The Traditional Chinese Medicinal Formula BDL301 Suppresses Tumor Growth by Inhibiting STAT3 Pathway and Inducing Apoptosis in Colorectal Cancer Cells

Abstract

The traditional Chinese medicinal formula BDL301 has been used to inhibit inflammation for hundreds of years. The development of colorectal cancer and chronic inflammation are closely related. In this study, we investigated whether BDL301 could inhibit tumor growth. We found that angiogenesis and tumor growth were both inhibited in vivo. In addition, apoptosis was induced and the signal transducer and activator of transcription-3 (STAT3) pathway were suppressed in the colorectal cancer cells in vitro and in vivo by BDL301. This study demonstrates that BDL301 exerted significant anticancer activity by inhibiting the STAT3 pathways and inducing apoptosis in colorectal cancer cells.

Introduction

Traditional Chinese medicine includes many medicinal formulas that are composed of a variety of Chinese herbal medicines, and are widely used for cancer treatment. However, basic research assessing their molecular composition and mechanism of action is lacking. BDL, a Chinese medicinal formula containing rhubarb, indigowoad, wine cornus, Divaricate Saposhnikovia, baikal skullcap, Mongolian milkvetch, and largehead atractylodes rhizome has been used to improve immunity and combat inflammation for hundreds of years. Additional research at Shanghai Changzheng Hospital demonstrated that it was an effective cancer treatment. Previous studies of BDL demonstrated that it could stimulate immune activity in normal mice (Niu et al., 2000). Furthermore, the administration of BDL could enhance the anticancer effects of 5-FU and prolong the survival of both tumor-bearing and tumor-free mice treated with 5-FU (Xu et al., 2005). In our article, quality-controlled BDL is called BDL301.

A large number of studies reported a link between chronic inflammation and the development of colorectal cancer (Grivennikov et al., 2010). Clinical studies demonstrated that the mucosa of patients with colorectal cancer exhibited signs typical of inflammation, and preliminary findings suggested that downregulating inflammatory factors could inhibit tumor growth and improve prognosis (Cui et al., 2010; Pendyala et al., 2011). In addition, patients with inflammatory bowel diseases are predisposed to the development of colon cancer (Klampfer, 2011). Therefore, inflammation is a potentially valuable target for cancer prevention and treatment (Porta et al., 2011). Thus, we investigated whether BDL301 could inhibit tumor growth since the medicinal formulas in BDL301 had been used to inhibit inflammation for hundreds of years.

Pathological angiogenesis is a hallmark of cancer, as well as various ischemic and inflammatory diseases (Carmeliet and Jain, 2000). Tumor-induced angiogenesis is initiated by the release of angiogenic cytokines from tumors and inflammatory cells (D'Amore and Thompson, 1987; Blood and Zetter, 1990; Leek et al., 1994; Sunderkötter et al., 1994). It was estimated that >90% cancer deaths occur due to angiogenesis, invasion, and the metastasis of cancer to vital organs. Angiogenesis is a key process that mediates metastasis (Jeon et al., 2010).

Signal transducer and activator of transcription (STATs) comprise a family of cytoplasmic transcription factors that play an important role in the Janus-activated kinase (JAK)-STAT signaling pathway. Upon stimulation by molecules such as IL-6, STATs translocate to the nucleus and regulate the expression of genes involved in inflammation. STATs are common targets of cancer treatments. The constitutive activation of STAT3 by JAK plays a major role in human cancers, and p-STAT3 plays an important role in stimulating the development of colorectal cancer (Garcia et al., 2001; Park et al., 2008). Elevated serum levels of proinflammatory cytokines such as IL-6 in colorectal carcinoma patients can activate NF-κB and STATs, which might serve as a prognostic tool (Knupfer et al., 2010; Klampfer, 2011).

In this study, we found that BDL301 effectively inhibited tumor growth and angiogenesis by targeting and downregulating STAT3 signaling pathways that play an important role in the progression of colorectal cancer. Besides, the results showed that inducing apoptosis by BDL301 might also be a primary mechanism for tumor regression.

Materials and Methods

Cell lines and in vivo experiments

The CT26 and HCT116 cell lines were directly obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. They were cultured in RPMI-1640 (HyClone) supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin (Gino Shanghai Biological Technology Co., Ltd.) at 37°C in a humidified atmosphere with a 95%:5% (v/v) mixture of air and CO₂.

BDL301 sample preparation

BDL301 is a formula containing granules of rhubarb emodin and chrysophanol (roots), indigowoad epigoitrin (roots), wine cornus loganin, Divaricate Saposhnikovia 4′-O-β-glucopyranosyl-5-O-methylvisamminol (roots), baikal skullcap (roots), Mongolian milkvetch (roots), and largehead atractylodes rhizome (roots). A total of 1.9 g of BDL301 was dissolved in 10 mL of saline at 100°C. The mixture was vortexed for 5 min, cooled to room temperature, and filtered through a 0.22-μm membrane before use. For analysis using high-performance liquid chromatography (HPLC), the sample was added to a 1:1 mixture of methanol (Chromatographically pure; Tedia Company) and acetonitrile (Analytically Pure, AR; Tedia Company). The mixture was centrifuged at 10,000 rpm for 20 min, and the supernatant was filtered through a 0.22-μm membrane before use.

BDL301 extract serum

Experiments were performed with male Sprague Dawley (SD) rats weighing ∼120 g. The rats were divided into two groups: orally treated with BDL301, and blank treated (control). The rats in the treatment group were treated with 15 mL of BDL301 twice daily for 3 days. The rats in the blank group were given the same amount of saline. One hour after the final administration, blood was collected aseptically from the rats. The blood was vortexed for 15 min at 2000 rpm after 2 h, then, the serum was carefully isolated, filtered through a 0.22-μm membrane, diluted in RPMI-1640 to a final concentration of 10%, and stored at −20°C.

Animal model

Mice were purchased from Shanghai Slac Laboratory Animal Co., Ltd., and housed in the animal facility at Shanghai Tenth People's Hospital, Tongji University. Mice were maintained under pathogen-free conditions, and all in vivo experiments were performed in accordance with the established institutional guidelines. The Animal Care Committee of Tongji University School of Medicine approved the study protocol. Experiments were performed using 5-week-old female BALB/c mice. Mice were given a subcutaneous injection of 1×10⁶ CT26 cells in 100 μL RPMI-1640 culture media to induce tumor growth. Ten mice were further divided randomly into two groups (five mice per group). One group was subcutaneously injected with CT26 cells treated with 0.5% BDL301 for 24 h; the time was selected based on a pilot experiment. The other group was subcutaneously injected with the same number of untreated CT26 cells. The initial body weight of each mouse was recorded. After 1 week, the mice were sacrificed, and the solid tumors were removed. Tumor weight was recorded, and tumor volume was measured using vernier calipers and the formula A×B ²×0.5, where A and B are the longest and shortest diameters of the tumor, respectively.

High-performance liquid chromatography

Little is known about the chemical composition of BDL301 and BDL301 extract serum (BES). Quality control is critically important to guarantee drug safety and consistency among batches during the development of botanical drugs, particularly for formulated preparations such as BDL301. In this study, some chemical markers of BDL301 and BES were quantified and compared with a biological reference preparation (BRP). The analyses were performed using an Agilent 1200 HPLC System equipped with a quaternionic pump, an autodegasser, a variable wavelength detector (VWD), an autosampler, and a column oven. Samples were separated on an Atlantis C18 column (5 μm, 250×4.6 mm).

All of the BRPs were purchased from the National Institute for Food and Drug Control, Beijing, China. For emodin and chrysophanol (batch lots [BLs] 110756-200110 and 100796-200716, respectively), the mobile phase was methanol and 0.1% phosphoric acid solution (AR; Tedia Company) (85:15, v/v), and the VWD was monitored at 254 nm. For epigoitrin (BL 111753-200601), the mobile phase was methanol (chromatographically pure [CP]) and 0.02% phosphoric acid solution (AR; Tedia Company) (10:90, v/v). The VWD was monitored at 245 nm. For loganin (BL 111640-200604), the mobile phase was methanol (CP) and water (ultrapure) (35:65, v/v), and the VWD was monitored at 240 nm. For 4′-O-β-glucopyranosyl-5-O-methylvisamminol (BL 111523-200405), the mobile phase was methanol CP in water (AR) (40:60, v/v), and the VWD was monitored at 254 nm. All analyses times were 150 min. The number of theoretical plates was not less than 2500. The mobile phase rate was 1.0 mL/min, and the column was maintained at room temperature (∼25°C), and the instrument was operated using an Agilent ChemStation.

Hematoxylin and Eosin staining

Tumor tissues were fixed in 4% paraformaldehyde (PFA) and stored at 4°C. Samples were warmed to room temperature for 10 min, and washed briefly in deionized water. Sections were stained with Harris Hematoxylin solution stain (Goodbio Technology Co., Ltd.) for 3–8 min, and washed with deionized water. Following treatment with 1% acid–alcohol for 30 s, the sections were washed with running tap water for 1 min. Next, a 0.6% bluing reagent was added, followed by a rinse with deionized water for 5 min. Finally, eosin–phloxine solution (Goodbio Technology Co., Ltd.) was added, and the slides were washed and stored in deionized water.

Proliferation, interference, and flow cytometry

CT26 cells in the logarithmic growth phase were trypsinized, resuspended to form a single-celled suspension, and 6000 cells were transferred to each well of a 96-well plate. Cells were treated with 0, 0.1%, 0.5%, and 1% BDL301 for 24 h. Then, the Cell Counting Kit-8 (CCK-8; Shanghai SunBio Medical Biotechnology) reagent was added, and the cells were incubated at 37°C for 1 h to allow the color to change. The optical density (OD) values were read using a microplate reader at 450 nm. Each experiment was performed five times independently.

CT26 and HCT116 cells were plated in six-well plates. After 24 h, cells in the control, 0.5% BDL301-treated, blank serum, and BES-treated groups were collected, washed twice in cold phosphate buffered saline (PBS), mixed with 100 μL of 1× binding buffer, and incubated at room temperature for 15 min with an Annexin-V/Propidium Iodide (BD Biosciences) double staining solution. Stained cells were analyzed by flow cytometry.

TUNEL assay

TUNEL assay was performed using the In Situ Cell Death Detection Kit from Roche as per the manufacturer's instructions. Briefly after dewaxing and rehydrating the tissue section, incubated the tissue section for 20 min with proteinase K working solution and with TUNEL reaction mixture for 1 h at 37°C. Then the samples were mounted in the ProLong^® Gold Antifade Reagent with DAPI. Fluorescent images were captured using a Nikon fluorescence microscope at 40× magnification. The total number of DAPI-positive cells and total number of TUNEL-positive cells were counted from five images from each sample. Each experiment was repeated three times.

Enzyme-linked immunosorbent assay

HCT116 and CT26 cells were cultured in 10% BES and blank serum and the media were carefully isolated after 24 h. The concentration of IL-6 in the 10% BES, 10% blank serum, and cell culture supernatants was detected using a human and rat IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Perotech) following the manufacturer's instructions.

Western blotting

Tumor tissues were snap frozen in liquid nitrogen, and pulverized. The tumor cells were resuspended in 2× loading buffer for 30 min, and cleared by centrifugation at 12,000 rpm for 1 min at 4°C. Protein extracts were incubated in 6× loading buffer (without bromophenol blue), incubated at 100°C for 10 min, and then cleared by centrifugation at 12,000 rpm for 1 min at 4°C. Protein concentrations were determined using the BCA protein assay (Thermo Scientific). Supernatants were then separated by SDS-PAGE, and analyzed by western blotting using primary antibodies against STAT3, p-STAT3 (Tyr705), SHP1, SHP2, NF-κB p65, p-IκBα (Ser32), β-actin (all diluted 1:1000; Cell Signaling Technology). Secondary antibodies were Goat anti Rabbit IRDye 800CW and Goat anti Mouse IRDye 700CW (Li-Cor).

Immunohistochemical staining for CD31, p-STAT3, STAT3, and P65

Isolated tumor tissues were fixed by immersion in 4% PFA for 48 h at 4°C and embedded in paraffin. The paraffin-embedded sections were placed on poly-l-lysine-coated slides. The slides were air-dried overnight at room temperature, covered, and stored at 4°C until immunostaining. Slides were immersed in 0.3% H₂O₂ for 25 min to abolish endogenous peroxidase activity, and then rinsed with PBS (pH 7.4). Slides were incubated in 5% bovine serum albumin for 20 min at room temperature to block nonspecific staining, and incubated with primary antibodies against CD31 (1:50 dilution; Santa Cruz Biotechnology, Inc.), STAT3, and p-STAT3 (Tyr705) (1:50 dilution, Cell Signaling Technology), P65 (1:200 dilution; Cell Signaling Technology) in a humidified chamber overnight at 4°C.

All slides were incubated with biotinylated secondary antibodies for 1 h at 37°C, followed by horseradish peroxidase-conjugated streptavidin for 50 min at room temperature, and detected using a DAB kit (DAKO). Harris Hematoxylin solution was applied for 3 min, and washed with deionized water. Following treatment with 1% acid alcohol for 30 s, the slides were washed in running tap water. Next, 0.6% bluing reagent was added, and samples were rinsed again in deionized water. The sections were examined under a microscope, and analyzed using ip-win32.

Statistical analysis

Data are presented as means±standard deviations, and statistical comparisons between the treated and control groups were performed using paired sample t-tests or one-way ANOVA. p-Values <0.05 were considered statistically significant.

Results

HPLC analysis of BRP, BDL301, BES, and blank serum

To quantify BDL301 and BES, we purchased pure standards for the five commercially available compounds. The chemical constituents of the individual herbs in BDL have been reported in Pharmacopoeia of The People's Republic of China, Volume I, 2005. Based on the HPLC retention time, UV absorption, and mass spectra of the standards, the corresponding compounds were identified and quantified. Emodin and chrysophanol in rhubarb, epigoitrin in indigowoad, loganin in wine cornus, and 4′-O-β-glucopyranosyl-5-O-methylvisamminol in Divaricate Saposhnikovia were quantified in BDL301, BES, and the blank serum by HPLC, as shown in Figures 1 and 2 and Table 1.

FIG. 1.

The quantitative of the main components in BDL301, BES, and blank serum. (A1–A5), (B1–B4), (C1–C4) Emodin, chrysophanol, epigoitrin, loganin, 4′-O-β-glucopyranosyl-5-O-methylvisamminol high-performance liquid chromatography figures of the biological reference preparation, BDL301 and BES. BES, BDL301 extract serum.

FIG. 2.

The molecular formula of the main components. (A1–A5) Emodin, chrysophanol, epigoitrin, loganin, 4′-O-β-glucopyranosyl-5-O-methylvisamminol by SciFinder.

Table 1.

The Quantification of Emodin, Chrysophanol, Epigoitrin, Loganin, 4′-O-β-Glucopyranosyl-5-O-Methylvisamminol in BDL301, BDL301 Extract Serum, and Blank Serum (n=2)

BDL301	Concentration (mg/mL)	Components	BDL301 (μg/mL)	BDL301-extract serum (μg/mL)	Blank serum (μg/mL)
Rhubarb	18	Emodin	3.33	0.653	<0.01
		Chrysophanol	3.87	0.151	<0.01
Indigowoad root	30	Epigoitrin	13.2	1.01	<0.01
Wine cornus	30	Loganin	353	2.05	<0.01
Divaricate Saposhnikovia	10	4′-O-β-glucopyranosyl-5-O-methylvisamminol	5.6	<0.01	<0.01
Baikal skullcap root	12	—	—	—	—
Mongolian milkvetch root	60	—	—	—	—
Largehead atractylodes rhizome	30	—	—	—	—

BDL301 inhibits the proliferation and induces apoptosis HCT116 and CT26 cells in vitro and in vivo

The CCK-8 assay was used to analyze the proliferation of control and BDL301-treated CT26 cells. The OD value at 450 nm represented the number of viable cells. Compared with the control group, the number of viable CT26 cells treated for 24 h with 0.5% and 1.0% BDL301 were significantly decreased (**p<0.005), whereas 0.5% BDL301 did not have a significant effect (^▿ p>0.05; Fig. 3). To assess the cell cycle distribution, HCT116 and CT26 cells were treated for 24 h with BES, and CT26 cells treated for 24 h with BDL301 were analyzed by flow cytometry. The results revealed a significant decrease in cells in the S phase, and apoptosis-inducing effect of BES on HCT116 and CT26. The TUNEL assay showed an increased total apoptosis cell number and percent apoptosis in the implantation tumors of the 0.5% BDL301-treated group, as shown in Figures 4 and 5 and Tables 2 –4.

FIG. 3.

A proliferation suppression curve of control group and BDL301 treatment in CT26 were conducted by the Cell Counting Kit-8 (CCK-8) assay. The OD value at 450 nm represented the viable cell numbers. All experiments were carried out five times independently (n=3, ^▿ p>0.05, **p<0.005). OD, optical density.

FIG. 4.

Effects of BDL301 on cell cycles. (A, B) A representative of BES influence on cell cycle distribution of HCT116, CT26 cells. (C, D) A representative of BES influence on cell cycle distribution of CT26 cells (n=3, *p<0.05, **p<0.005).

FIG. 5.

(A) Apoptosis-inducing effect of BES on HCT116 and CT26 detected by Annexin V-Propidium Iodide method. (B) The total numbers and percentage of TUNEL-positive cells were measured using TUNEL assay in the implantation tumors of the control and 0.5% BDL301-treated groups. A significant difference was found between the two groups (n=3, *p<0.05, **p<0.005).

Table 2.

Cell Cycles Analyses of CT26 Cells in Control and BDL301 for 24 H Groups

	CT26
Groups (n=3)	G₀/G₁ (%)	S (%)	G₂/M (%)
Control	46.60±0.38	45.10±0.29	8.30±0.22
BDL301	52.00±0.73	41.19±0.46	6.81±0.28
p	0.0025^a	0.0006^a	0.0124^b

p<0.005.

p<0.05.

Table 3.

Cell Cycle Analyses of HCT116 and CT26 Cells in Blank and BDL301 Extract Serum for 24 H Groups

	HCT116			CT26
Groups (n=3)	G₀/G₁ (%)	S (%)	G₂/M (%)	G₀/G₁ (%)	S (%)	G₂/M (%)
Blank	40.02±0.64	33.94±0.22	26.03±0.78	42.52±1.05	45.23±0.62	12.24±0.85
BDL301	42.47±0.50	30.12±0.64	27.40±0.20	44.72±0.53	42.17±0.35	13.08±0.79
p	0.015^a	0.007^a	0.078	0.109	0.011^a	0.200

p<0.05.

Table 4.

Total Apoptosis Cell Numbers and Percent Apoptosis in the Implantation Tumors of the Control and 0.5% BDL301-Treated Groups

Groups (n=3)	Total apoptosis cell numbers	Percent apoptosis
Control	86.4±27.4	0.0055±0.0017
BDL301	428.0±117.5	0.0273±0.0075
p	0.0028^a	0.0028^a

p<0.005.

BDL301 inhibits tumor growth and angiogenesis in CT26 mouse models

To observe angiogenesis and apoptosis, the implanted tumors were stained with Hematoxylin and Eosin (H&E), which revealed that the tumors in the treatment group had more apoptosis and necrosis than the control group. In addition, immunohistochemistry (IHC) revealed that the important pan-endothelial marker CD31 was downregulated in the treatment group compared with control, as shown in Figure 6C and E. The initial weights of the mice in the control and BDL301 treatment groups were 18.57±1.2 g and 18.72±0.6 g, respectively (^▿ p>0.05). There was no significant difference in body weight between the control and 0.5% BDL301-treated groups at baseline. After 1 week, the solid tumors were removed, and the implant tumor weights in the two groups were 1.24±0.26 g and 0.76±0.13 g, respectively (**p<0.005), and the tumor volume was 0.84±0.17 cm³ and 0.35±0.2 cm³, respectively (**p<0.005), as shown in Figure 6A and B.

FIG. 6.

BDL301 inhibits tumor growth and tumor angiogenesis and immunohistochemical staining of CD31, p-STAT3, STAT3, P65. (A, B) The weights of mice, implant tumor weight, and volume in control and BDL301 treatment groups (^▿ p>0.05, **p<0.005). (C) Hematoxylin and Eosin staining was used to visualize the formalin-fixed sections of the implantation tumors of the two groups. (D) Paraffin sections stained for p-STAT3, STAT3 in the CT26 mouse models (*p<0.05). (E) Paraffin sections stained for CD31 in the CT26 mouse models (*p<0.05). STAT3, Signal transducer and activator of transcription-3.

BDL301 inhibits p-STAT3 expression by stimulating SHP1 and SHP2 and inhibiting IL-6 secretion

To assess the effect of BDL301 on the STAT3 signaling pathway, we assessed the levels of STAT3, p-STAT3, SHP1, and SHP2 using western blotting and IHC. As shown in Figures 6D, 7, and 8, p-STAT3 levels were suppressed by BDL301 in a time-dependent manner, but STAT3 levels were unchanged. A similar effect was seen in the BES group (Fig. 8). There was a significant decrease in p-STAT3 24 h after treatment, suggesting that both BDL301 and BES might affect p-STAT3 expression. Furthermore, we also detected increased SHP1 and SHP2 expression. These results suggest that BDL301 could inhibit p-STAT3 expression by stimulating SHP1 and SHP2. An ELISA also revealed that BES suppressed IL-6 secretion into the cell culture media, as shown in Table 5.

FIG. 7.

Western blot analysis for STAT3, p-STAT3, P65, p-IκBα, SHP1, SHP2. (A, C) STAT3, p-STAT3, P65, p-P65, p-IκBα protein level were suppressed by BDL301 in a time-dependent manner in CT26. Accordingly, p-IκBα showed a lower level than the control group. (B, C) STAT3, p-STAT3, SHP1, SHP2, P65, p-IκBα protein level in CT26 mouse models (n=3, **p<0.005).

FIG. 8.

(A, B) STAT3, p-STAT3, p-P65, p-IκBα protein level in HCT116 and CT26 cells after 24 h treatment by blank serum and BES (n=3, *p<0.05, **p<0.005).

Table 5.

The Levels of IL-6 in the 10% BES, 10% Blank Serum and the Cell Culture Supernatant

Groups	10% original serum	HCT116	CT26
Blank serum	13.53±0.59	16.60±0.16	13.67±0.23
BES	14.17±0.23	15.44±0.23	13.86±0.20

BES, BDL301 extract serum.

BDL301 decreases p65 and p-P65 by inhibiting p-IκBα expression in vitro

To assess the effect of BDL301 on the IκBα/NF-κB signaling pathway in vitro, we evaluated the expression of p65, p-p65, and p-IκBα using western blotting or IHC. There was no significant change in the levels of P65 and p-IκBα observed in vivo by IHC and western blot (Fig. 7B and Supplementary Fig. S1; Supplementary Data are available online at www-liebertpub-com.web.bisu.edu.cn/dna), whereas the protein levels of p-P65 and p-IκBα were suppressed by BDL301 in a time-dependent manner (Fig. 7A). The same expression patterns were observed in the BES group (Fig. 8).

Discussion

In the current study, the traditional Chinese medicinal formula BDL301 and its extract serum inhibited the growth of HCT116 and CT26 cells and mouse CT26 tumor models. BDL301 suppressed the STAT3 signaling pathway and induced apoptosis. Components of BDL301 and BES were quantified by HPLC for quality control. Generally, the data suggested that the medicine we used in vitro (10% BES) could reach comparable concentrations in vivo (0.5% BDL301). 4′-O-β-glucopyranosyl-5-O-methylvisamminol was undetectable, demonstrating that its chemical properties changed after being metabolized (Li et al., 2010). This remains to be clarified in future studies.

Angiogenesis has traditionally been evaluated by measuring microvessel density on fixed tissues immunostained for a variety of endothelial markers, including CD31 (Hasan et al., 2002). In our study, analysis of the implanted tumors from the BDL301 treatment group exhibited more apoptosis and necrosis by H&E staining. In addition, the important pan-endothelial marker CD31 was downregulated, as assessed by western blotting and IHC, in the BDL301 treatment group, which was accompanied by reduced tumor weight and volume, apoptosis, and necrosis. This suggests that BDL blocked angiogenesis to suppress tumor growth.

The expression of STAT3 and p-STAT3 was increased significantly in invasive colorectal adenocarcinoma compared with normal intestinal epithelial tissue, and their expression was closely related to tumor proliferation and lymph node metastasis (Lassmann et al., 2007). Statistical analysis showed that the expression of p-STAT3 was closely related to the degree of tumor invasion in colorectal cancer (Kusaba et al., 2005). The phosphorylation of STAT3 plays a critical role in the transformation and proliferation of tumor cells (Aggarwal et al., 2006). Numerous protein tyrosine phosphatases have been implicated in STAT3 signaling, including SHP1 and SHP2, which downregulates STAT3 phosphorylation (Pandey et al., 2009).

Our results clearly demonstrate that the levels of p-STAT3 were suppressed by BDL301 and BES treatment in HCT116 and CT26 cells. SHP1 and SHP2 were upregulated, with a more significant increase in the expression of SHP2. In addition, the levels of IL-6 were higher in BES than the blank serum immediately after gavage administration, whereas the levels of secreted IL-6 in the culture supernatants of HCT116 and CT26 cells were obviously reduced after 24 h. These results suggest that BDL301 inhibits p-STAT3 expression by inducing SHP1, SHP2, and suppressing IL-6 secretion.

In addition, NF-κB signaling can upregulate the production of key proinflammatory cytokines and enzymes such as IL-6 (Barnes et al., 1997). The phosphorylation of IκBα is a central point where diverse stimuli converge to regulate NF-κB (Baldwin et al., 2001; Jeon et al., 2010). NF-κB is inactivated in the cytoplasm by binding to IκB inhibitory proteins. When phosphorylated on serine 32 and serine 36, IκBα is targeted for degradation by the ubiquitin/26S proteasome pathway, liberating the NF-κB heterodimer to translocate to the nucleus and bind DNA.

The IκB family includes IκBα, IκBβ, IκBγ, IκBɛ, Bcl-3, the precursors of NF-κB1 (p105) and NF-κB2 (p100), and the Drosophila protein Cactus. Only IκBα, IκBβ, and IκBγ contain the N-terminal regulatory regions that are required for stimulus-induced degradation, the key step in NF-κB activation. IκBα was the first IκB family member to be cloned, and it remains the best characterized (Karin et al., 2000). A previous study reported that IκB kinase α plays an NF-κB-independent role in the control of metastatogenesis, which is described as a bridge between inflammation and cancer (Greten and Karin, 2004; Karin, 2008). Consistent with the STAT3 signal pathway, the current study showed that the protein levels of p-P65 and p-IκBα were significantly suppressed by BDL301 and BES in vitro. Interestingly, there is not a difference between the control and BDL301 groups in vivo. Whether and how the IκBα/NF-κB signaling pathway is cooperating with the inhibition of STAT3 signaling pathway remains to be further studied.

These data demonstrate that BDL301 has significant anticancer activities by the STAT3 signaling pathways inhibition and the apoptosis inducing. Our study is the first to isolate BES from SD rats, and analyze and quantify the components of both BDL301 and BES by HPLC. Our results show, for the first time, that BDL301 could suppress tumor growth by inhibiting tumor angiogenesis in vitro and in vivo through targeting the STAT3 signaling pathways and inducing apoptosis. Nevertheless, the important chemical constituents of BDL301 are yet to be elucidated, and analysis of the individual herbs in BDL301 might be required.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Aggarwal

B.B.

, Sethi

, Ahn

K.S.

, Sandur

S.K.

, Pandey

M.K.

, Kunnumakkara

A.B.

, Sung

, and Ichikawa

(2006). Targeting signal-transducer-and-activator-of-transcription-3 for prevention and therapy of cancer: modern target but ancient solution. Ann N Y Acad Sci, 1091, 151–169.

Baldwin

A.S.

(2001). Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest, 107, 241–246.

Barnes

P.J.

, and Karin

(1997). Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med, 336, 1066–1071.

Blood

C.H.

, and Zetter

B.R.

(1990). Tumor interactions with the vasculature: angiogenesis and tumor metastasis. Biochim Biophys Acta, 1032, 89–118.

Carmeliet

, and Jain

R.K.

(2000). Angiogenesis in cancer and other diseases. Nature, 407, 249–257.

Cui

, Jin

, Hofseth

A.B.

, Pena

, Habiger

, Chumanevich

, Poudyal

, Nagarkatti

P.S.

, Singh

U.P.

, and Hofseth

L.J.

(2010). Resveratrol suppresses colitis and colon cancer associated with colitis. Cancer Prev Res (Phila), 3, 549–559.

D'amore

P.A.

, and Thompson

R.W.

(1987). Mechanisms of angiogenesis. Annu Rev Physiol, 49, 453–464.

Garcia

, Bowman

T.L.

, Niu

, Yu

, Minton

, Muro-Cacho

C.A.

, Cox

C.E.

, Falcone

, Fairclough

, Parsons

, Laudano

, Gazit

, Levitzki

, Kraker

, and Jove

(2001). Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene, 20, 2499–2513.

Greten

F.R.

, and Karin

(2004). The IKK/NF-kappaB activation pathway—a target for prevention and treatment of cancer. Cancer Lett, 206, 193–199.

10.

Grivennikov

S.I.

, Greten

F.R.

, and Karin

(2010). Immunity, inflammation, and cancer. Cell, 140, 883–899.

11.

Hasan

, Byers

, and Jayson

G.C.

(2002). Intra-tumoural microvessel density in human solid tumours. Br J Cancer, 86, 1566–1577.

12.

Jeon

K.I.

, Xu

, Aizawa

, Lim

J.H.

, Jono

, Kwon

D.S.

, Abe

, Berk

B.C.

, Li

J.D.

, and Yan

(2010). Vinpocetine inhibits NF-kappaB-dependent inflammation via an IKK-dependent but PDE-independent mechanism. Proc Natl Acad Sci U S A, 107, 9795–9800.

13.

Karin

, and Ben-Neriah

(2000). Phosphorylation meets ubiquitination: the control of NF-kappaB activity. Annu Rev Immunol, 18, 621–663.

14.

Karin

(2008). The IkappaB kinase—a bridge between inflammation and cancer. Cell Res, 18, 334–342.

15.

Klampfer

(2011). Cytokines, inflammation and colon cancer. Curr Cancer Drug Targets, 11, 451–464.

16.

Knupfer

, and Preiss

(2010). Serum interleukin-6 levels in colorectal cancer patients–a summary of published results. Int J Colorectal Dis, 25, 135–140.

17.

Kusaba

, Nakayama

, Yamazumi

, Yakata

, Yoshizaki

, Nagayasu

, and Sekine

(2005). Expression of p-STAT3 in human colorectal adenocarcinoma and adenoma; correlation with clinicopathological factors. J Clin Pathol, 58, 833–838.

18.

Lassmann

, Schuster

, Walch

, Göbel

, Jütting

, Makowiec

, Hopt

, and Werner

(2007). STAT3 mRNA and protein expression in colorectal cancer: effects on STAT3-inducible targets linked to cell survival and proliferation. J Clin Pathol, 60, 173–179.

19.

Leek

R.D.

, Harris

A.L.

, and Lewis

C.E.

(1994). Cytokine networks in solid human tumors: regulation of angiogenesis. J Leukoc Biol, 56, 423–435.

20.

Y.Y.

, Wang

, Chen

, Zhao

, Zhang

, Chai

Y.F.

, and Zhang

G.Q.

(2010). RRLC-TOF/MS in identification of constituents and metabolites of Radix Saposhnikoviae in rat plasma and urine. Academic Journal of Second Military Medical University, 31, 760–763.

21.

Niu

, Tan

, Turner

J.G.

, Brabham

J.G.

, Burdelya

L.G.

, Crucian

B.E.

, Wall-Apelt

, Zhao

R.J.

, and Yu

(2000). Bing de ling, a Chinese herbal formula, stimulates multifaceted immunologic responses in mice. DNA Cell Biol, 19, 515–520.

22.

Pandey

M.K.

, Sung

, Ahn

K.S.

, and Aggarwal

B.B.

(2009). Butein suppresses constitutive and inducible signal transducer and activator of transcription (STAT) 3 activation and STAT3-regulated gene products through the induction of a protein tyrosine phosphatase SHP-1. Mol Pharmacol, 75, 525–533.

23.

Park

J.K.

, Hong

, Kim

K.J.

, Lee

T.B.

, and Lim

S.C.

(2008). Significance of p-STAT3 expression in human colorectal adenocarcinoma. Oncol Rep, 20, 597–604.

24.

Pendyala

, Neff

L.M.

, Suárez-Fariñas

, and Holt

P.R.

(2011). Diet-induced weight loss reduces colorectal inflammation: implications for colorectal carcinogenesis. Am J Clin Nutr, 93, 234–242.

25.

Porta

, Riboldi

, and Sica

(2011). Mechanisms linking pathogens-associated inflammation and cancer. Cancer Lett, 305, 250–262.

26.

Sunderkötter

, Steinbrink

, Goebeler

, Bhardwaj

, and Sorg

(1994). Macrophages and angiogenesis. J Leukoc Biol, 55, 410–422.

27.

, Brabham

J.G.

, Zhang

, Munster

, Fields

, Zhao

R.J.

, and Yu

(2005). Chinese herbal formula, Bing De Ling, enhances antitumor effects and ameliorates weight loss induced by 5-fluorouracil in the mouse CT26 tumor model. DNA Cell Biol, 24, 470–475.

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