Leptin receptor 170 kDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance

[EN] To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2-dependent leptin signalling and signal transducer and activator of transcription 3 (STAT3) phosphorylation and reduce AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Deltoid and vastus lateralis muscle biopsies were obtained from 20 men: 10 non-obese control subjects (mean +/- s.d. age, 31 +/- 5 years; height, 184 +/- 9 cm; weight, 91 +/- 13 kg; and percentage body fat, 24.8 +/- 5.8%) and 10 obese (age, 30 +/- 7 years; height, 184 +/- 8 cm; weight, 115 +/- 8 kg; and percentage body fat, 34.9 +/- 5.1%). Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P < 0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPKalpha and ACCbeta phosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPKalpha and ACCbeta phosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P < 0.05). In summary, skeletal muscle leptin receptors and leptin signalling are reduced in obesity, particularly in the leg muscles.

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

[EN] 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.

Prediction of clinical toxicity in locally advanced head and neck cancer patients by radio-induced apoptosis in peripheral blood lymphocytes (PBLs)

[EN] Head and neck cancer is treated mainly by surgery and radiotherapy. Normal tissue toxicity due to x-ray exposure is a limiting factor for treatment success. Many efforts have been employed to develop predictive tests applied to clinical practice. Determination of lymphocyte radio-sensitivity by radio-induced apoptosis arises as a possible method to predict tissue toxicity due to radiotherapy. The aim of the present study was to analyze radio-induced apoptosis of peripheral blood lymphocytes in head and neck cancer patients and to explore their role in predicting radiation induced toxicity. Seventy nine consecutive patients suffering from head and neck cancer, diagnosed and treated in our institution, were included in the study. Toxicity was evaluated using the Radiation Therapy Oncology Group scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody. Radiation-induced apoptosis increased in order to radiation dose and fitted to a semi logarithmic model defined by two constants: α and β. α, as the origin of the curve in the Y axis determining the percentage of spontaneous cell death, and β, as the slope of the curve determining the percentage of cell death induced at a determined radiation dose, were obtained. β value was statistically associated to normal tissue toxicity in terms of severe xerostomia, as higher levels of apoptosis were observed in patients with low toxicity (p = 0.035; Exp(B) 0.224, I.C.95% (0.060-0.904)). These data agree with our previous results and suggest that it is possible to estimate the radiosensitivity of peripheral blood lymphocytes from patients determining the radiation induced apoptosis with annexin V/propidium iodide staining. β values observed define an individual radiosensitivity profile that could predict late toxicity due to radiotherapy in locally advanced head and neck cancer patients. Anyhow, prospective studies with different cancer types and higher number of patients are needed to validate these results.

Prediction of clinical toxicity in localized cervical carcinoma by radio-induced apoptosis study in peripheral blood lymphocytes (PBLs)

[EN] Background: Cervical cancer is treated mainly by surgery and radiotherapy. Toxicity due to radiation is a limiting factor for treatment success. Determination of lymphocyte radiosensitivity by radio-induced apoptosis arises as a possible method for predictive test development. The aim of this study was to analyze radio-induced apoptosis of peripheral blood lymphocytes. Methods: Ninety four consecutive patients suffering from cervical carcinoma, diagnosed and treated in our institution, and four healthy controls were included in the study. Toxicity was evaluated using the Lent-Soma scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24, 48 and 72 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide to determine early and late apoptosis. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody. Results: Radiation-induced apoptosis (RIA) increased with radiation dose and time of incubation. Data strongly fitted to a semi logarithmic model as follows: RIA = βln(Gy) + α. This mathematical model was defined by two constants: α, is the origin of the curve in the Y axis and determines the percentage of spontaneous cell death and β, is the slope of the curve and determines the percentage of cell death induced at a determined radiation dose (β = ΔRIA/Δln(Gy)). Higher β values (increased rate of RIA at given radiation doses) were observed in patients with low sexual toxicity (Exp(B) = 0.83, C.I. 95% (0.73-0.95), p = 0.007; Exp(B) = 0.88, C.I. 95% (0.82-0.94), p = 0.001; Exp(B) = 0.93, C.I. 95% (0.88-0.99), p = 0.026 for 24, 48 and 72 hours respectively). This relation was also found with rectal (Exp(B) = 0.89, C.I. 95% (0.81-0.98), p = 0.026; Exp(B) = 0.95, C.I. 95% (0.91-0.98), p = 0.013 for 48 and 72 hours respectively) and urinary (Exp(B) = 0.83, C.I. 95% (0.71-0.97), p = 0.021 for 24 hours) toxicity. Conclusion: Radiation induced apoptosis at different time points and radiation doses fitted to a semi logarithmic model defined by a mathematical equation that gives an individual value of radiosensitivity and could predict late toxicity due to radiotherapy. Other prospective studies with higher number of patients are needed to validate these results.