Genetically engineered pre-microRNA-34a prodrug suppresses orthotopic osteosarcoma xenograft tumor growth via the induction of apoptosis and cell cycle arrest.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children, and microRNA-34a (miR-34a) replacement therapy represents a new treatment strategy. This study was to define the effectiveness and safety profiles of a novel bioengineered miR-34a prodrug in orthotopic OS xenograft tumor mouse model. Highly purified pre-miR-34a prodrug significantly inhibited the proliferation of human 143B and MG-63 cells in a dose dependent manner and to much greater degrees than controls, which was attributed to induction of apoptosis and G2 cell cycle arrest. Inhibition of OS cell growth and invasion were associated with release of high levels of mature miR-34a from pre-miR-34a prodrug and consequently reduction of protein levels of many miR-34a target genes including SIRT1, BCL2, c-MET, and CDK6. Furthermore, intravenous administration of in vivo-jetPEI formulated miR-34a prodrug significantly reduced OS tumor growth in orthotopic xenograft mouse models. In addition, mouse blood chemistry profiles indicated that therapeutic doses of bioengineered miR-34a prodrug were well tolerated in these animals. The results demonstrated that bioengineered miR-34a prodrug was effective to control OS tumor growth which involved the induction of apoptosis and cell cycle arrest, supporting the development of bioengineered RNAs as a novel class of large molecule therapeutic agents.

Reducing bone cancer cell functions using selenium nanocomposites.

Cancer recurrence at the site of tumor resection remains a major threat to patient survival despite modern cancer therapeutic advances. Osteosarcoma, in particular, is a very aggressive primary bone cancer that commonly recurs after surgical resection, radiation, and chemotherapeutic treatment. The objective of the present in vitro study was to develop a material that could decrease bone cancer cell recurrence while promoting healthy bone cell functions. Selenium is a natural part of our diet which has shown promise for reducing cancer cell functions, inhibiting bacteria, and promoting healthy cells functions, yet, it has not been widely explored for osteosarcoma applications. For this purpose, due to their increased surface area, selenium nanoparticles (SeNP) were precipitated on a very common orthopedic tissue engineering material, poly-l-lactic acid (or PLLA). Selenium-coated PLLA materials were shown to selectively decrease long-term osteosarcoma cell density while promoting healthy, noncancerous, osteoblast functions (for example, up to two times more alkaline phosphatase activity on selenium coated compared to osteoblasts grown on typical tissue culture plates), suggesting they should be further studied for replacing tumorous bone tissue with healthy bone tissue. Importantly, results of this study were achieved without the use of chemotherapeutics or pharmaceutical agents, which have negative side effects.

Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in pediatric human sarcoma cell lines.

Pediatric sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). Our main objective was to study the effect of a nutrient mixture (NM) on activity of u-PA, MMPs and TIMPs in various human pediatric sarcomas. Human osteosarcoma MNNG-HOS, osteosarcoma U-2OS and rhabdomyosarcoma RD cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1,000 µg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. All sarcoma cell lines studied expressed u-PA, which was inhibited by NM in a dose-dependent manner. On gelatinase zymography, osteosarcoma MNNG-HOS showed a band corresponding to MMP-2 and induction of MMP-9 with PMA (100 ng/ml) treatment. U-2OS osteosarcoma cells showed strong bands corresponding to inactive MMP-2 and MMP-9 and faint bands corresponding to active MMP-2 and MMP-9 dimer; PMA treatment enhanced MMP-9 and MMP-9 dimer activity. Rhabdomyosarcoma showed MMP-2 and faint MMP-9 bands; PMA treatment enhanced MMP-9 expression. NM inhibited their expression in a dose-dependent manner. Activity of TIMPs was upregulated by NM in all cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPs. These findings suggest the therapeutic potential of NM in treatment of pediatric sarcomas.

Dietary selenium supplementation modifies breast tumor growth and metastasis.

The survival rate for breast cancer drops dramatically once the disease progresses to the metastatic stage. Selenium (Se) is an essential micronutrient credited with having high anticancer and chemopreventive properties. In our study, we investigated if dietary Se supplementation modified breast cancer development in vivo. Three diets supplemented with sodium selenite, methylseleninic acid (MSA) or selenomethionine (SeMet), as well as a Se-deficient and a Se-adequate diet were fed to mice before mammary gland inoculation of 4T1.2 cells. The primary tumor growth, the numbers of cancer cells present in lungs, hearts, livers, kidneys and femurs and several proinflammatory cytokines were measured. We found that inorganic selenite supplementation provided only short-term delay of tumor growth, whereas the two organic SeMet and MSA supplements provided more potent growth inhibition. These diets also affected cancer metastasis differently. Mice fed selenite developed the most extensive metastasis and had an increased incidence of kidney and bone metastasis. On the other hand, mice fed the SeMet diet showed the least amount of cancer growth at metastatic sites. The MSA diet also provided some protection against breast cancer metastasis although the effects were less significant than those of SeMet. The cytokine profiles indicated that serum levels of interlukin-2, interleukin-6, interferon γ and vascular endothelial growth factor were elevated in SeMet-supplemented mice. There was no significant difference in tumor growth and the patterns of metastasis between the Se-deficient and Se-adequate groups. Our data suggest that organic Se supplementation may reduce/delay breast cancer metastasis, while selenite may exacerbate it.

Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to Apo2L/TRAIL-induced apoptosis.

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.

Acompanhamento do estado nutricional de pacientes com osteosarcoma / Nutritional status follow-up of patients with osteosarcoma

Objetivo: avaliar a evolução do estado nutricional de pacientes com osteosarcoma em tratamento oncológico após um programa de orientação nutricional por via oral. Métodos: a avaliação nutricional foi efetuada por meio do peso, estatura, prega cutânea triciptal (PCT), circunferência braquial (CB) e circunferência muscular do braço (CMB). O diagnóstico nutricional foi baseado nos critérios da OMS, utilizando-se o índice de massa corporal (IMC). 20 pacientes foram acompanhados prospectivamente do 1º ao 4º mês da admissão. Resultados: houve aumento na prevalência de desnutrição com resultados semelhantes observados em relação aos indicadores de PCT, CB. A média da porcentagem de adequação do IMC caiu de 91±14 para 86±13. Conclusões: a conduta nutricional proposta não demonstrou reduzir as taxas globais de desnutrição dessa população. Entretanto, acreditamos que a intervenção, provavelmente, minimizou a magnitude do problema nutricional desse grupo. Concluímos que são necessários mais estudos avaliando outros métodos de terapia nutricional nesses pacientes.

[Cellular polarization]. / Polarización celular.

The principal ideas of Dr. Santiago Ramón y Cajal are commented. 1. Theory of the dynamic polarization. 2. Transmission of electric signals in the neuronal synapsis. 3. The static description is guided by a dynamic concern. 4. In cancer patients without hope, the positive effect of our metabolic treatment is impressive. 5. Two cases are presented that could avoid cardiac transplantation with our treatment. 6. The nice poem of Alfonso Camín, entitled "We went to bury him to a mountain" is presented.