Document Type

Thesis - University Access Only

Award Date

2011

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

Abstract

Skin cancer is the most prevalent of all cancer types and its incidence is expected to increase substantially. In the United States 11,790 deaths were estimated to result from cancer in 2010. The major causative agent for skin cancer is UV radiations reaching earth surface. UV radiation besides resulting in characteristic DNA damage also causes tumor promotion by inducing various signal transduction pathways which can lead to distinct cellular responses including cell proliferation, transformation and cell death. It has been proposed that the use of sunscreens alone are not sufficient to prevent skin cancer, thus there is a need for more effective ways to prevent this malignancy. In this regard, chemoprevention of skin cancer by natural compounds has gained importance in recent years. Chemoprevention involves the administration of chemical agents to prevent initiation, promotion and /or progression that occurs during neoplastic development. Honokiol and magnolol are plant lignans isolated from bark and seed cones of Magnolia officinalis, have been shown to have chemopreventive effects on chemically- induced skin cancer development. The objective of this investigation was to study the chemopreventive effects of honokiol and magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and to elucidate the possiblemechanism of their effects on skin tumor development in A431 human epidermoid carcinoma cells. Female SKH-1 mice were divided into different groups. Two groups of mice were used for honokiol single dose study, one control (200 μl of acetone) and other group treated with honokiol (30μg/ 200 μl of acetone). For honokiol and magnolol dose response studies, Group 1 received acetone (0.2 ml, topical) and Groups 2, 3 and 4 received honokiol/magnolol (30, 45 and 60 μgin 0.2 ml acetone, topical) respectively one hour before UV B treatment. Tumor initiation and promotion were carried out by UVB radiation (30 mJ/cm 2/day), 5 days a week for 25-30 weeks. Tumor counts and mice weights were taken weekly. At the end of study mice were euthanized and dorsal skin samples were collected. For in vitro studies, effects of honokiol and magnolol were determined in human epidermoid carcinoma A431 cells on cell proliferation and apoptosis, as majority of cancers were resulted because of disruption of these pathways. MTT assay was used to determine cell viability and cell proliferation was determined by using BrdU incorporation assay. For determining early and late stages of apoptosis, Annexin V /propidium iodide staining and TUNEL assay were used. Cell cycle progression is determined by propidium iodide staining. Westem blot was used for mechanistic studies for determining expression of various proteins on signaling pathways of apoptosis, STA T3 and RAF /MEK/ERK pathway and finally expression of various proteins involved in cell cycle progression. Pretreatment of honokiol decreased (36 - 78 %) tumor multiplicity compared to control and decreased tumor incidence significantly in 60 μg pretreated group. Mechanistic studies showed the possible involvement of caspase-3, caspase-8, caspase-9, poly-(-ADP-ribose) polymerase (PARP) and p53 activation (P< 0.05) leading to the induction of DNA fragmentation and apoptosis. Treatment of honokiol to A431 cells resulted in decreased cell viability and proliferation and increased apoptosis in concentration dependent manner. Honokiol induced cell cycle arrest at G0/G 1 phase at 24 hand decreased the expression of cyclin D1, D2 and CDK2 proteins and increased expression of p21 and p27 cell cycle inhibitors. Magnolol pretreated groups resulted in 27- 55% reduction in tumor multiplicity as compared to control group. Magnolol pretreated groups showed higher expression of cleaved caspase-3 and poly-(-ADP-ribose) polymerase (PARP). Magnolol pretreatment caused cell cycle arrest at G2/M phase. Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced O2/M phase cell cycle arrest in A431 cells at 12 hand decreased the expression of cell cycle proteins such as cyclin B 1, cyclin A, CDK4, Cdc2 and simultaneous increase in expression of Cip/p21, a cyclin-dependent kinase inhibitor. Mechanistic studies showed magnolol induced apoptosis in vivo and in vitro with an increased expression of caspase-8 and cleaved PARP. Our results indicate magnolol induces apoptosis by activation of extrinsic pathway. We further determined the effects of magnolol on signal transduction and activators of transcription 3 (STAT 3) and B- Raf/MEK/ERK/AKT pathways. Our Western blot results showed that phosho-signal transducers and activators of transcription 3 (Tyr 705 ) and B-Raf, MEK and AKT were down regulated by magnolol whereas magnolol induced phosphorylation of ERK. Pretreatment with honokiol/magnolol at concentrations in micrograms per application (compared with milligrams application of other potential chemopreventive agents) prevents UVB-induced skin cancer development, possibly by activating proapoptotic proteins through both intrinsic and / or extrinsic pathways. All these novel findings concerning mechanisms of honokiol and magnolol in A43 l skin cancer cells and its effects in animals on UVB- induced skin cancer development suggests that honokiol and magnolol could be a potent and safe chemopreventive agent for skin cancer.

Library of Congress Subject Headings

Skin -- Tumors -- Chemoprevention

Bioactive compounds

Honokiol

Magnolol

Format

application/pdf

Number of Pages

149

Publisher

South Dakota State University

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