The cytotoxic and antiproliferative effect of Polygala saponin XLIV on the human colorectal carcinoma cell line

Introduction

Colorectal cancer (CRC) ranks as the fourth leading cause of cancer-related deaths around the globe. The occurrence and progression of CRC are induced by multiple risk factor combinations, including age, gender, family, and personal history [1]. CRC conventional treatments contain chemotherapy and surgery. Chemotherapeutic agents can cause the death of cancer cells by initiating multiple molecular signaling pathways and inducing DNA damage, including cell cycle arrest and global translation inhibition. However, various studies showed that chemotherapeutic drugs have side effects such as drug resistance, cytotoxicity, and adverse reactions [2].

The role of natural compounds in drug discovery studies has never lost its importance and the number of the new candidates will always be increased [3], [4], [5], [6]. Numerous widely used chemotherapeutics originate from plant sources, such as etoposide, irinotecan, paclitaxel, and vincristine [7]. Moreover, diverse natural products are used to yield many effective compounds for the discovery of new anticancer drugs. Many of the natural products have exhibited excellent anticancer activities against tumors with slight side effects [8]. Previous studies reported that phytochemical compounds have extensive utilization prospects for CRC treatment.

Experimental studies reported that at least 150 natural saponins exhibited significant antitumor effects on cancer cells [8], [9], [10]. Saponins’ profound impacts on cancer cells have gained researchers’ interest. In recent studies, saponins exerted antitumor impacts by targeting the Wnt-β-catenin and JAK-STAT3 signaling pathways. These are crucial pathways in regulating colorectal cancer cell proliferation and metastasis [11].

Saponins are a class of natural compounds classified under the isoprenoids. Acyclic triterpene squalene is the precursor of all triterpenes, including tetracyclic (cycloartenol, lanostane, dammarane, and euphane) and pentacyclic (oleanane, ursane, lupane, hopane) triterpenes. Among them, cycloartenols are an intermediate in the biosynthesis of cucurbitacins and cholesterol. On the other hand, phytosterols, cardenolides, bufadienolides, ecdysteroids, spirostanols, and furostanols are biosynthesized by the shortening or elongation of their side chains. These compounds are among traditionally used medicinal plants’ most active constituent groups. Glycosidic forms of the triterpenes, spirostanols and furastanols have foaming properties due to their amphiphilic properties. Because of their soap-like foam-generating abilities, they are called saponins and saponosides. Although both of the terms are synonyms, saponin is used to define the monodesmosidic saponins such as cyclamin and parillin while saponoside is used for the bisdesmosidic saponosides such as gypsoside, sarsaparilloside. Either triterpenoid or steroidal saponins exert a variety of biological activities and have a wide range of pharmacological effects such antitumor, antifertility, immunomodulatory, antioxidant, anti-inflammatory, hypoglycemic, and therapeutic in cardiovascular diseases [12]. A recent report deals with the impact of the saponins on signal transduction pathways in cancer [11].

The present study has studied an oleanane-type triterpenoid saponin (Polygalasaponin XLIV, PS-XLIV) for its cytotoxic activity. PS-XLIV was isolated during the ongoing studies performed on the roots of Polygala vulgaris. As a continuation of the phytochemical studies carried out on Polygala species [13], [14], [15] recorded in the flora of Turkey, P. vulgaris was chosen for further studies concerning the presence of diverse metabolites and higher amounts of secondary metabolites of the polyploid species [16], [17], [18]. The structure of PS-XLIV (Figure 1) was identified as 3-O-β-D-glucopyranosyl-presenegenin-28-O-β-D-galactopyranosyl-(1→4)-β-D-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-{4-O-[E]-3,4-dmethoxycinnamoyl]}-β-D-fucopyranosyl ester (PS-XLIV) which was first reported from Polygala glomerata [19]. Z-isomer of the same saponin has been reported from the Securidaca inappendiculata and PS-XLIV [20]. Moreover, this hexaglycosidic as well as bisdesmosidic ester-type oleanane saponoside has a similar structure to those of senegins II and IV, and E- and Z-senegasaponin c exhibiting same glycosidation patterns on the same sapogenin (presenegenin) except ester moieties reported from Polygala senega var. latifolia [21].

Figure 1:

The structure of Polygalasaponin XLIV (PS-XLIV).

The primary aim of this study was to conclusively determine the cytotoxic impact of PS-XLIV, a potent saponin derived from Polygala species, on the human colorectal carcinoma cell line (HCT116). We also aimed to determine the anticancer and antiproliferative impacts of PS-XLIV via WNT3A, WNT11, STAT3, β-catenin, and Ki-67 distributions in HCT116 cells.

Methods

Results

Evaluation of the cell viability and growth assay

HCT116 cells were exposed to a range of PS-XLIV concentrations (10, 20, 50, 100, and 150 μg/mL) for 24 and 48 h. The appropriate concentration and duration time were determined for cancer cell viability-decreasing and non-toxic effects. IC50 value of PS-XLIV was 80 μg/mL for 48 h (Figure 2).

Figure 2:

Effect of PS-XLIV on the cell viability of HCT116 cells. HCT116 cells were treated with PS-XLIV in five different concentrations for 24 and 48 h (absorbance: 540 nm).

Cell morphology

HCT116 cells have adherent and epithelial morphology. After PS-XLIV treatment, the epithelial morphology of cells was protected; however, the number of cells was less than that of the control group cells (Figure 3).

Figure 3:

HCT116 cells were imaged under the inverted microscope. PS-XLIV-treated (A) and control (B) groups of HCT116 cells (magnification: ×20).

Immunohistochemical evaluation

Moderate WNT3A immunoreactivities were detected in PS-XLIV treated (Figure 4A) and control groups HCT116 cells (Figure 4B). This immunoreactivity was not statistically significant. WNT11 immunoreactivity was weak in HCT116 cells (Figure 4C) after being treated with PS-XLIV. The WNT11 H-SCORE value was significantly lower in PS-XLIV treated HCT116 cells (Figure 4C) in comparison to the control group (Figure 4D) (p<0.05), STAT3 immunoreactivities were weak and moderate in PS-XLIV treated and control groups HCT116 cells, respectively (Figure 4E and F). This dissimilarity between the two groups was statistically significant (p<0.05). Comparing the PS-XLIV-treated HCT116 cells with its control group, β-catenin immunoreactivity was lower (Figure 4G) than the control group cells (Figure 4H). Additionally, the H-SCORE for β-catenin immunoreactivity demonstrated a noteworthy decrease in the PS-XLIV treated group (Figure 4G) compared to the control group (Figure 4H) (p<0.05). In HCT116 cells, the PS-XLIV treated group exhibited significantly weaker Ki-67 immunoreactivity (Figure 4I) compared to the control group (Figure 4J), with a noteworthy difference (p<0.05).

Figure 4:

Immunoreactivities of WNT3A (a, b), WNT11 (c, d), STAT3 (e, f), β-catenin (g, h) and Ki-67 (i, j) in HCT116 cells after treatment with 80 μg/mL PS-XLIV (a, c, e, g, i), and standard culture conditions (b, d, f, h, j) for 48 h (magnification: ×10).

Discussion

Colorectal cancer has complicated pathological processes with multiple molecular signaling pathways. The inhibition or activation of various cell signaling pathways drives the initiation and progression of tumors. The extent of the anticancer effects of natural products has been examined against multiple signaling pathways in ongoing cancer investigations [23]. Mainly, saponins purified and characterized from different plants inhibit actions, including induction apoptosis, reducing invasion and proliferation in cancer cell types [11]. Our study evaluated the anticancer effects of triterpenoid saponin (PS-XLIV) isolated from P. vulgaris. We demonstrated the inhibitory acts of PS-XLIV on STAT3, Ki-67, and WNT/β-catenin signaling pathways in HCT116 cells.

The WNT/β-catenin signaling pathway is a vital intracellular cascade that regulates cell survival, fate, apoptosis, proliferation, and differentiation within tissues and organs [24]. Numerous experimental studies have reported the potential role of the WNT/β-catenin signaling pathway activation in colon cancer. In particular, WNT11 levels increased in different cancer types, including CRC [25]. It stimulates cancer cell invasion and metastasis. Stable high WNT11 expression promotes HCT116 cell proliferation, invasion, and metastasis, and WNT11 silencing decreases HCT116 cell migration [26], 27]. WNT11 uses β-catenin-dependent pathway signals in CRC [25]. Moreover, triterpenoid saponins effectively inhibit cell proliferation and promote apoptosis in both in vitro and in vivo models of CRC. Wang et al. [28] reported that β-catenin expression was reduced in the cytoplasm and nucleus by triterpenoid saponin and promoted the expression of apoptosis trigger protein, Bax [28]. Also, in vitro and in vivo studies indicated that ginsenosides from Korean Red ginseng reduced CMYC, CCND1, and LEF1 expressions by inhibiting the WNT/β-catenin signaling pathway [29]. Another study showed that ginsenoside isolated from Polygala ginseng upregulated p53 expression but downregulated β-catenin, c-myc, and TCF-4 protein in LNCaP prostate cancer cells [30]. In the current study, PS-XLIV-treated HCT116 cells showed statistically significant lower immunoreactivities for WNT11 than the group of control cells. Moreover, β-catenin immunoreactivities were significantly lower in PS-XLIV-treated HCT116 cells when compared with the group of control cells. These results suggested that PS-XLIV could be a potential plant-derived bioactive component for treating colorectal cancer by inhibiting the WNT11/β-catenin signaling pathway.

STAT3 is an important transcription factor associated with CRC initiation and progress. It excessively activates inflammatory mechanisms in CRC and augments cancer cell proliferation, angiogenesis, and metastasis. In CRC cells, upregulation of STAT3 promotes epithelial-mesenchymal transition and, therefore, aggressive cancer cell phenotype [31]. Wang et al. [32] demonstrated that saponins showed anticancer activities in liver cancer cells by reducing STAT3 expression. The present study results reported that STAT3 immunoreactivity was significantly lower in PS-XLIV-treated HCT116 cells compared to control group cells. Given STAT3’s critical role in colorectal cancer physiology, PS-XLIV can potentially protect by targeting the STAT3 protein signaling pathway.

The Ki-67 protein is an essential biomarker used to assess the proliferation of human tumor cells, providing crucial insights for effective diagnosis and treatment. Ki-67 protein has essential roles in mitotic and interphase cells, and its cellular dispersion changes during the progression of the cell cycle [33]. Li et al. showed that astragaloside IV from Radix Astragali, an active triterpenoid, suppressed tumor cell growth and decreased Ki-67 levels by regulating the MAPK/ERK molecular signaling pathway in U251 cells [34]. In agreement with a previous study, Ki-67 immunoreactivity was statistically significantly lower in PS-XLIV-treated HCT116 cells compared to our study’s control group of HCT116 cells. Therefore, it can be concluded that PS-XLIV inhibited proliferation in HCT116 cells.

In conclusion, the anticancer effects of triterpenoid saponin (PS-XLIV) isolated from P. vulgaris on colorectal cancer cells were shown for the first time. The mechanisms of this effect may involve the reduction of WNT11, β-catenin, STAT3, and Ki-67 distributions by PS-XLIV in HCT116 cells. The limitation of this study was that more signaling molecules could have been studied to assess and investigate the protective effects of the PS-XLIV against carcinogenesis. More research is crucial to confirm the anticancer properties of PS-XLIV through in vitro and in vivo molecular studies. Moreover, as a result of this study, it can be concluded that the other Polygala saponins representing oleanane-type bisdesmosidic ester structures are candidates for future studies on the same topic.