions are more extensive [27, 28]. It has been reported that the anti-tumor effect of baicalein was related to the phosphorylation of p38 MAPK [27, 29]. To further explore the possible mechanism of baicalein inhibiting renal cancer invasion, we detected the phosphorylation level of p38 in A498 cells. The results showed that baicalein could significantly inhibit the phosphorylation of p38 in renal carcinoma cells. SB203580 reduced baicalein-induced cell growth inhibition and apoptosis. Our data suggested that baicalein inhibited cell growth and promoted apoptosis through ERK and p38 MAPK pathways.

In conclusion, we demonstrated that baicalein can observably exhibit pro-apoptotic and metastasis inhibitory effect on renal cancer cells. Mechanically, we found ERK and p38 MAPK pathways account for the anti-tumor effects of baicalein. All these results provide evidence for the therapeutic potential of baicalein as renal cancer treatment.

In conclusion, baicalein can promote apoptosis and inhibit metastasis of renal carcinoma cells. Mechanistically, we found that ERK and p38 MAPK pathways were involved in the anti-tumor effect of baicalein. These results suggest that baicalein has potential therapeutic effect on renal cell carcinoma.

Funding

This work was supported by Shandong Provincial Key Research and Development Program (No.2019GSF108255).

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All data generated or analysed during this study are included in this published article.

Code availability

Not applicable

Authors' contributions

Z.G. and T.L. performed the experiments. R.L. and Y.Z. designed the study, analyzed and interpreted the results, wrote and edited the manuscript. L.M. and Z.Z. assisted with main experiments. L.Y. and Z.L. provided essential reagents and techniques for this study and reviewed the manuscript. All authors contributed to the article and approved the submitted version.

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Baicalein inhibits renal cancer cell growth and metastasis via ERK and p38 pathway

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