Advanced melanoma poses a major therapeutic challenge, and despite the development of recent promising therapeutic agents, resistance to treatment remains a problem. Until recently, despite low response rates, alkylating agents dacarbazine and temozolomide (TMZ) were the standard of care for the treatment of advanced melanoma. The cytotoxic effects of these agents relies upon the formation of alkylated base lesions such as O⁶-methylguanine (O⁶MeG), which is repaired by a protein implicated in TMZ resistance called O⁶-methylguanine-DNA methyltransferase (MGMT). Failure to resolve such alkylated bases results in DNA replication-associated double-strand breaks (DSBs). The type 1 insulin-like growth factor receptor (IGF-1R) mediates a number of characteristics common to cancers, including proliferation and survival, and evidence suggests it may also contribute to resistance to many anticancer agents. The aims of this study were to test whether melanoma cells could be sensitized to TMZ by small molecule IGF1R inhibitors, and to explore the mechanism of any chemosensitization. This study found an association between basal IGF1R phosphorylation and in vitro TMZ resistance in seven MGMT-proficient melanoma cell lines, suggesting that IGF1R activation may be linked with TMZ resistance. Furthermore, IGF1R inhibition caused dose-dependent sensitization of melanoma cells to TMZ, regardless of BRAF mutation status. This reduction in cell survival was not accompanied by an increase in apoptosis, but rather Chk2 activation and an accumulation of cells in the G2/M phase of the cell cycle, suggesting a possible effect of IGF1R inhibition on DNA repair. IGF1R depletion was found to increase MGMT protein levels and activity, but this effect was not seen in IGF1R inhibited cells. In addition, IGF1R inhibition was not epistatic with MGMT inhibition, and IGF1R inhibition reduced survival of TMZ treated MGMT-null cells. This suggested that TMZ sensitization by IGF1R inhibition was independent of MGMT. IGF1R inhibition did however cause an increase in the accumulation of TMZ-induced RPA foci, and delay in resolution of RAD51 foci. Together with the finding that IGF1R inhibition reduced survival in PARP inhibited melanoma cells, these results suggested that IGF1R inhibition influenced DSB repair by homologous recombination. Finally, the combination of IGF1R inhibition with TMZ was tested in a mouse model and was found to be tolerable. TMZ or IGF-1R inhibitor alone caused minor reduction in melanoma xenograft growth rates (rate reduction by 13% and 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from other treatment groups (p<0.05). The findings of this study suggest IGF1R inhibition as a possible option in overcoming alkylating drug resistance in melanoma.

Insulin and insulin-like growth factor 1 (IGF-1) and 2 (IGF-2) are related protein hormones with different but overlapping biological functions. All the hormones interact with a receptor within the insulin-IGF system (insulin receptor A and B, IGF-1 receptor), however with different affinity. The different interaction with individual receptors is just one of the main tools for regulation of the system that is essential for the proper functioning of the organism. Although the residues directly interacting with receptors are mainly located in A and B domains, the C and D domains probably play a role in receptor specificity. Here, we firstly focused on the impact of D domains of IGF-1 and 2 (D1 and D2 domains) and C domain of IGF- 2 (C2 domain). To probe the impact of C and D domains, we prepared insulin analogues containing a part of or an entire domain following a pattern seen in IGFs. The receptor-binding affinities of these analogues and their receptor autophosphorylation potentials were characterised. Our results revealed that the initial part of D1 domain has a detrimental effect on IR affinity that is only slightly enhanced by the rest of the D1 domain. D2 domain has rather neutral effect on IR affinity. We further showed that the addition of amino acids derived from the C2 domain to the...

OBJETIVO: avaliação da expressão gênicado receptor do fator de crescimento semelhante à insulina de (Insulin-Like Growth Factor- IGF) 1 em células da granulosa luteinizadas do cumulusde mulheres não obesas, com sensibilidade à insulina normal, com síndrome dos ovários policísticos (SOP) tratadas e não tratadas com metformina. MODELO DO ESTUDO: prospectivo, longitudinal, randomizado. PACIENTES E MÉTODOS: avaliamos 12 mulheres com ciclosovulatórios, 9 mulheres com SOP e 8 mulheres com SOP e tratadas com metformina, ao menos 8 semanas na dose de 1.700 mg/dia. Todos os grupos foram similares com relação ao peso, ao índice de massa corporal (IMC), à circunferência da cintura e com sensibilidade à insulina normal. Todas as mulheres foram submetidas à estimulação ovariana controlada com uso de agonista de GnRH em protocolo longo e gonadotrofinas para ciclos de FIV/ICSI. As células da granulosa do cumulusforam obtidas por microdissecção dos cinco maiores folículos pré-ovulatórios. A expressão gênica do receptor de IGF-1 foi determinada com técnica da Reação da Polimerase em Cadeia a partir da Transcrição Reversa (Reverse transcriptase - Polymerase Chain ReactionRT-PCR) emiquantitativa. Foram avaliadas as concentrações séricas e foliculares de estradiol, progesterona, testosterona, hormônio folículo estimulante (Follicle-Stimulating Hormone- FSH), hormônio luteinizante (Luteinizing Hormone - LH), Sex Hormone-Binding Globulin(SHBG), glicose, insulina e IGF-1. Para análise estatística, foram utilizados os testes: ANOVA, Newman-Keuls, coeficiente de Pearsone regressão linear múltipla, sendo considerado nível de significância de 5%. RESULTADOS: não foram observadas diferenças com relação à expressão gênica do receptor de IGF-1 nos três grupos analisados (P>0,05). O número de oócitos (20,4 vs. 13,1 vs.11,5, P= 0,009), os níveis séricos de estradiol (1.896,00 pcg/mL vs. 985,20 pcg/mL vs.908,10 pcg/mL,P = 0,03) e testosterona (1,43 ng/mL vs.0,89 ng/mL vs. 0,82 ng/mL pcg/mL,P = 0,02) foram maiores no grupo de mulheres com SOP não tratadas com metformina em comparação com as mulheres com ciclos ovulatórios e tratadas com metformina, respectivamente. As mulheres com ciclos ovulatórios (50.710±42.520 ng/mL) apresentaram maiores concentrações foliculares de progesterona quando comparados com as mulheres com SOP tratadas (13.660±5.212 ng/mL) e não tratadas com metformina (17.680±6.644 ng/mL) (P=0,01). Na avaliação da regressão múltipla, a testosterona sérica não sofreu influência da expressão gênica do receptor de IGF-1 ou do IMC. CONCLUSÕES: as altas concentrações séricas de estradiol e testosterona, maior número de oócitos no grupo de mulheres com SOP não tratadas com metformina nos levam a concluir que mulheres com SOP provavelmente têm uma maior sensibilidade à estimulação da esteroidogênese ovariana quando comparadas com mulheres sem essa doença, embora não tenhasido encontrada diferença na expressão do receptor de IGF-1 nos trêsgrupos analisados. A similaridade dos resultados deste estudo entre mulheres com SOP tratadas com metformina e com ciclos ovulatórios nos levam a \"hipotetizar\" que um dos possíveis mecanismos de ação da metformina no sistema IGF-1 nas células da granulosa do cumulus poderia ser por mecanismos pós-receptores. / OBJECTIVE: evaluation of the gene expression of the IGF-I receptor in luteinized cumulus granulosa cells from non-obese women with normal insulin sensitivity and with polycystic ovarian syndrome (PCOS)treated or nor with metformin. STUDY MODEL: prospective,longitudinal, randomized. PATIENTS AND METHODS: we evaluated 12 women withovulatory cycles and 9 women with PCOS who had been treated for at least 8 weeks with a metformin dose of 1700 mg/day. All groups were similar interms of weight, body mass index (BMI), and waist circumference and all had normal insulin sensitivity. All women were submitted to controlled ovarian stimulation with a GnRH agonist in a long protocol and with gonadotropins for IVF/ICSI cycles. Cumulus granulosa cells were obtained by microdissection of the five largest pre-ovulatory follicles. Gene expression of the IGF-1 receptor was determined by semiquantitative RT-PCR. Serum and follicular concentrations of estradiol, progesterone, testosterone, FSH, LH, insulin, SHBG, and IGF-1 were determined. Data were analyzed statistically by ANOVA and by the Newman-Keuls test and the Pearson coefficient and linear multiple regression were calculated, with the level of significance set at 5%. RESULTS: no difference in geneexpression of the IGF-I receptor were observed between the three groups studied (P>0.05). The number of oocytes (20.4 vs. 13.1 vs. 11.5, P= 0.009) and the serum levels of estradiol (1,896.00 pcg/mL vs. 985.20 pcg/mL vs.908.10 pcg/mL,P = 0.03) and testosterone (1.43 ng/mL vs.0.89 ng/mL vs. 0.82 ng/mL pcg/mL,P = 0.02) were higher in the group of women with PCOS not treated with metformin than in women with ovulatory cycles and in women treated with metformin, respectively. The women with ovulatory cycles (50.710±42.520 ng/mL) presented higher follicular concentrations of progesterone compared with women with PCOS treated (13.660±5.212 ng/mL) or not with metformin (17.680±6.644 ng/mL) (P=0.01). Multiple regression revealed that serum testosterone was not affected by the gene expression of the IGF-1 receptor or by BMI. CONCLUSIONS: the high serum concentrations of estradiol and testosterone and the larger number of oocytes in the group ofwomen with PCOS not treated with metformin lead us to conclude that women with PCOS probably have greater sensitivity to stimulation ofovarian steroidogenesis than women without the disease, although no difference was detected in the expression of the IGF-I receptor between the three groups studied. The similarity ofthe present results for the women with PCOS treated with metformin and for the women with ovulatory cycles leads us to hypothesize that one of the possible mechanisms of action of metformin on the IGF-1 system in cumulus granulosa cells may be of the post-receptor type.

The complicated problem of efficient stimulation of elastic fiber production in already developed human tissues has not yet been solved. The present study introduces sodium ascorbate (SA) as a stimulator of elastogenesis in cultures of different cell types including fibroblasts isolated from patients with elastopathy genetic diseases. We then elucidated mechanisms of elastogenic action of SA. SA exercises its net elastogenic effect only after being actively transported into the cell interior through two separate mechanisms. These are the “fast effect,” which reflects the greater stability of intracellular tropoelastin, and the “late effect,” which reflects the true enhancement of the elastin gene expression occurring after SA-induced activation of c-src tyrosine kinase and the consecutive phosphorylation of IGF-1 receptor, which triggers the downstream signals leading to activation of the elastin gene expression. In conclusion, for the first time we have established that SA is a potent stimulator of elastic fiber production.

The complicated problem of efficient stimulation of elastic fiber production in already developed human tissues has not yet been solved. The present study introduces sodium ascorbate (SA) as a stimulator of elastogenesis in cultures of different cell types including fibroblasts isolated from patients with elastopathy genetic diseases. We then elucidated mechanisms of elastogenic action of SA. SA exercises its net elastogenic effect only after being actively transported into the cell interior through two separate mechanisms. These are the “fast effect,” which reflects the greater stability of intracellular tropoelastin, and the “late effect,” which reflects the true enhancement of the elastin gene expression occurring after SA-induced activation of c-src tyrosine kinase and the consecutive phosphorylation of IGF-1 receptor, which triggers the downstream signals leading to activation of the elastin gene expression. In conclusion, for the first time we have established that SA is a potent stimulator of elastic fiber production.

OBJETIVO: avaliação da expressão gênicado receptor do fator de crescimento semelhante à insulina de (Insulin-Like Growth Factor- IGF) 1 em células da granulosa luteinizadas do cumulusde mulheres não obesas, com sensibilidade à insulina normal, com síndrome dos ovários policísticos (SOP) tratadas e não tratadas com metformina. MODELO DO ESTUDO: prospectivo, longitudinal, randomizado. PACIENTES E MÉTODOS: avaliamos 12 mulheres com ciclosovulatórios, 9 mulheres com SOP e 8 mulheres com SOP e tratadas com metformina, ao menos 8 semanas na dose de 1.700 mg/dia. Todos os grupos foram similares com relação ao peso, ao índice de massa corporal (IMC), à circunferência da cintura e com sensibilidade à insulina normal. Todas as mulheres foram submetidas à estimulação ovariana controlada com uso de agonista de GnRH em protocolo longo e gonadotrofinas para ciclos de FIV/ICSI. As células da granulosa do cumulusforam obtidas por microdissecção dos cinco maiores folículos pré-ovulatórios. A expressão gênica do receptor de IGF-1 foi determinada com técnica da Reação da Polimerase em Cadeia a partir da Transcrição Reversa (Reverse transcriptase - Polymerase Chain ReactionRT-PCR) emiquantitativa. Foram avaliadas as concentrações séricas e foliculares de estradiol, progesterona, testosterona, hormônio folículo estimulante (Follicle-Stimulating Hormone- FSH), hormônio luteinizante (Luteinizing Hormone - LH), Sex Hormone-Binding Globulin(SHBG), glicose, insulina e IGF-1. Para análise estatística, foram utilizados os testes: ANOVA, Newman-Keuls, coeficiente de Pearsone regressão linear múltipla, sendo considerado nível de significância de 5%. RESULTADOS: não foram observadas diferenças com relação à expressão gênica do receptor de IGF-1 nos três grupos analisados (P>0,05). O número de oócitos (20,4 vs. 13,1 vs.11,5, P= 0,009), os níveis séricos de estradiol (1.896,00 pcg/mL vs. 985,20 pcg/mL vs.908,10 pcg/mL,P = 0,03) e testosterona (1,43 ng/mL vs.0,89 ng/mL vs. 0,82 ng/mL pcg/mL,P = 0,02) foram maiores no grupo de mulheres com SOP não tratadas com metformina em comparação com as mulheres com ciclos ovulatórios e tratadas com metformina, respectivamente. As mulheres com ciclos ovulatórios (50.710±42.520 ng/mL) apresentaram maiores concentrações foliculares de progesterona quando comparados com as mulheres com SOP tratadas (13.660±5.212 ng/mL) e não tratadas com metformina (17.680±6.644 ng/mL) (P=0,01). Na avaliação da regressão múltipla, a testosterona sérica não sofreu influência da expressão gênica do receptor de IGF-1 ou do IMC. CONCLUSÕES: as altas concentrações séricas de estradiol e testosterona, maior número de oócitos no grupo de mulheres com SOP não tratadas com metformina nos levam a concluir que mulheres com SOP provavelmente têm uma maior sensibilidade à estimulação da esteroidogênese ovariana quando comparadas com mulheres sem essa doença, embora não tenhasido encontrada diferença na expressão do receptor de IGF-1 nos trêsgrupos analisados. A similaridade dos resultados deste estudo entre mulheres com SOP tratadas com metformina e com ciclos ovulatórios nos levam a \"hipotetizar\" que um dos possíveis mecanismos de ação da metformina no sistema IGF-1 nas células da granulosa do cumulus poderia ser por mecanismos pós-receptores. / OBJECTIVE: evaluation of the gene expression of the IGF-I receptor in luteinized cumulus granulosa cells from non-obese women with normal insulin sensitivity and with polycystic ovarian syndrome (PCOS)treated or nor with metformin. STUDY MODEL: prospective,longitudinal, randomized. PATIENTS AND METHODS: we evaluated 12 women withovulatory cycles and 9 women with PCOS who had been treated for at least 8 weeks with a metformin dose of 1700 mg/day. All groups were similar interms of weight, body mass index (BMI), and waist circumference and all had normal insulin sensitivity. All women were submitted to controlled ovarian stimulation with a GnRH agonist in a long protocol and with gonadotropins for IVF/ICSI cycles. Cumulus granulosa cells were obtained by microdissection of the five largest pre-ovulatory follicles. Gene expression of the IGF-1 receptor was determined by semiquantitative RT-PCR. Serum and follicular concentrations of estradiol, progesterone, testosterone, FSH, LH, insulin, SHBG, and IGF-1 were determined. Data were analyzed statistically by ANOVA and by the Newman-Keuls test and the Pearson coefficient and linear multiple regression were calculated, with the level of significance set at 5%. RESULTS: no difference in geneexpression of the IGF-I receptor were observed between the three groups studied (P>0.05). The number of oocytes (20.4 vs. 13.1 vs. 11.5, P= 0.009) and the serum levels of estradiol (1,896.00 pcg/mL vs. 985.20 pcg/mL vs.908.10 pcg/mL,P = 0.03) and testosterone (1.43 ng/mL vs.0.89 ng/mL vs. 0.82 ng/mL pcg/mL,P = 0.02) were higher in the group of women with PCOS not treated with metformin than in women with ovulatory cycles and in women treated with metformin, respectively. The women with ovulatory cycles (50.710±42.520 ng/mL) presented higher follicular concentrations of progesterone compared with women with PCOS treated (13.660±5.212 ng/mL) or not with metformin (17.680±6.644 ng/mL) (P=0.01). Multiple regression revealed that serum testosterone was not affected by the gene expression of the IGF-1 receptor or by BMI. CONCLUSIONS: the high serum concentrations of estradiol and testosterone and the larger number of oocytes in the group ofwomen with PCOS not treated with metformin lead us to conclude that women with PCOS probably have greater sensitivity to stimulation ofovarian steroidogenesis than women without the disease, although no difference was detected in the expression of the IGF-I receptor between the three groups studied. The similarity ofthe present results for the women with PCOS treated with metformin and for the women with ovulatory cycles leads us to hypothesize that one of the possible mechanisms of action of metformin on the IGF-1 system in cumulus granulosa cells may be of the post-receptor type.

ABSTRACT Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in Taiwan. The development of hepatocellular carcinoma is a multi-step process associated with alterations in genes expression such as activation of oncogenes and inactivation of tumor suppressor genes. Mutation/deletion of tumor suppressor gene p53 occurs in 40-50% HCC. Moreover, patients with p53 inactivation have significantly shorter survival after surgery. Inactivation of p53 leads to chromosome instability and may alter expression of its downstream target genes including 14-3-3s for cell cycle arrest or PUMA for apoptosis induction. In this thesis study, we employed five human hepatoma cell lines and ten surgical HCC samples containing paired normal and tumor tissues to investigate 14-3-3s and PUMA expression during liver carcinogenesis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that 14-3-3s mRNA expression was detected in well and poorly differentiated hepatoma cells except Mahlavu cells. Western blot analysis further validated such finding that 14-3-3s protein is not detectable in Mahlavu cell. In human surgical HCC tissues, qRT-PCR showed that 14-3-3s mRNA was elevated in 90% of HCC tissues. Western blot analysis indicated that 14-3-3s protein level was increased in 60% of HCC tissues. Finally, immunohistochemical analysis revealed that 14-3-3s was up-regulated in 50% of HCC tissues comparing with their adjacent non-tumor tissues. Together, these results indicated that 14-3-3s expression was up-regulated in HCC. qRT-PCR and western blot analysis indicated that PUMA mRNA and protein levels were decreased in human and rat hepatoma cells. In human surgical HCC tissues, qRT-PCR showed that PUMA mRNA was reduced in 60% of HCC tissues. Western blot analysis indicated that PUMA protein level was decreased in 100 of HCC tissues. Finally, immunohistochemical analysis revealed that PUMA was down-regulated in 70% of HCC tissues comparing with their adjacent non-tumor tissues. Together, these results indicated that PUMA expression was down-regulated in HCC. In the future, large-scale analysis using more HCC samples will be required to delineate the correlation of 14-3-3s/PUMA expression with clinical parameters of HCC.

Insulin-like growth factors 1 and 2 (IGF-1/2) are single-chain peptides exerting homology (in both amino-acid sequence and tertiary structure) to insulin. The main function of these peptides is promoting celular growth, proliferation and differentiation. Both insulin and insulin-like growth factors mediate their function through membrane receptors - insulin receptor (isoforms A and B) and IGF-1 receptor. All these receptors are members of the tyrosinkinase family of receptors and they exert the same subunit and domain composition. The activation of insulin and IGF-1 receptors is tightly associated with activation of two intracellular signaling pathways. The PI3-K/Akt pathway is involved in the glucose transport to the cell, induction of proliferation or inhibition of apoptosis, while the Ras/MAPK pathway is involved mainly in the induction of cell growth and differentiation. Due to the structure similarity in both the ligands and receptors, every ligand can activate different receptors (with different potency) and the signaling pathways associated with these receptors. Thus, the functions of IGFs and insulin, the same as their receptors, are overlapping. The distinct function of the concrete ligand can be distinguished by the different tissue distribution of both isoforms of insulin receptor and...