The role of bile acids in the increased incidence of colorectal cancer in cholecystectomized patients

doi:10.21203/rs.3.rs-3222463/v1

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The role of bile acids in the increased incidence of colorectal cancer in cholecystectomized patients

https://doi.org/10.21203/rs.3.rs-3222463/v1

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An increased incidence of colorectal cancer (CRC) has been observed among cholecystectomized patients. However, the exact mechanism is not fully understood. Therefore, this study aimed to compare the effects of hepatic and gallbladder bile on normal and cancerous colon cells and to separately examine the effects of the most common bile acids (BAs). The concentrations of BAs in hepatic and gallbladder bile were determined using high-performance liquid chromatography. The effects of bile and individual BAs (100, 300, 500, and 1000 µM) on the viability, proliferation, and metastatic potential of a normal cell line (FHC) and two adenocarcinoma (Caco-2 and FHC-116) cell lines were investigated using different cell assays. No difference was observed in the effects of hepatic and gallbladder bile on the proliferation of normal and adenocarcinoma cells. Among the BAs, taurine-conjugated BAs, especially taurocholic acid, time-dependently increased the proliferation of normal and adenocarcinoma cells and the metastatic potential of cancer cells. The study results showed that the increased risk of CRC in cholecystectomized patients is not due to bile composition changes, but elevated levels of taurine-conjugated BAs may contribute to the development or progression of CRC.

Biological sciences/Cancer

Biological sciences/Cancer/Gastrointestinal cancer

Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer

Bile acids (BAs) are responsible for many functions in the body. BAs are essential for the emulsification of dietary fats and facilitate digestion. Among the BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA) are primary BAs, which are conjugated with glycine or taurine in the liver. Secondary BAs result from the dehydroxylation of primary BAs due to the action of intestinal bacteria. Under normal circumstances, bile is periodically secreted into the duodenum and helps break down fats. Then, most of the bile is absorbed from the large intestine and returned to the liver. However, under pathological conditions, such as symptomatic cholelithiasis or gallbladder diseases, removal of the gallbladder may become necessary, causing changes in the composition and secretory properties of bile. Cholecystectomy increases the risk of certain gastrointestinal complications, such as bile duct stricture, choledocholithiasis, papillary stenosis, or the development of colorectal cancer (CRC) [1-6].

Colon cancer is one of the most common gastrointestinal cancers worldwide [7]. Several clinical studies have investigated the association between cholecystectomy and CRC [4-6,8,9]. The risk of colon cancer increases several times in cholecystectomized patients, especially in females [6,10]. Interestingly, cholecystectomy increases the risk of developing proximal right-sided colon tumors [11].

After cholecystectomy, the composition of bile changes and it is continuously released into the duodenum. Secondary BAs, such as deoxycholic acid (DCA) and lithocholic acid (LCA), have been reported to promote colon cancer progression [12-16]. Since the proportion of secondary BAs increases compared with that of primary BAs in the bile after cholecystectomy [17,18], this may explain why CRC is more likely to develop in cholecystectomized patients. However, other studies have found no difference in the composition of bile before and after cholecystectomy [19-21]. A recent review has reported that the altered metabolism of BAs and the change in the composition of the intestinal microbiome play a role in the development of CRC [22]. However, the exact mechanism is still completely unclear. Thus, this study aimed to compare the composition of hepatic and gallbladder bile and investigate their effects on normal and colon cancer cell lines.

Composition of hepatic and gallbladder bile

The composition of hepatic and gallbladder bile was compared using the high-performance liquid chromatography (HPLC) technique (Fig. 1a and b). In the gallbladder bile, glycine- and taurine-conjugated BAs were found in the highest amount, whereas, among the non-conjugated BAs, small amounts of CA, DCA, and CDCA were detected. The BA concentrations in the liver were roughly a third or a quarter of the BA concentrations measured in the gallbladder, and in the case of glycine-conjugated BAs, the proportions corresponded to the proportions found in the gallbladder. Among the taurine-conjugated BAs, only taurocholic acid (TCA) could be measured in detectable amounts, whereas the concentrations of non-conjugated BAs (CA, DCA, and CDCA) were less than 1 µg/mL. Additionally, the amount of ursodeoxycholic acid (UDCA), which is known to have a cell-protective function in many tissues, was examined [23]. This BA was not present in the gallbladder, and a small amount was detected in the liver.

Effects of human bile on the proliferation of normal and tumor-derived colon cell lines

The effects of bile treatment on the growth of normal (FHC) and cancerous (Caco-2 and HTC-116) colon cell lines were examined (Fig. 2). Since the amount and composition of BAs in the bile samples taken from each patient differed (Fig. 1b), the effect of each sample was examined separately. In the case of normal and Caco-2 cells, a significant increase in proliferation was detected in one case (sample 6). In this case, TCA, taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA) concentrations were higher than in other cases. In the case of the HTC-116 cell line, the highest degree of proliferation was observed in groups treated with sample 6, but other samples (samples 1, 2, and 4) also induced elevated cell growth. Interestingly, treatment with hepatic bile samples reduced the proliferation of normal and Caco-2 cells, except for samples 4 and 8 (containing a high amount of TCA), where an increase was observed. In the case of HTC-116 cells, proliferation was increased in almost all samples, except sample 3, where the BA concentration was very low. The average effect of the individual samples showed no difference compared to the control, either for hepatic (H Total) or gallbladder (GB Total) bile in the normal and Caco-2 cell lines.

However, for HTC-116 cells, the average effect of the samples showed a significant increase in the proliferation rate in both hepatic and gallbladder bile. These results showed no difference in the effects of hepatic and gallbladder bile on cell proliferation in either normal or adenocarcinoma cells. Thus, the different composition of hepatic and gallbladder bile is probably not responsible for the higher risk of CRC development or the accelerated cancer progression in cholecystectomized patients. However, differences in the effects of individual patient samples were observed. Cells proliferated more in bile samples with high concentrations of taurine-conjugated BAs, especially TCA. This suggests that individual BAs affect cell function differently.

Effects of BAs on the viability, proliferation, and metastatic potential of normal and tumor-derived colon cell lines

The effects of individual BAs on colon cells were examined to determine whether the effect of bile on the cell lines is attributed to the BAs. The six most common BAs found in human bile (glycine- and taurine-conjugated forms of CA, DCA, and CDCA) were selected, the presence of which was also confirmed using HPLC. Furthermore, the effect of the unconjugated DCA was investigated since several studies have described the carcinogenic effect of this BA in the colon [13,15,16]. BAs can be toxic to cells. Thus, the concentration at which cells retained their viability was first determined (Fig. 3a-c).

The effects of BAs were tested in four different concentrations (100, 300, 500, and 1000 µM) at 24, 48, and 72 h. Cytotoxicity studies showed that BAs caused a high degree of cell death at 500 and 1000 µM concentrations in both normal and adenocarcinoma cells. Interestingly, the toxic effect of the BAs on the cells during the 24–48 h treatment was greater in several groups than that detected during the 72 h treatment. This suggests that the cells have adapted to the changed conditions to some extent. Since higher concentrations of BAs proved toxic, subsequent experiments were performed with 300 µM BAs since this concentration is closest to physiological conditions [24].

In the case of the normal cell line, the 24 and 48 h BA treatments mostly decreased or did not affect cell proliferation. The 72 h incubation increased the rate of proliferation in the TCA- and TCDCA-treated groups (Fig. 4a). In adenocarcinoma cells, TCA, TCDCA, and DCA treatment increased cell proliferation at longer incubation times (48 and 72 h) in a time-dependent manner. Furthermore, in the case of HTC-116, glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), and TDCA also time-dependently enhanced the proliferation. Additionally, whether the proliferation induced by BAs is associated with increased cell adhesion was investigated (Fig. 4b). In the case of normal cells, TCA and TCDCA treatment increased cell adhesion, similar to proliferation studies. In Caco-2 cells, only TCA and DCA treatment caused a significant increase, while in the case of HTC-116 cells, cell adhesion increased in all treated groups.

The invasion, migration, and colony-forming ability of the adenocarcinoma cells were also investigated (Fig. 5a-e). The degree of cell migration and invasiveness largely determines the ability of cells to form metastases, whereas the colony-forming capacity of the cells is required for tumor initiation and progression. TCA, TCDCA, and DCA treatment increased the extent of invasion (Fig. 5a), migration (Fig. 5b and c), and the size of colonies (Fig. 5d and e) in both cell lines. Interestingly, TDCA did not affect or slightly reduced the metastatic potential of the cells. Among the glycine-conjugated BAs, GCDCA enhanced the invasion of both cell lines and migration of HTC-116 cells.

Colon diseases, especially CRC, are responsible for increased morbidity and mortality worldwide. CRC is the second leading cause of cancer death globally, and its incidence is steadily rising [7,25]. The relationship between CRC and BAs, especially DCA and LCA, has long been known [12-16]. DCA and LCA are secondary BAs produced from primary BAs under the influence of bacterial flora. During enterohepatic circulation, most of the primary and secondary BAs return to the liver, where they are conjugated with glycine or taurine and excreted in the bile. However, this process occurs differently in cholecystectomized patients as the composition of bile and its secretion into the duodenum change. Several studies have indicated that cholecystectomy promotes CRC development. However, the mechanism is still not well understood.

This study used different cell assays and showed that the different composition of hepatic and gallbladder bile alone is not responsible for the development or progression of CRC. However, a difference in the effects of individual BAs was observed. Previous studies highlighted the role of secondary BAs, especially DCA and LCA, in the development and progression of CRC. These studies have identified several intracellular pathways through which these BAs exert their effects. DCA has been shown to enhance the proliferation and metastatic potential of colon cells by the production of reactive oxygen species [26,27], induction of DNA damage and mutation [28,29], which causes genetic instability, or activation of a range of signal transduction pathways [13,30-34]. However, the study of other BAs was far behind that of DCA. This comprehensive study showed for the first time that TCA and TCDCA strongly increase the proliferation and metastatic potential of colon cells, indicating that the concentration of these BAs in the BA pool is likely a key factor in the development and progression of CRC.

The composition of hepatic and gallbladder bile was first examined using the HPLC technique. Glycine-conjugated BAs (glycocholic acid (GCA), GDCA, and GCDCA) and TCA were present in both hepatic and gallbladder bile. However, the concentrations of these BAs were orders of magnitude lower in the liver. In the case of TDCA and TCDCA, detectable amounts were found only in the gallbladder. The fact that taurine-conjugated BAs occur in a smaller proportion in cholecystectomized patients can presumably be explained by the different diets of these patients. The glycine:taurine ratio is greatly influenced by the protein and fat content of the food. High meat consumption increases taurine uptake and the amount of taurine-conjugated BAs in bile [35,36]. Therefore, the low taurine-conjugated BA ratio observed in cholecystectomized patients can presumably be explained by the special diet of these patients. In the case of non-conjugated BAs, roughly the same BAs are present in hepatic bile as in gallbladder bile, but in lower concentrations. The concentration difference between hepatic and gallbladder bile is not surprising since, after synthesis, BAs are stored and concentrated in the gallbladder, during which their concentration becomes approximately ten times higher than the concentration of BAs in the liver.

Next, the effects of hepatic and gallbladder bile on colon cells were compared. In the case of normal cells, one gallbladder sample (GB5) decreased the proliferation of the cells, whereas another sample (GB6) significantly increased it. Comparing the composition of the two samples, GB5 did not contain non-conjugated BAs, and the concentrations of TCA and TCDCA were relatively low. The concentration of taurine-conjugated BAs was extremely high in sample GB6. Sample GB6 also increased the proliferation of adenocarcinoma cells, whereas sample GB5 caused a decrease only in Caco-2 cells. Since the TCA concentration was still higher in GB5 than in the hepatic bile samples, this assumes that other factors play a role in the effect of this sample. These results emphasize the key role of taurine-conjugated BAs, especially TCA, in cell proliferation. This is also supported by the fact that, in the case of hepatic bile, where the samples did not contain TDCA and TCDCA, the increase was significant in those cases only where the concentration of TCA was relatively high (sample H4 and H8).

Furthermore, the effects of individual BAs were examined. TCA, TCDCA, and DCA treatments increased the rate of proliferation and adhesion in all three cell lines. Additionally, these BAs promoted colony formation and increased the migration and invasion capacity of cells. These results suggest that these BAs accelerate colon tumor progression by enhancing the proliferation and metastasis potentials of the cells. Among the glycine-conjugated BAs, GDCA and GCDCA exerted a similar effect on the HTC-116 cell line, but not on the Caco-2 cells. This discrepancy may be because the two cell lines do not express the same BA transporters or the BAs do not activate the same signaling pathways within the cells.

Although the role of DCA in CRC is well recognized, fewer results are reported regarding the role of taurine-conjugated BAs. Taurine is an essential amino acid. The human body cannot produce taurine only to a limited extent, so most of the taurine enters the body with food. Red meat, mollusks, and energy drinks contain large amounts of taurine, which is either excreted in the urine or bound to free BAs in the liver [36]. Diets rich in dark meat have been known to increase the chance of CRC development [37,38]. Since red meats are rich in taurine, high consumption of these foods increases the proportion of taurine-conjugated BAs in bile, which may contribute to the development of CRC, among other mechanisms. A recent review by Ridlon et al. described a novel mechanism for the role of TCA in CRC [39]. According to their hypothesis, during the bacterial metabolism of TCA, two potentially toxic substances are formed, namely, hydrogen sulfide and DCA, which are known to be involved in the biological processes of colon cancer. This hypothesis assumes that substances formed during the decomposition of TCA lead to CRC. However, our results showed that TCA also has a direct effect on colon cells, as shown in esophageal cancer or cholangiocarcinoma [40,41].

To the best of our knowledge, this is the first study to investigate the effects of hepatic and gallbladder bile on the development and progression of colon cancer. The results showed that although the composition of hepatic and gallbladder bile differs, this is not responsible for the higher incidence of colon cancer in cholecystectomized patients. Since bile is constantly released into the duodenum due to gallbladder removal, constant BA exposure can increase the risk of CRC development. Furthermore, the effects of the most common BAs found in human bile were comprehensively examined using one normal and two adenocarcinoma cell lines. The novelty of these results is that we showed that, in addition to DCA, taurine-conjugated BAs, TCA and TCDCA, also increase the proliferation of both normal and cancer cells, as well as the metastatic potential of cancer cells, which highlights the importance of taurine-conjugated BAs in the pathogenesis of CRC.

Chemicals and solutions

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and type I collagen were obtained from Thermo Fisher Scientific (Darmstadt, Germany). Technical Manual Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Rockville, MD, USA). GDCA was obtained from Cayman Chemical Company (Michigan, MI, USA). Methanol (liquid chromatography–mass spectrometry (LC–MS) grade), acetonitrile (LC–MS grade), and water (LC–MS grade) were obtained from VWR (Budapest, Hungary). Matrigel matrix was ordered from Corning (Glendale, AZ, US). All other laboratory chemicals were purchased from Sigma-Aldrich Kft. (Budapest, Hungary).

Cell lines and treatments

HTC-116, a human colon adenocarcinoma cell line, was obtained from Sigma-Aldrich Kft. (Budapest, Hungary). HTC-116 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F-12 Ham media supplemented with 15% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine, and 2% (v/v) antimycotics/antibiotics (AA). Caco-2, a human colon adenocarcinoma cell line, was a kind gift from Dr. Maria A. Deli (Biological Research Center, Szeged, Hungary). Caco-2 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 media supplemented with 15% (v/v) FBS, 1% (v/v) L-glutamine, and 2% (v/v) AA. FHC, a normal colon epithelial cell line, was obtained from American Type Culture Collection (Manassas, VA, United States). FHC cells were cultured in DMEM/F-12 Ham media supplemented with 15% (v/v) FBS, 10 mM HEPES (v/v) (for a final concentration of 25 mM), 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 100 ng/ml hydrocortisone, and 20 ng/mL human recombinant epidermal growth factor. The cells were incubated at 37°C and gassed with a mixture of 5% CO₂ and 95% air. The media were replaced every second day. Passage numbers were between 3 and 15 in each experiment.

For BA treatments, the cells were treated with seven different types of BAs (GCA, GDCA, GCDCA, TCA, TDCA, TCDCA, and DCA) in four different concentrations (100, 300, 500, and 1000 µM) for 24, 48, and 72 h. For human bile treatments, the cells were treated with gallbladder or hepatic bile. Gallbladder bile samples were obtained from the gallbladder of 6 cadavers (average age: 78.42 ± 4.7; male-to-female ratio: 3:3) in the Department of Pathology, University of Szeged. The cadavers had no known history of hepatic or biliary disease. Hepatic bile samples were obtained during routine endoscopic retrograde cholangiopancreatography procedure from patients (average age: 66 ± 5.6; male/female ratio: 2/6) who previously underwent cholecystectomy at the Department of Medicine, University of Szeged. Patients’ data are shown in Table 1. Bile samples were kept at −20°C until use. A small part of the samples was used for HPLC measurements, while the remaining bile samples were used for cell treatments for 24, 48, and 72 h in a 100× dilution. The cells were seeded in 25 cm² tissue culture flasks or 96-well plates and grown to 70%–80% of confluence two days before BA or human bile treatments.

Ethical approval

The clinical part of the study was carried out with the approval of the Ethics Committee of the University of Szeged (No. 5016), followed by the EU Member States’ Directive 2004/23/EC on presumed consent practice for tissue collection, the guidelines of the Helsinki Declaration, and the General Data Protection Regulation (EU) 2016/679. Written informed consent was obtained from all patients for sample and data collection.

Table 1. Clinicopathological characteristics and the level of bile acids (BAs) in the gallbladder and liver.

Clinicopathological characteristics and the level of bile acids in gallbadder and liver
Variable	Gallbladder (n=6)		Liver (n=8)
Variable	n	(%)	n	(%)
Gender Male Female	3 3	(50.50) (50.00)	2 6	(25.00) (75.00)
Age ˂65 ≥65	1 5	(16.66) (83.33)	2 6	(25.00) (75.00)
Cause of death Sepsis Acute left ventricular failure Stroke Tumour	1 3 1 1	(16.66) (50.02) (16.66) (16.66)	0 0 0 3	(0.0) (0.0) (0.0) (37.5)
Bile acids in human serum ( µg/mL)
GCA	8 048 ± 1996	(19.77)	929 ± 160	(20.37)
GDCA	7 733 ± 1790	(19.00)	799 ± 233	(17.51)
GCDCA	11 138 ± 4096	(27.37)	1 982 ± 736	(43.46)
TCA	5 144 ± 1890	(12.64)	848 ± 133	(18.59)
TDCA	3 303 ± 1142	(8.11)	0±0.0	(0.00)
TCDCA	4 877 ± 1474	(11.98)	0±0.0	(0.00)
DCA	34.5 ± 25.9	(0.09)	0.20 ± 0.08	(0.004)
CDCA	65.4 ± 41.3	(0.16)	0.65 ± 0.31	(0.014)
CA	354 ± 331	(0.88)	0.32 ± 0.1	(0.007)
UDCA	0±0.0	(0.00)	1.61	(0.035)
Total Bile Acids:	40 699.05		4 559.49

LC–MS measurements

The frozen samples were allowed to thaw at 4°C. Then, a dilution of 1:500 of the bile samples and a dilution of 1:200 of the liver samples were prepared in cold methanol:water 1:1. 100 µL of each sample was spiked with 10 µL methanol, containing 100 µg/mL internal standards (GCDA-D4 used for TCA, GCA, GCDA, and GDCA and DCA-D4 used for DCA, UDCA, CDCA, TDCA, and TCDCA, respectively). Samples were vortex-mixed (LSE W, Corning, NY, USA) for 1 min and then incubated for 30 min at −20°C. The incubated samples were centrifuged (Heraeus Fresco 17, Thermo Scientific, Waltham, MA, USA) at 13000 rpm for 15 min at 10°C. 5 µL of the supernatants was subjected to liquid chromatography–high-resolution mass spectrometry (LC–HRMS) analysis.

LC–HRMS measurements were performed using a Thermo Scientific Dionex Ultimate 3000 UHPLC System (Dionex) coupled to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer operating with a heated electrospray interface. BAs were separated using a Purospher STAR RP-18 Hibar HR 100 × 2.1 mm, 2 µm reversed-phase columns (Merck KGaA, Darmstadt, Germany) tempered at 40°C. Solvent A was water, and solvent B was methanol:acetonitrile 1:1, and both were supplemented with 0.1% formic acid. The flow rate was 300 µL/min, and the following gradient elution was applied: 0–1 min, 5% B; 12 min, 95% B; 15 min, 95% B; 15.5 min, 5% B; 20 min, 5% B.

The ion source had the following settings: the temperature of the probe heater and ion transfer capillary, spray voltage, sheath gas flow rate, auxiliary gas flow rate, and S-lens radio frequency level were set to 350°C, 275°C, 3.5 kV, 30 arbitrary units, 20 arbitrary units, and 50 arbitrary units, respectively. The mass spectrometer acquired data using parallel reaction monitoring in negative polarity mode at 17,500 (at m/z = 200) resolution and a minimum automatic gain control target of 2.00 × 105 with a maximum injection time of 100 ms. The isolation window was 0.4 m/z. Fragmentation was performed with a normalized collision energy of 60.

Cell adhesion

96-well tissue plates were coated with 40 µg/ml type I collagen (diluted in phosphate-buffered saline (PBS)) at 4°C. After 12 h of coating, the collagen solution was removed, and the wells were air-dried and then washed with PBS. The cells were serum-starved for 8 h. Then, 100 µl cell suspension (10⁵ cells/ml) was added to each coated well and incubated for 20 min at 37°C. The non-adherent cells were removed by washing the wells with a culture medium four times. After washing, the cells were incubated with different BAs for 24, 48, and 72 h. After treatments, 10 µl of MTT substrate was added to each well, and the cells were incubated for an additional 3 h. The MTT-treated cells were then lysed in dimethyl sulfoxide, and absorbance was measured at 560 nm using a FLUOstar OPTIMA Spectrophotometer (BMG LabTech, Ortenberg, Germany). All treatments were carried out in triplicate.

Proliferation

Cell proliferation was determined using the CCK-8 solution. Briefly, the cells (5 × 10³) were seeded in a sterile 96-well plate and incubated with BAs for 24, 48, and 72 h. After treatments, the cells were incubated with 10 µl of the CCK-8 solution for 3 h. Then, absorbance was measured at 450 nm using a FLUOstar OPTIMA Spectrophotometer (BMG LabTech, Ortenberg, Germany). All treatments were carried out in triplicate.

Viability

Cell viability was measured using Lactate Dehydrogenase (LDH) Cytotoxicity Colorimetric Assay Kit according to the manufacturer’s instructions. Briefly, 100 µl cell suspension (2 × 10⁴ cells) was seeded in a sterile 96-well tissue culture plate and incubated overnight to allow the cells to adhere. The following day, the cells were treated with 0.1% Triton X-100 or BAs. The LDH release induced by Triton X-100 was assigned to 100%. After incubation, 100 µl supernatant was carefully removed from each well and transferred into a new sterile 96-well plate. Then, 100 µl reaction mixture was added to each well and incubated for 30 min at room temperature. Absorbance was measured at 490 nm using a FLUOstar OPTIMA Spectrophotometer (BMG LabTech, Ortenberg, Germany) to determine LDH release. All treatments were carried out in triplicate. The average absorbance values of each triplicate were calculated, and the average value of the background control (200 µl assay medium) was subtracted from each sample to reduce background noise. Then, the percentage of cytotoxicity was calculated using the following formula: Cytotoxicity (%) = (exp. value − low control/high control − low control) × 100. For low control, 100 µl cell suspension plus 100 µl assay medium was added, and for high control, 100 µl cell suspension plus 100 µl Triton X-100 was added.

Migration

The cells (250,000 cells/well) were seeded in 24-well plates and allowed to adhere overnight. The following day, the confluent monolayer was gently scratched using P2 tips. Only wells containing even-sided and sharp-edged wounds were used for experiments. After washing, reagents were added to the wells. Pictures were taken before and after the 72 h BA treatment using an Olympus IX71 inverted microscope (Olympus, Budapest, Hungary). The digital images were analyzed using Image J to quantify the distance between the two edges of the wound, and the migration rate was expressed as the percentage of migration.

Clonogenic assay

The cells (1,000 cells/ well) were seeded in 6-well plates and allowed to adhere overnight. The following day, BAs were added to the wells, and the cells were incubated for 24, 48, and 72 h. After treatments, the normal culture medium was returned, and the cells were allowed to grow until day 9. Then, the media were removed, and the cells were washed with PBS, fixed with methanol–ethanol solutions (3:1 dilution), and stained with Giemsa. Pictures were taken before and after the 72 h BA treatment using an Olympus IX71 inverted microscope (Olympus, Budapest, Hungary). The digital images were analyzed using Image J.

Invasion assay

Matrigel pre-coated transwell inserts were used for invasion assays. 200 µl cell suspension (~ 2.5 × 10⁵/ml in serum-free medium) was loaded into the upper chamber of the transwell. In contrast, the lower chamber contained 750 µl complete medium with or without BAs. Then, the cells were incubated at 37°C for 24–72 h in a humidified incubator. The cells that migrated to the bottom surface of the transwell were fixed in formaldehyde (3.7% in PBS), permeabilized with 100% methanol, and stained with Giemsa. The non-invading cells on the upper surface of the membrane were gently scraped off using a cotton swab. Cell invasion was quantified by counting the average number of invaded cells in five randomly selected microscopic fields. All treatments were carried out in triplicate.

Statistical analysis

Quantitative variables were presented as mean ± standard error of the mean. Significant differences between groups using determined by ANOVA. P ≤ 0.05 was considered statistically significant.

Funding

This study was supported by the National Research, Development, and Innovation Office (SNN134497 to VV).

Authors’ contributions

EGperformed all of the experiments and analyzed the data. IM were involved in the wound healing, adhesion, and clonogenic assays; MV and ASz performed the HPLC-MS measurements. Human serum samples were provided by BV and ZSz. LT, ÁSz, DV and PH were involved in conceptualization and data interpretation. VV supervised the project and drafted the manuscript. All authors approved the final version of the manuscript.

Data availability statement

The data that support the findings of this study are openly available in figshare at https://figshare.com/, reference number 23500980.

Informed consent statement

Written informed consent was obtained from all patients and healthy volunteers involved in the study.

Competing interests

The authors declare that they have no competing interests.

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The role of bile acids in the increased incidence of colorectal cancer in cholecystectomized patients

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