Tesis sobre el tema "A375 Melanoma cells"

Das maligne Melanom zählt zu den aggressivsten und behandlungsresistentesten aller Krebsarten. In den letzten 20 Jahren hat sich die Rate der Melanom-Erkrankungen innerhalb der weißen Bevölkerung verdreifacht. Mittlerweile liegen eine Reihe von Untersuchungen zu den molekularbiologischen Mechanismen der Entwicklung und Progression des malignen Melanoms vor. Aktuelle Forschungsvorhaben beschäftigen sich vor allem mit der Identifizierung Melanom-spezifischer Biomarker, die diagnostische und prognostische Informationen liefern sowie die Entwicklung einer zielgerichteten, kombinierten und individualisierten Therapie des metastasierenden Melanoms ermöglichen. In diesem Kontext soll die vorliegende Arbeit einen weiteren Beitrag zum Verständnis der Metastasierungskaskade und der daran beteiligten Proteine leisten. Aufgrund der Überexpression in einer Reihe von Tumoren und seiner geringen Molmasse von lediglich 11,5 kDa bietet sich das S100A4-Protein als Marker mit hoher prognostischer Signifikanz für verschiedene Tumorentitäten an. Jedoch ist die Beteiligung von S100A4 bei der Ausbildung des invasiven Tumorphänotyps noch nicht vollständig aufgeklärt. S100A4 besitzt zahlreiche intra- und extrazelluläre Bindungspartner, wobei die Metastasierung scheinbar ausschließlich durch das extrazelluläre Protein beeinflusst wird. S100A4 wechselwirkt extrazellulär beispielsweise mit dem Rezeptor für fortgeschrittene Glykierungsendprodukte (RAGE). Ziel dieser Arbeit war es, speziell die Bedeutung von S100A4 und seiner Interaktion mit RAGE für das prometastatische Verhalten von Melanomzellen in vitro und in vivo näher zu charakterisieren. Darüber hinaus sollte die Beteiligung von S100A4 bei der Gehirn-Metastasierung untersucht werden, wobei insbesondere die Regulierung der Endothelzell-Permeabilität und der transendothelialen Migration der Melanomzellen im Vordergrund stand. Im Rahmen dieser Arbeit wurde gezeigt, dass S100A4 und die Interaktion mit RAGE die prometastatischen Eigenschaften der A375-Melanomzellen förderte. Zudem verringerte extrazelluläres S100A4 die Zell-Integrität von Gehirn-Endothelzellen und erleichterte somit die Durchdringung der Blut-Hirn-Schranke. Diese Erkenntnis lässt sich möglicherweise auf andere Blut-Gewebe-Schranken übertragen. Die In-vivo-Orientierungsstudie zeigte, dass S100A4- und RAGE-überexprimierende Zellen zu einer verstärkten disseminierten Metastasierung führten, wobei sich zwei unterschiedliche Verteilungsmuster ergaben. Darüber hinaus führten beide Zelllinien vereinzelt zur Bildung von Gehirnmetastasen, wodurch sich die intrakardiale Injektion durchaus als Modell für weitere Therapiestudien mit dem Augenmerk der S100A4-RAGE-stimulierten Metastasierung eignet. Die genauere Kenntnis regulativer Mechanismen bei der Synthese und Sekretion von S100A4 sowie die pathophysiologische Differenzierung der S100A4-Interaktion mit RAGE eröffnen neue Wege, die S100A4-vermittelten Effekte therapeutisch zu beeinflussen. Daraus lassen sich möglicherweise neue zielgerichtete Radionuklid-basierte Therapieansätze für das metastasierende Melanom ableiten.

This diploma thesis dealt with the comparison of different methods of amination of polycaprolcatone in terms of their effectiveness for tissue engineering. A polycaprolactone membrane was prepared by an electrospinning method, which was subsequently modified by three different amination methods. Selected types of amination were plasma polymerization with cyclopropylamine monomer, hybrid modification using plasma and N-allylmethylamine monomer, and chemical amination using aminolysis with diaminohexane. Surface amines were subsequently characterized by electron scanning microscopy (SEM), X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared spectroscopy (ATR-FTIR) and contact angle measurement. A cell culture designated A375 (Human malignant melanoma cell lines A375® CRL-1619®) was cultured on the thus modified membranes, which was analyzed by optical microscopy, and a proliferation assay was performed by determining the relative amount of ATP. Based on the experimental results, we can confirm the success for all types of amination. In terms of efficiency for tissue engineering, the amination method by plasma polymerization with the monomer cyclopropylamine has the most satisfactory results.

A study was undertaken to determine whether cytogenetic abnormalities can be identified in an invasive melanoma cell population that has been selected in vitro out of a larger cell population of low invasive potential. The selecting agent was a denuded human amniotic membrane situated within Mega-Membrane Invasion Culture System chambers. Invasive cells were collected, grown, and harvested for cytogenetic analysis. Metaphases of these cells were examined for chromosomal abnormalities and for evidence of gene amplification in the form of double minute chromosomes. Invasive cell lines evinced changes in their degree of aneuploidy which were not seen in parental control lines of the same passage number. Significant karyotypic abnormalities identified in invasive cell lines were an increased dosage of chromosome 7 and multiple 1q translocation marker chromosomes. Double minute chromosomes were found in up to 18% of invasive cell metaphases examined and in 3% of parental controls. The incidence of double minutes was found to decrease as a function of passage number.

碩士
嘉南藥理科技大學
化妝品科技研究所
95
This study is the first to investigate the anticancer effect of plumbagin in human melanoma A375.S2 cells. Plumabagin exhibited effective cell growth inhibition by inducing cancer cells to undergo S-G2/M phase arrest and apoptosis. Further investigation revealed that plumbagin’s inhibition of cell growth was also evident in a nude mice model. Blockade of cell cycle was associated with increased levels of p21, and reduced amounts of cyclinB1, cyclinA, cdc2 and cdc25C. Plumbagin also enhanced the levels of inactivated phosphorylated cdc2 and cdc25C. Plumbagin triggered the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activation. We also found the generation of ROS is a critical mediator in plumbagin-induced cell growth inhibition. Antioxidants vitamin C and catalase significantly decreased apoptosis. In addition, plumbagin also increased the activation of ASK and then enhanced the phosphorylation of JNK and ERK1/2, but not p38. Moreover, blocking ERK and JNK by specific inhibitors suppressed plumbagin-induced apoptosis. Taken together, these results imply a critical role for ROS and JNK in the plumbagin’s anticancer activity.

Tong, Kit Fong.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 91-104).
Abstracts in English and Chinese.
Abstract --- p.i
Acknowledgements --- p.vi
Table of Contents --- p.vii
List of Figures --- p.x
List of Tables --- p.xii
List of Abbreviations --- p.xiii
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Cancer --- p.2
Chapter 1.1.1 --- Tumor development --- p.2
Chapter 1.1.2 --- Cell cycle --- p.4
Chapter 1.1.3 --- Apoptosis --- p.9
Chapter 1.1.3.1 --- The extrinsic pathway --- p.14
Chapter 1.1.3.2 --- The intrinsic pathway --- p.16
Chapter 1.1.3.3 --- The Bcl-2 family proteins --- p.17
Chapter 1.1.3.4 --- Execution of apoptosis --- p.20
Chapter 1.1.4 --- Melanoma --- p.22
Chapter 1.2 --- Polyunsaturated fatty acids (PUFAs) --- p.24
Chapter 1.2.1 --- "Chemistry, classification, metabolic conversion and sources …" --- p.24
Chapter 1.2.2 --- Epidemiology studies --- p.27
Chapter 1.2.3 --- Docosahexaenoic acid (DHA) --- p.28
Chapter 1.2.3.1 --- Sources --- p.28
Chapter 1.2.3.2 --- DHA and cancer --- p.29
Chapter 1.3 --- Objectives --- p.33
Chapter Chapter 2 --- Materials and Methods --- p.34
Chapter 2.1 --- In vitro studies of DHA on growth and survival of human cancer cells --- p.34
Chapter 2.1.1 --- Cell cultures --- p.34
Chapter 2.1.2 --- Studies of growth inhibition of DHA on human cancer cells --- p.35
Chapter 2.1.2.1 --- MTT assay --- p.35
Chapter 2.1.2.2 --- Chemiluminescent-bromodeoxyuridine (Chemi-BrdU) immunoassay --- p.36
Chapter 2.1.3 --- Studies of growth inhibitory mechanism of DHA on A375 cells. --- p.38
Chapter 2.1.3.1 --- DNA -flow cytometry analysis --- p.38
Chapter 2.1.3.2 --- Western blot analysis --- p.39
Chapter 2.1.3.3 --- Caspase inhibitor studies --- p.42
Chapter 2.1.3.4 --- Mitochondrial membrane potential analysis --- p.42
Chapter 2.2 --- In vivo study of the anticancer effect of DHA on A375 cells --- p.44
Chapter 2.2.1 --- Animals --- p.44
Chapter 2.2.2 --- Cell inoculation and treatments --- p.44
Chapter 2.2.3 --- Western blot analysis --- p.45
Chapter 2.3 --- Statistical analysis --- p.46
Chapter Chapter 3 --- Results --- p.47
Chapter 3.1 --- In vitro studies of DHA on growth and survival of human canccr cells --- p.47
Chapter 3.1.1 --- DHA reduced proliferation and survival of human cancer cells --- p.47
Chapter 3.1.2 --- DHA modulated cell cycle of A375 cells --- p.52
Chapter 3.1.3 --- DHA induced apoptosis in A375 cells --- p.55
Chapter 3.1.4 --- Caspase activations were involved in the DHA-induced apoptosis in A375 cells --- p.59
Chapter 3.1.5 --- "Caspase 3´ة 6, 8 and 9 were activated in DHA-induced apoptosis of A375 cells" --- p.62
Chapter 3.1.6 --- DHA dissipated mitochondrial membrane potential in A375 cells --- p.66
Chapter 3.1.7 --- DHA triggered the mitochondrial pathway of apoptosis --- p.68
Chapter 3.1.8 --- DHA triggered the death receptor pathway of apoptosis --- p.71
Chapter 3.2 --- In vivo study of the anticancer effect of DHA on A375 cells --- p.74
Chapter 3.2.1 --- Effect of DHA on the growth ofA375 xenograft in athymic Bαlb/c mice --- p.74
Chapter 3.2.2 --- DR4 and TRAIL were upregulated by DHA treatment in A375 solid tumor --- p.77
Chapter Chapter 4 --- Discussion --- p.79
References --- p.91

碩士
亞洲大學
生物科技學系碩士班
98
Skin cancer is one of the common carcinomatous diseases. It is becoming increasingly common in younger populations.Recently, many reports indicated that increased intake of cruciferous vegetables may be effective in preventing the risk of cancer and to reduce the incidence of cancer. Benzyl isothiocyanate (BITC), a compound presented in cruciferous vegetables, had shown to induce cell cycle arrest and apoptosis in many cancer cells. There is no report to show BITC inhibited the growth of A375.S2 human skin cancer cells. In this study BITC affecting apoptosis in A375.S2 cells was investigated. MTT assay was used to measure cell viability of A375.S2 cells after BITC treatment. DNA damage was determined by DNA fragmentation assay, DAPI staining and Comet assay. Flow cytometric analysis was performed to investigate the levels of mitochondrial membrane potential (ΔΨm), the production of reactive oxygen species (ROS), intracellular Ca2+ release and cell cycle distribution. Finally, we used the flow cytometry to examine caspase-3 activity and Annexin V affinity assay for apoptosis. In western blotting assay, we found cytochrome c, AIF and Endo G were released from mitochondria and activated downstream pathway to cause cell apoptosis. Our results showed that BITC treatment for 24 h significantly reduced the cell survival with an IC50 of 10±0.5 μM. BITC induced cell cycle arrest at G2/ M phase in A375.S2 cells. Moreover, BITC also caused DNA damage, decreased ΔΨm and increased ROS and intracellular Ca2+ levels. These observations indicate that BITC could induce apoptosis in human melanoma A375.S2 cell.

碩士
輔英科技大學
生物技術系碩士班
98
Vanadate is an environmentally toxic metal with peculiar and sometimes contradictory cellular effects. Studies concerning the mechanisms of action of vanadate showed to induce gene expressions, oxidative burst, changes in cytosolic calcium, tyrosine phosphorylation, insulin-mimetic effects and also cytoskeletal alterations, but the regulation mechanisms remain to be elucidated. Hydrogen peroxide（H2O2）is a strong oxide which can induce stress in culture cells and play a role in the physiological mode in the processes of growth, differentiation and the death control. We found that human melanoma A375 cells were relatively more sensitive than human lung carcinoma NCI-H460 cells to the cytotoxic effects induced by H2O2 as well as sodium vanadate. In this study, the role of intracellular oxidative stress in the mechanism of action was studied using the H2O2, vanadate, and H2O2 plus vanadate in the three cells. The proliferation and viability as well as protein expressions of sub-toxic dosage H2O2 treated cells were detected in the prensence of different concentrations of vanadate for different durations. Our data demonstrated up to 1.25μM of vanadate had no effect on proliferation and viability of A375 cells, but cotreated cells with vanadate and H2O2 resulted in rapidly cell growth inhibition and apoptotic cells became more abundant. Vanadate seems to have synergistic effects to enhance cytotoxicity of H2O2 in A375 cells depending on the dose-and time. The fragmentation of DNA, cell cycle distribution and generation of ROS as well as of were observed by using of flow cytometry in H2O2 and vanadate treated cells for providing other cytotoxic mechanisms. However, the antioxidant N-acetyl cysteine, a reactive oxygen species inhibitor, greatly diminished the intracellular oxidation and protein phosphotyrosine accumulation as well as the H2O2 induced cytotoxicity which potentiated by vanadate. These results indicate a role for oxidative stress in the biological effects of PTP inhibitor vanadate in H2O2 treated cells.

Li, Wing Yan Kate.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 130-154).
Abstracts in English and Chinese.
Acknowledgements --- p.i
Abstract --- p.iii
Abstract (Chinese Version) --- p.vi
Table of Contents --- p.viii
List of Figures --- p.xiii
List of Abbreviations --- p.xv
Chapter Chapter 1 --- General Introduction
Chapter 1.1. --- Overview of cancer --- p.1
Chapter 1.2. --- Apoptosis and cancer --- p.4
Chapter 1.3. --- Roles and regulation of caspase-dependent apoptosis --- p.7
Chapter 1.3.1. --- Extrinsic death receptor pathway --- p.8
Chapter i. --- TNFR1 and TNFa --- p.13
Chapter ii. --- CD95/Fas and CD95 Ligand/FasL --- p.14
Chapter iii. --- "TRAIL-R1(DR4), TRAIL-R2 (DR5) and TRAIL" --- p.14
Chapter 1.3.2. --- Intrinsic mitochondrial pathway --- p.16
Chapter i. --- Bcl-2 family of proteins --- p.17
Chapter ii. --- Reactive Oxygen Species (ROS) --- p.19
Chapter 1.4. --- Phytochemicals from Traditional Chinese Medicine (TCM) as a source of new therapeutics --- p.22
Chapter 1.5. --- Biological effects of baicalein --- p.25
Chapter 1.5.1 --- Roles of baicalein as a lipoxygenase inhibitor --- p.28
Chapter 1.5.2 --- Dual roles of baicalein as an antioxidant and prooxidant --- p.28
Chapter 1.5.3 --- "Roles of baicalein as an anti-carcinogenic, anti-proliferative and anti-metastatic agent" --- p.29
Chapter 1.6. --- Aims of current study --- p.30
Chapter Chapter 2 --- Effects of Baicalein on Growth and Survival of Human Cancer Cells
Chapter 2.1 --- Introduction --- p.33
Chapter 2.2 --- Materials and Methods
Chapter 2.2.1 --- Cell culture --- p.35
Chapter 2.2.2 --- Measurement of growth and survival of various cell lines --- p.36
Chapter 2.2.3 --- Statistical analysis --- p.37
Chapter 2.3 --- Results
Chapter 2.3.1 --- Baicalein retards the growth and survival of human melanoma A375 and colorectal carcinoma Caco-2 --- p.37
Chapter 2.3.2 --- Baicalein reduces the growth and survival of melanoma A375 but not in normal skin fibroblast Hs68 cells --- p.40
Chapter 2.4 --- Discussion --- p.42
Chapter Chapter 3 --- Effects of Baicalein on Cell Cycle and the Apoptosis in Human Melanoma A375 Cells
Chapter 3.1 --- Introduction --- p.44
Chapter 3.2 --- Materials and Methods
Chapter 3.2.1 --- Determination of cell cycle changes and quantification of apoptosis --- p.51
Chapter 3.2.2 --- Immunoblotting --- p.52
Chapter 3.2.3 --- Inhibition of caspase-8 by caspase-8 inhibitor --- p.54
Chapter 3.2.4 --- Fluorometric measurement of caspase-3 activity --- p.54
Chapter 3.2.5 --- Statistical analysis --- p.55
Chapter 3.3 --- Results
Chapter 3.3.1 --- Baicalein induces S-phase arrest in cell cycle and triggers apoptosis --- p.55
Chapter 3.3.2 --- Baicalein induces proteolytic inactivation of PARP and activation of caspases --- p.59
Chapter 3.3.3 --- Caspase-8 is the major initiator caspase eliciting the baicalein-induced apoptosis --- p.62
Chapter 3.4 --- Discussion --- p.67
Chapter Chapter 4 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells
Chapter 4.1 --- Introduction --- p.72
Chapter 4.2 --- Materials and Methods
Chapter 4.2.1 --- Immunoblotting --- p.75
Chapter 4.2.2 --- Determination of sub-lethal dose of exogenous TRAIL --- p.76
Chapter 4.2.3 --- Determination of the combinatory effect of exogenous TRAIL and baicalein --- p.76
Chapter 4.2.4 --- Statistical analysis --- p.77
Chapter 4.3 --- Results
Chapter 4.3.1 --- Baicalein upregulates the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) --- p.77
Chapter 4.3.2 --- Baicalein sensitizes the melanoma cells to sub-lethal dose of exogenous TRAIL --- p.80
Chapter 4.4 --- Discussion --- p.84
Chapter Chapter 5 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells Cancer Cells
Chapter 5.1 --- Introduction --- p.88
Chapter 5.2 --- Materials and Methods
Chapter 5.2.1 --- Analysis of mitochondrial membrane potential --- p.94
Chapter 5.2.2 --- Fractionation of cell lysates into cytosolic and mitochondrial fractions for immunoblotting --- p.95
Chapter 5.2.3 --- Immunoblotting --- p.95
Chapter 5.2.4 --- Determination of cellular reactive oxygen species (ROS) production --- p.96
Chapter 5.2.5 --- Verification of ROS generation via the addition of Trolox´ёØ --- p.96
Chapter 5.2.6 --- Statistical analysis --- p.97
Chapter 5.3 --- Results
Chapter 5.3.1 --- Baicalein induces mitochondrial membrane depolarization --- p.97
Chapter 5.3.2 --- Cytochrome c is released in the baicalein-induced mitochondrial membrane depolarization --- p.100
Chapter 5.3.3 --- Baicalein does not elicit the intrinsic apoptotic pathway via modulation of some better-characterized Bcl-2 family proteins in A375 cells --- p.102
Chapter 5.3.4 --- Baicalein induces ROS production --- p.105
Chapter 5.3.5 --- Baicalein induces mitochondrial permeabilization via ROS-mediated mechanisms --- p.108
Chapter 5.4 --- Discussion --- p.112
Chapter Chapter 6 --- General Discussion --- p.119
References --- p.130

Melanoma is one of the most aggressive form of skin cancer, characterized by high mortality rate due to the metastatic potential of its cells. Several therapies have been approved during last few years. Recently, the melanoma molecular characterization led to development of drugs acting against specific targets. For instance, BRAF inhibitors (BRAFi) have been tested against BRAF-mutated melanoma. Unfortunately, all chemotherapy strategies failed for the resistance acquired by tumor cells. Poly (ADP-ribose) polymerase (PARP) enzymes are crucial in the DNA damage response and the PARP inhibition causes the accumulation of unrepaired DNA, inducing cell death. PARP inhibitors (PARPi) are commonly used in chemotherapy. The most used PARPi in cancer therapy is olaparib, although it can be quickly throwing outside the cells by the P-glycoprotein drug efflux transporter. For this reason, a new PARPi generation has been designed, such as AZD2461 that demonstrated a lower binding affinity to the P-glycoprotein than olaparib. In vitro and in vivo studies showed that the protein onconase (ONC) exerted an antitumor effect in different cancer types, due to its ribonuclease activity. Therefore, in this work, the ONC effects on A375 human melanoma cell line that harbors a mutation in the BRAF kinase gene, has been evaluated. A reduction in A375 cell viability has been observed with ONC treatment, while no cytotoxicity was registered in normal human melanocytes. In A375 cells a reduction in BrdU incorporation and a decrease in Ki67 protein expression occurred, suggesting that ONC elicits a cytostatic effect. In addition, an increase of both Annexin V-FITC fluorescence and cleaved PARP1 expression level were observed, suggesting that ONC induces apoptotic cell death. In order to investigate the ONC intracellular targets, changes in the proteome profile have been investigated using mass spectrometry. A decreased expression level of proteins involved in telomere elongation, in the mesenchymal cell development and in the ribosomal subunit biogenesis has been obtained. A decrease in protein synthesis in ONC-treated cells was also demonstrated. Besides ONC, AZD2461 PARPi can reduce A375 cell viability, conversely, no strong benefits were obtained when ONC was administered in combination with AZD2461, in comparison with each single drug treatment. ONC and AZD2461 have displayed an inhibitory effect on both TNF-α gene transcription and NF-κB DNA binding activity, but no additive effect was observed when they were used in combination. In order to obtain A375 cells resistant to AZD2461, cells were treated for two months with AZD2461, but this long treatment did not induce resistance. Nevertheless, the AZD2461 long-time treated cells resulted more responsive to the ONC pro-apoptotic action, if compared to the parental ones. Subsequently, resistance against the BRAFi dabrafenib has been induced in A375 since long time treatment with increasing drug concentrations of dabrafenib induced a sorting of an A375 resistant cell (A375DR) subpopulation. A375DR cells displayed activation of the cancer stem cells markers CD133 and NANOG and increase expression of epithelial-mesenchymal transition-related proteins N-cadherin and nuclear β-catenin, in comparison with A375 parental (A375P) cells. In addition, A375DR showed an increase in the ERK1/2 phosphorylation level, suggesting a reactivation of the MAPK pathway. By comparing A375P and A375DR, ONC treatment can inhibit the total viability and the proliferation rate in both cell subpopulations and induce apoptotic cell death. Moreover, among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IκB kinase phosphorylation level, as well as metalloproteinase-2 activity in both cell subpopulations. Finally, ONC decreased cell colony formation, migration and invasion capability more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts malignant phenotype of A375 cells especially in BRAFi resistant cells and could become a helpful tool for therapy of melanoma recurrence.

碩士
中國醫藥學院
中西醫結合研究所
91
Shikonin is one of the components of Lithospermum erythrorhizon (Chinese herb medicine) used for skin infection and allergy for many generations in the Chinese population. In this study, shikonin was used to determine the inhibition of N-acetylation of 2-aminofluorence (2-AF) in human malignant melanoma cell line (A357.S2). The amounts of N-acetylation and non-N-acetylation of 2-AF were measured by high performance liquid chromatography. The results demonstrated that N-acetylation of AF from examined systems were decreased by shikonin in a dose-dependent manner. We also found out that this effect of shikonin on N-aectylation of AF also was time-course dependent. Apparently shikonin affect N-acetylation of AF in human malignant melanoma cell line (A357.S2). We investigated how shikonin affects human malignant melanoma cells (A375.S2) for determining the inhibition of cell growth, morphological changes, DNA fragmentation, and cell cycle by using flow cytometric assay and DNA gel electrophoresis. After exposure of the cells to shikonin which resulted in cell cycle arrest and cell death through apoptosis. This effect is also dose-dependent manner. Exposure of A375.S2 cells to shikonin induced expression of the cyclins and cyclin-dependent kinases (CDK) activity. These studies demonstrated that cyclins and CDKs play a key role in the inhibition of shikonin-induced apoptosis and cell cycle arrest in A375.S2 cells. This is the first findings to show shikonin affect human malignant melanoma cell lines (A375.S2).

碩士
亞洲大學
生物科技學系碩士班
98
According to epidemiological studies, dietary intake of cruciferous vegetables may be protective against the risk of cancer. Isothiocyanates (ITCs) are found in cruciferous vegetables, including broccoli, cabbage, watercress and cauliflower. Recent studies had been shown that ITCs are classes of naturally occurring chemopreventive and possibly chemotherapeutic agents. Phenethyl isothiocyanate (PEITC) is one of the most common researched ITCs. The recent studies reported that PEITC can induce cancer cell growth inhibition or apoptosis of human prostate, leukemia and hepatoma cancer cells. However, the molecular mechanism and signaling pathway of PEITC-induced apoptosis in human malignant melanoma A375.S2 cells are still not clear. In this study, apoptosis induced by PEITC in A375.S2 cells was investigated. PEITC induced cell death in time- and dose-dependent manners was found in MTT assay. Cell cycle analysis, reactive oxygen species (ROS) generation, intercellular Ca2+ detection, the change of level mitochondria membrane potential (ΔΨm) and caspase-3 activity in A375.S2 cells were detected by flow cytometry after exposure to PEITC. Chromatin condensation and DNA damage were determined by DAPI staining, Comet assay and DNA gel electrophoresis. The results showed that PEITC caused chromatin condensation, DNA damage, cell cycle G2/M phase arrest, promoted ROS generation, stimulated Ca2+ release, loss of ΔΨm and activated caspase-3 activity lead to cell apoptosis in human malignant melanoma A375.S2 cells.

博士
高雄醫學大學
藥學研究所
99
Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) chemicals are antitumor antibiotics inhibiting nucleic acid synthesis. An indole carboxylate-PBD hybrid with six-carbon spacer structure (IN6CPBD) has been previously demonstrated to induce melanoma cell apoptosis and reduce metastasis in mouse lungs. This study aimed at investigating the efficacy of the other hybrid compound with four-carbon spacer (IN4CPBD) and elucidating its anti-metastatic mechanism. Human melanoma A375 cells with IN4CPBD treatment underwent cytotoxicity and apoptosis-associated assays. Transwell migration assay, Western blotting, and ELISA were used for mechanistic study. IN4CPBD exhibited potent melanoma cytotoxicity through interrupting G1/S cell cycle progression, increasing DNA fragmentation and hypodipoidic DNA contents, and reducing mitochondrial membrane potential. Caspase activity elevation suggested that both intrinsic and extrinsic pathways were involved in IN4CPBD-induced melanoma apoptosis. IN4CPBD up-regulated p53 and p21, thereby concomitantly derailing the equilibrium between Bcl-2 and Bax levels. Transwell migration assay demonstrated that stromal cell-derived factor-1???? (SDF1-??) stimulated A375 cell motility, while kinase inhibitors treatment confirmed that Rho/ROCK, Akt, ERK1/2, and p38 MAPK pathways were involved in SDF1-??-enhanced melanoma migration. IN4CPBD not only abolished the SDF1-??-enhanced chemotactic motility but also suppressed constitutive MMP-9 and VEGF expression. Mechanistically, IN4CPBD down-regulated Akt, ERK1/2, and p38 MAPK total proteins and MYPT1 phosphorylation. In conclusion, beyond the fact that IN4CPBD induces melanoma cell apoptosis at cytotoxic dose, the interruption in the VEGF expression and the SDF1-??-related signaling at cytostatic dose may partially constitute the rationale for its in vivo anti-metastatic potency.

碩士
高雄醫學大學
香粧品學系碩士班
100
Melanoma, a malignant tumor of melanocytes, has one of the worldwide fastest growing incidences among cancers and is the deadliest form of skin cancer. We identified that 7-hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv.rosa-plena to be a bio-active agent against human melanoma A375.S2 cells. Cell proliferation assay displayed minor cytotoxicities on normal human skin cells including, epidermal keratinocytes and melanocytes and dermal fibroblasts. It also demonstrated the strongest anticancer effect of 7-HDNF on A375.S2 cells to exhibit a dose-dependent behavior. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining to confirm the cellular membrane asymmetry losing. One-dimensional flow cytometric analysis showed that 7-HDNF increased cellular DNA population in cell cycle at G2/M phase. With acridine orange (AO) staining, we found that 7-HDNF induced patterns of autophagy unexpectedly, such as the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). Through western blot, protein expressions were discovered to verify apoptosis and autophagy response mechanisms sharing some common associated pathways. In addition to, 7-HDNF presented the high-quality anti-migratory activity in wound healing assay. Within this study, we confirmed that there were dual cell death machineries via apoptotic and autophagic inductions in 7-HDNF-treated A375.S2 cells to be a potential effective chemotherapeutic agent.

碩士
中山醫學大學
醫學研究所
96
Malignant melanoma is an increasing common malignancy and with high mortality. Melanoma in situ and early stage melanoma could be cured if early diagnosis and surgical excision. Unless chemotherapy or immunotherapy, the prognosis is quite poor in patient of late staging and highly invasive malignancy. But the side effects of chemotherapy or immunotherapy are severe, such as nausea, vomiting, hair loss and bone marrow suppression. Danthron is one of the extracts of Rhubarb that is a traditional Chinese herb. Danthron, Emodin, Aloe-emodin, and Rhein are all active compounds of Rhubarb. Researchers find that Emodin, Aloe-emodin, and Rhein can suppress cell growth via arresting cell cycle. They also can induce apoptosis of a lot of cancer cell lines via mitochondrial pathway and inhibit metastasis. This research is being probed into effects of Danthron on the induction of cell cycle arrest and apoptosis in human malignant melanoma cell line (A375.S2). The result suggests that Danthron can kill A375.S2 cells associated with G0/G1 phase arrest and induction apoptosis. DNA damage was found by DAPI stain and Comet assay. Flow cytometric assay and real time PCR showed that Danthron can decrease mitochondrial membrane potential and let releasing cytochrome c, AIF and Endo G to cytoplasm. Danthron increased expression of Bax and suppressed expression of Bcl-xL/Bcl-2 then active caspase-9 and caspase-3. Finally, it induced apoptosis of A375.S2 cells through caspase-dependent or caspase-independent pathway. Elevation of intracellular calcium concentration may decrease mitochondrial membrane potential and increase reactive oxygen species. Otherwise, Danthron could make cytochrome c to release via activating caspase-8 and Bid/tBid. In conclusion, Danthron can induce apoptosis through mitochondrial, endoplasmic reticulum and death receptor pathway in human malignant melanoma cell line (A375.S2).

碩士
高雄醫學大學
醫學系生物化學科碩士班
104
Lemongrass one kind of herb, is widely used as a flavoring of food, and is used to lessen pains by it’s analgesic and anti-inflammatory properties. Previous research indicated citral is the major effective compound of lemongrass and has the potential to induce apoptosis of cells. The present study is to explore the effects of lemongrass oil and citral on human malignant melanoma cell line (A375) and explore the underline mechanism. Results showed that lemongrass oil and citral inhibited A375 cells growth in both time-dependent and dose-dependent manner. At the concentrations ranged from 30μg/ml to 50 μg/ml, either lemongrass oil or citral could stimulate production of reactive oxygen species (ROS), arrest cell cycle progress in G2/M phase by decreasing the synthesis of cyclin B1 and cdc25c, and induce apoptosis of human malignant melanoma A375 cells. Moreover, the expressions of antiapoptotic protein of Bcl-2 and Bcl-XL were down-regulated, whereas expressions of proapoptotic protein Bax, caspase-9, and caspase-3 were up-regulated.Moreover, Fas ligand and receptor expressions were also up-regulated which could activate caspase-3. Changes of those leaded to occurence of apoptosis.These results suggest that lemongrass oil and citral may have the potential in prevention against human malignant melanoma. However, further studies are needed to accumulate enough evidences to support this viewpoint.

碩士
靜宜大學
食品營養研究所
94
Recently, there is increasing interest in finding natural antioxidant from plants to protect human body against cancer. Carcinoma is the major death cause in Taiwan. Graptopetalum paraguayense E. Walther is a traditional Chinese herbal medicine. Our in-vitro study had showed that Graptopetalum paraguayense E. Walther (GP) extracts had antioxidant,antimutagenic,and tyrosinase inhibitory activities. In this study, we will examine the effects of 50% ethanolic extract from GP (GE50) on the growth of human malignant cell lines (A375. S2). The results demonstrated that GE50 can inhibit the cell growth, induce cell cycle arrest on G2/M phase. The morphology, DAPI staining and DNA fragmenation assay were conducted and they indicated that GE50 induced A375. S2 cells apoptosis. Western boltting analysis, GE50 decrease the protein level of cyclin A、cyclin B、cdc2 and cdc25c, and increased the level of CHK1、CHK2、Wee1、p53 and p21 of cells. Flow cytometry assay demonstrated that GE50 induced the change of mitochondria membrance potential and calcium concentration. In ER (endoplasmic reticulum) stress, GE50 can increase the expression of GRP-78 and GADD153. GE50 inhibited the expression of Bcl-2 and promoted the level of Bax. Furthermore, GE50 can increase the expression of cytochrome c、 caspase-9、caspase-3 and caspase-7. GE50 also can inhibit the invasiveness of human malignant cells by suppressing MMP-2 and MMP-9 expression. GE50 can increase superoxide dismutase and decreased the catalase、glutathion peroxidase activity and glutathion levels, however, GE50 increased malondialdehyde levels in A375. S2 cells. The results of this study we suggested that GE50 might inhibite cell growth and induced G2/M phase arrest. GE50 induced apoptosis throught the activation of p53、Bax , induced calcium concentration, which lead to change on mitochondria membrance permeability and release of cytochrome c. Therefore, caspase-9、caspase-3、caspase-7 、GRP-78 and GADD153 are activited and decrease MMP-2 and MMP-9 expression. Furthermore, our results demonstrate that GE50 can change GSH antioxidant system. The current evidence suggests that Graptopetalum paraguayense E. Walther can inhibite the cell growth, induce apoptosis and inhibit invasiveness.

碩士
亞洲大學
生物科技學系碩士班
97
The purposes of this study were to evaluate the inhibitory potential of 500 mg/L ethanol extract from the seed coat of black soybean (Glycine max (L.) Merrill, Tainan No. 3) (BSSCEE) toward the activity of tyrosinase, otherwise human malignant melanoma A375.S2 cell line was used to assess the in vitro effect of BSSCEE on the melanogenesis. The results of inhibitory effects on mushroom tyrosinase activity showed that BSSCEE could inhibit the activity of mushroom tyrosinase. At the same concentration (1.66 mg/ml) between BSSCEE and 500 mg/L ethanol extract from the whole black soybean (WSSEE), the inhibition rate of BSSCEE better than WSSEE exceed in double. Indicate that the most of inhibitors on mushroom tyrosinase were existed in the seed coat of black soybean. Otherwise the inhibition rate was depend on the concentration of BSSCEE and the inhibition mechanism was belong to mixed-type inhibition. The results of cell culture were showed that there was the tyrosinase ability to catalyze L-dopa during human malignant melanoma A375.S2 cell line. After treated with different concentrations of BSSCEE, using three different concentrations of L-dopa (0.05, 0.25 and 0.5 mg/ml) to evaluate. Among all of results the highest two concentrations of BSSCEE (20 μg/ml and 30 μg/ml) showed the more significant inhibitory effect on human malignant melanoma A375.S2 cell line.