5-Fluorouracil-loaded poly(ε-caprolactone) nanoparticles combined with phage E gene therapy as a new strategy against colon cancer

This work aimed to develop a new therapeutic approach to increase the efficacy of 5-fluorouracil (5-FU) in the treatment of advanced or recurrent colon cancer. 5-FU-loaded biodegradable poly(ε-caprolactone) nanoparticles (PCL NPs) were combined with the cytotoxic suicide gene E (combined therapy). The SW480 human cancer cell line was used to assay the combined therapeutic strategy. This cell line was established from a primary adenocarcinoma of the colon and is characterized by an intrinsically high resistance to apoptosis that correlates with its resistance to 5-FU. 5-FU was absorbed into the matrix of the PCL NPs during synthesis using the interfacial polymer disposition method. The antitumor activity of gene E from the phage ϕX174 was tested by generating a stable clone (SW480/12/E). In addition, the localization of E protein and its activity in mitochondria were analyzed. We found that the incorporation of 5-FU into PCL NPs (which show no cytotoxicity alone), significantly improved the drug's anticancer activity, reducing the proliferation rate of colon cancer cells by up to 40-fold when compared with the nonincorporated drug alone. Furthermore, E gene expression sensitized colon cancer cells to the cytotoxic action of the 5-FU-based nanomedicine. Our findings demonstrate that despite the inherent resistance of SW480 to apoptosis, E gene activity is mediated by an apoptotic phenomenon that includes modulation of caspase-9 and caspase-3 expression and intense mitochondrial damage. Finally, a strongly synergistic antiproliferative effect was observed in colon cancer cells when E gene expression was combined with the activity of the 5-FU-loaded PCL NPs, thereby indicating the potential therapeutic value of the combined therapy.

8-weeks training program attenuates mitochondrial oxidative stress in the liver of emotionally stressed rats

In recent years it has been shown that emotional stress induced by immobilization may change the balance between pro-oxidant and antioxidant factors inducing oxidative damage. On the other hand, contradictory views exist concerning the role of physical activity on redox metabolism. Consequently, the present work was designed to assess the influence of an 8-week moderate swimming training program in emotionally stressed rats. Sixty 1-month-old male albino Wistar rats weighing 125-135 g were used in this experimental study. They were divided into three groups, as Control (lot A; n=20), Stressed (lot B; n=20) and Stressed & Exercised (lot C; n=20). Rats were stressed by placing the animals in a 25 x 7 cm plastic bottle 1 h/day, 5 days a week for 8 weeks. Protein carbonyl content values in liver homogenates were significantly increased in stressed animals (0.58+/-0.02 vs 0.86+/-0.03; p=0.018) which clearly indicated that emotional stress was associated with oxidative stress. Ultrastructural alterations, predominantly mitochondrial swelling and the decrease of cristae number observed by electron microscopy represented direct evidence of membrane injury. The most striking feature of our study was that we also found differences between stressed rats and stressed rats that performed our 8 week training program. Consequently our results highlight the potential benefit of a moderate training program to reduce oxidative damage induced by emotional stress since it attenuated protein oxidation and mitochondrial alterations.

Peripheral inflammation increases the damage in animal models of nigrostriatal dopaminergic neurodegeneration: possible implication in Parkinson's disease incidence.

Inflammatory processes described in Parkinson’s disease (PD) and its animal models appear to be important in the progression of the pathogenesis, or even a triggering factor. Here we review that peripheral inflammation enhances the degeneration of the nigrostriatal dopaminergic system induced by different insults; different peripheral inflammations have been used, such as IL-1β and the ulcerative colitis model, as well as insults to the dopaminergic system such as 6-hydroxydopamine or lipopolysaccharide. In all cases, an increased loss of dopaminergic neurons was described; inflammation in the substantia nigra increased, displaying a great activation of microglia along with an increase in the production of cytokines such as IL-1β and TNF-α. Increased permeability or disruption of the BBB, with overexpression of the ICAM-1 adhesion molecule and infiltration of circulating monocytes into the substantia nigra, is also involved, since the depletion of circulating monocytes prevents the effects of peripheral inflammation. Data are reviewed in relation to epidemiological studies of PD.

Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease.

Introduction. Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia. Methods. We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q10 levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX™ and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker™ Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells. Results. We found reduced levels of coenzyme Q10, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy. Conclusions. These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.

Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients.

Background. DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Methods. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results. Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. Conclusions. An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

INTRODUCTION Radiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects. METHODS Intrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments. RESULTS Radiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects. CONCLUSION After radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

BACKGROUND. Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. METHODS. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. RESULTS. Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. CONCLUSIONS. A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Mitochondrial haplogroups and polymorphisms reveal no association with sporadic prostate cancer in a southern European population.

BACKGROUND It is known that mitochondria play an important role in certain cancers (prostate, renal, breast, or colorectal) and coronary disease. These organelles play an essential role in apoptosis and the production of reactive oxygen species; in addition, mtDNA also reveals the history of populations and ancient human migration. All these events and variations in the mitochondrial genome are thought to cause some cancers, including prostate cancer, and also help us to group individuals into common origin groups. The aim of the present study is to analyze the different haplogroups and variations in the sequence in the mitochondrial genome of a southern European population consisting of subjects affected (n = 239) and non-affected (n = 150) by sporadic prostate cancer. METHODOLOGY AND PRINCIPAL FINDINGS Using primer extension analysis and DNA sequencing, we identified the nine major European haplogroups and CR polymorphisms. The frequencies of the haplogroups did not differ between patients and control cohorts, whereas the CR polymorphism T16356C was significantly higher in patients with PC compared to the controls (p = 0.029). PSA, staging, and Gleason score were associated with none of the nine major European haplogroups. The CR polymorphisms G16129A (p = 0.007) and T16224C (p = 0.022) were significantly associated with Gleason score, whereas T16311C (p = 0.046) was linked with T-stage. CONCLUSIONS AND SIGNIFICANCE Our results do not suggest that mtDNA haplogroups could be involved in sporadic prostate cancer etiology and pathogenesis as previous studies performed in middle Europe population. Although some significant associations have been obtained in studying CR polymorphisms, further studies should be performed to validate these results.

The MRC1/CD68 Ratio Is Positively Associated with Adipose Tissue Lipogenesis and with Muscle Mitochondrial Gene Expression in Humans.

BACKGROUND Alternative macrophages (M2) express the cluster differentiation (CD) 206 (MCR1) at high levels. Decreased M2 in adipose tissue is known to be associated with obesity and inflammation-related metabolic disturbances. Here we aimed to investigate MCR1 relative to CD68 (total macrophages) gene expression in association with adipogenic and mitochondrial genes, which were measured in human visceral [VWAT, n = 147] and subcutaneous adipose tissue [SWAT, n = 76] and in rectus abdominis muscle (n = 23). The effects of surgery-induced weight loss were also longitudinally evaluated (n = 6). RESULTS MCR1 and CD68 gene expression levels were similar in VWAT and SWAT. A higher proportion of CD206 relative to total CD68 was present in subjects with less body fat and lower fasting glucose concentrations. The ratio MCR1/CD68was positively associated with IRS1gene expression and with the expression of lipogenic genes such as ACACA, FASN and THRSP, even after adjusting for BMI. The ratio MCR1/CD68 in SWAT increased significantly after the surgery-induced weight loss (+44.7%; p = 0.005) in parallel to the expression of adipogenic genes. In addition, SWAT MCR1/CD68ratio was significantly associated with muscle mitochondrial gene expression (PPARGC1A, TFAM and MT-CO3). AT CD206 was confirmed by immunohistochemistry to be specific of macrophages, especially abundant in crown-like structures. CONCLUSION A decreased ratio MCR1/CD68 is linked to adipose tissue and muscle mitochondrial dysfunction at least at the level of expression of adipogenic and mitochondrial genes.

Incidence of liver damage of uncertain origin in HIV patients not co-infected with HCV/HBV.

BACKGROUND AND AIMS Several studies have reported that a significant number of HIV patients not co-infected with HCV/HBV develop liver damage of uncertain origin (LDUO). The objective of our study was to evaluate the incidence of and risk factors for the development of LDUO in HIV infected patients not co-infected with HCV/HBV. METHODS Prospective longitudinal study that included HIV-infected patients free of previous liver damage and viral hepatitis B or C co-infections. Patients were followed up at 6-monthly intervals. Liver stiffness was measured at each visit. Abnormal liver stiffness (ALS) was defined as a liver stiffness value greater than 7.2 kPa at two consecutive measurements. For patients who developed ALS, a protocol was followed to diagnose the cause of liver damage. Those patients who could not be diagnosed with any specific cause of liver disease were diagnosed as LDUO and liver biopsy was proposed. RESULTS 210 patients matched the inclusion criteria and were included. 198 patients completed the study. After a median (Q1-Q3) follow-up of 18 (IQR 12-26) months, 21 patients (10.6%) developed ALS. Of these, fifteen patients were diagnosed as LDUO. The incidence of LDUO was 7.64 cases/100 patient-years. Histological studies were performed on ten (66.6%) patients and all showed liver steatosis. A higher HOMA-IR value and body mass index were independently associated with the development of LDUO. CONCLUSION We found a high incidence of LDUO in HIV-infected patients associated with metabolic risk factors. The leading cause of LDUO in our study was non-alcoholic fatty liver disease.

Diets based on virgin olive oil or fish oil but not on sunflower oil prevent age-related alveolar bone resorption by mitochondrial-related mechanisms.

BACKGROUND/OBJECTIVES Aging enhances frequency of chronic diseases like cardiovascular diseases or periodontitis. Here we reproduced an age-dependent model of the periodontium, a fully physiological approach to periodontal conditions, to evaluate the impact of dietary fat type on gingival tissue of young (6 months old) and old (24 months old) rats. METHODS/FINDINGS Animals were fed life-long on diets based on monounsaturated fatty acids (MUFA) as virgin olive oil, n-6 polyunsaturated fatty acids (n-6PUFA), as sunflower oil, or n-3PUFA, as fish oil. Age-related alveolar bone loss was higher in n-6PUFA fed rats, probably as a consequence of the ablation of the cell capacity to adapt to aging. Gene expression analysis suggests that MUFA or n-3PUFA allowed mitochondria to maintain an adequate turnover through induction of biogenesis, autophagy and the antioxidant systems, and avoiding mitochondrial electron transport system alterations. CONCLUSIONS The main finding is that the enhanced alveolar bone loss associated to age may be targeted by an appropriate dietary treatment. The mechanisms involved in this phenomenon are related with an ablation of the cell capacity to adapt to aging. Thus, MUFA or n-3PUFA might allow mitochondrial maintaining turnover through biogenesis or autophagy. They might also be able to induce the corresponding antioxidant systems to counteract age-related oxidative stress, and do not inhibit mitochondrial electron transport chain. From the nutritional and clinical point of view, it is noteworthy that the potential treatments to attenuate alveolar bone loss (a feature of periodontal disease) associated to age could be similar to some of the proposed for the prevention and treatment of cardiovascular diseases, a group of pathologies recently associated with age-related periodontitis.

Use of the comet test to assess DNA damage in children with ataxia-telangiectasia and their relatives.

The electrophoresis of cells in alkaline medium (comet assay) is a valid technique for quantifying DNA damage in patients with ataxia-telangiectasia and their relatives.