RETRACTED: Antiproliferative Activity of Triterpene Glycoside Nutrient from Monk Fruit in Colorectal Cancer and Throat Cancer

: Colorectal cancer and throat cancer are the world’s most prevalent neoplastic diseases, and a serious threat to human health. Plant triterpene glycosides have demonstrated antitumor activity. In this study, we investigated potential anticancer effects of mogroside IVe, a triterpenoid glycoside from monk fruit, using in vitro and in vivo models of colorectal and laryngeal cancer. The effects of mogroside IVe on the proliferation of colorectal cancer HT29 cells and throat cancer Hep-2 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression levels of p53, phosphorylated ERK1/2, and MMP-9 were analyzed by western blotting and immunohistochemistry. The results indicated that mogroside IVe inhibited, in a dose-dependent manner, the proliferation of HT29 and Hep-2 cells in culture and in xenografted mice, which was accompanied by the upregulation of tumor suppressor p53, and downregulation of matrix metallopeptidase 9 (MMP-9) and phosphorylated extracellular signal-regulated kinases (ERK)1/2. This study revealed the suppressive activity of mogroside IVe towards colorectal and throat cancers and identified the underlying mechanisms, suggesting that mogroside IVe may be potentially used as a biologically-active phytochemical supplement for treating colorectal and throat cancers.


Introduction
Natural products play an important role in contemporary cancer therapy [1,2], and a substantial number of clinically-used chemicals are derived from plants [3][4][5]. Monk fruit (Siraitia grosvenorii) is a cucurbitaceous herb widely planted in the Guangxi province of China, which produces high-potency sweeteners increasingly popular in the food industry as additives in low-calorie drinks or foods [6]. A study on adverse effect of monk fruit has shown that its extracts and individual compounds are essentially non-toxic [7]. Mogroside IVe, a triterpenoid glycoside from monk fruit, consists of an aglycone (mogrol) and four glucose groups [8]. However, despite the presence of glucose residues, mogroside IVe exerts an antihyperglycemic effect and could regulate blood sugar levels in diabetic patients [9]. Mogroside IVe is responsible, in part, for the intense sweetness of the fruit and is estimated to be about 392 times sweeter than sucrose [10].
Colorectal cancer and throat cancer are the world's most prevalent neoplastic diseases and a serious threat to human health [11,12]. Diet and exercise have been established as important factors in cancer prevention, and many triterpene glycosides have demonstrated antitumor activity [13][14][15]. However, the pharmacological properties of mogroside IVe in preventing colorectal and laryngeal cancers remain unknown. As no negative side effects associated with the ingestion of monk fruit extracts have been reported, in 2014, Food and Drug Administration (FDA) acknowledged mogrosides as Generally Recognized as Safe (GRAS) and approved their use as general-purpose food sweeteners, indicating their safety as pharmacological agents. In this study, we investigated potential anticancer effects of mogroside IVe, including the ability to induce cancer cell apoptosis and inhibit proliferation using in vitro and in vivo models.

Identification of Mogroside IVe
The chemical structure of mogroside IVe was confirmed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) analysis as previously described, with slight modifications [16]. LC-ESI-MS was performed in positive ion mode at the scan range of 100-1500 m/z; 0.1% formic acid was used as a mobile phase additive.

Measurement of Cell Proliferation
HT29 and Hep-2 cells were seeded into 96-well plates (1ˆ10 4 cells/well) and treated with vehicle (PBS) or increasing concentrations (0-250 µmol/L) of mogroside IVe for 48 h. Cell viability was assessed by MTT assay; the absorbance was read at a wavelength of 570 nm using a spectrophotometer as previously described [14].

Mouse Studies
All animal experiments were performed in accordance with institutional guidelines approved by the Beijing University of Agriculture. Xenograft tumor models were established by implanting 1ˆ10 7 HT29 or Hep-2 cells subcutaneously into male BALB/c nude mice (eight weeks old, 25-30 g) purchased from Charles River Ltd. (Beijing, China). Then, mice were randomly assigned into four groups (n = 8 per group): three treatment groups received intravenous injections of mogroside IVe (2 mg/kg, 10 mg/kg, and 30 mg/kg) three times per week for five weeks, while the control group received saline. Tumor size was measured with a vernier caliper and monitored twice weekly, andtumor volume was calculated as V = (LˆW 2 )/2, where L is the length and W is the width of the tumor. At day 35 post-implantation, mice were euthanized by intraperitoneal injection of sodium pentobarbital (150-200 mg/kg), and tumors were harvested for further analysis.

Immunoblotting and Immunohistochemistry
The level of protein expression in cultured cells was determined by immunoblotting as previously described [17,18] using primary antibodies against MMP-9 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), p53, phospho-ERK1/2, total ERK, and β-actin (all Sigma-Aldrich, St. Louis, MO, USA). Protein expression in xenografted tumors was analyzed by immunohistochemistry. Tumor tissues were fixed immediately after harvesting in 10% phosphate-buffered formalin, embedded in paraffin, and tissue sections were stained with the antibodies indicated above.

Apoptosis Assessment
Tumor sections prepared as above were analyzed for cell apoptosis by the TUNEL assay using the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Mannheim, Germany) as described previously [19,20]. After TUNEL staining, tumor sections were mounted using Vectashield supplemented with 4 1 -6-diamidino-2-phenylindole (DAPI; H-1200, Vector Laboratories, Burlingame, CA, USA) for nuclei detection and the images were acquired under a confocal laser scanning microscope.

Statistical Analysis
All in vitro experiments were repeated at least three times. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was determined by Student's t-test, and p values < 0.05 were considered statistically significant.

Identification of Mogroside IVe
The structure of mogroside IVe was confirmed by mass spectrometry. tissues were fixed immediately after harvesting in 10% phosphate-buffered formalin, embedded in paraffin, and tissue sections were stained with the antibodies indicated above.

Apoptosis Assessment
Tumor sections prepared as above were analyzed for cell apoptosis by the TUNEL assay using the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Mannheim, Germany) as described previously [19,20]. After TUNEL staining, tumor sections were mounted using Vectashield supplemented with 4′-6-diamidino-2-phenylindole (DAPI; H-1200, Vector Laboratories, Burlingame, CA, USA) for nuclei detection and the images were acquired under a confocal laser scanning microscope.

Statistical Analysis
All in vitro experiments were repeated at least three times. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was determined by Student's t-test, and p values < 0.05 were considered statistically significant.

Identification of Mogroside IVe
The structure of mogroside IVe was confirmed by mass spectrometry.

Mogroside IVe Inhibits the Proliferation of HT29 and Hep-2 Cells
The effect of mogroside IVe on cell proliferation was measured by MTT assay. HT29 and Hep-2 cells were cultured in the presence of different concentrations (0-250 μmol/L) of mogroside IVe for 48 h. The results demonstrated that mogroside IVe inhibited the proliferation of both HT29 and Hep-

Mogroside IVe Inhibits the Proliferation of HT29 and Hep-2 Cells
The effect of mogroside IVe on cell proliferation was measured by MTT assay. HT29 and Hep-2 cells were cultured in the presence of different concentrations (0-250 µmol/L) of mogroside IVe for 48 h. The results demonstrated that mogroside IVe inhibited the proliferation of both HT29 and Hep-2 cells in a dose-dependent manner (Figure 2b,c). Figure 2d shows significant morphological changes induced by mogroside IVe in both cell lines. While control (PBS-treated) cells were similar in size and regularly shaped, some of mogroside IVe-treated cells were detached from the surface and lysed, as evidenced by the presence of cell debris, or formed apoptotic bodies.

Effect of Mogroside IVe on the Phosphorylation of ERK1/2, and the Expression of p53 and MMP-9
Next, we examined the mechanism underlying mogroside IVe inhibition of cancer cell proliferation. As tumor suppressor protein p53, extracellular signal-regulated kinasesERK1/2, and matrix metallopeptidase 9 (MMP-9) are implicated in cancer cell proliferation and metastasis, we examined their expression in HT29 and Hep-2 cells. The results indicated that mogroside IVe, in a dose-dependent manner, significantly suppressed the phosphorylation of ERK1/2 (Figure 3a,b), while upregulating the expression of p53 (Figure 3c,d) and downregulating that of MMP-9 ( Figure  3e,f). These findings strongly suggested that phospho-ERK1/2, p53, and MMP-9 may be molecular targets of mogroside IVe.  Next, we examined the mechanism underlying mogroside IVe inhibition of cancer cell proliferation. As tumor suppressor protein p53, extracellular signal-regulated kinasesERK1/2, and matrix metallopeptidase 9 (MMP-9) are implicated in cancer cell proliferation and metastasis, we examined their expression in HT29 and Hep-2 cells. The results indicated that mogroside IVe, in a dose-dependent manner, significantly suppressed the phosphorylation of ERK1/2 (Figure 3a,b), while upregulating the expression of p53 (Figure 3c,d) and downregulating that of MMP-9 (Figure 3e,f).

Anticancer Effect of Mogroside IVe in Mice
To investigate the effect of mogroside IVe on tumor growth in vivo, we developed an animal xenograft model of HT29-and Hep-2 cell-derived tumors. In HT29 and Hep-2 xenografted mice, mogroside IVe showed statistically significant inhibition of tumor growth (Figure 4a,b). After five weeks of treatment with mogroside IVe, all mice were euthanized, and the tumors were weighed. Consistent with the tumor volumes estimated by external measurements, tumor weight in mogroside IVe-treated mice was significantly less than that in control mice by day 35. The results presented in Figure 4, confirmed that the treatment with mogroside IVe significantly inhibited the growth of HT29-and Hep-2-derived tumors. (e,f) Mogroside IVe inhibited MMP-9 expression in HT29 (e) and Hep-2 (f) cells. The data represent the mean˘SD of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control.

Anticancer Effect of Mogroside IVe in Mice
To investigate the effect of mogroside IVe on tumor growth in vivo, we developed an animal xenograft model of HT29-and Hep-2 cell-derived tumors. In HT29 and Hep-2 xenografted mice, mogroside IVe showed statistically significant inhibition of tumor growth (Figure 4a,b). After five weeks of treatment with mogroside IVe, all mice were euthanized, and the tumors were weighed. Consistent with the tumor volumes estimated by external measurements, tumor weight in mogroside IVe-treated mice was significantly less than that in control mice by day 35. The results presented in Figure 4, confirmed that the treatment with mogroside IVe significantly inhibited the growth of HT29-and Hep-2-derived tumors. Statistical significance between control and treated animals were evaluated by Student's t-test (n = 8). ** p < 0.01; *** p < 0.001 compared to control.

Mogroside IVe Induces Apoptosis of Tumor Cells in Mice
The induction of apoptosis in cancer cells is an important aspect of tumor-suppressing activity exerted by anticancer agents. As revealed by the TUNEL assay, mogroside IVe-treated mice demonstrated a significant increase in the number of apoptotic bodies (stained red) in cancer cell xenografts compared with control (Figure 5a,b), suggesting that the suppression of tumor growth in mice injected with mogroside IVe was due to increased apoptosis of tumor cells.
To investigate the mechanism by which mogroside IVe induced apoptosis in HT29-and Hep-2derived tumors, they were evaluated by immunohistochemistry, which revealed significantly increased p53 expression in the tumors from mogroside IVe-treated mice compared with those from the control group (Figure 5c,d). This observation indicated that mogroside IVe induced tumor cell apoptosis by promoting p53 expression.

Mogroside IVe Induces Apoptosis of Tumor Cells in Mice
The induction of apoptosis in cancer cells is an important aspect of tumor-suppressing activity exerted by anticancer agents. As revealed by the TUNEL assay, mogroside IVe-treated mice demonstrated a significant increase in the number of apoptotic bodies (stained red) in cancer cell xenografts compared with control (Figure 5a,b), suggesting that the suppression of tumor growth in mice injected with mogroside IVe was due to increased apoptosis of tumor cells.

Effect of Mogroside IVe on ERK1/2 Phosphorylation and MMP-9 Expression in Vivo
ERK1/2 phosphorylation-dependent signaling is required to support the invasion of HT29 and Hep-2 cells through activation of the downstream pathways [21,22]. MMP-9 is a zinc-dependent protease implicated in cancer cell invasion and metastasis [23]. Immunohistochemistry analysis indicated that mogroside IVe markedly decreased the levels of phospho-ERK1/2 and downregulated MMP-9 expression in the tumors of treated mice in a dose-dependent manner ( Figure 6). To investigate the mechanism by which mogroside IVe induced apoptosis in HT29-and Hep-2-derived tumors, they were evaluated by immunohistochemistry, which revealed significantly increased p53 expression in the tumors from mogroside IVe-treated mice compared with those from the control group (Figure 5c,d). This observation indicated that mogroside IVe induced tumor cell apoptosis by promoting p53 expression.

Effect of Mogroside IVe on ERK1/2 Phosphorylation and MMP-9 Expression in Vivo
ERK1/2 phosphorylation-dependent signaling is required to support the invasion of HT29 and Hep-2 cells through activation of the downstream pathways [21,22]. MMP-9 is a zinc-dependent protease implicated in cancer cell invasion and metastasis [23]. Immunohistochemistry analysis indicated that mogroside IVe markedly decreased the levels of phospho-ERK1/2 and downregulated MMP-9 expression in the tumors of treated mice in a dose-dependent manner ( Figure 6).

Discussion
Our results indicate that mogroside IVe, a triterpenoid glycoside from monk fruit used as a strong sweetener, has a potential to prevent the development of colorectal and laryngeal cancers because it significantly inhibited the proliferation of HT29 and Hep-2 cells in vitro (Figure 2b-d), and, consistently, demonstrated an anticancer effect in mice with HT29-and Hep-2-derived xenografted tumors (Figure 4).
The TUNEL assay showed that the ability of mogroside IVe to suppress tumor growth may be based, at least in part, on the induction of apoptosis in cancer cells (Figure 5a,b). Previous studies have indicated that many apoptotic signals associated with cell death in HT29 and Hep-2 cells are mediated by p53 because enhanced p53 expression induces tumor cell apoptosis [24][25][26][27]. In agreement with these findings, in our xenograft model mogroside IVe significantly increased the expression level of p53 in tumors (Figure 5c,d), which was corroborated by the in vitro results (Figure  3c,d). Overall, these data suggest that mogroside IVe induced p53-mediated apoptosis in HT29 and Hep-2 cancer cells.
Cell invasion is a critical step in cancer progression as it facilitates metastasis [28][29][30], which is a critical factor in determining the survival of cancer patients [31][32][33]. MMP-9 is thought to play an important role in HT29 and Hep-2 cancer cell migration and invasion [34,35], and our findings indicated that mogroside IVe treatment downregulatedMMP-9 expression in HT29 and Hep-2 cells

Discussion
Our results indicate that mogroside IVe, a triterpenoid glycoside from monk fruit used as a strong sweetener, has a potential to prevent the development of colorectal and laryngeal cancers because it significantly inhibited the proliferation of HT29 and Hep-2 cells in vitro (Figure 2b-d), and, consistently, demonstrated an anticancer effect in mice with HT29-and Hep-2-derived xenografted tumors (Figure 4).
The TUNEL assay showed that the ability of mogroside IVe to suppress tumor growth may be based, at least in part, on the induction of apoptosis in cancer cells (Figure 5a,b). Previous studies have indicated that many apoptotic signals associated with cell death in HT29 and Hep-2 cells are mediated by p53 because enhanced p53 expression induces tumor cell apoptosis [24][25][26][27]. In agreement with these findings, in our xenograft model mogroside IVe significantly increased the expression level of p53 in tumors (Figure 5c,d), which was corroborated by the in vitro results (Figure 3c,d). Overall, these data suggest that mogroside IVe induced p53-mediated apoptosis in HT29 and Hep-2 cancer cells.
Cell invasion is a critical step in cancer progression as it facilitates metastasis [28][29][30], which is a critical factor in determining the survival of cancer patients [31][32][33]. MMP-9 is thought to play an important role in HT29 and Hep-2 cancer cell migration and invasion [34,35], and our findings indicated that mogroside IVe treatment downregulatedMMP-9 expression in HT29 and Hep-2 cells both in vitro (Figure 3e,f) and in vivo (Figure 6c,d). These results were supported by the observation that mogroside IVe inhibited the phosphorylation of ERK1/2 (Figure 3a,b and Figure 6a,b), which acts as an upstream regulator of MMP-9 expression [36][37][38][39]. Together, these findings suggest that mogroside IVe may suppress MMP-9 expression via inhibition of ERK1/2 phosphorylation-dependent activation. As MMP-9 is considered to play a critical role in the migration and invasion of HT29 and Hep-2 cancer cells, we can speculate that mogroside IVe may also inhibit cancer invasion and metastasis. However, future in vitro and in vivo studies are required to test this hypothesis and confirm that mogroside IVe can prevent or reduce cancer cell migration and invasion.
In conclusion, we demonstrated that mogroside IVe has the ability to suppress the proliferation of colorectal cancer and throat cancer cells by inducing apoptosis through upregulation of p53, and downregulation of p-ERK1/2 and MMP-9 levels, strongly indicating anticancer activity. Thus, we highlighted, for the first time, the role and mechanism of mogroside IVe as a phytochemical with anti-colorectal cancer and anti-throat cancer activity. As mogroside IVe does not have side effects and is used as a sweetener in many low-calorie foods and drinks, its application as a dietary supplement may have benefits over conventional drugs in terms of cancer prevention effects and user compliance.