Engagement with tNOX (ENOX2) to Inhibit SIRT1 and Activate p53-Dependent and -Independent Apoptotic Pathways by Novel 4,11-Diaminoanthra[2,3-b]furan-5,10-diones in Hepatocellular Carcinoma Cells

Abstract

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver and is among the top three causes of cancer-associated death worldwide. However, the clinical use of chemotherapy for HCC has been limited by various challenges, emphasizing the urgent need for novel agents with improved anticancer properties. We recently synthesized and characterized a series of 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives that exhibit potent apoptotic activity against an array of cancer cell lines, including variants with multidrug resistance. Their effect on liver cancer cells, however, was unknown. Here, we investigated three selected 4,11-diaminoanthra[2,3-b]furan-5,10-dione derivatives (compounds 1–3) for their cytotoxicity and the underlying molecular mechanisms in wild-type or p53-deficient HCC cells. Cytotoxicity was determined by WST-1 assays and cell impedance measurements and apoptosis was analyzed by flow cytometry. The interaction between compounds and tumor-associated NADH oxidase (tNOX, ENOX2) was studied by cellular thermal shift assay (CETSA). We found that compound 1 and 2 induced significant cytotoxicity in both HepG2 and Hep3B lines. CETSA revealed that compounds 1 and 2 directly engaged with tNOX, leading to a decrease in the cellular NAD+/NADH ratio. This decreased the NAD+-dependent activity of Sirtuin 1 (SIRT1) deacetylase. In p53-wild-type HepG2 cells, p53 acetylation/activation was enhanced, possibly due to the reduction in SIRT1 activity, and apoptosis was observed. In p53-deficient Hep3B cells, the reduction in SIRT1 activity increased the acetylation of c-Myc, thereby reactivating the TRAIL pathway and, ultimately leading to apoptosis. These compounds thus trigger apoptosis in both cell types, but via different pathways. Taken together, our data show that derivatives 1 and 2 of 4,11-diaminoanthra[2,3-b]furan-5,10-diones engage with tNOX and inhibit its oxidase activity. This results in cytotoxicity via apoptosis through tNOX-SIRT1 axis to enhance the acetylation of p53 or c-Myc in HCC cells, depending on their p53 status.

SIRT1 in the Development and Treatment of Hepatocellular Carcinoma

“…In p53 WT HCC cells, these anti-cancer compounds bound and downregulated tNOX, which decreased intracellular NAD, and consequently suppressed SIRT1 activity. Decreased SIRT1 activity led to increased p53 acetylation and activation, which upregulated its downstream target, pro-apoptotic Bak and thus increased apoptosis (246). Augmented p53 acetylation promoted by SIRT1 inhibition was also associated with activation of PUMA, which upregulates Bak and prompts apoptosis (246, 247).…”

“…tNOX reduction reestablished non-cancer phenotypes, such as decreased migration, and amplified sensitivity to stress-induced apoptosis. Accumulating evidence suggests that suppressing tNOX may improve patient prognosis (246, 248–250).…”

“…In HCC cells, SIRT1 induced the expression of oncogenic c-Myc, which in turn increased β-catenin mRNA and protein expression and amplified the transcription and expression of the β-catenin target genes survinin and cyclin D1. Therefore, SIRT1 overexpression promoted oncogenesis via c-Myc activation (39, 246) and protected against p53 induced apoptosis (26).…”

Cardiac glycoside sensitized hepatocellular carcinoma cells to TRAIL via ROS generation, p38MAPK, mitochondrial transition, and autophagy mediation

“…Most hepatocellular carcinoma exhibits resistance to TRAIL. Therefore, combination therapies may yield high therapeutic potential in HCC management …”

“…Therefore, combination therapies may yield high therapeutic potential in HCC management. 19 Additionally, reactive oxygen species (ROS) also play vital roles in intrinsic apoptosis by triggering mitochondrial damage. 20,21 ROS generation can damage multiple macromolecules, impair cellular functions, and lead to apoptotic or necrotic cell death.…”