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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 519-525

Baicalein and Ly294002 induces liver cancer cells apoptosis via regulating phosphatidyl inositol 3-kinase/Akt signaling pathway


1 Department of Hepatobiliary Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, P.R. China
2 Department of Obstetrics and Gynecology, The Affiliated TCM Hospital of Southwest Medical University, Luzhou, Sichuan, P.R. China
3 Experimental Medicine Center, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, P.R. China
4 Cancer Research Center of Southwest Medical University, Luzhou, Sichuan, P.R. China

Date of Web Publication29-Jun-2018

Correspondence Address:
Jiyi Xia
Cancer Research Center of Southwest Medical University, Luzhou, Sichuan
P.R. China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.235356

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 > Abstract 

Aim: The aim of this study is to investigate the mechanism of baicalein in inducing human liver cell line SMMC-7721 apoptosis.
Materials and Methods: Twenty micromoles baicalein or 10 μM LY294002 was adopted to treat SMMC-7721 cells. Cell proliferation was tested by cell counting kit-8 assay. Cell cycle was determined by flow cytometry and cyclin D1 expression. Cell apoptosis was detected by annexin V/propidium iodide double staining. Phosphatidyl inositol 3-kinase (PI3K)/Akt signaling pathway was assessed by real-time polymerase chain reaction and Western blot.
Results: Baicalein suppressed liver cancer cell SMMC-7721 proliferation and induced cell apoptosis together with LY294002. Baicalein blocked cell cycle in G0–G1 phase and downregulated cyclin D1 level. Baicalein and LY294002 significantly suppressed PI3K/Akt signaling pathway-related molecule activity at both mRNA and protein levels (P < 0.05).
Conclusion: Baicalein can inhibit liver cancer cell proliferation and promote cell apoptosis by affecting PI3K/Akt signaling pathway together with LY294002.

Keywords: Akt, baicalein, liver cancer, LY294002, phosphatidyl inositol 3-kinase


How to cite this article:
He K, Yu X, Wang X, Tang L, Cao Y, Xia J, Cheng J. Baicalein and Ly294002 induces liver cancer cells apoptosis via regulating phosphatidyl inositol 3-kinase/Akt signaling pathway. J Can Res Ther 2018;14, Suppl S2:519-25

How to cite this URL:
He K, Yu X, Wang X, Tang L, Cao Y, Xia J, Cheng J. Baicalein and Ly294002 induces liver cancer cells apoptosis via regulating phosphatidyl inositol 3-kinase/Akt signaling pathway. J Can Res Ther [serial online] 2018 [cited 2019 Jul 19];14:519-25. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/519/235356


 > Introduction Top


Liver cancer is a common form of malignant tumor, with high incidence and mortality.[1] Although it is a critical threat to patient's health, there is still a lack of effective treatment for liver cancer.[2] Easy to metastasis, recurrence, and resistance to chemotherapy further unsatisfy clinical curative effect and prognosis. Moreover, the treatment options for liver cancer are extremely limited because its mechanisms of pathogenesis are not completely known. Ascertaining liver cancer origin and development thus to identify novel targets for therapy is of great significance.[3],[4],[5] Inhibiting liver cancer cell proliferation and promoting cell apoptosis are the key research directions for liver cancer therapy.[6],[7]

Phosphatidyl inositol 3-kinase (PI3K)/Akt signaling pathway is involved in a variety of biological processes, such as malignant cancer cells proliferation, differentiation, apoptosis, angiogenesis, invasion, and metastasis. Multiple drugs that can induce liver cancer cell apoptosis may interfere PI3K/Akt signaling pathway. PI3K and Akt, also known as protein kinase B (PKB), are the key molecules of the signaling pathway.[8] Numerous growth factor receptors' activation, such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and insulin receptor, can activate PI3K, which elevates 3-phosphoinositide phosphatide content on the cytoplasmic membrane. Akt is partly activated after binding with 3-phosphoinositide phospholipids while PKB kinase activated by 3-phosphoinositide phospholipids causes Akt phosphorylation and fully activation. Akt activation can trigger ribosomal S6 kinase to promote cell proliferation and Bcl-2 expression that inhibits apoptosis.

Flavonoid is a type of widely used Chinese herbal medicine. Epidemiological studies indicate that an increased intake of dietary flavonoids is associated with a decreased risk of some cancers.[9],[10] Baicalein, a bioactive flavonoid derived from the root of Scutellaria baicalensis, has been widely used to treat inflammation, cardiovascular diseases, and infections.[11] Baicalein alone, or together with other agents, can also inhibit cancer cell growth in breast cancer, leukemia, and colon cancer.[12],[13],[14] However, to date, there is still no study conducted with regard to the antiproliferation effect of baicalein in liver cancer. LY294002 is a specific inhibitor of PI3K, which can completely suppress the catalytic activity of the p110 subunit of PI3K.[15] This study intended to investigate baicalein and LY294002 impact on human liver cancer cell line SMMC-7721 apoptosis.


 > Materials and Methods Top


Cell line and reagents

Human liver cancer cell line SMMC-7221 was got from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 20 mmol/L NaHCO3, 20 mmol/L hydroxyethylpiperazine ethane sulfonic acid, 100 U/ml penicillin, and 100 g/ml streptomycin and cultured at 37°C and 5% CO2. Baicalein was purchased from Sigma and dissolved in dimethyl sulfoxide. LY294002 was got from Alexis Pharmaceuticals. TRIzol was bought from Invitrogen. Cell cycle detection kit was from Keygentec. Reverse transcription kit was purchased from Fermentas. Primary antibodies for Bax (No. 5023), Bcl-2 (No. 3498), cyclin D1 (No. 2978), GAPDH (No. 5174), ERK1/2 (No. 4695), p-ERK1/2 (No. 4094), GSK-3β (No. 9315), p-GSK-3β (No. 9322), Akt (No. 4685), and p-Akt (No. 4060) were purchased from Cell Signaling Technology. Horseradish peroxidase-tagged secondary antibody was from Western Instrument Science and Technology Co., Ltd. Cell counting kit-8 (CCK-8) was got from Biotend.

Cell counting kit-8 assay

SMMC-7221 cells in suspension were seeded in a 96-well plate. Different concentrations of baicalein or LY294002 were adopted to treat cells for 4 h with three replicates. Then, 10 μl CCK-8 reagent was added to the well for 4 h to form formazan. At last, the plate was read at 450–490 nm on a microplate reader.

Cell cycle detection

SMMC-7221 cells in logarithmic phase were seeded in a 12-well plate and treated with baicalein or LY294002. After collected and washed by phosphate-buffered saline (PBS) for twice, the cells were added to 1 ml 70% precooled ethanol at 4°C overnight. Then, the cells were washed by PBS and treated with 100 mg/L ribonuclease at 37°C for 30 min. After stained by 50 mg/L propidium iodide (PI) at 4°C avoid of light for 30 min, the cells were detected on flow cytometry with the excitation wavelength at 488 nm. The primary result was analyzed by cell cycle matching software to record hypodiploid peak, namely sub-G1 phase, G0/G1 phase, S phase, and G2/M phase. All the experiments were repeated for three times.

Annexin V/propidium iodide assay

After treated by 20 μM baicalein or 10 μM LY294002 for 24 h, SMMC-7221 cells were collected and washed by PBS for twice. Then, the cells were resuspended in 400 μl 1X binding buffer and added with 5 μl annexin V-FITC avoid of light for 15 min. Then, the cells were added with 10 μl PI and incubated avoid of light for 5 min. The cells were then tested for early and late apoptosis on flow cytometry. The results were analyzed by CellQuest software (Becton-Dickinson, San Jose, CA, USA). All the experiments were repeated for three times.

Real-time polymerase chain reaction

Total RNA was extracted using TRIzol and reverse transcripted to cDNA using K1622 kit (Thermo Fermentas). The primers used were designed by Primer 6.0. Real-time polymerase chain reaction (PCR) was applied to test target gene expression. Reaction condition was 55°C for 1 min, followed by 40 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 45s. GAPDH was used as internal reference. 2−△Ct was applied to calculate relative expression level. The primers sequences were as follows: Akt, forward, 5'-AGCGACGTGGCTATTGTGAAG-3', reverse, 5'-GCCATCATTCTTGAGGAGGAAGT-3'. ERK1/2, forward, 5'-AATCACACGGTAGACACTGAAATGCC-3', reverse, 5'-CATCATCCCATCTAAAATGTCCCCTG-3'. GSK-3β, forward, 5'-GGCAGCATGAAAGTTAGCAGA-3', reverse, 5'-GGCGACCAGTTCTCCTGAATC-3'. Cyclin D1, forward, 5'-GCTGCGAAGTGGAAACCATC-3', reverse, 5'-CCTCCTTCTGCACACATTTGAA-3'. GAPDH, forward, 5'-GCACCGTCAAGGCTGAGAAC-3', reverse, 5'-TGGTGAAGACGCCAGTGGA-3'.

Western blot

SMMC-7221 cells were cracked on ice for 20 min after added with protease inhibitor and lysis to extract protein. After centrifuged at 12,000 rpm, the supernatant was moved to a new eppendorf tube and the protein concentration was determined by bicinchoninic acid. A total of 20 μg protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. After blocked by 5% skimmed milk for 1.5 h, the membrane was incubated in primary antibody for 2 h. Then, the membrane was incubated with secondary antibody for 30 min and washed by PBS with Tween 20. At last, the membrane was developed by diaminobenzidine and detected using the Enhanced chemiluminescence (ECL) reagents. All experiments were repeated for three times.

Statistical analysis

All the data were presented as mean ± standard deviation and analyzed using IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. (Armonk, NY: IBM Corp.). Data comparison was performed using t-test or one-way ANOVA. P <0.05 was considered as statistical difference.


 > Results Top


Baicalein suppressed SMMC-7721 proliferation and induced cell apoptosis together with LY294002

To investigate the inhibitory effect of baicalein and LY294002 on SMMC-7721 cells, different concentrations of baicalein (1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, and 300 μM) or LY294002 (1 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 30 μM) were applied to treat SMMC-7721 cells for 24 h. CCK-8 assay revealed that SMMC-7721 cell proliferation was significantly suppressed by baicalein or LY294002 with dose dependence (P < 0.05) [Figure 1]a. To further investigate the pro-apoptotic effect of baicalein and LY294002 on liver cancer cells, annexin V/PI staining demonstrated that baicalein and LY294002 could increase numbers of cells in early and late phases of apoptosis status through a synergistic effect. Western blot detection showed that pro-apoptotic factor Bax was upregulated and apoptosis suppressor Bcl-2 protein was declined after baicalein or LY294002 treatment. Baicalein together with LY294002 more obviously elevated Bax/Bcl-2 ratio compared with single baicalein treatment [Figure 1]c.
Figure 1: Baicalein and U0126 suppressed SMMC-7721 cell proliferation and induced cell apoptosis by restraining cell cycle. (a) SMMC-7721 cell viability detected by cell counting kit-8. (b) SMMC-7721 cell apoptosis tested by annexin V/propidium iodide double staining. (c) Bax and Bcl-2 protein expression determined by real time polymerase chain reaction. *P < 0.05, compared with control

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Baicalein blocked SMMC-7721 cell cycle

Since PI3K/Akt signaling pathway can regulate cell cycle, we intended to explore baicalein and LY294002 impact on SMMC-7721 cell cycle. Flow cytometry results presented that compared with control, cell content obviously increased in G0–G1 phase and declined in S phase after 20 μM baicalein treatment for 24 h. LY294002 addition markedly reduced cell content in S phase compared with single baicalein group [Figure 2]a. As a specific marker of cell cycle, cyclin D1 mRNA and protein expression in SMMC-7721 cells treated by baicalein was significantly declined [Figure 2]b and [Figure 2]c. LY294002 further downregulated cyclin D1 expression compared with single baicalein treatment.
Figure 2: Baicalein blocked SMMC-7721 cell cycle. (a) SMMC-7721 cell cycle detected by flow cytometry. (b) Cyclin D1 mRNA expression tested by real time polymerase chain reaction. (c) Cyclin D1 protein level determined by Western blot. *P < 0.05, compared with control

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Baicalein and LY294002 affected phosphatidyl inositol 3-kinase/Akt signaling pathway at mRNA level

Since previous research reported that baicalein can mediate PI3K/Akt signaling pathway, we tested baicalein and LY294002 effect on PI3K/Akt signaling pathway in SMMC-7721 at mRNA level. Real-time PCR results showed that both baicalein and LY294002 obviously declined Akt, ERK1/2, and GSK-3β levels in SMMC-7721 cells. Their combination further reduced Akt, ERK1/2, and GSK-3β mRNA levels in SMMC-7721 cells [Figure 3].
Figure 3: Baicalein and LY294002 affected phosphatidyl inositol 3-kinase/Akt signaling pathway at mRNA level. *P < 0.05, compared with control

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Baicalein and LY294002 suppressed phosphatidyl inositol 3-kinase/Akt signaling pathway at protein level

Moreover, we tested PI3K/Akt signaling pathway-related protein expression in SMMC-7721 cells after baicalein treatment. Western blot revealed that ERK1/2 and GSK-3β phosphorylation levels but not Akt phosphorylation were markedly reduced in SMMC-7721 cells treated by baicalein. Baicalein together with LY294002 more obviously suppressed PI3K/Akt signaling pathway-related proteins expression compared with single baicalein treatment [Figure 4].
Figure 4: Baicalein and LY294002 suppressed phosphatidyl inositol 3-kinase/Akt signaling pathway at protein level. *P < 0.05, compared with control

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 > Discussion Top


Liver cancer is one of the most common malignant cancers, which is ranked 3rd in deaths from various malignant cancers.[16] Liver cancer mostly occurs in hepatic disease and liver cirrhosis, leading to poor hepatic function.[17] Therefore, patients often cannot tolerate an operation or endure traditional chemotherapeutics in enough doses. Furthermore, liver cancer is easy to appear resistance to traditional chemotherapeutics.[18] Therefore, identification of natural chemotherapeutics without harmful effects has become one of the strategies to improve curative effect of liver cancer.[19]

Baicalein, a flavonoid found in S. baicalensis Georgi, exhibits various biological effects, including anti-inflammatory and antitumor activity.[20],[21] Although Scutellaria is reported to have almost no toxicity to animals and humans, baicalein so far shows no or very little toxicity to normal myeloid cells and also no impact on the viability of normal human epithelial cells at doses that are toxic to malignant cells.[22],[23] In contrast, baicalein has been shown to inhibit growth of numerous human cancer cell lines.[24],[25] Baicalein also possesses a direct cytotoxicity to a large panel of human malignant cell lines by inducing apoptotic cell death. Our results demonstrated that baicalein has therapeutic potential against liver cancers.

Cell cycle abnormal rapid transformation or checkpoint destruction may cause liver cancer cell growth, leading to hepatocellular carcinoma occurrence and development. Cyclin D1 and cyclin E play a key role in the G1 phase of cell cycle. Their overexpression can accelerate G1 phase transmitting to S phase.[26],[27],[28] P21 and p27 are suppressor genes in regulating cell cycle, which may block G1/S transmission by binding with cyclin-dependent kinase and cell cycle protein complexes to inhibit cell proliferation.[29] Our cell cycle results showed that baicalein blocked SMMC-7721 cell cycle and downregulated cyclin D1 level, indicating that baicalein may restrain liver cancer proliferation through mediating cell cycle.

Treatment with baicalein has been reported to inhibit inflammatory-signaling pathways involving ERK, Akt, and NF-κB activities in cancer cells.[30] PI3K/Akt signaling pathway widely exists in eukaryotic cells, which can transmit the extracellular signal of PDGF, IGF, and EGF to the nucleus. Therefore, it plays a key role in the process of metabolism, cell proliferation, and apoptosis.[5],[8] PI3K activation can elevate 3-phosphoinositide phospholipids content on the cytoplasmic membrane.[10] Akt can be activated by 3-phosphoinositide phospholipids directly or indirectly.[11] Akt activation can further activate ribosomal S6 kinase, promoting protein synthesis and cell proliferation. Akt also can promote Bcl-2 expression and inhibit cell apoptosis, thus blocking mitochondrial cytochrome C release.[12],[13] In our experiment, Bax/Bcl-2 ratio increased after baicalein treatment, indicating that blocking Akt may suppress Bcl-2 expression.

As a target of PI3K/Akt signaling pathway, ERK can regulate some transcription factors' activity through phosphorylation, such as Elkl, EtsI, Sapla, c-Myc, Tal, STATS, which indirectly activates rapid response genes FOS and JUN to promote AP-1 activity. These transcription factors further regulate their respective target genes to affect cell metabolism, function, and proliferation.[29],[31],[32] p-ERK in the nuclear upregulates its downstream factor c-fos expression, thus promotes c-fos and c-jun polymerization to form AP-1. PCNA, a downstream target gene of AP-1, is a cofactor of DNA polymerase. It is involved in DNA synthesis and plays an important role in regulating cell cycle, which can be treated as an import index for cell proliferation activity evaluation.[33] Our data revealed that baicalein can suppress ERK phosphorylation at both mRNA and protein level, and LY294002 further strengthened such effects. It suggested that baicalein can regulate ERK level though mediating PI3K/Akt signaling pathway.

GSK-3β is another target of PI3K/Akt signaling pathway. It also can regulate Wnt/β-catenin signaling pathway by forming degrading complex with adenomatous polyposis coli and Axin. The later can phosphorylate β-catenin and cause it degradation. GSK-3β phosphorylation may promote β-catenin activation, leading to cancer cell proliferation and differentiation.[34],[35],[36] It has been reported that GSK-3β can promote colorectal cancer cell line Colo320 apoptosis under 5-fluorouracil effect. Our results also presented that baicalein can suppress GSK-3β phosphorylation, and such inhibitory effect was enhanced by LY294002 treatment. It further confirmed that baicalein can induce liver cancer cell SMMC-7721 by mediating PI3K/Akt signaling pathway.


 > Conclusion Top


Baicalein can induce SMMC-7721 cell apoptosis through regulating PI3K/Akt signaling pathway. Combination of baicalein and LY294002 further restrained liver cancer cell proliferation and induced cell apoptosis, providing theoretical basis for liver cancer treatment.

Acknowledgement

This work supported by tThe fund of science&Technology Department of Sichuan province (14JC01383-LH53), The fund of science&Technology Department of Sichuan province (14JC01353-LH67), The fund of Science&Technology Department of Luzhou city (2014-S-44) and The mutual fund of Science&Technology Department of Sichuan province (2015sx-0002).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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