# CNOT2 /c-Myc/STAT3 signaling is critically involved in glycolysis mediated apoptosis of benzyl isothiocyanate in hepatocellular carcinoma

## TL;DR

This study shows how benzyl isothiocyanate from cruciferous vegetables triggers cancer cell death in liver cancer by disrupting a key signaling pathway involved in energy metabolism.

## Contribution

The study identifies the CNOT2/c-Myc/STAT3 signaling axis as a critical mediator of BITC-induced apoptosis through glycolysis inhibition in hepatocellular carcinoma.

## Key findings

- BITC suppresses HCC cell proliferation and induces apoptosis by inhibiting glycolytic enzymes like HK2, PKM2, and LDH.
- CNOT2 knockdown or c-Myc overexpression reverses BITC's effects, indicating their role in BITC's mechanism.
- BITC disrupts interactions between CNOT2 and STAT3 or c-Myc, suggesting a disruption of the signaling pathway.

## Abstract

Although benzyl isothiocyanate (BITC), a major compound found in cruciferous vegetables, has been reported to exert antitumor effects in various cancers, its apoptotic mechanism remains unclear. This study aimed to elucidate the apoptotic mechanism of BITC by investigating its role in inhibiting Warburg effect in hepatocellular carcinoma (HCC) cells. BITC suppressed cell proliferation, increased the sub-G1 population, Annexin V/PI and reduced the expression of pro-poly (ADP-ribose) polymerase (pro-PARP), pro-caspase-3, CCR4-NOT transcription complex subunit 2 (CNOT2), c-Myc, signal transducer and activator of transcription 3 (STAT3), and phosphorylated Janus kinase 1 (p-JAK1) in SK-Hep1 and Huh7 HCC cell lines. Notably, knockdown of STAT3 or its upstream regulator CNOT2 further enhanced BITC-induced apoptosis, as evidenced by decreased pro-PARP and pro-caspase-3 expression in SK-Hep1 cells. Additionally, BITC attenuated the expression of hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), and lactate dehydrogenase (LDH) along with reduced LDH production and glucose in SK-Hep1 and Huh7 cells. However, treatment of pyruvate or overexpression of CNOT2 or c-Myc reversed the capacity of BITC to reduce the expression of HK2, pro-caspase-3, and pro-PARP in SK-Hep1 cells. Immunoprecipitation assays further revealed that BITC disrupted the interactions between CNOT2 and STAT3 or c-Myc. Collectively, these findings suggest that the CNOT2/c-Myc/STAT3 signaling axis plays a critical role in glycolysis mediated apoptosis of BITC in HCC cells.

The online version contains supplementary material available at 10.1038/s41598-026-38416-8.