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mTOR signal pathway
Biomedicine & Pharmacotherapy 120 (2019) 109443
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Rosmarinic acid inhibits proliferation and invasion of hepatocellular carcinoma cells SMMC 7721 via PI3K/AKT/mTOR signal pathway
T
Li Wanga, Huiyu Yangb, , Chen Wangc, Xiaoxin Shid, Kunkun Lie ⁎
a
Department of medical administration, Henan Provincial People’s Hospital, Department of medical administration of of Central China Fuwai Hospital, Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, 450003, Henan Province, China b Gastroenterology department, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450000, Henan Province, China c Department of neck, shoulder, waist and leg, Zhengzhou orthopaedic Hospital, Zhengzhou, 450000, China d Health examination centre, Henan Provincial People’s Hospital, Health examination centre of of Central China Fuwai Hospital, Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, 450003, Henan Province, China e Gastroenterology department, Zhengzhou Central Hospital, Zhengzhou, 450000, Henan Province, China
ARTICLE INFO
ABSTRACT
Keywords: Rosmarinic acid Hepatocellular carcinoma Proliferation Apoptosis PI3K/AKT/mTOR signal pathway
Objective: To investigate the effect of rosmarinic acid (RosA) on hepatocellular carcinoma cell in vivo and in vitro and to explore its possible mechanism of anti-hepatocarcinoma. Methods: The hepatocellular carcinoma cell line SMMC-7721 was treated with different concentrations of RosA (0, 20, 50, 100 μmol/L) to detect cell proliferation, cell cycle, apoptosis and invasion.PI3K pathway-specific activator IGF-1 was used to explore whether the mechanism for RosA action relates to PI3K/AKT signal pathway.Nude mice inoculated with SMMC-7721 cells were treated with different doses of RosA (0, 5, 10 and 20 mg/kg) to detect the tumor formation of cancer cells in vivo. Results: RosA significantly inhibited the proliferation of SMMC-7721 cells and induced G1 arrest and apoptosis in a dose-dependent manner. RosA might inhibit cell invasion by regulating epithelial-mesenchymal transition. Rescue experiments showed that IGF-1 could reverse the inhibition of PI3K/AKT/mTOR signal pathway by RosA and the effect on proliferation, apoptosis, cell cycle, invasion and EMT by IGF-1 in SMMC-7721 cells;RosA could inhibit tumor formation of SMMC-7721 cells in vivo. Conclusion: RosA can inhibit the proliferation and invasion of hepatocellular carcinoma cell in vitro and inhibit tumour growth in vivo and the mechanism may relate to inhibiting the activation of PI3K/AKT signal pathway.
1. Introduction Primary hepatocellular carcinoma (PHC) is the second most common malignant tumors that has a high mortality rate [1,2], and there is still no effective treatment for advanced PHC [3]. Studies have found that the natural active ingredients of various traditional Chinese medicines play active roles in the treatment of various diseases including tumors in recent years [4,5]. Rosmarinic acid (RosA) is an active ingredient of traditional Chinese medicines such as rosemary, perilla, and Prunella vulgaris that have proved to have a variety of biological activities including antibacterial, anti-oxidant, anti-inflammatory and anti-tumor [6,7]. Studies have confirmed that RosA inhibits the proliferation of small cell lung cancer cells (NCI-H82), human prostate cancer cells (DU-145), and human chronic myeloid leukemia cells (K-562) in vitro [8]. However, there are currently no
research on the effect of RosA on liver cancer cells. Phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase (AKT)/mTOR signal pathway is highly activated in a variety of tumors including hepatocellular carcinoma [9], and the inhibition of PI3K/AKT/mTOR signal pathway can inhibit the growth, invasion and metastasis of various tumors such as colon cancer [8], hepatocellular carcinoma [10] and breast cancer [11]. The core of the PI3K/AKT pathway is the continued activation of AKT, which further regulates cell cycle, apoptosis, metastasis, and angiogenesis [12]. Therefore, PI3K/ Akt signal pathway is a target for a variety of anti-tumor drugs. Studies have shown that one of the mechanisms by which RosA protects hippocampal neurons is to regulate the activity of AKT [13]. We speculated that the mechanism that activation on tumors of RosA may be related to the PI3K/AKT signal pathway. Therefore, this study aims to observe the effect of RosA on liver cancer cell SMMC-7721 in vivo and in vitro and to
⁎ Corresponding author at: Gastroenterology department, the First Affiliated Hospital of Zhengzhou University, NO.1 Jianshe East Road, Jinshui District, Zhengzhou, 450000, Henan Province, China. E-mail address: [email protected] (H. Yang).
https://doi.org/10.1016/j.biopha.2019.109443 Received 28 May 2019; Received in revised form 27 August 2019; Accepted 6 September 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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explore whether its mechanism is related to PI3K/AKT/mTOR signal pathway, in order to provide new ideas for the study of clinical antihepatocarcinoma drugs.
containing 15% fetal calf serum was added to the lower chamber. 3 duplicate holes were set in each group. After 24 h, the Matrigel and the cells in the upper chamber were wiped off. The cells were fixed with 4% paraformaldehyde for 24 h, and then stained with 0.1% crystal violet for 40 min. Five fields were randomly selected under the microscope to take pictures and count the number of cells invading the bottom of the chamber.
2. Materials and methods 2.1. Cells and animals The human hepatocellular carcinoma cell line SMMC-7721 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). SMMC-7721 was placed in RPMI1640 medium (Gibco, USA) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin (Roswell Park Memorial Institute), and incubated at 37 ℃ in a 5% CO2 humid environment. Male BALB/c nude mice (5 weeks) were purchased from the Experimental Animal Center of Zhengzhou University. All experiments were carried out with the approval of the administrative panel at Ethic Committee of Henan Provincial People's Hospital.
2.6. Animal experiments Nude mice were randomly divided into 0 mg/kg RosA group, 5 mg/ kg RosA group, 10 mg/kg RosA group and 20 mg/kg RosA group. Nude mice were injected subcutaneously in the inguinal to inoculate SMMC7721 cells. 5 mg/kg RosA group, 10 mg/kg RosA group and 20 mg/kg RosA group were given 5, 10 and 20 mg/kg of RosA for 5 days, respectively, once a day. The 0 mg/kg RosA group was given the same amount of vehicle. Tumor long diameter (a) and short diameter (b) were measured and recorded at 1, 3, 6, 9, 12, 15, 18, 21d after modeling, and tumor volume was calculated as :tumor volume = (a × b2)/ 2.
2.2. Cytotoxicity test and drug treatment The cells were seeded at 1 × 105 /well into 96-well plates for 12 h, and gradient concentrations (0–500 μmol/L) of RosA (Maohong Biotech, China) were added to each well to treat the cells. Five replicate wells were set for each concentration. After 24 h,48 h and 72 h, cell viability was measured by adding 10 μl of CCK-8 reagent (Biyuntian Biotechnology Co., Ltd., China) respectively. The optical density (OD) of each well was measured at a wavelength of 450 nm. The proper concentrations that without significant toxicity to cells at 24 h and with significant effect on cells at 48 h were selected for following experiments. The cells were inoculated separately into complete medium with selected gradient dose of RosA,6 duplicate holes were set in each group.
2.7. H&E staining The animals were sacrificed and the tumor tissue was removed. Paraffin sections were prepared after tumor tissue was fixed overnight with 4% formaldehyde. After paraffin sections were stained with conventional hematoxylin-eosin (H&E), the pathological changes of tumor tissues were observed under light microscope. 2.8. TUNEL assay Apoptosis in tumor tissues was measured according to the TUNEL kit instructions. In brief, tumor tissue was fixed with 4% paraformaldehyde, and incubated with 0.5% Triton X-100 for 15 min. After treatment with 2% hydrogen peroxide for 5 min, the Tunel reaction solution was added dropwise for 1 h. Counterstaining was performed with 0.05% DAB staining solution.300 cells were counted per slice under the microscope, the positive rate of apoptosis = the number of positive cells / 300 × 100%.
2.3. Cloning formation experiment 200 cells were inoculated to a Petri dish containing RPMI-1640 medium and 4 μg/mL of heparin for culture after being treated with RosA for 48 h. After 14 days, it was stained with Giemsa solution. The number of colonies containing > 50 cells was counted under a microscope.
2.9. Western blot analysis(WB)
2.4. Cell apoptosis rate and cell cycle detection by flow cytometry(FCM)
The total protein was extracted after cell lysis, and the concentration was determined by BCA method (Biyuntian Technology Co., Ltd., China). After 10% SDS-PAGE electrophoresis, PVDF (Millipore, USA) transfection, and blocking, the corresponding primary antibody was
After being treated with RosA for 48 h, SMMC-7721 cells in logarithmic growth phase were seeded into 6-well plates, and the medium were discarded when cells grew to 85% confluence. The cells were collected, mixed with 5 μl of FITC-Annexin V, and incubated for 15 min at room temperature in the dark. Added 5 μl of PI stain and incubated for 5 min in the dark. The apoptosis rate of the cells was measured by FCM. Three replicate wells were set up in each group and three independent replicates were performed. After being treated with RosA for 48 h, cells were digested with trypsin and collected, and fixed by adding 70% ethanol at 4 °C overnight. After washing with PBS, 1 mg/mL RNaseA was added and incubated for 30 min at room temperature. After incubation for 15 min at room temperature in the dark, the cell cycle was detected by FCM. 2.5. Cell invasion ability detection by Transwell assay After being treated with RosA for 48 h, the cells were transferred to serum-free medium for starvation for 24 h. The cells were harvested by trypsin digestion without EDTA. After being washed with PBS, the cells were resuspended in RPMI1640 medium to adjust the cell density to 1 × 10^6 cells/mL. The cell suspension(200 μl) was added to the upper chamber of a transwell chamber (Corning, USA), and complete medium
Fig. 1. Cytotoxic effect of RosA on SMMC-7721. 2
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Fig. 2. Effect of RosA on proliferation, cell cycle and apoptosis of SMMC-7721 cells. (A)The proliferation of SMMC-7721 cells detected by colony formation assay and the expression of Ki67, PCNA by WB. (B) The proportion of SMMC-7721 cells in the G0/G1, S and G2/M phases analyzed by FCM and the expression of Cyclin D1, Cyclin E by WB. (C) The apoptosis rate of SMMC-7721 cells determined by FCM and the expression of Bax and Bcl-2 by WB. *P < 0.05 vs 0 μg/mL RosA.
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added and incubated overnight at 4 °C. Taken β-actin as an internal reference. After being washed,added in the secondary antibody for incubation for 90 min. Washed the membrane with TBST,added ECL luminescent reagen for development. The relative expression levels of the proteins of interest were analyzed using Quantity One gel image analysis software.
2.11. Statistics and analysis SPSS software (ver.07 for Windows; SPSS Inc., Chicago, IL, USA). The results were represented as mean ± SEM. Comparative studies were analyzed by Student’s t-test or one- or two-way ANOVA test followed by post-hoc test with p values less than 0.05 considered as statistically significant.
2.10. The effect of RosA on PI3K/AKT/mTOR signal pathway and Rescue experiments
3. Results 3.1. Cytotoxic effect of RosA on SMMC-7721
To explore the mechanism of effect of RosA on SMMC-7721 cells,the expression of proteins related to PI3K/AKT signal pathway were detected by WB.To validate that the effect mechanism of RosA is related to PI3K/ AKT signal pathway,the rescue experiments were conducted:Control group,RosA group,IGF-1 group and IGF-1+RosA group of 7721 cells were set and performed the cell experiments above. Before being conducted, cells in IGF-1 and IGF-1 + RosA groups were pre-treated with the PI3K pathway-specific activator IGF-1(10 nmol/ml) for 12 h, and then cells in RosA and IGF-1+RosA group were treated with RosA (100 μg/ mL). Correspondingly, nude mice also were divided into the control group,RosA group,IGF-1 group and IGF-1+RosA group.The mice in IGF1 and IGF-1+RosA group were inoculate 7721 cells with IGF-1(10 nmol/ ml), and the other two groups were inoculate 7721 cells without IGF-1. Then the mice in RosA and IGF-1+RosA were treated with 20 mg/kg of RosA for 5 days (once a day),the other two groups were treated with normal saline of same volume.
CCK-8 assay results showed that the activity of SMMC-7721 decreased with the increase of RosA concentration. At 24 h, when the concentration of RosA reached 200 μg/mL, the cell activity fell below 80%; At 48 h, the cell activity fell to about 50% for 100 μg/mL, and at 72 h, the cell activity fell obviously for most of concentrations (Fig. 1). Therefore, the concentrations (0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/ mL) that were effective and were not serious toxic to SMMC-7721 cells were selected for subsequent experiments. 3.2. Effects of RosA on proliferation,cell cycle and apoptosis Colony formation assay showed that the number of cell clones in the 20, 50 and 100 μg/mL RosA groups were significantly reduced compared with the 0 μg/mL group (Fig. 2A). At the same time, RosA (20, 50, 100 μg/mL) significantly inhibited the expression of cell proliferation marker proteins Ki67 and PCNA (Fig. 2D), suggesting that RosA
Fig. 3. Effect of RosA on invasion and EMT of SMMC-7721 cells. (A) The invasion ability of SMMC-7721 cells analyzed by transwell assay,Bar = 100 μm. (B)The expression of MMP-2, MMP-9, E-cadherin, N-cadherin and Vimentin analyzed by WB. (C) Comparison showed by histogram. *P < 0.05 vs 0 μg/mL RosA. 4
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can inhibit the proliferation of liver cancer cells. The results of FCM cell cycle detection showed that the number of SMMC-7721 cells in G0/G1 phase in both the 50 μg/mL group and 100 μg/mL group were significantly higher than the 0 μg/mL group, while the number of cells in the G2/M phase were significantly lower (Fig. 2B). And the expression of Cyclin D1 and Cyclin E were higher in the 50 μg/mL and 100 μg/mL groups compared with the 0 μg/mL group (Fig. 2D). These results revealed that RosA may inhibit the proliferation of SMMC-7721 cells by inducing G1 arrest. FCM results showed that the apoptotic rate was significantly higher in the 50 μg/mL and 100 μg/mL group than the 0 μg/mL group (Fig. 2C). The expression levels of the apoptotic marker protein Bax were significantly higher, while the expression levels of Bcl-2 were significantly lower in the 20 μg/mL, 50 μg/mL and 100 μg/mL groups than the 0 μg/mL group (Fig. 2D), suggesting that RosA can induce apoptosis of SMMC-7721 cells. 3.3. Effect of RosA on cell invasion and epithelial-mesenchymal transition (EMT) Transwell assay was used to analyze the effect of RosA on SMMC7721 cell invasion. It was found that 50 μg/mL and 100 μg/mL RosA significantly inhibited SMMC-7721 cell invasion (Fig. 3A). Meanwhile, the expression levels of MMP-2 and MMP-9 were significantly lower in the 50 μg/mL and 100 μg/mL groups compared to the 0 μg/mL group (Fig. 3B). EMT is a key link to regulate tumor invasion and metastasis [14].Then the effect of RosA on the expression of the epithelial phenotype marker E-cadherin and the interstitial phenotype markers Vimentin and N-cadherin. It was found that 50 μg/mL and 100 μg/mL of RosA promoted the expression of E-cadherin in SMMC-7721 cells, while inhibited the expression of Vimentin and N-cadherin (Fig. 3B). These results suggested that RosA may inhibit SMMC-7721 cell invasion by regulating EMT. 3.4. Anti-cancer efficiency of RosA in vivo Nude mice were used to perform transplanted tumor formation experiments of hepatocellular carcinoma cells. After inoculation of SMMC-7721 cells in nude mice, the volume of xenografts in each group gradually increased (Fig. 4A). At the end of feeding, the tumor volume and weights were significantly lower in the 10 mg/kg and 20 mg/kg groups compared to the 0 mg/kg group (Fig. 4B). HE staining showed that as the dose of RosA increased, the cell density in the tumor tissue gradually decreased (Fig. 4C). TUNEL analysis showed that, the apoptosis rate in the 10 mg/kg and 20 mg/kg groups were significantly higher than the 0 mg/kg group (Fig. 4D). 3.5. Effect of RosA on PI3K/AKT/mTOR signal pathway The expression level of proteins relate to PI3K/AKT/mTOR signal pathway in vitro and in vivo were showed in Fig. 5. It was found that RosA (20 μg/mL, 50 μg/mL and 100 μg/mL) significantly inhibited the expression of p-PI3K, p-AKT and p-mTOR without affecting the expression of total PI3K, AKT and mTOR proteins in vitro (Fig. 5A), and so did the RosA (5 mg/kg,10 mg/kg and 20 mg/kg) in vivo (Fig. 5B), which suggesting that RosA can inhibit the activation of the PI3K/AKT/mTOR signal pathway both in vitro and in vivo.
Fig. 4. RosA inhibited the growth of hepatocellular carcinoma cells in vivo. (A)Tumor volume. (B) Representative tumorigenic pictures and the analysis of tumor weight. (C) The pathological changes of the tumor was observed by HE staining. Bar = 100 μm. (D) The apoptosis of tumor tissues was observed by TUNEL assay. Bar = 50 μm. n = 5.*P < 0.05, **P < 0.01 vs 0 mg/kg RosA.
could attenuate the inhibition of the PI3K/AKT/mTOR signal pathway.Further results of cell proliferation,cell apoptosis,cell cycle and cell invasion experiments revealed that IGF-1 could reverse the inhibition effect of RosA on cell proliferation (Fig. 6B), cell apoptosis (Fig. 6C), G1 arrest in cell cycle (Fig. 6D), cell invasion (Fig. 6E). Correspondingly,the results of proteins expression,cell proliferation and cell apoptosis in transplanted tumor tissue of mice showed that IGF-1 could reverse the inhibition effect of RosA in vivo (Fig. 7).These results indicated that the effect of RosA on the biological characteristics of
3.6. PI3K/AKT/mTOR activator IGF-1 reversed the effect of RosA on SMMC-7721 cells Firstly, the expression levels of proteins related to PI3K/AKT/mTOR signal pathway were detected. It was found that the expression levels of p-PI3K, p-AKT and p-mTOR proteins were significantly higher in the IGF-1 + RosA group than RosA group (Fig. 6A), suggesting that IGF-1 5
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Fig. 5. The expression of proteins relate to PI3K/AKT/mTOR signal pathway. (A) Expression levels of proteins in vitro; (B) Expression levels of proteins in vivo; *P < 0.05, **P < 0.01 vs 0 mg/kg RosA.
SMMC-7721 cells is related to the inhibition of PI3K/AKT/mTOR signal pathway.
[18], Bcl-2 inhibits apoptosis by inhibiting the release of cytochrome C, Bax induces apoptosis by promoting cytochrome c release and a variety of caspase-dependent signal pathways [19,20]. In this study, it was found that RosA caused decrease of Cyclin D1, Cyclin E and Bcl-2 expression and increase the apoptosis rate and Bax expression in SMMC7721 cells, suggesting that RosA may inhibit cell proliferation and promote cell apptosis by regulating the G1/S arrest and expression of relate proteins.Moreover, it also was observed that RosA promoted cell apoptosis in vivo. So it could be speculated that RosA can promote apoptosis of hepatocellular carcinoma cells and inhibit the growth. Invasion and metastasis of tumor cells are the main cause of cancer treatment failure and poor prognosis. Matrix metalloproteinases 2 (MMP-2) and MMP-9 are hallmarks of tumor cell invasion and metastasis [21]. Moreover, EMT is an important biological process for migration and invasion of malignant tumor [22], which has been confirmed in a variety of malignant tumors including gastric cancer [23], breast cancer [24], hepatocellular carcinoma [25]. This process is accompanied by down-regulation of epithelial marker E-cadherin and upregulation of Vimentin and N-cadherin etc. And MMPs could also promote EMT by degradation of proteins relate to linking cell and
4. Discussion As a natural source of polyphenolic hydroxy compounds, RosA has been proved to have anti-cancer activity [15]. Han et al. [16] showed that RosA can inhibit the Warburg effect and growth of human gastric cancer cell MKN45 in vitro. In this paper,it was found that RosA treatment of hepatocellular carcinoma cells SMMC-7721 could significantly inhibit the cell proliferation, and the effect was dose-independent. Additionally, RosA could also inhibit the growth of subcutaneous tumors of hepatocellular carcinoma cells in nude mice. These results suggested that RosA may slow the development of hepatocellular carcinoma by inhibiting the proliferation of cancer cells and the growth of subcutaneous tumors. Abnormal rapid switching of the cell cycle or destruction of the checkpoint will lead to uncontrolled growth of cancer cells [17], which regulate by abnormal expression of Cyclin D1 and Cyclin E. Moreover, the interaction between Bcl-2 and Bax plays a decisive role in apoptosis 6
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extracellular matrix. In this study, it was found that RosA treatment could inhibit the cell invasion and the expression of MMP-2 and MMP9, promote the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin, suggesting that RosA can inhibit the invasion ability of hepatocellular carcinoma cells by MMPs and EMT. The PI3K/AKT signal pathway is one of the most major signal pathways closely relate to the proliferation, apoptosis and invasion of most tumor cells. Molecular targeted therapy for PI3K/AKT signal pathway has become a hot topic [26]. Studies have shown that AKT is activated in tumor tissues of gastric cancer [27], breast cancer [28], and hepatocellular carcinoma [29]. Activation of AKT can phosphorylate a variety of downstream target molecules, such as the Bcl-2 family, glycogen synthase kinase 3 (GSK3) and S6 protein kinase, which have important effect on cell survival, apoptosis and motility. Gao et al. [30] found that activation of PI3K/AKT/mTOR signal pathway in ovarian cancer cells promoted cell proliferation by increasing the cyclin D1 expression and G1 phase proportion. Maseki et al. [31] reported that the AKT/GSK3β/Snail signal pathway mediated the proliferation and invasion of head and neck squamous cell carcinoma.The present researches show that activated PI3K/Akt could extricate and up-regulate NF-κB to increase the metastasis ability of tumor cells [32]. At the same time, phosphorylated Akt could activate the MMPs and p21 to enhance the invasion of cancer cells [33]. There also are some researches proved that the activation of PI3K/Akt could promote the phosphorylation of Vimentin who relate to EMT [34]. The results in our study showed that RosA treatment significantly inhibited the expression levels of p-PI3K, p-AKT and p-mTOR without effecting total PI3K, AKT and mTOR proteins, so it could be speculated that the effect of RosA on SMMC-7721 may be mediated by activation of the PI3K/AKT/mTOR signal pathway,including the growth inhibition,G1 arrest and apoptosis promotion effects by regulating Bax protein and cyclin D1, inhibition effect of migration and invasion by regulation MMPs, Vimentin and EMT. To validate this mechanism, IGF-1, activator of PI3K/AKT/mTOR signal pathway, was used for rescue experiments.The results showed that IGF1 could reverse the inhibition of PI3K/AKT/mTOR signal pathway by RosA, as well as reverse the effect of RosA on cell proliferation, apoptosis, invasion and EMT. Moreover, in vivo experiments also showed that RosA inhibited tumor growth and inhibited the expression of related proteins of PI3K/AKT/mTOR signal pathway. Therefore, it is likely that the anti-proliferation and invasion, and pro-apoptotic effects of RosA on the hepatocellular carcinoma cell line SMMC-7221 are at least partially achieved by inhibiting the PI3K/AKT/mTOR signal pathway. In conclusion, RosA inhibits the proliferation and invasion and induce apoptosis of hepatocellular carcinoma cell line SMMC-7721, which may be related to the inhibition of PI3K/AKT/mTOR signal pathway. Additionally, RosA inhibits the growth of SMMC-7721 cells in vivo, which may provide a new theoretical basis for the application of RosA anticancer effect. Convention on biodiversity All animal experiments and cell experiments were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All researches were under the framework of the United Nations Convention on Biodiversity.
Fig. 6. IGF-1 reversed the effect of RosA on PI3K/AKT/mTOR signal pathway and SMMC-7721cells in vitro. (A)The expression of PI3K/AKT/mTOR pathway proteins in SMMC-7721 cells detected by WB. (B) The proliferation of SMMC-7721 cells detected by colony formation assay. (C) The cell cycle distribution of SMMC-7721 cells analyzed by FCM. (D) The apoptosis of SMMC-7721 cells analyzed by FCM. (E) The invasive ability of SMMC-7721 cells was detected by transwell assay. Scale bar = 100 μm.*P < 0.05 vs control group; #P < 0.05 vs RosA group.
Declaration of Competing Interest 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.
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Fig. 7. IGF-1 reversed the effect of RosA on PI3K/AKT/mTOR signal pathway and SMMC-7721cells in vivo. (A) Change of tumor volume and comparison of tumor weight at the end of experiments. (B) The apoptosis of tumour tissue analyzed by TUNEL. (C) The expression of PI3K/AKT/mTOR pathway proteins in tumour tissue detected by WB. Scale bar = 50 μm.*P < 0.05 vs control group; #P < 0.05 vs RosA group.
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Rosmarinic acid inhibits proliferation and invasion of hepatocellular carcinoma cells SMMC 7721 via PI3K/AKT/mTOR signal pathway
T
Li Wanga, Huiyu Yangb, , Chen Wangc, Xiaoxin Shid, Kunkun Lie ⁎
a
Department of medical administration, Henan Provincial People’s Hospital, Department of medical administration of of Central China Fuwai Hospital, Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, 450003, Henan Province, China b Gastroenterology department, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450000, Henan Province, China c Department of neck, shoulder, waist and leg, Zhengzhou orthopaedic Hospital, Zhengzhou, 450000, China d Health examination centre, Henan Provincial People’s Hospital, Health examination centre of of Central China Fuwai Hospital, Central China Fuwai Hospital of Zhengzhou University, Zhengzhou, 450003, Henan Province, China e Gastroenterology department, Zhengzhou Central Hospital, Zhengzhou, 450000, Henan Province, China
ARTICLE INFO
ABSTRACT
Keywords: Rosmarinic acid Hepatocellular carcinoma Proliferation Apoptosis PI3K/AKT/mTOR signal pathway
Objective: To investigate the effect of rosmarinic acid (RosA) on hepatocellular carcinoma cell in vivo and in vitro and to explore its possible mechanism of anti-hepatocarcinoma. Methods: The hepatocellular carcinoma cell line SMMC-7721 was treated with different concentrations of RosA (0, 20, 50, 100 μmol/L) to detect cell proliferation, cell cycle, apoptosis and invasion.PI3K pathway-specific activator IGF-1 was used to explore whether the mechanism for RosA action relates to PI3K/AKT signal pathway.Nude mice inoculated with SMMC-7721 cells were treated with different doses of RosA (0, 5, 10 and 20 mg/kg) to detect the tumor formation of cancer cells in vivo. Results: RosA significantly inhibited the proliferation of SMMC-7721 cells and induced G1 arrest and apoptosis in a dose-dependent manner. RosA might inhibit cell invasion by regulating epithelial-mesenchymal transition. Rescue experiments showed that IGF-1 could reverse the inhibition of PI3K/AKT/mTOR signal pathway by RosA and the effect on proliferation, apoptosis, cell cycle, invasion and EMT by IGF-1 in SMMC-7721 cells;RosA could inhibit tumor formation of SMMC-7721 cells in vivo. Conclusion: RosA can inhibit the proliferation and invasion of hepatocellular carcinoma cell in vitro and inhibit tumour growth in vivo and the mechanism may relate to inhibiting the activation of PI3K/AKT signal pathway.
1. Introduction Primary hepatocellular carcinoma (PHC) is the second most common malignant tumors that has a high mortality rate [1,2], and there is still no effective treatment for advanced PHC [3]. Studies have found that the natural active ingredients of various traditional Chinese medicines play active roles in the treatment of various diseases including tumors in recent years [4,5]. Rosmarinic acid (RosA) is an active ingredient of traditional Chinese medicines such as rosemary, perilla, and Prunella vulgaris that have proved to have a variety of biological activities including antibacterial, anti-oxidant, anti-inflammatory and anti-tumor [6,7]. Studies have confirmed that RosA inhibits the proliferation of small cell lung cancer cells (NCI-H82), human prostate cancer cells (DU-145), and human chronic myeloid leukemia cells (K-562) in vitro [8]. However, there are currently no
research on the effect of RosA on liver cancer cells. Phosphatidylinositol 3-kinase (PI3K)/serine-threonine protein kinase (AKT)/mTOR signal pathway is highly activated in a variety of tumors including hepatocellular carcinoma [9], and the inhibition of PI3K/AKT/mTOR signal pathway can inhibit the growth, invasion and metastasis of various tumors such as colon cancer [8], hepatocellular carcinoma [10] and breast cancer [11]. The core of the PI3K/AKT pathway is the continued activation of AKT, which further regulates cell cycle, apoptosis, metastasis, and angiogenesis [12]. Therefore, PI3K/ Akt signal pathway is a target for a variety of anti-tumor drugs. Studies have shown that one of the mechanisms by which RosA protects hippocampal neurons is to regulate the activity of AKT [13]. We speculated that the mechanism that activation on tumors of RosA may be related to the PI3K/AKT signal pathway. Therefore, this study aims to observe the effect of RosA on liver cancer cell SMMC-7721 in vivo and in vitro and to
⁎ Corresponding author at: Gastroenterology department, the First Affiliated Hospital of Zhengzhou University, NO.1 Jianshe East Road, Jinshui District, Zhengzhou, 450000, Henan Province, China. E-mail address: [email protected] (H. Yang).
https://doi.org/10.1016/j.biopha.2019.109443 Received 28 May 2019; Received in revised form 27 August 2019; Accepted 6 September 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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explore whether its mechanism is related to PI3K/AKT/mTOR signal pathway, in order to provide new ideas for the study of clinical antihepatocarcinoma drugs.
containing 15% fetal calf serum was added to the lower chamber. 3 duplicate holes were set in each group. After 24 h, the Matrigel and the cells in the upper chamber were wiped off. The cells were fixed with 4% paraformaldehyde for 24 h, and then stained with 0.1% crystal violet for 40 min. Five fields were randomly selected under the microscope to take pictures and count the number of cells invading the bottom of the chamber.
2. Materials and methods 2.1. Cells and animals The human hepatocellular carcinoma cell line SMMC-7721 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). SMMC-7721 was placed in RPMI1640 medium (Gibco, USA) containing 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin (Roswell Park Memorial Institute), and incubated at 37 ℃ in a 5% CO2 humid environment. Male BALB/c nude mice (5 weeks) were purchased from the Experimental Animal Center of Zhengzhou University. All experiments were carried out with the approval of the administrative panel at Ethic Committee of Henan Provincial People's Hospital.
2.6. Animal experiments Nude mice were randomly divided into 0 mg/kg RosA group, 5 mg/ kg RosA group, 10 mg/kg RosA group and 20 mg/kg RosA group. Nude mice were injected subcutaneously in the inguinal to inoculate SMMC7721 cells. 5 mg/kg RosA group, 10 mg/kg RosA group and 20 mg/kg RosA group were given 5, 10 and 20 mg/kg of RosA for 5 days, respectively, once a day. The 0 mg/kg RosA group was given the same amount of vehicle. Tumor long diameter (a) and short diameter (b) were measured and recorded at 1, 3, 6, 9, 12, 15, 18, 21d after modeling, and tumor volume was calculated as :tumor volume = (a × b2)/ 2.
2.2. Cytotoxicity test and drug treatment The cells were seeded at 1 × 105 /well into 96-well plates for 12 h, and gradient concentrations (0–500 μmol/L) of RosA (Maohong Biotech, China) were added to each well to treat the cells. Five replicate wells were set for each concentration. After 24 h,48 h and 72 h, cell viability was measured by adding 10 μl of CCK-8 reagent (Biyuntian Biotechnology Co., Ltd., China) respectively. The optical density (OD) of each well was measured at a wavelength of 450 nm. The proper concentrations that without significant toxicity to cells at 24 h and with significant effect on cells at 48 h were selected for following experiments. The cells were inoculated separately into complete medium with selected gradient dose of RosA,6 duplicate holes were set in each group.
2.7. H&E staining The animals were sacrificed and the tumor tissue was removed. Paraffin sections were prepared after tumor tissue was fixed overnight with 4% formaldehyde. After paraffin sections were stained with conventional hematoxylin-eosin (H&E), the pathological changes of tumor tissues were observed under light microscope. 2.8. TUNEL assay Apoptosis in tumor tissues was measured according to the TUNEL kit instructions. In brief, tumor tissue was fixed with 4% paraformaldehyde, and incubated with 0.5% Triton X-100 for 15 min. After treatment with 2% hydrogen peroxide for 5 min, the Tunel reaction solution was added dropwise for 1 h. Counterstaining was performed with 0.05% DAB staining solution.300 cells were counted per slice under the microscope, the positive rate of apoptosis = the number of positive cells / 300 × 100%.
2.3. Cloning formation experiment 200 cells were inoculated to a Petri dish containing RPMI-1640 medium and 4 μg/mL of heparin for culture after being treated with RosA for 48 h. After 14 days, it was stained with Giemsa solution. The number of colonies containing > 50 cells was counted under a microscope.
2.9. Western blot analysis(WB)
2.4. Cell apoptosis rate and cell cycle detection by flow cytometry(FCM)
The total protein was extracted after cell lysis, and the concentration was determined by BCA method (Biyuntian Technology Co., Ltd., China). After 10% SDS-PAGE electrophoresis, PVDF (Millipore, USA) transfection, and blocking, the corresponding primary antibody was
After being treated with RosA for 48 h, SMMC-7721 cells in logarithmic growth phase were seeded into 6-well plates, and the medium were discarded when cells grew to 85% confluence. The cells were collected, mixed with 5 μl of FITC-Annexin V, and incubated for 15 min at room temperature in the dark. Added 5 μl of PI stain and incubated for 5 min in the dark. The apoptosis rate of the cells was measured by FCM. Three replicate wells were set up in each group and three independent replicates were performed. After being treated with RosA for 48 h, cells were digested with trypsin and collected, and fixed by adding 70% ethanol at 4 °C overnight. After washing with PBS, 1 mg/mL RNaseA was added and incubated for 30 min at room temperature. After incubation for 15 min at room temperature in the dark, the cell cycle was detected by FCM. 2.5. Cell invasion ability detection by Transwell assay After being treated with RosA for 48 h, the cells were transferred to serum-free medium for starvation for 24 h. The cells were harvested by trypsin digestion without EDTA. After being washed with PBS, the cells were resuspended in RPMI1640 medium to adjust the cell density to 1 × 10^6 cells/mL. The cell suspension(200 μl) was added to the upper chamber of a transwell chamber (Corning, USA), and complete medium
Fig. 1. Cytotoxic effect of RosA on SMMC-7721. 2
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Fig. 2. Effect of RosA on proliferation, cell cycle and apoptosis of SMMC-7721 cells. (A)The proliferation of SMMC-7721 cells detected by colony formation assay and the expression of Ki67, PCNA by WB. (B) The proportion of SMMC-7721 cells in the G0/G1, S and G2/M phases analyzed by FCM and the expression of Cyclin D1, Cyclin E by WB. (C) The apoptosis rate of SMMC-7721 cells determined by FCM and the expression of Bax and Bcl-2 by WB. *P < 0.05 vs 0 μg/mL RosA.
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added and incubated overnight at 4 °C. Taken β-actin as an internal reference. After being washed,added in the secondary antibody for incubation for 90 min. Washed the membrane with TBST,added ECL luminescent reagen for development. The relative expression levels of the proteins of interest were analyzed using Quantity One gel image analysis software.
2.11. Statistics and analysis SPSS software (ver.07 for Windows; SPSS Inc., Chicago, IL, USA). The results were represented as mean ± SEM. Comparative studies were analyzed by Student’s t-test or one- or two-way ANOVA test followed by post-hoc test with p values less than 0.05 considered as statistically significant.
2.10. The effect of RosA on PI3K/AKT/mTOR signal pathway and Rescue experiments
3. Results 3.1. Cytotoxic effect of RosA on SMMC-7721
To explore the mechanism of effect of RosA on SMMC-7721 cells,the expression of proteins related to PI3K/AKT signal pathway were detected by WB.To validate that the effect mechanism of RosA is related to PI3K/ AKT signal pathway,the rescue experiments were conducted:Control group,RosA group,IGF-1 group and IGF-1+RosA group of 7721 cells were set and performed the cell experiments above. Before being conducted, cells in IGF-1 and IGF-1 + RosA groups were pre-treated with the PI3K pathway-specific activator IGF-1(10 nmol/ml) for 12 h, and then cells in RosA and IGF-1+RosA group were treated with RosA (100 μg/ mL). Correspondingly, nude mice also were divided into the control group,RosA group,IGF-1 group and IGF-1+RosA group.The mice in IGF1 and IGF-1+RosA group were inoculate 7721 cells with IGF-1(10 nmol/ ml), and the other two groups were inoculate 7721 cells without IGF-1. Then the mice in RosA and IGF-1+RosA were treated with 20 mg/kg of RosA for 5 days (once a day),the other two groups were treated with normal saline of same volume.
CCK-8 assay results showed that the activity of SMMC-7721 decreased with the increase of RosA concentration. At 24 h, when the concentration of RosA reached 200 μg/mL, the cell activity fell below 80%; At 48 h, the cell activity fell to about 50% for 100 μg/mL, and at 72 h, the cell activity fell obviously for most of concentrations (Fig. 1). Therefore, the concentrations (0 μg/mL, 20 μg/mL, 50 μg/mL, 100 μg/ mL) that were effective and were not serious toxic to SMMC-7721 cells were selected for subsequent experiments. 3.2. Effects of RosA on proliferation,cell cycle and apoptosis Colony formation assay showed that the number of cell clones in the 20, 50 and 100 μg/mL RosA groups were significantly reduced compared with the 0 μg/mL group (Fig. 2A). At the same time, RosA (20, 50, 100 μg/mL) significantly inhibited the expression of cell proliferation marker proteins Ki67 and PCNA (Fig. 2D), suggesting that RosA
Fig. 3. Effect of RosA on invasion and EMT of SMMC-7721 cells. (A) The invasion ability of SMMC-7721 cells analyzed by transwell assay,Bar = 100 μm. (B)The expression of MMP-2, MMP-9, E-cadherin, N-cadherin and Vimentin analyzed by WB. (C) Comparison showed by histogram. *P < 0.05 vs 0 μg/mL RosA. 4
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can inhibit the proliferation of liver cancer cells. The results of FCM cell cycle detection showed that the number of SMMC-7721 cells in G0/G1 phase in both the 50 μg/mL group and 100 μg/mL group were significantly higher than the 0 μg/mL group, while the number of cells in the G2/M phase were significantly lower (Fig. 2B). And the expression of Cyclin D1 and Cyclin E were higher in the 50 μg/mL and 100 μg/mL groups compared with the 0 μg/mL group (Fig. 2D). These results revealed that RosA may inhibit the proliferation of SMMC-7721 cells by inducing G1 arrest. FCM results showed that the apoptotic rate was significantly higher in the 50 μg/mL and 100 μg/mL group than the 0 μg/mL group (Fig. 2C). The expression levels of the apoptotic marker protein Bax were significantly higher, while the expression levels of Bcl-2 were significantly lower in the 20 μg/mL, 50 μg/mL and 100 μg/mL groups than the 0 μg/mL group (Fig. 2D), suggesting that RosA can induce apoptosis of SMMC-7721 cells. 3.3. Effect of RosA on cell invasion and epithelial-mesenchymal transition (EMT) Transwell assay was used to analyze the effect of RosA on SMMC7721 cell invasion. It was found that 50 μg/mL and 100 μg/mL RosA significantly inhibited SMMC-7721 cell invasion (Fig. 3A). Meanwhile, the expression levels of MMP-2 and MMP-9 were significantly lower in the 50 μg/mL and 100 μg/mL groups compared to the 0 μg/mL group (Fig. 3B). EMT is a key link to regulate tumor invasion and metastasis [14].Then the effect of RosA on the expression of the epithelial phenotype marker E-cadherin and the interstitial phenotype markers Vimentin and N-cadherin. It was found that 50 μg/mL and 100 μg/mL of RosA promoted the expression of E-cadherin in SMMC-7721 cells, while inhibited the expression of Vimentin and N-cadherin (Fig. 3B). These results suggested that RosA may inhibit SMMC-7721 cell invasion by regulating EMT. 3.4. Anti-cancer efficiency of RosA in vivo Nude mice were used to perform transplanted tumor formation experiments of hepatocellular carcinoma cells. After inoculation of SMMC-7721 cells in nude mice, the volume of xenografts in each group gradually increased (Fig. 4A). At the end of feeding, the tumor volume and weights were significantly lower in the 10 mg/kg and 20 mg/kg groups compared to the 0 mg/kg group (Fig. 4B). HE staining showed that as the dose of RosA increased, the cell density in the tumor tissue gradually decreased (Fig. 4C). TUNEL analysis showed that, the apoptosis rate in the 10 mg/kg and 20 mg/kg groups were significantly higher than the 0 mg/kg group (Fig. 4D). 3.5. Effect of RosA on PI3K/AKT/mTOR signal pathway The expression level of proteins relate to PI3K/AKT/mTOR signal pathway in vitro and in vivo were showed in Fig. 5. It was found that RosA (20 μg/mL, 50 μg/mL and 100 μg/mL) significantly inhibited the expression of p-PI3K, p-AKT and p-mTOR without affecting the expression of total PI3K, AKT and mTOR proteins in vitro (Fig. 5A), and so did the RosA (5 mg/kg,10 mg/kg and 20 mg/kg) in vivo (Fig. 5B), which suggesting that RosA can inhibit the activation of the PI3K/AKT/mTOR signal pathway both in vitro and in vivo.
Fig. 4. RosA inhibited the growth of hepatocellular carcinoma cells in vivo. (A)Tumor volume. (B) Representative tumorigenic pictures and the analysis of tumor weight. (C) The pathological changes of the tumor was observed by HE staining. Bar = 100 μm. (D) The apoptosis of tumor tissues was observed by TUNEL assay. Bar = 50 μm. n = 5.*P < 0.05, **P < 0.01 vs 0 mg/kg RosA.
could attenuate the inhibition of the PI3K/AKT/mTOR signal pathway.Further results of cell proliferation,cell apoptosis,cell cycle and cell invasion experiments revealed that IGF-1 could reverse the inhibition effect of RosA on cell proliferation (Fig. 6B), cell apoptosis (Fig. 6C), G1 arrest in cell cycle (Fig. 6D), cell invasion (Fig. 6E). Correspondingly,the results of proteins expression,cell proliferation and cell apoptosis in transplanted tumor tissue of mice showed that IGF-1 could reverse the inhibition effect of RosA in vivo (Fig. 7).These results indicated that the effect of RosA on the biological characteristics of
3.6. PI3K/AKT/mTOR activator IGF-1 reversed the effect of RosA on SMMC-7721 cells Firstly, the expression levels of proteins related to PI3K/AKT/mTOR signal pathway were detected. It was found that the expression levels of p-PI3K, p-AKT and p-mTOR proteins were significantly higher in the IGF-1 + RosA group than RosA group (Fig. 6A), suggesting that IGF-1 5
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Fig. 5. The expression of proteins relate to PI3K/AKT/mTOR signal pathway. (A) Expression levels of proteins in vitro; (B) Expression levels of proteins in vivo; *P < 0.05, **P < 0.01 vs 0 mg/kg RosA.
SMMC-7721 cells is related to the inhibition of PI3K/AKT/mTOR signal pathway.
[18], Bcl-2 inhibits apoptosis by inhibiting the release of cytochrome C, Bax induces apoptosis by promoting cytochrome c release and a variety of caspase-dependent signal pathways [19,20]. In this study, it was found that RosA caused decrease of Cyclin D1, Cyclin E and Bcl-2 expression and increase the apoptosis rate and Bax expression in SMMC7721 cells, suggesting that RosA may inhibit cell proliferation and promote cell apptosis by regulating the G1/S arrest and expression of relate proteins.Moreover, it also was observed that RosA promoted cell apoptosis in vivo. So it could be speculated that RosA can promote apoptosis of hepatocellular carcinoma cells and inhibit the growth. Invasion and metastasis of tumor cells are the main cause of cancer treatment failure and poor prognosis. Matrix metalloproteinases 2 (MMP-2) and MMP-9 are hallmarks of tumor cell invasion and metastasis [21]. Moreover, EMT is an important biological process for migration and invasion of malignant tumor [22], which has been confirmed in a variety of malignant tumors including gastric cancer [23], breast cancer [24], hepatocellular carcinoma [25]. This process is accompanied by down-regulation of epithelial marker E-cadherin and upregulation of Vimentin and N-cadherin etc. And MMPs could also promote EMT by degradation of proteins relate to linking cell and
4. Discussion As a natural source of polyphenolic hydroxy compounds, RosA has been proved to have anti-cancer activity [15]. Han et al. [16] showed that RosA can inhibit the Warburg effect and growth of human gastric cancer cell MKN45 in vitro. In this paper,it was found that RosA treatment of hepatocellular carcinoma cells SMMC-7721 could significantly inhibit the cell proliferation, and the effect was dose-independent. Additionally, RosA could also inhibit the growth of subcutaneous tumors of hepatocellular carcinoma cells in nude mice. These results suggested that RosA may slow the development of hepatocellular carcinoma by inhibiting the proliferation of cancer cells and the growth of subcutaneous tumors. Abnormal rapid switching of the cell cycle or destruction of the checkpoint will lead to uncontrolled growth of cancer cells [17], which regulate by abnormal expression of Cyclin D1 and Cyclin E. Moreover, the interaction between Bcl-2 and Bax plays a decisive role in apoptosis 6
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extracellular matrix. In this study, it was found that RosA treatment could inhibit the cell invasion and the expression of MMP-2 and MMP9, promote the expression of E-cadherin and decreased the expression of N-cadherin and Vimentin, suggesting that RosA can inhibit the invasion ability of hepatocellular carcinoma cells by MMPs and EMT. The PI3K/AKT signal pathway is one of the most major signal pathways closely relate to the proliferation, apoptosis and invasion of most tumor cells. Molecular targeted therapy for PI3K/AKT signal pathway has become a hot topic [26]. Studies have shown that AKT is activated in tumor tissues of gastric cancer [27], breast cancer [28], and hepatocellular carcinoma [29]. Activation of AKT can phosphorylate a variety of downstream target molecules, such as the Bcl-2 family, glycogen synthase kinase 3 (GSK3) and S6 protein kinase, which have important effect on cell survival, apoptosis and motility. Gao et al. [30] found that activation of PI3K/AKT/mTOR signal pathway in ovarian cancer cells promoted cell proliferation by increasing the cyclin D1 expression and G1 phase proportion. Maseki et al. [31] reported that the AKT/GSK3β/Snail signal pathway mediated the proliferation and invasion of head and neck squamous cell carcinoma.The present researches show that activated PI3K/Akt could extricate and up-regulate NF-κB to increase the metastasis ability of tumor cells [32]. At the same time, phosphorylated Akt could activate the MMPs and p21 to enhance the invasion of cancer cells [33]. There also are some researches proved that the activation of PI3K/Akt could promote the phosphorylation of Vimentin who relate to EMT [34]. The results in our study showed that RosA treatment significantly inhibited the expression levels of p-PI3K, p-AKT and p-mTOR without effecting total PI3K, AKT and mTOR proteins, so it could be speculated that the effect of RosA on SMMC-7721 may be mediated by activation of the PI3K/AKT/mTOR signal pathway,including the growth inhibition,G1 arrest and apoptosis promotion effects by regulating Bax protein and cyclin D1, inhibition effect of migration and invasion by regulation MMPs, Vimentin and EMT. To validate this mechanism, IGF-1, activator of PI3K/AKT/mTOR signal pathway, was used for rescue experiments.The results showed that IGF1 could reverse the inhibition of PI3K/AKT/mTOR signal pathway by RosA, as well as reverse the effect of RosA on cell proliferation, apoptosis, invasion and EMT. Moreover, in vivo experiments also showed that RosA inhibited tumor growth and inhibited the expression of related proteins of PI3K/AKT/mTOR signal pathway. Therefore, it is likely that the anti-proliferation and invasion, and pro-apoptotic effects of RosA on the hepatocellular carcinoma cell line SMMC-7221 are at least partially achieved by inhibiting the PI3K/AKT/mTOR signal pathway. In conclusion, RosA inhibits the proliferation and invasion and induce apoptosis of hepatocellular carcinoma cell line SMMC-7721, which may be related to the inhibition of PI3K/AKT/mTOR signal pathway. Additionally, RosA inhibits the growth of SMMC-7721 cells in vivo, which may provide a new theoretical basis for the application of RosA anticancer effect. Convention on biodiversity All animal experiments and cell experiments were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All researches were under the framework of the United Nations Convention on Biodiversity.
Fig. 6. IGF-1 reversed the effect of RosA on PI3K/AKT/mTOR signal pathway and SMMC-7721cells in vitro. (A)The expression of PI3K/AKT/mTOR pathway proteins in SMMC-7721 cells detected by WB. (B) The proliferation of SMMC-7721 cells detected by colony formation assay. (C) The cell cycle distribution of SMMC-7721 cells analyzed by FCM. (D) The apoptosis of SMMC-7721 cells analyzed by FCM. (E) The invasive ability of SMMC-7721 cells was detected by transwell assay. Scale bar = 100 μm.*P < 0.05 vs control group; #P < 0.05 vs RosA group.
Declaration of Competing Interest 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.
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Fig. 7. IGF-1 reversed the effect of RosA on PI3K/AKT/mTOR signal pathway and SMMC-7721cells in vivo. (A) Change of tumor volume and comparison of tumor weight at the end of experiments. (B) The apoptosis of tumour tissue analyzed by TUNEL. (C) The expression of PI3K/AKT/mTOR pathway proteins in tumour tissue detected by WB. Scale bar = 50 μm.*P < 0.05 vs control group; #P < 0.05 vs RosA group.
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