Authors: Yan Song Xin Sun WeiLiang Bai WenYue Ji
Publish Date: 2013/02/13
Volume: 270, Issue: 4, Pages: 1397-1404
Abstract
A novel drug named Dasatinib is a highly potent ATPcompetitive orally active dual Src/Abl kinase inhibitor with antiproliferative activity against solid tumors and CML chronic myeloid leukaemia cell lines Dasatinib has been shown to have preclinical activity against human prostate breast pancreatic lung and head and neck cancer To determine whether Dasatinib can inhibit the growth of laryngeal squamous cell carcinoma in the present study we investigated the antitumor effect of Dasatinib on Hep2 cells Hep2 cells were treated with different concentrations of Dasatinib for different time Cell proliferation cell cycle distribution and cell apoptosis were evaluated using MTT assay flow cytometry and fluorescent microscopy It was found that Dasatinib exhibited significant efficacy in growth inhibition cell cycle arrest at G0/G1 phase and apoptosis induction in a dose and timedependent manner Measuring the modulation of regulators in the cell cycle apoptosis and signal transductions by western blot analysis showed that the effect of Dasatinib was due to suppression of the expression of Bax Bcl2 Caspase3 and Caspase8 Moreover in vivo studies were performed in a nude mouse xenograft model the new prescription DDP + Dasatinib was better than DDP alone in terms of therapeutic efficacy In conclusion the antitumor effect of Dasatinib on Hep2 cells was due to the induction of cell cycle arrest as well as apoptosis The possible mechanisms underlying the action might be attributed to the suppression of Src phosphorylation This investigation suggests a potential clinical application of Dasatinib for the treatment of laryngeal cancer patientsLaryngeal squamous cell carcinoma is one of the most common squamous cell carcinomas of the head and neck Laryngeal carcinoma ranks the 11th common kind of cancer in men worldwide with a tendency toward an increasing occurrence of new cases and deaths annually 1 More than 95 of all laryngeal cancers are squamous cell carcinomas The traditional treatment for squamous cell carcinoma of the larynx was surgery usually total laryngectomy in advanced cases followed by radiotherapy During the last decades combined modality approaches have been developed and chemotherapy has been confirmed as a major component of these treatment approaches to enhance locoregional disease control reduce distant metastasis and improve survival in patients with advanced laryngeal cancer 2 Therefore the therapeutic strategies which are able to produce greater efficacy and preserve larynx as well are essential to this concept 3 However potential complications and side effects of this drug limit its application in Laryngeal carcinoma chemotherapy which may account for the failure of chemotherapy in patients with advanced laryngeal carcinoma In this regard novel effective chemotherapeutic agents are desperately needed in the treatment of laryngeal cancer to improve survival and to enhance larynx preservationSrc is the prototypic member of a family of nine nonreceptor tyrosine kinases SFKs Src family kinases which include Src Lyn Fyn Lck Hck Fgr Blk Yrk and Yes that has a key role in many cellular signaling pathways 4 SFKs regulate four main cellular functions that ultimately control the behavior of transformed cells cell proliferation adhesion invasion and motility 5 Src is overexpressed in head and neck squamous cell carcinoma relative to control tissues Src offers particularly a promising molecular target for anticancer therapy so we want to use Src kinase inhibitor to inhibit the signaling pathways and to decrease the growth of laryngeal carcinomaA novel drug named Dasatinib BMS354825 SPRYCEL® BristolMyers Squibb is a highly potent ATPcompetitive orally active dual Src/Abl kinase inhibitor with antiproliferative activity against solid tumors and CML chronic myeloid leukaemia cell lines It sensitively inhibits all members of the Src family including cSrc Lck Fyn and Yes At higher concentrations 3–28 nmol/L Dasatinib also inhibits the Src kinases Abl cKit PDGFR and EphA2 6 Dasatinib has been shown to have preclinical activity against human prostate breast pancreatic lung head and neck cancer mesotheliomas and sarcomas dependent on Src kinase 7To date no study has been conducted in the evaluation of the effect of Dasatinib on human laryngeal cancer cells As constitutive activation of PSrc has been determined in head and neck squamous cell carcinoma and blockade of Src signaling has been shown to effectively inhibit cell growth and induce apoptosis in head and neck squamous cell carcinoma cells we hypothesized that Dasatinib would be useful in treating human laryngeal cancer cells To test this hypothesis we designed the present study to examine the antitumor effect of Dasatinib in vitro and in vivo on human laryngeal cancer cells including the effects on cell growth cell cycle distribution apoptosis and the expression of proteins relevant to the regulation of cell cycle and apoptosis pathwayDasatinib Mr 4880 was obtained from BristolMyers Squibb For in vitro use Dasatinib was dissolved in DMSO at a concentration of 10 mg/mL The solution was aliquoted and kept at −20 °C until use Frequent freezethawing was avoided For in vivo use Dasatinib was dissolved in citrate buffer and this solution was kept at 0–4 °C for up to 14 days Cisplatin EMD Biosciences Inc was dissolved in 10 mg/ml of DMSO for both in vitro and in vivo use 345dimethylthiazol2yl25diphenyl tetrazoliumbromide MTT was from Fluka USA Propidium iodide PI was from Biosharp USA Annexin VFITC apoptosis detection kit was from Jingmei Biotech Co Ltd Shenzhen China Bcl2 Bax caspase3 caspase8 and actin primary antibodies as well as the secondary antibody were obtained from Santa Cruz Biotechnology Inc CA USA Antiphosphorylated Src antibody pSrc Tyr 416 and total Src antibody were both obtained from Cell Signaling Technologies Dulbecco’s modified Eagle’s medium DMEM and fetal calf serum FCS were obtained from Gibco BRL Grand Island NY USA Other laboratory reagents were obtained from Sigma USA Fluorescence microscopy Olympus Japan and flow cytometry FACScalibur Becton–Dickinson USA were employedTwenty male nude mice 3 months old weighing 20 ± 2 g were obtained from China Medical University animal facility and fed with purified water and a commercial stock diet in an airconditioned room at 20–22 °C All the animals were treated softly and equally All animals were killed by cervical dislocation method All the research was approved by The Institutional Animal Care and Use Committee of China Medical UniversityHuman laryngeal cancer line Hep2 was bought from Cell Bank Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai and cultured in RPMI 1640 supplemented with 10 FBS 100 U/ml penicillin G and 100 μg/ml streptomycin Cultures were maintained in a 5 CO2 humidified atmosphere a 37 °C
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