Ivana Grivicich

Role of serum S100B as a predictive marker of fatal outcome following isolated severe head injury or multitrauma in males.

Abstract Background: Severe traumatic brain injury (TBI) is associated with a 30%–70% mortality rate. S100B has been proposed as a biomarker for indicating outcome after TBI. Nevertheless, controversy has arisen concerning the predictive value of S100B for severe TBI in the context of multitrauma. Therefore, our aim was to determine whether S100B serum levels correlate with primary outcome following isolated severe TBI or multitrauma in males. Methods: Twenty-three consecutive male patients (age 18–65years), victims of severe TBI [Glasgow Coma Scale (GCS) 3–8] (10 isolated TBI and 13 multitrauma with TBI) and a control group consisting of eight healthy volunteers were enrolled in this prospective study. Clinical outcome variables of severe TBI comprised: survival, time to intensive care unit (ICU) discharge, and neurological assessment [Glasgow Outcome Scale (GOS) at ICU discharge]. Venous blood samples were taken at admission in the ICU (study entry), 24h later, and 7days later. Serum S100B concentration was measured by an immunoluminometric assay. Results: At study entry (mean time 10.9h after injury), mean S100B concentrations were significantly increased in the patient with TBI (1.448μg/L) compared with the control group (0.037μg/L) and patients with fatal outcome had higher mean S100B (2.10μg/L) concentrations when compared with survivors (0.85μg/L). In fact, there was a significant correlation between higher initial S100B concentrations and fatal outcome (Spearmans =0.485, p=0.019). However, there was no correlation between higher S100B concentrations and the presence of multitrauma. The specificity of S100B in predicting mortality according to the cut-off of 0.79μg/L was 73% at study entry. Conclusions: Increased serum S100B levels constitute a valid predictor of unfavourable outcome in severe TBI, regardless of the presence of associated multitrauma. Clin Chem Lab Med 2006;44:1234–42.

5-Fluorouracil and its active metabolite FdUMP cause DNA damage in human SW620 colon adenocarcinoma cell line.

5‐Fluorouracil (5‐FU) is an antineoplasic drug widely used to treat cancer. Its cytotoxic effect has been principally ascribed to the misincorporation of fluoronucleotides into DNA and RNA during their synthesis, and the inhibition of thymidylate synthase (TS) by FdUMP (one of the 5‐FU active metabolites), which leads to nucleotide pool imbalance. In the present study, we compared the ability of 5‐FU and FdUMP to induce apoptosis and to influence the cell cycle progression in human colon SW620 adenocarcinoma cells in regards to their genotoxic and clastogenic activities. Our study demonstrates that 5‐FU induces SSB, DSB and apoptosis earlier than FdUMP. Interestingly, while both drugs are able to induce apoptosis, their effect on the cell cycle progression differed. Indeed, 5‐FU induces an arrest in G1/S while FdUMP causes an arrest in G2/M. Independently of the temporal difference in strand breaks and apoptosis induction, as well as the differential cell cycle modulation, both drugs presented similar clastogenic effects. The different pattern of cell cycle arrest suggests that the two drugs induce different types of primary DNA lesions that could lead to the activation of different checkpoints and recruit different DNA repair pathways. Copyright

Radioresistance of human glioma spheroids and expression of HSP70, p53 and EGFr

BackgroundRadiation therapy is routinely prescribed for high-grade malignant gliomas. However, the efficacy of this therapeutic modality is often limited by the occurrence of radioresistance, reflected as a diminished susceptibility of the irradiated cells to undergo cell death. Thus, cells have evolved an elegant system in response to ionizing radiation induced DNA damage, where p53, Hsp70 and/or EGFr may play an important role in the process. In the present study, we investigated whether the content of p53, Hsp70 and EGFr are associated to glioblastoma (GBM) cell radioresistance.MethodsSpheroids from U-87MG and MO59J cell lines as well as spheroids derived from primary culture of tumor tissue of one GBM patient (UGBM1) were irradiated (5, 10 and 20 Gy), their relative radioresistance were established and the p53, Hsp70 and EGFr contents were immunohistochemically determined. Moreover, we investigated whether EGFr-phospho-Akt and EGFr-MEK-ERK pathways can induce GBM radioresistance using inhibitors of activation of ERK (PD098059) and Akt (wortmannin).ResultsAt 5 Gy irradiation UGBM1 and U-87MG spheroids showed growth inhibition whereas the MO59J spheroid was relatively radioresistant. Overall, no significant changes in p53 and Hsp70 expression were found following 5 Gy irradiation treatment in all spheroids studied. The only difference observed in Hsp70 content was the periphery distribution in MO59J spheroids. However, 5 Gy treatment induced a significant increase on the EGFr levels in MO59J spheroids. Furthermore, treatment with inhibitors of activation of ERK (PD098059) and Akt (wortmannin) leads to radiosensitization of MO59J spheroids.ConclusionsThese results indicate that the PI3K-Akt and MEK-ERK pathways triggered by EGFr confer GBM radioresistance.

Relationship between oxidative stress levels and activation state on a hepatic stellate cell line

Abstract: Background/Aims: Oxidative stress plays an important role in liver fibrosis. Under pathological conditions, hepatic stellate cells (HSC) undergo an activation process, developing a myofibroblast‐like phenotype from the lipocyte phenotype. In this study, we determined the levels of oxidative stress and proliferation in different activation states of an experimental model of mouse HSC, the GRX cell line. These cells can be induced in vitro to display a more activated state or a quiescent phenotype.

Phase II study of pentosan polysulfate (PPS) in patients with AIDS-related Kaposi's sarcoma.

Aims and background To evaluate the response rate, toxicity and survival of patients with AIDS-related Kaposis sarcoma (AIDS-KS) treated in a phase II clinical trial of pentosan polysulpate (PPS), an inhibitor of basic-fibroblast growth factor (b-FGF) which blocks the growth of Kaposis sarcoma cells both in culture and in animal models. Patients and methods Between March 1992 and March 1994 16 homosexual males with histopathologically confirmed AIDS-KS were accrued for this phase II clinical trial. PPS was administered at the dose of 25 mg/m2 q6 hrs at day 1, followed by 25 mg/m2 q12 hrs daily by a subcutaneous injection. The number of patients to be included in the trial was calculated according to the two-stage Gehan method. Toxicity was graded according to the NCI Common Toxicity Criteria, while responses were evaluated according to the WHO Criteria adapted for KS lesions. Patients were all homosexual males, median age 35 (27-43) years, performance status (WHO) 1 (0-2), NYU stage II-IV and prior therapy included vincristine and etoposide (3 cases), local irradiation (4 cases) and meges-trol acetate (2 cases). Concomitant AZT (zidovudine) was given to 3 patients, while DDI (dideoxyinosine) was administered in one case. Results A median of 5 (3-11) weeks of therapy was administered to the patients. Pain at the injection site and low grade fever were the only toxicities observed. Drug-related effects on coagulation parameters or thrombocytopenia were not observed in the trial. One objective response (6%) was documented, which lasted for 9 weeks, while stable disease was observed in three patients, lasting for 11, 9 and 5 weeks, respectively. Conclusion This is the first observation of objective antitumor activity with a b-FGF inhibitor in patients with AIDS-KS. Considering its novelty and the lack of significant toxicity, the authors suggest that this experimental approach deserves further evaluation.

Irinotecan/5-fluorouracil combination induces alterations in mitochondrial membrane potential and caspases on colon cancer cell lines.

The combination of irinotecan (CPT-11) and 5-fluorouracil (5-FU) is currently used in the treatment of advanced colorectal carcinoma. When compared to both agents alone, CPT-11 followed by 5-FU treatment demonstrated a synergistic effect. This observation can be related to increased in apoptosis induction after caspase activation. Several studies have demonstrated that changes in mitochondrial membrane potential occur earlier in apoptosis. In this study, we verified whether the collapse in mitochondrial membrane and the activation of caspases is responsible for increased apoptosis observed with CPT-11/5-FU treatment. Thus, HT-29 and SNU-C4 human colon carcinoma cell lines were exposed for 24 h to each drug alone, and to various combinations and treatment sequences, and assessed for colony formation, changes in the mitochondrial membrane potential, and the activities of caspase-3, -8, and -9. The CPT-11/5-FU treatment induced apoptosis in both cell lines; however, the most pronounced effect was observed in HT-29 cells. In these cells, both caspase-3 and -9 were involved in the activation of apoptosis after CPT-11/5-FU treatment. Moreover, in these cells, a reduction of 50% in mitochondrial membrane potential was observed with this treatment. On the other hand, in the SNU-C4 cell line in addition to caspase-3 and-9, caspase-8 seems to be important to apoptosis after CPT-11/5-FU treatment. Furthermore, in this cell line we did not observe alterations in mitochondrial membrane potential. In spite of the differences among the cell lines, these results indicated that the increase in apoptosis in HT-29 cells observed with CPT-11 followed by 5-FU treatment could be explained by a disruption in mitochondria membrane potential that induced caspases activation.

DNA repair pathways involved in repair of lesions induced by 5-fluorouracil and its active metabolite FdUMP

5-Fluorouracil (5-FU) is an antitumor antimetabolite that can be converted into fluoronucleotides and FdUMP. Fluoronucleotides are incorporated into DNA and RNA, while FdUMP results in nucleotide pool imbalance. Saccharomyces cerevisiae is unable to convert 5-FU into FdUMP, making yeast a unique model system to study the cellular effects of 5-FU and FdUMP independently. A panel of repair-deficient yeast strains was used to identify the DNA repair pathways needed for repair of lesions generated by 5-FU or FdUMP. This included yeast deficient in base excision repair (BER), nucleotide excision repair (NER), translesion synthesis (TLS), mismatch repair (MMR), post-replication repair (PRR), homologous recombination (HR) and non-homologous end-joining (NHEJ). The results revealed an important role of BER, since BER-mutants (ntg1, ntg2, apn1, apn2) showed pronounced sensitivity to both 5-FU and FdUMP. MMR mutants also showed high sensitivity to both compounds. In contrast, deficiencies in NER, NHEJ and TLS repair had only minor influence on the sensitivity to FU and FdUMP. Interestingly, deficiencies in HR (rad52) and PPR (rad6, rad18) were associated with increased sensitivity to 5-FU, but not to FdUMP. Taken together, our study reveals an important contribution of DNA repair pathways on the sensitivity to 5-FU and its active metabolite FdUMP. Importantly, the repair mechanisms differed for the 2 antimetabolites since lesions induced by 5-FU were repaired by BER, MMR, HR and PRR, while only BER and MMR were required for repair of FdUMP-induced lesions.

Synchronous gastrointestinal stromal tumors (GIST) and other primary cancers: Case series of a single institution experience

BACKGROUND Gastrointestinal stromal tumors (GISTs) are rare mesenchymal neoplasm affecting the gastrointestinal tract. The incidental occurrence of mesenchymal tumors and other primary tumors has not been well described in literature. OBJECTIVE The aim of this study was to evaluate the clinical and pathologic features of GIST occurring synchronously with other primary tumors. METHODS Forty-three patients with diagnosis of GIST treated surgically with curative intent at our institution from 1998 to 2006 were included. The patient clinical data and pathological reports were reviewed. RESULTS Of the 43 patients, there were 6 (14%) cases of synchronous GIST and other primary tumors discovered as coincidental findings. The synchronous GISTs analyzed were located in the stomach (50%) and small intestine (50%), size ranging from 0.7 to 7.6 cm (median 3.35 cm). Five (83%) of the concurrent primary tumors were from gastrointestinal origin and only one (17%) patient presented with concurrent breast cancer and GIST. The synchronous GISTs immunofenotype shows positivity for CD117 and CD34 (100%), smooth-muscle actin (SMA) (67%), S100 (50%) and desmin (33%). Whereas staining for cytokeratin AE1/AE3 and PDGF were all negative. According to GIST risk category for aggressive behavior three were classified as very low, one intermediate and two high. CONCLUSIONS The synchronous occurrence of GISTs and other primary neoplasm is not an uncommon entity and usually they are discovery incidentally. Epithelial tumors of the gastrointestinal tract are the most associated with concomitant GISTs. Further studies are required to clarify the molecular and genetic mechanisms of carcinogenesis and progression associating GIST and synchronous tumors.

The Irinotecan/5-Fluorouracil Combination Induces Apoptosis and Enhances Manganese Superoxide Dismutase Activity in HT-29 Human Colon Carcinoma Cells

Background: We examined whether induction of apoptosis and Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activities were involved in the greater cytotoxicity of the irinotecan (CPT-11)/5-fluorouracil (5-FU) combination for human colon cancer cells when compared to both drugs alone. Methods: HT-29 and SNU-C4 human colon carcinoma cell lines were treated with 5-FU and CPT-11, then apoptosis was evaluated by flow cytometry and SOD activities were determined by polyacrylamide gel electrophoresis. Results: Enhanced apoptosis of HT-29 cells was observed with all treatments containing 5-FU in SNU-C4 cells; however, in HT-29 cells, apoptosis was enhanced only with the CPT-11/5-FU combination. In the SNU-C4 cell line, none of the treatments exerted a significant effect on Cu,Zn-SOD or Mn-SOD activity. However, in HT-29 cells, the CPT-11/5-FU combination enhanced Mn-SOD activity when compared to cells treated with CPT-11 alone. Nevertheless, the combined treatment did not interfere with Cu,Zn-SOD activity. Conclusion: Treatment with the CPT-11/5-FU combination may promote in HT-29 cell apoptosis by enhancing Mn-SOD activity.

Increased serum sFas and TNFα following isolated severe head injury in males

Objectives: Severe traumatic brain injury (TBI) is associated with a 30–70% mortality rate. Nevertheless, controversy has been raised concerning the prognostic value of biomarkers following severe TBI. Therefore, our aim was to determine whether sFas or TNFα serum levels correlate with primary outcome following isolated severe TBI. Methods: Seventeen consecutive male patients, victims of isolated severe TBI (Glasgow Coma Scale score 3–8) and a control group consisting of 6 healthy male volunteers were enrolled in this prospective study. Clinical outcome variables of severe TBI comprised: survival, time for intensive care unit (ICU) discharge, and neurological assessment by Glasgow Outcome Scale at ICU discharge. Venous blood samples were taken at admission in the ICU. Serum sFas and TNFα concentrations were measured by ELISA assays. Results: At admission in the ICU (mean time 10.2 h after injury), mean sFas and TNFα concentrations were significantly increased in the TBI (0.105 and 24.275 ρg/l, respectively) compared with the control group (0.047 and 15.475 ρg/l, respectively). However, no significant correlation was found between higher serum sFas or TNFα concentrations and fatal outcome. Conclusions: Increased serum sFas and TNFα levels following isolated severe TBI did not predict fatal outcome.