*Article* **Anti-Cancer Effects of a New Herbal Medicine PSY by Inhibiting the STAT3 Signaling Pathway in Colorectal Cancer Cells and Its Phytochemical Analysis**

**Sanghee Han 1, Hail Kim 1, Min Young Lee 2, Junhee Lee 1,2, Kwang Seok Ahn 1, In Jin Ha 1,2,\* and Seok-Geun Lee 1,2,3,\***


**Abstract:** Colorectal cancer (CRC) is an inflammation-associated common cancer worldwide. Paejangsan and Mori Cortex Radicis have been traditionally used for treating intestinal inflammatory diseases in Korea and China. In the present study, we developed a new herbal formula as an alternative to CRC treatments, which is composed of two main components of Paejangsan (Patriniae Radix (Paejang in Korean) and Coix Seed (Yiyiin in Korean)), and Mori Cortex Radicis (Sangbekpi in Korean) based on the addition and subtraction theory in traditional medicine, hence the name PSY, and explored the potential therapeutic effects of the new formula PSY in human CRC cells by analyzing viability, cell cycle and apoptosis. We found that PSY ethanol extract (EtOH-Ex), but not water extract, significantly suppressed the viability of human CRC cells, and synergistically decreased the cell proliferation compared to each treatment of Patriniae Radix and Coix Seed extract (PY) or Mori Cortex Radicis extract (S), suggesting the combination of PY and S in a 10-to-3 ratio for the formula PSY. PSY EtOH-Ex in the combination ratio reduced cell viability but induced cell cycle arrest at the G2/M and sub-G1 phases as well as apoptosis in CRC cells. In addition, the experimental results of Western blotting, immunofluorescence staining and reporter assays showed that PSY also inhibited STAT3 by reducing its phosphorylation and nuclear localization, which resulted in lowering STAT3 mediated transcriptional activation. In addition, PSY regulated upstream signaling molecules of STAT3 by inactivating JAK2 and Src and increasing SHP1. Moreover, the chemical profiles of PSY from UPLC-ESI-QTOF MS/MS analysis revealed 38 phytochemicals, including seven organic acids, eight iridoids, two lignans, twelve prenylflavonoids, eight fatty acids, and one carbohydrate. Furthermore, 21 potentially bioactive compounds were highly enriched in the PSY EtOH-Ex compared to the water extract. Together, these results indicate that PSY suppresses the proliferation of CRC cells by inhibiting the STAT3 signaling pathway, suggesting PSY as a potential therapeutic agent for treating CRC and 21 EtOH-Ex-enriched phytochemicals as anti-cancer drug candidates which may act by inhibiting STAT3.

**Keywords:** Patriniae Radix; Mori Cortex Radicis; Coix Seed; PSY; colorectal cancer; STAT3

Colorectal cancer (CRC) is the third most common cancer in the world, the third most diagnosed cancer among men, and the second most common among women. Overall, CRC ranks third in terms of incidence but second in terms of mortality [1]. Incidence rates have steadily risen in many countries in eastern Europe, southeastern and south-central Asia, and South America. Risk factors include the consumption of red or processed meat and heavy alcohol consumption, whereas adequate consumption of whole grains, fiber,

**Citation:** Han, S.; Kim, H.; Lee, M.Y.; Lee, J.; Ahn, K.S.; Ha, I.J.; Lee, S.-G. Anti-Cancer Effects of a New Herbal Medicine PSY by Inhibiting the STAT3 Signaling Pathway in Colorectal Cancer Cells and Its Phytochemical Analysis. *Int. J. Mol. Sci.* **2022**, *23*, 14826. https://doi.org/ 10.3390/ijms232314826

Academic Editors: Barbara De Filippis, Alessandra Ammazzalorso and Marialuigia Fantacuzzi

Received: 12 October 2022 Accepted: 22 November 2022 Published: 27 November 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and dairy products decreases the risk [2]. Primary prevention remains a key strategy for reducing the increasing global burden of CRC. The largest proportion of CRC cases has been linked to environmental mutations rather than heritable genetics [2]. Inflammatory bowel disease (IBD) is an important risk factor for colon cancer. In this regard, colitis-associated cancer is a CRC subtype associated with IBD, is challenging to treat, and has high mortality [3]. Moreover, CRC exhibits constitutive activation of NF-κB and STAT3, transcription factors that influence interactions between tumor cells and tumor microenvironment and play integrated roles in cancer-promoting inflammation [4]. Further, inflammation is associated with tumor cell proliferation, survival, metastasis, angiogenesis, and chemoresistance and may also affect the efficacy of CRC therapies, including STAT3 inhibitors [4,5]. However, NF-κB is also involved in anti-tumor immune responses, and in contrast, STAT3 restrains the NF-κB-mediated anti-tumor immunity [4]. Thus, STAT3 has been suggested as a more promising target for cancer therapy by redirecting inflammation [4,5]. Recent studies have also indicated that CRC progression occurs by activating tumorigenic JAK/STAT3 signaling [5,6].

Although many cancer medications such as oxaliplatin for treating CRC are available, better options have been requested for cancer patients because of the low efficacy and severe side effects of conventional chemotherapies [7,8]. Thus, medicinal plants and dietary phytochemicals have attracted great attention due to strong beliefs that as these substances are edible they have minimal toxicity [9].

Roots of *Patrinia scabiosaefolia* Fisch. (Patriniae Radix (PR)) and seeds of *Coix lacrymajobi* L. var. *ma-yuen* Stapf (Coix Seed (CS)) are the main components of Paejangsan which has been traditionally used to treat intestinal inflammatory diseases in Korea and China [10]. The root bark of *Morus alba* L. (Mori Cortex Radicis (MCR)) has been used for inflammationrelated diseases in the intestine and lungs in traditional Korean and Chinese medicine [10]. Recent studies have also shown that the extract of each medicinal plant inhibits the proliferation of CRC cells [11–16]. Moreover, a basic theory of herbal combination in traditional medicine is the addition and subtraction theory that adds or/and removes one or more herbal medicines or dosages from an original foundational formula, thus generating another new formula for personalized medicine or better therapeutic effects [17]. Based on the information, we hypothesized that the addition of MCR to the main components of Paejangsan generating a new herbal formula might have anti-cancer properties for CRC. Therefore, we have in the present study developed a new herbal formula as an alternative to CRC treatments, which is comprised of PR (Paejang in Korean), MCR (Sangbekpi in Korean), and CS (Yiyiin in Korean), hence the name PSY, and have investigated whether this formula could be a potential therapeutic intervention for treating CRC.

#### **2. Results and Discussion**

#### *2.1. PSY Synergistically Suppresses Cell Viability in Human CRC Cells*

Since we aimed to develop PSY as a potential therapeutic intervention for CRC based on the addition and subtraction theory, we first examined whether PSY ethanol extract (EtOH-Ex) would synergistically affect the viability of human CRC cells compared to two main components of Paejangsan (PR and CS combination: PY) and MCR (S). Human CRC cells were treated with PY EtOH-Ex for 72 h, and cell viability was determined. As shown in Figure 1a, PY decreased cell viability in a dose-dependent manner in all CRC cell lines, but IC50 values in each cell line were quite high. In addition, the results indicated that HT-29 cells are quite resistant to PY (Figure 1a). We then performed the combination of IC30 (125 μg/mL) and IC50 (200 μg/mL) concentrations of PY in HCT116 with various concentrations of S EtOH-Ex to evaluate the potential effect of the combination and determine the best combination ratio. As shown in Figure 1b, an increase of S together with 125 μg/mL or 200 μg/mL of PY reduced cell viability in a dose-dependent manner in all CRC cell lines including HCT116. In addition, combinations of 37.5 μg/mL of S with 125 μg/mL of PY and 60 μg/mL of S with 200 μg/mL of PY were the lowest concentrations showing significant effects in all CRC cells including HT-29 (Figure 1b). These results

suggested the combination of PY and S in a 10-to-3 ratio for the formula PSY. Additionally, the increase of PSY in the combination ratio (PY:S = 10:3) dramatically and synergistically suppressed the cell viability of all CRC cell lines tested compared to PY or S treatment (Figure 1c). However, PSY water extract (Water-Ex) did not show any significant effect (Figure 1d). These results indicated that PSY is potent to suppress the viability of CRC cells, and we further evaluated PSY EtOH-Ex in the combination ratio (PY:S = 10:3) as a potential anti-CRC agent.

**Figure 1.** Effects of PSY extracts on the viability of human CRC cells. (**a**) Human CRC cells were treated with ethanol extract (EtOH-Ex) of Patriniae Radix and Coix Seed (PY) for 72 h as indicated. Cell viability was determined by MTT assays. IC50 values of PY EtOH-Ex in CRC cell lines were determined. (**b**) CRC cells were treated with five concentrations of Mori Cortex Radicis (S) EtOH-Ex in combination with PY EtOH-Ex (125 and 200 μg/mL) for 72 h. The ratio of PY and S was from 10-to-1 to 10-to-5. (**c**) CRC cells were treated with PSY EtOH-Ex in a 10-to-3 combination ratio and compared to each treatment of PY or S EtOH-Ex. (**d**) CRC cells were treated with PSY water extract (Water-Ex) in the combined ratio as indicated. Data are presented as the mean ± standard error of the mean (SEM) of results from at least three independent experiments performed in triplicates. \*, *p* < 0.05, \*\*, *p* < 0.01, \*\*\*, *p* < 0.001.
