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Scutellarin inhibits human renal cancer cell proliferation and migration via upregulation of PTEN
Biomedicine & Pharmacotherapy 107 (2018) 1505–1513
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Scutellarin inhibits human renal cancer cell proliferation and migration via upregulation of PTEN
T
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Wenting Denga, , Wei Hanb, Tao Fana, Xiaoku Wanga, Zhao Chenga, Bo Wana, Jinlian Chena a b
College of Pharmacy, Xi’an Medical University, No. 1 Xinwang Road of Weiyang District, 710021, Xi’an, Shaanxi, China Department of Medical Equipment, Shaanxi Provincial People's Hospital, No. 256 Youyi West Road, 710068, Xi’an, Shaanxi, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Scutellarin Renal cancer PTEN P13K/AKT/mTOR Proliferation
Background: Scutellarin is a naturally flavone glycoside that has been shown to exhibit anti-proliferative and anti-apoptotic activities among various human malignancies. However, the anti-cancer effect of Scutellarin in Renal cell carcinoma (RCC) and the underlying mechanism remains unclear. Methods and materials: RCC cell lines ACHN and 786-O were treated with different concentrations (0–210 μM) of Scutellarin in vitro. Cell viability and proliferation were investigated by MTT and colony formation assays. Cell invasion and migration were detected by Transwell assays. Cell apoptosis and cell cycle distribution was measured by flow cytometry. Western blot was used to investigate the expression levels of crucial proteins. Xenograft tumor model was established to evaluate tumor growth in vivo. Results: Scutellarin significantly inhibited RCC cell proliferation in a dose- and time- dependent manner. Treatment of RCC cells with Scutellarin (30, 60, and 90 μM) markedly induced apoptosis and cell cycle arrested at G0/G1 phase in a concentration-dependent characteristic. Cell invasion and migration capacities of RCC cells were also dose-dependently suppressed by Scutellarin treatment. Western blot assays revealed that the crucial proteins including cyclin D1, CDK2, Bcl2, MMP-2, and MMP-9 were significantly reduced while Bax, cleaved caspase 3 and p21 were increased by Scutellarin in RCC cells. In vivo assay indicated that Scutellarin possessed anti-cancer effect on xenograft without triggering toxic effect. Mechanically, Scutellarin dramatically increased the protein level of phosphatase and tensin homologue (PTEN) and inhibited the activity of P13K/AKT/mTOR signaling. Ectopic expression of PTEN enhanced the inhibitory effect of Scutellarin on RCC proliferation while knockdown of PTEN abrogated it through regulating its downstream P13K/AKT/mTOR signaling pathway. Conclusion: Scutellarin inhibited RCC cell proliferation and invasion partially by enhancing the expression of PTEN through inhibition of P13K/AKT/mTOR pathway, suggesting that Scutellarin might serve as a potential therapeutic agent in RCC treatment.
1. Introduction Renal cell carcinoma (RCC) is the most common urological cancer worldwide, of which clear cell renal cell carcinoma (ccRCC) accounts for approximately 70% of RCC [1,2]. Though surgical resection is still considered as the major option for local RCC treatment, about 20%–50% of patients develop metastases and recurrence after radical nephrectomy [3]. The prognosis of patients with advanced stage remains unfavorable as the 5 year survival is fewer than 10% [4]. Until now, the current drug applications for aggressive RCC treatment are limited due to the acquirement of drug resistance [5]. Therefore, it is an urgent need to identify effective biomarkers and safe agents to improve the therapeutic outcome of RCC. Scutellarin (SC), 4,5,6-trihydroxylflavone-7-O-glucuronoside, is a ⁎
flavonoid monomer compound isolated from a variety of medicinal herbs Scutellaria barbata D. Don and Erigeron breviscapus (vant) Hand Mass [6,7]. Studies have suggested that Scutellarin as the primary active ingredients in these herbs displays diverse physiological actions of anti-inflammatory, antioxiadant scavenging of free radicals, and antiapoptotic [8,9]. For instance, Yuan et al. have demonstrated that Scutellarin regulates the migration and morphological transformation of activated microglia in cerebral ischemia rat model [10]. Long et al. have shown that Scutellarin significantly inhibits cell apoptosis and morphologic impairments on type II diabetes of rats [11]. Niu et al. have indicated that Scutellarin prevents diosbulbin B-induced liber injury by attenuating hepatic inflammation and liver oxidative stress [12]. Notably, Scutellarin has been reported to play crucial suppressive roles in the progression of tumoregenesis, such as colorectal cancer
Corresponding author. E-mail address: [email protected] (W. Deng).
https://doi.org/10.1016/j.biopha.2018.08.127 Received 8 May 2018; Received in revised form 18 August 2018; Accepted 24 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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2.2. Cell transfection
[13], breast cancer [14], hepatocellular carcinoma [15], and human tongue carcinoma [16]. However, the function of Scutellarin on RCC progression and the underlying regulatory mechanisms still remains unclear. Phosphatase and tensin homolog (PTEN) serves as a dual specificity protein phosphatase that is identified as a tumor suppressor in various human malignancies [17]. The lipid phosphatase activity of PTEN contributes to the dephosphorylation of the second messenger lipid phosphatidylinositol 3, 4, 5-trisphosphate (PIP3), which in turn facilitates itself to counteract the activity of phosphatidylinositol-3-kinase (P13 K) and phosphorylation of AKT [18]. P13 K/AKT signaling plays crucial roles in diverse cellular processes during tumorigenesis including cell proliferation, cell survival, angiogenesis, and differentiation [19]. Since PTEN is the only known lipid phosphatase that antagonize P13 K/AKT signaling transduction, accordingly, it has been well documented that frequently deletions and mutations of PTEN result in a substantial influence on multiple tumor progressions [20]. Thus, we hypothesized that the PTEN/P13 K/AKT may be involved in the anti-tumor processes of Scutellarin in RCC. The aim of this study was to explore the effects of Scutellarin on the progression of RCC and elucidate the possible molecular signaling pathways involved. We found that Scutellarin exerted suppressive role on the proliferation, invasion, and tumorigenesis on RCC cells both in vitro and in vivo via upregulating PTEN through inactivation of P13 K/ AKT signaling pathway.
The siRNA targeting human PTEN mRNA (si- PTEN) and the negative control (si-NC) were purchased from GenePharma Co., Ltd (Shanghai, China). To generate PTEN overexpressing plasmid, human cDNAs for PTEN was synthesized and cloned into pcDNA3.1 plasmid (pcDNA- PTEN, GenePharma). ACHN and 786-O cells were transfected with nucleotides using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction and incubated for 24 h before treatment with Scutellarin. The siRNA or overexpression efficiency was determined by western blot.
2.3. Cell proliferation assay The cellular viability of RCC cells was tested by Cell Counting kit-8 (CCK-8) assay. ACHN and 786-O cells were seeded into 96-well plates at the concentration of 5 × 103 cells per well and cultured to 70–80% confluence, cells were treated with different concentrations (0, 30, 60, 90, 120, 150, 180, and 210 μM) of Scutellarin dissolved in DMSO for 24, 48, 72 h at the indicated time point, 10 μl of CCK-8 solution (SigmaAldrich) was added into each well and incubated for 2 h. The absorbance was measured at a wavelength of 490 nm by a microplate reader (Thermo Fisher Scientific, MA, USA). The half-maximal inhibitory concentration (IC50) value was determined by nonlinear regression analysis using GraphPad Prim 6.0 (GraphPad Software, CA, USA). Moreover, ACHN and 786-O were transfected with pcDNA- PTEN or siPTEN for 24, 48 and 72 h and then treated with 60 μM Scutellarin for another 24 h, cell viability was measured as described above.
2. Material and methods 2.1. Chemical preparation and cell culture The chemical formula of Scutellarin is C21H18O12 (Fig. 1A). Scutellarin (purity ≥ 98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Human RCC cell lines (ACHN and 786-O) and the normal renal proximal tubule epithelial cell line HK-2 were obtained from the Cell bank of the Chinese Academy of Science (Shanghai, China) and cultured in RPMI-1640 medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% ampicillin/streptomycin at 37℃ with 5% CO2.
2.4. Colony formation assay After treating with Scutellarin (30, 60, and 90 μM) for 24 h, ACHN and 786-O cells were collected and seeded into 6-well plated at the density of 1000 cells per well. After incubation for two weeks, cells were washed and fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet solution. The number of colony was photographed and calculated under an optical microscope (Olympus, Tokyo, Japan).
Fig. 1. Scutellarin inhibits proliferation of RCC cells in vitro. (A) Chemical structure of Scutellarin. The chemical formula of Scutellarin is C21H18O12. (B) The effect of Scutellarin (30, 60, 90, 120, 150, 180, and 210 μM) on ACHN, 786-O, and HK-2 cell viability for 24, 48, and 72 h was determined by CCK-8 assay. (C) IC50 value of the effect of Scutellarin on ACHN and 786-O cells for 24, 48, and 72 h. (D) Clonogenic survival effects on ACHN and 786-O cells after treatment with Scutellarin (30, 60, and 90 μM) for 24 h were measured by colony formation assay. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. control group. 1506
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equivalent amount of DMSO in normal saline (Vehicle); Mice in lower and higher concentration groups were administered with Scutellarin intraperitoneally at the dose of 30 mg/kg and 60 mg/kg every day for 30 days, respectively. The tumor volume was calculated once a week using the formula V = (width2 × length)/2. After 6 weeks, the mice were sacrificed by cervical luxation, and the weight of isolated tumor was measured.
2.5. Cell cycle analysis ACHN and 786-O cells were treated with Scutellarin (30, 60, and 90 μM) for 24 h. For cell cycle analysis, cells were collected and fixed with ice-cold 70% ethanol overnight at 4℃. After rinsing, cells were incubated with 50 mg/ml propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/ml RNase A (Sigma-Aldrich) for 30 min at room temperature in the dark. Cell cycle distribution was measured by flow cytomety (FACSCalibur, BD Bioscience, CA, USA).
2.10. Plasma pharmacokinetic study In pharmacokinetic study, blood samples (500 μl) from caudal vein of tumor mice were collected immediately at 0.33, 0.67, 1, 1.5, 2, 2.5, 3, 5, 8, 12, 17 and 24 h after first (single) and last (daily) treatment of Scutellarin (at the dose of 30 mg/kg) using heparinized tubes. The plasma fraction was immediately separated by centrifugation and stored at -20℃ until analysis. Scutellarin concentration in plasma was determined by high-performance liquid chromatography (HPLC, Agilent, USA) method according to a previous study [21].Pharmacokinetic parameters of single and daily dose treatment including the maximum observed plasma concentration (Cmax) and time to maximum observed plasma concentration (Tmax) were obtained from experimental detection; area under plasma concentration curve from time 0 to 24 h (AUC0-24) was calculated by the linear trapezoidal method.
2.6. Apoptosis assay The cell apoptosis was performed by using the Annexin V-FITC/PI apoptosis detection kit (BD, USA) according to the manufacturer’s protocol. Cells were washed twice with ice cold PBS and then resuspended in 500 μl of 1 × binding buffer at a concentration of 1 × 105 cells. And then cells were stained with 5 μl of Annexin V-FITC and 5 μl of PI solution and incubated for 10 min in the dark at room temperature. Apoptosis rate was detected by FACSCalibur and analyzed by Flowjo software. 2.7. Western blot assay After treatment with Scutellarin (30, 60, and 90 μM) for 24 h, RCC cells were harvested and lysed with RIPA buffer (Beyotime). Protein concentrations were measured by BCA protein assay (Beyotime). Equal amounts of protein were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrotransferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After blocking in 5% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies cyclin D1, CDK2, p21, Bcl2, Bax, cleaved caspase 3, MMP-2, MMP-9, PTEN, phosphorylated (p)-AKT, total (t)-AKT, t-mTOR, p-mTOR, and βactin (Santa Cruz Biotechnology, TX, USA) at 4℃ overnight. Following by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at 37℃. Protein bands were visualized with an enhanced chemiluminescence (ECL) system (Invitrogen, USA) and analyzed by ImageJ software (NIH, MD, USA). βactin was used as internal control.
2.11. Immunohistochemistry Isolated tumor tissues were fixed in 4% paraformaldehyde, dehydrated with an ethanol gradient, embedded in paraffin, and cut into 4 μm thick sections. For immunohistochemistry (IHC), the sections were deparaffinized and subjected to a series of rehydration. After heating in sodium citrate buffer for antigen retrieval, the slides were incubated with anti- PTEN antibody (Cell Signaling Technology, USA) overnight at 4℃, followed by incubation with biotinylated -conjugated secondary antibody and streptavidin- conjugated HRP. The protein expressions were visualized with 3,3′-diaminobenzidine (DAB) tetrahydrochloride reagent. 2.12. Statistical analysis All data were expressed as the mean ± standard deviation (SD) and analyzed using GraphPad Prim 6.0 (GraphPad software, La Jolla, CA, USA). Two-tailed Student’s t-test was used to compare the difference between two groups and two-way ANOVA analysis was used for multiple comparisons. P < 0.05 was considered as statistically significant. Each experiment was performed independently at least in triplicate.
2.8. Invasion and migration assay Cell invasion and migration abilities were determined using Transwell assay (8 μm pore size, Corning Incorporated, NY, USA). After treatment with Scutellarin (30, 60, and 90 μM) for 24 h, ACHN and 786O cells were harvested and resuspended in serum free medium. A total of 1 × 105 cells were seeded in the upper chamber. For invasion assay, the upper chamber was pre-coated with Matrigel. 600 μl of RPMI-160 medium with 10% FBS was added to the lower chamber as a chemotactic gradient. After incubation at 37℃ for 24 h, cells remaining on the upper chamber were removed and the cells that invaded onto the lower surface were stained with 0.1% crystal violet. The number of invaded or migrated cells was counted in 10 random fields from per group.
3. Results 3.1. Scutellarin inhibits proliferation of RCC cells in vitro To identify the effect of Scutellarin on RCC cell growth, increasing concentrations of Scutellarin (0, 30, 60, 90, 120, 150, 180, and 210 μM) were used to treat the RCC cell lines (ACHN and 786-O cells) and the normal renal proximal tubule epithelial cell line HK-2 for 24, 48, and 72 h. CCK-8 assay revealed that cell viability of ACHN and 786-O cells were significantly reduced in a time- and dose- dependent manner, however, the Scutellarin treatment showed little viability inhibition on the normal human renal cell line HK-2 (Fig. 1B). The IC50 values of Scutellarin in ACHN and 786-O were 185.6 ± 7.8 μM and 183.6 ± 8.5 μM for 24 h, 149.3 ± 6.9 μM and 131.5 ± 8.9 μM for 48 h, 92.5 ± 5.7 μM and 106.7 ± 6.8 μM for 72 h (Fig. 1C), suggesting that ACHN and 786-O cells exhibited similar sensitivity to Scutellarin. Thus, according to the IC50 value of Scutellarin on ACHN and 786-O cells, cells treated with Scutellarin at the concentrations 30, 60, and 90 μM for 24 h was selected as the optimal cell treatment for further mechanistic investigation. Colony formation assay was employed to
2.9. In vivo tumorigenicity assay 5 weeks old male BALB/c nude mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University, Xi’an, China. All mice were maintained in a pathogen-free environment with water and food ad libitum. The animal experiment was approved by the Ethics Experimental Animal Care Committee of Xian Medical College, and in accordance with the Declaration of Helsinki. Xenograft tumor model was established as follows: 1 × 106 of ACHN cells in 200 μl of PBS was subcutaneously injected into right flank of each mouse. After tumors showed, nude mice were randomly divided into 3 groups (n = 5 in each group). Mice in vehicle group were received 1507
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Fig. 2. Scutellarin suppressed invasion and migration of RCC cells in vitro. Cell invasive (A) and migrated (B) capacities in ACHN and 786-O cells exposed to Scutellarin (30, 60, and 90 μM) for 24 h were determined by Transwell assays. (C) The expressions of MMP-2 and MMP-9 in ACHN and 786-O cells exposed to Scutellarin (30, 60, and 90 μM) for 24 h were calculated by western blot. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. control group.
measure the long-term effect of Scutellarin on RCC cell proliferative capacity. Compared to control group, the number of colonies was dramatically decreased in ACHN and 786-O cells with Scutellarin treatment in a dose-dependent manner (Fig. 1D). These data suggested that Scutellarin possessed significantly inhibitory effect on RCC cell proliferation.
migration of cancer cells were also investigated in this study. Western blot assay revealed that the expression levels of MMP-2 and MMP-9 were decreased in a dose dependent characteristic in Scutellarin treatment groups in comparison to control groups (Fig. 2C). These findings illustrated that Scutellarin could suppress the invasion and migration of RCC cells in vitro.
3.2. Scutellarin suppressed invasion and migration of RCC cells in vitro
3.3. Scutellarin induced apoptosis and cell cycle arrest in RCC cells
The functions of Scutellarin on the invasion and migration capacities of RCC cells were assessed by Transwell invasion/migration assay. As shown in Fig. 2A, the number of ACHN and 786-O cells that invaded through the filter into the bottom chamber was dose-dependent reduced in Scutellarin treatment groups when compared to control group (all P < 0.01). When treated with Scutellarin at the dose of 30 μM, the invasion capacities in ACHN and 786-O cells were reduced to 75.6% and 78.3%, respectively, while the invasion reduced to 23.5% and 27.7%, respectively, in the 90 μM Scutellarin group. Similarly, Scutellarin also inhibited the migrated capacity of ACHN and 786-O cells in a dose-dependent manner (Fig. 2B). In the 30 μM Scutellarin group, the migration capacities of ACHN and 786-O cells were decreased to 76.8% and 74.9%, respectively, while in the 90 μM Scutellarin group, the invasion capacities of ACHN and 786-O cells were decreased to 27.9% and 25.7%, respectively. Subsequently, MMPs (matrx metalloproteinases) that are involved in the invasion and
It has been widely demonstrated that the alternation of apoptosis and cell cycle are also correlated with cell proliferation during tumor progression. Next, flow cytometry was undertaken to determine whether Scutellarin treatment could impact the apoptosis and cell cycle distribution of RCC cells. Annexin V-FITC/PI double staining revealed that after treatment with Scutellarin (30, 60, and 90 μM) for 24 h, the apoptosis rates in ACHN and 786-O cells were remarkably enhanced dose-dependently when compared to the control groups (Fig. 3A). In addition, flow cytometry analysis also exhibited that the percentage of cells in G0/G1 phase was considerably increased after treatment with Scutellarin (30, 60, and 90 μM), and showed a concentration dependent manner (Fig. 3B). Whereas, the proportions of ACHN and 786-O cells in S phase were significantly reduced after exposure to Scutellarin. In order to explore the underlying molecular mechanism that Scutellarin induced apoptosis and G0/G1cell cycle arrest, the crucial protein expressions involved in apoptosis and cell cycle regulation including p21, 1508
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Fig. 3. Scutellarin induced apoptosis and cell cycle arrest in RCC cells. (A) Apoptosis rate of ACHN and 786-O cells exposed to Scutellarin (30, 60, and 90 μM) for 24 h was detected by AnnexinV-FITC/PI staining. (B) Cell cycle phase distribution of ACHN and 786-O cells treatment with Scutellarin (60, 90, and 120 μM) for 24 h was measured by flow cytometry. (C) The crucial protein levels involved in cell apoptosis and cell cycle were analyzed by western blot. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. control group.
constructed to up- or down- the expression of PTEN. Western blot assay demonstrated that the increased expression level of PTEN induced by Scutellarin treatment (60 μM) was further enhanced by PTEN overexpressing plasmid transfection, but abrogated by PTEN-siRNA transfection in both ACHN and 786-O cells (Fig. 4B). Moreover, Scutellarin decreased the levels of p-AKT and p-mTOR substantially in RCC cells, and co-treatment with PTEN inhibited the P13K/AKT/mTOR signaling (Fig. 4B). Conversely, knockdown of PTEN expression apparently recovered the suppressive effect of Scutellarin on P13K/AKT/mTOR signaling. CCK-8 assay showed that, when compared to NC group, ectopic expression of PTEN mimicked the inhibitory effect of Scutellarin on RCC cell proliferation and even increased the anti-proliferative effects of Scutellarin in RCC cells (Fig. 4C, P < 0.05), in contrast, knockdown of PTEN significantly promoted RCC cell proliferative rate and abrogated the inhibitory effect of Scutellarin on RCC cell proliferation (Fig. 4C, P < 0.05); In addition, flow cytometry assay revealed that exogenous expression of PTEN markedly enhanced the apoptotic effect induced by Scutellarin (Fig. 4D, P < 0.05); whereas, the apoptosis rate was obviously reversed with PTEN knockdown in Scutellarin treated RCC cells. Thus, these findings suggested that PTEN/P13K/AKT/mTOR signaling pathway might play a crucial role in Scutellarin mediated anti-tumor effect in RCC cells.
CDK2, cyclin D1, cleaved caspase 3 (c-casp3), Bax, and Bcl2 were determined by western blot. As shown in Fig. 2C, the expression levels of CDK2, cyclin D1, and Bcl2 were markedly reduced in Scutellarin treatment groups, which showed a dose dependent feature. Conversely, Scutellarin gradually concentration-dependently increased the expression levels p21, cleaved caspase 3, and Bax. Thus, these results demonstrated that Scutellarin inhibited RCC cell proliferation through inducing apoptosis and G0/G1 cell cycle arrest.
3.4. Anti-tumor effect of Scutellarin on PTEN-mediated P13K/AKT/mTOR pathway To elucidate the underlying mechanism of Scutellarin mediated RCC repression, PTEN/P13 K/AKT/mTOR signaling pathway involved in cancer progression was investigated in this study. Western blot revealed that the expression levels of PTEN increased dose-dependently after treatment with various concentrations of Scutellarin for 24 h (Fig. 4A). Furthermore, the crucial components of P13 K/AKT signaling such as phosphorylated (p)-AKT and p-mTOR were also dramatically reduced with Scutellarin treatment in a dose-dependent manner, though no obvious effect was found for total (t)-AKT and t-mTOR. To explore the role of PTEN in the Scutellarin mediated anti-tumor effect in RCC progression, PTEN overexpression plasmid and PTEN-siRNA was 1509
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Fig. 4. Anti-tumor effect of Scutellarin on PTEN-mediated P13 K/AKT/mTOR pathway in RCC cells. (A) The effect of Scutellarin (30, 60, and 90 μM) on the protein expressions of PTEN/ P13 K/AKT/mTOR pathway in ACHN and 786-O cells was measured by western blot. (B) The effect of Scutellarin on PTEN/P13 K/AKT/mTOR pathway in ACHN and 786-O cells transfected with PTEN overexpression plasmid or PTEN-siRNA was detected by western blot. The anti-tumor effect of Scutellarin affected by ectopic expression of PTEN or PTEN knockdown on cell viability (C) and apoptosis (D) in ACHN and 786-O cells were analyzed by CCK-8 and flow cyctometry, respectively. NC group: treatment with empty vector + siNC; Scutellarin group: treatment with Scutellarin (60 μM) + empty vector + si-NC; PTEN group: treatment with PTEN overexpressing plasmid + si-NC; Scutellarin + PTEN group: treatment with Scutellarin (60 μM) + PTEN overexpressing plasmid + siNC; siPTEN group: treatment with empty vector + siPTEN; Scutellarin + siPTEN group: Scutellarin (60 μM) + empty vector + siPTEN. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. NC group. #P < 0.05 vs. Scutellarin group.
determined after single and daily treatment of Scutellarin at the dose of 30 mg/kg, and we found that the plasma concentration curve of the daily dose was similar to the single dose of the Scutellarin (Fig. 5F). Scutellarin was rapidly absorbed by intraperitoneally injection and Cmax of Scutellarin at dose of 4.16 ± 0.55 μg/ml and 4.35 ± 0.72 μg/ ml were observed at Tmax of 0.82 ± 0.36 h and 0.75 ± 0.47 h for single and daily Scutellarein treatment, respectively, followed by a rapid initial descending phase and a slower terminal phase. After 24 h, the plasma concentrations of Scutellarein for single and daily dose treatment were reduced to 0.17 ± 0.16 μg/ml and 0.21 ± 0.17 μg/ml, respectively, indicating that no accumulation of Scutellarein in nude mice when given intraperitoneally every 24 h. Moreover, the AUC0-24 represented the bioavailability in the single dose group was 47.65 ± 8.77 μg*h/ml, which was comparable to the daily dose treatment (48.18 ± 11.32).
3.5. Scutellarin inhibits tumor growth in vivo Xenograft mouse model was established to further determine whether Scutellarin could suppress the growth of RCC in vivo. No mice died or significant difference of whole body was observed between untreated group and experimental groups (Vehicle, 30 mg/kg, 60 mg/kg group), indicating that Scutellarin administration caused non-toxic side effect (Fig. 5A). However, tumor volume and weight were remarkably reduced with Scutellarin treatment when compared to NC group (Fig. 5B and C). Moreover, the high dose of Scutellarin administration (60 mg/ kg) was more efficiently in suppressing tumor growth than the low dose treatment (30 mg/kg). Western blot assay further demonstrated that Scutellarin administration significantly enhanced the expression of PTEN but downregulated p-AKT and p-mTOR in dissected tumor tissues (Fig. 5E). Consistently, IHC staining showed that up-regulation of PTEN expression was found in xenograft tumor tissues after treatment with Scutellarin (Fig. 5D), suggesting that Scutellarin repressed RCC growth in vivo might be through regulating PTEN/P13 K/AKT/mTOR signaling pathway. Furthermore, Scutellarin plasma concentration was
4. Discussion RCC as a highly risk and mortality malignancy is particularly 1510
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Fig. 5. Scutellarin inhibits tumor growth in vivo. Whole body weight (A) and tumor volume (B) were monitored during the detectable period in nude mice. (C) Tumor weight was measured after xenograft was harvested. (D) IHC staining for PTEN of Scutellarin-treated RCC cell tumors (×200). Scale bar: 50 μm. (E) The expression levels of PTEN/P13 K/AKT/mTOR pathway in xenograft tumor tissues were quantified by western blot. (F) Plasma concentration-time curve of Scutellarin after intraperitoneally administration of single and daily dose of Scutellarin (dose at 30 mg/kg) in nude mice. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. vehicle group.
demonstrated that Scutellarin treatment destroyed the balance between Bcl-2 and Bax, and resulted in the caspase-3 mediated apoptosis in a concentration-dependent manner. The dysregulation transition from G0/G1 phase to S phase contributes to the progression of oncogenesis [31]. Amplification of cyclin D1 results in the activation of cyclin-dependent kinase (CDK2, CDK4, and CKD6) and suppression of cell cycle inhibitors (p21, p27, and p57) [32]. The present study revealed that the proportion of RCC cells delayed at G0/G1 phase was gradually increased when treatment with increased concentration of Scutellarin, displaying cell cycle arrest effect. Furthermore, the downregulation of CyclinD1 and CDK2, which could facilitate G1 to S transition, and upregulation of p21, which could result in G0/G1 phase arrest, were observed in RCC cells in a dosedependent characteristic. However, Lin et al. have illustrated that Scutellarin induced G2/M cell cycle arrest by suppressing the expression of CDK1 and cyclin B in Hela cells [33]. Gao et al. have reported that Scutellarin promoted cell cycle arrest at G2/M phase by downregulating CDK2 and cyclin B1 in prostate cancer cells [34]. Therefore, we supposed that among diverse cancer types, Scutellarin might trigger different molecular mechanisms to exert its anti-cancer effect. Metastatic dissemination that includes invasion and migration is a complex process and cause the majority of cancer related mortality in human malignancies [35]. Extracellular matrix metalloproteinases (MMPs) are family of zinc dependent endopeptydases that is associated with matrix remodeling and plays crucial roles in the tumor survival and progression [36]. Accumulating evidence has suggested that the increased expressions and activations of important members of the MMP family such as MMP-2 and MMP-9 affect various aspects of tumor development, including enhancement of cell proliferation, invasion, migration, angiogenesis, and tissue repair [37]. In the present study, Transwell assay demonstrated that Scutellarin has the ability to inhibit RCC invasion and migration in a dose-dependent manner, and this is the biological phenomenon associated with the inhibition of expressions of MMP-2 and MMP-9 in RCC cells. Similarly, previous studies have suggested that Scutellarin repressed invasion and migration of human hepatocellular carcinoma by down-regulating the STAT3/ Girdin/AKT signaling [38]. Furthermore, Li et al. have demonstrated that Scutellarin suppressed cell invasion and migration via decreasing MMP-2 and MMP-9 expression levels in human tongue carcinoma xenograft [16]. Collectively, these results demonstrated that blocking the capacity of cancer metastasis might be an effectively strategy for Scutellarin-mediated anti-tumor effect.
resistant to traditional radio- or chemo- therapeutic methods, thus, the better understanding of the molecular mechanism of RCC pathogenesis contributes to the improvement of novel target agents in clinical practice [22,23]. However, the outcome of patients with advanced RCC still remains limited. Various active ingredients extracted from traditional Chinese medicinal herbs have been widely used to prevent RCC progression. For instance, Silibinin could inhibit RCC growth through inducing caspase-dependent apoptosis [24]. Aeginetia indica Linn has been proved to possess suppressive effect on cancer cell-induced growth and metastasis in RCC cells [25]. Recently, accumulating evidence has suggested that Scutellarin as the principal active component of flavonoid possess the anti-tumor activity. Feng et al. have suggested that Scutellarin inhibits the growth of B-lymphoma Namalwa cells in vitro and suppressed lymphoma growth in Namalwa cell-xenotransplanted mice in vivo [26]. Nevertheless, the effect of Scutellarin on RCC progression has not yet elucidated. In the current study, we demonstrated that Scutellarin suppressed proliferation of RCC cells in a time- and concentration- dependent characteristic. Furthermore, our data also revealed that Scutellarin induced apoptosis and G1/G0 phase arrest, inhibited invasion and migration in RCC cells by modulating the proteins involved in survival and metastasis. Using xenograft as a model, the current study furthermore revealed that Scutellarin effectively inhibited the capacity of tumor growth in vivo without enhancing the toxicity. Apoptosis is a critical biochemical process that maintains the homeostasis in multicellular animals through eliminating the potential harmful DNA-damaged cells under normal conditions [27]. Whereas, the impairment in the balance of anti- and pro- apoptotic proteins may result in the promotion of tumorigenesis and induction of therapeutic resistant to tumor treatment [28]. In a variety of human cancers, the elevated expression of anti-apoptotic Bcl-2 protein and the downregulated pro-apoptotic proteins such as Bax were closely associated with the susceptibility of cell apoptosis [29]. Once cell death program is activated, apoptotic factors are quickly released from mitochondrial membrane to subsequently initiate caspase cascade. [30] Scutellarin was confirmed to promote apoptosis in many cancer cells through mitochondrial signaling and caspase-dependent signaling pathway. Previous studies have found that in human colorectal cancer, Scutellarin could inhibit the growth and induce apoptosis of cancer cells by p53 pathway [13]. In HepG2 hepatocellular carcinoma cells, Scutellarin has been shown to promote apoptosis through downregulating STAT3 transcriptional targets Bcl-xl and Mcl-1 [15]. Consistently, our findings 1511
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Previous studies and our findings revealed that Scutellarin selectively inhibited cancer cell proliferation without causing significant toxicity to normal cells or tissues, indicating that Scutellarin might interact with some specific targets related to tumorigenesis [38,39]. PTEN plays pivotal roles in the mediation of cell growth, cell cycle transition, apoptosis, migration, and chemotherapeutic resistance in multiple tumor types, including RCC [40]. Thus, targeting PTEN is considered as a potential biomarker for tumor therapy. For example, Tian et al. have suggested that the inhibitory effect of tetrandrine on human osteosarcoma cell proliferation is mediated by the upregulation of PTEN signaling [41]. Wu et al. have reported that Oridonin functions as a promising anticancer drug against colon cancer cell growth through reducing the phosphorylation of PTEN [42]. Meng et al. have also reported that knockdown of PTEN reverses the inhibitory effect of evodiamine on osteosarcoma cell proliferation [43]. In this study, the possible mechanism by which Scutellarin exerted anti-cancer effects in RCC was investigated. Consistent with previous studies, we demonstrated that Scutellarin strengthened the expression of PTEN concentration-dependently both in RCC cells and in xenograft tumors. Ectopic expression of PTEN enhanced the inhibitory effect of Scutellarin on RCC proliferation, whereas, which was apparently abrogated with PTEN knockdown. PTEN is the key antagonist of the P13 K/AKT cell-survival signaling pathway, overacitvation of P13 K/AKT induced by the functional loss of PTEN results in the proliferation and resistance to apoptosis in tumor cells through multiple signaling mechanisms, such as phosphorylating its downstream substrate mTOR kinases [44]. Phosphatidylinositol 3kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is the most frequently mutate pathway that is responsible for the carcinogenesis, progression, and metastasis [45]. Thus, accumulating evidence have elucidated that the activation of P13 K/AKT/mTOR signaling provides various potential targets for tumor therapy [46]. The approved targeted drugs for metastatic RCC including pazopanib, brivanib, temsirolimus, and ramucirumab that target VEGF or P13 K/AKT/ mTOR have been widely shown some degree of efficacy. Furthermore, other novel therapeutic approaches such as monoclonal antibodies and peptide vaccines have also been explored and developed for different types of RCC treatment [47]. Our data revealed that upregulation of PTEN expression was observed in Scutellarin-induced RCC cells through invactivation of P13K/AKT/mTOR signaling transduction. Exogenous PTEN expression even strengthened the inhibition effect of Scutellarin on P13 K/AKT/mTOR signaling, whereas, this inhibitory effect was obviously abrogated with PTEN knockdown. Thus, our findings implied that PTEN might be a potent target for Scutellarin to deactivate the P13 K/AKT/mTOR signaling in RCC progression. The pharmacokinetic study conducted after single and daily treatments also supported the antitumor effect of Scutellarin as evidenced in nude mice after intraperitoneal injection. The plasma concentration-time curves exhibited similar pattern between the single dose and daily dose of Scutellarin, suggesting that there is no accumulation of Scutellarin in mice when given intraperitoneally every 24 h. Furthermore, a previous study reported by Huang et al. has emphasized that rapid and efficient bioavaiability of intraperitoneal injection of scutellarin than oral administration is observed in male SD rats [21]. Thus, the current results indicated that intraperitoneal injection of Scutellarin at dose of 30 mg/ kg was safe for successive administration. In conclusion, we demonstrated that Scutellarin inhibited cell proliferation, induced apoptosis and cell cycle arrest, suppressed invasion and migration might through PTEN-mediated repression of P13K/AKT/ mTOR pathway in RCC cells, indicating that Scutellarin could be considered as a promising anti-tumor agent against RCC progression. Further investigations are still needed to elucidate the potential targets of Scutellarin and uncover the regulatory mechanisms of how Scutellarin suppresses the RCC development.
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Scutellarin inhibits human renal cancer cell proliferation and migration via upregulation of PTEN
T
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Wenting Denga, , Wei Hanb, Tao Fana, Xiaoku Wanga, Zhao Chenga, Bo Wana, Jinlian Chena a b
College of Pharmacy, Xi’an Medical University, No. 1 Xinwang Road of Weiyang District, 710021, Xi’an, Shaanxi, China Department of Medical Equipment, Shaanxi Provincial People's Hospital, No. 256 Youyi West Road, 710068, Xi’an, Shaanxi, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Scutellarin Renal cancer PTEN P13K/AKT/mTOR Proliferation
Background: Scutellarin is a naturally flavone glycoside that has been shown to exhibit anti-proliferative and anti-apoptotic activities among various human malignancies. However, the anti-cancer effect of Scutellarin in Renal cell carcinoma (RCC) and the underlying mechanism remains unclear. Methods and materials: RCC cell lines ACHN and 786-O were treated with different concentrations (0–210 μM) of Scutellarin in vitro. Cell viability and proliferation were investigated by MTT and colony formation assays. Cell invasion and migration were detected by Transwell assays. Cell apoptosis and cell cycle distribution was measured by flow cytometry. Western blot was used to investigate the expression levels of crucial proteins. Xenograft tumor model was established to evaluate tumor growth in vivo. Results: Scutellarin significantly inhibited RCC cell proliferation in a dose- and time- dependent manner. Treatment of RCC cells with Scutellarin (30, 60, and 90 μM) markedly induced apoptosis and cell cycle arrested at G0/G1 phase in a concentration-dependent characteristic. Cell invasion and migration capacities of RCC cells were also dose-dependently suppressed by Scutellarin treatment. Western blot assays revealed that the crucial proteins including cyclin D1, CDK2, Bcl2, MMP-2, and MMP-9 were significantly reduced while Bax, cleaved caspase 3 and p21 were increased by Scutellarin in RCC cells. In vivo assay indicated that Scutellarin possessed anti-cancer effect on xenograft without triggering toxic effect. Mechanically, Scutellarin dramatically increased the protein level of phosphatase and tensin homologue (PTEN) and inhibited the activity of P13K/AKT/mTOR signaling. Ectopic expression of PTEN enhanced the inhibitory effect of Scutellarin on RCC proliferation while knockdown of PTEN abrogated it through regulating its downstream P13K/AKT/mTOR signaling pathway. Conclusion: Scutellarin inhibited RCC cell proliferation and invasion partially by enhancing the expression of PTEN through inhibition of P13K/AKT/mTOR pathway, suggesting that Scutellarin might serve as a potential therapeutic agent in RCC treatment.
1. Introduction Renal cell carcinoma (RCC) is the most common urological cancer worldwide, of which clear cell renal cell carcinoma (ccRCC) accounts for approximately 70% of RCC [1,2]. Though surgical resection is still considered as the major option for local RCC treatment, about 20%–50% of patients develop metastases and recurrence after radical nephrectomy [3]. The prognosis of patients with advanced stage remains unfavorable as the 5 year survival is fewer than 10% [4]. Until now, the current drug applications for aggressive RCC treatment are limited due to the acquirement of drug resistance [5]. Therefore, it is an urgent need to identify effective biomarkers and safe agents to improve the therapeutic outcome of RCC. Scutellarin (SC), 4,5,6-trihydroxylflavone-7-O-glucuronoside, is a ⁎
flavonoid monomer compound isolated from a variety of medicinal herbs Scutellaria barbata D. Don and Erigeron breviscapus (vant) Hand Mass [6,7]. Studies have suggested that Scutellarin as the primary active ingredients in these herbs displays diverse physiological actions of anti-inflammatory, antioxiadant scavenging of free radicals, and antiapoptotic [8,9]. For instance, Yuan et al. have demonstrated that Scutellarin regulates the migration and morphological transformation of activated microglia in cerebral ischemia rat model [10]. Long et al. have shown that Scutellarin significantly inhibits cell apoptosis and morphologic impairments on type II diabetes of rats [11]. Niu et al. have indicated that Scutellarin prevents diosbulbin B-induced liber injury by attenuating hepatic inflammation and liver oxidative stress [12]. Notably, Scutellarin has been reported to play crucial suppressive roles in the progression of tumoregenesis, such as colorectal cancer
Corresponding author. E-mail address: [email protected] (W. Deng).
https://doi.org/10.1016/j.biopha.2018.08.127 Received 8 May 2018; Received in revised form 18 August 2018; Accepted 24 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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2.2. Cell transfection
[13], breast cancer [14], hepatocellular carcinoma [15], and human tongue carcinoma [16]. However, the function of Scutellarin on RCC progression and the underlying regulatory mechanisms still remains unclear. Phosphatase and tensin homolog (PTEN) serves as a dual specificity protein phosphatase that is identified as a tumor suppressor in various human malignancies [17]. The lipid phosphatase activity of PTEN contributes to the dephosphorylation of the second messenger lipid phosphatidylinositol 3, 4, 5-trisphosphate (PIP3), which in turn facilitates itself to counteract the activity of phosphatidylinositol-3-kinase (P13 K) and phosphorylation of AKT [18]. P13 K/AKT signaling plays crucial roles in diverse cellular processes during tumorigenesis including cell proliferation, cell survival, angiogenesis, and differentiation [19]. Since PTEN is the only known lipid phosphatase that antagonize P13 K/AKT signaling transduction, accordingly, it has been well documented that frequently deletions and mutations of PTEN result in a substantial influence on multiple tumor progressions [20]. Thus, we hypothesized that the PTEN/P13 K/AKT may be involved in the anti-tumor processes of Scutellarin in RCC. The aim of this study was to explore the effects of Scutellarin on the progression of RCC and elucidate the possible molecular signaling pathways involved. We found that Scutellarin exerted suppressive role on the proliferation, invasion, and tumorigenesis on RCC cells both in vitro and in vivo via upregulating PTEN through inactivation of P13 K/ AKT signaling pathway.
The siRNA targeting human PTEN mRNA (si- PTEN) and the negative control (si-NC) were purchased from GenePharma Co., Ltd (Shanghai, China). To generate PTEN overexpressing plasmid, human cDNAs for PTEN was synthesized and cloned into pcDNA3.1 plasmid (pcDNA- PTEN, GenePharma). ACHN and 786-O cells were transfected with nucleotides using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction and incubated for 24 h before treatment with Scutellarin. The siRNA or overexpression efficiency was determined by western blot.
2.3. Cell proliferation assay The cellular viability of RCC cells was tested by Cell Counting kit-8 (CCK-8) assay. ACHN and 786-O cells were seeded into 96-well plates at the concentration of 5 × 103 cells per well and cultured to 70–80% confluence, cells were treated with different concentrations (0, 30, 60, 90, 120, 150, 180, and 210 μM) of Scutellarin dissolved in DMSO for 24, 48, 72 h at the indicated time point, 10 μl of CCK-8 solution (SigmaAldrich) was added into each well and incubated for 2 h. The absorbance was measured at a wavelength of 490 nm by a microplate reader (Thermo Fisher Scientific, MA, USA). The half-maximal inhibitory concentration (IC50) value was determined by nonlinear regression analysis using GraphPad Prim 6.0 (GraphPad Software, CA, USA). Moreover, ACHN and 786-O were transfected with pcDNA- PTEN or siPTEN for 24, 48 and 72 h and then treated with 60 μM Scutellarin for another 24 h, cell viability was measured as described above.
2. Material and methods 2.1. Chemical preparation and cell culture The chemical formula of Scutellarin is C21H18O12 (Fig. 1A). Scutellarin (purity ≥ 98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Human RCC cell lines (ACHN and 786-O) and the normal renal proximal tubule epithelial cell line HK-2 were obtained from the Cell bank of the Chinese Academy of Science (Shanghai, China) and cultured in RPMI-1640 medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% ampicillin/streptomycin at 37℃ with 5% CO2.
2.4. Colony formation assay After treating with Scutellarin (30, 60, and 90 μM) for 24 h, ACHN and 786-O cells were collected and seeded into 6-well plated at the density of 1000 cells per well. After incubation for two weeks, cells were washed and fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet solution. The number of colony was photographed and calculated under an optical microscope (Olympus, Tokyo, Japan).
Fig. 1. Scutellarin inhibits proliferation of RCC cells in vitro. (A) Chemical structure of Scutellarin. The chemical formula of Scutellarin is C21H18O12. (B) The effect of Scutellarin (30, 60, 90, 120, 150, 180, and 210 μM) on ACHN, 786-O, and HK-2 cell viability for 24, 48, and 72 h was determined by CCK-8 assay. (C) IC50 value of the effect of Scutellarin on ACHN and 786-O cells for 24, 48, and 72 h. (D) Clonogenic survival effects on ACHN and 786-O cells after treatment with Scutellarin (30, 60, and 90 μM) for 24 h were measured by colony formation assay. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. control group. 1506
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equivalent amount of DMSO in normal saline (Vehicle); Mice in lower and higher concentration groups were administered with Scutellarin intraperitoneally at the dose of 30 mg/kg and 60 mg/kg every day for 30 days, respectively. The tumor volume was calculated once a week using the formula V = (width2 × length)/2. After 6 weeks, the mice were sacrificed by cervical luxation, and the weight of isolated tumor was measured.
2.5. Cell cycle analysis ACHN and 786-O cells were treated with Scutellarin (30, 60, and 90 μM) for 24 h. For cell cycle analysis, cells were collected and fixed with ice-cold 70% ethanol overnight at 4℃. After rinsing, cells were incubated with 50 mg/ml propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/ml RNase A (Sigma-Aldrich) for 30 min at room temperature in the dark. Cell cycle distribution was measured by flow cytomety (FACSCalibur, BD Bioscience, CA, USA).
2.10. Plasma pharmacokinetic study In pharmacokinetic study, blood samples (500 μl) from caudal vein of tumor mice were collected immediately at 0.33, 0.67, 1, 1.5, 2, 2.5, 3, 5, 8, 12, 17 and 24 h after first (single) and last (daily) treatment of Scutellarin (at the dose of 30 mg/kg) using heparinized tubes. The plasma fraction was immediately separated by centrifugation and stored at -20℃ until analysis. Scutellarin concentration in plasma was determined by high-performance liquid chromatography (HPLC, Agilent, USA) method according to a previous study [21].Pharmacokinetic parameters of single and daily dose treatment including the maximum observed plasma concentration (Cmax) and time to maximum observed plasma concentration (Tmax) were obtained from experimental detection; area under plasma concentration curve from time 0 to 24 h (AUC0-24) was calculated by the linear trapezoidal method.
2.6. Apoptosis assay The cell apoptosis was performed by using the Annexin V-FITC/PI apoptosis detection kit (BD, USA) according to the manufacturer’s protocol. Cells were washed twice with ice cold PBS and then resuspended in 500 μl of 1 × binding buffer at a concentration of 1 × 105 cells. And then cells were stained with 5 μl of Annexin V-FITC and 5 μl of PI solution and incubated for 10 min in the dark at room temperature. Apoptosis rate was detected by FACSCalibur and analyzed by Flowjo software. 2.7. Western blot assay After treatment with Scutellarin (30, 60, and 90 μM) for 24 h, RCC cells were harvested and lysed with RIPA buffer (Beyotime). Protein concentrations were measured by BCA protein assay (Beyotime). Equal amounts of protein were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrotransferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After blocking in 5% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies cyclin D1, CDK2, p21, Bcl2, Bax, cleaved caspase 3, MMP-2, MMP-9, PTEN, phosphorylated (p)-AKT, total (t)-AKT, t-mTOR, p-mTOR, and βactin (Santa Cruz Biotechnology, TX, USA) at 4℃ overnight. Following by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at 37℃. Protein bands were visualized with an enhanced chemiluminescence (ECL) system (Invitrogen, USA) and analyzed by ImageJ software (NIH, MD, USA). βactin was used as internal control.
2.11. Immunohistochemistry Isolated tumor tissues were fixed in 4% paraformaldehyde, dehydrated with an ethanol gradient, embedded in paraffin, and cut into 4 μm thick sections. For immunohistochemistry (IHC), the sections were deparaffinized and subjected to a series of rehydration. After heating in sodium citrate buffer for antigen retrieval, the slides were incubated with anti- PTEN antibody (Cell Signaling Technology, USA) overnight at 4℃, followed by incubation with biotinylated -conjugated secondary antibody and streptavidin- conjugated HRP. The protein expressions were visualized with 3,3′-diaminobenzidine (DAB) tetrahydrochloride reagent. 2.12. Statistical analysis All data were expressed as the mean ± standard deviation (SD) and analyzed using GraphPad Prim 6.0 (GraphPad software, La Jolla, CA, USA). Two-tailed Student’s t-test was used to compare the difference between two groups and two-way ANOVA analysis was used for multiple comparisons. P < 0.05 was considered as statistically significant. Each experiment was performed independently at least in triplicate.
2.8. Invasion and migration assay Cell invasion and migration abilities were determined using Transwell assay (8 μm pore size, Corning Incorporated, NY, USA). After treatment with Scutellarin (30, 60, and 90 μM) for 24 h, ACHN and 786O cells were harvested and resuspended in serum free medium. A total of 1 × 105 cells were seeded in the upper chamber. For invasion assay, the upper chamber was pre-coated with Matrigel. 600 μl of RPMI-160 medium with 10% FBS was added to the lower chamber as a chemotactic gradient. After incubation at 37℃ for 24 h, cells remaining on the upper chamber were removed and the cells that invaded onto the lower surface were stained with 0.1% crystal violet. The number of invaded or migrated cells was counted in 10 random fields from per group.
3. Results 3.1. Scutellarin inhibits proliferation of RCC cells in vitro To identify the effect of Scutellarin on RCC cell growth, increasing concentrations of Scutellarin (0, 30, 60, 90, 120, 150, 180, and 210 μM) were used to treat the RCC cell lines (ACHN and 786-O cells) and the normal renal proximal tubule epithelial cell line HK-2 for 24, 48, and 72 h. CCK-8 assay revealed that cell viability of ACHN and 786-O cells were significantly reduced in a time- and dose- dependent manner, however, the Scutellarin treatment showed little viability inhibition on the normal human renal cell line HK-2 (Fig. 1B). The IC50 values of Scutellarin in ACHN and 786-O were 185.6 ± 7.8 μM and 183.6 ± 8.5 μM for 24 h, 149.3 ± 6.9 μM and 131.5 ± 8.9 μM for 48 h, 92.5 ± 5.7 μM and 106.7 ± 6.8 μM for 72 h (Fig. 1C), suggesting that ACHN and 786-O cells exhibited similar sensitivity to Scutellarin. Thus, according to the IC50 value of Scutellarin on ACHN and 786-O cells, cells treated with Scutellarin at the concentrations 30, 60, and 90 μM for 24 h was selected as the optimal cell treatment for further mechanistic investigation. Colony formation assay was employed to
2.9. In vivo tumorigenicity assay 5 weeks old male BALB/c nude mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University, Xi’an, China. All mice were maintained in a pathogen-free environment with water and food ad libitum. The animal experiment was approved by the Ethics Experimental Animal Care Committee of Xian Medical College, and in accordance with the Declaration of Helsinki. Xenograft tumor model was established as follows: 1 × 106 of ACHN cells in 200 μl of PBS was subcutaneously injected into right flank of each mouse. After tumors showed, nude mice were randomly divided into 3 groups (n = 5 in each group). Mice in vehicle group were received 1507
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Fig. 2. Scutellarin suppressed invasion and migration of RCC cells in vitro. Cell invasive (A) and migrated (B) capacities in ACHN and 786-O cells exposed to Scutellarin (30, 60, and 90 μM) for 24 h were determined by Transwell assays. (C) The expressions of MMP-2 and MMP-9 in ACHN and 786-O cells exposed to Scutellarin (30, 60, and 90 μM) for 24 h were calculated by western blot. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. control group.
measure the long-term effect of Scutellarin on RCC cell proliferative capacity. Compared to control group, the number of colonies was dramatically decreased in ACHN and 786-O cells with Scutellarin treatment in a dose-dependent manner (Fig. 1D). These data suggested that Scutellarin possessed significantly inhibitory effect on RCC cell proliferation.
migration of cancer cells were also investigated in this study. Western blot assay revealed that the expression levels of MMP-2 and MMP-9 were decreased in a dose dependent characteristic in Scutellarin treatment groups in comparison to control groups (Fig. 2C). These findings illustrated that Scutellarin could suppress the invasion and migration of RCC cells in vitro.
3.2. Scutellarin suppressed invasion and migration of RCC cells in vitro
3.3. Scutellarin induced apoptosis and cell cycle arrest in RCC cells
The functions of Scutellarin on the invasion and migration capacities of RCC cells were assessed by Transwell invasion/migration assay. As shown in Fig. 2A, the number of ACHN and 786-O cells that invaded through the filter into the bottom chamber was dose-dependent reduced in Scutellarin treatment groups when compared to control group (all P < 0.01). When treated with Scutellarin at the dose of 30 μM, the invasion capacities in ACHN and 786-O cells were reduced to 75.6% and 78.3%, respectively, while the invasion reduced to 23.5% and 27.7%, respectively, in the 90 μM Scutellarin group. Similarly, Scutellarin also inhibited the migrated capacity of ACHN and 786-O cells in a dose-dependent manner (Fig. 2B). In the 30 μM Scutellarin group, the migration capacities of ACHN and 786-O cells were decreased to 76.8% and 74.9%, respectively, while in the 90 μM Scutellarin group, the invasion capacities of ACHN and 786-O cells were decreased to 27.9% and 25.7%, respectively. Subsequently, MMPs (matrx metalloproteinases) that are involved in the invasion and
It has been widely demonstrated that the alternation of apoptosis and cell cycle are also correlated with cell proliferation during tumor progression. Next, flow cytometry was undertaken to determine whether Scutellarin treatment could impact the apoptosis and cell cycle distribution of RCC cells. Annexin V-FITC/PI double staining revealed that after treatment with Scutellarin (30, 60, and 90 μM) for 24 h, the apoptosis rates in ACHN and 786-O cells were remarkably enhanced dose-dependently when compared to the control groups (Fig. 3A). In addition, flow cytometry analysis also exhibited that the percentage of cells in G0/G1 phase was considerably increased after treatment with Scutellarin (30, 60, and 90 μM), and showed a concentration dependent manner (Fig. 3B). Whereas, the proportions of ACHN and 786-O cells in S phase were significantly reduced after exposure to Scutellarin. In order to explore the underlying molecular mechanism that Scutellarin induced apoptosis and G0/G1cell cycle arrest, the crucial protein expressions involved in apoptosis and cell cycle regulation including p21, 1508
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Fig. 3. Scutellarin induced apoptosis and cell cycle arrest in RCC cells. (A) Apoptosis rate of ACHN and 786-O cells exposed to Scutellarin (30, 60, and 90 μM) for 24 h was detected by AnnexinV-FITC/PI staining. (B) Cell cycle phase distribution of ACHN and 786-O cells treatment with Scutellarin (60, 90, and 120 μM) for 24 h was measured by flow cytometry. (C) The crucial protein levels involved in cell apoptosis and cell cycle were analyzed by western blot. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. control group.
constructed to up- or down- the expression of PTEN. Western blot assay demonstrated that the increased expression level of PTEN induced by Scutellarin treatment (60 μM) was further enhanced by PTEN overexpressing plasmid transfection, but abrogated by PTEN-siRNA transfection in both ACHN and 786-O cells (Fig. 4B). Moreover, Scutellarin decreased the levels of p-AKT and p-mTOR substantially in RCC cells, and co-treatment with PTEN inhibited the P13K/AKT/mTOR signaling (Fig. 4B). Conversely, knockdown of PTEN expression apparently recovered the suppressive effect of Scutellarin on P13K/AKT/mTOR signaling. CCK-8 assay showed that, when compared to NC group, ectopic expression of PTEN mimicked the inhibitory effect of Scutellarin on RCC cell proliferation and even increased the anti-proliferative effects of Scutellarin in RCC cells (Fig. 4C, P < 0.05), in contrast, knockdown of PTEN significantly promoted RCC cell proliferative rate and abrogated the inhibitory effect of Scutellarin on RCC cell proliferation (Fig. 4C, P < 0.05); In addition, flow cytometry assay revealed that exogenous expression of PTEN markedly enhanced the apoptotic effect induced by Scutellarin (Fig. 4D, P < 0.05); whereas, the apoptosis rate was obviously reversed with PTEN knockdown in Scutellarin treated RCC cells. Thus, these findings suggested that PTEN/P13K/AKT/mTOR signaling pathway might play a crucial role in Scutellarin mediated anti-tumor effect in RCC cells.
CDK2, cyclin D1, cleaved caspase 3 (c-casp3), Bax, and Bcl2 were determined by western blot. As shown in Fig. 2C, the expression levels of CDK2, cyclin D1, and Bcl2 were markedly reduced in Scutellarin treatment groups, which showed a dose dependent feature. Conversely, Scutellarin gradually concentration-dependently increased the expression levels p21, cleaved caspase 3, and Bax. Thus, these results demonstrated that Scutellarin inhibited RCC cell proliferation through inducing apoptosis and G0/G1 cell cycle arrest.
3.4. Anti-tumor effect of Scutellarin on PTEN-mediated P13K/AKT/mTOR pathway To elucidate the underlying mechanism of Scutellarin mediated RCC repression, PTEN/P13 K/AKT/mTOR signaling pathway involved in cancer progression was investigated in this study. Western blot revealed that the expression levels of PTEN increased dose-dependently after treatment with various concentrations of Scutellarin for 24 h (Fig. 4A). Furthermore, the crucial components of P13 K/AKT signaling such as phosphorylated (p)-AKT and p-mTOR were also dramatically reduced with Scutellarin treatment in a dose-dependent manner, though no obvious effect was found for total (t)-AKT and t-mTOR. To explore the role of PTEN in the Scutellarin mediated anti-tumor effect in RCC progression, PTEN overexpression plasmid and PTEN-siRNA was 1509
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Fig. 4. Anti-tumor effect of Scutellarin on PTEN-mediated P13 K/AKT/mTOR pathway in RCC cells. (A) The effect of Scutellarin (30, 60, and 90 μM) on the protein expressions of PTEN/ P13 K/AKT/mTOR pathway in ACHN and 786-O cells was measured by western blot. (B) The effect of Scutellarin on PTEN/P13 K/AKT/mTOR pathway in ACHN and 786-O cells transfected with PTEN overexpression plasmid or PTEN-siRNA was detected by western blot. The anti-tumor effect of Scutellarin affected by ectopic expression of PTEN or PTEN knockdown on cell viability (C) and apoptosis (D) in ACHN and 786-O cells were analyzed by CCK-8 and flow cyctometry, respectively. NC group: treatment with empty vector + siNC; Scutellarin group: treatment with Scutellarin (60 μM) + empty vector + si-NC; PTEN group: treatment with PTEN overexpressing plasmid + si-NC; Scutellarin + PTEN group: treatment with Scutellarin (60 μM) + PTEN overexpressing plasmid + siNC; siPTEN group: treatment with empty vector + siPTEN; Scutellarin + siPTEN group: Scutellarin (60 μM) + empty vector + siPTEN. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. NC group. #P < 0.05 vs. Scutellarin group.
determined after single and daily treatment of Scutellarin at the dose of 30 mg/kg, and we found that the plasma concentration curve of the daily dose was similar to the single dose of the Scutellarin (Fig. 5F). Scutellarin was rapidly absorbed by intraperitoneally injection and Cmax of Scutellarin at dose of 4.16 ± 0.55 μg/ml and 4.35 ± 0.72 μg/ ml were observed at Tmax of 0.82 ± 0.36 h and 0.75 ± 0.47 h for single and daily Scutellarein treatment, respectively, followed by a rapid initial descending phase and a slower terminal phase. After 24 h, the plasma concentrations of Scutellarein for single and daily dose treatment were reduced to 0.17 ± 0.16 μg/ml and 0.21 ± 0.17 μg/ml, respectively, indicating that no accumulation of Scutellarein in nude mice when given intraperitoneally every 24 h. Moreover, the AUC0-24 represented the bioavailability in the single dose group was 47.65 ± 8.77 μg*h/ml, which was comparable to the daily dose treatment (48.18 ± 11.32).
3.5. Scutellarin inhibits tumor growth in vivo Xenograft mouse model was established to further determine whether Scutellarin could suppress the growth of RCC in vivo. No mice died or significant difference of whole body was observed between untreated group and experimental groups (Vehicle, 30 mg/kg, 60 mg/kg group), indicating that Scutellarin administration caused non-toxic side effect (Fig. 5A). However, tumor volume and weight were remarkably reduced with Scutellarin treatment when compared to NC group (Fig. 5B and C). Moreover, the high dose of Scutellarin administration (60 mg/ kg) was more efficiently in suppressing tumor growth than the low dose treatment (30 mg/kg). Western blot assay further demonstrated that Scutellarin administration significantly enhanced the expression of PTEN but downregulated p-AKT and p-mTOR in dissected tumor tissues (Fig. 5E). Consistently, IHC staining showed that up-regulation of PTEN expression was found in xenograft tumor tissues after treatment with Scutellarin (Fig. 5D), suggesting that Scutellarin repressed RCC growth in vivo might be through regulating PTEN/P13 K/AKT/mTOR signaling pathway. Furthermore, Scutellarin plasma concentration was
4. Discussion RCC as a highly risk and mortality malignancy is particularly 1510
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Fig. 5. Scutellarin inhibits tumor growth in vivo. Whole body weight (A) and tumor volume (B) were monitored during the detectable period in nude mice. (C) Tumor weight was measured after xenograft was harvested. (D) IHC staining for PTEN of Scutellarin-treated RCC cell tumors (×200). Scale bar: 50 μm. (E) The expression levels of PTEN/P13 K/AKT/mTOR pathway in xenograft tumor tissues were quantified by western blot. (F) Plasma concentration-time curve of Scutellarin after intraperitoneally administration of single and daily dose of Scutellarin (dose at 30 mg/kg) in nude mice. Data are expressed as the mean ± SD (n = 3 from independent experiments). *P < 0.05, **P < 0.01 vs. vehicle group.
demonstrated that Scutellarin treatment destroyed the balance between Bcl-2 and Bax, and resulted in the caspase-3 mediated apoptosis in a concentration-dependent manner. The dysregulation transition from G0/G1 phase to S phase contributes to the progression of oncogenesis [31]. Amplification of cyclin D1 results in the activation of cyclin-dependent kinase (CDK2, CDK4, and CKD6) and suppression of cell cycle inhibitors (p21, p27, and p57) [32]. The present study revealed that the proportion of RCC cells delayed at G0/G1 phase was gradually increased when treatment with increased concentration of Scutellarin, displaying cell cycle arrest effect. Furthermore, the downregulation of CyclinD1 and CDK2, which could facilitate G1 to S transition, and upregulation of p21, which could result in G0/G1 phase arrest, were observed in RCC cells in a dosedependent characteristic. However, Lin et al. have illustrated that Scutellarin induced G2/M cell cycle arrest by suppressing the expression of CDK1 and cyclin B in Hela cells [33]. Gao et al. have reported that Scutellarin promoted cell cycle arrest at G2/M phase by downregulating CDK2 and cyclin B1 in prostate cancer cells [34]. Therefore, we supposed that among diverse cancer types, Scutellarin might trigger different molecular mechanisms to exert its anti-cancer effect. Metastatic dissemination that includes invasion and migration is a complex process and cause the majority of cancer related mortality in human malignancies [35]. Extracellular matrix metalloproteinases (MMPs) are family of zinc dependent endopeptydases that is associated with matrix remodeling and plays crucial roles in the tumor survival and progression [36]. Accumulating evidence has suggested that the increased expressions and activations of important members of the MMP family such as MMP-2 and MMP-9 affect various aspects of tumor development, including enhancement of cell proliferation, invasion, migration, angiogenesis, and tissue repair [37]. In the present study, Transwell assay demonstrated that Scutellarin has the ability to inhibit RCC invasion and migration in a dose-dependent manner, and this is the biological phenomenon associated with the inhibition of expressions of MMP-2 and MMP-9 in RCC cells. Similarly, previous studies have suggested that Scutellarin repressed invasion and migration of human hepatocellular carcinoma by down-regulating the STAT3/ Girdin/AKT signaling [38]. Furthermore, Li et al. have demonstrated that Scutellarin suppressed cell invasion and migration via decreasing MMP-2 and MMP-9 expression levels in human tongue carcinoma xenograft [16]. Collectively, these results demonstrated that blocking the capacity of cancer metastasis might be an effectively strategy for Scutellarin-mediated anti-tumor effect.
resistant to traditional radio- or chemo- therapeutic methods, thus, the better understanding of the molecular mechanism of RCC pathogenesis contributes to the improvement of novel target agents in clinical practice [22,23]. However, the outcome of patients with advanced RCC still remains limited. Various active ingredients extracted from traditional Chinese medicinal herbs have been widely used to prevent RCC progression. For instance, Silibinin could inhibit RCC growth through inducing caspase-dependent apoptosis [24]. Aeginetia indica Linn has been proved to possess suppressive effect on cancer cell-induced growth and metastasis in RCC cells [25]. Recently, accumulating evidence has suggested that Scutellarin as the principal active component of flavonoid possess the anti-tumor activity. Feng et al. have suggested that Scutellarin inhibits the growth of B-lymphoma Namalwa cells in vitro and suppressed lymphoma growth in Namalwa cell-xenotransplanted mice in vivo [26]. Nevertheless, the effect of Scutellarin on RCC progression has not yet elucidated. In the current study, we demonstrated that Scutellarin suppressed proliferation of RCC cells in a time- and concentration- dependent characteristic. Furthermore, our data also revealed that Scutellarin induced apoptosis and G1/G0 phase arrest, inhibited invasion and migration in RCC cells by modulating the proteins involved in survival and metastasis. Using xenograft as a model, the current study furthermore revealed that Scutellarin effectively inhibited the capacity of tumor growth in vivo without enhancing the toxicity. Apoptosis is a critical biochemical process that maintains the homeostasis in multicellular animals through eliminating the potential harmful DNA-damaged cells under normal conditions [27]. Whereas, the impairment in the balance of anti- and pro- apoptotic proteins may result in the promotion of tumorigenesis and induction of therapeutic resistant to tumor treatment [28]. In a variety of human cancers, the elevated expression of anti-apoptotic Bcl-2 protein and the downregulated pro-apoptotic proteins such as Bax were closely associated with the susceptibility of cell apoptosis [29]. Once cell death program is activated, apoptotic factors are quickly released from mitochondrial membrane to subsequently initiate caspase cascade. [30] Scutellarin was confirmed to promote apoptosis in many cancer cells through mitochondrial signaling and caspase-dependent signaling pathway. Previous studies have found that in human colorectal cancer, Scutellarin could inhibit the growth and induce apoptosis of cancer cells by p53 pathway [13]. In HepG2 hepatocellular carcinoma cells, Scutellarin has been shown to promote apoptosis through downregulating STAT3 transcriptional targets Bcl-xl and Mcl-1 [15]. Consistently, our findings 1511
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Previous studies and our findings revealed that Scutellarin selectively inhibited cancer cell proliferation without causing significant toxicity to normal cells or tissues, indicating that Scutellarin might interact with some specific targets related to tumorigenesis [38,39]. PTEN plays pivotal roles in the mediation of cell growth, cell cycle transition, apoptosis, migration, and chemotherapeutic resistance in multiple tumor types, including RCC [40]. Thus, targeting PTEN is considered as a potential biomarker for tumor therapy. For example, Tian et al. have suggested that the inhibitory effect of tetrandrine on human osteosarcoma cell proliferation is mediated by the upregulation of PTEN signaling [41]. Wu et al. have reported that Oridonin functions as a promising anticancer drug against colon cancer cell growth through reducing the phosphorylation of PTEN [42]. Meng et al. have also reported that knockdown of PTEN reverses the inhibitory effect of evodiamine on osteosarcoma cell proliferation [43]. In this study, the possible mechanism by which Scutellarin exerted anti-cancer effects in RCC was investigated. Consistent with previous studies, we demonstrated that Scutellarin strengthened the expression of PTEN concentration-dependently both in RCC cells and in xenograft tumors. Ectopic expression of PTEN enhanced the inhibitory effect of Scutellarin on RCC proliferation, whereas, which was apparently abrogated with PTEN knockdown. PTEN is the key antagonist of the P13 K/AKT cell-survival signaling pathway, overacitvation of P13 K/AKT induced by the functional loss of PTEN results in the proliferation and resistance to apoptosis in tumor cells through multiple signaling mechanisms, such as phosphorylating its downstream substrate mTOR kinases [44]. Phosphatidylinositol 3kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is the most frequently mutate pathway that is responsible for the carcinogenesis, progression, and metastasis [45]. Thus, accumulating evidence have elucidated that the activation of P13 K/AKT/mTOR signaling provides various potential targets for tumor therapy [46]. The approved targeted drugs for metastatic RCC including pazopanib, brivanib, temsirolimus, and ramucirumab that target VEGF or P13 K/AKT/ mTOR have been widely shown some degree of efficacy. Furthermore, other novel therapeutic approaches such as monoclonal antibodies and peptide vaccines have also been explored and developed for different types of RCC treatment [47]. Our data revealed that upregulation of PTEN expression was observed in Scutellarin-induced RCC cells through invactivation of P13K/AKT/mTOR signaling transduction. Exogenous PTEN expression even strengthened the inhibition effect of Scutellarin on P13 K/AKT/mTOR signaling, whereas, this inhibitory effect was obviously abrogated with PTEN knockdown. Thus, our findings implied that PTEN might be a potent target for Scutellarin to deactivate the P13 K/AKT/mTOR signaling in RCC progression. The pharmacokinetic study conducted after single and daily treatments also supported the antitumor effect of Scutellarin as evidenced in nude mice after intraperitoneal injection. The plasma concentration-time curves exhibited similar pattern between the single dose and daily dose of Scutellarin, suggesting that there is no accumulation of Scutellarin in mice when given intraperitoneally every 24 h. Furthermore, a previous study reported by Huang et al. has emphasized that rapid and efficient bioavaiability of intraperitoneal injection of scutellarin than oral administration is observed in male SD rats [21]. Thus, the current results indicated that intraperitoneal injection of Scutellarin at dose of 30 mg/ kg was safe for successive administration. In conclusion, we demonstrated that Scutellarin inhibited cell proliferation, induced apoptosis and cell cycle arrest, suppressed invasion and migration might through PTEN-mediated repression of P13K/AKT/ mTOR pathway in RCC cells, indicating that Scutellarin could be considered as a promising anti-tumor agent against RCC progression. Further investigations are still needed to elucidate the potential targets of Scutellarin and uncover the regulatory mechanisms of how Scutellarin suppresses the RCC development.
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