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BMC Complementary and
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Research article                                                                                                                       Open Access
Anticancer activity of extracts derived from the mature roots of
Scutellaria baicalensis on human malignant brain tumor cells
Adrienne C Scheck1,2, Krya Perry1,3, Nicole C Hank1 and W Dennis Clark*3

Address: 1Neuro-Oncology Research, Barrow Neurological Institute of St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA,
2Neurosurgery Research, Barrow Neurological Institute of St. Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA and 3School of Life

Sciences, Arizona State University, Tempe, AZ 85287-4501, USA
Email: Adrienne C Scheck - adrienne.scheck@chw.edu; Krya Perry - Krya.Perry@sccmail.maricopa.edu; Nicole C Hank - nicole.hank@chw.edu;
W Dennis Clark* - wdclark@usbotanicals.net
* Corresponding author




Published: 16 August 2006                                                                 Received: 27 February 2006
                                                                                          Accepted: 16 August 2006
BMC Complementary and Alternative Medicine 2006, 6:27   doi:10.1186/1472-6882-6-27
This article is available from: http://www.biomedcentral.com/1472-6882/6/27
 2006 Scheck et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.




                 Abstract
                 Background: Flavonoid-rich extracts from the mature roots of Scutellaria baicalensis have been
                 shown to exhibit antiproliferative effects on various cancer cell lines. We assessed the ability of an
                 ethanolic extract of S. baicalensis root to inhibit the proliferation of malignant glioma cells.
                 Methods: Cell lines derived from primary and recurrent brain tumors from the same patient and
                 cells selected for resistance to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea
                 (BCNU) were used to identify antiproliferative effects of this extract when used alone and in
                 conjunction with BCNU.
                 Results and discussion: Results indicated that Scutellaria baicalensis not only inhibits cellular
                 growth in recurrent and drug resistant brain tumor cell lines, but also demonstrates an increased
                 inhibitory effect when used in conjunction with BCNU.
                 Conclusion: The results of this study support the efficacy of S. baicalensis as an anticancer agent
                 for glioblastomas multiforme and a potential adjuvant treatment to current chemotherapeutic
                 agents used in the treatment of both primary and recurrent GBMs. Further studies of the effects
                 of individual flavonoids alone and in combination with each other and with currently used therapies
                 are needed.




Background                                                                    mens that include surgery, radiation and chemotherapy.
Malignant gliomas are one of the more lethal forms of                         Complete surgical removal of the tumor is typically not
cancer. An estimated 18,000 new cases of brain and cen-                       achieved due to the infiltrative nature of these tumors and
tral nervous system tumors are diagnosed each year and                        while radiation and chemotherapy kill the majority of the
approximately 13,000 people die of their disease in the                       remaining tumor cells, the rapid recurrence of these
United States alone [1]. Those diagnosed with the most                        tumors suggest the presence within the primary tumor of
malignant form of astrocytoma (glioblastoma multi-                            a subpopulation of cells intrinsically resistant to therapy
forme, GBM) have a dismal prognosis. The median sur-                          and capable of survival and growth within the tumor bed
vival rate of one year has remained essentially unchanged                     following therapy [2,3]. When these tumors recur, they
for a number of years despite aggressive treatment regi-                      are typically refractory to additional courses of the same


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therapies. Improvement in the survival and quality of life        removed from the market due to concerns about contam-
of glioma patients requires the design of new therapies or        ination [35]. Subsequent work has shown that flavonoids
therapeutic combinations that are effective and preferably        from S. baicalensis were likely to have been at least one of
have fewer side effects than those presently available.           the active ingredients in this herbal mixture [31], and S.
                                                                  baicalensis extract and its constituents have been shown to
One promising new source of therapeutic agents has been           cause reduced expression of the androgen receptor and
discovered in plant secondary metabolites, irregularly            androgen regulated genes in prostate carcinoma [20].
occurring compounds that characterize certain plants or           Recent studies have also shown that the flavonoid-rich
plant groups [4]. Recent interest in these secondary              extract from the roots of S. baicalensis exhibit antiprolifer-
metabolites has been focused upon their medicinal prop-           ative effects upon prostate, squamous, colon, breast, lung,
erties [5]. For example, flavonoids are a large group of aro-     and liver carcinomas, as well as various leukemia cell lines
matic plant secondary metabolites that are produced in            [16,21,36,37]; however, there have been no studies con-
the plant for the purpose of protection from photosyn-            ducted in brain tumors despite suggestions that compo-
thetic stress, reactive oxygen species (ROS), wounds and          nents of this extract can have effects on microglia in the
herbivores. Studies of flavonoids have produced the most          brain [38]. We, therefore, tested an extract from the root
compelling data for the antitumor activities of plant sec-        of S. baicalensis to determine if it had antiproliferative
ondary metabolites in various types of cancers [6], and           effects on cells from human malignant brain tumors
several flavonoids have been shown to inhibit cancer              alone or in combination with 1,3-bis(2-chloroethyl)-1-
development while exhibiting antioxidant activities in            nitrosourea (BCNU, carmustine), an alkylating agent
various animal models [7-11]. Furthermore, some studies           commonly used in the treatment of human brain tumors.
suggest that the most promising use of these compounds
may be as an adjuvant to currently used therapies [12,13].        Methods
                                                                  Plant material extraction
Numerous cancer research studies have been conducted              Secondary metabolites, including flavonoids and other
using traditional medicinal plants in an effort to discover       phenolic compounds, were extracted from ground mature
new therapeutic agents that lack the toxic side effects asso-     roots of S. baicalensis (common name Huang Qin) using
ciated with current chemotherapeutic agents. One of the           95% ethanol. Commercially available roots from a Cali-
more versatile plants used as a source of flavonoids is the       fornia importer of Chinese herbal medicines (Win Hop
root of the traditional Chinese medicinal herb Baikal             Fung, Los Angeles) were obtained and ground into a fine
skullcap (Scutellaria baicalensis), a member of the mint          powder using a Scientific ApparatusTM soil mill. The root
family [14]. Traditionally, the dried roots of S. baicalensis     powder was stirred for 24 hours in a 95% ethanol solution
were extracted and used in a Chinese herbal medicine              in order to extract the secondary metabolites and flavo-
"Huang Qin" to treat a variety of ailments [15], and Scutel-      noids. The crude extract was filtered through a PTFE mem-
laria baicalensis has remained an important herb in both          brane filter using a Swinney (Millipore Corp., Bedford,
Chinese and Japanese traditional prescriptions, such as           MA) filtering device. The extract was dried and quantified
"Xiao-Chai-Hu-Tang" which is used in the treatment of             for the total amount of crude extract. A stock solution was
viral hepatitis and a variety of tumors [16-18]. Various fla-     prepared at 25 mg of solid per ml in absolute ethanol and
vonoids isolated from this traditional Chinese medicinal          was further diluted with sterile water immediately before
plant were shown to have antiandrogenic and growth                treatment of the cells to achieve a concentration of 10 mg/
inhibitory activity against prostate cancer cells in vitro and    ml in a 40% (v/v) ethanol solution.
in vivo [19-26]. In addition, extracts and isolated flavo-
noids from this herb have been shown to relieve oxidative         Cell culture
stress and immune dysfunction associated with the onset           Cell lines grown from primary and recurrent GBMs from
and progress of cancer [8]. Studies have also demon-              three patients (Table 1) were cultured using previously
strated that flavonoids from S. baicalensis have the ability      established protocols [39]. Primary tumors are designated
to arrest the cell cycle of tumor cell lines that are resistant   with a 2-letter code (ME, DI) or the last 2 digits of the year
to multiple chemotherapeutic drugs [27] and act as inhib-         followed by a 2-letter code (00WA). The recurrent tumors
itors of key steps necessary for the progression of tumor         from the same patient receive the same code with the
angiogenesis [28].                                                addition of an "R" (ME/MER, DI/DIR). Following surgery
                                                                  for their primary tumor, both patients from whom we
More recently, Scutellaria baicalensis was used as a compo-       received recurrent tumor samples received 1,3-bis(2-chlo-
nent of PCSPES, an herbal mixture that showed efficacy in         roethyl)-1-nitrosourea (BCNU, carmustine) and radiation
laboratory trials for prostate cancer, small-cell lung cancer     therapy. Despite therapy, both tumors recurred and the
and acute myeloid leukemia [29-34]. Despite these prom-           patients underwent additional surgery. Tumor cell line
ising results in human trials, this herbal mixture was            00WA (Table 1) was selected for use in this study as a low-


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Table 1: Patient information

 Tumor codea      Ageb    Sex    Diagnosisc        Treatmentd          Days between primary and secondary surgery             Survival (days)

    DI/DIR         38      F        GBM        Irradiation and BCNU                            111                                  426
    ME/MER         49      F        GBM        Irradiation and BCNU                            138                                  285
    00WA           39      M        GBM                  NAe                                   NA                                   NAvf

 a The tumor code is a random 2 letter code. Recurrent tumor from the same patient receives the same code with the addition of R. For each
 patient the primary and recurrent tumors were diagnosed by the same neuropathologist.
 b Age in years at diagnosis of primary tumor.
 c GBM = glioblastoma multiforme.
 d BCNU = 1,3-bis(2-chloroethyl)-1-nitrosourea.
 e NA = not applicable since this was a primary tumor.
 f NAv = not available

passage cell line derived from a primary tumor and serial                 tion. Cells were diluted and divided into aliquots contain-
passaged 5 times. Normal glial cells (HJ) grown from a                    ing approximately 1.5  103 cells. Aliquots were treated
sample obtained from a crainiotomy done for treatment                     with a single dose of 0 (untreated), 10, 25, 50, 75, 100,
of trauma were also cultured following previously estab-                  150 and 200 g/ml of the extract in MAB 87/3 medium
lished protocols [39]. All cell lines were cultured in Way-               supplemented with 20% FBS for 1 hour. Following incu-
mouth's MAB 87/3 (Mediatech, Herndon, Virginia)                           bation, cells were washed three times with fresh medium.
containing 20% Fetal Bovine Serum (FBS; Intergen Co.,                     Cells were resuspended in the medium and aliquots of 1.5
Purchase, NY) and incubated at 37C in a humidified                       ml containing approximately 2  103 cells were dispensed
atmosphere of 5% CO2. To select for the subpopulation of                  into three 60 mm culture dishes. Cells were cultured for at
cells resistant to 10 g/ml BCNU, cells were treated with                  least 6 divisions, approximately 14 days, before they were
gradually increasing concentrations of drug as described                  fixed with methanol and stained with 4% Giemsa stain.
[40,41]. Cells were washed with MAB without serum; they                   Colonies, defined as 50 or more cells, were counted and
were then mock-treated using MAB alone or treated with                    plotted as a percentage of the control (untreated) colo-
increasing concentrations of BCNU in MAB (2.5, 5.0, 7.5,                  nies.
10 g/ml) for 1 hour at 37C with 5% CO2. Cells were
subsequently washed and fed with MAB containing 20%                       Trypan blue exclusion cell viability assay
FBS. The cells were treated for three consecutive days then               The cells were plated at approximately 1  105 cells per
allowed to grow. Treatment was repeated several times                     well in 6-well tissue culture plates in 2 ml of medium and
until the cells in culture were resistant to 10 g/ml of                   incubated at 37C at 5% CO2. After 24 hours, the
BCNU (ME Drug/MER Drug and DI Drug/DIR Drug) as                           medium was removed and replaced with fresh medium
evidenced by the absence of cell death compared to that                   plus 20% FBS and supplemented with S. baicalensis extract
observed in the mock-treated cultures. Cells were re-                     (0, 25, 50, 100, 150, and 200 g/ml) or a combination of
treated with 10 g/ml BCNU every 810 passages to                          S. baicalensis extract and BCNU (2.5 or 5 g/ml). Cells
maintain the resistant phenotype.                                         were harvested 48 hours after treatment by digestion with
                                                                          0.25% trypsin-EDTA solution at 37C for 23 minutes.
AlamarBlueTM metabolic assay                                              The cells were stained with trypan blue and live cells were
Cells were seeded at a density of approximately 1  4  103               enumerated. Cell counts were expressed as mean  stand-
cells in 200 l per well. Following a 24-hour incubation                   ard deviation (SD).
period, cells were treated with 100 g/ml of S. baicalensis
extract containing 0.4% ethanol (v/v) in 200 l of                         Statistical analysis
medium. Mock treatment consisted of a 0.4% (v/v) solu-                    All results are expressed as mean  standard deviation
tion of ethanol in 200 l of medium. Cellular metabolic                    (SD). Statistical differences between correlated samples
activity was assayed using AlamarBlueTM (Serotec, Raleigh,                were evaluated using Student's t-test and noted to be sig-
NC) and the manufacturer's protocol at 0, 1, 3, 7, 10, and                nificantly different where p < 0.05. Student t-test calcula-
15 days following treatment. The fluorescent reading was                  tions were assessed on the VassarStats Statistical
performed using a microtiter plate reader (485 nm  exci-                  Computation website [42]. Composite treatments were
tation, 595  nm emission).                                                compared using one-way analysis of variances (ANOVA)
                                                                          and considered significantly different where probability
Colony forming assay                                                      values were found to be equal to or less than 0.05. All
A Colony Forming Assay (CFA) was used to verify that the                  ANOVA tests, as well as mean and SD calculations, were
cells remaining metabolically active following treatment                  performed using GraphPad Prism (GraphPad Software,
with S. baicalensis extract were also capable of prolifera-               Inc., San Diego, USA).


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Results                                                         treated with S. baicalensis extract compared with mock- or
Effects of S. baicalensis on glioma cell metabolic activity     ethanol treated cells. There was no increase in metabolic
The metabolic inhibitory effects of an extract from S. bai-     activity exhibited in the ME and MER glioma cell lines, as
calensis were studied in cell lines cultured from paired pri-   well as subpopulations of these cell lines that were
mary (ME) and recurrent (MER) glioblastoma multiforme           selected for BCNU resistance (ME Drug and MER Drug)
(GBM) tumors obtained from a single patient, as well as         for the 15 day duration of the experiment (Figure 1),
primary and recurrent cells that have been selected in vitro    while the untreated glioma cells (controls) exhibited
for resistance to 10 g/ml of BCNU (ME Drug/MER                  exponential increases in metabolic activity. As an addi-
Drug). Metabolic activity was assessed at 0, 1, 3, 7, 10, and   tional control, glioma cells were treated with a 0.4% (v/v)
15 days following treatment with 100 g/ml of the extract        ethanol solution (solvent vehicle). Although there was
using an AlamarBlue metabolic assay. All wells were sub-        some difference in fluorescence between untreated and
confluent when the experiment was begun to allow for            ethanol treated MER cells at day 7 (Figure 1D) and 10
changes in cell growth. In this way, we would see a differ-     (Figure 1C), there was good growth in all four cell lines
ence regardless of whether the effect of the extract was        and the ethanol-treated cells showed no significant
cytotoxic or cytostatic. There was a significant (p < .05; n    decrease in metabolic activity compared to the untreated
= 5) difference in the metabolic activity exhibited by cells    control cells by the fifteenth day of culture. Microscopic




Time
Figure
     course
         1    treatment of ME series cells with S. baicalensis extract
Time course treatment of ME series cells with S. baicalensis extract. Metabolic activity was measured using AlamarBlueTM.
Wells were subconfluent on Day 0. Cells were left untreated (-), mock treated with 0.4% ethanol (-) or treated with a
0.4% ethanol solution containing 100 g/ml S. baicalensis extract (-). Data is mean  SD using 5 replicates. (A) cells from
primary tumor (ME), (B) ME cells selected for resistance to 10 g/ml BCNU (ME drug), (C) cells from recurrent tumor (MER),
(D) cells from recurrent tumor selected for resistance to 10 g/ml BCNU (MER drug).




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examination showed extensive cytopathic effect (CPE) in           To demonstrate that the effect of the S. baicalensis extract
cells treated with the S. baicalensis extract (data not           on glioma cells was not a result of long-term in vitro culti-
shown).                                                           vation of the cells, we tested the extract on cell line 00WA
                                                                  after 5 serial passages in vitro. This was the minimum
Effects of S. baicalensis on glioma cell viability and            number of passages required to obtain enough cells to
proliferation                                                     perform the assay. Cells were treated with varying concen-
Colony forming assays were done to demonstrate a dose             trations of the S. baicalensis extract as described in the
response to the S. baicalensis extract and to show that the       Methods section, and the percentage of viable cells was
effect is cytopathic and not cytostatic. Cells from the           determined by trypan blue exclusion 48 hours later. Fig-
paired primary and recurrent tumors ME/MER and DI/                ure 3 shows that the extract caused a dose-dependent
DIR prior to and following selection for resistance to 10         reduction in viability similar to that observed in the ME/
g/ml BCNU were treated with 0  200 g/ml S. baicalensis           MER and DI/DIR cell line series.
extract as described in the methods section. Cells were
allowed to grow to form colonies of at least 50 cells. As         Effects of S. baicalensis on normal glial cells
seen in Figure 2, cells from all 8 cell lines showed a dose-      To assess whether the extract demonstrates toxicity in nor-
dependent response to the S. baicalensis extract.                 mal cells, normal human glial cells (HJ) were plated and
                                                                  allowed to grow until they were close to confluence. These
                                                                  cells were then cultured in the presence and absence of
                                                                  various concentrations of the extract for 48 hours. As
                                                                  shown in Figure 4, normal glial cells treated with 25100
                                                                  g/ml of S. baicalensis extract for 48 hours displayed no
                                                                  significant change (p > .05; n = 3) in metabolic activity
                                                                  assayed using AlamarBlue and compared to the control
                                                                  (untreated) cells. For comparison, a recurrent glioma cell
                                                                  line (MER) was also treated with 25-100 g/ml of the
                                                                  extract for 48 hours. The glioma cells treated with S. bai-
                                                                  calensis extract at concentrations of 50 and 100 g/ml dis-
                                                                  played a significant reduction (p < .05, n = 3) in metabolic
                                                                  activity when compared to the metabolic activity of the
                                                                  control (untreated) cells.




Figure 2 baicalensis
Dose-dependent
Scutellaria         inhibition
                        extractof glioma cell proliferation by
Dose-dependent inhibition of glioma cell proliferation by
Scutellaria baicalensis extract. Glioma cell lines from primary
tumors (ME and DI) and recurrent (MER and DIR) tumors, as
well as BCNU resistant (designated "Drug") glioma cell lines      Figure
                                                                  Dose-dependent
                                                                  cell line 3
                                                                            by an extract
                                                                                     growth
                                                                                          from
                                                                                             inhibition
                                                                                               S. baicalensis
                                                                                                        of a low-passage glioma
were cultured with various concentrations of S. baicalensis       Dose-dependent growth inhibition of a low-passage glioma
extract (0200 g/ml). After treatment, cells were incubated       cell line by an extract from S. baicalensis. The viability of cell
for 14 days to allow for colony growth. Colonies of at least      line 00WA following treatment with 0200 g/ml of S. bai-
50 cells were counted using a 4% Giemsa stain. Results were       calensis extract was tested by trypan blue exclusion at serial
normalized to the control (untreated cells). Data show            passage 5. Results are the mean  SD of 2 replicates and data
means  SD of 3 replicates.                                       were normalized to the control.




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