Thesis (M.Phil.) - University of Queensland, 2005. / Includes bibliography.

O desenvolvimento de novas estratégias terapêuticas ao melanoma é de particular importância devido à sua baixa resposta aos tratamentos tradicionais. No presente trabalho, utilizamos modelo de melanoma murino experimental para estudarmos os efeitos da fosfoetanolamina (PEA) sintética sobre o desenvolvimento deste tumor. Nossos resultados demonstram que o fosfomonoéster apresentou efeito inibidor da proliferação de células da linhagem B16F10 in vitro, induzindo apoptose após estimulação por 24 a 72h. In vivo, o tratamento (via oral) de animais portadores de melanoma com diferentes doses de PEA (10, 20 e 40mg/Kg), durante 10 ou 20 dias consecutivos, resultou em volumes tumorais pelo menos 70% menores que o de animais controle e diferenças macroscópicas consideráveis. PEA induziu, de maneira dose-dependente, aumento da apoptose e diminuição da proliferação de células tumorais. O tratamento resultou em alterações hematológicas como aumento do número de plaquetas, eritrócitos e leucócitos. Dentre os leucócitos, observou-se uma maior proporção de linfócitos e monócitos após 10 e 20 dias de tratamento, respectivamente. Em adição, PEA induziu uma maior produção da citocina pró-inflamatória IL-6 e das citocinas anti-inflamatórias IL-10 e TGF- e menores níveis da citocina pró-inflamatória IFN-. Os níveis de IL-1, IL-12p70 e IL-17 não foram alterados com o tratamento. Nossos resultados demonstram um papel inibidor da PEA sobre a progressão do melanoma, contribuindo para um melhor entendimento de sua atividade anti-tumoral. / The low responsiveness of melanoma to traditional treatments together with its increasing incidence makes the development of new therapeutic strategies against this type of cancer extremely important. In this study, we used a murine melanoma model to evaluate the effects of synthetic phosphoethanolamine (PEA) on the development of this tumor. In vitro, PEA had an inhibitory effect on the proliferation of B16F10 cells, inducing apoptosis after 24 to 72h stimulation. In vivo, oral treatment of melanoma-bearing animals with different doses of PEA (10, 20 e 40mg/Kg) during 10 or 20 consecutive days resulted in reduced tumor volumes (at least 70% compared to the control) and in expressive macroscopic differences. PEA also induced a dose-dependent increase of apoptosis and decrease in tumor cell proliferation. The treatment also resulted in hematological changes, such as increased numbers of platelets, erythrocytes and leukocytes. Among leukocytes, we observed a higher proportion of lymphocytes and monocytes after 10 and 20 days of treatment, respectively. In addition, PEA induced higher levels of the pro-inflammatory cytokine IL-6 and of the anti-inflammatory cytokines IL-10 and TGF-, and it also induced a lower production of the pro-inflammatory cytokine IFN-. No differences were observed in the levels of IL-1, TNF-, IL-12p70 and IL-17 upon treatment. Our results demonstrate an inhibitory role of PEA in the development of melanoma, contributing to a better understanding of its antitumoral activity.

Huai-Hua-Jin-Yin-Jiu, a traditional Chinese medicine (TCM) formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos, was used for treating melanoma in ancient China. Our group has previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects, and that inhibiting STAT3 signaling contributes to the action mechanisms. STAT3 activation promotes the formation of an immunosuppressive microenvironment of melanoma. Regulation of the miR-let- 7/CCR7 pathway is a strategy for treating metastatic melanoma. Chrysoeriol is a flavonoid identified in SLE. The compound has anti-tumor properties, but there is no report about its effects on melanoma. The first aim of this study is to determine whether SLE inhibits melanoma progression by reprogramming tumor microenvironment; the second aim is to explore the involvement of miR-let-7a/f- CCR7 signaling in the anti-metastatic effects of SLE; and the third aim is to elucidate the anti-melanoma mode and mechanisms of action of chrysoeriol. Results showed that SLE suppresses melanoma growth and angiogenesis in mice. SLE inhibits STAT3 signaling, and alters immune cell compositions and molecules involved in STAT3 signaling in melanoma tissues. Cell co-culture experiments showed that SLE inhibits STAT3 signaling in melanoma cells and splenic lymphocytes. Over-activation of STAT3 in melanoma cells diminishes SLE's effects in altering compositions of immune cells and STAT3 signaling molecules in the co- culture system. Small RNA sequencing showed that SLE upregulates miR-let-7a/f levels in B16F10 melanomas. RT-qPCR analyses confirms these results. SLE elevates miR-let- 7a/f levels, and inhibits CCR7 (a target gene of miR-let-7a/f) signaling in melanoma cells. In a lung metastasis mouse model, SLE inhibits melanoma metastasis, elevates miR-let-7a/f levels, and suppresses CCR7 signaling. Knockdown of miR-let-7a/f diminishes the effects of SLE on CCR7 signaling and melanoma cell invasion; and overexpression of CCR7 lessens the effects of SLE on melanoma cell invasion. Chrysoeriol shows in vitro and in vivo anti-melanoma effects. Molecular dynamics and microscale thermophoresis assays demonstrate that chrysoeriol directly binds to Src. Chrysoeriol suppresses the Src/STAT3 pathway in melanoma cells and tissues. Chrysoeriol's anti-proliferative effects are diminished by STAT3 over- activation in melanoma cells. Chrysoeriol also has inhibitory effects in vemurafenib- resistant melanoma cell and animal models. RNA-seq analyses shows that syndecan-3 mRNA level is lower in vemurafenib-resistant cells than in vemurafenib-sensitive cells, and chrysoeriol reverses this vemurafenib resistance-associated downregulation. RT-qPCR and Western blotting analyses confirmed the results. In conclusion, we have demonstrated that reprograming immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. Regulation of the miR-let-7a/f-CCR7 pathway is another mechanism underlying the anti-melanoma effects of SLE. Chrysoeriol is identified to be one of the active components responsible for the anti- melanoma effects of SLE. Inhibiting the Src/STAT3 pathway contributes to the anti- melanoma mechanisms of chrysoeriol. Chrysoeriol is able to overcome vemurafenib resistance in melanoma, and upregulation of syndecan-3 is involved in the action mechanisms. This study provides further pharmacological and chemical justifications for the use of the formula SL in treating melanoma, and suggests that SLE and SLE- derived compounds have the potential to be developed as modern alternative and/or complimentary agents for melanoma management

Huai-Hua-Jin-Yin-Jiu, a traditional Chinese medicine (TCM) formula (SL) comprising Sophorae Flos and Lonicerae Japonicae Flos, was used for treating melanoma in ancient China. Our group has previously shown that an ethanolic extract of SL (SLE) possesses anti-melanoma effects, and that inhibiting STAT3 signaling contributes to the action mechanisms. STAT3 activation promotes the formation of an immunosuppressive microenvironment of melanoma. Regulation of the miR-let- 7/CCR7 pathway is a strategy for treating metastatic melanoma. Chrysoeriol is a flavonoid identified in SLE. The compound has anti-tumor properties, but there is no report about its effects on melanoma. The first aim of this study is to determine whether SLE inhibits melanoma progression by reprogramming tumor microenvironment; the second aim is to explore the involvement of miR-let-7a/f- CCR7 signaling in the anti-metastatic effects of SLE; and the third aim is to elucidate the anti-melanoma mode and mechanisms of action of chrysoeriol. Results showed that SLE suppresses melanoma growth and angiogenesis in mice. SLE inhibits STAT3 signaling, and alters immune cell compositions and molecules involved in STAT3 signaling in melanoma tissues. Cell co-culture experiments showed that SLE inhibits STAT3 signaling in melanoma cells and splenic lymphocytes. Over-activation of STAT3 in melanoma cells diminishes SLE's effects in altering compositions of immune cells and STAT3 signaling molecules in the co- culture system. Small RNA sequencing showed that SLE upregulates miR-let-7a/f levels in B16F10 melanomas. RT-qPCR analyses confirms these results. SLE elevates miR-let- 7a/f levels, and inhibits CCR7 (a target gene of miR-let-7a/f) signaling in melanoma cells. In a lung metastasis mouse model, SLE inhibits melanoma metastasis, elevates miR-let-7a/f levels, and suppresses CCR7 signaling. Knockdown of miR-let-7a/f diminishes the effects of SLE on CCR7 signaling and melanoma cell invasion; and overexpression of CCR7 lessens the effects of SLE on melanoma cell invasion. Chrysoeriol shows in vitro and in vivo anti-melanoma effects. Molecular dynamics and microscale thermophoresis assays demonstrate that chrysoeriol directly binds to Src. Chrysoeriol suppresses the Src/STAT3 pathway in melanoma cells and tissues. Chrysoeriol's anti-proliferative effects are diminished by STAT3 over- activation in melanoma cells. Chrysoeriol also has inhibitory effects in vemurafenib- resistant melanoma cell and animal models. RNA-seq analyses shows that syndecan-3 mRNA level is lower in vemurafenib-resistant cells than in vemurafenib-sensitive cells, and chrysoeriol reverses this vemurafenib resistance-associated downregulation. RT-qPCR and Western blotting analyses confirmed the results. In conclusion, we have demonstrated that reprograming immune microenvironment, partially mediated by inhibiting STAT3 signaling, contributes to the anti-melanoma mechanisms of SLE. Regulation of the miR-let-7a/f-CCR7 pathway is another mechanism underlying the anti-melanoma effects of SLE. Chrysoeriol is identified to be one of the active components responsible for the anti- melanoma effects of SLE. Inhibiting the Src/STAT3 pathway contributes to the anti- melanoma mechanisms of chrysoeriol. Chrysoeriol is able to overcome vemurafenib resistance in melanoma, and upregulation of syndecan-3 is involved in the action mechanisms. This study provides further pharmacological and chemical justifications for the use of the formula SL in treating melanoma, and suggests that SLE and SLE- derived compounds have the potential to be developed as modern alternative and/or complimentary agents for melanoma management

目的 黑色素瘤是一种高度恶性肿瘤，极易耐药。而肿瘤细胞的免疫逃逸很可能是产生耐药的原因之一。参茋扶正注射液（SQFZ）广泛应用于联合放化疗药物治疗癌症，且具有一定的临床疗效基础。通过研究参茋扶正注射液（SQFZ）对A375细胞分泌的免疫抑制因子的作用和影响和研究SQFZ是否可以提高jurkat细胞的功能，并间接对肿瘤细胞起到杀伤作用。探讨SQFZ对抑制黑色素瘤细胞A375免疫逃逸的影响和作用机制，以及SQFZ用于治疗黑色素瘤的可能性。 方法 外培养黑色素瘤细胞A375，应用不同的SQFZ干预细胞。 1. 采用MTT技术检测SQFZ对A375的直接作用； 2. 采用Real-Time PCR技术检测SQFZ对A375细胞表达免疫抑制因子IL-10、TGF-beta、VEGF的影响； 3. 用ELISA检测SQFZ对A375细胞分泌IL-10、TGF-beta、VEGF的影响； 4. 建立transwell小室模型，检测SQFZ对jurkat T细胞迁徙功能的影响； 5. 利用transwell小室，建立共培养体系, 检测SQFZ对jurkat T细胞分泌细胞因子功能的影响。 结果 1. SQFZ浓度为1280μg/mL时，对A375细胞有抑制作用，与空白组比较，差异具有统计学意义（p<0.01），但抑制率低于20%，无研究意义； 2. 不同浓度（1280μg/mL、640μg/mL)SQFZ干预A375细胞24h，48h后，可以降低IL-10、TGF-beta 在mRNA水平的表达，与对照组比较结果有统计学意义(p<0.05），并且抑制呈时间剂量依赖关系；SQFZ(l280μg/mL）干预A375細胞48h后，VEGF在mRNA水平的表达降低，与对照组比较结果有统计学 意义（p<0.05); 3. 经不同浓度SQFZ处理24h后，A375细胞IL-10在SQFZ浓度为640μg/mL表达降低，；VEGF-beta、VEGF在SQFZ浓度为1280μg/mL、640μg/mL 时，蛋白水平表达降低，以上结果均与对照组比结果有统计学意义（p<0 .05); 4. 经不同浓度SQFZ处理A375细胞48h后，jurkat T细胞相对迁徙面积和穿膜细胞数变化显著，与对照组比较结果具有统计学意义（p

HVEM est une molécule exprimée à la surface des mélanomes (M) qui pourrait contribuer à la croissance tumorale en se liant à BTLA, un co-récepteur inhibiteur exprimé par les lymphocytes T infiltrants les tumeurs (TILs). Nos objectifs étaient 1-d’explorer la valeur pronostique de l’expression d’HVEM par les M; 2-de confirmer l’existence d’une interaction HVEM/BTLA in vivo à l’interface mélanome-TILs; 3- de comprendre les mécanismes de régulation d’HVEM..Méthodes: L’expression d’HVEM a été analysée par immunohistochimie et corrélée à la survie globale des patients. Ces résultats ont été renforcés par l'étude de données transcriptomiques du TCGA. L’analyse de l’expression d’HVEM à la surface des cellules de M et de BTLA à la surface des TILs a été réalisée par cytométrie en flux et co-immunoflorescence afin de confirmer l’existence d’interactions HVEM-BTLA in vivo. Enfin, les mécanismes de régulation d’HVEM ont été explorés par des analyses bio-informatiques couplées à des techniques de siRNA.Résultats : Les patients ayant une expression élevée d’HVEM par leur M avaient une survie globale significativement plus réduite que ceux ayant une expression faible d’HVEM à la fois en immunohistochimie (p= 0,0124) et en transcriptomique (p=0,0282).D’un point de vue mécanistique: HVEM exprimé par les M interagissait avec BTLA exprimé par les TILs et l’expression d’HVEM n’était ni liée au statut mutationnel, ni induite par l’interféron gamma . Nous avons également montré que les gènes co-exprimés avec HVEM formaient une signature d’agressivité et que MITF contrôlait l’expression d’HVEM.Conclusion : HVEM ou BTLA pourraient être des cibles thérapeutiques de choix dans le M. / Purpose: Upon engagement with HVEM, BTLA triggers inhibitory signals in T cells. Using melanoma, we correlated HVEM expression with clinical outcomes and confirmed the occurrence of HVEM-BTLA interaction inside melanoma. Moreover, we determined regulatory mechanisms accounting for the complementary pattern of expression we observed for HVEM and PD-L1.Experimental design: HVEM expression levels were analyzed by immunohistochemistry in melanoma metastases and correlated with overall survival (OS). Coincident expression of HVEM and its ligand BTLA was studied in tumor cells and tumor-infiltrated-lymphocytes (TILs) by flow cytometry and co-immunofluorescence. Candidate genes controlling HVEM expression on melanoma were defined by bioinformatics studies and validated by siRNA.Result: Patients with high HVEM expression on melanoma cells had a significantly poorer OS than those with a low expression as documented by immunohistochemistry (p=0.0124) and TCGA (p=0.0282). From a mechanistic point of view, we showed (1) that HVEM expressed at the surface of melanoma cells interacts with BTLA expressed by TILs, (2) that HVEM expression is neither linked to the melanoma mutational status nor inducible by IFN 3) that genes co-expressed with HVEM are associated with an aggressive gene signature, and (4) that MITF controls HVEM expression.Conclusion: In contrast to PD-L1, HVEM expression is constitutive and correlated with the expression of genes involved in aggressive features. Therefore, high HVEM expression on melanoma cells is a pejorative prognostic marker, substantiating the use of checkpoint inhibitors directed at HVEM and BTLA in the treatment of melanoma

Thesis (General Surgery)) -- University of Limpopo, 2012.

Thesis (Ph. D.)--Marshall University, 2003. / Title from document title page. Document formatted into pages; contains xiii, 153 p. including illustrations. Includes abstract. Includes bibliographical references (p. 125-153).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.