El reclutamiento en los estudios clínicos, clave para mejorar los ingresos económicos en investigación biomédica / Recruitment in clinical studies key for improve income in biomedical research

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The multikinase inhibitor Sorafenib induces apoptosis and sensitises endometrial cancer cells to TRAIL by different mechanisms.

Sorafenib induces apoptosis and enhances Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-induced cell killing of tumoural cells. We have investigated the effects of the multikinase inhibitor Sorafenib alone or in combination with TRAIL and agonistic Fas antibodies on endometrial carcinoma cells. We have also focused on the search of the differential molecular mechanisms by which Sorafenib induces cell death and the ones involved in sensitisation to TRAIL. In the present study, we show that Sorafenib induces apoptosis of both endometrial cancer cell lines and human primary cultures and sensitises these cells to TRAIL and agonistic Fas antibodies (aFas)-induced apoptosis. However, Raf/MEK/ERK inhibition by Sorafenib was not responsible for Sorafenib cell death or TRAIL sensitisation of endometrial cancer cells. Sorafenib treatment correlated with a downregulation of both FLICE-Inhibitory Protein (FLIP) and myeloid cell leukaemia-1 (Mcl-1), caused by a proteasomal degradation of both proteins. We evaluated the contribution of FLIP and Mcl-1 downregulation in apoptosis triggered by Sorafenib alone or Sorafenib plus TRAIL. Interestingly, cell death caused by Sorafenib was mediated by downregulation of Mcl-1, but not by FLIP. In contrast, we found that Sorafenib sensitisation of endometrial carcinoma cells to TRAIL- and Fas-induced apoptosis was dependent on FLIP but not on Mcl-1 downregulation. Altogether, we discern the dual mechanisms by which Sorafenib causes cell death from those involved in death receptor sensitisation.

Molecular pathology of endometrial carcinoma: practical aspects from the diagnostic and therapeutic viewpoints.

This article reviews the main molecular alterations involved in endometrial carcinoma. Five molecular features (microsatellite instability, and mutations in the PTEN, k-RAS, PIK3CA and beta-catenin genes) are characteristic of endometrioid carcinomas, whereas non-endometrioid carcinomas show alterations of p53, loss of heterozygosity (LOH) on several chromosomes, as well as other molecular alterations (STK15, p16, E-cadherin and C-erb B2). The review also covers the phenomenon of apoptosis resistance, as well as the results obtained from cDNA array studies, and the perspectives for targeted therapies. A group of practical applications of molecular pathology techniques are also mentioned: diagnosis of hereditary non-polyposis colon cancer syndrome in patients with endometrial carcinoma; evaluation of precursor lesions; prognosis; diagnosis, particularly for synchronous endometrioid carcinomas of the uterus and the ovaries; and targeted therapies.

Effect of proteasome inhibitors on proliferation and apoptosis of human cutaneous melanoma-derived cell lines.

BACKGROUND: Cutaneous malignant melanoma is an aggressive type of skin cancer which causes disproportionate mortality in young and middle-aged adults. Once disseminated, melanoma can be considered an incurable disease, highly resistant to standard antineoplastic treatment, such as chemotherapy or radiation therapy. The proteasome represents a novel target for cancer therapy that can potentially be used in melanoma. OBJECTIVES: To assess the effect of four structurally different proteasome inhibitors on human cutaneous melanoma-derived cell lines. METHODS: Sixteen human cutaneous melanoma-derived cell lines which are original were obtained from patients who were treated by two of the authors. Cells were cultured, exposed to proteasome inhibitors (bortezomib, ALLN, MG-132 and epoxomicin) and then assayed for cell cycle and cell death analyses. RESULTS: Proteasome inhibitors inhibited the in vitro growth of melanoma cells, and this effect was due to a reduction in cell proliferation rate and an induction of both caspase-dependent and caspase-independent cell death. Moreover, release of apoptosis-inducing factor was observed in the presence of the broad-specificity caspase inhibitor BAF (Boc-D-fmk). In addition, the four different proteasome inhibitors induced caspase 2 processing. CONCLUSIONS: This study provides information regarding the in vitro effects of proteasome inhibitors on melanoma cell lines, and the molecular mechanisms involved. It also gives support to the future use of such inhibitors in the treatment of patients with melanoma, either administered alone or in combination with other drugs.

CK2 controls TRAIL and Fas sensitivity by regulating FLIP levels in endometrial carcinoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising antineoplastic agent because of its ability to selectively kill tumoral cells. However, some cancer cells are resistant to TRAIL-induced apoptosis. We have previously demonstrated that in endometrial carcinoma cells such resistance is caused by elevated FLICE-inhibitory protein (FLIP) levels. The present study focuses on the mechanisms by which FLIP could be modulated to sensitize endometrial carcinoma cells to TRAIL-induced apoptosis. We find that inhibition of casein kinase (CK2) sensitizes endometrial carcinoma cells to TRAIL- and Fas-induced apoptosis. CK2 inhibition correlates with a reduction of FLIP protein, suggesting that CK2 regulates resistance to TRAIL and Fas by controlling FLIP levels. FLIP downregulation correlates with a reduction of mRNA and is prevented by addition of the MG-132, suggesting that CK2 inhibition results in a proteasome-mediated degradation of FLIP. Consistently, forced expression of FLIP restores resistance to TRAIL and Fas. Moreover, knockdown of either FADD or caspase-8 abrogates apoptosis triggered by inhibition of CK2, indicating that CK2 sensitization requires formation of functional DISC. Finally, because of the possible role of both TRAIL and CK2 in cancer therapy, we demonstrate that CK2 inhibition sensitizes primary endometrial carcinoma explants to TRAIL apoptosis. In conclusion, we demonstrate that CK2 regulates endometrial carcinoma cell sensitivity to TRAIL and Fas by regulating FLIP levels.

An acquired inhibitor that produced a delay of fibrinopeptide B release in an asymptomatic patient.

An asymptomatic, 29-year-old woman was referred to our hospital before surgery because in the basic study of hemostasis she showed a prolonged thrombin time (TT) and a normal reptilase time (RT). She had not received any anticoagulants so, to account for these abnormal results the presence of an inhibitor or a dysfibrinogenemia was suspected. A 1:1 mixture of the patient's plasma with control plasma did not correct the TT. Dysfibrinogenemia was excluded because the defibrinated plasma retained the inhibitory activity when mixed with normal plasma. When 0.02 mg/ml of Protamine Sulphate (a concentration that neutralizes 1 U/mL of heparin in normal plasma) was added to the patient's plasma, the inhibitory activity did not disappear. IgG from the patient and from normal serum was isolated. The patient's IgG was able to prolong the TT of a normal plasma and of a purified fibrinogen. The patient IgG did not impair the catalytic activity of thrombin, because no difference was observed in the hydrolysis of S-2238 by 1 U NIH human thrombin with normal or patient IgG. The time course of the thrombin-mediated fibrinopeptide-release from normal fibrinogen with the patient's IgG, showed a delay in the fibrinopeptide B (FPB) release without affecting the fibrinopeptide A (FPA) release. This patient has an IgG antibody that delays fibrinopeptide B release of fibrinogen.

Homozygotes for prothrombin gene 20210 A allele in a thrombophilic family without clinical manifestations of venous thromboembolism.

BACKGROUND AND OBJECTIVE: A new genetic risk factor for venous thromboembolism has recently been described which involves a G to A transition at position 20210 in the 3' untranslated region of the prothrombin gene. To date, only a few homozygotes for this mutation have been reported and in most of cases, they suffered from thrombotic disease. Here, we describe a pedigree including both heterozygous and homozygous subjects for prothrombin (PT) 20210 A. DESIGN AND METHODS: This family was recruited in 1996 as part of our GAIT (Genetic Analysis of Idiopathic Thrombophilia) project. To qualify for the GAIT study, a pedigree was required to have at least 10 living individuals in three or more generations (i.e. extended pedigree). The pedigrees were selected through probands with idiopathic thrombophilia. A complete set of plasma and DNA determinations related to hemostasis was performed on this family. RESULTS: The plasma studies yielded normal results in all of the individuals. The family members who had a history of thromboembolism were heterozygous carriers of the PT 20210 A variant. In addition, 4 relatives who were heterozygous, and two who were homozygous for this A allele, failed to show clinical manifestations. These two homozygotes were 51 and 19 years old. INTERPRETATION AND CONCLUSIONS: This case exemplifies the complexity of thrombotic disease since individuals homozygous for a mutant gene do not exhibit symptoms while heterozygous individuals often do exhibit the disease. This case suggests that the new genetic risk factor for thrombosis (i.e. PT 20210 A) may not be as strong as most of the previously described genetic risk factors.

The prothrombin 20210A allele is the most prevalent genetic risk factor for venous thromboembolism in the Spanish population.

We investigated the prevalence of the new recently reported mutation in the prothrombin gene (20210 A) in a sample of 116 unrelated patients with venous thromboembolism. We found 20 heterozygous carriers (17.2%, CI 95% 10.4-21.1). In comparison, we observed 13 carriers among 201 healthy unmatched controls (6.5%, CI 3.5-10.8). The 20210 A mutation seems to increase the risk of venous thrombosis 3-fold (odds ratio 3.1, 95% CI 1.4-6.6). Considering only patients with a first event (n = 62) the OR was 2.0 (p = 0.18, NS) while those with recurrent events (n = 54) showed an OR of 5.9 (95% CI 2.5-14.4). A majority of heterozygous patients (55%) presented a second thrombophilic factor and 60% of affected females had their first event before 30 years of age, while on oral contraceptive treatment. The prevalence found in this study for healthy people is the highest reported to date. The 20210 A variant appears to be the most prevalent genetic risk factor among patients with thrombosis in our geographical area.

Low levels of tissue factor pathway inhibitor (TFPI) in two out of three members of a family with thrombophilia.

We describe a family with severe thrombosis of early onset in two siblings and in their father. Low levels of TFPI were detected in the two siblings (50% and 45% respectively) but their parents have normal TFPI levels. All other hemostatic proteins associated with thrombophilia, as well as blood lipids were within the normal range. The release of TFPI, after heparin administration, was proportionally reduced in the two siblings as compared with controls. The study of this family does not permit to conclude that low levels of TFPI are responsible for their thrombophilia. In the future the study of more families with similar characteristics may allow to know the inheritance of TFPI and whether there is a causal relationship between low levels of TFPI and thrombosis.