Document Type

Dissertation - University Access Only

Award Date

2012

Degree Name

Doctor of Philosophy (PhD)

Department / School

Pharmaceutical Sciences

First Advisor

Chandradhar Dwivedi

Abstract

Honokiol is a plant lignan extracted from the magnolia bark tree. Previous studies from our laboratory showed its chemopreventive effects in a UVB (Ultraviolet B)induced skin cancer model, as well as pro-apoptotic effects in A431 squamous cancer cell line. Studies from other groups have reported antineoplastic effects in different cancer models as well. In this study, the antineoplastic effects ofhonokiol were evaluated in vivo and in vitro against UACC-62 melanoma cell line that harbors mutated BRAF, CDK4i, PTEN and against SKMEL-2 melanoma cell line that presents NRAF, and TP53 mutated genes. For the in vivo studies, nude male mice were injected subcutaneously with 5x10 6 UACC-62 or SKMEL-2 cells. The next day animals were randomized into control and treatment groups, each group containing 20 animals. The control group received 250 μl sesame oil intraperitoneally (IP), the treatment groups received honokiol dissolved in sesame oil; 1.25 mg (IP) in the UACC-62 group and 50 mg/kg (IP) in the SKMEL-2 injected mice. Animals were treated three times a week, for 5-7 weeks. Honokiol doses were slightly different in the two in vivo experiments. For the first experiment it was used a fixed dose (1.25 mg/dose), and for the second experiment honokiol was administered according to each animal weight ( 50 mg/kg), as it was thought this approach will address the small differences in animals weights. Tumors' incidence, multiplicity and volume were recorded every three days, mice's weights and external signs of toxicity were also closely monitored. Tumor tissues were collected for histopathology, western blots, and PCR arrays for studying gene expression of apoptosis related genes. For the in vitro studies, both cell lines were evaluated for cell viability, proliferation, apoptosis by TUNEL assay, cell cycle arrest by propidium iodide staining, and western blots for proteins expressions. Honokiol treatment significantly reduced (P< 0.05) tumor volumes in both xenograft experiments. In the SKMEL-2 cell line xenograft at the end of the experiment there was a 3 7.4% average reduction in tumor volume in the honokiol treated animals. At termination of the UACC-62 cell line xenograft experiment there was an average inhibition in tumor volume of 34.8%. Animals' weight gain was similar among vehicle and honokiol treated animals. No evident signs of toxicity were observed in the honokiol treated animals. From the PCR arrays in tumors we observed that in the SKMEL-2 tumors, honokiol caused a significant up-regulation of: CARD6 (Caspase recruitment domain protein 6), DFF A (DNA fragmentation factor subunit alpha), L TBR (Lymphotoxin beta receptor) and CD27 genes. Other genes that were also up-regulated by at least 1.5 fold although not significantly were: BCL2L 1, BCL2L 10, Casp 10, Casp 14, F ASLG and TNFSRl lB. These results suggest an increase in the apoptosis machinery caused by the honokiol treatment on SKMEL-2 cells xenografts. In the UACC-62 cells tumors, significant up-regulation of the following genes was observed: BAG4 (Bcl-2-associated athanogene (BAG) family), BAKl (Bcl-2 homologous antagonist/killer), BAX (Bcl-2- associated X protein), BCL2 (B-cell lymphoma 2), BCL2L 1 (BCL2-like 1 ), BNIP2 (BCL2/adenovirus E 1 B 19 kDa protein-interacting protein 2) , CARD8 (Caspaseassociated recruitment domain-8), CASP 10 (Caspase 10), CASP2 (Caspase 2) , CASP6 (Caspase 6) , CASP7 (Caspase 7), F ADD (Fas-Associated protein with Death Domain), TNFRSF25 (Tumor necrosis factor receptor superfamily member 25), TP53 (Tumor protein p53), TP53BP2 (Tumor protein p53 binding protein 2), TRAF2 (TNF receptorassociated factor 2), RPL13A (60S ribosomal protein Ll3a) and down regulation of BNIP3 (Bcl-2/adenovirus ElB 19-kDa-interacting protein 3). These results suggest that honokiol caused apoptosis through the extrinsic and intrinsic pathways as observed from the up-regulation of the caspases genes and the up-regulation on the tumor necrosis factor receptor superfamily members. Additionally honokiol in vivo may increase the response of the p53 apoptotic pathway. The increase in the TP53 gene expression is consistent with an increase in the p53 protein expression in UACC-62 cells upon honokiol treatment observed by western blot. From the in vitro experiments we observed that honokiol significantly decreased cell viability and proliferation in both cell lines (P < 0.05). Honokiol caused cell cycle arrest in GJ G 1 phase in UACC-62 cell line , while it caused accumulation of cells in the G2/M phase in SKMEL-2 cell line. Honokiol significantly increased the DNA fragmentation in both cell lines. Honokiol strongly decreased the expressions of cyclinD 1, cyclin D2 and cyclin B 1 while increasing the cleavage of caspase 3, caspase 8, caspase 9 and P ARP in both cell lines. In UACC-62 honokiol decreased the expressions of CDK2, CDK4, cyclin E, cdc2p34, p21 and p27 these results are consistent with the Go/G1 cell cycle arrest observed. In SKMEL-2, honokiol decreased the expression of CDK4, PCNA and increased the expression of p21, these effects on proteins expressions may explain the G2/M cell cycle arrest observed in SKMEL-2 cell line. All these novel findings suggest that honokiol is an excellent candidate for further investigation for the treatment of human malignant melanoma.

Library of Congress Subject Headings

Antineoplastic agents 
Melanoma -- Treatment 
Honokiol

Publisher

South Dakota State University

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Rights Statement

In Copyright