Resistance to various chemotherapeutic agents is a major problem in success of cancer chemotherapy. One of the primary reasons of development of multidrug resistance (MDR) is the overexpression of ATP binding cassette (ABC) transporter proteins. Breast cancer resistance protein (BCRP) belongs to ABC transporter family and encoded by ABCG2 gene. BCRP is mainly expressed in MDR1 (P-glycoprotein) lacking breast cancer cells. Overexpression of BCRP leads to efflux of chemotherapeutic agents at higher rates, therefore, decreased levels of intracellular drug accumulation. Despite the fact that several chemical modulators claim to restore BCRP-mediated increased drug efflux, these modulators were shown to display various side effects, precluding their clinical use. Therefore, to reverse BCRPmediated MDR phenotype by a modulator with minimum cytotoxicity may increase clinical benefits and minimize side effects.

No description available.

OBJECTIVE: To determine the effect of clinically used zoledronate (ZOL) and docetaxel on breast cancer cells and bone biology under both bone remodeling stages and the rate of tumor dissemination and state of dormancy. METHODS: The effect of clinically used zoledronate (ZOL) was examined on MDA-MB-231 and MDA-BO cells in a roller tube system under bone resorption and formation conditions. Three groups; calvaria alone, calvarial co-cultured with tumor cells, and calvaria with tumor cells treated with four repeat doses of 2 µM of ZOL were cultured for 8, 14 and 20 days. The formation groups were supplemented with 150 µg/ml ascorbic acid. Cell counts were performed on trypsinized calvaria harvested at 2, 8 and 14 days. Media was changed every 2 days and the changed media was re-seeded in a 24-well for 20 days. To test the impact of chemotherapy agents on cancer-bone metastasis the effect of 10 µM of docetaxel was tested on breast cancer cells under formation and resorption conditions using the above design. Tumor burden was assessed at 8 days. RESULTS: Tumor burden: no statistically significant difference between ZOL treated and untreated groups under resorption and formation conditions in both cell lines. Exposure to docetaxel revealed that ~30% of the cells were affected by chemotherapy in formation model, while ~70% was affected in resorption model in both cell types. Dissemination model: the dissemination rate for MDA and BO cells under formation condition is significantly less than for resorption conditions. Fluorescent microscopy: MDA and BO tumor-calvaria were treated with Ki 67 antibody showed that under bone resorption conditions the cancer-bone cells colony were predominantly in proliferation stage while under formation conditions cancer cells were in dormancy. Confocal microscopy: observation confirmed the relation of the mode of cancer cell attachment to bone endosteal cell layer with the dormancy and cell proliferation states. CONCLUSIONS: Both cancer cell lines showed resistance to ZOL under formation and resorption conditions. Drug resistance to docetaxel was more evident under formation condition, where cells are dormant and not proliferating. The dissemination rate is significantly higher in resorption condition, suggesting that cells in formation are dormant with lower dissemination rate. / 2019-07-23T00:00:00Z

Orientador: Karina Gottardello Zecchin / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-19T22:07:17Z (GMT). No. of bitstreams: 1 Gomes_CamillaBorgesFerreira_M.pdf: 2172456 bytes, checksum: 5dd9bb49385077ce4213db57e518b145 (MD5) Previous issue date: 2012 / Resumo: O comprometimento ósseo em pacientes com câncer é responsável por significativas morbidade e redução da qualidade de vida, principalmente quando se trata de câncer de mama, no qual o esqueleto é o principal sítio de desenvolvimento das metástases. Bisfosfonatos (BPs) contendo ou não nitrogênio são os principais agentes usados no combate à perda óssea como na osteoporose, uma vez que induzem apoptose de osteoclastos. Recentemente foram demonstradas ações antitumoral e antiangiogência dos BPs, in vitro e in vivo. Desse modo, o objetivo deste trabalho foi analisar e comparar os efeitos antitumorais e antiangiogênicos de BP que não contêm nitrogênio (clodronato, CLO) e BP contendo nitrogênio (ácido zoledrônico, ZOL), em linhagem celular derivada de câncer de mama, MCF-7, e em culturas de células endoteliais. Os resultados mostram que ZOL reduziu a proliferação das MCF-7 de maneira dose-dependente, com aumento do número de células nas fases G0/G1, sem alterar a viabilidade. Paralemamente, CLO não mostrou nenhum efeito sobre as células tumorais. O tratamento de células endoteliais de coelho, RAEC, com ZOL ou CLO reduziu a proliferação celular derivada, sem alterar sua viabilidade. Células endoteliais humanas HUVEC mostraram 15% de redução da viabilidade quando tratadas com ZOL, enquanto CLO não mostrou efeitos citotóxicos. A incubação de células HUVEC com meio condicionado por células MCF-7 tratadas com ZOL culminou na redução da proliferação das células endoteliais e na redução da formação de estruturas semelhantes a vasos, in vitro. Esse mesmo meio condicionado pelas células tumorais tratadas com ZOL resultou em menor proliferação de células provenientes de aorta murina, em ensaio ex vivo. Ensaios de imunoabsorbância revelaram diminuição nos níveis de VEGFA total nesse meio condicionado, assim como aumento da quantidade da isoforma antiangiogênica VEGFA165b, após tratamento das MCF-7 com ZOL. Por último, o tratamento das células MCF-7 com ZOL modulou a expressão de VEGFA total e de suas isoformas. Em conjunto, estes achados mostram que ZOL exibe ação antitumoral e antiangiogênica em células de câncer de mama e em células endoteliais. Tais dados contribuem para um maior esclarecimento sobre os mecanismos de ação dos BPs, permitindo buscas por melhorias no uso desses agentes como coadjuvantes na quimioterapia, a fim de minimizar a morbidade decorrente de metástases ósseas em tumores malignos / Abstract: Patients with cancer frequently develop bone metastasis, resulting in considerable morbidity and affecting quality of life. This is particularly important considering breast cancer, in which skeletal is the main site for metastasis. Non-nitrogen-containing and nitrogen-containing bisphosphonates (BPs) are the mainly agents to treat bone loss due to osteoporosis, Paget disease, bone metastasis, multiple myeloma, hypercalcemia and osteogenesis imperfecta. BPs impair bone loss by inducing apoptosis in osteoclasts. Recently it was shown anti-tumoral and anti-angiogenic effects of BPs, in vitro and in vivo. The present study aim to analyze and compare the anti-angiogenic pathway of the non-nitrogen-containing BP, clodronate (CLO), and the nitrogen-containing BP, zoledronic acid (ZOL), using estrogen receptor-positive breast cancer cell line, MCF-7, and endothelial cells. ZOL treatment reduced MCF-7 cell proliferation and induced cell cycle arrest in a dose-dependent manner, while CLO did not affect these cells. Treatment with ZOL or CLO also reduced RAEC cells proliferation, without changes in cell viability. HUVEC cells showed reduced viability after incubation with ZOL, but not with CLO. The conditioned medium by MCF-7 ZOL-treated cells reduced the formation of blood vessels in vitro and proliferation of HUVEC cells, together with lower endothelial cells proliferation derived from mouse aortic rings. Enzyme-linked immunoabsorbent assay showed reduction of ~25% in VEGFA levels, and increased amount of VEGFA165b isoform in the conditioned medium by ZOL-treated MCF-7 cells. The nitrogen-containing BP also modulated the expression of mRNAs for VEGF in MCF-7 treated cells. In all assays, CLO showed less anti-angiogenic properties when compared with ZOL. Data provided from this study amplify the knowledge of BPs actions, contributing for new approaches of bone metastases in malignant tumors / Mestrado / Estomatologia / Mestre em Estomatopatologia

Background: Osteoporosis has become a major problem worldwide as the population ages. An osteoporotic fracture is associated with a high rate of morbidity and mortality. Data on the prevalence, risk factors and outcome of osteoporotic fractures in South Africa remains sparse. Method: A retrospective audit was undertaken in all patients attending the Endocrine Clinic at Groote Schuur Hospital between March 2019 and March 2020 for the treatment of osteoporosis. Patients folders were reviewed to obtain the following information: demographic data, risk factors, laboratory investigations, treatment, baseline and follow up DEXA scans. Results: 264 patients were evaluated, average age 65.7 ± 12.3 years, 92.8% (n=245) were female. Common risk factors included smoking (50.8%, n=134), vitamin D deficiency (29.2%, n=77), steroid use (21.6%, n=57) and primary hyperparathyroidism (15.2%, n=40). A fragility fracture was diagnosed in 68.6% (n=181) - vertebral only (54.7%, n=99), hip only (14.9%, n=27), vertebral and hip (10.5%, n=19), wrist (7.2%, n=13) and other (12.7%, n=23). Bisphosphonates were used by 75% (n=198) of patients at the time of enrolment. Of these, 80.8% (n=160) received intravenous zoledonic acid alone, 6.1% (n=12) received oral alendronate alone and 13.1% (n=26) initially received alendronate followed by intravenous zoledronic acid. Over 5.2 years there was an improvement in bone mineral density (BMD) of 4.4% at the lumbar spine, while there was slight worsening of BMD at the femoral neck (- 0.17%). A fracture whilst on treatment occurred in 10.6% (n=21) of patients. Conclusion: The majority of patients with osteoporosis at Groote Schuur Hospital had a fragility fracture at diagnosis with a vertebral fracture being most common. Bisphosphonate treatment showed a measurable improvement in BMD at the lumbar spine, however, there was no improvement at the femoral neck. Despite this, few patients had a symptomatic vertebral or hip fracture whilst on treatment.

The availability of a once-a-year zoledronic acid infusion heralds a new era in the management of osteoporosis. It virtually eliminates the problem of poor compliance with orally administered bisphosphonates and, because it bypasses the gastrointestinal tract, it is not associated with gastrointestinal side effects. Zoledronic acid is effective for the treatment and prevention of postmenopausal osteoporosis, and for the treatment of osteoporosis in men, and glucocorticoid- induced osteoporosis. When administered within three months of a hip fracture, it reduces mortality and the risk of subsequent fractures. It is remarkably free of serious adverse effects. After administration of the intravenous infusion, about 18% of bisphosphonate-naïve patients experience an acute-phase reaction, including low-grade temperature, aches, and pains. This is reduced to about 9% in those who have been treated with oral bisphosphonates, and is further reduced by the concomitant and subsequent administration of acetaminophen. The likelihood and magnitude of the acute-phase reaction is less after the second infusion. Other adverse effects are similar to those encountered with other bisphosphonates. Because it is mostly excreted by the kidneys, zoledronic acid should not be administered to patients with a creatinine clearance less than 35 mL/min. It should not be administered to patients with hypocalcemia.

O objetivo do presente trabalho foi avaliar a microarquitetura, caracterizando a rede de canais e o sistema lacunocanalicular, e também, as propriedades biomecânicas, do osso cortical de ratos Wistar tratados com Ácido Zoledrônico. Vinte e um ratos machos foram divididos da seguinte forma: 12 animais alocados no grupo de animais submetidos ao experimento no quarto mês de idade e 9 alocados no grupo de animais submetidos ao experimento no nono mês de idade. Dentro destes grupos, os mesmos foram divididos em grupos controle e teste, sendo que no grupo teste os animais receberam três doses, no esquema de 7,5 g/kg, semanalmente. Após o uso da medicação, três animais foram eutanasiados nos respectivos períodos, após a última dose: 14 dias, 42 dias e 75 dias. Todos os animais tiveram suas mandíbulas, tíbias e fêmures dissecados. As tíbias e mandíbulas foram descalcificadas e processadas para inclusão em parafina, enquanto que, os fêmures foram armazenados para os testes biomecânicos. Imagens histológicas digitalizadas, provenientes das mandíbulas e tíbias, foram analisadas utilizando o processo de segmentação interativa, acessando as características da microarquitetura da rede de canais. O processo de reconstrução tridimensional, para caracterização do sistema lacunocanalicular, foi realizado nos cortes seriados proveniente das tíbias. Os fêmures foram analisados com os ensaios de microdureza, compressão e flexão em três pontos. Os resultados revelaram que a microarquitetura da rede de canais não foi alterada nas mandíbulas, no entanto, nas tíbias, houve diferença significante apenas nos animais de 4 meses de idade, nos quesitos área dos canais (p 0.0031), desvio-padrão da área (p 0.0338), perímetro dos canais (p 0.0068), desvio padrão do perímetro (p 0.0297) e porcentagem de matriz (p 0.0235). Os testes biomecânicos revelaram resultados que indicavam uma melhora nas características analisadas, que foi correlacionada apenas com o aumento da idade e não com o uso do ácido zoledrônico. A visualização tridimensional possibilitou analisar a distribuição dos osteócitos e dos canais, caracterizando o sistema lacunocanalicular, e constatou-se que nos animais de 4 meses o uso da droga causou uma descontinuidade na estabilidade da distribuição dos osteócitos. Conclui-se que o ácido zoledrônico não interferiu na microarquitetura da rede de canais do osso cortical das mandíbulas, enquanto que nas tíbias, nos animais jovens, houve uma modificação na microarquitetura e no sistema lacunocanalicular, revelando uma topologia mais heterogênea quando comparadas ao grupo controle. As propriedades biomecânicas não sofreram alterações quanto ao uso da droga, sendo evidenciada melhora nos parâmetros analisados relacionada apenas ao aumento de idade nos animais. / The aim of this study was to evaluate the microstructure, characterizing the bone channels network and the lacunocanalicular system, also, the biomechanical properties of the cortical bone of Wistar rats treated with Zoledronic Acid. Twenty-one male rats were separated as follows: 4-month-old (12 animals) and 9 month-old (9 animals). For each age they were divided in control and test group, and the test group received a 7.5 g/kg dose of the drug, once a week, for 3 weeks. In the 4 month-old test group they were euthanized at 14, 42 and 75 days after the drug injection and in the 9-month-old test group they were sacrificed at 14 and 75 days after medication. All animals had their mandible, tibia and femur dissected, and then, the mandible and tibia were decalcified and embedded in paraffin, and, the femur were stored for mechanical tests. Histological images were analyzed through developed algorithms using the SCILAB mathematical environment, getting percentage of bone matrix, ROI areas, ROI perimeters, their standard deviations and Lacunarity, as well, the images were accessed and processed to tridimensional visualization, to characterize the lacunocanalicular system. The femurs were analyzed by microhardness, three point bending and compression testing. The results revealed that the bone channels network in the mandible was not affected by the drug, whereas the tibia in the 4-month-old animals revealed a statistical difference between control and test groups, with the test group revealing more heterogeneity in the channels topography, as seen in results of bone matrix (p=0.0235), ROI area (p=0.0031), ROI area standard-deviation (p=0.0338), ROI perimeter (p=0.0068) and ROI perimeter standard-deviation (p=0.0297). The biomechanical tests showed an increased in strength, and that this event was age-related and not depending on the use of the drug. The tridimensional visualization allowed to analyze the osteocyte distribution in relation to the channels position, characterizing the lacunocanalicular system, and, it was found that the 4-month-old animals the drug caused a discontinuity in the stability of osteocyte distribution. In conclusion, the zoledronic acid not interfere in the bone channels network in the cortical bone of the mandible, however, in the tibia of the young animals, theres been an alteration in bone channels network and in the lacunocanalicular system, revealing more heterogeneity in the topography in these animals. The biomechanical properties were not altered after the use of the drugs, and the increased in strength being age-related.

A terapia com bisfosfonatos tem sido frequentemente empregada no tratamento de doenças metabólicas do osso e neoplasias malignas. O objetivo deste estudo foi avaliar através de análise histológica e histomorfométrica em cortes não descalcificados, a influência dos bisfosfonatos nitrogenados endovenosos associados ou não a dexametasona sobre a osseointegração de implantes instalados em tíbias de 27 ratos Wistar. Ácido zoledrônico e dexametasona foram administrados por via subcutânea nos animais dos grupos experimentais. Os animais foram acompanhados por 7, 14 e 28 dias. Nossos resultados mostraram que não houve falha na osseointegração em nenhum animal avaliado e que não foram observadas diferenças estatisticamente significantes entre os 3 grupos com relação à quantidade de contato entre osso e implante e presença de osso em áreas pré determinadas, nos tempos observados. No entanto nossas observações histológicas revelaram que nos animais tratados com bisfosfonatos associados ou não com dexametasona, aos 14 e 28 dias após a colocação do implante não ocorreu o fenômeno de remodelação da cortical óssea, ao contrário do grupo controle. Concluímos que a terapia com bisfosfonatos associada ou não com dexametasona não impediu a osseointegração do implante com o osso mas inibiu severamente a remodelação da cortical óssea pré existente. / Bisphosphonate therapy has been often employed in the treatment of metabolic bone diseases and malignancies. The aim of this study was to evaluate through histological and histomorphometric analysis the influence of intravenous nitrogen-containing bisphosphonates alone or combined with dexamethasone on the osseointegration of implants placed in the tibia of 27 male Wistar rats in non decalcified samples. Zoledronic acid and dexamethasone were administered through sub cutaneous injections. The animals were followed through 7, 14, and 28 days. Our results showed that there was no failure in osseointegration in any animal, and that there were no statistically significant differences among the 3 groups regarding the amount of bone-implant contact and peri-implant bone density in predetermined areas. However our histological observations revealed that animals treated with bisphosphonates, associated or not with dexamethasone, at 14 and 28 days after implant placement has not occurred the phenomenon of cortical bone remodeling, unlike control group. We conclude that bisphosphonate therapy associated or not with dexamethasone did not prevent osseointegration of the implants but severely inhibited the remodeling of pre-existing cortical bone.

A terapia com bisfosfonatos tem sido frequentemente empregada no tratamento de doenças metabólicas do osso e neoplasias malignas. O objetivo deste estudo foi avaliar através de análise histológica e histomorfométrica em cortes não descalcificados, a influência dos bisfosfonatos nitrogenados endovenosos associados ou não a dexametasona sobre a osseointegração de implantes instalados em tíbias de 27 ratos Wistar. Ácido zoledrônico e dexametasona foram administrados por via subcutânea nos animais dos grupos experimentais. Os animais foram acompanhados por 7, 14 e 28 dias. Nossos resultados mostraram que não houve falha na osseointegração em nenhum animal avaliado e que não foram observadas diferenças estatisticamente significantes entre os 3 grupos com relação à quantidade de contato entre osso e implante e presença de osso em áreas pré determinadas, nos tempos observados. No entanto nossas observações histológicas revelaram que nos animais tratados com bisfosfonatos associados ou não com dexametasona, aos 14 e 28 dias após a colocação do implante não ocorreu o fenômeno de remodelação da cortical óssea, ao contrário do grupo controle. Concluímos que a terapia com bisfosfonatos associada ou não com dexametasona não impediu a osseointegração do implante com o osso mas inibiu severamente a remodelação da cortical óssea pré existente. / Bisphosphonate therapy has been often employed in the treatment of metabolic bone diseases and malignancies. The aim of this study was to evaluate through histological and histomorphometric analysis the influence of intravenous nitrogen-containing bisphosphonates alone or combined with dexamethasone on the osseointegration of implants placed in the tibia of 27 male Wistar rats in non decalcified samples. Zoledronic acid and dexamethasone were administered through sub cutaneous injections. The animals were followed through 7, 14, and 28 days. Our results showed that there was no failure in osseointegration in any animal, and that there were no statistically significant differences among the 3 groups regarding the amount of bone-implant contact and peri-implant bone density in predetermined areas. However our histological observations revealed that animals treated with bisphosphonates, associated or not with dexamethasone, at 14 and 28 days after implant placement has not occurred the phenomenon of cortical bone remodeling, unlike control group. We conclude that bisphosphonate therapy associated or not with dexamethasone did not prevent osseointegration of the implants but severely inhibited the remodeling of pre-existing cortical bone.

O objetivo do presente trabalho foi avaliar a microarquitetura, caracterizando a rede de canais e o sistema lacunocanalicular, e também, as propriedades biomecânicas, do osso cortical de ratos Wistar tratados com Ácido Zoledrônico. Vinte e um ratos machos foram divididos da seguinte forma: 12 animais alocados no grupo de animais submetidos ao experimento no quarto mês de idade e 9 alocados no grupo de animais submetidos ao experimento no nono mês de idade. Dentro destes grupos, os mesmos foram divididos em grupos controle e teste, sendo que no grupo teste os animais receberam três doses, no esquema de 7,5 g/kg, semanalmente. Após o uso da medicação, três animais foram eutanasiados nos respectivos períodos, após a última dose: 14 dias, 42 dias e 75 dias. Todos os animais tiveram suas mandíbulas, tíbias e fêmures dissecados. As tíbias e mandíbulas foram descalcificadas e processadas para inclusão em parafina, enquanto que, os fêmures foram armazenados para os testes biomecânicos. Imagens histológicas digitalizadas, provenientes das mandíbulas e tíbias, foram analisadas utilizando o processo de segmentação interativa, acessando as características da microarquitetura da rede de canais. O processo de reconstrução tridimensional, para caracterização do sistema lacunocanalicular, foi realizado nos cortes seriados proveniente das tíbias. Os fêmures foram analisados com os ensaios de microdureza, compressão e flexão em três pontos. Os resultados revelaram que a microarquitetura da rede de canais não foi alterada nas mandíbulas, no entanto, nas tíbias, houve diferença significante apenas nos animais de 4 meses de idade, nos quesitos área dos canais (p 0.0031), desvio-padrão da área (p 0.0338), perímetro dos canais (p 0.0068), desvio padrão do perímetro (p 0.0297) e porcentagem de matriz (p 0.0235). Os testes biomecânicos revelaram resultados que indicavam uma melhora nas características analisadas, que foi correlacionada apenas com o aumento da idade e não com o uso do ácido zoledrônico. A visualização tridimensional possibilitou analisar a distribuição dos osteócitos e dos canais, caracterizando o sistema lacunocanalicular, e constatou-se que nos animais de 4 meses o uso da droga causou uma descontinuidade na estabilidade da distribuição dos osteócitos. Conclui-se que o ácido zoledrônico não interferiu na microarquitetura da rede de canais do osso cortical das mandíbulas, enquanto que nas tíbias, nos animais jovens, houve uma modificação na microarquitetura e no sistema lacunocanalicular, revelando uma topologia mais heterogênea quando comparadas ao grupo controle. As propriedades biomecânicas não sofreram alterações quanto ao uso da droga, sendo evidenciada melhora nos parâmetros analisados relacionada apenas ao aumento de idade nos animais. / The aim of this study was to evaluate the microstructure, characterizing the bone channels network and the lacunocanalicular system, also, the biomechanical properties of the cortical bone of Wistar rats treated with Zoledronic Acid. Twenty-one male rats were separated as follows: 4-month-old (12 animals) and 9 month-old (9 animals). For each age they were divided in control and test group, and the test group received a 7.5 g/kg dose of the drug, once a week, for 3 weeks. In the 4 month-old test group they were euthanized at 14, 42 and 75 days after the drug injection and in the 9-month-old test group they were sacrificed at 14 and 75 days after medication. All animals had their mandible, tibia and femur dissected, and then, the mandible and tibia were decalcified and embedded in paraffin, and, the femur were stored for mechanical tests. Histological images were analyzed through developed algorithms using the SCILAB mathematical environment, getting percentage of bone matrix, ROI areas, ROI perimeters, their standard deviations and Lacunarity, as well, the images were accessed and processed to tridimensional visualization, to characterize the lacunocanalicular system. The femurs were analyzed by microhardness, three point bending and compression testing. The results revealed that the bone channels network in the mandible was not affected by the drug, whereas the tibia in the 4-month-old animals revealed a statistical difference between control and test groups, with the test group revealing more heterogeneity in the channels topography, as seen in results of bone matrix (p=0.0235), ROI area (p=0.0031), ROI area standard-deviation (p=0.0338), ROI perimeter (p=0.0068) and ROI perimeter standard-deviation (p=0.0297). The biomechanical tests showed an increased in strength, and that this event was age-related and not depending on the use of the drug. The tridimensional visualization allowed to analyze the osteocyte distribution in relation to the channels position, characterizing the lacunocanalicular system, and, it was found that the 4-month-old animals the drug caused a discontinuity in the stability of osteocyte distribution. In conclusion, the zoledronic acid not interfere in the bone channels network in the cortical bone of the mandible, however, in the tibia of the young animals, theres been an alteration in bone channels network and in the lacunocanalicular system, revealing more heterogeneity in the topography in these animals. The biomechanical properties were not altered after the use of the drugs, and the increased in strength being age-related.