Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer / Modelo de enxerto tumoral ortotópico em camundongos para avaliar os efeitos do calcitriol em câncer de mama

Abstract Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. Objectives To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. Methods Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 μg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. Results Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. Conclusions Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Resumo Os efeitos antiproliferativos de calcitriol foram observados em xenotransplantes de linhagens celulares de câncer de mama, entretanto, não foram ainda investigados em enxertos tumorais, consistindo de implantes em animais de amostras de câncer de mama recém-coletadas. Objetivos Estabelecer modelo de enxerto tumoral, a partir de amostra de câncer de mama recém-coletada e diretamente implantada em camundongos nude, para estudar o efeito do calcitriol. Métodos Amostras de câncer de mama de 12 pacientes foram implantadas ortotopicamente em camundongos nude. Os animais foram tratados com injeção intratumoral semanal de calcitriol 3 μg/Kg, a qual foi previamente associada com indução de pico sérico de calcitriol dentro do intervalo de nível terapêutico. Resultados A taxa de sucesso de pega do enxerto foi de 25%. Os enxertos tumorais foram estabelecidos de tumores agressivos com alta taxa de proliferação (HER2 positivo ou grau histológico 3) e as características do tumor original foram preservadas. O calcitriol induziu fortemente a expressão do gene alvo, CYP24A1, indicando que a via genômica da vitamina D está ativa nos enxertos tumorais, entretanto, não se observou diferenças na expressão de marcadores de proliferação e apoptose (incorporação de BrdU, expressão de Ki67, CDKN1A, CDKN1B e BCL2) nestas amostras altamente proliferativas. Conclusões Os enxertos tumorais parecem ser um modelo promissor para explorar outros esquemas e doses de calcitriol, considerando a heterogeneidade da doença e interações com o microambiente.

Gene trio signatures as molecular markers to predict response to doxorubicin cyclophosphamide neoadjuvant chemotherapy in breast cancerpatients

In breast cancer patients submitted to neoadjuvant chemotherapy (4 cycles of doxorubicin and cyclophosphamide, AC), expression of groups of three genes (gene trio signatures) could distinguish responsive from non-responsive tumors, as demonstrated by cDNA microarray profiling in a previous study by our group. In the current study, we determined if the expression of the same genes would retain the predictive strength, when analyzed by a more accessible technique (real-time RT-PCR). We evaluated 28 samples already analyzed by cDNA microarray, as a technical validation procedure, and 14 tumors, as an independent biological validation set. All patients received neoadjuvant chemotherapy (4 AC). Among five trio combinations previously identified, defined by nine genes individually investigated (BZRP, CLPTM1,MTSS1, NOTCH1, NUP210, PRSS11, RPL37A, SMYD2, and XLHSRF-1), the most accurate were established by RPL37A, XLHSRF-1based trios, with NOTCH1 or NUP210. Both trios correctly separated 86 percent of tumors (87 percent sensitivity and 80 percent specificity for predicting response), according to their response to chemotherapy (82 percent in a leave-one-out cross-validation method). Using the pre-established features obtained by linear discriminant analysis, 71 percent samples from the biological validation set were also correctly classified by both trios (72 percent sensitivity; 66 percent specificity). Furthermore, we explored other gene combinations to achieve a higher accuracy in the technical validation group (as a training set). A new trio, MTSS1, RPL37 and SMYD2, correctly classified 93 percent of samples from the technical validation group (95 percent sensitivity and 80 percent specificity; 86 percent accuracy by the cross-validation method) and 79 percent from the biological validation group (72 percent sensitivity and 100 percent specificity). Therefore, the combined expression of MTSS1, RPL37 and SMYD2, as evaluated by real-time RT-PCR, is a potential candidate to predict response to neoadjuvant doxorubicin and cyclophosphamide in breast cancer patients.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR á mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 æM 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of á-casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR á, as well as RXR a, á and g expression was low in both HC11 and HC11ras cells. In addition, RAR á mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1)

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of ß1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5 percent dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha2 (63.8 ± 11.3 percent positive cells), alpha3 (93.3 ± 7.0 percent), alpha5 (50.4 ± 12.0 percent) and alpha6 (34.1 ± 4.9 percent) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0 percent) and fibronectin (40.0 ± 2.0 percent) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4 percent vs DMSO: 70.7 ± 2.5 percent) while simultaneously reducing alpha5 (24.2 ± 19 percent) and alpha6 (14.3 ± 10.8 percent) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha3 and alpha5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 ± 2 cells vs DMSO: 64 ± 6 cells), was blocked by an antibody against alpha6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells

Antiproliferative effects of 1, 25-dihydroxyvitamin D3 on breast cells: a mini review

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, is an important regulator of calcium homeostasis, exerts antiproliferative effects on various cell systems and can induce differentiation in some kinds of hematopoietic cells. These effects are triggered by its receptor, vitamin D receptor (VDR), a phosphoprotein member of the nuclear receptor superfamily, which functions as a transcriptional factor. VDR binds as a heterodimer with retinoid X receptor (R X R) to hexameric repeats, characterized as vitamin D-responsive elements present in the regulatory region of target genes such as osteocalcin, osteopontin, calbindin-D28K, calbindin-D9K, p21WAF1/CIP1, TGF-ß2 and vitamin D 24-hydroxylase. Many factors such as glucocorticoids, estrogens, retinoids, proliferation rate and cell transformation can modulate VDR levels. VDR is expressed in mammary tissue and breast cancer cells, which are potential targets to hormone action. Besides having antiproliferative properties, vitamin D might also reduce the invasiveness of cancer cells and act as an anti-angiogenesis agent. All of these antitumoral features suggest that the properties of vitamin D could be explored for chemopreventive and therapeutic purposes in cancer. However, hypercalcemia is an undesirable side effect associated with pharmacological doses of 1,25-(OH)2D3. Some promising 1,25-(OH)2D3 analogs have been developed, which are less hypercalcemic in spite of being potent antiproliferative agents. They represent a new field of investigation

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Integrin receptors and TGF-Beta expression in chronic myeloid leukemia cells

To understand relationiship between transforming growth factor beta-1 (TGF-ß1) and the integrin profile presented by chronic myeloid leukemia cells, we have studied, using Northen analysis, the expression of TGF-ß1 messenger RNA (TGF-ß mRNA) in myeloid cell lines and in patient with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In addition we determined the positivity for alfa4 and alfa5 integrin moleculas in those cell using specific monoclonal antibodies and flow cytometry. CML patients (N=3) presented mean values of alfa4 higher (alfa4: 60 ñ 20 per cent); alfa5: 70 ñ 41 per cent) than AML (N=10) blast cells (alfa4: 25 ñ 23 per cent); alfa5: 18 ñ 16 per cent). Northern analysis revealed an almost four-fold higher expression of TGF-ß mRNA in K562 (derived from a patient with chronic myeloid leukemia) compared to the myeloblastic cell line HL60. The highest TGF-ß mRNA levels were seen in the U937 lineage. CML leukemic cells (N=3) showed high TGF-ß mRNA levels comparable to the levels expressed by K562 which was paralleled by high ß1 integrin mRNA. AML blast cells presented a variable degree of expression of TGF-ß mRNA when compared to HL60. One patient with acute megakaryoblastic leukemia (FAB subtype M7), usually associated with myelofibrosis, presented the highest TGF-ß mRNA levels. We conclude that studing TGF-ß1 and its mechanisms of action will help in understanding fibrosis in leukemic patients, and perhaps to design treatments for such conditions

Pressäo no ouvido médio e óxido nitroso (N2O) / Middle ear pressure and nitrous oxide

O objetivo do trabalho foi verificar a pressäo gerada no ouvido médio durante a administraçäo de N2), se há relaçäo com o tempo e se ocorre difusäo dos gases pela tuba auditiva. Dezesseis pacientes de ambos os sexos, ASA 1 e submetidos a septoplastias foram estudados. Medicaçäo pré-anestésica: midazolam (0,1 mg/kg). Induçäo com droperidol (0,1 mg/kg), alfentanil (0,02 mg/kg), propofol (1-2 mg/kg) para tubagem orotraqueal. Os pacientes foram divididos: Grupo I (n=8), mantidos com O2 + isoflurano e grupo N (n=8), com N2O: O2 + isoflurano. A hipotensäo arterial foi induzida com injeçöes de metropolol e droperidol. As PAS e PAD foram medidas a cada 5 minutos, com observaçäo contínua e FC e ECG. Para verificar a interferência do N2) sobre a pressäo no ouvido médio, foram feitas impedanciometrias e aferiçöes do reflexo estapediano antes da induçäo da anestesia e a cada dois minutos após a intubaçäo traqueal. Houve diferença sifnificativa (p>0.01) quanto a PAS, PAD e FC iniciais e os menores valores de manutençäo, nos dois grupos. No grupo I, a pressäo no ouvido médio manteve-e inalterada. No grupo N, a pressäo no ouvido médio apresentou elevaçäo gradual, pico e declínio brusco. Ao se interromper a anestesia, com a recuperaçäo do reflexo da deglutiçäo a pressäo cai próximo a zero. O reflexo estapediano foi abolido nos dois grupos. Conclusöes: O N2O näo altera a funçäo da tuba auditiva normal. Com aumento da pressäo no interior do ouvido médio até 258 mm H2O (2.54 kPa) em média, a tuba auditiva abre-se, equalizando a pressäo com a atmosfera. Em pacientes anteriormente submetidos a timpanoplastia ou mastoidectomia fechada, o N2O deve ser evitado poi pode desorgaizar os elementos anatômicos reconstruídos. Naqueles já submetidos a estapedectomia, o N2O deve ser contra-indicado em quaisquer circunstâncias, pela maior labilidade dos elementos anatômicos de ligaçäo, a nível da janela oval, às variaçöes pressóricas observadas, podendo provocar dano auditivo neurossensorial irreversível. Os resultados näo permitiram relacionar a pressäo no ouvido médio com o tempo de administraçäo do N2O. O isoflurano + oxigênio näo alteram as pressöes do ouvido médio e näo interferem com a funçäo tubária. As técnicas anestésicas empregadas abolem o reflexo estapediano

Bloqueio intercostal na linha axilar média para pequenas operaçöes de mama / Intercostal nerve block at the axillary midline for minor procedures of the breast

Descreve-se uma técnica de bloqueios dos nervos intercostais, realizados na linha axilar média com bupivacaína a 0,5% com epinefrina a 1:200.000. Foram realizados bloqueios a nível da segunda a sexta costelas: a área de analgesia conferida foi estudada considerando-se somente a regiäo mamária, dividida em quadrantes. Observou-se que a analgesia interessou o quadrante superior externo, inferiores externo e interno e a regiäo periareolar do quadrante superior interno. O bloqueio do quadrante súpero-interno, por vezes, é insuficiente por razöes anatômicas. A técnica é recomendada para pacientes em regime ambulatorial

Diazepam como hipotensor ocular / Diazepam as ocular hypotensor

Estudaram-se 34 pacientes, submetidos a operaçöes intra-oculares, com idade variando entre 50 e 74 anos, sem nenhuma medicaçäo sistêmica ou local prévia. As pressöes intra-oculares foram registradas com tonômetro de aplanaçäo, antes e cinco minutos após a injeçäo de diazepam (0.2 mg.kg-1) por via venosa. Houve significativa queda da pressäo intra-ocular após a injeçäo do benzodiazepínico, que concluíram-se ser a melhor droga para proporcionar hipotensäo rápida durante operaçöes oculares. Os prováveis mecanismos dessa açäo säo discutidos

Passagem placentária de drogas / Placental transfer of drugs

Tecem-se consideraçöes sobre a anatomia e fisiologia da placenta, e os mecanismos de transferência de gases (oxigênio e gás carbônico) e nutrientes. Faz-se um resumo dos processos utilizados pela placenta na difusäo de drogas e dos fatores mais importantes que alteram esta transferência, aqueles intrínsecos a sua fisiologia, bem como os ligados às drogas. A equaçäo de Fick, bem como os fatores que influenciam os fluxos sangüíneos umbilical säo abordados, de forma correlacionada à clínica e aos anestésicos comumente empregados. Alguns dados sobre a distribuiçäo fetal de drogas e os efeitos dos principais anestésicos e drogas comumente empregados em clínica médica e obstétrica säo focalizados

Uma nova ambulância para transporte de doentes e acidentados / A new ambulance for transportation of patients and accident victims

Apresenta-se um novo veículo para transporte de pacientes acidentados, com aumento de 30% da área interna útil e teto elevado, que possibilita massagem cardíaca externa e maior facilidade para manutençäo de via venosa. As condiçöes de segurança para os socorristas, durante o transporte, foram aumentadas pela utilizaçäo de assentos escamoteáveis dotados de cintos de segurança. Foi acrescida uma maca destinada especialmente ao transporte de suspeitos de fraturas. Os testes, realizados com pessoal treinado, confirmaram a possibilidade de realizaçäo contínua e efetiva das medidas de reanimaçäo durante o transporte. A possibilidade de execuçäo de massagem cardíaca externa e demais medidas de reanimaçäo cardiopulmonar, pela reduçäo da necessidade de desenvolver grandes velocidades, aumentou significativamente a segurança dos socorristas e dos pacientes

Bases farmacológicas para o uso de éter venoso em anestesia / Pharmacological basis of intravenous diethyl ether anesthesia

Apresentam-se as bases farmacocinéticas para o emprego do éter a 5% em soluçäo de glicose por via venosa. Descrevem-se os princípios para os cálculos das quantidades a serem administradas, de acordo com os princípios expostos por Brody e desenvolvidos por Lowe para a anestesia quantitativa. A técnica preconizada é descrita com detalhes, quando se utiliza ventilaçäo com oxigênio puro e com misturas de óxido nitroso. As vantagens e as desvantagens säo descritas, ressaltando-se a segurança associada ao baixo custo de sua aplicaçäo

O uso da via aerea esofageana. / The use of esophageal obturator airway

A via aerea com obturador esofageano (VAOE) e um dispositivo que facilita a intubacao e obliteracao do esofago e atraves de uma mascara facial, permite a insuflacao de ar ou oxigenio que ventila os pulmoes. O dispositivo evita a regurgitacao de conteudo gastrico e a insuflacao de ar indevida ao estomago.O metodo descrito pode ser usado por pessoal medico ou paramedico nao treinado em intubacao traqueal sendo de facil manipulacao e apredizado.As complicacoes sao raras e evitaveis, principalmente se a tecnica e aplicada adequadamente. O tubo nao deve ser retirado sem o devido esvaziamento do balonete esofagico e, cuidados devem ser tomados para evitar intubacao traqueal inadvertida. Nao ha experiencia sobre sua aplicacao em criancas, nao sendo recomendavel o seu uso em pacientes abaixo de 16 anos. Os autores utilizaram o dispositivo em 10 pacientes adultos, observando o tempo para sua instalacao e a eficacia da ventilacao boca-tubo e concluiram que o metodo permite efetiva ventilacao pulmonar em 5 a 10 segundos