Aqueous-soluble components of sporoderm-removed Ganoderma lucidum spore powder promote ferroptosis in oral squamous cell carcinoma

Objective: Ferroptosis is a novel cell death process which displays a promising role in cancer treatment.However, clinically available drugs targeting ferroptosis are rarely used, and yet there are no studies reporting on inducing ferroptosis via Chinese herbal extracts. Here we explored the tumor inhibition effects of Ganoderma lucidum(G. lucidum) on oral squamous cell carcinoma(OSCC). Specifically, we aimed to clarify the biological mechanism of components in the dietary, aqueous-soluble sporoderm-removed G. lucidum spore powder(A-GSP).Methods: Preliminary transcriptome analysis revealed the significant enrichment of the ferroptosis pathway.Cellular Fe2+, glutathione(GSH), malondialdehyde(MDA), reactive oxygen species(ROS) and lipid peroxide levels were measured to identify ferroptosis occurrence. Western blotting was used to measure ferroptosis-related proteins. Changes in mitochondria morphology and function were observed with transmission electron microscopy(TEM) and ATP detection assays. Ferroptosis inhibitor ferrostatin-1 was then used to verify the anti-tumor effects of A-GSP. Finally, nude mice xenograft models of oral cancer confirmed that A-GSP inhibited tumor growth.Results: A-GSP promoted ferroptosis in oral cancer cells by inducing Fe2+influx, GSH depletion, as well as lipid peroxide and ROS accumulation. Ferroptosis-related proteins exhibited corresponding changes, particularly Acylco A synthetase long chain family member 4(ACSL4) increase and glutathione peroxidase 4(GPX4) decrease. A-GSP considerably lowered mitochondrial volume and ridge number, while significantly decreasing ATP production. Ferrostatin-1 reversed all of these A-GSP-induced changes. In vivo, A-GSP exerted a ferroptosismediated tumor-suppressing effect without observable adverse reactions.Conclusions: Our findings demonstrate the therapeutic potential of A-GSP for treating patients with OSCC by targeting ferroptosis.

Tumor necrosis factor a accelerates Hep-2 cells proliferation by suppressing TRPP2 expression

TRPP2, a Ca^(2+)-permeable non-selective cation channel, has been shown to negatively regulate cell cycle, but the mechanism underlying this regulation is unknown. Tumor necrosis factor a(TNF-α) is a proinflammatory cytokine extensively involved in immune system regulation, cell proliferation and cell survival. However, the effects and mechanisms for the role of TNF-αin laryngeal cancer remain unclear. Here, we demonstrated using western blot analyses and intracellular Ca^(2+) concentration measurements that TNF-α treatment suppressed both TRPP2 expression and ATP-induced Ca^(2+) release in a laryngeal cancer cell line(Hep-2). Knockdown of TRPP2 by a specific siRNA significantly decreased ATP-induced Ca^(2+) release and abolished the effect of TNF-α on the ATP-induced Ca^(2+) release. TNF-α treatment also enhanced Hep-2 cell proliferation and growth, as determined using cell counting and flow cytometry cell cycle assays. Moreover, TNF-α treatment down-regulated phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK) and phosphorylated eukaryotic translation initiation factor(p-eIF2α)expression levels, without affecting PERK and eIF2 a expression levels in Hep-2 cells. We concluded that suppressing TRPP2 expression and TRPP2-mediated Ca^(2+) signaling may be one mechanism underlying TNF-α-enhanced Hep-2 cell proliferation.These results offer new insights into the mechanisms of TNF-α-mediated laryngeal cancer cell proliferation, and provide evidences showing a potential role of TNF-α in the development of laryngeal cancer.