Ethanolic extract of gabirobeira (Campomanesia adamantium) leaves reduces proliferation of osteosarcoma cells in vitro

Osteosarcoma is the most commonly diagnosed malignant bone tumor in humans, with a higher incidence in children and young people. It is highly aggressive and has a high metastatic potential. Its treatment is based on both chemotherapy and surgical intervention. However, currently used chemotherapeutic agents, such as doxorubicin, have several adverse effects on the patient. Therefore, there is a growing demand for new chemotherapeutic agents that stimulate new researches, such as those involving compounds extracted from plants, such as the gabirobeira. In this study, we aimed to evaluate the cytotoxic effects of ethanolic extract, both crude and ethyl acetate, of gabirobeira leaves on osteosarcoma cells in vitro. Cytotoxicity was evaluated using the Trypan blue exclusion method and the IC50 values were calculated using the tetrazolium reduction method. The ethanolic extract of gabirobeira leaves showed a cytotoxic effect on osteosarcoma cells in vitro. The group treated with the crude extract at 1. 0& 956;L mL-1 concentration for 48 hours showed higher cytotoxicity and the lowest IC50 value for this extract was found in the 24 to 48 hours interval. The ethanolic extract of gabirobeira leaves is cytotoxic for osteosarcoma cells.

A novel strategy combining magnetic particle hyperthermia pulses with enhanced performance binary ferrite carriers for effective in vitro manipulation of primary human osteogenic sarcoma cells.

The present study examines the heating efficiency of a combination of manganese or cobalt ferrites in a binary (Co- or Mn-) ferrite nanoparticle form with magnetite, covered with citric acid to improve biocompatibility. The nanoparticle synthesis is based on the aqueous co-precipitation of proper salts, a facile, low-cost, environmentally friendly and high yield synthetic approach. By detailed structural and magnetic characterisation, the direct influence of structural and magnetic features on magnetic hyperthermia concludes to optimum heating efficiency. At a second stage, best performing magnetic nanoparticles undergo in vitro testing in three cell lines: one cancer cell line and two reference healthy cell lines. Both binary ferrite (MnFe2O4/Fe3O4 and CoFe2O4/Fe3O4) appear to be internalised and well tolerated by the cells while a versatile hyperthermia protocol is attempted in an effort to further improve their in vitro performance. Within this protocol, hyperthermia sequences are split in two runs with an intermediate 48 h time interval cell incubation stage while in each run a variable field mode (single or multiple pulses) is applied. Single-pulse field mode represents a typical hyperthermia application scheme where cells undergo the thermal shock continuously. On the other hand multiple-pulses mode refers to multiple, much shorter in duration AC field changes (field ON/OFFs), at each hyperthermia run, resulting eventually in high heating rate and much more harmful cell treatment. Consequently, we propose a novel series of improved performance heat mediators based on ferrite structures which show maximum efficiency at cancer cells when combined with a versatile multiple-pulse hyperthermia module.

[Histopathological diagnostic concordance in bone and soft tissue sarcomas between two comprehensive cancer centers from eastern and western Europe: a collaborative experience]. / La concordance du diagnostic histopathologique des sarcomes osseux et des tissus mous entre deux centres anticancéreux de l'Europe de l'Est et de l'Ouest : une expérience de collaboration.

INTRODUCTION: This study aims to assess the degree of concordance of histological diagnosis of bone and soft tissue sarcomas between a Comprehensive Cancer Center (CCC) of Eastern Europe - not specialized in this area of pathology - and an important CCC of Western Europe, which is one of the coordinators of a clinical reference network in sarcoma pathology. The goal is to have an overview of the sarcomatous pathology in a region of Eastern Europe and to discover diagnostic discrepancies between the two centers, while determining their cause. MATERIALS AND METHODS: The initial diagnosis was compared with the revised diagnosis on 110 specimens from 88 patients with bone or soft tissue sarcomas from East-European CCC, in a one-year period of time. RESULTS: Complete diagnostic agreement was observed in 55 cases (62.5%), a partial agreement in 23 cases (26.1%) and a major disagreement in 10 cases (11.4%). Major discrepancies of the histological type was observed in only 3 cases (3.4%): one case of discordance benign/malignant and 2 cases of discordance mesenchymal/non mesenchymal. Minor histological discrepancies - not affecting the management of the patient - were observed in 18 cases (20.4%). A major discordance in grading - potentially changing the management of the patient - was noted in 7 cases (7.9%), and a minor discrepancy in 5 cases (5.7%). DISCUSSIONS: Some histological types were clearly overdiagnosed, like "adult fibrosarcomas" and "malignant peripheral nerve sheet tumors" (MPNST), mostly converted after the audit into "undifferentiated spindle cell sarcomas" or other types of sarcomas. Some "unclassified" sarcomas and "undifferentiated pleomorphic sarcomas" could be re-classified with the aid of an extensive panel of antibodies. Overall, immunohistochemistry was responsible, but not in exclusivity, for half of the minor discrepancies, and for 2 out of 3 cases of major histological discrepancies. Otherwise, the main cause of discrepancies was the difficulties in the interpretation of the morphology. Molecular biology was decisive in one case. Most grading discrepancies resulted from the appreciation of the mitotic index. CONCLUSIONS: The profile of the sarcomatous pathology in the northwest region of Romania does not appear to differ significantly from other parts of Europe or the world, but a prospective epidemiological study would be necessary to confirm this assessment. The expansion of immunohistochemical antibody panel, the over-specialization of pathologists and, in the future, the establishment of a national network of referral centers in sarcoma pathology, are required for a high level of histological diagnosis in Eastern Europe. A periodic external audit, continuing this trans-European collaboration between the two centers, would be beneficial for monitoring progress.

[Pathological assessment of bone sarcomas]. / Pathologische Begutachtung von Knochensarkomen.

Bone tumors are very rare. Diagnosis and treatment is an interdisciplinary task for experienced radiologists, pathologist, and surgeons that is ideally performed in specialized centers. For optimal processing of bone specimens, basic laboratory equipment and special techniques are required. The cornerstone of the histological diagnosis remains H&E staining, supplemented by special stains, immunohistochemistry, and molecular techniques. For an appropriate diagnosis, data on clinical history, age, location, topography within bone, and imaging are required. Major differences between histological and radiological diagnosis have to be clarified before starting treatment (e.g., by involving a reference registry).

Induction of cell cycle arrest and apoptosis in human osteosarcoma U-2 OS cells by Solanum lyratum extracts.

This research focused on a Chinese herb medicine, Solanum lyratum Thunb (Solanaceae) by ethanol extracts (SLE) for investigating the molecular anticancer mechanism in vitro for exploring the means of cell death through the effects on mitochondrial function. We found that SLE induced cytotoxic effects in human osteosacroma U-2 OS cells, and these effects include cell morphological changes, a decrease of the percentage of viable cells and induction of apoptosis. The results suggest that cell death induced by SLE is closely related to apoptosis based on the observations of DAPI staining and sub-G1 phase in U-2 OS cells. Flow cytometric assays also showed that SLE promoted the production of reactive oxygen species and nitric oxide but decreased the levels of mitochondrial membrane potential and promoted the activations of caspase-8 and -9 in U-2 OS cells. SLE inhibited the level of Bcl-2 but promoted the Bax level, and both proteins led to the release of cytochrome c from mitochondria to cytosol and activation of caspase-9 and -3, resulting in the apoptotic death which is mediated through the mitochondrial pathway. Taken together, SLE was demonstrated to be effective in killing U-2 OS osteosacroma cells via the ROS-promoted and mitochondria- and caspase-dependent apoptotic pathways.

Different expression of CD146 in human normal and osteosarcoma cell lines.

The CD146 cell membrane adhesion molecule is highly expressed on the cell surface of several tumours. The level of its expression has been found to correlate directly with tumour progression and metastatic potential, thus establishing CD146 as an important candidate of tumour growth and metastasis. In order to characterize its expression in human osteosarcoma (OS) cell lines, we have examined the CD146 expression at protein and RNA levels in both normal and tumour osteoblast-like cell lines by several methods. Our results indicate that CD146 protein is expressed at low levels in normal osteoblast cells whereas it is highly expressed in all OS cell lines analysed, (SaOS, MG-63, U-2OS). Moreover, CD146 overexpression was partially reduced in shYY1 cells, where the Yin Yang 1 transcription factor, also found over-expressed in human OS cells, has been silenced.

Synergistic increase in osteosarcoma cell sensitivity to photodynamic therapy with aminolevulinic acid hexyl ester in the presence of hyperthermia.

OBJECTIVE: We observed that two osteosarcoma cell lines from the same tumor displayed marked differences in their sensitivities to photodynamic therapy (PDT) with aminolevulinic acid hexyl ester (hALA-PDT). We investigated why these two closely related lines had different hALA-PDT sensitivities and whether the PDT phototoxicity of the less sensitive cell line could be increased by a simultaneous application of hyperthermia (HT). METHODS: Flow cytometry was used to evaluate the intracellular accumulation of protoporphyrin IX (PpIX), a metabolic product of aminolevulinic acid, in two human mandibular osteosarcoma cell lines (HOSM-1 and HOSM-2) treated with HT, hALA-PDT, or hALA-PDT combined with HT (PDT + HT). With hALA-PDT, cells treated with 0.2 mM hALA were irradiated with a light dose of 10-80 J/cm(2) from a near-infrared irradiator. With PDT + HT, the cells were treated as for hALA-PDT except that the temperature was raised to 43.5 degrees C during irradiation. RESULTS: At 6 h after hALA treatment, HOSM-2 cells carried about 1.53-fold more PpIX than HOSM-1 cells. With hALA-PDT, the survival rate for HOSM-1 cells treated with 80 J/cm(2) irradiation was 35.7%, while that for HOSM-2 cells treated with 40-80 J/cm(2) was below 12%. With PDT + HT, the survival rate for HOSM-1 and HOSM-2 cells treated with 80 J/cm(2) irradiation was 14.1% and 10.7%, respectively. CONCLUSION: A combination therapy comprising hALA-PDT + HT treatment may be very useful for the treatment of tumors containing cells that are insensitive to hALA-PDT, such as the HOSM-1 cell line described in this study.

Identification of prognostic markers in bone sarcomas using proton-decoupled phosphorus magnetic resonance spectroscopy.

It has been hypothesized that the (31)Phosphorus ((31)P) nuclear magnetic resonance spectrum from certain tumors may provide prognostic information. The goal of the present study was to identify prognostic metabolic markers by using proton-decoupled phosphorus magnetic resonance spectroscopic imaging ((31)P MRSI). Twenty patients with bone [osteogenic (OS) and Ewing's (ES) and/or primitive neuroectodermal tumor (PNET)] sarcoma, treated with chemotherapy and surgery or with chemotherapy alone, underwent (31)P MRSI studies pre- and post-therapy. The studies were performed on a 1.5 Tesla General Electric (GE) clinical scanner equipped with a stand-alone proton decoupler and a dual (1)H/(31)P surface coil pair. The limited sensitivity of the (31)P nucleus required that a large soft tissue component of the disease be located within 10 cm (maximum distance) of the body surface and the use of a highly sensitive coil placed near the skin surface. Proton decoupling and nuclear Overhauser enhancement were used to improve the spectral resolution and signal:noise ratio. Baseline (31)P spectral features and metabolic changes with treatment were compared with treatment outcome. The patients were categorized depending on survival as event-free survivors or those who died. The pretreatment nucleoside triphosphate:inorganic phosphate (NTP:P(i)) ratio, an index of tumor bioenergetic status, was significant (P = 0.003) in differentiating event-free survivors versus those who died. The pretreatment NTP:P(i) was higher in patients who were destined to undergo a durable event-free survival compared with those who died. The results are promising, although a prospective study is necessary for confirmation. (31)P MRSI appears to be a useful tool for the prediction of survival before therapy in bone sarcomas.

Vitamin K2 regulation of bone homeostasis is mediated by the steroid and xenobiotic receptor SXR.

Vitamin K2 is a critical nutrient required for blood clotting that also plays an important role in bone formation. Vitamin K2 supplementation up-regulates the expression of bone markers, increases bone density in vivo, and is used clinically in the management of osteoporosis. The mechanism of vitamin K2 action in bone formation was thought to involve its normal role as an essential cofactor for gamma-carboxylation of bone matrix proteins. However, there is evidence that suggests vitamin K2 also has a transcriptional regulatory function. Vitamin K2 bound to and activated the orphan nuclear receptor SXR and induced expression of the SXR target gene, CYP3A4, identifying it as a bona fide SXR ligand. Vitamin K2 treatment of osteosarcoma cells increased mRNA levels for the osteoblast markers bone alkaline phosphatase, osteoprotegerin, osteopontin, and matrix Gla protein. The known SXR activators rifampicin and hyperforin induced this panel of bone markers to an extent similar to vitamin K2. Vitamin K2 was able to induce bone markers in primary osteocytes isolated from wild-type murine calvaria but not in cells isolated from mice deficient in the SXR ortholog PXR. We infer that vitamin K2 is a transcriptional regulator of bone-specific genes that acts through SXR to favor the expression of osteoblastic markers. Thus, SXR has a novel role as a mediator of bone homeostasis in addition to its role as a xenobiotic sensor. An important implication of this work is that a subset of SXR activators may function as effective therapeutic agents for the management of osteoporosis.

Molecular characterization of gene expression changes in ROS 17/2.8 cells cultured in diffusion chambers in vivo.

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3-10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3-28 days and by 56 days was 4-5 fold above control levels. In 3-10 days, c-jun expression increased 1.6-1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10-56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to

[Prostaglandins of series E in morphologic variants of osteogenic sarcoma and evaluation of the degree of therapeutic pathomorphism of the tumor]. / Prostaglandiny serii E v razlichnykh morfologicheskikh variantakh osteogennoi sarkomy i otsenke stepeni lechebnogo patomorfoza opukholi.

The content of prostaglandins E (PGE) is studied in osteogenic sarcomas from 191 patients. The level of PGE after a neo-adjuvant therapy of osteogenic sarcoma depends upon the individual susceptibility to the drug. Inverse correlation is found between the PGE content in the tumor and the degree of therapeutic pathomorphosis when adriamycin and methotrexate are used before the operation. Single transfusion of the allogenic bone-marrow suspension results in a considerable decrease of the PGE level in the osteogenic sarcoma. Possible mechanisms of action of different therapeutical methods on the PGE level as well as use of the drugs influencing arachidonic acid metabolism in the tumor are discussed.

Bone formation in vitro and in nude mice by human osteosarcoma cells.

Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.