Baicalein Induces Apoptosis in SCC-4 Human Tongue Cancer Cells via a Ca2+-dependent Mitochondrial Pathway

Abstract.

Background: The effects of baicalein on SCC-4

human tongue cancer cells were examined to better understand

its effect on apoptosis and associated possible signal pathways

in vitro. Materials and Methods: Apoptosis induction, reactive

oxygen species (ROS), cytoplasmic Ca

, mitochondrial

membrane potential (MMP) and caspase-3 activity were

analyzed using the flow cytometric assay. Apoptosis-associated

proteins, such as p53, BAX, BCL-2, cytochrome c, caspase-3

and -9, EndoG and AIF were determined by Western blotting.

Results: Our results showed that baicalein promoted the levels

of p53, BAX, cytochrome c, capase-3 and -9 and reduced the

level of BCL-2, which were associated with the induction of

apoptotic cell death of SCC-4 cells. A release of cytochrome c

from mitochondria into cytosol was demonstrated and an

activation of caspase-3, which led to the occurrence of

apoptosis in SCC-4 cells treated with baicalein as determined

by Western blot. In order to understand the role of Ca

in the

induction of apoptosis, cells were pre-treated with BAPTA

(intracellular calcium chelator) and baicalein. It was shown

that the MMP was restored, and the level of cytoplasmic Ca

suppressed, the proportion of cells undergoing apoptosis was

also markedly diminished. Our data suggest that cellular Ca

modulates baicalein-induced cell death via a Ca

-dependent

mitochondrial death pathway in SCC-4 cells.

Many studies have demonstrated that flavonoids from natural

plants exhibit a variety of biological activities, such as the

anti-inflammatory, antioxidant, antitumor and antiviral actions (1).

Baicalein (5,6,7-trihydroxyflavone) is a flavonid derived from

the root of Scutellaria baicalensis Georgi, a medicinal plant

traditionally used in Chinese herbal medicine (2). It was

reported that baicalein ameliorated all the considered

inflammatory symptoms in dextran sulfate sodium-induced

colitis in mice in vivo experiments (3). Many experiments also

showed that baicalein is a free-radical scavenger, an

antioxidant (4-7) and exhibits a cytoprotective effects (8-11).

Baicalein was also protective against benzo[·]pyrene and

aflatoxin B1-induced genotoxicity (12). Baicalein has been

reported to be an antiinflammatory agent (13) and an

inhibitor of prostaglandin E2 (14). Recently, it was reported

that baicalein inhibited hydrogen peroxide-induced apoptosis

via ROS-dependent heme oxygenase 1 gene expression (15).

It is well-known that the best strategy for killing cancer

cells is to induce cancer cell apoptosis. Antitumor agents can

trigger apoptosis and then lead to rapid elimination of tumor

cells (16, 17). Interference with the apoptotic process is

considered a crucial part of cancer prevention and therapy

(18). Another important factor is the interactions among

BCL-2 family proteins (pro-apoptotic and anti-apoptotic

proteins) which are also involved in cell death or survival (19,

20). Although many reports have demonstrated that baicalein

induced apoptosis in human cancer cells including breast

cancer (21), hepatoblastoma (22), prostate cancer (23) and

gastric cancer cells (24), there is no available information to

address the mechanism of baicalein-induced apoptosis in

SCC-4 human tongue cancer cells. The aim of the present

study was to investigate the effects and the role of Ca

and

caspase-3 in the induction of apoptosis by baicalein in SCC-4

human tongue cancer cells.

Correspondence to: Jing-Gung Chung, Ph.D., Department of

Microbiology, School of Medicine, China Medical University, No 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C. Tel: +88 642205 3366, Fax: +88 642205 3764, e-mail: jgchung@mail.cmu.edu.tw

Key Words: Baicalein, reactive oxygen species, cytochrome c,

cytoplasmic Ca2+_{, caspase-3, mitochondrial death pathway,} apoptosis, SCC-4 cells.

Baicalein Induces Apoptosis in SCC-4 Human Tongue Cancer

Cells via a Ca

2+

_{-dependent Mitochondrial Pathway}

YUH-TZY LIN

, JAI-SING YANG

, HUI-JU LIN

, TZU-WEI TAN

, NOU-YING TANG

,

JO-HUA CHAING

, YUNG-HSIEN CHANG

, HSU-FENG LU

and JING-GUNG CHUNG

1

_{Department of Nursing and Management, Jen-Teh Junior College of Medicine, Taiwan;}

Departments of

Pharmacology and

Biological Science and Technology,

School of Chinese Medicine,

6

_{Graduate Institute of Integration Chinese and Western Medicine, China Medical University, Taichung, Taiwan;}

_{Department of Ophthalmology, China Medical University Hospital, Taichung, Taiwan;}

4

_{Department of Life Science, Tunghai University, Taichung, Taiwan;}

7

_{Department of Clinical Pathology, Cheng Hsin Rehabilitation Medical Center, Taipei, Taiwan;}

_{Department of Biotechnology, Asia University, Wufeng, Taichung County 41354, Taiwan, R.O.C.}

USA). Caspase-3 activity assay kits were bought from Boehringer Mannheim (Mannhein, Germany).

SCC-4 human tongue cancer cell line. The SCC-4 cell line was

purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC). SCC-4 cells have been cultured for several generations and have been checked for viability as described previously. The cells were placed into 75 cm3_tissue culture flasks and grown at 37ÆC under a humidified 5% CO₂and 95% air at one atmosphere in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin (10 ng/ml penicillin and 10 ng/ml streptomycin) and 1% glutamine (25).

Baicalein effect on caspase-3 activity. The SCC-4 cells were plated

in 6-well plates at a density of 5x105_{cells/well and grown for 24} hours. Then cells were grown in 25 and 75 ÌM baicalein, with DMSO (solvent) alone for the control regimen, at 37ÆC in a humidified 5% CO₂ for 24 hours. The caspase-3 activity was analyzed by flow cytometry (Becton Dickinson FACS Calibur) as described elsewhere (26-28).

Baicalein effect on reactive oxygen species (ROS). The SCC-4 cells were

treated with or without 0, 25 or 75 ÌM of baicalein (or pre-treated with 10 ÌM caspase inhibitor Ac-DEVD-CHO for 3 hours) for 24 hours to detect the changes of ROS. The cells were harvested and washed twice, re-suspended in 500 Ìl of 2,7-dichlorodihydrofluorescein diacetate (10 ÌM) (DCFH-DA, Sigma) and incubated at 37ÆC for 30 min, before being analyzed by flow cytometry (26-28).

Baicalein effect on Ca2+_{concentrations. The SCC-4 cells were treated}

with or without 0, 25 or 75 ÌM of baicalein for 24 hours then to detect the changes in Ca2+_{concentrations, and then they were} harvested and washed twice for re-suspension in Indo 1/AM (3 Ìg/ml) (Calbiochem, La Jolla, CA, USA), incubated at 37ÆC for 30 min, and then analyzed by flow cytometry (26-28).

Baicalein effect on mitochondrial membrane potential (MMP).

Cells were treated with 75 ÌM baicalein for 24 hours. The cells were harvested and washed twice, re-suspended in 500 Ìl of DiOC₆ (4 mol/L), incubated at 37ÆC for 30 min, and then analyzed by flow cytometry (26-28).

Effect of baicalein on cytoplasmic Ca2+_{, MMP, and proportion of} apoptosis in human tongue cancer SCC-4 cells pretreated with BAPTA. The level of Ca2+_{, MMP and apoptosis of the SCC-4 cells} was determined by flow cytometry (Becton Dickinson FACS Calibur) using the Indo 1/AM (Calbiochem). Cells were pre-treated with or without 10 ÌM BAPTA-AM (intracellular calcium chelator) for 3 hours then with 25 and 75 ÌM baicalein for 24 hours. The cells were harvested and washed twice, for apoptosis

analysis and for re-suspension in Indo 1/AM (3 Ìg/ml), incubated at 37ÆC for 30 min, and then analyzed by flow cytometry (26-28) for Ca2+_{concentration, MMP and apoptosis.}

Effect of baicalein on the expressions of p53, BAX, BCL-2, cytochrome c, caspase-3 and -9, EndoG and AIF. The total proteins were

collected from SCC-4 cells treated with or without 0, 25 or 75 ÌM of baicalein for 48 hours. Subsequently, p53, BAX, BCL-2, cytochrome

c, caspase-3 and -9, EndoG and AIF were measured by sodium

dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The levels of cytochrome c in the mitochondria and in the cytosol were also determined. The SCC-4 cells after treatment with or without 75 ÌM baicalein were harvested and disrupted. They were then centrifuged to obtain cytosolic and mitochondria fractions and underwent further examination in regards to the level of cytochrome c by Western blot as described elsewhere (28, 29).

Statistical analysis. Statistical calculations of the data were

performed using an unpaired Student’s t-test and Tukey’s test. Statistical significance was set at p<0.05.

Results

Induction of apoptosis by baicalein. After SCC-4 cells were

treated with 75 ÌM baicalein for 0, 6, 12, 24, 48 and 72

hours, apoptosis was detected by PI staining method and

then analyzed by flow cytometry. As shown in Figure 1,

baicalein induced apoptosis in a time-dependent manner.

Effect of baicalein on caspase-3 activity. Caspase-3 activity

increased in baicalein-treated SCC-4 cells and increasing

baicalein dose led to increasing caspase-3 activity, as shown

in Table I.

Effect of baicalein on ROS. The increase of the levels of

ROS was detected in 24 hours after cells being treated with

cells. PI staining analysis for the effects of baicalein on human tongue cancer SCC-4 cell’s apoptosis. The SCC-4 cells were incubated with 75 ÌM baicalein for 6, 12, 24, 48 and 72 h and apoptosis was determined by flow cytometric analysis as described in Materials and Methods. Data represents mean±S.D. of three experiments (*p<0.05).

baicalein and increasing dose led to an increase in ROS

production (Table I).

Effect of baicalein on the levels of cytoplasmic Ca

from

human tongue cancer SCC-4 cells. The increase of the levels

of cytoplasmic Ca

was detected 24 hours after treatment

with baicalein, dose-dependently (Table I).

Effect of baicalein on the MMP levels. Mitochondria

membrane potential (MMP) declined in SCC-4 cells treated

with 25 and 75 ÌM baicalein (Table I).

Effect of Ac-DEVD-CHO on baicalein promoted caspase-3

activity in human tongue cancer SCC-4 cells. SCC-4 cells

were pretreated with or without Ac-DEVD-CHO (inhibitor

of caspase-3) followed by treatment with 75 ÌM baicalein

for 24 h. Caspase-3 activity was increased in

baicalein-treated cells of SCC-4, but decreased in caspase-3 inhibitor

(Ac-DEVD-CHO) pretreatment in baicalein-treated

SCC-4 cells, as shown in Figure 2A.

Effects of BAPTA on baicalein-induced changes in levels of

cytoplasmic Ca

, MMP and apoptosis of SCC-4 human tongue

cancer cells. The changes of the levels of cytoplasmic Ca

,

for 3 h then treated with 75 ÌM baicalein before cells were harvested for caspase-3 activity, Ca2+_{concentration, MMP levels and apoptosis determinations} as described in Materials and Methods. Data represents mean±S.D. of three experiments. *p<0.05. (Panel A: caspase-3 activity; panel B: Ca2+ concentration; panel C: MMP levels; and panel D: apoptosis).

Table I. Flow cytometric analysis of intracellular ROS and Ca2+_levels, MMP and caspase-3 activity in SCC-4 cells treated with baicalein.

Baicalein (ÌM) % of control

ROS Ca2+ _{MMP Caspase-3}

0 1.2±0.6 4.8±1.2 96.0±6.8 2.9±0.8

25 19.6±2.9b _32.4±2.8b _78.4±7.2b _26.6±3.4b 75 54.8±6.8a _61.2±5.2a _54.2±4.8a _64.2±7.4a The SCC-4 cells (5x105 _{cells/ml) were treated with various} concentrations of baicalein. The zero concentration was defined as control. The stained cells were determined by flow cytometry as described in the Materials and Methods section. Values are means±SD (n=3). Dates not sharing the same letter are significantly different by Tukey’s test (*p<0.05).

MMP and apoptosis in baicalein-treated SCC-4 cells were

greatly affected by pretreatment with BAPTA. The decline of

MMP induced by baicalein was reversed by BAPTA. After

cells were pretreated with BAPTA, the increase in cytoplasmic

Ca

was suppressed and the proportion of apoptosis was also

markedly diminished (Figure 2B, C and D).

(Figure 4).

Discussion

Although many experiments have shown that baicalein

induced apoptosis in human cancer cell lines there is no

available information regarding the effects of baicalein on

SCC-4 human tongue cancer cells. In this study, we

demonstrated that baicalein induced apoptosis in SCC-4

cells via a Ca

-associated mitochondrial and

caspase-3-dependent pathway. Our results from Western blotting

demonstrated that baicalein promoted production of p53,

BAX, cytochrome c, EndoG and AIF and reduced the levels

of BCL-2 which led to the disruption of mitochondrial

membrane potential (MMP) and the release of cytochrome

c from the mitochondria to the cytosol. This finding also

points out that baicalein may be useful in clinical trials in

the future for tongue cancer patients.

Our data indicated that baicalein-induced apoptosis also

involved a decrease of the MMP, which is in agreement with

the reports showing that apoptosis is accompanied by a loss

of MMP (30, 31) and exposure of phosphatidylserine at the

surface of the cell (32-35). It is well-known that

mitochondrial alterations constitute a critical event of the

apoptotic cascade (36). Our results also showed that

baicalein promoted cytochrome c release (Figure 4) which

is in agreement with other reports which indicated that the

reduction of MMP is an early reversible step of apoptosis,

followed by cytochrome c release in many cell types based

on the DiOC

uptake as shown by flow cytometric analysis

(37). Therefore, baicalein induced apoptosis in SCC-4 cells

through a mitochondria-dependent pathway.

Many investigators have demonstrated that baicalein

induced apoptosis in human cancer cells and the

apoptogenic action of baicalein was associated with

caspase activation, mitochondrial dysfunction and

modulation of BCL-2 family proteins (21-24). However,

information regarding the role of Ca

in the induction of

apoptosis caused by baicalein is scarce. Based on our

results, baicalein elevated cytoplasmic Ca

which is

involved in the induction of apoptosis (based on

pretreatment with BAPTA (Ca

chelator) in

baicalein-treated SCC-4 cells), MMP was restored, the level of

cytoplasmic Ca

was reduced and the proportion of

proteins in SCC-4 human tongue cancer cells. Representative Western blot showing changes on the levels of p53, BAX, BCL-2, cytochrome c, caspase-9, caspase-3, EndoG and AIF in human tongue cancer SCC-4 cells after treated with baicalein. The SCC-4 cells (5x106_{/ml) were treated with 0, 25} and 75 ÌM baicalein for 48 h, then total protein was prepared and determined as described in Materials and Methods, then followed by evaluation of the protein levels (Panel A: p53, BAX and BCL-2; panel B: caspase-9, caspase-3, EndoG and AIF). Expressions were estimated by Western blotting as described in Materials and Methods.

apoptosis was also markedly diminished. This conclusion

is confirmed by other reports demonstrating that under

normal conditions, the Ca

influx increases and cells die

(38). Furthermore, other investigators also showed that

Co

blocks Ca

influx, resulting in the protection of the

cells despite high ROS levels (21). Our results also showed

baicalein induced ROS production (Table I). It is

well-known that in normal amounts ROS is involved in

maintaining human physiological functions, however

overproduction of ROS can be detrimental and has been

shown to participate in the etiology of several human

diseases such as cancer, inflammation and diabetes.

It is also reported that flavonols may interact directly with

a Ca

channel which leads to prevention of its opening

that is responsible for the final demise of the cell (39).

Whether or not they may prevent the signaling mechanism

between high ROS levels and the opening of the Ca

channel needs further investigation. We summarize the

possible signal pathway (Figure 5) demonstrating that

baicalein induced apoptosis through a mitochondrial and

caspase-3-dependent pathway and that AIF and EndoG are

involved in these events.

Acknowledgements

This work was supported by grant CMU95-127 from China Medical University, Taichung, Taiwan, R.O.C.

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Received June 21, 2007

Revised October 4, 2007

Accepted October 16, 2007