Berberine Kills Colorectal Cancer Cells by Triggering Iron-Dependent Cell Death

Colorectal cancer remains the third most common and second deadliest cancer globally, with existing therapies limited by resistance and systemic toxicity. This study investigates whether berberine (BBR), an over-the-counter plant-derived alkaloid with established anti-CRC properties, can suppress tumor growth by inducing ferroptosis — a regulated, iron-dependent cell death mode increasingly recognized as a cancer vulnerability — and whether this process disrupts mitochondrial energy metabolism through a defined molecular axis.

Using human HCT116 and murine CT26 CRC cell lines treated at 10, 20, and 40 µM BBR, the team demonstrated dose-dependent inhibition of proliferation (IC50 ~20 µM at 48 h), colony formation, and invasion. Ferroptosis was confirmed by elevated malondialdehyde, 4-hydroxynonenal, lactate dehydrogenase, and Fe2+, alongside depleted glutathione and reduced GPX4, SLC7A11, and FTH1 protein expression. Transmission electron microscopy revealed characteristic mitochondrial shrinkage and cristae loss. Ferroptosis specificity was validated using the inhibitor ferrostatin-1, which reversed BBR-induced cell death, while apoptosis and necroptosis inhibitors did not.

A graph convolutional network-based drug 'on-target' pathway algorithm identified Gli1 — a Hedgehog pathway transcription factor aberrantly activated in CRC — as a top predicted BBR target. Direct binding was confirmed by surface plasmon resonance (KD = 0.652 µM) and cellular thermal shift assay (ΔT = 2.3°C). Mechanistically, BBR inhibited Gli1, which in turn suppressed downstream STAT3, a transcription factor shown to bind promoter elements of ferroptosis negative regulators GPX4, SLC7A11, and FTH1. This Gli1/STAT3-FNR axis represents a novel regulatory pathway connecting Hedgehog signaling to ferroptosis resistance in CRC.

On the energy metabolism front, BBR significantly reduced intracellular ATP levels, oxygen consumption rate, mitochondrial membrane potential, and ATPase (total, Na+/K+, Ca2+/Mg2+) activities in treated CRC cells. These findings establish a mechanistic link: ferroptosis-driven lipid peroxidation products such as 4-HNE inactivate electron transport chain complexes, glutathione depletion depletes TCA cycle intermediates, and Fe2+ overload drives Fenton reactions damaging mitochondrial DNA and metabolic enzymes — collectively producing profound bioenergetic failure.

In BALB/c mouse xenograft models, BBR at 80 mg/kg significantly reduced tumor volume and weight, decreased tumor expression of Gli1, STAT3, GPX4, SLC7A11, and FTH1, and showed no significant organ histopathology or hematological toxicity. These results support translational potential of BBR as a ferroptosis-inducing, metabolically disruptive anti-CRC agent targeting a previously uncharacterized upstream regulatory hub.