An Indirubin Derivative, Indirubin-3′-Monoxime Suppresses Oral Cancer Tumorigenesis through the Downregulation of Survivin

Wan-Yu Lo,Nai-Wen Chang,A R M Ruhul Amin

doi:10.1371/journal.pone.0070198

Abstract

Oral cancer is the fourth most common cause of death from cancer in Taiwanese men. Indirubin-3′-monoxime (I3M), a potent cyclin-dependent kinase inhibitor, has therapeutic effects in other cancer cells. In this study, we carried out in vitro assays to test cell viability, cell cycle progression, apoptosis, cell migration and invasion in this cancer type. In addition, using an oral tumorigenic animal model, we examined target gene and protein expression using real time qPCR, immunoblotting and immunohistochemical staining. Our results demonstrate that I3M has an anti-proliferative effect in both Cal-27 and HSC-3 oral cancer cell lines and that treatment of Cal-27 and HSC-3 cells with I3M results in apoptosis through the activation of cytochrome c. In addition, I3M interrupts the cell cycle in Cal-27 cells in a dose-dependent manner by arresting cells in the G2/M phase. We also found that I3M suppresses migration and invasion in Cal-27 cells by inhibiting the expression of focal adhesion kinase, urokinase-type plasminogen inhibitor, and matrix metalloproteinase 9. Moreover, we identified survivin as a target protein in I3M-treated oral cancer cells. Using an oral cancer mouse model, we demonstrate that topical application of an adhesive gel composed of I3M and poly(vinyl alcohol) (I3M/PVA) has dose-dependent anti-tumorigenic effects. Following treatment, the expression of survivin protein and mRNA was downregulated in cancerous tissues. Furthermore, plasma survivin levels were also reduced in the I3M-treated mice. These results suggest that topical application of I3M, a drug synthesized from indirubin, which is found in Qing-Dai – has therapeutic potential for treating oral cancer.

Highlights

Oral squamous cell carcinoma (OSCC) accounts for approximately 90% of oral malignancies
The human oral cancer cell line Cal-27 and the cell line HSC-3 were purchased from the Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute (FIRDI) (Hsinchu, Taiwan)
Indirubin, and I3M were first tested for growthinhibition activity using the oral cancer cell lines Cal-27 and HSC-3, and the 50% inhibitory concentration (IC50) values for these three substances were determined following 24 h drug treatment (Table S2)

Summary

Introduction

Oral squamous cell carcinoma (OSCC) accounts for approximately 90% of oral malignancies. 274,000 new cases are diagnosed annually worldwide, and despite improved diagnostic and therapeutic methods, patients only have a 50% survival rate over 5 years [1]. Betel-quid chewing, alcohol use, and smokeless tobacco products constitute the major risk factors for oral cancer. Current treatment options for oral cancer include surgery, radiotherapy, and chemotherapy, the 5-year survival rate for oral cancer remains one of the lowest among common malignant neoplasms [2]. Oral cancer is the sixth most common cancer in Taiwan and the fourth most common cause of death from cancer among Taiwanese men since 2006 [3]. The identification of new agents and novel targets for the treatment of oral cancer needed to improve clinical management of this disease

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Conclusion