AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells

  • Authors:
    • Chingju Lin
    • Shih-Chang Tsai
    • Michael T. Tseng
    • Shu-Fen Peng
    • Sheng-Chu Kuo
    • Meng-Wei Lin
    • Yuan-Man Hsu
    • Miau-Rong Lee
    • Sakae Amagaya
    • Wen-Wen Huang
    • Tian-Shung Wu
    • Jai-Sing Yang
  • View Affiliations

  • Published online on: January 23, 2013     https://doi.org/10.3892/ijo.2013.1791
  • Pages: 993-1000
Metrics: HTML 0 views | PDF 0 views     Cited By (CrossRef): 0 citations

Abstract

Baicalin is one of the major compounds in the traditional Chinese medicinal herb from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin inhibited cell survival as shown by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. Baicalin did not induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay. Baicalin induced the acidic vesicular organelle cell autophagy marker, manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by 3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore, protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT were downregulated in T24 cells after baicalin treatment. In conclusion, baicalin triggered cell autophagy through the AKT signaling pathway in T24 cells.

Introduction

The morphological processes leading to cell death include apoptosis, necrosis and autophagy (13). Autophagy (or called self-eating) is a process maintaining cellular homeostasis (4,5). When the cells undergo cellular damage, autophagy is required for the promotion of cellular survival (4,6). Autophagy involves the autophagosome formation (a double-membrane structure), which fuses with a lysosome to form an autophagolysosome, finally resulting in degradation of the captured proteins or organelles by lysosomal enzymes (7,8). Several reports have shown that autophagy-related (Atg) proteins and microtubule-associated protein 1 light chain 3 (LC3) are major proteins involved in autophagy processes (911). Induction of Atg and LC3 protein levels has been linked with altering a variety of cellular signaling pathways, such as adenosine monophosphate-activated protein kinase (AMPK) (1214), mitogen-activated protein kinase (MAPK) (15,16) and PI3K/Akt pathways (17,18). Previous studies indicated that suppression of PI3K/Akt is involved in regulating autophagy formation (1921).

Chinese herbs are used for treatment of diseases in Taiwan and in China for a long time (22,23). Baicalin is one of the major flavonoids (molecular formula: C21H18O11; Fig. 1) in the traditional Chinese medicinal herb ‘Huang qin’ (Scutellaria baicalensis Georgi) (24,25). The baicalin exhibits many different pharmacological actions such as anti-oxidant (26), photo-protective (27), neural protective (28,29), anti-depressant (30), anti-inflammatory (31,32), anti-viral (33,34), anti-hepatotoxicity (35,36) and anticancer effects (3739). Baicalin induces CA46 Burkitt lymphoma cell apoptosis through inhibiting the PI3K/Akt kinase activity (40). Baicalin induces apoptosis in SW620 colorectal cancer cells in vitro and anticancer activity in HCT-116 cells in vivo(41,42), and Zheng et al demonstrated that baicalin induces apoptosis in leukemia HL-60/ADR cells through inhibiting the PI3K/Akt kinase (43). Our previous study demonstrated that baicalin induced apoptosis in leukemia HL-60 cells through ER stress and mitochondrial-dependent pathways (44). Recently, Zhang et al pointed out that baicalin induces autophagy in human hepatocellular carcinoma SMMC-7721 cells (45); however, there is no evidence to show the effects of baicalin on the induction of autophagy in human bladder cancer T24 cells. In the present study, we investigated the pharmacological effects of the baicalin on inhibition of cell growth and induction of cell autophagy in T24 cells. Our results indicated that baicalin might contribute to cell autophagy via the Akt pathway in T24 cells.

Materials and methods

Chemicals and reagents

Acridine orange (AO), Baicalin, 3- methyladenine (3-MA), monodansylcadaverine (MDC) and tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin and trypsin-EDTA were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). AKT kinase assay kit was obtained from Cell Signaling Technology (Danvers, MA, USA). Tdt-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay kit was purchased from Roche Diagnostics (GmBH, Mannheim, Germany). Caspase-3 activity assay kit was purchased from R&D Systems Inc. (Minneapolis, MN, USA). The primary antibodies against Atg 5, Atg 7 and Atg 12, Beclin, LC3-II, AKT and phospho-AKT (Ser473) were purchased from Cell Signaling Technology. Antibody against β-actin was obtained from Sigma Chemical Co. All peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The enhanced chemiluminescence (ECL) detection kit was obtained from Pierce Chemical (Rockford, IL, USA).

Cell culture

The T24 human bladder cancer cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were grown in McCoy’s 5a medium fortified with 10% FBS, 2 mM L-glutamine and penicillin/streptomycin and incubated at 37°C under a humidified 5% CO2 atmosphere (46).

Cell viability and morphology

The cell viability was assessed by the MTT assay. Briefly, the T24 cells were cultured in a 96-well plate at the density of 1×104 cells/per well and were incubated with 0, 50, 100, 150 and 200 μM of baicalin for 24 h. For the study of autophagy inhibition, cells were pre-treated with 3-MA (10 mM) for 1 h before the 24-h treatment of baicalin with indicated concentrations. At the end of baicalin treatment, culture medium containing MTT (0.5 mg/ml) was added to each well after washing the cells. The cells were then incubated at 37°C for 4 h and the supernatant was removed. The formed blue formazan crystals in viable T24 cells were dissolved with isopropanol/0.04 N HCl, followed by measurement of the absorbance of each well at 570 nm with the ELISA reader with a reference wavelength of 620 nm. All experiments were performed in triplicate. The cell viability of each treatment was expressed as percentage of the control. The morphological examination of autophagic vacuoles in baicalin-treated cells was determined under a phase-contrast microscope (18).

Trypan blue exclusion assay for cell death

Trypan blue exclusion assay was used to evaluate cell death induced by baicalin treatment. T24 cells in a 24-well plate (2.5×105 cells/per well) were incubated with 0, 50, 100, 150 and 200 μM of baicalin. After 24 h, cells were stained with 0.25% trypan blue solution and the numbers of dead cells were determined by Countess Automated Cell Counter (Invitrogen/Life Technologies) (18).

TUNEL staining

TUNEL staining was performed to detect apoptotic cells according to the manufacturer’s protocol (in situ cell death detection kit; Roche Diagnostics). T24 cells in a 24-well plate (2.5×105 cells/per well) were exposed to 0, 50, 100, 150 and 200 μM of baicalin for 24 h. At the end of the incubation, cells were collected, fixed with 70% ethanol and washed twice with ice-cold PSB. After incubated in the dark for 30 min at 37°C in 100 μl of TdT-containing solution, the T24 samples were washed once before flow cytometry analysis of the TUNEL-positive cells using a FACSCalibur (Becton-Dickinson). The median fluorescence intensity was quantified by CellQuest software (18).

Caspase-3 activity assays

The caspase-3 activity assay was performed according to the manufacturer’s instructions (Caspase Colorimetric Kit; R&D Systems Inc.). Briefly, after a 24-h incubation with 0, 50, 100, 150 and 200 μM of baicalin, T24 cells (∼1×107/75-T flask) were harvested. The collected cells were then lysed in the lysis buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT], followed by centrifugation to collect total proteins in the supernatant. The cell lysate containing 50 μg proteins were then incubated for 1 h at 37°C with caspase-3 specific substrate (Ac-DEVD-pNA) in the reaction buffer. The caspase-3 activity was determined by measuring OD405 of the released pNA (18).

Detection of acidic vesicular organelles (AVO) with acridine orange (AO) and acidic autophagic vacuoles with monodan-sylcadaverine (MDC)

T24 cells were seeded on sterile coverslips in tissue culture plates with a density of 5×104 cells/per coverslip. After 0 or 200 μM of baicalin treatment for 24 h, cells were stained with either acridine orange (AO) or 0.1 mM monodansylcadaverine (MDC) at 37°C for 10 min. After three washes with PBS, cells were immediately visualized by fluorescence microscopy (Nikon, Melville, NY, USA) for the detection of acidic vesicular organelles and MDC-positive autophagic vacuoles (18,47).

Western blot analysis

T24 cells (1×107/75-T flask) were treated with 0, 50, 100, 150 and 200 μM of baicalin for 24 h, then harvested, lysed and the total proteins were collected by SDS sample buffer. In brief, ∼30 μg of protein from each treatment was resolved on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a nitro-cellulose membrane. The transferred membranes were blocked in 5% non-fat dry milk in 20 mM Tris-buffered saline/0.05% Tween-20 for 1 h at room temperature followed by incubation with primary antibodies against indicated autophagic-associated proteins or AKT and autophagy pathway-related proteins at 4°C overnight. At the end of incubation, membranes were washed with Tris-buffered saline/Tween-20 and incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). The blots were developed by a chemiluminescence kit (Millipore, Bedford, MA, USA), followed by X-ray film exposure. Each membrane was stripped and reprobed with anti-β-actin antibody to ensure equal protein loading during the experiment (18).

In vitro AKT kinase assay

In brief, T24 cells (1×107/75-T flask) were treated with 0, 50, 100, 150 and 200 μM of baicalin for 6 h. Cells were lysed in ice-cold lysis buffer provided by the kit. The 200 μg of protein from each time-point of treatment was immuno-precipitated with 2 μg of anti-AKT antibody overnight. Immuno-precipitates were extensively washed and then incubated with 1 μg of GSK-3 α/β fusion protein substrate in 50 μl of kinase buffer for 30 min at 30°C. Reactions were stopped by SDS loading buffer and samples were separated on 12% SDS-PAGE. The phospho-GSK-3 α/β (Ser219) was detected by immunoblotting (18).

Statistical analysis

All the statistical results are presented as the mean ± SEM for the indicated numbers of separate experiments. Statistical analyses of data were done using one-way ANOVA followed by Student’s t-test and *P<0.05, **P<0.01, ***P<0.001 were considered statistically significant (18).

Results

Baicalin decreased the viability of T24 human bladder cancer cells

The human bladder cancer cells T24 were treated with baicalin (0, 50, 100, 150 and 200 μM) for 24 h. Results from the MTT assay showed that even though 50 μM of baicalin did not reduce cell viability, increased concentrations of baicalin treatment (100, 150 and 200 μM) significantly led to decrease of cell viability in T24 cells in a concentration-dependent manner (Fig. 2A). Fig. 2B showed that baicalin increased the number of cell death at 100, 150 and 200 μM in a concentration-dependent manner using the trypan blue exclusion assay.

Baicalin induces caspase-independent cell death in T24 cells

To verify whether baicalin induced apoptosis in T24 cells, cells were treated with baicalin (0, 50, 100, 150 and 200 μM) for 24 h before subjected to TUNEL staining. Fig. 3A indicated that the percentages of TUNEL-positive cells in baicalin-treated groups were <5%. In addition, to further examine whether the cell death caused by baicalin treatment was mediated through caspase-3 activation, protein samples collected from T24 cells after baicalin (0, 50, 100, 150 and 200 μM) treatment were analyzed for caspase-3 activity. The caspase-3 activity assay showed no changes in baicalin-treated cells regardless of the baicalin concentrations (Fig. 3B). Our results demonstrated that apoptosis and the activation of caspase-3 were not involved in baicalin-induced cell death.

Baicalin induces cell autophagy in T24 cells

We further investigated whether the cell death caused by baicalin treatment was mediated by autophagy. T24 cells were treated with baicalin (0, 50, 100, 150 and 200 μM) for 24 h and the formation of autophagic vacuoles was examined under a phase contrast microscope. As shown in Fig. 4A, 100 and 200 μM of baicalin treatment induced the formation of autophagic vacuoles, while baicalin at control and 50 μM induced hardly any formation of autophagic vacuoles. In addition, the amount of autophagic vacuole formation was significantly elevated in a concentration-dependent manner in higher baicalin concentration groups (≥100 μM) (Fig. 4B). Especially, upon the challenge of 200 μM baicalin for 24 h, ∼40% of cells manifested autophagic vacuoles.

One of the hallmarks of autophagic cell death is the cytosolic acidic vesicular organells (AVO) (48). Through the staining of acridine orange (AO), a lysotropic dye that emits bright red fluorescence inside the low pH acidic vesicles, AVO were noticeably observed in the cytoplasm of baicalin-treated T24 cells (200 μM of baicalin) when compared to the control group by the fluorescence microscopy. In addition, as the concentration of baicalin increased, the measured AO intensity became stronger (Fig. 5A). Furthermore, we confirmed the autophagic cell death caused by baicalin treatment using monodansylcadaverine (MDC) staining. MDC is another widely used fluorescent marker that preferentially accumulates in autophagic vacuoles. As shown in Fig. 5B, T24 cells treated 200 μM of baicalin for 24 h clearly showed autophagic vacuoles, while very few autophagic vacuoles were observed in the control group. Again, the MDC intensity increased as the baicalin concentration increased. Our results indicated that autophagy was the mechanism underlying baicalin-induced cell death.

Baicalin regulates the autophagy-associated protein levels in T24 cells

It has been shown that the autophagic cell death is associated with the elevations of autophagosome formation protein levels. Those proteins includes light-chain-3 (LC-3), Atg complex (Atg 5, Atg 7 and Atg 12) and Beclin-1 (18,49). As examined by western blot analysis, baicalin increased the protein expression of Atg 5, Atg 7 and Atg 12, Beclin-1 and LC-3 II (Fig. 6A). For example, when compared with the control group, the respective protein levels of Atg 5, Atg 7 and Atg 12, Beclin-1 and LC-3 II were 2.1-, 2.5-, 2.5-, 1.8- and 2.8-fold higher after treated with 200 μM of baicalin for 24 h.

Among the afore-mentioned proteins, the microtubule-associated protein light-chain 3 (LC-3) is a reliable autophagic membrane marker for the detection of early autophagosome formation (50,51). The conversion of LC-3I to LC-3II is indicative of autophagic activity. We next examined whether 3-methyladenine (3-MA), a commonly used reagent that inhibits autophagy by blocking autophagosome formation via the inhibition of type III phosphatidylinositol 3-kinases (PI-3K), could attenuate the elevated LC-3 II expression induced by baicalin. As shown in Fig. 6B, 200 μM of baicalin treatment upregulated the LC-3 II protein levels to 2.8-fold when compared to the control group. Nevertheless, 3-MA pretreatment (10 mM) decreased the LC-3II expression level to 1.8-fold in the presence of 200 μM of baicalin. The quantitative data from the numbers of autophagic vacuoles also indicated that the baicalin-induced autophagic vacuoles formation was sharply diminished upon 3-MA pretreatment (Fig. 6C). The experimental results (Fig. 6) indicated that baicalin induced autophagic cell death through upregulation of proteins associated with autophagosome formation and it could be attenuated by 3-MA.

Baicalin blocks the AKT signaling in T24 cells

The AKT activity has been demonstrated to contribute to autophagic cell death (6,40). We next performed western blot analysis and AKT kinase activity assay to investigate whether the AKT signaling was involved in the baicalin-induced autophagic cell death in T24 cells. The present study showed that baicalin decreased the phosphor-AKT (Ser473) protein levels in T24 cells in a concentration-dependent manner (Fig. 7A). In addition, baicalin inhibited AKT kinase activity and the inhibition was concentration-dependent (Fig. 7B). Our data implied that baicalin induced cell autophagy in T24 cells through blocking the AKT signaling.

Discussion

Previous studies have showed that Scutellaria baicalensis Gerogi containing over 30 different kinds of flavonoids (24,52,53), including baicalin, baicalein, oroxylin A and wogonin (5355). It was reported that ethanol extracts of Scutellaria baicalensis Gerogi prevent oxidative damage (56,57) and has anti-inflammation (58,59) and anti-angiogenesis effects (60). In addition, Scutellaria baicalensis Gerogi extract triggers G2/M arrest and caspase-dependent apoptosis by modulating ERK pathway in HSC-T6 cells (58). In the present study, we focused on the baicalin from Scutellaria baicalensis Gerogi for their anticancer effect on human bladder cancer T24 cells. Baicalin is a natural flavonoids compound with anticancer activity and low toxicity against normal cells (61,62). Previous reports showed that baicalin exerted anti-proliferative ability and induced apoptotic effects in many cancer cell lines (CA46, SW620, HCT-116, HL-60/ADR and HL-60) (40,41,43,44,63,64). In this study, we investigated the anticancer effects of baicalin on T24 human bladder cancer cells in vitro. Our results showed that baicalin exerted a significant anti-proliferative effect on T24 cells (Fig. 2A). Baicalin is a new anticancer agent and has apoptotic effect on T24 cells. However, the apoptotic TUNEL-positive cells and caspase-3 activity did not change in baicalin-treated T24 cells (Fig. 3). Data suggested that there may be another mechanism involved in baicalin-induced cell death in T24 cells.

Many studies have suggested the autophagy has a cancer suppressor role (65). Several traditional Chinese medicines such as arsenic trioxide (AS2O3) (66), berberine (67), bufalin (18) and kaempferol (68) have been demonstrated to induce autophagy and to exert anticancer activity in cancer cells. Intriguingly, baicalin-induced autophagy in T24 cells was demonstrated by autophagic vesicle formation (Fig. 4). Baicalin induced autophagy generation shown by larger bright-red AO-stained vacuoles (Fig. 5A) and induction of the LC3 cleavage (Fig. 6A). In contrast, protein levels of the LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in T24 cells after baicalin treatment (Fig. 6A). When T24 cells were pre-treated with 3-MA followed by treatment with baicalin, LC3 protein cleavage (Fig. 6B) and autophagic vesicle formation (Fig. 6C) were significantly decreased compared with the baicalin alone treatment group. Our results demonstrated that baicalin-induced cell death possibly involved autophagy, and is the first detailed evidence that baicalin induced autophagy in T24 cells. Our findings are in agreement with previous studies that baicalin induced autophagy in SMMC-7721 cells (45).

Akt serine/threonine kinase [also called protein kinase B (PKB)] is one of the most regularly activated protein kinases in human bladder cancer (6971). Activation of Akt is associated with anti-apoptosis, cell proliferation and cellular energy metabolism (72). The AKT pathway is frequently activated in human bladder cancer cells. Askham et al demonstrated that the AKT1 G49A (E17K) mutation led to constitutive AKT1 activation and was found in 4.8% bladder cancer cell lines and 2.7% bladder tumors (73). Regulating the Akt pathway is potentially essential for developing therapeutic inhibitors in human bladder cancer. Dickstein et al demonstrated that the AKT inhibitor AZ7328 has synergistic effect on inducing apoptosis with autophagy inhibitors in human bladder cancer cells (74). Wu et al demonstrated that PI-3 kinase inhibitor LY294002 inhibits cell proliferation and sensitizes doxorubicin in human bladder cancer cells (75). Our study demonstrated baicalin induced autophagy accompanied with downregulation of phospho-AKT (Ser473) protein level (Fig. 7A) and Akt kinase activity (Fig. 7B). Previous studies demonstrated that baicalin induced apoptotic cell death through inhibiting the AKT signaling pathway in CA46 Burkitt lymphoma and leukemia HL-60/ADR cells (40,76). In the present study, the result showed that the AKT pathway is associated with the induction of autophagy in baicalin-treated T24 cells.

The molecular mechanisms underlying the inhibitory effect of baicalin on T24 cell proliferation are summarized in Fig. 8. In conclusion, baicalin induces autophagy through the Akt signaling pathway in T24 human bladder cancer cells. Our findings imply that baicalin may be used as a novel anticancer drug candidate for the treatment of human bladder cancer.

Acknowledgements

We thank the grant-in-aid DOH101-TD-C-111-005 from Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence. This study was supported by the grant from the National Science Council, Republic of China (Taiwan). This study was also supported in part by grant from China Medical University (CMU101-S-27) awarded to J.-S. Yang.

References

1 

Golstein P and Kroemer G: Cell death by necrosis: towards a molecular definition. Trends Biochem Sci. 32:37–43. 2007. View Article : Google Scholar : PubMed/NCBI

2 

Bustamante-Marin X, Quiroga C, Lavandero S, Reyes JG and Moreno RD: Apoptosis, necrosis and autophagy are influenced by metabolic energy sources in cultured rat spermatocytes. Apoptosis. 17:539–550. 2012.PubMed/NCBI

3 

Edinger AL and Thompson CB: Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol. 16:663–669. 2004. View Article : Google Scholar : PubMed/NCBI

4 

Mah LY and Ryan KM: Autophagy and cancer. Cold Spring Harb Perspect Biol. 4:a0088212012.PubMed/NCBI

5 

Lockshin RA and Zakeri Z: Cell death in health and disease. J Cell Mol Med. 11:1214–1224. 2007. View Article : Google Scholar

6 

Glick D, Barth S and Macleod KF: Autophagy: cellular and molecular mechanisms. J Pathol. 221:3–12. 2010. View Article : Google Scholar : PubMed/NCBI

7 

Kaminskyy V and Zhivotovsky B: Proteases in autophagy. Biochim Biophys Acta. 1824:44–50. 2012. View Article : Google Scholar : PubMed/NCBI

8 

Chen Y and Yu L: Autophagic lysosome reformation. Exp Cell Res. 19:142–146. 2013. View Article : Google Scholar

9 

Mizushima N: The role of the Atg1/ULK1 complex in autophagy regulation. Curr Opin Cell Biol. 22:132–139. 2010. View Article : Google Scholar : PubMed/NCBI

10 

Reggiori F: 1. Membrane origin for autophagy. Curr Top Dev Biol. 74:1–30. 2006. View Article : Google Scholar : PubMed/NCBI

11 

Tanida I, Ueno T and Kominami E: LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 36:2503–2518. 2004. View Article : Google Scholar : PubMed/NCBI

12 

Yang WL, Perillo W, Liou D, Marambaud P and Wang P: AMPK inhibitor compound C suppresses cell proliferation by induction of apoptosis and autophagy in human colorectal cancer cells. J Surg Oncol. 106:680–688. 2012. View Article : Google Scholar : PubMed/NCBI

13 

Ge W, Guo R and Ren J: AMP-dependent kinase and autophagic flux are involved in aldehyde dehydrogenase-2-induced protection against cardiac toxicity of ethanol. Free Radic Biol Med. 51:1736–1748. 2011. View Article : Google Scholar

14 

Wu Y, Li X, Zhu JX, et al: Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson’s disease. Neurosignals. 19:163–174. 2011.PubMed/NCBI

15 

Li ZY, Yang Y, Ming M and Liu B: Mitochondrial ROS generation for regulation of autophagic pathways in cancer. Biochem Biophys Res Commun. 414:5–8. 2011. View Article : Google Scholar : PubMed/NCBI

16 

Dagda RK, Zhu J, Kulich SM and Chu CT: Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson’s disease. Autophagy. 4:770–782. 2008.PubMed/NCBI

17 

Nishiyama Y, Shimada Y, Yokoi T, et al: Akt inactivation induces endoplasmic reticulum stress-independent autophagy in fibroblasts from patients with Pompe disease. Mol Genet Metab. 107:490–495. 2012. View Article : Google Scholar

18 

Tsai SC, Yang JS, Peng SF, et al: Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells. Int J Oncol. 41:1431–1442. 2012.

19 

Chen J, Crawford R and Xiao Y: Vertical inhibition of the PI3K/Akt/mTOR pathway for the treatment of osteoarthritis. J Cell Biochem. Aug 28–2012.(Epub ahead of print). View Article : Google Scholar

20 

Zeng T, Zhang CL, Song FY, et al: PI3K/Akt pathway activation was involved in acute ethanol-induced fatty liver in mice. Toxicology. 296:56–66. 2012. View Article : Google Scholar : PubMed/NCBI

21 

Martelli AM, Evangelisti C, Follo MY, et al: Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells. Curr Med Chem. 18:2715–2726. 2011. View Article : Google Scholar : PubMed/NCBI

22 

Hugel HM, Jackson N, May BH and Xue CC: Chinese herbs for dementia diseases. Mini Rev Med Chem. 12:371–379. 2012. View Article : Google Scholar : PubMed/NCBI

23 

Chen S, Wu T, Kong X and Yuan H: Chinese medicinal herbs for measles. Cochrane Database Syst Rev. Nov 9–2011.CD005531 View Article : Google Scholar

24 

Yuan Y, Shuai L, Chen S, Huang L, Qin S and Yang Z: Flavonoids and antioxidative enzymes in temperature-challenged roots of Scutellaria baicalensis Georgi. Z Naturforsch C. 67:77–85. 2012. View Article : Google Scholar : PubMed/NCBI

25 

Ma AT, Zhong XH, Liu ZM, et al: Protective effects of baicalin against bromocriptine induced abortion in mice. Am J Chin Med. 37:85–95. 2009. View Article : Google Scholar : PubMed/NCBI

26 

Waisundara VY, Siu SY, Hsu A, Huang D and Tan BK: Baicalin upregulates the genetic expression of antioxidant enzymes in Type-2 diabetic Goto-Kakizaki rats. Life Sci. 88:1016–1025. 2011. View Article : Google Scholar : PubMed/NCBI

27 

Bing-Rong Z, Song-Liang J, Xiao EC, et al: Protective effect of the Baicalin against DNA damage induced by ultraviolet B irradiation to mouse epidermis. Photodermatol Photoimmunol Photomed. 24:175–182. 2008. View Article : Google Scholar : PubMed/NCBI

28 

Li HY, Hu J, Zhao S, et al: Comparative study of the effect of baicalin and its natural analogs on neurons with oxygen and glucose deprivation involving innate immune reaction of TLR2/TNFalpha. J Biomed Biotechnol. Mar 21–2012.(Epub). 267890 View Article : Google Scholar

29 

Cao Y, Mao X, Sun C, et al: Baicalin attenuates global cerebral ischemia/reperfusion injury in gerbils via anti-oxidative and anti-apoptotic pathways. Brain Res Bull. 85:396–402. 2011. View Article : Google Scholar : PubMed/NCBI

30 

de Carvalho RS, Duarte FS and de Lima TC: Involvement of GABAergic non-benzodiazepine sites in the anxiolytic-like and sedative effects of the flavonoid baicalein in mice. Behav Brain Res. 221:75–82. 2011.PubMed/NCBI

31 

Zhu J, Wang J, Sheng Y, et al: Baicalin improves survival in a murine model of polymicrobial sepsis via suppressing inflammatory response and lymphocyte apoptosis. PLoS One. 7:e355232012. View Article : Google Scholar : PubMed/NCBI

32 

Fu S, Sun C, Tao X and Ren Y: Anti-inflammatory effects of active constituents extracted from Chinese medicinal herbs against Propionibacterium acnes. Nat Prod Res. 26:1746–1749. 2012. View Article : Google Scholar

33 

Chu ZY, Chu M and Teng Y: Effect of baicalin on in vivo anti-virus. Zhongguo Zhong Yao Za Zhi. 32:2413–2415. 2007.(In Chinese).

34 

Kitamura K, Honda M, Yoshizaki H, et al: Baicalin, an inhibitor of HIV-1 production in vitro. Antiviral Res. 37:131–140. 1998. View Article : Google Scholar : PubMed/NCBI

35 

Qiao H, Han H, Hong D, Ren Z, Chen Y and Zhou C: Protective effects of baicalin on carbon tetrachloride induced liver injury by activating PPARgamma and inhibiting TGFbeta1. Pharm Biol. 49:38–45. 2011. View Article : Google Scholar : PubMed/NCBI

36 

Hwang JM, Wang CJ, Chou FP, et al: Protective effect of baicalin on tert-butyl hydroperoxide-induced rat hepatotoxicity. Arch Toxicol. 79:102–109. 2005. View Article : Google Scholar : PubMed/NCBI

37 

Chiu YW, Lin TH, Huang WS, et al: Baicalein inhibits the migration and invasive properties of human hepatoma cells. Toxicol Appl Pharmacol. 255:316–326. 2011. View Article : Google Scholar : PubMed/NCBI

38 

Shieh DE, Cheng HY, Yen MH, Chiang LC and Lin CC: Baicalin-induced apoptosis is mediated by Bcl-2-dependent, but not p53-dependent, pathway in human leukemia cell lines. Am J Chin Med. 34:245–261. 2006. View Article : Google Scholar : PubMed/NCBI

39 

Motoo Y and Sawabu N: Antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines. Cancer Lett. 86:91–95. 1994. View Article : Google Scholar : PubMed/NCBI

40 

Huang Y, Hu J, Zheng J, et al: Down-regulation of the PI3K/Akt signaling pathway and induction of apoptosis in CA46 Burkitt lymphoma cells by baicalin. J Exp Clin Cancer Res. 31:482012. View Article : Google Scholar : PubMed/NCBI

41 

Chen WC, Kuo TH, Tzeng YS and Tsai YC: Baicalin induces apoptosis in SW620 human colorectal carcinoma cells in vitro and suppresses tumor growth in vivo. Molecules. 17:3844–3857. 2012. View Article : Google Scholar : PubMed/NCBI

42 

Lee DH, Kim C, Zhang L and Lee YJ: Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells. Biochem Pharmacol. 75:2020–2033. 2008. View Article : Google Scholar : PubMed/NCBI

43 

Zheng J, Hu JD, Chen YY, et al: Baicalin induces apoptosis in leukemia HL-60/ADR cells via possible down-regulation of the PI3K/Akt signaling pathway. Asian Pac J Cancer Prev. 13:1119–1124. 2012. View Article : Google Scholar : PubMed/NCBI

44 

Lu HF, Hsueh SC, Ho YT, et al: ROS mediates baicalin-induced apoptosis in human promyelocytic leukemia HL-60 cells through the expression of the Gadd153 and mitochondrial-dependent pathway. Anticancer Res. 27:117–125. 2007.

45 

Zhang X, Tang X, Liu H, Li L, Hou Q and Gao J: Autophagy induced by baicalin involves downregulation of CD147 in SMMC-7721 cells in vitro. Oncol Rep. 27:1128–1134. 2012.PubMed/NCBI

46 

Huang WW, Yang JS, Pai SJ, et al: Bufalin induces G(0)/G(1) phase arrest through inhibiting the levels of cyclin D, cyclin E, CDK2 and CDK4, and triggers apoptosis via mitochondrial signaling pathway in T24 human bladder cancer cells. Mutat Res. 732:26–33. 2012. View Article : Google Scholar

47 

Kim JY, Cho TJ, Woo BH, et al: Curcumin-induced autophagy contributes to the decreased survival of oral cancer cells. Arch Oral Biol. 57:1018–1025. 2012. View Article : Google Scholar : PubMed/NCBI

48 

Munafo DB and Colombo MI: A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci. 114:3619–3629. 2001.PubMed/NCBI

49 

McCoy F, Hurwitz J, McTavish N, et al: Obatoclax induces Atg7-dependent autophagy independent of beclin-1 and BAX/BAK. Cell Death Dis. 1:e1082010. View Article : Google Scholar : PubMed/NCBI

50 

Barth S, Glick D and Macleod KF: Autophagy: assays and artifacts. J Pathol. 221:117–124. 2010. View Article : Google Scholar : PubMed/NCBI

51 

Mizushima N and Yoshimori T: How to interpret LC3 immuno-blotting. Autophagy. 3:542–545. 2007. View Article : Google Scholar : PubMed/NCBI

52 

Yu K, Gong Y, Lin Z and Cheng Y: Quantitative analysis and chromatographic fingerprinting for the quality evaluation of Scutellaria baicalensis Georgi using capillary electrophoresis. J Pharm Biomed Anal. 43:540–548. 2007. View Article : Google Scholar

53 

Zhou XQ, Liang H, Lu XH, Cai SQ, Wang B and Zhao YY: Flavonoids from Scutellaria baicalensis and their bioactivities. Beijing Da Xue Xue Bao. 41:578–584. 2009.(In Chinese).

54 

Liu B, Shi RB and Zhu LJ: HPLC fingerprint of flavonoids of Kushen Tang and its correlation to Scutellaria baicalensis and Sophora flavescens. Zhongguo Zhong Yao Za Zhi. 32:1631–1634. 2007.(In Chinese).

55 

Kim YH, Jeong DW, Kim YC, Sohn DH, Park ES and Lee HS: Pharmacokinetics of baicalein, baicalin and wogonin after oral administration of a standardized extract of Scutellaria baicalensis, PF-2405 in rats. Arch Pharm Res. 30:260–265. 2007. View Article : Google Scholar : PubMed/NCBI

56 

Hirunuma M, Shoyama Y, Sasaki K, et al: Flavone-catalyzed apoptosis in Scutellaria baicalensis. Phytochemistry. 72:752–760. 2011. View Article : Google Scholar : PubMed/NCBI

57 

Choi J, Conrad CC, Malakowsky CA, Talent JM, Yuan CS and Gracy RW: Flavones from Scutellaria baicalensis Georgi attenuate apoptosis and protein oxidation in neuronal cell lines. Biochim Biophys Acta. 1571:201–210. 2002. View Article : Google Scholar : PubMed/NCBI

58 

Pan TL, Wang PW, Leu YL, Wu TH and Wu TS: Inhibitory effects of Scutellaria baicalensis extract on hepatic stellate cells through inducing G2/M cell cycle arrest and activating ERK-dependent apoptosis via Bax and caspase pathway. J Ethnopharmacol. 139:829–837. 2012. View Article : Google Scholar

59 

Li HB, Jiang Y and Chen F: Separation methods used for Scutellaria baicalensis active components. J Chromatogr B Analyt Technol Biomed Life Sci. 812:277–290. 2004. View Article : Google Scholar : PubMed/NCBI

60 

Wang S, Zheng Z, Weng Y, et al: Angiogenesis and anti-angiogenesis activity of Chinese medicinal herbal extracts. Life Sci. 74:2467–2478. 2004. View Article : Google Scholar : PubMed/NCBI

61 

Zhu H, Wang Z, Xing Y, et al: Baicalin reduces the permeability of the blood-brain barrier during hypoxia in vitro by increasing the expression of tight junction proteins in brain microvascular endothelial cells. J Ethnopharmacol. 141:714–720. 2012. View Article : Google Scholar

62 

Hu Q, Noor M, Wong YF, et al: In vitro anti-fibrotic activities of herbal compounds and herbs. Nephrol Dial Transplant. 24:3033–3041. 2009. View Article : Google Scholar : PubMed/NCBI

63 

Yang BL, Chen HJ, Chen YG, et al: Inhibitory effects of baicalin on orthotopic xenografts of colorectal cancer cells that are deficient in a mismatch repair gene in nude mice. Int J Colorectal Dis. Aug 23–2012.(Epub ahead of print).

64 

Ren X, Li CL, Wang HX, et al: Molecular mechanism of HL-60 cell apoptosis induced by baicalin. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 20:847–851. 2012.(In Chinese).

65 

Carew JS, Kelly KR and Nawrocki ST: Autophagy as a target for cancer therapy: new developments. Cancer Manag Res. 4:357–365. 2012.

66 

Goussetis DJ, Altman JK, Glaser H, McNeer JL, Tallman MS and Platanias LC: Autophagy is a critical mechanism for the induction of the antileukemic effects of arsenic trioxide. J Biol Chem. 285:29989–29997. 2010. View Article : Google Scholar : PubMed/NCBI

67 

Wang N, Feng Y, Zhu M, et al: Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: the cellular mechanism. J Cell Biochem. 111:1426–1436. 2010. View Article : Google Scholar : PubMed/NCBI

68 

Filomeni G, Desideri E, Cardaci S, et al: Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment. Autophagy. 6:202–216. 2010. View Article : Google Scholar

69 

Shahjee HM, Koch KR, Guo L, Zhang CO and Keay SK: Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells. J Exp Clin Cancer Res. 29:1602010. View Article : Google Scholar : PubMed/NCBI

70 

Chen M, Cassidy A, Gu J, et al: Genetic variations in PI3K-AKT-mTOR pathway and bladder cancer risk. Carcinogenesis. 30:2047–2052. 2009. View Article : Google Scholar : PubMed/NCBI

71 

Champelovier P, El Atifi M, Mantel F, et al: In vitro tumoral progression of human bladder carcinoma: role for TGFbeta. Eur Urol. 48:846–851. 2005. View Article : Google Scholar : PubMed/NCBI

72 

Stueckle TA, Lu Y, Davis ME, et al: Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells. Toxicol Appl Pharmacol. 261:204–216. 2012. View Article : Google Scholar

73 

Askham JM, Platt F, Chambers PA, Snowden H, Taylor CF and Knowles MA: AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K. Oncogene. 29:150–155. 2010. View Article : Google Scholar : PubMed/NCBI

74 

Dickstein RJ, Nitti G, Dinney CP, Davies BR, Kamat AM and McConkey DJ: Autophagy limits the cytotoxic effects of the AKT inhibitor AZ7328 in human bladder cancer cells. Cancer Biol Ther. 13:1325–1338. 2012. View Article : Google Scholar : PubMed/NCBI

75 

Wu D, Tao J, Xu B, et al: Phosphatidylinositol 3-kinase inhibitor LY294002 suppresses proliferation and sensitizes doxorubicin chemotherapy in bladder cancer cells. Urol Int. 86:346–354. 2011. View Article : Google Scholar

76 

Zheng J, Hu JD, Huang Y and Chen BY: Effects of baicalin on proliferation and apoptosis of adriamycin-resistant human leukemia HL-60/ADR cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 17:1198–1202. 2009.(In Chinese).

Related Articles

Journal Cover

March 2013
Volume 42 Issue 3

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Lin, C., Tsai, S., Tseng, M.T., Peng, S., Kuo, S., Lin, M. ... Yang, J. (2013). AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells. International Journal of Oncology, 42, 993-1000. https://doi.org/10.3892/ijo.2013.1791
MLA
Lin, C., Tsai, S., Tseng, M. T., Peng, S., Kuo, S., Lin, M., Hsu, Y., Lee, M., Amagaya, S., Huang, W., Wu, T., Yang, J."AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells". International Journal of Oncology 42.3 (2013): 993-1000.
Chicago
Lin, C., Tsai, S., Tseng, M. T., Peng, S., Kuo, S., Lin, M., Hsu, Y., Lee, M., Amagaya, S., Huang, W., Wu, T., Yang, J."AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells". International Journal of Oncology 42, no. 3 (2013): 993-1000. https://doi.org/10.3892/ijo.2013.1791