الفهرس 
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Abstract
Background : Head and neck squamous cell carcinoma (HNSCC), and laryngeal squamous cell carcinoma (LSCC) in particular, are always recorded to have unfavorable prognosis due to local invasion, regional lymph node and distant metastases, and resistance to conventional chemoradiotherapy. Regarding the new model of carcinogenesis, the Cancer Stem Cells (CSCs) are potentially responsible for the genesis, sustained growth, metastatic tumor spread, and even the chemoradioresistance. Most of the current larynx cancer therapies are generally aimed at the global mass of tumor, targeting the non-tumorigenic cells, and unfortunately sparing the tumorigenic CSCs. It therefore appears reasonable to consider that the novel treatment approaches to larynx cancer should be directed against CSCs. Recently, phytochemicals and herbs have been suggested to be potential sources of therapeutics for CSCs eradication. The current study introduced two renowned herbal ingredients, namely Honokiol and the green tea extract (Epigallocatechin-3-gallate) as LSCC treatment options because of their easy accessibility, selectivity and reduced incidence of side effects. The molecular mechanisms underpinning their cytotoxic effects on CSCs were further elucidated. Purpose:The present study was carried out to characterize the immunomagnetically sorted CD44high cells, and to compare between the anti-tumor effects of EGCG and HNK on either HEp-2 parental cells or CD44high CSCs, with respect to growth inhibition, apoptosis/necrosis induction, as well as expression of genes involved in stemness, self-renewal pathways, apoptosis, and cell cycle. Materials & methods: The CD44high cells were isolated from HEp-2 laryngeal cancer cell line, and characterized for their functional features of self-renewal and clonogenicity, by tumorsphere and colony formation assays in comparison to CD44low cells. Moreover, the expression levels of the stem-cell markers and the key self-renewal pathways genes in CD44high and CD44low cells were measured by real-time qRT-PCR.Thereafter, treatment of HEp-2 parental cells and CD44high CSCs with either EGCG or HNK was commenced, then their anti-proliferative and apoptosis/necrosis-inducing impacts, by MTT assay and flow cytometry analysis, respectively, were investigated. Finally, the underlying mechanisms of EGCG and HNK potential anti-cancer effects were assessed by real-time qRT-PCR screening for expression of genes involved in stemness, self-renewal, cell cycle, and apoptosis. Results: In vitro self-renewal capacity of CD44high cells was observed by inducing three-dimensional tumorspheres formation. The statistical analysis revealed elevated expression levels of the stem-cell markers and the key self-renewal pathways genes in CD44high cells that significantly exceeded those in CD44low cells. Both EGCG and HNK decreased the viability of parental and CD44high cells in a dose-dependent manner, but the statistical analysis showed that growth inhibitory and apoptotic efficiencies of EGCG on CD44high CSCs exceeded those of HNK.However, the growth inhibitory, late apoptotic, and necrotic efficiencies of HNK were more pronounced than those of EGCG on parental cells.Significant suppression of Notch1 was prominent in EGCG-treated CD44high cells, but HNK showed higher proficiency in inhibiting β-catenin and PTCH-1. In CSCs, EGCG triggered significant suppression of BCL-2, and upregulation of CASP-3, that exceeded those in parental cells. However, HNK induced more prominent suppression of BCL-2, as well as upregulation of BAX and CASP-9 in parental cells rather than CSCs. Increased BAX expression was more evident in HNK-treated parental cells than either HNK-treated CSCs or EGCG-treated parental cells. p53 was preeminently upregulated in HNK-treated CSCs and especially parental cells, in contrast to its unexpected downregulation in HEp-2 cells treated with EGCG.Both AIFM1 and EndoG were significantly inhibited upon treatment of HEp-2 cells with either EGCG or HNK. No observable changes were detected in expression levels of CDKN1A in EGCG-treated cells, although HNK could significantly upregulate CDKN1A in CSCs. Conclusion:CD44high LSCC cells were highly enriched for CSC properties. EGCG harbored a preeminent selective targeting potential on the LSCC CSCs, mainly by apoptosis induction. However, HNK conferred its cytotoxic impacts on the LSCC tumor bulk as a whole, mostly by necrosis induction, especially with its higher doses, but HNK-triggered apoptosis with lower doses was rather noticeable. EGCG outshined HNK as a potent suppressor of Notch signaling pathway. Nonetheless, HNK privileged in downregulating β-catenin and Sonic hedgehog (SHH) signaling pathways. Both EGCG and HNK engaged the BCL-2 family-regulated intrinsic mitochondrial apoptotic pathway in LSCC cells, but EGCG induced apoptosis via caspase-dependent, p53-independent pathway, whereas HNK triggered apoptosis via caspase- and p53-dependent pathway.