FOXO1, a Potential Therapeutic Target, Regulates Autophagic Flux, Oxidative Stress, Mitochondrial Dysfunction, and Apoptosis in Human Cholangiocarcinoma QBC939 Cells

Abstract

Background/Aims: Autophagy is an evolutionarily conserved catabolic mechanism to maintain energy homeostasis and to remove damaged cellular components, which plays an important role in the survival of various cells. Inhibiting autophagy is often applied as a new strategy to halt the growth of cancer cells. Methods: The effect of FOXO1 gene on cellular function and apoptosis and its underlying mechanisms were investigated in cultured QBC939 cells by the methylthiazoletetrazolium (MTT) assay, western blot, DCFDA mitochondrial membrane potential, and ATP content measurement. FOXO1 siRNA was applied to down-regulate FOXO1 expression in QBC939 cells. Results: Here we reported that FOXO1, acetylation of FOXO1 (Ac-FOXO1) and the following interaction between Ac-FOXO1 and Atg7 regulated the basal and serum starvation (SS)-induced autophagy as evidenced by light chain 3 (LC3) accumulation and p62 degration. Either treatment with FOXO1 siRNA or resveratrol, a sirt1 agonist, inhibited autophagic flux, resulting in oxidative stress, mitochondrial dysfunction (MtD) and apoptosis in QBC939 cells, which were attenuated by enhancing autophagy with rapamycin. On the contrary, inhibiting autophagic flux with 3-MA worsened all these effects in QBC939 cells. Conclusions: Taken together, our study for the first time identified FOXO1 as a potential therapeutic target to cure against human cholangiocarcinoma via regulation of autophagy, oxidative stress and MtD.

Therapeutic Potential of Autophagy Modulation in Cholangiocarcinoma

“…Another potential therapeutic target associated with autophagic flux in CCA is FOXO1. FOXO1 expression and transcriptional activity are involved in promoting cellular autophagy, and the interaction of acetylated FOXO1 with ATG7 regulates basal and starvation-induced autophagy in CCA cells [30]. Cytoplasmic accumulation of FOXO1 is associated with increased proliferation in cholangiocytes [129] and pharmacological inhibition of acetylated FOXO1, which results in autophagy inhibition, leads to apoptosis induction and reduced viability of CCA cells [30].…”

“…FOXO1 expression and transcriptional activity are involved in promoting cellular autophagy, and the interaction of acetylated FOXO1 with ATG7 regulates basal and starvation-induced autophagy in CCA cells [30]. Cytoplasmic accumulation of FOXO1 is associated with increased proliferation in cholangiocytes [129] and pharmacological inhibition of acetylated FOXO1, which results in autophagy inhibition, leads to apoptosis induction and reduced viability of CCA cells [30]. Epigenetic alterations are frequent in CCA, such as miR-124, which was found significantly downregulated in the tumor tissue of patients and in CCA cell lines, and its administration in vitro induced cytotoxic autophagy in CCA cells [29], supporting a protumoral role of epigenomic-mediated inhibition of autophagy.…”

“…Several natural compounds are under evaluation in CCA preclinical models. Salinomycin (a naturally occurring polyether antibiotic [138]), capsaicin (a major pungent component of chili pepper [139]), oblongifolin C (a natural small molecule extracted from Garcinia yunnanensis Hu [140]) and resveratrol (a natural phenol, phytoalexin, produced by plants against infections [30]) have shown anticancer efficacy on CCA models by different mechanisms: inhibiting autophagosome fusion to lysosomes, promoting mTOR activation and blocking ATG7 activation, respectively. Two class III PI3K inhibitors that block initiation of autophagy (3-MA and wortmannin [106]) and Mdivi1 (selective Drp-1 inhibitor [141]), which interferes with mitochondrial activity, have also shown efficacy on CCA.…”

“…Autophagy has been shown to act as a tumor promoter as well as a tumor suppressor in cancer, depending on the cell context, and autophagy modulation has arisen as a promising therapeutic strategy to treat cancer [20][21][22][23][24][25]. Even though the molecular mechanisms of autophagy regulation of tumor biology are not fully understood, multiple reports are showing promising therapeutic potential in combination with other drugs, such as chemotherapy [26].In CCA, several reports released during the last decades have shown how autophagy deregulation is associated with malignant cells compared with normal cholangiocytes in clinical samples, correlating with metastatic disease and poor prognosis [27][28][29][30][31][32][33], and how autophagy modulation shows anticancer efficacy in preclinical models.This review collects clinical and preclinical scientific reports involving autophagy modulation in CCA, putting all puzzle pieces together to try to shed light on the current knowledge of this therapeutic strategy for treating this aggressive disease.…”

“…In CCA, several reports released during the last decades have shown how autophagy deregulation is associated with malignant cells compared with normal cholangiocytes in clinical samples, correlating with metastatic disease and poor prognosis [27][28][29][30][31][32][33], and how autophagy modulation shows anticancer efficacy in preclinical models.…”