Cellular dynamics as a marker of normal-to-cancer transition in human cells.

Normal-to-cancer (NTC) transition is known to be closely associated to cell´s biomechanical properties which are dependent on the dynamics of the intracellular medium. This study probes different human cancer cells (breast, prostate and lung), concomitantly to their healthy counterparts, aiming at characterising the dynamical profile of water in distinct cellular locations, for each type of cell, and how it changes between normal and cancer states. An increased plasticity of the cytomatrix is observed upon normal-to-malignant transformation, the lung carcinoma cells displaying the highest flexibility followed by prostate and breast cancers. Also, lung cells show a distinct behaviour relative to breast and prostate, with a higher influence from hydration water motions and localised fast rotations upon NTC transformation. Quasielastic neutron scattering techniques allowed to accurately distinguish the different dynamical processes taking place within these highly heterogeneous cellular systems. The results thus obtained suggest that intracellular water dynamics may be regarded as a specific reporter of the cellular conditions-either healthy or malignant.

Physical activity measured with an accelerometer in dogs following extracapsular stabilisation to treat cranial cruciate ligament rupture.

OBJECTIVES: To quantify the physical activity levels in dogs with cranial cruciate ligament rupture before and after lateral fabellar suture stabilisation surgery. MATERIALS AND METHODS: Seventeen dogs (mean weight, 12.3±5.1 kg) with unilateral cranial cruciate ligament rupture were fitted with an accelerometer for seven consecutive days at four different time points: before surgery (T0), one (T1), three (T3) and six (T6) months after surgery. The total activity and times spent in sedentary activity, light to moderate activity and vigorous activity were recorded by the accelerometer, and preoperative and postoperative data were compared. At all time points, dogs underwent clinical evaluations (lameness score, stifle pain score and thigh circumference) and their owners were asked to respond to questionnaires to subjectively score the physical activity and quality of life of the dogs. RESULTS: At the four time points, the dogs spent between 21.2 and 21.4 hours on sedentary behaviour, 2.3 and 2.5 hours performing light to moderate activity, and 13 to 15 minutes performing vigorous activity. There was no increase in physical activity variables or decrease in sedentary behaviour over time. Lameness scores, pain score and dogs' quality of life improved significantly during the postoperative period. At T6, 17 (100%) of 17 dogs presented no lameness, 16 (94%) of 17 dogs presented no stifle pain, 16 (94%) of 17 owners rated the quality of life as very good and excellent, and 16 (100%) of 16 owners reported a total return to normal activity levels. CLINICAL SIGNIFICANCE: The clinical recovery after extracapsular stabilisation of the stifle joint was not associated with a spontaneous increase in physical activity or a decrease in sedentary behaviour.

Water dynamics in human cancer and non-cancer tissues.

Normal-to-malignant transformation is a poorly understood process associated with cellular biomechanical properties. These are strongly dependent on the dynamical behaviour of water, known to play a fundamental role in normal cellular activity and in the maintenance of the three-dimensional architecture of the tissue and the functional state of biopolymers. In this study, quasi-elastic neutron scattering was used to probe the dynamical behaviour of water in human cancer specimens and their respective surrounding normal tissue from breast and tongue, as an innovative approach for identifying particular features of malignancy. This methodology has been successfully used by the authors in human cells and was the first study of human tissues by neutron scattering techniques. A larger flexibility was observed for breast versus tongue tissues. Additionally, different dynamics were found for malignant and non-malignant specimens, depending on the tissue: higher plasticity for breast invasive cancer versus the normal, and an opposite effect for tongue. The data were interpreted in the light of two different water populations within the samples: one displaying bulk-like dynamics (extracellular and intracellular/cytoplasmic) and another with constrained flexibility (extracellular/interstitial and intracellular/hydration layers).

Role of intracellular water in the normal-to-cancer transition in human cells-insights from quasi-elastic neutron scattering.

The transition from normal to malignant state in human cells is still a poorly understood process. Changes in the dynamical activity of intracellular water between healthy and cancerous human cells were probed as an innovative approach for unveiling particular features of malignancy and identifying specific reporters of cancer. Androgen-unresponsive prostate and triple-negative breast carcinomas were studied as well as osteosarcoma, using the technique of quasi-elastic neutron scattering. The cancerous cells showed a considerably higher plasticity relative to their healthy counterparts, this being more significant for the mammary adenocarcinoma. Also, the data evidence that the prostate cancer cells display the highest plasticity when compared to triple-negative mammary cancer and osteosarcoma, the latter being remarkably less flexible. Furthermore, the results suggest differences between the flexibility of different types of intracellular water molecules in normal and cancerous cells, as well as the number of molecules involved in the different modes of motion. The dynamics of hydration water molecules remain virtually unaffected when going from healthy to cancer cells, while cytoplasmic water (particularly the rotational motions) undergoes significant changes upon normal-to-cancer transition. The results obtained along this study can potentially help to understand the variations in cellular dynamics underlying carcinogenesis and tumor metastasis, with an emphasis on intracellular water.

Novel platinum-based anticancer drug: a complete vibrational study.

The introduction of cisplatin to oncology, in the 1970s, marked the onset of the search for novel and improved metal-based anticancer drugs. Polynuclear PtII and PdII complexes with linear alkylamines as bridging ligands are a class of potential antineoplastic agents that have shown promising cytotoxicity against low-prognosis human cancers, such as metastatic breast adenocarcinoma and osteosarcoma. The present study reports an analysis of [µ-N,N'-bis(3-aminopropyl)butane-1,4-diamine-κ4N,N':N'',N''']bis[dichloridoplatinum(II)], [Pt2Cl4(C10H26N4)], denoted Pt2Spm (Spm is spermine), by vibrational spectroscopy coupled to theoretical calculations. Within the latter, the Density Functional Theory (DFT - mPW1PW/6-31G*) and Effective Core Potential (ECP - LANL2DZ) approaches were used, in order to ensure the most accurate representation of the molecule and achieve a maximum agreement with the experimental data. The solid-state geometry of Pt2Spm corresponds to Ci symmetry, displaying 132 vibrational modes. A complete assignment of the experimental vibrational profile of the system was attained through the combined application of complementary Raman, FT-IR and Inelastic Neutron Scattering (INS) techniques. INS allowed an unequivocal identification of the CH2 and NH2 rocking modes, not clearly detected by the optical techniques, while Raman measurements led to a clear discrimination of the Pt-N stretching frequencies from the two distinct Pt-N moieties within the chelate. The metal-to-metal distances calculated for the molecule under study were found to allow the establishment of effective inter- and intrastrand crosslinks with DNA. These results will hopefully help to clarify the mode of action of the compound, at the molecular level, contributing to the development of improved cisplatin-like chemotherapeutic drugs having a higher efficacy and specificity coupled to lower acquired resistance and deleterious side effects.

Intracellular water - an overlooked drug target? Cisplatin impact in cancer cells probed by neutrons.

The first neutron scattering study on human nucleated cells is reported, addressing the subject of solvent-slaving to a drug by probing intracellular water upon drug exposure. Inelastic and quasi-elastic neutron scattering spectroscopy with isotope labelling was applied for monitoring interfacial water response to the anticancer drug cisplatin, in the low prognosis human metastatic breast cancer cells MDA-MB-231. Optical vibrational data were also obtained for lyophilised cells. Concentration-dependent dynamical changes evidencing a progressive mobility reduction were unveiled between untreated and cisplatin-exposed samples, concurrent with variations in the native organisation of water molecules within the intracellular medium as a consequence of drug action. The results thus obtained yielded a clear picture of the intracellular water response to cisplatin and constitute the first reported experimental proof of a drug impact on the cytomatrix by neutron techniques. This is an innovative way of tackling a drug's pharmacodynamics, searching for alternative targets of drug action.

Raman microspectroscopy for probing the impact of a dietary antioxidant on human breast cancer cells.

Breast cancer is the second most common type of cancer worldwide and the most frequent among women, being the fifth cause of death from neoplastic disease. Since this is an oxidative-stress related neoplasia, it is largely preventable. A dietary isoflavone abundant in soybean - daidzein - is currently being investigated owing to its chemopreventive and/or chemotherapeutic properties towards the human MDA-MB-231 (metastatic, estrogen-unresponsive) and MCF-7 (estrogen-responsive) breast cancer cell lines. Biological assays for evaluation of antitumour and anti-invasive activities were combined with state-of-the-art vibrational microspectroscopy techniques. At 50 and 100 µM concentrations and 48 h incubation time, daidzein was found to induce a marked decrease in cell viability (ca. 50%) for MDA-MB-231 and MCF-7 cells (respectively ca. 50% and 42%) and 40% inhibition of cell migration. MicroRaman analysis of fixed cells upon exposure to this isoflavone unveiled its metabolic impact on both cell lines. Multivariate data analysis (unsupervised PCA) led to a clear discrimination between the control and DAID-exposed cells, with distinctive effects on their biochemical profile, particularly regarding DNA, lipids and protein components, in a cell-dependent way. This is the first reported study on the impact of dietary antioxidants on cancer cells by microRaman techniques.

Chemotherapeutic response to cisplatin-like drugs in human breast cancer cells probed by vibrational microspectroscopy.

Studies of drug-cell interactions in cancer model systems are essential in the preclinical stage of rational drug design, which relies on a thorough understanding of the mechanisms underlying cytotoxic activity and biological effects, at a molecular level. This study aimed at applying complementary vibrational spectroscopy methods to evaluate the cellular impact of two Pt(ii) and Pd(ii) dinuclear chelates with spermine (Pt2Spm and Pd2Spm), using cisplatin (cis-Pt(NH3)2Cl2) as a reference compound. Their effects on cellular metabolism were monitored in a human triple-negative metastatic breast cancer cell line (MDA-MB-231) by Raman and synchrotron-radiation infrared microspectroscopies, for different drug concentrations (2-8 µM) at 48 h exposure. Multivariate data analysis was applied (unsupervised PCA), unveiling drug- and concentration-dependent effects: apart from discrimination between control and drug-treated cells, a clear separation was obtained for the different agents studied - mononuclear vs. polynuclear, and Pt(ii) vs. Pd(ii). Spectral biomarkers of drug action were identified, as well as the cellular response to the chemotherapeutic insult. The main effect of the tested compounds was found to be on DNA, lipids and proteins, the Pd(ii) agent having a more significant impact on proteins while its Pt(ii) homologue affected the cellular lipid content at lower concentrations, which suggests the occurrence of distinct and unconventional pathways of cytotoxicity for these dinuclear polyamine complexes. Raman and FTIR microspectroscopies were confirmed as powerful non-invasive techniques to obtain unique spectral signatures of the biochemical impact and physiological reaction of cells to anticancer agents.

New strategies against prostate cancer--Pt(II)-based chemotherapy.

Prostate cancer is the second most common cancer worldwide and the sixth cause of cancer-related death in men. When hormone therapy fails to control tumour growth, castration-resistant prostate cancer (CRPC) occurs and chemotherapy drugs must be administered. Since 2004, docetaxel administration is the standard of care in metastatic CRPC, although it presents severe limitations such as acquired resistance and poor prognosis. An analogue (cabazitaxel) was approved by the FDA in 2010 as a second-line chemotherapeutic agent. Novel immuno- and hormonal therapy agents, as well as tumour vaccines, have been recently developed, but new strategies are still needed for effectively handling this type of neoplasia. Platinum compounds, in particular, have been the object of a growing interest, despite the former belief that they should have modest activity against prostate cancer. Compounds such as carboplatin, oxaliplatin or satraplatin, either alone or in combination, have lately shown promising results. In order to overcome the deleterious side-effects usually associated to these metal-based agents, several approaches have been followed with a view to optimise drug delivery and targeting, some of which showed considerable success in CRPC. Platinum drugs may therefore have an important role in the chemotherapeutic management of human metastatic castration-resistant prostate cancer, mostly in second-line strategies. The present review addresses the most relevant studies on platinum-based antineoplastic agents towards CRPC in the last decade--from first--and second-generation complexes to newly developed compounds.