thiostrepton and Carcinoma--Squamous-Cell

We have previously reported that forkhead box M1 (FoxM1) transcription factor was overexpressed in laryngeal squamous cell carcinoma (LSCC) and was associated with development of LSCC. However, there are limited studies regarding the functional significance of FoxM1 and FoxM1 inhibitor thiostrepton in LSCC. Therefore, the aim of this study was to examine both in vitro and in vivo activity of FoxM1 inhibitor thiostrepton against LSCC cell line and nude mice.. Cell viability was studied by CCK-8 assay. Cell growth was evaluated by CFSE staining and cell cycle analysis. Apoptosis was measured by flow cytometry. The mRNA and protein expression were detected by quantitative real-time RT-PCR, Western blot and immunohistochemical staining. Xenograft model of tumor formation was used to investigate how thiostrepton influences tumorigenesis in vivo.. Overexpression of FoxM1 in LSCC cells was down-regulated by thiostrepton in a dose-dependent manner. Thiostrepton caused dose- and time-dependent suppression of cell viability of LSCC. Moreover, thiostrepton induced cell cycle arrest at S phase at early time and inhibited DNA synthesis in LSCC cells in a dose- and time-dependent manner by down-regulation of cyclin D1 and cyclin E1. Thiostrepton also induced dose- and time-dependent apoptosis of LSCC cells by down-regulation of Bcl-2, up-regulation of Bax and p53, and inducing release of cytochrome c accompanied by activation of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. In addition, z-VAD-fmk, a universal inhibitor of caspases, prevented activation of cleavage caspase-3 and abrogates cell death induced by thiostrepton treatment. Furthermore, FADD and cleaved caspase-8 were activated, and expression of cIAP1, XIAP and survivin were inhibited by thiostrepton. Finally, treatment of LSCC cell line xenografts with thiostrepton resulted in tumorigenesis inhibition of tumors in nude mice by reducing proliferation and inducing apoptosis of LSCC cells.. Collectively, our finding suggest that targeting FoxM1 by thiostrepton inhibit growth and induce apoptosis of LSCC through mitochondrial- and caspase-dependent intrinsic pathway and Fas-dependent extrinsic pathway as well as IAP family. Thiostrepton may represent a novel lead compound for targeted therapy of LSCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Inhibitor of Apoptosis Proteins; Laryngeal Neoplasms; Mice; Mice, Nude; Molecular Targeted Therapy; Random Allocation; Real-Time Polymerase Chain Reaction; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Thiostrepton; Time Factors; Xenograft Model Antitumor Assays

Cellular quiescence is a reversible growth arrest in which cells retain their ability to enter into and exit from the proliferative cycle. This study investigates the hypothesis that cell growth-state specific oxidative stress response regulates radiosensitivity of cancer cells. Results showed that quiescent (low proliferative index; >75% G1 phase and lower RNA content) Cal27 and FaDu human head and neck squamous cell carcinoma (HNSCC) are radioresistant compared to proliferating cells. Quiescent cells exhibited a three to tenfold increase in mRNA levels of Mn-superoxide dismutase (MnSOD), dual oxidase 2 (DUOX2) and dual-specificity phosphatase 1 (DUSP1), while mRNA levels of catalase (CAT), peroxiredoxin 3 (PRDX3) and C-C motif ligand 5 (CCL5) were approximately two to threefold lower compared to proliferating cells. mRNA levels of forkhead box M1 (FOXM1) showed the largest decrease in quiescent cells at approximately 18-fold. Surprisingly, radiation treatment resulted in a distinct gene expression pattern that is specific to proliferating and quiescent cells. Specifically, FOXM1 expression increased two to threefold in irradiated quiescent cells, while the same treatment had no net effect on FOXM1 mRNA expression in proliferating cells. RNA interference and pharmacological-based downregulation of FOXM1 abrogated radioresistance of quiescent cells. Furthermore, radioresistance of quiescent cells was associated with an increase in glucose consumption and expression of glucose-6-phosphate dehydrogenase (G6PD). Knockdown of FOXM1 resulted in a significant decrease in G6PD expression, and pharmacological-inhibition of G6PD sensitized quiescent cells to radiation. Taken together, these results suggest that targeting FOXM1 may overcome radioresistance of quiescent HNSCC.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Pentose Phosphate Pathway; Radiation Tolerance; Squamous Cell Carcinoma of Head and Neck; Thiostrepton