BMC Complementary and Alternative Medicine

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.


Background
Malignant gliomas are one of the more lethal forms of cancer. An estimated 18,000 new cases of brain and central nervous system tumors are diagnosed each year and approximately 13,000 people die of their disease in the United States alone [1]. Those diagnosed with the most malignant form of astrocytoma (glioblastoma multiforme, GBM) have a dismal prognosis. The median survival rate of one year has remained essentially unchanged for a number of years despite aggressive treatment regi-mens that include surgery, radiation and chemotherapy. Complete surgical removal of the tumor is typically not achieved due to the infiltrative nature of these tumors and while radiation and chemotherapy kill the majority of the remaining tumor cells, the rapid recurrence of these tumors suggest the presence within the primary tumor of a subpopulation of cells intrinsically resistant to therapy and capable of survival and growth within the tumor bed following therapy [2,3]. When these tumors recur, they are typically refractory to additional courses of the same therapies. Improvement in the survival and quality of life of glioma patients requires the design of new therapies or therapeutic combinations that are effective and preferably have fewer side effects than those presently available.
One promising new source of therapeutic agents has been discovered in plant secondary metabolites, irregularly occurring compounds that characterize certain plants or plant groups [4]. Recent interest in these secondary metabolites has been focused upon their medicinal properties [5]. For example, flavonoids are a large group of aromatic plant secondary metabolites that are produced in the plant for the purpose of protection from photosynthetic stress, reactive oxygen species (ROS), wounds and herbivores. Studies of flavonoids have produced the most compelling data for the antitumor activities of plant secondary metabolites in various types of cancers [6], and several flavonoids have been shown to inhibit cancer development while exhibiting antioxidant activities in various animal models [7][8][9][10][11]. Furthermore, some studies suggest that the most promising use of these compounds may be as an adjuvant to currently used therapies [12,13].
Numerous cancer research studies have been conducted using traditional medicinal plants in an effort to discover new therapeutic agents that lack the toxic side effects associated with current chemotherapeutic agents. One of the more versatile plants used as a source of flavonoids is the root of the traditional Chinese medicinal herb Baikal skullcap (Scutellaria baicalensis), a member of the mint family [14]. Traditionally, the dried roots of S. baicalensis were extracted and used in a Chinese herbal medicine "Huang Qin" to treat a variety of ailments [15], and Scutellaria baicalensis has remained an important herb in both Chinese and Japanese traditional prescriptions, such as "Xiao-Chai-Hu-Tang" which is used in the treatment of viral hepatitis and a variety of tumors [16][17][18]. Various flavonoids isolated from this traditional Chinese medicinal plant were shown to have antiandrogenic and growth inhibitory activity against prostate cancer cells in vitro and in vivo [19][20][21][22][23][24][25][26]. In addition, extracts and isolated flavonoids from this herb have been shown to relieve oxidative stress and immune dysfunction associated with the onset and progress of cancer [8]. Studies have also demonstrated that flavonoids from S. baicalensis have the ability to arrest the cell cycle of tumor cell lines that are resistant to multiple chemotherapeutic drugs [27] and act as inhibitors of key steps necessary for the progression of tumor angiogenesis [28].
More recently, Scutellaria baicalensis was used as a component of PCSPES, an herbal mixture that showed efficacy in laboratory trials for prostate cancer, small-cell lung cancer and acute myeloid leukemia [29][30][31][32][33][34]. Despite these promising results in human trials, this herbal mixture was removed from the market due to concerns about contamination [35]. Subsequent work has shown that flavonoids from S. baicalensis were likely to have been at least one of the active ingredients in this herbal mixture [31], and S. baicalensis extract and its constituents have been shown to cause reduced expression of the androgen receptor and androgen regulated genes in prostate carcinoma [20]. Recent studies have also shown that the flavonoid-rich extract from the roots of S. baicalensis exhibit antiproliferative effects upon prostate, squamous, colon, breast, lung, and liver carcinomas, as well as various leukemia cell lines [16,21,36,37]; however, there have been no studies conducted in brain tumors despite suggestions that components of this extract can have effects on microglia in the brain [38]. We, therefore, tested an extract from the root of S. baicalensis to determine if it had antiproliferative effects on cells from human malignant brain tumors alone or in combination with 1,3-bis(2-chloroethyl)-1nitrosourea (BCNU, carmustine), an alkylating agent commonly used in the treatment of human brain tumors.

Plant material extraction
Secondary metabolites, including flavonoids and other phenolic compounds, were extracted from ground mature roots of S. baicalensis (common name Huang Qin) using 95% ethanol. Commercially available roots from a California importer of Chinese herbal medicines (Win Hop Fung, Los Angeles) were obtained and ground into a fine powder using a Scientific Apparatus™ soil mill. The root powder was stirred for 24 hours in a 95% ethanol solution in order to extract the secondary metabolites and flavonoids. The crude extract was filtered through a PTFE membrane filter using a Swinney (Millipore Corp., Bedford, MA) filtering device. The extract was dried and quantified for the total amount of crude extract. A stock solution was prepared at 25 mg of solid per ml in absolute ethanol and was further diluted with sterile water immediately before treatment of the cells to achieve a concentration of 10 mg/ ml in a 40% (v/v) ethanol solution.

Cell culture
Cell lines grown from primary and recurrent GBMs from three patients (Table 1) were cultured using previously established protocols [39]. Primary tumors are designated with a 2-letter code (ME, DI) or the last 2 digits of the year followed by a 2-letter code (00WA). The recurrent tumors from the same patient receive the same code with the addition of an "R" (ME/MER, DI/DIR). Following surgery for their primary tumor, both patients from whom we received recurrent tumor samples received 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine) and radiation therapy. Despite therapy, both tumors recurred and the patients underwent additional surgery. Tumor cell line 00WA (Table 1) was selected for use in this study as a low-passage cell line derived from a primary tumor and serial passaged 5 times. Normal glial cells (HJ) grown from a sample obtained from a crainiotomy done for treatment of trauma were also cultured following previously established protocols [39]. All cell lines were cultured in Waymouth's MAB 87/3 (Mediatech, Herndon, Virginia) containing 20% Fetal Bovine Serum (FBS; Intergen Co., Purchase, NY) and incubated at 37°C in a humidified atmosphere of 5% CO 2 . To select for the subpopulation of cells resistant to 10 μg/ml BCNU, cells were treated with gradually increasing concentrations of drug as described [40,41]. Cells were washed with MAB without serum; they were then mock-treated using MAB alone or treated with increasing concentrations of BCNU in MAB (2.5, 5.0, 7.5, 10 μg/ml) for 1 hour at 37°C with 5% CO 2 . Cells were subsequently washed and fed with MAB containing 20% FBS. The cells were treated for three consecutive days then allowed to grow. Treatment was repeated several times until the cells in culture were resistant to 10 μg/ml of BCNU (ME Drug/MER Drug and DI Drug/DIR Drug) as evidenced by the absence of cell death compared to that observed in the mock-treated cultures. Cells were retreated with 10 μg/ml BCNU every 8-10 passages to maintain the resistant phenotype.

AlamarBlue™ metabolic assay
Cells were seeded at a density of approximately 1 -4 × 10 3 cells in 200 μl per well. Following a 24-hour incubation period, cells were treated with 100 μg/ml of S. baicalensis extract containing 0.4% ethanol (v/v) in 200 μl of medium. Mock treatment consisted of a 0.4% (v/v) solution of ethanol in 200 μl of medium. Cellular metabolic activity was assayed using AlamarBlue™ (Serotec, Raleigh, NC) and the manufacturer's protocol at 0, 1, 3, 7, 10, and 15 days following treatment. The fluorescent reading was performed using a microtiter plate reader (485 nm λ excitation, 595 λ nm emission).

Colony forming assay
A Colony Forming Assay (CFA) was used to verify that the cells remaining metabolically active following treatment with S. baicalensis extract were also capable of prolifera-tion. Cells were diluted and divided into aliquots containing approximately 1.5 × 10 3 cells. Aliquots were treated with a single dose of 0 (untreated), 10, 25, 50, 75, 100, 150 and 200 μg/ml of the extract in MAB 87/3 medium supplemented with 20% FBS for 1 hour. Following incubation, cells were washed three times with fresh medium. Cells were resuspended in the medium and aliquots of 1.5 ml containing approximately 2 × 10 3 cells were dispensed into three 60 mm culture dishes. Cells were cultured for at least 6 divisions, approximately 14 days, before they were fixed with methanol and stained with 4% Giemsa stain. Colonies, defined as 50 or more cells, were counted and plotted as a percentage of the control (untreated) colonies.

Trypan blue exclusion cell viability assay
The cells were plated at approximately 1 × 10 5 cells per well in 6-well tissue culture plates in 2 ml of medium and incubated at 37°C at 5% CO 2 . After 24 hours, the medium was removed and replaced with fresh medium plus 20% FBS and supplemented with S. baicalensis extract (0, 25, 50, 100, 150, and 200 μg/ml) or a combination of S. baicalensis extract and BCNU (2.5 or 5 μg/ml). Cells were harvested 48 hours after treatment by digestion with 0.25% trypsin-EDTA solution at 37°C for 2-3 minutes. The cells were stained with trypan blue and live cells were enumerated. Cell counts were expressed as mean ± standard deviation (SD).

Statistical analysis
All results are expressed as mean ± standard deviation (SD). Statistical differences between correlated samples were evaluated using Student's t-test and noted to be significantly different where p < 0.05. Student t-test calculations were assessed on the VassarStats Statistical Computation website [42]. Composite treatments were compared using one-way analysis of variances (ANOVA) and considered significantly different where probability values were found to be equal to or less than 0.05. All ANOVA tests, as well as mean and SD calculations, were performed using GraphPad Prism (GraphPad Software, Inc., San Diego, USA).

Effects of S. baicalensis on glioma cell metabolic activity
The metabolic inhibitory effects of an extract from S. baicalensis were studied in cell lines cultured from paired primary (ME) and recurrent (MER) glioblastoma multiforme (GBM) tumors obtained from a single patient, as well as primary and recurrent cells that have been selected in vitro for resistance to 10 μg/ml of BCNU (ME Drug/MER Drug). Metabolic activity was assessed at 0, 1, 3, 7, 10, and 15 days following treatment with 100 μg/ml of the extract using an AlamarBlue metabolic assay. All wells were subconfluent when the experiment was begun to allow for changes in cell growth. In this way, we would see a difference regardless of whether the effect of the extract was cytotoxic or cytostatic. There was a significant (p < .05; n = 5) difference in the metabolic activity exhibited by cells treated with S. baicalensis extract compared with mock-or ethanol treated cells. There was no increase in metabolic activity exhibited in the ME and MER glioma cell lines, as well as subpopulations of these cell lines that were selected for BCNU resistance (ME Drug and MER Drug) for the 15 day duration of the experiment (Figure 1), while the untreated glioma cells (controls) exhibited exponential increases in metabolic activity. As an additional control, glioma cells were treated with a 0.4% (v/v) ethanol solution (solvent vehicle). Although there was some difference in fluorescence between untreated and ethanol treated MER cells at day 7 ( Figure 1D) and 10 ( Figure 1C), there was good growth in all four cell lines and the ethanol-treated cells showed no significant decrease in metabolic activity compared to the untreated control cells by the fifteenth day of culture. Microscopic Time course treatment of ME series cells with S. baicalensis extract examination showed extensive cytopathic effect (CPE) in cells treated with the S. baicalensis extract (data not shown).

Effects of S. baicalensis on glioma cell viability and proliferation
Colony forming assays were done to demonstrate a dose response to the S. baicalensis extract and to show that the effect is cytopathic and not cytostatic. Cells from the paired primary and recurrent tumors ME/MER and DI/ DIR prior to and following selection for resistance to 10 μg/ml BCNU were treated with 0 -200 μg/ml S. baicalensis extract as described in the methods section. Cells were allowed to grow to form colonies of at least 50 cells. As seen in Figure 2, cells from all 8 cell lines showed a dosedependent response to the S. baicalensis extract.
To demonstrate that the effect of the S. baicalensis extract on glioma cells was not a result of long-term in vitro cultivation of the cells, we tested the extract on cell line 00WA after 5 serial passages in vitro. This was the minimum number of passages required to obtain enough cells to perform the assay. Cells were treated with varying concentrations of the S. baicalensis extract as described in the Methods section, and the percentage of viable cells was determined by trypan blue exclusion 48 hours later. Figure 3 shows that the extract caused a dose-dependent reduction in viability similar to that observed in the ME/ MER and DI/DIR cell line series.

Effects of S. baicalensis on normal glial cells
To assess whether the extract demonstrates toxicity in normal 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 25-100 μg/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 μg/ml of the extract for 48 hours. The glioma cells treated with S. baicalensis extract at concentrations of 50 and 100 μg/ml displayed a significant reduction (p < .05, n = 3) in metabolic activity when compared to the metabolic activity of the control (untreated) cells.

Combination treatment with S. baicalensis and BCNU
To determine if the S. baicalensis extract could potentiate the effects of conventional chemotherapeutic agents, we tested the effect of combined treatment using 50 μg/ml of S. baicalensis extract and 2.5 μg/ml of BCNU on the viabil-ity of cells from primary and recurrent cells selected for resistance to 10 μg/ml BCNU ( Figure 5A). As expected, 2.5 μg/ml of BCNU alone had no effect on these cells and 50 μg/ml of S. baicalensis extract reduced viability to 15-30% of control; however, when used in combination survival was reduced substantially, particularly in the DI and DIR cell lines. The differences between the individual treatments and the combined treatment were found to be significant in all but the ME drug cells by one-way ANOVA (ME Drug: effects of BCNU and the S. baicalensis extract again indicated an increase in the number of detached (floating) cells with atypical morphologies when compared to cells treated with either BCNU or the extract alone ( Figure 6).
The effect of combined treatment on low passage, BCNU naive 00WA cells showed a similar significant potentiation of the drug effect, although the overall effect was less than that observed with the ME and DI cell series ( Figure  5B). BCNU or S. baicalensis extract alone reduced viability to 75%; however, combination treatment resulted in a reduction of viability to 30% of the control, mock-treated cells (p = 0.0141 F = 24.20 by one-way ANOVA).

Discussion
Treatment of malignant brain tumors typically includes surgery, radiation and chemotherapy; however, this tumor typically recurs following therapy and the recurrent tumor is often refractory to additional therapy. The identification of a novel therapy that is effective against recurrent tumor could substantially impact the morbidity and median survival of patients with this disease. The purpose of this study was to investigate the effects of plant secondary metabolites from Scutellaria baicalensis on cells from primary and recurrent gliomas prior to, and following, selection for therapy resistance.
There are many reports of the medicinal utility of the Chinese herb S. baicalensis for the treatment of a host of diseases. Although there have been numerous studies on the effects of S. baicalensis extracts and isolated flavonoids on cancer cells, few have studied their effects on glioma cells. Further, there are no studies examining the effect of extracts of this herb on cells from tumors that have recurred following standard therapies. We, therefore, tested an ethanol extract from S. baicalensis for its ability to inhibit the growth of cells from primary gliomas, cells from recurrent gliomas from the same patient and cells selected for resistance to BCNU therapy in vitro. We also tested cells cultured from primary gliomas after only 5 serial passages in vitro. Our work demonstrated that the S. baicalensis extract caused a dose-dependent inhibition of growth of all of these cell lines, irrespective of whether they were from a primary or recurrent tumor. Reduced metabolic activity due to the S. baicalensis extract was demonstrated using AlamarBlue™, and cytotoxicity was demonstrated by colony forming assays as well as by trypan blue exclusion and microscopic examination. A dose of 50 μg/ml of the extract typically reduced the population of glioma cells by at least 50% of the control (untreated) population; however, the same dose of extract had little, if any, effect on HJ cells, the cultured normal glia. In fact, 25 -100 μg/ml of S. baicalensis extract did not induce the same inhibitory effects in HJ cells that were observed in all of the glioma cells tested. The normal glial cells also failed to undergo the typical morphological changes seen following treatment of the glioma cells. This suggests that the S. baicalensis extract may not affect normal cells to the extent that it affects tumor cells, thus warranting in vivo studies.
The effect of the S. baicalensis extract on cells from recurrent tumor is of particular interest. Recurrent tumor generally arises from cells in the primary tumor that survived treatment with radiation and chemotherapy; thus, recurrent tumor is often refractile to further therapies. Our data demonstrates that not only are the cells from recurrent tumor sensitive to the effects of the S. baicalensis extract, but cells from primary and recurrent tumor selected for resistance to 10 μg/ml BCNU are sensitive to lower doses when given in combination with S. baicalensis extract. This suggests that this extract may have its greatest utility when used in combination with currently available therapies. This work is similar to previous studies showing that S. baicalensis extracts may reduce tumor growth and proliferation when applied to chemotherapy and radiation resistant tumors in various forms of cancer [18,[43][44][45]. One postulated mechanism for this is the inhibition of extracellular signal-regulated kinase (ERK). Pathways involving ERK are activated in most GBMs [46], and inhibition of ERK has been shown to inhibit growth of GBMs and medulloblastomas alone and in combination with temozolomide [47,48]. The effects of extracts and isolated flavonoids are not all antiproliferative. Choi et al [49] found that an aqueous extract from S. baicalensis reduced the apoptotic death induced in neuronal HT-22 cells exposed to H 2 O 2 by increased Bcl-2 and reducing Bax levels. In addition, baicalein, a major component of the S. baicalensis extract, can exert either pro-or anti-apoptotic activity depending on the cell type. For example, in addition to its antiproliferative effects, baicalein has been shown to prevent the loss of viability and apoptosis induced in the human glioma cell line A172 by cisplatin [50]. Regardless of this, our data has demonstrated a potential role for the use of S. baicalensis as an adjuvant therapy in the treatment of human malignant brain tumors, particularly recurrent tumors.

Conclusion
In summary, 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 in progress.