Aplicação da biotecnologia na obtenção de triterpenos quinonametídeos bioativos utilizando Salacia campestris (Cambess.) Walp. (Celastraceae) como modelo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Antiproliferative effect of Pterogyne nitens on melanoma cells

As part of our program of bioprospecting for novel antitumor drug prototypes, twenty extracts and fractions obtained from Pterogyne nitens Tul. (Fabaceae, Caesalpinioideae) were screened for antiproliferative activity against B16F10 murine melanoma cells, by the MTT colorimetric assay. The strongest activity was found in EtOAc fractions from the flowers (IC50 = 0.35 µg/mL), fruits (IC50 = 0.34 µg/mL), leaves (IC50 = 0.33 µg/mL) and stems (IC50 = 0.29 µg/mL). Analysis by TLC and HPLC-DAD showed the presence of guanidine alkaloids, flavones and flavonols in the bioactive samples. Additionally, a phytochemical study of the EtOAc fraction of the stems afforded quercetin (1) and isoquercitrin (2), two flavonols with antiproliferative activity previously described in the literature. On the basis of these results, it can be concluded that P. nitens inhibits the growth of melanoma cells in vitro. Further investigations will be needed to assess the usefulness of the samples under study for the treatment of neoplasms and to characterize other bioactive compounds. Keywords: antiproliferative; Pterogyne nitens; Caesalpinioideae; melanoma; flavonoids; Fabaceae.

Pyrostegia venusta heptane extract containing saturated aliphatic hydrocarbons induces apoptosis on B16F10-Nex2 melanoma cells and displays antitumor activity in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In Vitro PLK1 Inhibition by BI 2536 Decreases Proliferation and Induces Cell-Cycle Arrest in Melanoma Cells

Melanoma is one of the most treatment-resistant malignancies and regardless of new therapeutic tactics the outcome remains dismal. Polo-like kinase 1 (PLK1) has been shown to be over-expressed in a variety of tumors, becoming an attractive target for cancer management. In the present study we tested the in vitro antitumor activities of BI 2536, a selective inhibitor of PLK1, against two melanoma cell lines. Our results showed that nanomolar concentrations (10-150 nmol/L) of the drug significantly decreased cell proliferation and clonogenicity, promoting cell cycle arrest in G2/M. Targeting the cell cycle offers an attractive potential cancer-treatment option. Herein we show that PLK1 inhibition may be a feasible approach for the impairment of tumor progression and dissemination. This in vitro profile of melanoma cell growth inhibition by PLK1 modulation may be an interesting model to be tested in association with first-line antineoplasic agents in melanomas.

Boron uptake in normal melanocytes and melanoma cells and boron biodistribution study in mice bearing B16F10 melanoma for boron neutron capture therapy

Information on B-10 distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The B-10 concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the B-10 concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of B-10 uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.

Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.

Individualisierte Krebstherapie : präklinischer „proof of concept“ einer mutationsspezifischen Immuntherapie

Da nicht-synonyme tumorspezifische Punktmutationen nur in malignen Geweben vorkommen und das veränderte Proteinprodukt vom Immunsystem als „fremd“ erkannt werden kann, stellen diese einen bisher ungenutzten Pool von Zielstrukturen für die Immuntherapie dar. Menschliche Tumore können individuell bis zu tausenden nicht-synonymer Punktmutationen in ihrem Genom tragen, welche nicht der zentralen Immuntoleranz unterliegen. Ziel der vorliegenden Arbeit war die Hypothese zu untersuchen, dass das Immunsystem in der Lage sein sollte, mutierte Epitope auf Tumorzellen zu erkennen und zu klären, ob auf dieser Basis eine wirksame mRNA (RNA) basierte anti-tumorale Vakzinierung etabliert werden kann. Hierzu wurde von Ugur Sahin und Kollegen, das gesamte Genom des murinen B16-F10 Melanoms sequenziert und bioinformatisch analysiert. Im Rahmen der NGS Sequenzierung wurden mehr als 500 nicht-synonyme Punktmutationen identifiziert, von welchen 50 Mutationen selektiert und durch Sanger Sequenzierung validiert wurden. rnNach der Etablierung des immunologischen Testsysteme war eine Hauptfragestellung dieser Arbeit, die selektierten nicht-synonyme Punktmutationen in einem in vivo Ansatz systematisch auf Antigenität zu testen. Für diese Studien wurden mutierte Sequenzen in einer Länge von 27 Aminosäuren genutzt, in denen die mutierte Aminosäure zentral positioniert war. Durch die Länge der Peptide können prinzipiell alle möglichen MHC Klasse-I und -II Epitope abgedeckt werden, welche die Mutation enthalten. Eine Grundidee des Projektes Ansatzes ist es, einen auf in vitro transkribierter RNA basierten oligotopen Impfstoff zu entwickeln. Daher wurden die Impfungen naiver Mäuse sowohl mit langen Peptiden, als auch in einem unabhängigen Ansatz mit peptidkodierender RNA durchgeführt. Die Immunphänotypisierung der Impfstoff induzierten T-Zellen zeigte, dass insgesamt 16 der 50 (32%) mutierten Sequenzen eine T-Zellreaktivität induzierten. rnDie Verwendung der vorhergesagten Epitope in therapeutischen Vakzinierungsstudien bestätigten die Hypothese das mutierte Neo-Epitope potente Zielstrukturen einer anti-tumoralen Impftherapie darstellen können. So wurde in therapeutischen Tumorstudien gezeigt, dass auf Basis von RNA 9 von 12 bestätigten Epitopen einen anti-tumoralen Effekt zeigte.rnÜberaschenderweise wurde bei einem MHC Klasse-II restringierten mutiertem Epitop (Mut-30) sowohl in einem subkutanen, als auch in einem unabhängigen therapeutischen Lungenmetastasen Modell ein starker anti-tumoraler Effekt auf B16-F10 beobachtet, der dieses Epitop als neues immundominantes Epitop für das B16-F10 Melanom etabliert. Um den immunologischen Mechanismus hinter diesem Effekt näher zu untersuchen wurde in verschieden Experimenten die Rolle von CD4+, CD8+ sowie NK-Zellen zu verschieden Zeitpunkten der Tumorentwicklung untersucht. Die Analyse des Tumorgewebes ergab, eine signifikante erhöhte Frequenz von NK-Zellen in den mit Mut-30 RNA vakzinierten Tieren. Das NK Zellen in der frühen Phase der Therapie eine entscheidende Rolle spielen wurde anhand von Depletionsstudien bestätigt. Daran anschließend wurde gezeigt, dass im fortgeschrittenen Tumorstadium die NK Zellen keinen weiteren relevanten Beitrag zum anti-tumoralen Effekt der RNA Vakzinierung leisten, sondern die Vakzine induzierte adaptive Immunantwort. Durch die Isolierung von Lymphozyten aus dem Tumorgewebe und deren Einsatz als Effektorzellen im IFN-γ ELISPOT wurde nachgewiesen, dass Mut-30 spezifische T-Zellen das Tumorgewebe infiltrieren und dort u.a. IFN-γ sekretieren. Dass diese spezifische IFN-γ Ausschüttung für den beobachteten antitumoralen Effekt eine zentrale Rolle einnimmt wurde unter der Verwendung von IFN-γ -/- K.O. Mäusen bestätigt.rnDas Konzept der individuellen RNA basierten mutationsspezifischen Vakzine sieht vor, nicht nur mit einem mutations-spezifischen Epitop, sondern mit mehreren RNA-kodierten Mutationen Patienten zu impfen um der Entstehung von „escape“-Mutanten entgegenzuwirken. Da es nur Erfahrung mit der Herstellung und Verabreichung von Monotop-RNA gab, also RNA die für ein Epitop kodiert, war eine wichtige Fragestellungen, inwieweit Oligotope, welche die mutierten Sequenzen sequentiell durch Linker verbunden als Fusionsprotein kodieren, Immunantworten induzieren können. Hierzu wurden Pentatope mit variierender Position des einzelnen Epitopes hinsichtlich ihrer in vivo induzierten T-Zellreaktivitäten charakterisiert. Die Experimente zeigten, dass es möglich ist, unabhängig von der Position im Pentatop eine Immunantwort gegen ein Epitop zu induzieren. Des weiteren wurde beobachtet, dass die induzierten T-Zellfrequenzen nach Pentatop Vakzinierung im Vergleich zur Nutzung von Monotopen signifikant gesteigert werden kann.rnZusammenfassend wurde im Rahmen der vorliegenden Arbeit präklinisch erstmalig nachgewiesen, dass nicht-synonyme Mutationen eine numerisch relevante Quelle von Zielstrukturen für die anti-tumorale Immuntherapie darstellen. Überraschenderweise zeigte sich eine dominante Induktion MHC-II restringierter Immunantworten, welche partiell in der Lage waren massive Tumorabstoßungsreaktionen zu induzieren. Im Sinne einer Translation der gewonnenen Erkenntnisse wurde ein RNA basiertes Oligotop-Format etabliert, welches Eingang in die klinische Testung des Konzeptes fand.rn

MMP19 is upregulated during melanoma progression and increases invasion of melanoma cells

During the progression of cutaneous melanomas, matrix metalloproteinases (MMPs) facilitate the tumour cells to traverse the basement membrane and invade the dermis. In this study, we analysed the expression of MMP19 in the course of melanoma progression. Although MMP19 was absent in melanocytes and melanoma cells of early stages of melanoma development, its expression was strongly upregulated in the neighbouring keratinocytes that may facilitate the vertical outgrowth of melanoma cells. In contrast to early stages, MMP19 was upregulated during the vertical growth phase of melanoma and in metastases. The upregulation of MMP19 in melanoma of Clark levels IV and V correlates with that of MMP2 and also simultaneously with ceased expression of E-cadherin. To reveal whether MMP19 facilitates the invasion of melanomas, we examined adhesion and migratory capacity of selected melanoma cell lines. Melanoma cell lines with low expression of MMP19 exhibited increased adhesion to various substrates and lower migration in comparison with the cell line with higher expression of MMP19. Moreover, ectopic expression of MMP19 could restore the migratory capacity of melanoma cells with low endogenous level of MMP19. These results suggest that the increase of MMP19 expression hallmarks the progression of cutaneous melanoma and might augment melanoma growth by promoting the invasion of tumour cells.

Antisense-mediated melanoma inhibitor of apoptosis protein downregulation sensitizes G361 melanoma cells to cisplatin

Malignant melanoma is an aggressive form of skin cancer that is highly resistant to conventional therapies. The melanoma inhibitor of apoptosis protein is a potent inhibitor of apoptosis and is overexpressed in melanoma cells, but undetectable in most normal tissues including melanocytes. We designed 20-mer phosphorothioate antisense oligonucleotides complementary to five putatively single-stranded sites on the melanoma inhibitor of apoptosis protein mRNA and investigated their ability to sensitize G361 melanoma cells to cisplatin. Inhibition of melanoma inhibitor of apoptosis protein mRNA and protein expression were measured by real-time polymerase chain reaction and immunoblotting. Cell viability and apoptosis were quantitated by colorimetric viability assays and by annexin V staining, respectively. Oligonucleotide M706 was identified as the most efficient antisense sequence which downregulated melanoma inhibitor of apoptosis protein mRNA and protein levels in G361 cells by 68 and 78%, respectively. The specificity of target downregulation was confirmed using scrambled sequence control oligonucleotides that only marginally decreased melanoma inhibitor of apoptosis protein expression. Whereas downregulation of melanoma inhibitor of apoptosis protein moderately inhibited cell growth by 26%, in combination with cisplatin, this resulted in a supra-additive effect with almost 57% reduction in G361 cell viability compared with cisplatin alone (17%) (P<0.05). Cell death was mainly due to apoptosis as demonstrated by a 3- to 4-fold increase in annexin V-positive cells and typical morphological changes compared with controls. In summary, we describe a new antisense oligonucleotide that efficiently downregulates melanoma inhibitor of apoptosis protein expression and sensitizes melanoma cells to cisplatin.

Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

Anticancer activity of anandamide in human cutaneous melanoma cells

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8±0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.

IL-2 REGULATION OF IL-24 EXPRESSION LEADS TO GROWTH SUPPRESSION OF MELANOMA CELLS

Melanoma is known to be highly resistant to chemotherapy. Treatment with high dose IL-2 has shown significant clinical benefit in a minority of metastatic melanoma patients and has lead to long term survival in a few cases. However, this treatment is associated with excessive multiorgan toxicities, which severely limits its use. We hypothesize that one mechanism of effective IL-2 therapy is through the direct upregulation of IL-24 production in melanoma tumors and subsequent IL-24 mediated tumor growth suppression. Five melanoma cell lines were treated with high dose recombinant hIL-2 at 1000U/ml. Three of the cell lines (A375, WM1341, WM793) showed statistically significant increases in their levels of IL-24 protein when measured by Western blotting, while the remaining two lines (WM35, MeWo) remained negative for IL-24 message and protein. This increase in IL-24 was abolished by either preincubating with an anti-IL-2 antibody or by blocking the IL-2 receptor directly with antibodies against the receptor chains. We also demonstrated by ELISA that these three cell lines secrete IL-24 protein in higher amounts when stimulated with IL-2 than do untreated cells. These cells were found to contain IL-2R beta and gamma message by RT-PCR and also expressed higher levels of IL-24 when treated with IL-15, which shares the IL-2R beta chain. Thus we propose that IL-2 is signaling through IL-2R beta on some melanoma cells to upregulate IL-24 protein expression. To address the biological function of IL-2 in melanoma cells, five cell lines were treated with IL-2 and cell viability determined. Cell growth was found to be significantly decreased by day 4 in the IL-24 positive cell lines while no effect on growth was seen in WM35 or MeWo. Incubating the cells with anti-IL-24 antibody or transfecting with IL-24 siRNA effectively negated the growth suppression seen with IL-2. These data support our hypothesis that in addition to its immunotherapeutic effects, IL-2 also acts directly on some melanoma tumors and that the IL-24 and IL-2R beta status of a tumor may be useful in predicting patient response to high dose IL-2.

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

Endogenous nitric oxide regulates p53 signaling and DNA damage-induced apoptosis in melanoma cells

Nitric oxide is involved in a multitude of processes including regulation of vascular tone, neurotransmission, immunity, and cancer. Evidence suggests that nitric oxide exhibits anti-apoptotic activity in melanoma cells. Our laboratory showed that tumor expression of inducible nitric oxide synthase correlated strongly with poor survival in stage III and IV melanoma patients, suggesting an antagonistic role for nitric oxide in melanoma response to therapy. Therefore, the hypothesis that endogenously produced nitric oxide antagonizes chemotherapy-induced apoptosis was formed. Using cisplatin as a model for DNA damage in melanoma cell lines, the capacity of nitric oxide to regulate cell growth and apoptotic responses to cisplatin treatment was examined. The depletion of endogenously generated nitric oxide resulted in changes in cell cycle regulation and enhanced cisplatin-induced apoptosis in melanoma cells. Since nitric oxide was shown to be involved in the regulation of p53 stability, conformation and DNA binding activity, whether signaling through wild-type p53 in melanoma cells is regulated by nitric oxide was tested. Cisplatin-induced p53 accumulation and p21Waf1/Cip1/Sdi1 expression in nitric oxide-depleted melanoma cells were found to be strongly suppressed. When p53 binding to the p21Waf1/Cip1/Sdi1 promoter was examined, it was found that nitric oxide depletion significantly reduced the cisplatin-induced formation of p53-DNA complexes. These results suggest that nitric oxide is required for activation of wild-type p53 after DNA damage in melanoma cells. Finally, whether signaling through p53 controls melanoma response to DNA damage was examined. Transfection of a plasmid containing a dominant negative form of mutated p53 inhibited p21 Waf1/Cip1/Sdi1 expression and concomitantly enhanced apoptosis after cisplatin treatment. These data suggest that the induction of wild-type p53 protects melanoma cells against DNA damage via the up-regulation of p21 Waf1/Cip1/Sdi1. Together, these data strongly support the model that endogenous nitric oxide is required for p53 activation and p21Waf1/Cip1/Sdi1 expression after DNA damage, which can enhance melanoma resistance to therapy. Thus, in context of melanoma cells with wild-type p53 , low levels of endogenous constitutively-produced nitric oxide appear to facilitate the activation of p53 in response to DNA damage, thereby allowing for cell cycle arrest via p21Waf1/Cip1/Sdi1 induction, adequate DNA repair, and ultimately enhanced resistance to apoptosis. ^