BCL2 adenovirus

Alcohol abuse is a global health problem that contributes to a large number of chronic liver diseases. High amounts of TGF-β, a potent profibrotic cytokine, contribute to disease progression. Here, researchers show that ethanol itself has no effect on hepatocyte apoptosis, whereas TGF-β augments cell death. Combination treatment resulted in substantial hepatocyte apoptosis, which was also recapitulated in human HCC liver tissue treated ex vivo. Alcohol enhanced the TGF-β proapoptotic gene signature. Potential mechanisms underlying pathway crosstalk involve SMAD and non-SMAD/AKT signaling. Inhibition of CYP2E1 and ADH activity did not prevent this effect, implying that it was not a result of alcohol metabolism. Consistent with this, the ethanol metabolite acetaldehyde did not mimic this effect, and glutathione supplementation did not prevent the superinduction of cell death. In contrast, blocking the activity of GSK-3β, a downstream mediator of AKT signaling, rescued the robust apoptotic response triggered by ethanol and TGF-β. This study provides new information on the crosstalk between ethanol and TGF-β. These findings suggest that ethanol directly leads to an enhancement of the proapoptotic function of TGF-β in hepatocytes, which may have implications for patients with chronic alcoholic liver disease.

Here, the researchers used a biosensor chip to track changes in cell morphology, adhesion, cell-cell interactions, and membrane function in real time by measuring the impedance of mouse hepatocytes in response to ethanol and TGF-β (Figure 1a). While TGF-β and ethanol alone only slightly reduced cell impedance, their combined application effectively reduced cell impedance. Since TGF-β is an initiator of epithelial cell apoptosis, caspase-3 activity was next measured (Figure 1b). Interestingly, after 48 hours, only TGF-β led to caspase-3 activation in single treatment (Figure 1b). This activity was further significantly increased after the combined application of ethanol and TGF-β. By investigating the pathways leading to apoptosis, the researchers found by immunoblotting that mitochondrial cytochrome C release was strongest upon combined treatment (72 hours, Figure 1d), further suggesting that ethanol sensitizes hepatocytes to TGF-β-mediated apoptosis. Next, cell nuclei were stained for Annexin-V using Hoechst 33342. Consistent with previous results (Figure 1b), the strongest positivity for Annexin-V was detected upon combined treatment (Figure 1e). BCL2 has been shown to counteract TGF-β-mediated apoptosis. Therefore, the researchers transfected hepatocytes with an adenoviral vector expressing Bcl2 (Ad-Bcl2). BCL2 overexpression completely suppressed TGF-β-induced cell death and prevented ethanol-dependent superinduction (Figure 1f).

Figure 1. Ethanol plus TGF-β exert a strong apoptotic response in mouse hepatocytes and human HCC. (Gaitantzi H, et al., 2018)