Possible cytotoxic effect of allantoin on oral squamous cell carcinoma stemness (in vivo and in vitro model.

Cancer stem cells (CSCs) and associated tumor microenvironment (TM) have been proposed to mediate cancer progression, and therapeutic resistance rendering them valuable therapeutic targets in head and neck squamous cell carcinomas (HNSCC). Oral CSCs drive their tumorigenic effect via stromal fibroblasts. Phytochemicals like allantoin are extensively used in wound healing modulating fibroblast activity. The present study aimed to evaluate whether allantoin can modulate CSCs and/or TM and subsequently contribute to sensitization of cancer cells to chemotherapeutic agents. Hep2, OECs, DPSC cell lines were used to test the cytotoxicity of allantoin on cancer, oral epithelial cells, and normal dental pulp stem cells, respectively. The effect of allantoin and/or 5-fluorouracil (5FU) on cell cycle, apoptosis and expression of stem cell and apoptotic markers was tested in vitro. Further validation was performed using in vivo 4-nitroquinoline 1-oxide model of tongue squamous cell carcinoma model with assessment of cytotoxic effect of allantoin by flow cytometry, immunohistochemical staining, western-blot and RT-PCR. Allantoin was safe on both normal oral epithelial cells and dental pulp stem cells. Allantoin showed a statistically significant and dose dependent increase in 5FU apoptotic effect via reduction of cancer stem cell markers CD133 and CD44. Regarding cell cycle arrest, there was no statistically significant difference with addition of allantoin to 5FU. In vivo, allantoin down-regulated SOX2, NANOG, ALDH and OCT4 gene expression in tongue squamous cell carcinoma. Additionally, allantoin reduced the expression IL-6, TGF-Β, PDGF, α- SMA and CD163. Our results proposed that high concentration of allantoin drives its antitumorigenic effect. This was mediated by modulating stromal component of TM via down-regulation of trans-differentiation of cancer associated fibroblasts and tumor associated macrophages. These findings shed the light on potential use of allantoin as cytotoxic drug either alone or combined with other chemotherapeutic drugs.