Herbal Extract SH003 Suppresses Tumor Growth and Metastasis of MDA-MB-231 Breast Cancer Cells by Inhibiting STAT3-IL-6 Signaling

Cancer inflammation promotes cancer progression, resulting in a high risk of cancer. Here, we demonstrate that our new herbal extract, SH003, suppresses both tumor growth and metastasis of MDA-MB-231 breast cancer cells via inhibiting STAT3-IL-6 signaling path. Our new herbal formula, SH003, mixed extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz, suppressed MDA-MB-231 tumor growth and lung metastasis in vivo and reduced the viability and metastatic abilities of MDA-MB-231 cells in vitro. Furthermore, SH003 inhibited STAT3 activation, which resulted in a reduction of IL-6 production. Therefore, we conclude that SH003 suppresses highly metastatic breast cancer growth and metastasis by inhibiting STAT3-IL-6 signaling path.


Introduction
Triple-negative breast cancer (TNBC) constituting 10-20% in breast cancer is highly metastasizing and recurrent with poorer prognoses [1][2][3]. Although TNBC is sensitive to chemotherapies, TNBC metastases frequently occur and shorten 5-year survival rates of patients [1,3]. Furthermore, target therapies for TNBC remain yet to be clearly elucidated in clinical trials. Recent studies in cancer therapeutics revisit a traditional herbal medicine, since herbal extracts or mixtures based on the traditional medicines have shown anticancer effects with no or less side effects compared to other anticancer therapeutics including chemical compounds and targeting antibodies [4][5][6]. Anticancer effects of herbal extracts from Astragalus membranaceus (Am), Angelica gigas (Ag), or Trichosanthes Kirilowii Maximowicz (Tk) have been revealed in different cancer cell types such as leukemia, hepat-ocellular carcinoma, colon cancer, non-small-cell lung cancer, and gastric cancer cells [7][8][9][10][11][12][13]. Furthermore, extracts from a mixture of Am and Ag have been shown to affect various diseases including hematologic diseases or endocrine disorders [14][15][16].
On the basis of the traditional medicine, SH003 was extracted from the herbal mixtures of Am, Ag, and Tk. SH003 showed anticancer effects on different breast cancer cells without affecting normal epithelial cell viability, both in vitro and in vivo. Moreover, SH003 suppresses MDA-MB-231 growth and metastasis by inhibiting STAT3-IL-6 pathway, thereby suggesting that SH003 may be useful for treating TNBC.

Reagents, Preparation of SH003, and Cell
Lines. SH003 consists of Am, Ag, and Tk, which is based on the principle of the traditional medicine. All extracts were provided from Hanpoong Pharm and Foods Company (Jeonju, Republic of Korea) manufactured by the Good Manufacturing Product (GMP). Dried extracts were dissolved in 30% ethanol to prepare a stock solution of 20 mg/mL. The stock solution was stored at −80 ∘ C. HPLC and UPLC were performed to confirm characteristics of herbal mixtures including each component (Hanpoong Pharm and Foods Company). Breast cancer cell lines, MCF-7 (hormone-positive), T47D (hormone-positive), SKBR-3 (HER-2-positive), BT-20 (TNBC, noninvasive), and MDA-MB-231 (TNBC, highly metastatic) were cultured in DMEM medium with 10% fetal bovine serum and 1% antibiotics. Rat normal intestinal epithelial cells (RIEs) were also cultured in the same condition as above. GBL-60 cells (kindly provided by Dr. Sun Ha Paek at Seoul National University Hospital, Seoul, Republic of Korea) isolated from the brain of a patient who suffered from brain-metastasized breast cancer were also cultured in DMEM, which was approved by an Institutional Review Board at the Seoul National University Hospital [31].

Cell Viability Assay and Flow
Cytometry. Cells were seeded on 96-well plates and treated with different herbal extracts for 24 hours to 72 hours. Cell viability was measured by MTT assays. Absorbance was read at 570 nm on the ELISA reader (Molecular Devices, Palo Alto, CA, USA). Cells were seeded in 6-well plates and treated with each extract for 24 hours. Cells were then harvested and stained with propidium iodide (PI, 50 g/mL) at room temperature in the dark. PI-positive cells were detected using FACSCalibur (BD Biosciences, San Jose, CA, USA).

Cell Migration, Invasion
Assay, and Anchorage-Independent Assay. Cell migration was measured by scratching assays. Cells were seeded in 6-well plates and then scratched. 24 hours after treatments with herbal extracts, migrated cell numbers were counted. For invasion assays, cells were cultured in the upper chambers precoated with matrigels and treated with each extract for 24 hours. After swapping the upper chamber carefully, invaded cell numbers in four fields randomly chosen were counted. For anchorage-independent assays, cells were cultured on soft agar plates and treated with extracts every second day. At day 15, cells were stained with 0.5% crystal violet to be visualized and colonies were counted with photomicroscope.

Results
3.1. HPLC Analysis of SH003. SH003 was extracted from the mixture of three different herbs (Figure 1(a)). A characterization of SH003 was based on retention times and UV spectra of standard chemicals at wavelengths of 260 nm (formononetin), 280 nm (decursin), and 330 nm (nodakenin): formononetin (3.6 min) for Am, decursin (6.1 min) for Ag, and nodakenin (11.0 min) for Ag (Figure 1(b)). However, we failed to detect an index compound for Tk. We assumed that technical limitations might cause that failure.  (Figure 2(a)). In addition, SH003 did not affect body weights of mice until 34 days (Figure 2(b)). When tumor tissues were stained with hematoxylin and eosin, we found that tumor cohort treated with SH003, compared to that with control, was well differentiated (Figure 2(c)). Tumor tissues were then stained with anti-CD31 antibodies to detect tumor vessels because tumor angiogenesis is a bridge for distant metastasis [35]. SH003 compared to the control reduced vessel numbers in tumor burdens by approximately 79% (Figures 2(c) and 2(d)). Thus, our data indicate that SH003 inhibits tumor growth.

SH003 Inhibits
Next, we conducted in vivo experimental metastasis assays to examine SH003 effect on a distant metastasis. When metastatic tumor colonies on lungs were counted, SH003 compared to control strongly reduced colony numbers by approximately 100% (Figure 2(e)). Thus, our data indicate that SH003 inhibits MDA-MB-231 tumor growth and metastasis, in vivo. cancer cell lines, SH003 much strongly inhibited MDA-MB-231 cell viability at 500 g/mL. When MDA-MB-231 cells were treated with SH003 at 500 g/mL for 72 hours, percentages of viable MDA-MB-231 cells were approximately 9.8% (Figure 3(a)). In addition, SH003 highly increased PI-positive apoptotic cell numbers (Figure 3(b)). Accordingly, SH003 caused PARP cleavages, whereas single components did not affect it (Figure 3(c)). In addition, SH003 did not affect normal rat intestinal epithelial cell viabilities, while an extract from either Ag or Tk reduced cell viability (Figure 3(d)). Those indicate that SH003 ameliorates adverse effects of each component of SH003. Thus, our data indicate that SH003 but not each component uniquely inhibits MDA-MB-231 cell proliferation via apoptosis without affecting normal cell viability.

SH003 Inhibits Cell Proliferation, Migration, Invasion, and
Anchorage-Independent Growth. We next examined whether SH003 affects migratory abilities of MDA-MB-231 cells. 50 g/mL of SH003 inhibited MDA-MB-231 cell migration by approximately 40% (Figure 4(a)). When we examined an invasiveness of MDA-MB-231 cells, SH003 at 50 g/mL inhibited cell invasion by 30% (Figure 4(b)). Next, in the soft agar assays, SH003 at 500 g/mL inhibited anchorageindependent growth of MDA-MB-231 by 95% (Figure 4(c)). Thus, our data indicate that SH003 inhibits in vitro metastatic abilities of MDA-MB-231 cells such as cell migration, invasion, and anchorage-independent growth. effects of SH003 on MDA-MB-231 cells, we next examined intracellular signaling pathway. Cells were treated with each extract at 50 g/mL ( Figure 5(a)) or 500 g/mL ( Figure 5(b)) for 15 minutes and subjected to the western blots. While phosphorylation of EGFR and SRC was partly reduced by 50 g/mL of SH003 or each component (Am, Ag, and Tk), STAT3 phosphorylation was strongly and selectively inhibited by SH003. Furthermore, STAT3 phosphorylation was also selectively inhibited by SH003 at 500 g/mL, while each component at 500 g/mL did not repress it. Therefore, we assumed that SH003 selectively blocked STAT3 phosphorylation.

SH003 Inhibits Expression of STAT3 Target
Genes and IL-6 Production. As SH003 suppressed STAT3 activation, we next examined whether SH003 affects expression patterns of STAT3-dependent genes. SH003 at 500 g/mL inhibited protein expression levels of STAT3-dependent genes such as Cyclin D, MMP-9, VEGF, and Survivin, while 50 g/mL of SH003 only decreased levels of Cyclin D1 and MMP-9 (Figures 6(c) and 6(d)). Those data indicated that expression patterns of those genes might be restricted by STAT3 transcriptional activity and that SH003 effect on those genes was not selective. As shown in Figure 6(c), we found that SH003 at 50 g/mL or 500 g/mL decreased IL-6 mRNA level by approximately 65% and 68%, respectively. Next, when MDA-MB-231 cells were treated with SH003 at 50 g/mL or 500 g/mL, their cultured media were subjected to ELISA assays. SH003 significantly inhibited secreted IL-6 level by approximately 33.5% and 38.6%, respectively ( Figure 6(d)).

Discussion
TNBC is highly metastasizing with a severe recurrence rate, causing a death of patients [1][2][3][36][37][38]. Nevertheless, TNBC is yet clearly curable. Traditional herbal medicines are revisited in cancer biology because those have less adverse effects but better anticancer effects [4,5]. In this study, we found that SH003 strongly suppressed tumor growth and metastasis of MDA-MB-231 cells defined as TNBC by inhibiting STAT3 activity. Thus, our new herbal extract SH003 appears to be useful for TNBC treatment. SH003 is extracted from the mixture of Am, Ag, and Tk. Our in vitro studies demonstrate that the extract from either Ag or Tk is highly toxic in normal intestinal epithelial cells, while our data and previous reports have shown that the extract from Am, Ag, or Tk inhibited cancer cell growth [7,[10][11][12][13]. However, SH003 ameliorated this adverse effect and effectively inhibited tumor growth and metastatic abilities of MDA-MB-231, highly metastatic TNBC cell line, in vitro. Furthermore, SH003 suppressed in vivo MDA-MB-231 growth and metastasis with no effect on body weights. Thus, SH003 is safe and effective, both in vivo and in vitro.
STAT3 is crucial for cancer development and metastasis as well as cancer inflammation [39][40][41][42][43] and frequently activated in different types of cancers such as breast, lung, renal, prostate, pancreatic, colon, gastric, cervical, and ovarian cancers [44][45][46][47]. SH003 inhibited STAT3 transcriptional activity, while each component did not affect it. Interestingly, 50 g/mL of SH003 reduced expression levels of MMP-9 and Cyclin D1 with no alterations of Survivin and VEGF, whereas 500 g/mL of SH003 reduced all we tested. Furthermore, each component also reduced protein expression of those genes. As SH003 uniquely inhibited STAT3-dependent IL-6 expression, our data suggest that SH003 may selectively target STAT3-IL-6 pathway. Meanwhile, we could not exclude a possibility that SH003 is likely to target other molecules beyond STAT3 to suppress MDA-MB-231 cell growth and metastatic abilities. In addition, it remains to be defined how SH003 has this selective effect.

Conclusions
In conclusion, we provide evidence that anticancer effect of SH003 on MDA-MB-231 cells results from the inhibition of STAT3-dependent IL-6 production. As STAT3 is mutated in different cancer types, it is worth testing if SH003 is able to target those types of cancer cells.