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Alternative Title: Anti-cancer activity of biologically active substances from a Seaweed and a marine algae associated microorganism: Induction of Apoptosis accompanied by Cell Cycle Arrest and Endoplasmic Reticulum Stress by extract of Saccharina japonica in SK-Hep1 Human Hepatocellular Carcinoma Cells: Anti-angiogenic effect of Toluhydroquinone, a Secondary Metabolite from Marine fungi Phoma herbarum isolated from a Marine Algae Hypnea saidana on Human Umbilical Vein Endothelial Cells

Abstract: Many of fascinating marine natural products have been reported and some of them are originated from secondary metabolites of marine microorganisms. Some of these secondary metabolites have a potential as chemotherapeutic agents for the treatment of a wide variety of diseases. Toluhydroquinone (2-methylbenzene-1,4-diol), a secondary metabolites, was been isolated from a marine fungi Phoma herbarum (Class: Dothideomycete). Activity of toluhydroquinone is not known. In this study, the anti-angiogenic effects of toluhydroquinone were investigated in vitro. Angiogenesis is a crucial step in the growth and metastasis of cancer. We analyzed the effect of toluhydroquinone on HUVEC cells. Toluhydroquinone had no effect on human umbilical vein endothelial cells (HUVECs) viability. However, toluhydroquinone treatment resulted in a dose-dependent inhibition of tube formation after plating HUVECs on Matrigel and induced inhibition of HUVEC migration in a time dependent manner. More importantly, the invasion activity of HUVECs was significantly inhibited by toluhydroquinone. In conclusion, The results indicated that toluhydroquinone has an ability to inhibit diverse cell behaviours of HUVECs and the effects may be mediated at different levels of the tumor growth. Therefore, toluhydroquinone is proposed to be a potent anti-angiogenesis drug candidate.

Keyword: biologically active substances from a Seaweed and a marine algae associated microorganism Induction of Apoptosis accompanied by Cell Cycle Arrest and Endoplasmic Reticulum Stress by extract of Saccharina japonica Anti-angiogenic effect of Toluhydroquinone

URI: https://repository.pknu.ac.kr:8443/handle/2021.oak/9209
http://pknu.dcollection.net/jsp/common/DcLoOrgPer.jsp?sItemId=000001965555

Table Of Contents: (SECTION Ⅰ)
Introduction 1

Materials and Methods
Seaweed material 5
Extraction and fractionation 5
Cell culture 6
Cell viability assay 6
Caspase inhibitor assay 7
Western Blot analysis 7
Detection of intracellular Ca2+ 8
FACS analysis 8
Immunofluorescence of cleaved caspase-3 protein 8
TUNEL assay 9
Statistical analysis 9

Results
Cell death of SK-Hep1 by n-hexane extract of S. japonica 11
Induction of caspases mediated aoptosis in the extract treated cells 13
Apoptosis induced by S. japonica n-hexane extract is mediated by both caspase-dependent and independent pathways 16
Induction of ER-stress and cell cycle arrest by S. japonica n-hexane Extract 18
Effect of DNA fragmentation on SK-Hep1 cells by S. japonica n-hexane extract 20

Discussion 22

국문초록 24

References 25

(SECTION Ⅱ)
Introduction 30

Materials and Methods
Fungal isolation and culture 33
Extraction and isolation 33
Physicochemical data of compound 33
Drugs and reagents 34
Cell culture 34
Cell proliferation assay 34
Tube-like structure formation assay (Morphogenesis assay) 35
Migration assay 35
Matrigel invasion assay 36

Results
The effect of toluhydroquinone on HUVECs viability 37
The effect of toluhydroquinone on HUVECs tube-like structure 39
Toluhydroquinone inhibited HUVECs migration 42
Suppression of HUVECs invasion by toluhydroquione 44

Discussion 46

국문초록 48

Acknowledgement 49

References 50

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