SNU449 cells are less sensitive to alcohol metabolism induced toxicity
First, we determined differences in ADH activity and ALDH expression between Huh-7 and SNU449 cell lines. We observed that advanced grade HCC cell line SNU449 displays a 1.86-fold lower ADH activity in comparison to Huh-7 cells, an early grade HCC cell line (Fig. 1A). We also compared the ALDH expression in both Huh-7 and SNU449 using flow cytometry and observed lower number of ALDH positive cells in SNU449. This observation was further confirmed by the lower mean fluorescence intensity (MFI) observed in the case of SNU449 cells (Fig. 1B and C). Cell lines were cultured according to the protocol described in Fig. 1D, showing a 1-month alcohol withdrawal period after CAE. Our results show that CAE and WD had no effect on the ADH activity profile of Huh-7 and SNU449 cells (Suppl Fig. 1A and 1B). When Huh-7 cells were exposed to alcohol, the number of ALDH positive cells as well MFI displayed an increasing trend in CAE that was absent after WD (Suppl Fig. 1C and 1E). Interestingly, when SNU449 cells underwent 270 mM alcohol treatment significant increase of ALDH positive cells and elevated MFI was observed with a recovery seen in conditions of WD. (Suppl Fig. 1D and 1F). Thus, it seems that the increase in ALDH expression seen in SNU449 exposed to 270 mM alcohol reflect a mechanism of tolerance for a quick elimination of the toxic molecule acetaldehyde. The tolerance may be a protective factor and could influence cell survival.
Chronic alcohol exposure induces an aggressive phenotype while withdrawal reverses this effect
Trypan blue staining and counting of Huh-7 cells followed by alcohol exposure and WD revealed that CAE leads to the dose-dependent reduction of cell number. In comparison to untreated/control cells, the decrease of cell number was 18.40% and 52.54% at the 80 mM and 270 mM dose, respectively (Fig. 1E). Similarly, in the case of SNU449 cells, we observed a dose response in the effect of CAE on cell number with no effect at the 80 mM dose, a 35.49% reduction at the 160 mM dose and 77.60% reduction at the 270 mM dose (Fig. 1H). WD partially restored the cell number both in Huh-7 and SNU449.
To confirm the reduction in cell number by CAE observed by Trypan blue staining, cell viability assay was performed according to protocol mentioned in supplementary materials and methods. Cell viability was assessed by MTT assay at 24, 48, 72, 96 h time points. In Huh-7 cells we observed 59.54% decrease in viability in the 270 mM CAE condition (Fig. 1F). The reduction in cell viability at 270 mM CAE in SNU449 cells was 77.49% which was significantly higher in comparison to Huh-7 (Fig. 1I).
Cell mortality was also assessed by propidium iodide staining followed by flow cytometry. Figure 1G shows a dose-dependent increase in the dead cell count observed in Huh-7 cells after CAE. A partial recovery was observed after WD. Strikingly, CAE did not impact the cell mortality in SNU449 cells (Fig. 1J). We also tested the effect of CAE and WD on the cell cycle in Huh-7 and SNU449 cells and observed no effect (Suppl Fig. 2A and 2B).
Indeed, the cell count (Fig. 1H) and the MTT colorimetric assay (Fig. 1I) show a significant decrease in 270 mM CAE treated SNU449 cell line but dead cell did not increase significantly (Fig. 1J). In our cell cultures, we do not observe any cell death. The cells are adherent and alive but less numerous. The most probable hypothesis seems to be a slowing down of cell proliferation.
Withdrawal reverses aggressive phenotype induced by alcohol in Huh-7 cell line
Acquisition of aggressive phenotype in cancer cells can be reflected by a change in cell morphology in which circularity index serves as a quantifiable trait. Fluorescence microscopy was used to investigate the morphological changes in Huh-7 and SNU449 cell lines under CAE and WD conditions. Cells were stained with phalloidin conjugated with Alexafluor 488 to visualize cytoskeleton and DAPI to stain the nucleus. Circularity index of each cell line was determined under untreated conditions. We observed that Huh-7 cells presented a higher circulatory index than SNU449 (Fig. 2A and B). When Huh-7 cells underwent CAE, a significant dose-dependent decrease of circularity was observed: 9.21% in 80 mM, 36.84% in 160 mM, and 47.37% in 270 mM ethanol concentration. Interestingly, WD partially reversed the change in circularity index induced by CAE in Huh-7 cells. The shortfall in circularity index was 7.89% in WD 80 mM, 13.16% in WD 160 mM, and 21.05% in the case of WD 270 mM. Thus, the results presented here suggest that Huh-7, an early grade cancer cells acquire phenotype similar to that of aggressive cells upon exposure to alcohol with partial recovery when alcohol exposure is withdrawn (Fig. 2C and D ). However, it is noteworthy that CAE/WD did not have a significant impact on the circularity index of SNU449 cells (Fig. 2E and F). Thus, CAE can induce early grade cancer cells such as Huh-7 to acquire a phenotype resembling SNU449, an advanced cancer cell while WD can be beneficial as it can reverse the CAE modifications though partially.
Chronic alcohol exposure affects invasive and migratory capabilities
Migration and invasion assays were performed to further confirm our earlier observations. Both Huh-7 and SNU449 display cells migration and invasion capabilities. We compared their migratory capability and observed that SNU449 cells presented a higher migration capability than Huh-7 supporting its tumour grades (Fig. 3A). Enhanced migration capability in Huh-7 due to CAE was dose-dependent. Indeed, results showed an increase of 335.62% and 401.05% compared to control for CAE 160 mM and CAE 270 mM, respectively, while only a minimal increase was observed in the CAE 80 mM which was not statistically significant (Fig. 3B). Strikingly, WD significantly restored the migratory phenotype at a comparable level of untreated cells (Fig. 3B). Chronic alcohol exposure and WD did not change the migratory capability of SNU449 cells (Fig. 3C).
Invasive abilities of cancer cell lines correlate highly with their matrix metalloproteinase (MMP) activity. Here, we quantified the global MMP activity of Huh-7 and SNU449 cells to investigate the effect of CAE and WD on their invasive potential. Results of our experiments revealed that CAE induced the MMP activity in Huh-7 cells while WD resulted in reduced MMP activity. The Huh-7 cells exposed to CAE 160 mM displayed an elevated global MMP level by 1526 ± 584.8% while 270 mM CAE resulted in 1144 ± 224.4% increase in comparison to CAE untreated cells. Increase of global MMP levels upon CAE did not follow a dose-response pattern (Fig. 3D). Similarly, global MMP activity in SNU449 cells was also quantified in cells which have undergone CAE and WD. In the case of SNU449 cells, CAE did influence the global MMP levels with significant increase observed only in 160 mM (164.5 ± 23.55%) and 270 mM (190.2 ± 19.66%) CAE while WD totally reversed the effect of CAE and restored the global MMP levels comparable to those of untreated cells (Fig. 3E).
As expected, increased MMP activity results in enhanced invasive capabilities. Mechanistic assays using modified Boyden chamber were performed to assess the invasive capabilities of Huh-7 and SNU449 upon CAE and WD. Since Huh-7 is an early grade and SNU449, an advanced grade cell line, invasion assay was performed to compare the invasive capabilities of both cell lines. Results show that SNU449 cells displayed significantly higher invasive capability in comparison to Huh-7 (Fig. 3F). However, when Huh-7 cells were exposed to our protocol, significant (468.27%) increase in invasive cell number was observed upon 270 mM CAE while WD restored the invasive cell number comparable to control. (Fig. 3G). In the case of SNU449 cells, data represented in Fig. 3H show that CAE significantly increased the invasiveness. While a dose-dependent increase in the number of migrating cells was observed upon 160 mM and 270 mM CAE (Fig. 3H). Strikingly, WD did not show a significant reduction of invasive cells in SNU449 80 mM CAE while invasive cell number was significantly lower in WD after 160 mM CAE and WD after 270 mM CAE (Fig. 3H). The data presented here further emphasized that CAE promoted cancer cell aggressiveness by significantly enhancing the migratory and invasive potential while WD had a beneficial effect with a recovery of almost all parameters.
Modulation of cancer stem cell markers expression by CAE and WD
Cancer stem cell (CSC) markers have often been described as markers for tumour aggressiveness and their expression often correlates with poor prognosis. Here, we studied the expression of CD133, CD44, CD90, CD24, and CD73 in Huh-7 and SNU449 cells in the conditions of CAE as well as WD using flow cytometry and RT-qPCR (Suppl. Figure 3 and 4). Flow cytometry analysis of Huh-7 cells revealed that CAE induces the expression of CD133, CD44, CD90, and CD24 while CD73 expression remained unaffected and WD restores the expression levels comparable to those of untreated cells. CAE of Huh-7 cells resulted in increased CD133 positive cell number in a dose-dependent manner. Data presented here show that number of CD133 positive cells increased by 1.9-fold in 80 mM, 2.0-fold in 160 mM, and 3.3-fold in 270 mM CAE. WD partially restored the CD133 positive cell number though not comparable to untreated cells (Fig. 4A). The modulation of CD133 positive cell number after CAE and WD was also reflected in mean fluorescence intensity (Suppl Fig. 5A) suggestive of increased expression. In a similar way, expression of CD133 was also tested in SNU449 cells and observed that CAE and WD did not have a significant effect on the CD133 positive cell number (Fig. 4B) as well as on the mean fluorescence intensity (Suppl Fig. 5B).
CAE and WD also impacted the expression of other CSC marker’s namely CD44, CD90, and CD24 in Huh-7 cells but not in SNU449 cells (Fig. 4). CAE of Huh-7 cells resulted in a 7.6-fold increase of CD44 positive cells at 80 mM while a 6.8-fold increase was seen at the 270 mM dose. CD90 expression was similarly impacted by CAE, 270 mM CAE resulted in five-fold increase of CD90 positive cells. Significant impact on the CD24 positive cell number after CAE was observed in Huh-7 cells while CD73 positive cell number remained unaffected (Fig. 4C, E, G, I). Along with variation in the number of positively stained cells, enhanced MFI suggesting increase of expression was also noted (Suppl Fig. 5C, E, G, I). As observed in the case of CD133, WD partially reversed the effects (Fig. 4C, E, G, I). Interestingly, we observed that in SNU449 cells CAE did not have a profound impact on CD44, CD90, CD24, and CD73 positive cell number (Fig. 4D, F, H, J) as well as on MFI (Suppl Fig. 5D, F, H, J). Results presented here show that CAE has a profound impact on the expression of CSC markers in Huh-7 but not in SNU449 suggesting that Huh-7 cells can acquire characteristics of advanced stage cancer cells under conditions of CAE.
CAE in Huh-7 cells selectively inhibits GSK3β and activates AKT and ERK signaling pathways
We investigated the activation of signaling pathways regulating cell invasion with western blot experiments. Densitometry analysis of the immunoblots revealed a 4.141 ± 0.167-fold activation and 3.368 ± 0.355-fold activation of inhibitory site at Ser9 of GSK3β in CAE 160 mM and CAE 270 mM, respectively, compared to control condition. We observed also that CAE 270 mM induced a 1.676 ± 0.120 fold-activation of AKT compared to control condition. Our experiments demonstrated also an increase of ERK 1/2 phosphorylation in CAE conditions (fold-activation of 5.600 ± 0.775, 5.533 ± 1.506, and 4.888 ± 0.750 for CAE 80 mM, CAE 160 mM, and CAE 270 mM, respectively). Very interestingly, WD reversed the phosphorylation modifications induced by CAE. CAE and WD had no effect on mTOR phosphorylation and on PI3K protein levels (Fig. 5A and 5B). Results are schematically summarized in Fig. 5C.
CD133 expression is higher in HCC patients with alcohol use disorder.
Baseline characteristics of HCC patients are described in Table 1. No clinically relevant differences between groups were observed for median age, sex, BCLC stage, and Child–Pugh liver function class. Concerning biochemical characteristics, our clinical study highlighted a higher rate of alphafoetoprotein, bilirubin, and ALAT in non-alcoholic HCC in compared to alcoholic HCC.
Analysis of CD133 CSC markers by RT-qPCR on tumoural and peri-tumoural samples from HCC patients demonstrated that expression of CD133 is significantly higher in tumour samples in comparison to peri-tumoural samples (Suppl Fig. 6A). We found this same result for the CD90 CSC marker expression (Suppl Fig. 6B). Interestingly, when tumoral samples were segregated based on the etiology of the tumour we observed that CD133 expression was 2.35-fold higher in tumours whose etiology was related to excessive alcohol consumption (Fig. 6A). Segregation according to HCC etiology does not show difference on CD90 CSC marker expression (Fig. 6B).