Medicinal properties of Angelica archangelica root extract: Cytotoxicity in breast cancer cells and its protective effects against in vivo tumor development.

OBJECTIVE: Although Angelica archangelica is a medicinal and aromatic plant with a long history of use for both medicinal and food purposes, there are no studies regarding the antineoplastic activity of its root. This study aimed to evaluate the cytotoxicity and antitumor effects of the crude extract of A. archangelica root (CEAA) on breast cancer. METHODS: The cytotoxicity of CEAA against breast adenocarcinoma cells (4T1 and MCF-7) was evaluated by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Morphological and biochemical changes were detected by Hoechst 33342/propidium iodide (PI) and annexin V/PI staining. Cytosolic calcium mobilization was evaluated in cells staining with FURA-4NW. Immunoblotting was used to determine the effect of CEAA on anti- and pro-apoptotic proteins (Bcl-2 and Bax, respectively). The 4T1 cell-challenged mice were used for in vivo assay. RESULTS: Using ultra-high-performance liquid chromatography-mass spectrometry analysis, angelicin, a constituent of the roots and leaves of A. archangelica, was found to be the major constituent of the CEAA evaluated in this study (73 µg/mL). The CEAA was cytotoxic for both breast cancer cell lines studied but not for human fibroblasts. Treatment of 4T1 cells with the CEAA increased Bax protein levels accompanied by decreased Bcl-2 expression, in the presence of cleaved caspase-3 and cytosolic calcium mobilization, suggesting mitochondrial involvement in breast cancer cell death induced by the CEAA in this cell line. No changes on the Bcl-2/Bax ratio were observed in CEAA-treated MCF7 cells. Gavage administration of the CEAA (500 mg/kg) to 4T1 cell-challenged mice significantly decreased tumor growth when compared with untreated animals. CONCLUSION: Altogether, our data show the antitumor potential of the CEAA against breast cancer cells in vitro and in vivo. Further research is necessary to better elucidate the pharmacological application of the CEAA in breast cancer therapy.

Correlation of tokuhashi and tomita scores with the prognosis in metastatic breast cancer / Correlação dos escores de tokuhashi e tomita com o prognóstico em tumor metastático de mama / Correlación de las puntuaciones de tokuhashi y tomita com el pronóstico en tumor metastásico de mama

ABSTRACT Objective: The purpose of the present study was to evaluate the concordance between the Tokuhashi and Tomita scores with the prognosis of patients with vertebral metastases due to breast tumor, treated at the outpatient clinic of the Universidade Estadual de Campinas (UNICAMP). Methods: Twenty-nine patients with vertebral metastases from breast tumor were retrospectively evaluated. Twenty patients were surgically treated and received adjuvant therapy and only nine received conservative (chemotherapy/radiotherapy) or palliative/support treatment, depending on Tokuhashi and Tomita scores. Results: In this study, all selected patients were females with vertebral metastasis due to breast tumor; mean age of 57.6 years (SD = 11.8 years). The accuracy of the Tokuhashi scale was 62.1% and that of Tomita 72.4%. In addition, the Tomita scale concentrates the majority of the patients' classifications for more than 12 months (69%), indicating a good relation with the long-term prognosis (> 12 months). None of the evaluated characteristics - age or surgery - statistically influenced the survival of patients with primary breast tumor (p > 0.05). Conclusion: The Tokuhashi and Tomita scores showed good accuracy in relation to the prognosis of patients with spinal metastasis due to breast tumor.

RESUMO Objetivo: O objetivo do presente estudo é avaliar a concordância entre os escores de Tokuhashi e Tomita com o prognóstico das pacientes acometidas por metástases vertebrais por tumor de mama, atendidas no ambulatório da Universidade Estadual de Campinas (UNICAMP). Métodos: Foram avaliadas retrospectivamente 29 pacientes com metástases vertebrais de tumor de mama. Vinte pacientes foram tratadas com cirurgia e terapia adjuvante e apenas nove receberam tratamento conservador (quimioterapia/radioterapia) ou paliativo/suporte, dependendo dos escores de Tokuhashi e Tomita. Resultados: No estudo, foram selecionadas pacientes do sexo feminino acometidas por metástase vertebral decorrente de tumor de mama; média de idade de 57,6 anos (DP = 11,8 anos). A acurácia da escala de Tokuhashi foi de 62,1% e a de Tomita, 72,4%. Além disso, a escala de Tomita concentra a maioria das classificações das pacientes do grupo por mais de 12 meses (69%), indicando boa relação com o prognóstico a longo prazo (>12 meses). Nenhuma das características avaliadas - idade ou cirurgia - influenciou estatisticamente a sobrevida das pacientes com tumor primário de mama (p > 0,05). Conclusão: Os escores de Tokuhashi e de Tomita apresentaram boa acurácia com relação ao prognóstico das pacientes acometidas por metástase na coluna vertebral decorrente de tumor de mama.

RESUMEN Objetivo: El objetivo de este estudio es evaluar la concordancia entre las puntuaciones de Tokuhashi y Tomita con el pronóstico de las pacientes que tienen metástasis vertebrales por tumor de mama, tratadas en el ambulatorio de la Universidade Estadual de Campinas (Unicamp). Métodos: Se evaluaron retrospectivamente 29 pacientes con metástasis vertebrales por tumor de mama. Veinte pacientes fueron sometidas a tratamiento quirúrgico y terapia adyuvante y sólo nueve recibieron tratamiento conservador (quimioterapia/radioterapia) o paliativo/de apoyo, dependiendo de las puntuaciones de Tokuhashi y Tomita. Resultados: En el estudio, se seleccionaron pacientes del sexo femenino con metástasis vertebral derivada de tumor de mama; edad promedio de 57,6 años (DE = 11,8 años). La exactitud de la escala Tokuhashi fue del 62,1% y la de Tomita, el 72,4%. Además, la escala Tomita concentra la mayoría de las clasificaciones de las pacientes del grupo por más de 12 meses (69%), lo que indica una buena relación con el pronóstico a largo plazo (> 12 meses). Ninguna de las características evaluadas - edad o cirugía - influenció estadísticamente la supervivencia de las pacientes con tumor primario de mama (p > 0,05). Conclusión: Las puntuaciones de Tokuhashi y Tomita mostraron buena exactitud con relación al pronóstico de las pacientes que tienen metástasis de columna vertebral resultantes del tumor de mama.

Cafestol, a diterpene molecule found in coffee, induces leukemia cell death.

To evaluate the antitumor properties of Cafestol four leukemia cell lines were used (NB4, K562, HL60 and KG1). Cafestol exhibited the highest cytotoxicity against HL60 and KG1 cells, as evidenced by the accumulation of cells in the sub-G1 fraction, mitochondrial membrane potential reduction, accumulation of cleaved caspase-3 and phosphatidylserine externalization. An increase in CD11b and CD15 differentiation markers with attenuated ROS generation was also observed in Cafestol-treated HL60 cells. These results were similar to those obtained following exposure of the same cell line to cytarabine (Ara-C), an antileukemic drug. Cafestol and Ara-C reduced the clonogenic potential of HL60 cells by 100%, but Cafestol spared murine colony forming unit- granulocyte/macrophage (CFU-GM), which retained their clonogenicity. The co-treatment of Cafestol and Ara-C reduced HL60 cell viability compared with both drugs administered alone. In conclusion, despite the distinct molecular mechanisms involved in the activity of Cafestol and Ara-C, a similar cytotoxicity towards leukemia cells was observed, which suggests a need for prophylactic-therapeutic pre-clinical studies regarding the anticancer properties of Cafestol.

The biphosphinic paladacycle complex induces melanoma cell death through lysosomal-mitochondrial axis modulation and impaired autophagy.

Recently, palladium complexes have been extensively studied as cyclization of these complexes by cyclometallation reactions increased their stability making them promising antitumor compounds. In this study, we have investigated apoptosis induced by the Biphosphinic Paladacycle Complex (BPC11) and possible cross talk between apoptosis and autophagy in cell line models of metastatic (Tm5) and non-metastatic (4C11-) melanoma. The BPC11-induced cell death in melanoma involved the lysosomal-mitochondrial axis, which is characterized by LMP, CatB activation and increased Bax protein levels following its translocation to mitochondria. Mitochondrial hyperpolarization, followed by membrane potential dissipation and cleavage of caspase-3, also resulted in cell death after 24 h of incubation. We also found that BPC11-mediated LC3II formation and increased p62 protein levels, suggesting blocked autophagy, probably due to LMP. Interestingly, the treatment of Tm5 and 4C11(-) cells with 3-methyladenine (3-MA), an inhibitor of the initial stage of autophagy, potentiated the effects of BPC11. We conclude that BPC11 is an anti-melanoma agent and that autophagy may be acting as a mechanism of melanoma cells resistance. Also, these data highlight the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties.

Correlation between actual survival and Tokuhashi and Tomita scores in spine metastases / Correlação entre a sobrevida real e os escores de Tokuhashi e Tomita em metástases de coluna vertebral / Correlación entre la supervivencia real y las puntuaciones de Tokuhashi y Tomita en las metástasis de la columna vertebral

OBJECTIVE: To evaluate the accuracy of the scores of Tokuhashi and Tomita and the actual survival of patients with vertebral metastases. METHODS: A retrospective assessment of 45 patients with spinal metastases. Thirty-one patients underwent surgical treatment and adjuvant therapy and 14 received conservative treatment (chemotherapy/radiotherapy) or palliative/supportive, depending on the scores of Tokuhashi and Tomita. RESULTS: In the study, 80% of patients were female and the mean age was 57.8 years (SD=11.3 years). The most frequent primary tumors were breast and prostate (68.9%). The accuracy of Tokuhashi scale was 53.4% and the Tomita, 64.5%. The concentration of Tomita range of correct classification was in the category of survival > 12 months (57.8%), while the Tokuhashi scale presented some adjustment in the other categories, < 6 months (15.6%) and 6 to 12 months (2.2%). The histological type of the primary tumor was the only variable that statistically influenced the survival time of patients (p<0.001), and patients with lung or liver tumor (most aggressive) presented a risk of death 9.89 times higher than patients with primary tumors of breast or prostate (less aggressive) (95% CI: 3.10 to 31.57). CONCLUSION: The Tokuhashi and Tomita scores showed good accuracy with respect to the actual survival of patients with tumor metastasis in the spine.

OBJETIVO: Avaliar a acurácia entre os escores de Tokuhashi e Tomita e a sobrevida real dos pacientes acometidos por metástase vertebral. MÉTODOS: Foram avaliados retrospectivamente 45 pacientes com metástases vertebrais. Trinta e um pacientes foram submetidos a tratamento cirúrgico e a terapia adjuvante e 14 apenas receberam tratamento conservador (quimioterapia/radioterapia) ou paliativo/de suporte, dependendo da pontuação dos escores de Tokuhashi e Tomita. RESULTADOS: No estudo, 80% dos pacientes eram do sexo feminino e a média de idade foi 57,8 anos (DP=11,3 anos). Os tumores primários mais encontrados foram de mama e próstata (68,9%). A acurácia da escala de Tokuhashi foi de 53,4% e a de Tomita, de 64,5%. A concentração dos acertos de classificação da escala de Tomita foi na categoria sobrevida > 12 meses (57,8%), enquanto a escala de Tokuhashi apresentou algum acerto nas demais categorias, < 6 meses (15,6%) e de 6 a 12 meses (2,2%). O tipo histológico do tumor primário foi a única variável que influenciou estatisticamente o tempo de sobrevida dos pacientes (p<0,001), sendo que pacientes com tumor de pulmão ou fígado (mais agressivos) apresentaram risco de vida 9,89 vezes maior que os pacientes com tumor primário de mama ou próstata (menos agressivos) (IC 95%: 3,10 a 31,57). CONCLUSÃO: Os escores de Tokuhashi e de Tomita apresentaram boa acurácia com relação à sobrevida real dos pacientes acometidos por metástases na coluna vertebral.

OBJETIVO: Evaluar la exactitud de las puntuaciones de Tokuhashi y Tomita y la supervivencia real de pacientes con metástasis vertebrales. MÉTODOS: Se evaluaron retrospectivamente 45 pacientes con metástasis vertebrales. Treinta y un pacientes fueron sometidos a tratamiento quirúrgico y terapia adyuvante y 14 recibieron sólo tratamiento conservador (quimioterapia/radioterapia) o paliativo/de apoyo, dependiendo de las puntuaciones de Tokuhashi y Tomita. RESULTADOS: En el estudio, el 80% de los pacientes eran mujeres y edad media fue de 57,8 años (DE = 11,3 años). Los tumores primarios más frecuentes fueron mama y próstata (68,9%). La exactitud de la escala Tokuhashi fue de 53,4% y la de Tomita, de 64,5%. La concentración de aciertos de clasificación en la escala de Tomita fue en la categoría de supervivencia > 12 meses (57,8%), mientras que la escala Tokuhashi presentó algunos ajustes en las otras categorías < 6 meses (15,6%) y de 6 a 12 meses (2,2%). El tipo histológico del tumor primario fue la única variable que influyó estadísticamente el tiempo de supervivencia de los pacientes (p < 0,001), y los pacientes con tumor de pulmón o hígado (más agresivos) presentaron riesgo de vida 9,89 veces mayor que los pacientes con tumores primarios de mama o de próstata (menos agresivos) (IC del 95%: 3,10 a 31,57). CONCLUSIÓN: Las puntuaciones de Tokuhashi y Tomita mostraron una buena precisión con respecto a la supervivencia real de pacientes con metástasis en la columna vertebral.

Palladacycle (BPC) antitumour activity against resistant and metastatic cell lines: the relationship with cytosolic calcium mobilisation and cathepsin B activity.

The search for new compounds that induce p53-independent apoptosis is the focus of many studies in cancer biology because these compounds could be more specific and would overcome chemotherapy resistance. In this study, we evaluated the in vitro antitumour activity of a Biphosphinic Palladacycle Complex (BPC) and extended preclinical studies to an in vivo model. Saos-2 cells, a p53-null human osteosarcoma drug-resistant cell line, were treated with BPC in the presence or absence of a cathepsin B inhibitor and a calcium chelator (CA074 and BAPTA-AM, respectively), and several parameters related to apoptosis were evaluated. Preclinical studies were performed with mice that were intravenously inoculated with murine melanoma B16F10-Nex2 cells and treated intraperitoneally (i.p.) with BPC (8 mg/kg/day) for ten consecutive days, when lung metastatic nodules were counted. In vitro data show that BPC induces cell death in Saos-2 cells mainly by apoptosis, which was accompanied by the effector caspase-3 activation. These events are most likely related to Bax translocation and increased cytosolic calcium mobilisation, mainly from intracellular compartments. Lysosomal Membrane Permeabilisation (LMP) was also observed after 12 h of BPC exposure. Interestingly, BAPTA-AM and CA074 significantly decreased BPC cytotoxicity, suggesting that both calcium and cathepsin B are required for BPC antitumour activity. In vivo studies demonstrated that BPC protects mice against murine metastatic melanoma. In conclusion, BPC complex is an effective anticancer compound against metastatic murine melanoma. This complex is cytotoxic to the drug-resistant osteosarcoma Saos-2 human tumour cells by inducing apoptosis triggered by calcium signalling and a lysosomal-dependent pathway.

Use of the vascularized iliac-crest flap in musculoskeletal lesions.

Bone loss was in the past treated by several methods, such as bone distraction and the use of nonvascularized or tissue-bank bone grafts. With the advent of modern microsurgical techniques, the vascularized bone flap has been used with good results; it resolves local nutritional problems, repairs soft tissue that is often damaged by severe trauma, and treats bone loss due to tumors, pseudarthroses, and osteomyelitis. This paper reports the authors' experience with the use of vascularized iliac-crest flaps to treat orthopedic pathologies in five patients with traumatic bone loss (<10 cm), three with osteomyelitis, and three with atrophic nonunion. In all cases, the same surgeon obtained a vascularized iliac-crest flap with a pedicle based on the deep iliac circumflex artery. All flaps consolidated within a mean period of 3 months. These findings demonstrate that the use of an iliac-crest flap is a treatment option in cases of bone loss and that it is associated with good functional results and minimal donor-site morbidity.

Evolução dos questionários Oswestry 2. 0 e do componente físico (PCS) do SF-36 durante o primeiro ano de pós-operatório de artrodese da coluna lombar em doenças degenerativas / Evolution of Oswestry 2. 0 questionnaire and the physical component (PCS) of SF-36 during the first postoperative year of lumbar spine fusion in degenerative diseases / Evolución del cuestionario Oswestry 2. 0 y del componente físico (PCS) del SF-36 durante el primer año de postoperatorio de fusión lumbar en enfermidades degenerativas

OBJETIVO: Avaliar a melhora clínica dos pacientes submetidos a descompressão e artrodese lombar póstero-lateral por doença degenerativa, avaliando a evolução dos escores do questionário Oswestry 2.0 e pelo componente físico (PCS) da escala SF-36. MÉTODOS: Estudo prospectivo com 19 pacientes consecutivos com diagnóstico de doença degenerativa de disco (hérnia de disco, estenose lombar ou espondilolistese degenerativa) submetidos à descompressão e artrodese póstero-lateral. Todos os pacientes incluídos em nosso estudo responderam aos questionários Oswestry 2.0 e SF-36 em cinco momentos diferentes: no período pré-operatório e após 45, 90, 180 e 360 dias de cirurgia. RESULTADOS: Os escores dos questionários Oswestry 2.0 e do componente físico (PCS) do SF-36 dos 19 pacientes mostraram melhora significativa (p<0,001 e p=0,004 respectivamente) ao longo de um ano de seguimento pós-operatório. Não houve diferença significativa de melhora desses escores quando feita comparação entre os diagnósticos (hérnia de disco, estenose lombar ou espondilolistese degenerativa), o sexo dos pacientes e entre os indivíduos com idade até cinquenta anos e aqueles acima de cinquenta anos. CONCLUSÃO: Houve uma melhora dos escores nos questionários de Oswestry 2.0 e do componente físico (PCS) do SF-36 nos pacientes com doença degenerativa da coluna lombar sem resposta ao tratamento conservador, que foram submetidos a tratamento cirúrgico com descompressão e artrodese após um ano de seguimento pós-operatório.

OBJECTIVE: Assess the clinical improvement of patients undergoing decompression and posterolateral lumbar arthrodesis for degenerative diseases, evaluating the evolution of the scores of Oswestry 2.0 questionnaire and the physical component (PCS) of the SF-36 scale. METHODS: Prospective study of 19 patients with degenerative disc disease (disc herniation, lumbar stenosis or degenerative spondylolisthesis) that underwent decompression and posterolateral arthrodesis. All patients included in our study answered the questionnaire Oswestry 2.0 and the PCS of SF-36 at five different occasions: in the preoperative period and 45, 90, 180 and 360 days after surgery. RESULTS: The scores of Oswestry 2.0 questionnaire and the physical component (PCS) of the SF-36 of the patients showed significant improvement (p<0.001 and p=0.004 respectively) over a year of postoperative follow-up. There was no significant difference in improvement in these scores when comparing the diagnoses made (disc herniation, lumbar stenosis or degenerative spondylolisthesis), sex of patients, and individuals aged up to fifty years and those over fifty years. CONCLUSION: There was an improvement in Oswestry scores and physical component (PCS) of the SF-36 in patients with degenerative lumbar spine unresponsive to conservative treatment that underwent surgical decompression and arthrodesis after one year of postoperative follow-up.

OBJETIVO: Evaluar la mejoría clínica de los pacientes sometidos a descompresión y artrodesis lumbar posterolateral para las enfermedades degenerativas, valoración de la evolución de las puntuaciones del cuestionario Oswestry 2.0 y el componente físico (PCS) de la escala SF-36. MÉTODOS: Evaluación de 19 pacientes con enfermedad degenerativa del disco (hernia de disco, estenosis lumbar o espondilolistesis degenerativa) sometidos a descompresión y artrodesis posterolateral. Todos los pacientes incluidos en nuestro estudio respondieron al cuestionario de Oswestry 2.0 e al SF-36 en cinco ocasiones distintas: Período preoperatorio y 45, 90, 180 y 360 días después de la cirugía. RESULTADOS: Las puntuaciones del cuestionario Oswestry y el componente físico (PCS) de la SF-36 de los 19 pacientes mostraron una mejoría significativa (p<0,001 y p=0,004, respectivamente) a lo largo de un año de seguimiento postoperatorio. No hubo diferencias significativas en la mejoría de los puntajes cuando se compararon los diagnósticos realizados (hernia de disco, estenosis lumbar y espondilolistesis degenerativa), sexo de los pacientes e individuos hasta la edad de cincuenta años y los mayores de cincuenta años. CONCLUSIÓN: Se observó una mejoría en las puntuaciones en los cuestionarios de Oswestry 2.0 y el componente físico (PCS) de la SF-36 en pacientes con enfermedad degenerativa de la columna lumbar que no respondieron al tratamiento conservador y fueron sometidos a cirugía de descompresión y artrodesis en el seguimiento después de un año de la cirugía.

Autophagy inhibited Ehrlich ascitic tumor cells apoptosis induced by the nitrostyrene derivative compounds: relationship with cytosolic calcium mobilization.

Apoptosis induction is often associated with increased autophagy, indicating interplay between these two important cellular events in cell death and survival. In this study, the programmed cell death and autophagy induced by two nitrostyrene derivative compounds (NTS1 and NTS2) was studied using the tumorigenic Ehrlich ascitic tumor (EAT) cells. EAT cells were highly sensitive to NTS1 and NTS2 cytotoxicity in a dose-dependent manner. NTS1 and NTS2 IC(50) was less than 15.0µM post 12h incubation. Apoptosis was primarily induced by both compounds, as demonstrated by an increase in Annexin-V positive cells, concurrently with cytochrome c release from mitochondria to cytosol and caspase-3 activation. Although cytosolic Ca(2+) mobilization is involved in autophagy as well as apoptosis in response to cellular stress in many cancer cell types, from the two nitrostyrene derivative compounds studied, mainly NTS1 mobilized this ion and disparate autophagy in EAT cells. These results suggest that EAT induced cell death by NTS1 and NTS2 involved a Ca(2+)-dependent and a Ca(2+)-independent pathways, respectively. In accordance with these results, the treatment of EAT cells with 3 methyladenine (3-MA), an autophagy inhibitor; significantly increased the number of apoptotic cells after NTS1 treatment, suggesting that pharmacological modulation of autophagy augments the NTS1 efficacy. Thus, we denote the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties.

A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells.

BACKGROUND: Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd(2) [S((-))C(2), N-dmpa](2) (µ-dppe)Cl(2)} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. METHODS: B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. RESULTS: Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. CONCLUSIONS: The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.