A panel of protein kinases has been demonstrated as potential targets in Tan IIA-treated tumor cells, such as PI3K/Akt, MAPK, and mTOR40,44,45

A panel of protein kinases has been demonstrated as potential targets in Tan IIA-treated tumor cells, such as PI3K/Akt, MAPK, and mTOR40,44,45. exhibited a significant anti-tumor effect on OSCC cells both in vitro and in vivo, including reduction of Aurora B and histone H3 phosphorylation, induction of G2/M cell cycle arrest, increase the populace of polyploid cells, and promotion of apoptosis. The in vivo mouse model revealed that Tan IIA delayed tumor growth of OSCC cells. Tan IIA alone or in combination with radiation overcame radioresistance in OSCC xenograft tumors. Taken together, our data show that Tan IIA is an Aurora B kinase inhibitor with therapeutic potentials for malignancy treatment. test). T, tumor; N, adjacent non-tumor tissue. B IB analysis of Aurora B expression in hTERT-OME and OSCC cell lines. C Cell viability of SCC25 (left) and CAL27 (right) cells expressing shGFP or shAurora B. Top, IB analysis of Aurora B expression. Bottom, MTS analysis of cell viability. D Colony formation of CAL27 and SCC25 cells expressing shGFP or shAurora B; of ?49.979, indicating that Tan 18α-Glycyrrhetinic acid IIA could inhibit Aurora B by competitively binding at the ATP-binding site (Fig. ?(Fig.2D).2D). As shown in Fig. ?Fig.2E,2E, the furan oxygen of this compound was predicted to form hydrogen bonding conversation with the backbone nitrogen of A173 in the hinge region. In the mean time, an aromatic hydrogen bond was exhibited between E171 and this furan substructure. In the solvent accessible region, the positively charged amino of K180 also tended to form hydrogen bonding with the electro-withdraw carbonyl group of Tan IIA, which further strengthened the affinity. In addition, Tan IIA adopted broad hydrophobic interactions with other residues in the pocket, such as L170 and L223. These results suggested that Tan IIA could be an ATP-competitive inhibitor of Aurora B. To validate this molecular modeling data, we next decided the conversation between Aurora B and Tan IIA. Our data showed that this Tan IIA-conjugated Sepharose 4B beads, but not the Sepharose 4B beads only, can bind with Aurora B in vitro (Fig. ?(Fig.2F).2F). Moreover, with the increase of ATP concentration, the conversation between Tan IIA and Aurora B was decreased, indicating there is a competition binding between Tan IIA and ATP with Aurora B protein (Fig. ?(Fig.2G).2G). In addition, the in vitro Aurora B kinase assay showed that Tan IIA inhibited Aurora B kinase activity in a dose-dependent manner, as the phosphorylation of histone H3 on Ser10 and survivin on Thr117 were significantly decreased with the graded increase of Tan IIA concentration (Fig. ?(Fig.2H2H and Supplementary Fig. 1D). Furthermore, even though Aurora C kinase has redundant functions and substrates as Aurora B, the in vitro kinase assay showed that Tan IIA could not inhibit Aurora C activity significantly (Supplementary Fig. 1E). All of these data show that Tan IIA is an Aurora B kinase inhibitor. Tan IIA suppresses OSCC cells and exhibits little cytotoxicity We next decided whether Tan IIA exerted any cytotoxic effect on immortalized oral epithelial cells. The hTERT-OME cells were treated with numerous doses 18α-Glycyrrhetinic acid of Tan IIA for 24, 48, and 72?h, cell viability was examined by MTS assay. The result showed that Tan IIA experienced no significant cytotoxicity against hTERT-OME cells while concentrations were <20?M (Fig. ?(Fig.3A).3A). Moreover, the MTS data revealed that Tan IIA did not reduce the cell viability of a panel of immortalized non-tumor cells, including immortalized lung epithelial cells HBE and NL20, immortalized lung fibroblast cell MRC5, immortalized hepatocytes LO2, colon epithelial cell FHC, and esophageal epithelial cell Het-1A (Supplementary Fig. 2). To further confirm the anti-tumor effect of Tan IIA on Mouse monoclonal to CD23. The CD23 antigen is the low affinity IgE Fc receptor, which is a 49 kDa protein with 38 and 28 kDa fragments. It is expressed on most mature, conventional B cells and can also be found on the surface of T cells, macrophages, platelets and EBV transformed B lymphoblasts. Expression of CD23 has been detected in neoplastic cells from cases of B cell chronic Lymphocytic leukemia. CD23 is expressed by B cells in the follicular mantle but not by proliferating germinal centre cells. CD23 is also expressed by eosinophils. OSCC, we examined the inhibitory effect of Tan IIA on numerous OSCC cell lines, including CAL27, SCC25, and SCC15. The results revealed that Tan IIA reduced the cell viability of OSCC cells in a dose-dependent manner, and Tan IIA inhibited CAL27 cells most significantly (Fig. ?(Fig.3B).3B). The results of colony formation assay indicated that CAL27 cells created the largest size of colony in soft agar. Treatment with Tan IIA reduced both colony number and colony size, and 5?M Tan IIA almost blocked the colony formation of these tested OSCC cells (Fig. ?(Fig.3C).3C). Edu incorporation assay showed that Tan IIA reduced the efficacy of Edu incorporation in 18α-Glycyrrhetinic acid CAL27, SCC25, and SCC15 cells (Fig. 3DCG). These results indicate that Tan IIA inhibits OSCC cells dose-dependently but exhibits no significant toxicity 18α-Glycyrrhetinic acid against immortalized oral epithelial cells. Open in a separate windows Fig. 3 Tan IIA suppresses OSCC cells.A MTS assay analyzes the cell viability of hTERT-OME cells with Tan IIA treatment for various time points. B MTS assay analyzes the cell viability.