Deguelin Induces Both Apoptosis And Autophagy In Head And Neck Squamous Cell Carcinoma Cells

【Objective】Part Ⅰ: To analysis the anti-tumor ability of deguelin in vivo and in vitro.Part Ⅱ: To study the mechanism of heat shock protein (Hsp)90in deguelininduced head and neck squamous cell carcinoma (HNSCC) cell apoptosis.Part Ⅲ: To investigate mechanism of adenosine monophosphate-activated proteinkinase (AMPK) in deguelin induced HNSCC cell apoptosis and autophagy.【Methods】Part Ⅰ: Cell culture of HNSCC cell line Hep-2cells, human epidermalsquamous carcinoma cell line A-253cells, the human tongue squamous cellcarcinoma cell line SCC-9cells, immunology, molecular biology, pathology andother advanced technologies, such as nude mice Hep-2xenograft model to establishthe method of in vivo and in vitro studies of the cell culture, MTT, siRNAtechnology, immunoblotting, and immunofluorescence.Part Ⅱ: Cell culture of HNSCC cell line Hep-2cells, human epidermalsquamous carcinoma cell line A-253cells, the human tongue squamous cellcarcinoma cell line SCC-9cells, focus on Western blot test, IP experiments of Akt,Survivin and Cdk4.Part Ⅲ: Cell culture of HNSCC cell line Hep-2cells, MTT, siRNA technology,immunoblotting, immunofluorescence, Western blot experiments, IP experiments, and siRNA experiments.【Results】Part Ⅰ: Deguelin promote human laryngeal carcinoma cell line Hep-2cell deathand apoptosis; deguelin promote human epidermal squamous carcinoma cell lineA-253cells, the human tongue squamous cell carcinoma cell line SCC-9cell death;deguelin increased apoptosis by upregulating the Hep-2cells caspase-3activity,while down the expression of BCL-XL; low dose of deguelin markedly enhanced5-FU-induced death of Hep-2cells; low doses of deguelin significantly promote the5-FU-induced apoptosis of Hep-2cells; deguelin reduce the volume of Hep-2cellsxenograft tumors in nude mice, and to improve the survival rate of mice.Part Ⅱ: Deguelin inhibit Akt signaling pathway in human laryngeal squamouscarcinoma cell line Hep-2cells; deguelin block Akt and Hsp90combined, whilepromoting Akt and ubiquitination combined in Hep-2cells; MG-132reversed the thedeguelin induced Akt and Hsp90downregulation in Hep-2cells; deguelin inhibit theexpression of Survivin and Cdk4in Hep-2cells; in Hep-2cells, deguelin block Hsp90and Survivin and Cdk4inter-related, and promotion of the association betweenubiquitin Survivin and Cdk4; MG-132reversed deguelin induced Akt degradation inHep-2cells; deguelin has a similar inhibitory effect on Akt, Cdk4, and Survivinexpression in human epidermal squamous carcinoma cell line A-253cells andhuman tongue scales-cell carcinoma cell line SCC-9cells.Part Ⅲ: Fumonisin B1inhibit deguelin induced ceramide synthesis in laryngealsquamous cell carcinoma cell line Hep-2cells; in Hep-2cells, associated with C6ceramide strengthen deguelin induced cell death and apoptosis, but fumonisins B1reduced deguelin induced cell death and apoptosis; deguelin activation LKB1,AMPK, ACC phosphorylation in Hep-2cells; AMPKɑ1RNAi inhibit deguelin induced AMPK phosphorylation in Hep-2cells; AMPK activation enhance deguelininduced Hep-2cell death; C6ceramide enhanced deguelin induced AMPKphosphorylation in Hep-2cells; Z-VADfmk and3-MA reduce deguelin inducedHep-2cells death; deguelin induce phosphorylation of Ulk and LC3B expression inHep-2cells; deguelin promote combination between Ulk and AMPK with each other;AMPK ɑ1RNAi suppress deguelin induced Ulk phosphorylation and LC3Bexpression in Hep-2cells.【Conclusions】Part Ⅰ: Deguelin induce HNSCC cell death and apoptosis, the mechanism maybe to increase the activity of caspase-3, at the same time decrease the expression ofBcl-xL, eventually induce laryngeal squamous cell carcinoma cell line Hep-2cellapoptosis; low dose of deguelin can enhance the sensitivity to5-FU of Hep-2cells.Deguelin show significant anti-tumor effects in xenograft models of Hep-2cells.Part Ⅱ: Deguelin induce the degradation of Akt by blocking Hsp90and Aktbond to each other, and promoting the combination of ubiquitin and Akt, therebyenhance apoptosis in HNSCC cells. In HNSCC cells, deguelin block combination ofHsp90with Survivin and Cdk4, while promoting the ubiquitination bond withSurvivin and Cdk4, thereby inducing degradation of Survivin and Cdk4, increaseapoptosis.Part Ⅲ: Deguelin induced ceramide synthesis in laryngeal squamous cellcarcinoma cell line Hep-2cells, thereby further activate the LKB1-AMPK-ACCphosphorylation, and ultimately increase deguelin induced cell death and apoptosis.Deguelin induce AMPK phosphorylation and further activation of AMPK/Ulk1dependent autophagy pathway, and increase LC3B expression, ultimately enhancedeguelin induced Hep-2cells death and autophagy.