Targeting FoxM1 by thiostrepton inhibits growth and induces apoptosis of laryngeal squamous cell carcinoma.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Otolaryngology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Rd, Chongqing, 400016, People's Republic of China.
Lizhu
L
Jiang
Xiaosong
X
Wu
Peng
P
Wang
Taoyu
T
Wen
Chao
C
Yu
Lei
L
Wei
Hongyan
H
Chen
D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D005434:Flow Cytometry; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D007822:Laryngeal Neoplasms / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D058990:Molecular Targeted Therapy / Q000379:methods; D011897:Random Allocation; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D000077195:Squamous Cell Carcinoma of Head and Neck; D013883:Thiostrepton / Q000008:administration & dosage / Q000494:pharmacology*; D013997:Time Factors; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; C107585:FOXM1 protein, human; D000072278:Forkhead Box Protein M1; D051858:Forkhead Transcription Factors; D051034:Inhibitor of Apoptosis Proteins; D020169:Caspases; D013883:Thiostrepton