Proteomic profiles in hyperandrogenic syndromes.

BACKGROUND: Polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) represent the most common causes of hyperandrogenism. Although the etiopathogeneses of these syndromes are different, they share many clinical and biochemical signs, such as hirsutism, acne, and chronic anovulation. Experimental data have shown that peripheral T-lymphocytes function as molecular sensors, being able to record molecular signals either at staminal and mature cell levels, or hormones at systemic levels. METHODS: Twenty PCOS women and 10 CAH with 21-hydroxylase deficiency, aged between 18-35 yr, were studied. T-cells purified from all patients and 20 healthy donors have been analyzed by 2-dimensional gel electrophoresis. Silver-stained proteomic map of each patient was compared with a control map obtained by pooling protein samples of the 20 healthy subjects. RESULTS: Spots of interest were identified by peptide mass fingerprint. Computer analysis evidenced several peptidic spots significantly modulated in all patients examined. Some proteins were modulated in both syndromes, others only in PCOS or in CAH. These proteins are involved in many physiological processes as the functional state of immune system, the regulation of the cytoskeleton structure, the oxidative stress, the coagulation process, and the insulin resistance. CONCLUSION: Identification of the physiological function of these proteins could help to understand ethiopathogenetic mechanisms of hyperandrogenic syndromes and its complications.

Modulation of proteomic profile in H295R adrenocortical cell line induced by mitotane.

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p'-DDD), is a compound that represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remains still unknown. In this study, we have already shown a greater impairment in the first steps of the steroidogenesis and recognized a little effect on cell cycle. We also evaluated the variation of proteomic profile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction after treatment with mitotane. In total cell extracts, triose phosphate isomerase, alpha-enolase, D-3-phosphoglycerate dehydrogenase, peroxiredoxin II and VI, heat shock protein 27, prohibitin, histidine triad nucleotide-binding protein, and profilin-1 showed a different expression. In the mitochondrial fraction, the following proteins appeared to be down regulated: aldolase A, peroxiredoxin I, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-beta isoform II, heat shock cognate 71 kDa protein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heat shock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic study on the mitotane effects on ACC. It permits to identify some protein classes affected by the drug involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis.

Mitotane increases the radiotherapy inhibitory effect and induces G2-arrest in combined treatment on both H295R and SW13 adrenocortical cell lines.

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (o,p'-DDD) is an agent with adrenotoxic effect, which is able to block cortisol synthesis. This drug and radiotherapy are used also in adrenal cancer treatment even if their biological action in this neoplasia remains unknown. We investigated the effects of o,p'-DDD and ionizing radiations (IR) on cell growth inhibition and cell cycle perturbation in H295R and SW13 adrenocortical cancer cells. Both cell lines were irradiated at a 6 Gy dose and were treated with o,p'-DDD 10(-5) M separately and with IR/o,p'-DDD in combination. This combination treatment induced an irreversible inhibition of cell growth in both adrenocortical cancer cells. Cell cycle analysis showed that IR alone and IR/o,p'-DDD in combination induced the cell accumulation in the G2 phase. At 120 h after IR, the cells were able to recover the IR-induced G2 block while cells treated with IR/o,p'-DDD were still arrested in G2 phase. In order to study the molecular mechanism involved in the G2 irreversible arrest, we have considered the H295R cell line showing the highest inhibition of cell proliferation associated with a noteworthy G2 arrest. In these cells, cyclin B1 and Cdk2 proteins were examined by western blot and Cdk2 kinase activity measured by assay kit. The H295R cells treated with IR/o,p'-DDD shared an increase in cyclin B1 amount as the coimmunoprecipitation of Cdc2-cyclin B1 complex. The kinase activity also shows an increase in the treated cells with combination therapy. Moreover, in these cells, sequence analysis of p53 revealed a large deletion of exons 8 and 9. The same irreversible block on G2 phase, induced by IR/o,p'-DDD treatment, happened in H295R cells with restored wild-type p53 suggesting that this mechanism is not mediated by p53 pathway.

c-myc antisense oligodeoxynucleotides enhance the efficacy of cisplatin in melanoma chemotherapy in vitro and in nude mice.

This study was designed to assess the efficacy of a new antimelanoma therapeutic strategy that relies on the use of a c-myc antisense 15-mer phosphorothioate oligodeoxynucleotide ([S]ODN), in combination with cisplatin (cis-diamminedichloroplatinum; DDP), which is currently used in the clinical management of melanoma patients. Proliferation and colony formation of melanoma cells were both inhibited by the DDP/c-myc antisense [S]ODN combination to a greater extent than that observed with either agent alone. Inhibition was most effective when DDP was followed by c-myc antisense [S]ODNs. Cell cycle flow cytometric analysis of cells exposed to the two agents either alone or in combination demonstrated that (a) c-myc antisense [S]ODNs induced an accumulation of cells in S phase and apoptosis in a fraction of the cells, detectable at day 5 after the beginning of treatment; (b) DDP induced a block in G2-M phase detectable at day 1, which was partially recovered, and apoptosis similar in extent to that induced by c-myc antisense [S]ODNs; and (c) DDP and c-myc antisense [S]ODNs together induced arrest in G2-M phase, which was maximum at day 3, i.e., delayed as compared to the block induced by DDP. The combination induced a higher percentage of apoptosis, evident at day 3 from the start of treatment, that correlated with a marked reduction in Bcl-2 expression. Mice bearing human melanoma xenografts and treated sequentially with DDP and c-myc antisense [S]ODNs showed a higher inhibition of tumor growth, reduction in the number of lung metastases, and increase in life span compared with those treated with either agent alone. Together, these data lend support to the development of anticancer therapies involving oncogene-targeted antisense ODNs and conventional antineoplastic drugs.

Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27(kip1) levels.

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G(1) phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27(kip1) levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27(kip1) in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G(1) arrest and activates apoptosis by increasing p27(kip1) content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G(1) checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.

Fungal infections in patients undergoing bone marrow transplantation: an approach to a rational management protocol.

Candida sp. and Aspergillus sp. are the most common fungal pathogens causing infection in bone marrow transplant recipients and represent an increasing cause of morbidity and mortality. At this time there is no generally accepted rule for the antifungal management of these complications. Antifungal drugs in immunocompromised patients are usually administered for prophylaxis, for therapy of specific infections or for empirical or preemptive therapy. The present article reports schedules of administrations and pediatric and adult dosages of the main antifungal drugs presently available, (fluconazole, itraconazole, amphotericin B deoxycholate, lipid formulations of amphotericin B and flucytosine), together with their spectrum of action and main toxicities. Thereafter, the available information about prevention and treatment of fungal infections in bone marrow transplant recipients is summarized. Briefly, fluconazole remains the drug of choice for prevention of Candida infections in bone marrow transplant recipients, while itraconazole has been seldomly used for this indication, due to erratic oral absorption. However, new itraconazole formulations are being studied, that might disclose new clinical perspectives, due to improved bioavailability. The duration of prophylaxis is still an open issue. Resistance to the new azoles may become a problem in the near future. For this reason, it is likely that the approach to the use of these new drugs should be similar to the one commonly used for antibacterial drugs, i.e. based on pathogen-related, drug-related and host-related factors. Mainly due to lack of diagnostic tools, very little studies have been performed for prevention of aspergillosis. Available data seem to show that there might be a role for low-dose intravenous amphotericin B, which has shown to be effective for secondary prophylaxis. Itraconazole and intranasal amphotericin B have been studied, as well. Although fluconazole and itraconazole (in the rare instances in which the oral route is reliable) can also have therapeutic indications, both for empirical and for specific therapy, amphotericin B (with or without flucytosine) remains the main therapeutic option. New antifungal drugs and new supportive strategies (i.e. role of hematopoietic growth factors) are in the research pipeline and will hopefully disclose new perspectives in the near future.

Antifungal prophylaxis with azole derivatives.

In recent years, several reports have underlined the increasing role of fungal infections as a cause of morbidity and mortality in hospitalised patients. For this reason, and also in light of the high mortality rate associated with these infections, chemoprophylaxis has been advocated by several authors. The available evidence suggests that both fluconazole and itraconazole are able to decrease candida colonisation and infection, when compared with placebo or with nonabsorbable antifungals. Data seem also to suggest that a decrease in fungus-related mortality can be achieved with prophylaxis, although with little effect on overall mortality, probably because of the importance of severe underlying diseases. Itraconazole proved to be effective in the prevention of fungal infections, including invasive aspergillosis, although with increased incidence of side-effects, often leading to treatment discontinuation. The other side of the coin is that antifungal prophylaxis might have untoward effects, such as the selection of triazole-resistant Candida strains or the induction of resistance. In addition, some authors have suggested that the use of triazoles might modulate the pattern of infecting organisms in cancer patients, increasing the risk of both aspergillosis and bacteremia. In conclusion, antifungal prophylaxis with triazole antifungals should be used with caution, only in patients at high risk for invasive fungal infections. These include allogeneic bone marrow transplant patients (especially those with mismatched or unrelated donors), acute myeloid leukaemia patients treated with high-dose cytarabine (C-ara), very-low-birth-weight infants, patients with chronic granulomatous disease, and high-risk surgical and intensive-care unit patients.

Effects of poly(ADP-ribose) polymerase inhibition on cell death and chromosome damage induced by VP16 and bleomycin.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein involved in cellular response to various genotoxic agents. To understand the role of PARP in the mechanisms which lead from specific DNA damage to cell death, we studied the effects of PARP inhibition in human lymphoblasts damaged with bleomycin (BLM) and VP16. These agents can induce DNA breakage but through different mechanisms, enabling the study of the different effects of PARP in inducing apoptosis in damaged cells. We demonstrate that in lymphoblasts VP16 treatment induces apoptosis to a greater extent than BLM treatment, and that PARP inhibition reduces VP16-induced apoptosis whereas it has no effect on BLM-induced apoptosis. After VP16 treatment with PARP inhibition, a reduction in the depletion of the proliferative compartment and a G2/M phase arrest are observed. Therefore, the increase in cell viability and the reduction in chromosome damage may both be the result of a prolonged DNA repair time. Hence, PARP appears to play a significant role in VP16-induced apoptosis and not in BLM-induced apoptosis. Since apoptosis is important in tumor treatment these findings might be useful when considering the combined employment of PARP inhibition with antineoplastic drugs.

EGF-R expression in ductal breast cancer: proliferation and prognostic implications.

We studied epidermal growth factor receptor (EGF-R) expression in relation to steroid receptor status, flow cytometric DNA content and S-phase fraction (%S) in a selected case series of 129 ductal primary operable breast cancer to determine the possible role of EGF-R in prognosis assessment. EGF-R expression was positively related with proliferation activity, suggesting that EGF-R could be involved in the regulation of breast cancer cell growth. We found about 80% of highly proliferating DNA aneuploid tumors in the EGF-R positive category, while the EGF-R negative tumors showed a lower frequency of highly proliferating DNA aneuploid tumors (57%), confirming the important role of EGF-R in breast cancer aggressiveness and progression. No relationship between EGF-R expression and steroid receptor status was observed. To better understand how EGF-R and estrogen receptor (ER) operate together to stimulate breast cancer cell growth, we analyzed the %S in the two groups of ER negative (ER-) and ER positive (ER+) tumors, stratifying the patients on the basis of EGF-R tumor positivity. Here breast tumor proliferation activity seems mainly to be induced by the stimulus of EGF-R, the %S values of the EGF-R negative tumors in the ER- and ER+ groups being 6.1 and 6.9%, respectively. Instead, the median %S of EGF-R positive tumors was 10% in the ER- class and 14% in the ER+ group. The analysis of the percentages of 5-year patient disease free survival were 84% for patients with EGF-R negative tumors and 61% for patients with EGF-R positive lesions, respectively. The data reported here further show the crucial role of EGF-R in breast cancer cell growth and that the EGF-R overexpression is indicative of a poor prognosis.

Biological indicators of aggressiveness in T1 ductal invasive breast cancer.

BACKGROUND: Apoptosis plays an important role in the maintenance of tissue homeostasis. When defective, this process could contribute to the pathogenesis and the progression of tumors. On this basis, we investigated the combined effect of Bcl-2 and Bax expression, known regulators of apoptotic processes, in the activation of apoptosis in breast cancer. Their relationship with DNA content and proliferative activity was also studied in order to more accurately define breast cancer patients' prognosis and treatment. MATERIALS AND METHODS: In this study we investigated 76 T1 ductal invasive breast cancers and 76 normal epithelium samples for Bcl-2 and Bax expression by immunohistochemistry, for apoptosis by tunel assay and for DNA content and proliferative activity by flow cytometry. RESULTS: High levels of Bcl-2 were associated with prevention of apoptosis. Conversely high Bax expression was found to be related to apoptosis. DNA ploidy was strictly related to the proliferative activity. In addition most of the tumors showing high Bcl-2 expression were aneuploid. CONCLUSION: This report suggests that Bax over-expression could accelerate apoptotic cell death by counteracting the ability of Bcl-2 to inhibit apoptosis. These data also suggest that the ratio Bcl-2/Bax and their relationship with the activation of apoptosis could be used as predictive indicators of breast cancer patients' prognosis and response to conventional therapy.

[Designing a therapeutic education program for patients with lymphedema: live with lymphedema]. / Conception d'un programme d'éducation thérapeutique pour les patients porteurs de lymphœdème: « vivre avec un lymphœdème ¼

BACKGROUND: Lymphedema is a chronic condition considered to be rare in its primary form and potentially frequent in women after breast surgery for cancer: 27,000 new cases annually. Therapeutic management is a serious challenge. In France, the health authorities (Haute Autorité de santé [HAS]) have recently proposed that appropriate management practices for lymphedema include "patient education". The HAS and the National institute for health care prevention and education also published a methodology guide devoted to structuring a therapeutic education program for patients with chronic disease. Current hospital regulations state that this education program is part of the care to be delivered to patients with chronic disease and that it must comply with the national directives. The purpose of our present work was to present the concept and the contents of a patient education program entitled "Live with lymphedema" designed for patients with lymphedema and developed within the inpatient-outpatient network GRANTED in Sud-Isère. METHODS: A standard detailed educative approach was applied. It was designed after the educational program for patients with lower limb arterial occlusive disease authorized by the Rhône-Alpes regional health agency. It was adapted to the specific problematic of patients with lymphedema, including medical management, rehabilitation, dermatology and nutritional aspects. It was developed in cooperation with patients and favors local associative actions. RESULTS: The specifically structured program included three therapeutic education consultations and five workshops. Less than one year after its institution, more than 30 patients have participated in the program. DISCUSSION: We report a structured patient education program designed for patients with lymphedema. This program was authorized by the Rhône-Alpes regional health agency in March 2011 and is in compliance with the national directives and HAS guidelines.

New rehabilitation program for intermittent claudication: Interval training with active recovery: pilot study.

BACKGROUND: Peripheral arterial disease (PAD) is one of the complications of atherosclerosis. Intermittent claudication is the second stage of PAD. In controlled studies on patients with Stage II PAD, intensive rehabilitation training has proved effective for improving the walking distance in this population. The objective of this prospective study was to determine the effects of treadmill interval training followed by active recovery (low-intensity exercise). METHODS AND RESULTS: Eleven patients with Stage II peripheral arterial disease were included in a rehabilitation program (mean age 68.3±10.3 years) for five days a week during two weeks including global exercises, exercises below and above the level of injury. The interval training program consisted of treadmill training for 30minutes twice a day (morning and evening) with a progressively increased intensity: the first week speed was increased and the second week slope was increased. Each session included five six-minute cycles. Each cycle was made of three minutes of active workout followed by three minutes of active recovery. RESULTS: All patients improved their walking distance, from a mean of 610 m (120-1930) at the beginning of the program to a mean of 1252 m (320-2870) at the end (P=0.003). All patients were very motivated by the rehabilitation training program No adverse event was reported. CONCLUSION: This study showed that an interval training program with active recovery was effective and safe for patients with Stage II peripheral arterial disease, the patients' motivation was high. This study must now be validated by a clinical trial.