Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts.

BACKGROUND: Viscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album "mother tinctures", MT) prepared from different host trees. METHODS: Samples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD. RESULTS: Chemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3. CONCLUSION: Our results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

Effect of music therapy on stress in chemically dependent people: a quasi-experimental study / Efeito da musicoterapia sobre o estresse de dependentes químicos: estudo quase-experimental / Efecto de la musicoterapia sobre el estrés de dependientes químicos: estudio cuasi-experimental

ABSTRACT Objective: to evaluate the effect of music therapy on the stress of chemically dependent people. Method: quasi-experimental study conducted at a philanthropic institution with 18 chemically dependent people undergoing treatment. Salivary cortisol (stress hormone) was collected in three moments: before, 60 minutes after, and 120 minutes after a music therapy group intervention. Statistical analysis adopted a significance level of p < 0.05 and used the Wilcoxon and Kruskal-Wallis non-parametric tests. Results: after 60 minutes of intervention, there was a statistically significant reduction in mean salivary cortisol levels (p < 0.001). A reduction was also noted after 120 minutes, but without statistical significance (p = 0.139). Conclusion: a single session of 60 minutes of group music therapy was able to reduce stress (salivary cortisol levels) of chemically dependent people.

RESUMO Objetivo: avaliar o efeito da musicoterapia sobre o estresse de dependentes químicos. Método: estudo quase-experimental realizado em instituição filantrópica com 18 dependentes químicos em tratamento. O cortisol salivar (hormônio do estresse) foi coletado antes, 60 e 120 minutos após única intervenção musicoterapêutica realizada em grupo. A análise estatística adotou nível de significância de p < 0,05 mediante aplicação dos testes não paramétricos de Wilcoxon e de Kruskal-Wallis. Resultados: após 60 minutos da intervenção musicoterapêutica, houve redução estatisticamente significante nas médias dos níveis de cortisol salivar (p < 0,001). Após 120 minutos, também houve redução, mas sem significância estatística (p = 0,139). Conclusão: sessão única de 60 minutos de musicoterapia em grupo mostrou-se capaz de reduzir o estresse (níveis de cortisol salivar) de dependentes químicos.

RESUMEN Objetivo: evaluar el efecto de la musicoterapia sobre el estrés de dependientes químicos. Método: estudio cuasi-experimental realizado en institución filantrópica con 18 dependientes químicos en tratamiento. El cortisol salivar (hormona del estrés) fue recogido antes, 60 y 120 minutos después de una única intervención musicoterapéutica realizada en grupo. El análisis estadístico adoptó un nivel de significancia de p <0,05 mediante la aplicación de las pruebas no paramétricas de Wilcoxon y de Kruskal-Wallis. Resultados: después de 60 minutos de la intervención musicoterapéutica, hubo reducción estadísticamente significativa en las medias de los niveles de cortisol salivar (p <0,001). Después de 120 minutos, también hubo reducción, pero sin significancia estadística (p = 0,139). Conclusión: uma única sesión de 60 minutos de musicoterapia en grupo se mostró capaz de reducir el estrés (niveles de cortisol salivar) de dependientes químicos.

Loss of constitutive ABCB1 expression in breast cancer associated with worse prognosis.

ABCB1 gene encodes an adenosine 5'-triphosphate-binding cassette transporter, which not only confers multidrug resistance phenotype in malignant cells, but is also present in several nonmalignant tissues. For the last thirty years, ABCB1 expression in breast cancer has been described by many authors, but the extent of expression differs among the studies, and there is no consensus regarding its potential role in carcinogenesis or in the tumor response to antineoplastic drugs. This study aimed to characterize the expression of ABCB1 in breast tumors as a function of genetic, clinical, and histopathological variables. The ABCB1 expression was also evaluated in nonmalignant mammary tissues adjacent to tumors and in benign lesions. The detection of ABCB1 protein was performed by immunohistochemistry in tissue specimens of excised breasts obtained from a prospective cohort of Brazilian women with breast cancer. The association of ABCB1 protein levels with ABCB1 mRNA, gene polymorphisms, and clinical and histopathological variables was also evaluated. The Kaplan-Meier curves and multivariate Cox regression analyses were conducted to identify independent predictors of disease-free survival of patients with breast cancer. ABCB1 was detected in 86.3% (656) of breast tumors, 98.8% (606) of nonmalignant mammary tissue adjacent to tumors, and 100% (28) of benign lesions. Reduced ABCB1 protein levels in breast tumors was associated with triple-negative subtype (adjusted odds ratio [ORadj] =0.24; 95% confidence interval [CI] =0.13-0.45), lymph node status < pN2 (ORadj =0.27; 95% CI =0.10-0.71), tumor size >2 cm (ORadj =0.55; 95% CI =0.32-0.93), and hypertensive status (ORadj =0.42; 95% CI =0.24-0.73), and it was significantly associated with shorter disease-free survival, either for all breast cancer patients (p log-rank =0.012; hazard ratio [HR] =3.46; 95% CI =1.21-9.91) or for those with triple-negative tumors (p log-rank =0.007; HR =11.41; 95% CI =1.29-100.67). The loss of constitutive ABCB1 expression in breast cancer, especially in triple-negative tumors, seems to indicate a subgroup of worse prognosis.

Glycosylation in Cancer: Interplay between Multidrug Resistance and Epithelial-to-Mesenchymal Transition?

The expression of unusual glycan structures is a hallmark of cancer progression, and their functional roles in cancer biology have been extensively investigated in epithelial-to-mesenchymal transition (EMT) models. EMT is a physiological process involved in embryonic development and wound healing. It is characterized by loss of epithelial cell polarity and cell adhesion, permitting cell migration, and thus formation of new epithelia. However, this process is unwanted when occurring outside their physiological limit, resulting in fibrosis of organs and progression of cancer and metastasis. Several studies observed that EMT is related to the acquisition of multidrug resistance (MDR) phenotype, a condition in which cancer cells acquire resistance to multiple different drugs, which has virtually nothing in common. However, although some studies suggested interplay between these two apparently distinct phenomena, almost nothing is known about this possible relationship. A common pathway to them is the need for glycosylation, a post-translational modification that can alter biological function. Thus, this review intends to compile the main facts obtained until now in these two areas, as an effort to unravel the relationship between EMT and MDR.

Direct electric current treatment modifies mitochondrial function and lipid body content in the A549 cancer cell line.

Electrochemical therapy (EChT) entails treatment of solid tumors with direct electric current (DC). This work evaluated the specific effects of anodic flow generated by DC on biochemical and metabolic features of the A549 human lung cancer cell line. Apoptosis was evaluated on the basis of caspase-3 activity and mitochondrial transmembrane potential dissipation. Cell morphology was analyzed using transmission electron microscopy, and lipid droplets were studied through morphometric analysis and X-ray qualitative elemental microanalysis. High-resolution respirometry was used to assess mitochondrial respiratory parameters. Results indicated A549 viability decreased in a dose-dependent manner with a prominent drop between 18 and 24h after treatment (p<0.001), together with a two-fold increase in caspase-3 activity. AF-treatment induced a significantly increase (p<0.01) in the cell number with disrupted mitochondrial transmembrane potential. Furthermore, treated cells demonstrated important ultrastructural mitochondria damage and a three-fold increase in the cytoplasmic lipid bodies' number, quantified by morphometrical analyses. Conversely, 24h after treatment, the cells presented a two-fold increase of residual oxygen consumption, accounting for 45.3% of basal oxygen consumption. These results show remarkable alterations promoted by anodic flow on human lung cancer cells which are possibly involved with the antitumoral effects of EChT.

Ouabain-induced alterations in ABCB1 of mesenteric lymph nodes and thymocytes of rats and mice.

Ouabain is a glycoside with immunomodulating properties, and recent studies have suggested its use in adjuvant therapy for cancer treatment. Ouabain is known to modulate the immune system in vitro, and previous studies have revealed that ouabain can modulate the expression and activity of ABCB1, a protein associated with multidrug resistance present in immune system. Therefore, the present study investigated alterations in the expression and activity of ABCB1 in the thymi, peripheral blood monocytes and lymph nodes of Wistar rats and Swiss mice treated acutely or chronically with ouabain. A decrease of almost 45% in the monocyte count and an increase of 55% in the basophil count were observed. A significant decrease (75% reduction) in the amount of cells with ABCB1 activity was found in the thymocytes of ouabain-treated rats and mice. The possible implications of these results for cancer treatment are discussed.

Effects of Angiotensin, vasopressin and aldosterone on proliferation of mcf-7 cells and their sensitivity to Doxorubicin.

Breast cancer is one of the leading causes of death among women and the renin-angiotensin system (RAS) has been associated with breast tumor growth and metastasis. Inhibition of the RAS limits such effects and several efforts have been made to develop new inexpensive strategies for breast cancer treatment. We herein provide additional evidence that breast cancer chemotherapy can be influenced by losartan and PD123319, antagonists of angiotensin receptors AT1 and AT2, respectively. Perhaps the most important result was that this occurred without interfering with the expression or activity of the multidrug resistance-associated protein, ABCC1, which is associated with defensive cellular mechanisms. Moreover, they increased intracellular doxorubicin accumulation, which could increase cytotoxicity.

Modulation of ABCC1 and ABCG2 proteins by ouabain in human breast cancer cells.

ABCC1 and ABCG2 are two transporters associated with multi-drug resistance to cancer chemotherapy. Ouabain is a cardiotonic steroid, currently considered as a hormone associated with arterial hypertension. Previous studies have suggested that ouabain can modulate ABCB1 and ABCC1 expression in cancer and renal cell lines. The present study investigated the effects of physiological concentrations of ouabain on the expression and activity of ABCC1 and ABCG2 in two human breast cancer cell lines, MCF7 and MDA-MB-231, the first known to be responsive to estrogens. Cell viability and proliferation assays showed that 1 µM ouabain reduced proliferation of MCF7, but not if MDA-MB-231 cells. On the other hand, 10 nM ouabain increased proliferation of MDA-MB-231, but not of MCF7 cells. Ouabain (10 nM) prevented the cytotoxic effects of doxorubicin in MCF7 cells, but not in MDA-MB-231 cells. Treatment of cells under different ouabain concentrations for 24 h did not cause any significant effects in the expression of ABCG2 or ABCC1 in either cell line. However, the activity of ABCC1 was increased when MCF7 and MDA-MB-231 cells were treated with 10 mM and 1 nM ouabain respectively. These results claim attention to the possibility that breast cancer patients with high levels of endogenous ouabain may have different responses to chemotherapy.

Betulinic acid does not modulate the activity of P-gp/ABCB1 or MRP1/ABCC1 in a non-tumoral renal cell line: Possible utility in multidrug resistance cancer chemotherapy.

Multidrug resistance (MDR) is a multifactorial phenomenon considered to be the main cause of failure in cancer chemotherapy. One of the underlying mechanisms of MDR is the overexpression of membrane transporter proteins, such as P-glycoprotein (P-gp/ABCB1) and multidrug resistance-associated protein 1 (MRP1/ABCC1). As these proteins are also expressed in normal tissues, considerable attention has been dedicated to the search for cytotoxic drugs that are not substrates for these proteins. This study investigated the effects of betulinic acid (BA) on the activity of ABCB1 and ABCC1 in Ma-104, a non-tumoral renal cell line constitutively expressing both proteins. The results indicated that concentrations of BA with low cytotoxicity to Ma-104 did not alter the activity of ABCB1 or ABCC1, nor did BA interfere with the accumulation of a classic chemotherapeutic, methotrexate. This suggests it would also be a good choice for use in drug cocktails. The lack of effect of BA on ABCB1 and ABCC1, as well as its antitumoral properties, suggest that this triterpene is a viable chemotherapeutic agent for MDR tumors.

A light in multidrug resistance: photodynamic treatment of multidrug-resistant tumors.

The major drawback of cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells, which are cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors. In the last decade, a number of authors have studied the effects of photodynamic therapy (PDT), a combination of visible light with photosensitizing agents, on MDR cells. The results, although still inconclusive, have raised the possibility of treating MDR tumors by PDT. This review examines the growing literature concerning the responses of MDR cells to PDT, while stressing the need for the development of new photosensitizers that possess the necessary characteristics for the photodynamic treatment of this class of tumor.