Cytotoxic Effect of Cholesterol Metabolites on Human Colonic Tumor (Caco-2) and Non-Tumor (CCD-18Co) Cells and Their Potential Implication in Colorectal Carcinogenesis

: Unabsorbed cholesterol, along with that of bile secretions and flaked colon cells, can be metabolized by colonic microbiota. The generated metabolites have been proposed as promoters of colorectal cancer (CRC). In this study, the cytotoxicity (MTT assay) of the main commercially available cholesterol-derived metabolites (coprostanol, cholestanol, coprostanone, and cholestenone) on human colon cancer (Caco-2) and non-tumor (CCD-18Co) cells was evaluated at different physio-logically relevant concentrations (9.4–300 µM) and incubation times (24–72 h). In general, the metabolites that most reduced cell viability were coprostanone (54–85% in Caco-2 and 20–81% in CCD-18Co) and cholestenone (17–91% in Caco-2 and 14–81% in CCD-18Co). These two metabolites are the most hydrophobic, thus reflecting a possible relationship between hydrophobicity and cytotoxicity. Moreover, cholestenone (IC 50 at 72 h: 5 ± 1 µg/mL) should be considered cytotoxic on CCD-18Co cells (non-tumor cells) since it shows an IC 50 close to the one considered toxic (<4 µg/mL). Furthermore, CCD-18Co cells are more vulnerable to the cytotoxic effect of cholesterol metabolites. Possible compensatory responses, attenuating the reduction in cell viability caused by cholesterol metabolites, were observed, however these reactions could favor inflammation, resistance to apoptosis, and cellular proliferation, likely contributing to the development of CRC. In conclusion, cholesterol metabolites, mainly the most hydrophobic, could act as promoters of CRC through their cytotoxic activity.


Introduction
Colorectal cancer (CRC) was the second most prevalent cancer in the world in 2018 and the second cause of cancer-related death worldwide [1]. Some authors propose that diets rich in fats and proteins of animal origin are related to an increased risk of CRC through an increase in the production of cholesterol-derived metabolites by the intestinal microbiota [2,3]. Since cytotoxicity may be a mechanism in cancer induction [4], the objective of the present study was to evaluate the cytotoxic activity of the main commercially available cholesterol-derived metabolites (coprostanol, cholestanol, coprostanone, and cholestenone) on undifferentiated human colonic epithelial adenocarcinoma cells (Caco-2) and non-tumor human colon fibroblasts (CCD-18Co).

Cholestanol
The incubation of cholestanol for 24 h, at concentrations of 18.75 µM or above, reduces statistically (p < 0.05) CCD-18Co cells viability (28-41% vs. control), whilst at 48 h a cell viability reduction at all concentrations is observed (21-30% vs. control). At 72 h, a reduction in the viability of CCD-18Co cells is not observed, which could indicate a possible compensatory response. This response is verified by observing that the reductions in cell viability at 300 µM decrease with time (41, 28, and 2% vs. control at 24, 48, and 72 h, respectively). In addition, in Caco-2 cells, cholestanol at 24 h reduces the cell viability from 37.5 µM (20-24% vs. control), without effect at 48 and 72 h. Therefore, compensatory response is again observed (earlier than in CCD-18Co cells), which is confirmed with cell viability values after 72 h at 150 (104% vs. control) and 300 µM (108% vs. control) higher than 24 h (77 and 79% vs. control, respectively). On the other hand, the CCD-18Co cells are more susceptible since the reduction in cell viability at 150 and 300 µM after 24 h is greater than that observed in Caco-2 cells (39 vs. 23% control; 41 vs. 21% control, respectively).

Coprostanone
Coprostanone at 24 h significantly reduced the viability of CCD-18Co cells from 75 µM (35-56% vs. control), at 48 h from 150 µM (48-75% vs. control), and at 72 h at all concentrations except 18.75 µM (20-81% vs. control). A time-response relationship was observed, with an increase in the reduction of viability with time at 150 and 300 µM. In Caco-2 cells, coprostanone did not reduce cell viability at 24-48 h, and only at 72 h a reduction of cell viability from 150 µM (54-85% vs. control) was observed.

Cholestenone
In general, the incubation of cholestenone at 24 h reduced CCD-18Co (14-73% vs. control) and Caco-2 cells (17-71% vs. control) viability at all concentrations investigated in a dose-dependent manner. The effect at 48 h was slightly lower for both cell lines (18-81% and 23-88% vs. control, respectively), since the minimum dose required was 18.75 µM. At 72 h, cholestanone produced a similar cell viability reduction in CCD-18Co cells (39-79% vs. control) compared to 48 h, but in Caco-2 cells a dose ≥37.5 µM was necessary to produce a cell viability reduction (39-91% vs. control). The effect of the cholestenone on cell viability was time-dependent for certain conditions. In this regard, in the CCD-18Co cells there is a higher reduction of cell viability at 9.4 and 18.75 µM at 72 h, and in the Caco-2 cells at 75, 150 and 300 µM at 72 h. In addition, it appears that Caco-2 cells give a compensatory response at 9.4 and 18.75 µM with a trend of lower cell viability decrease at 48 and 72 h.

5-Fluorouracil
5-FU reduced the cell viability of both cell lines at all incubation times (CCD18-Co: 10-36%; Caco-2: 30-36% vs. control). The reduction in the viability of Caco-2 cells was not time-dependent, while in the non-tumor line the effect increased with time (24-48 < 72 h). Regarding the sensitivity of the cell lines, only at 24 h was the reduction in cell viability more pronounced in the Caco-2 vs. CCD-18Co cells (36 vs. 10% control), although these differences were not statistically significant (p < 0.05) at 48 and 72 h.

Discussion
The cholesterol that reaches the colon can be metabolized by the colonic microbiota (mainly bacteria of the genus Eubacterium and Bacteroides) [5]. There are studies which have observed that subjects with CRC present a significantly higher fecal concentration (vs. control) of cholesterol metabolites (21.1 vs. 14.5 mg/g dry feces) [3]. The authors attribute these observations to the abundance of anaerobic bacteria capable of metabolizing cholesterol to potential CRC promoters, and attribute the differences in intestinal flora to the composition of the diet [2,3]. The cytotoxicity may be a key mechanism in cancer induction [4], therefore, in the present study, the cytotoxic activity of the coprostanol, cholestanol, coprostanone, and cholestenone on Caco-2 and CCD-18Co cells was evaluated.
A pure compound is cytotoxic when the half maximal inhibitory concentration (IC50) value is less than 4 µg/mL after 48-72 h of incubation [6]. Based on this, cholestenone (IC50 at 72 h: 5 ± 1 µg/mL) would be considered cytotoxic on CCD-18Co cells. Studies in rats indicate that cholestenone induces nuclear aberrations [7] and exchange of sister chromatids [8] in the colonic epithelium. These data suggest that cholestenone could be the metabolite with the greatest capacity to promote CRC. Furthermore, coprostanone is the second most cytotoxic metabolite (IC50 at 72 h: 15.9-46.8 µg/mL). Therefore, the two most cytotoxic metabolites are, in turn, the most hydrophobic. Probably the greater hydrophobicity favors the ability to alter the structure of the cell membrane and thereby generate cell damage, as occurs with secondary bile acids (promoters of colorectal carcinogenesis, structurally similar to cholesterol metabolites) [9]. In contrast, coprostanol and cholestanol are the metabolites with the lowest cytotoxic activity. In the case of coprostanol, the equatorial position of the C-3 hydroxyl group reduces the ability of coprostanol to bind to the cell membranes of the colon, which would facilitate its elimination through the feces [10].
On other hand, it was observed that CCD-18Co cells are more sensitive to the cytotoxicity of cholesterol metabolites. In other studies, it was also observed that the viability of certain tumor lines (human esophageal, gastric and colorrectal, and mouse colorectal) is not affected by cholestanol [11,12], observing, instead, a slight reduction in the viability of non-tumor human esophageal cells [11]. If cholesterol metabolites exert their cytotoxicity by inducing oxidative stress, such as secondary bile acids [13], one possible reason why CCD-18Co cells are more vulnerable to metabolite cytotoxicity can be related to the antioxidant status. The Caco-2 cells are thought to exhibit high intracellular ferritin (antioxidant) levels due to exposure to the heme group through mucosal bleeding and dietary protein during neoplasia [14]. So, the ferritin content in Caco-2 cells is likely to be higher than that of CCD-18Co cells, which would provide protection against possible oxidative damage of cholesterol metabolites. In addition, the composition of the plasma membrane could also be responsible for the difference in the sensitivity of cell lines to metabolites. During colon carcinogenesis, tumor cells undergo changes that allow them to acquire resistance to hydrophobic cytotoxic agents, through an increase in the expression of adenosine triphosphate-binding cassette transporters (ABC), membrane proteins that induce the efflux of substances toxic to cells [15]. Given that these transporters are involved in the efflux of cholesterol and plant sterols from the enterocyte to the intestinal lumen [16], it would be plausible to think that they may also act as transporters for metabolites, making Caco-2 cells less sensitive to their toxicity. In summary, Caco-2 cells are less sensitive to the cytotoxicity of cholesterol metabolites due to their higher antioxidant status and expression in ABC transporters.
It was observed that the reduction in cell viability decreased in some cases with time, which could indicate that the cells adopt a compensatory response. As occurs when cells are exposed to secondary bile acids, the toxic effect of metabolites could lead to the activation of nuclear factor-kappa B (NF-κB) [17]. It is known that NF-κB is a transcriptional regulator that increases the expression of genes that encode antioxidant enzymes and ABC transporter, stimulates cell proliferation, induces resistance to apoptosis, and is related to inflammatory processes [18]. Therefore, a hyperproliferative and antioxidant response, together with an increase in the expression of ABC protein and resistance to apoptosis, would explain the lower reduction in cell viability observed. Furthermore, the increase in cell proliferation, resistance to apoptosis and inflammation could be the mechanisms through which cholesterol metabolites are involved in colorectal tumorigenesis, as occurs with secondary bile acids [13]. Furthermore, it was observed that the compensatory response in Caco-2 cells could occur much earlier than in the non-tumor line. The higher proliferative activity of Caco-2 cells, together with their higher antioxidant status [14] and the higher expression of ABC proteins [15], would explain why the compensatory response of Caco-2 cells occurred earlier than in CCD-18Co cells.
Untreated cells were the control and 5-FU (25 µM), a well-known cytotoxic agent on human colon cancer cells [19]. The cytotoxic activity of metabolites on Caco-2 and CCD-18Co cells was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Briefly, after treatment with metabolites, the culture medium was removed and 90 µL of phosphate-buffered saline (PBS) and 10 µL of MTT (0.5 mg/mL in PBS) added. After 4 h of incubation, the MTT solution was removed and the formazan salts solubilized with 10 µL of dimethylsulfoxide. Formazan formation was determined by spectrophotometry at 570 nm with background subtraction at 690 nm.

Conclusions
The metabolites produced by intestinal bacteria from cholesterol, mainly those of a hydrophobic nature (cholestenone and coprostanone), could be involved in colorectal carcinogenesis through their cytotoxic activity. More in-depth biochemical and molecular assays are needed to decipher the specific mechanisms involved in their deleterious activity.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the Informed Consent Statement.

Abbreviations
The following abbreviations are used in this manuscript: