# Deactivation of CK1α enhances the anti-cancer effects of salinomycin in colorectal cancer HCT116 cells

## TL;DR

Combining salinomycin and D4476 significantly reduces colorectal cancer cell growth by triggering cell death and blocking cell survival pathways.

## Contribution

The study reveals a synergistic effect of salinomycin and D4476 in inducing ferroptosis and inhibiting autophagy in colorectal cancer cells.

## Key findings

- The combination of salinomycin and D4476 inhibits HCT116 cell growth more effectively than either compound alone.
- The treatment diminishes autophagic flux and ferroptosis by suppressing NRF2 and PSAT1 expression.
- The combination synergistically depletes GSH and increases MDA production, indicating oxidative stress.

## Abstract

Salinomycin (Sal), an ion-carrier antibiotic, effectively suppresses cancer growth and metastasis through autophagy or ferroptosis induction, thereby overcoming drug resistance. D4476, a selective inhibitor of casein kinase 1 alpha (CK1α), also inhibits the growth of tumors. However, their combined effect on colorectal cancer (CRC) cell growth and the mechanism underlying this effect remain unknown. This study evaluated the impact of Sal and D4476 on HCT116 CRC cells growth, ferroptosis, and autophagy by utilizing MTT assays, real-time PCR, Scratch Wound Healing Assay, reduced glutathione (GSH), and lipid peroxidation assays. It was discovered that Sal in combination with D4476 inhibited cell growth and triggered ferroptosis in a time- and dosage-dependent way, with nuclear factor E2-related factor 2 (NRF2) expression decreasing. Nevertheless, the level of phosphohydroxythreonine aminotransferase 1 (PSAT1) expression is higher in the Sal-D4476 combination compared to Sal alone. In addition, this combination resulted in a synergistic depletion of GSH and production of MDA, as well as an inhibition of autophagic flux by upregulating the gene expressions of Beclin1, LC3βII, and p62. In conclusion, the combination of Sal and D4476 suppressed the growth of HCT116 CRC cells by inducing ferroptosis and inhibiting autophagic flux. This research could lead to a novel method of using Sal in the clinic as a new antitumor drug, particularly when combined with other therapies that target the p62-NRF2 axis, such as D4476.

•The combination of salinomycin and D4476 markedly inhibited HCT116 cell proliferation, exceeding the effects of each compound individually.•Salinomycin and D4476 induced ferroptosis, diminishing the expression of NRF2 and PSAT1.•The integrated therapy markedly diminished autophagic flux.•Salinomycin and D4476 may obstruct the antioxidant p62-NRF2-PSAT1 pathway.

The combination of salinomycin and D4476 markedly inhibited HCT116 cell proliferation, exceeding the effects of each compound individually.

Salinomycin and D4476 induced ferroptosis, diminishing the expression of NRF2 and PSAT1.

The integrated therapy markedly diminished autophagic flux.

Salinomycin and D4476 may obstruct the antioxidant p62-NRF2-PSAT1 pathway.