Here we report the chemotherapeutic effect of honokiol a phytochemical from plant about human head and neck squamous cell carcinoma (HNSCC). of honokiol by oral gavage (100 mg/kg body weight) significantly (< 0.01-0.001) inhibited the growth of SCC-1 and FaDu xenografts in athymic nude mice which was associated with: (i) inhibition Danshensu of tumor cell proliferation (ii) induction of apoptosis (iii) reduced expressions of cyclins and Cdks and (iv) inhibition of EGFR signaling pathway. Molecular docking analysis of honokiol in EGFR binding site indicated the chemotherapeutic effect of honokiol against HNSCC is definitely mediated through its firm binding with EGFR which is better than that of gefitinib a popular drug for HNSCC treatment. flower. The diverse biological and pharmacological activities such as anti-inflammatory antifungal anti-oxidative and anti-carcinogenic of honokiol have been investigated in recent years [15-19]. The chemotherapeutic and chemopreventive effects of honokiol have been reported previously in several tumor models including skin breast melanoma non-small cell lung malignancy and prostate [15-19]. Anti-carcinogenic effect of honokiol was also identified against HNSCC cells using and models and EGFR was recognized as a molecular target . However the anti-carcinogenic potential of honokiol with definitive EGFR binding using molecular docking analysis and molecular mechanism has not been explored in HNSCC. We hypothesize that honokiol inhibits the growth of HNSCC cells by focusing on and binding securely with EGFR. To test our hypothesis we assessed the chemotherapeutic effect of honokiol on HNSCC cell lines derived from different sub-sites such as larynx (UM-SCC5) pharynx (FaDu) tongue (OSC19) and oral cavity (UM-SCC1) . RESULTS Honokiol inhibits cell viability of HNSCC cells The effect of honokiol on viability of HNSCC cells SCC-1 SCC-5 OSC-19 and Danshensu FaDu were Danshensu identified Danshensu using an MTT assay. The cells were treated with different concentrations of honokiol (0 20 40 and 60 μM) for 24 48 and 72 h. A dose- and time-dependent inhibition in viability of HNSCC cells was observed as demonstrated in Number ?Number1.1. The reduction in the viability of the SCC-1 and FaDu cells observed after treatment with honokiol ranged respectively from 16% to 89% (< 0.001) and 15% to 94% (< 0.001) after 72 h (Figure ?(Figure1).1). Under identical conditions similar effects were observed on treatment of SCC-5 and Danshensu OSC-19 cells with honokiol. Number 1 treatment of HNSCC cells with honokiol inhibits the cell viability inside a dose- and time-dependent manner Treatment of HNSCC cells with honokiol induces apoptosis FACS analysis was performed to quantitate the percentage of apoptosis in HNSCC cells. As honokiol induced inhibition of cell viability was almost similar in all the four cell lines analyzed FaDu and SCC-1 cell lines were selected for further investigation. FaDu SGK and SCC-1 cell lines were treated with numerous doses of honokiol and quantitative analysis of apoptosis was decided using the Alexa488 Apoptotic Cell Detection Kit using circulation cytometry as detailed previously . The number of cells undergoing apoptosis was decided in terms of the percentage of early-stage and late-stage apoptotic cells which are shown in lower right (LR) and upper right (UR) quadrants of the FACS histogram respectively (Physique ?(Figure2A) 2 and as detailed previously . Treatment of the FaDu and SCC-1 cells with honokiol for 48 h resulted in a significant induction of apoptotic cell death in both cell lines. The percentages of total apoptotic cells (UR+LR quadrants) in FaDu cells after honokiol treatment ranged from 18.1% (20 μM) to 44.4% (60 μM) compared to only 7.8% in non-honokiol-treated control cells. Comparable range of apoptotic cell death after honokiol treatment was observed in SCC-1 cells (Physique ?(Figure2A2A). Physique 2 A Malignancy cell apoptosis is usually tightly regulated by functions of the proteins of Bcl-2 family and proteins of Bcl-2 family act as promoters or inhibitors of cell death [22-24]. Western blot analysis and subsequently measurement of band densities revealed that treatment of FaDu and SCC-1 cells with honokiol (0 20 40 60 μM) for 48 h led to a dose-dependent reduction in the appearance of anti-apoptotic protein Bcl-2 whereas the appearance of pro-apoptotic protein Bax was elevated with increasing dosages of honokiol treatment (Body ?(Figure2B).2B). As.