Personalized dendritic cell vaccination in cancer therapy: An evidence-based research study.

Background: Although chemotherapy is considered to be the golden standard, it does not come without a price. Toxicities and resistance are frequently limiting its effectiveness. Immunotherapy has emerged as a safer therapeutic alternative but still has a long way until it has proven to be of equal efficacy. A type of immunotherapy is dendritic cell (DC) vaccination. Aims and Objectives: We have developed a novel platform for the generation of autologous DCs that have been activated against peptides that are personalized for each patient individually. The aim of the study was to clinically evaluate this platform. Materials and Methods: Our platform and our algorithm for the determination of the immunogenic peptides has been tested. DC generation was verified both morphologically and by CD80/86 expression. Peptide antigenicity was determined using a number of T-cell epitope prediction algorithms. Response to therapy was evaluated using response evaluation criteria in solid tumors (RECIST) criteria by the doctors involved. Immune status was also evaluated before and after DC vaccination and correlated with circulated tumor cell count. Results: It was found that DC vaccine increased immune activation while correlated with decreased circulating tumor cell counts. Clinical evaluation by the determination of immune markers may be a superior tool than using RECIST criteria. Conclusion: Dendritic cell therapies could prove to be a valuable tool in cancer treatment.

A novel platform for the production of autologous human antibodies.

At Research Genetic Cancer Centre, we have developed a novel method for the production of human monoclonal antibodies against a specific antigen of our choice (c-met) using isolated human blood cells. By mimicking nature, dendritic, CD4 and CD19 cells from healthy volunteers were driven towards Th2 immunity. Cell activation was succeeded by a cytokine cocktail, and IgG production was promoted by IgG class switching factors. IgG secretion was determined using both enzyme linked immunosorbent assay (ELISA) and Western blot as well as immunoglobulin heavy chain gamma polypeptide gene expression. Secreted antibody was further purified by affinity column chromatography against c-met peptide. Anti-c-met activity was determined using the purified antibody as primary antibody for c-met detection by ELISA, Western blot and flow cytometry. Finally, anti-c-met antibody efficiency was determined by MCF-7 viability assay. Plasma cell formation and IgG secretion took place after 6 days of culture. Plasma cells produced anti-c-met IgG antibody that significantly decreased MCF-7 breast cancer cell proliferation. To our knowledge, this is the first platform of its kind, generating fully human antibodies-on-demand using patient's own cells, bringing personalized, targeted therapy for cancer one step closer.

A Novel Method for Colorectal Cancer Screening Based on Circulating Tumor Cells and Machine Learning.

Colorectal cancer is one of the most common types of cancer, and it can have a high mortality rate if left untreated or undiagnosed. The fact that CRC becomes symptomatic at advanced stages highlights the importance of early screening. The reference screening method for CRC is colonoscopy, an invasive, time-consuming procedure that requires sedation or anesthesia and is recommended from a certain age and above. The aim of this study was to build a machine learning classifier that can distinguish cancer from non-cancer samples. For this, circulating tumor cells were enumerated using flow cytometry. Their numbers were used as a training set for building an optimized SVM classifier that was subsequently used on a blind set. The SVM classifier's accuracy on the blind samples was found to be 90.0%, sensitivity was 80.0%, specificity was 100.0%, precision was 100.0% and AUC was 0.98. Finally, in order to test the generalizability of our method, we also compared the performances of different classifiers developed by various machine learning models, using over-sampling datasets generated by the SMOTE algorithm. The results showed that SVM achieved the best performances according to the validation accuracy metric. Overall, our results demonstrate that CTCs enumerated by flow cytometry can provide significant information, which can be used in machine learning algorithms to successfully discriminate between healthy and colorectal cancer patients. The clinical significance of this method could be the development of a simple, fast, non-invasive cancer screening tool based on blood CTC enumeration by flow cytometry and machine learning algorithms.

Cancer Comprehensive Analysis in Gastric Carcinoma: Benefits and New Perspectives.

Gastric cancer is one of the most common and deadly cancers worldwide. Screening tests as well as tools for prediction of treatment outcomes and prognosis have been developed, but they have many limitations. The integration of liquid biopsy provided new aspects in screening and diagnosis of gastric cancer. In the present study, we used different techniques, studying the genetic and epigenetic profile of circulating tumor cells. We aimed to acquire all the available information, compare it with already existing studies, and evaluate the benefit of this approach. A blood sample was isolated from 2 gastric cancer patients at stages III-IV, followed by the isolation of CTCs. The circulating tumor cells were used for array comparative genomic hybridization, next-generation sequencing, and whole gene expression microarrays. Different variants were detected, while the microsatellite instability status was stable in both cases. The tumor mutational burden was low to medium. Gene expression assays revealed that >100 genes were overexpressed compared to noncancer samples. Amplifications of X chromosome were also observed in both cases, by using array comparative genomic hybridization. Although there are several techniques for cancer screening, prediction of therapy outcomes, and prognosis, the application of a complete comprehensive cancer panel, combining the study of variants, fusions, chromosomal abnormalities, and gene expression, is more appropriate. Information provided by the above techniques might contribute in designing more efficient treatment protocols and screening tools. Despite the limitation of samples, the data are encouraging, and further study is needed so that they can be used at clinical level.

Increased breast cancer cell sensitivity to cisplatin using a novel small molecule inhibitor.

BACKGROUND: Although cisplatin is used for the treatment of more than half of cancer patients, its use is restricted by serious side effects as well as the development of cisplatin-resistant cancer cells, limiting its use. In RGCC we have synthesized an intermediate molecule in an ERK inhibitor synthesis process. AIMS AND OBJECTIVES: The aim of the study was to evaluate the effects of combined cisplatin plus RGCC molecule treatment on MCF-7 and MDAMB231 breast cancer cell viability, proliferation, ability to form clones and migrate, as well as the effects on cell cycle and gene expression. MATERIALS AND METHODS: Cell viability and proliferation were measured by Crystal violet exclusion dye and MTT respectively. Clone formation and wound healing assays were also used for clone formation and cell migration evaluation. Cell cycle was studied by flow cytometry, expression of genes was evaluated by PCR and protein expression was evaluated by western blot. RESULTS: It was found that combination therapy decreased cell viability and proliferation, caused growth arrest, decreased cancer cell invasiveness and the ability to form clones as well as perturbed the expression of genes involved in ERK, cell cycle and cell death pathways. Conclusion: Although the exact mechanism of action of the combination therapy remains to be investigated, it was found that it is more effective than cisplatin monotherapy. Our findings could potentially lead to a new therapeutic regime for the treatment of cancer.

Novel antibody against TMX2 and its effects on breast cancer cells.

Although monoclonal antibodies are promising, a truly fully human antibody is yet to be produced. Current human antibodies have the human sequence, but are produced in either transgenic animals or in phages. The aim of this paper was to produce a truly human antibody directed against an epitope of our choice, secreted by human plasma cells. The target protein was TMX2 one of the least studied disulfide isomerases. IgG and anti-TMX2 antibody were determined by both Elisa and western blot. TMX2 KD was evaluated by Surface Plasmon Resonance. TMX2 localization was determined by flow cytometry in MCF-7 cells. Efficiency was evaluated by MTT. Gene expression was evaluated by PCR. We have managed to produce two fully human antibodies directed against TMX2 protein. TMX2 protein was found both in the cytoplasm and cell membrane of breast cancer cells. RGCC TMX2 antibody recognizing an extracellular epitope increased cell proliferation. RGCC TMX2 antibody recognizing an intracellular epitope decreased cell proliferation and gene expression related to cancer survival, differentiation and metastasis. These findings suggest this platform is very promising for novel personalized therapies. TMX2 could be a novel target for cancer treatment.

Novel small molecule decreases cell proliferation, migration, clone formation, and gene expression through ERK inhibition in MCF-7 and MDA-MB-231 breast cancer cell lines.

The Ras-Raf-MEK1/2-ERK1/2 pathway is an attractive target for the development of anticancer agents because of the high prevalence of ERK activation in human cancers. However, resistance is often developed despite initial clinical response, most likely because of activation of cross-talk pathways. In Research Genetic Cancer Center (RGCC), we are in the process of synthesizing a novel ERK inhibitor, targeting the final stage of the pathway, thus minimizing cross-talk. We have synthesized an intermediate molecule -RGCC416 - and tested its biological activity. MCF-7 and MDA-MB-231 cells were used. Cell viability was measured by crystal violet and cell proliferation by the methyl tetrazolium assay using various compound concentrations. Cell migration and colony formation were determined to assess the ability of invasion and single cancer cell growth, respectively. Expression of genes linked to MAPK/PI3K pathways was determined by PCR. ERK and phospho-ERK levels were determined in both the cytoplasm and the nucleus by western blot. It was found that although the compound did not affect viability, it significantly decreased cell proliferation, migration, and colony formation in both cell lines. In MDA-MB-231, this is possibly through retaining phospho-ERK to the cytoplasm, where it cannot activate cancer-associated genes. There was no difference in ERK levels in MCF-7 cells. This could be because of the different pathways that these cells utilize for survival. We have synthesized a molecule, which could be a promising ERK inhibitor, leading to possible novel treatment options for breast cancer patients.

The use of serum extracellular vesicles for novel small molecule inhibitor cell delivery.

In Research Genetic Cancer Center (RGCC), we are in the process of synthesizing a novel ERK inhibitor. We have currently synthesized an intermediate molecule, RGCC169, that needed to be tested to confirm we are using the appropriate synthetic pathways. Because of the limited solubility the compound exhibits, a strategy had to be devised for the free entrance of the molecule into the cell. Extracellular vesicles (EVs) were isolated by polyethylene glycol precipitation and identified by western blot and scanning electron microscopy. Loading was determined by high-performance liquid chromatography, differential scanning calorimetry, and scanning electron microscopy. EV uptake was determined by fluorescent microscopy. The effect of EV-encapsulated RGCC169 was determined by MTT viability assay on MCF7 cells. RGCC169 was incorporated into EVs as shown by high-performance liquid chromatography (26.6%) and scanning electron microscopy. Differential scanning calorimetry peaks shifted from 100.84 to 108.79°C upon encapsulation. EVs were taken up by cells as evident from CD63 fluorescent signal inside the cell's cytoplasm. RGCC169 decreased MCF proliferation (93.5±2.2, P=0.02). EV-encapsulated RGCC169 decreased cell proliferation even further (93.5±2.2 vs. 81.6±2.8, P=0.0002). RGCC169 was successfully loaded into serum EVs possibly by incorporation into the lipid membrane. EVs were taken up by MCF7 cells possibly by endocytic pathways. Although RGCC169 significantly reduced MCF7 viability at 3 µmol/l, the same concentration of RGCC169 encapsulated into EVs decreased cells viability even further. Our findings validate the correctness of our methods and are very promising for the achievement of our final goal, that is, the synthesis of a novel cytotoxic agent.

Bortezomib overcomes MGMT-related resistance of glioblastoma cell lines to temozolomide in a schedule-dependent manner.

Development of drug resistance after standard chemotherapy for glioblastoma multiforme (GBM) with temozolomide (TMZ) is associated with poor prognosis of GBM patients and is at least partially mediated by a direct DNA repair pathway involving O6-methylguanine methyltransferase (MGMT). This enzyme is under post-translational control by a multisubunit proteolytic cellular machinery, the 26S proteasome. Inhibition of the proteasome by bortezomib (BZ), a boronic acid dipeptide already in clinical use for the treatment of myeloma, has been demonstrated to induce growth arrest and apoptosis in GBM cells. In this study we investigated the effect of sequential treatment with BZ and TMZ on cell proliferation-viability and apoptosis of the human T98G and U87 GBM cell lines. We also tested for an effect of treatment on MGMT expression and important upstream regulators of the latter, including nuclear factor kappa B (NFκB), p44/42 mitogen-activated protein kinase (MAPK), p53, signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1α (HIF-1α). The sequence of drug administration for maximal cytotoxicity favored BZ prior to TMZ in T98G cells while the opposite was the case for U87 cells. Maximal efficacy was associated with downregulation of MGMT, reduced IκBα-mediated proteasome-dependent nuclear accumulation of NFκB, attenuation of p44/42 MAPK, AKT and STAT3 activation, and stabilization of p53 and inactive HIF-1α. Collectively, these results suggest that proteasome inhibition by BZ overcomes MGMT-mediated GBM chemoresistance, with scheduling of administration being critical for obtaining the maximal tumoricidal effect of combination with TMZ.

MGMT repletion after treatment of glioblastoma cells with temozolomide and O6-benzylguanine implicates NFκB and mutant p53.

The DNA repair enzyme O6-methylguanine methyltransferase (MGMT) is a major determinant of glioma resistance to alkylating agents. Several strategies have been used to induce sensitization to alkylator-based treatments, including the direct MGMT inhibitor O6-benzylguanine (BG). However, replenishment of MGMT is often observed after the withdrawal of combined schedules of temozolomide (TMZ) and BG, thus preventing further treatment efficacy. In this study we investigated the potential mechanisms of resistance to combination treatment with TMZ and BG in the MGMT-proficient, p53-mutated (mt p53) T98G glioblastoma (GBM) cell line, looking for an effect on nuclear factor kappa B (NFκB) and mt p53, which are both transcriptional regulators of MGMT. The administration of TMZ alone led to minimal inhibition of T98G cell viability which was, however, enhanced with the addition of BG. This effect coincided with reduced expression of MGMT protein and transcript levels, and a decrease in cellular amount of NFκB and mt p53. However, withdrawal of the drugs led to an increase in cell viability, which was in parallel with repletion of MGMT protein and transcript levels and was also accompanied by elevated protein levels of NFκB and mt p53. Overall, these results suggest that NFκB and mt p53 induction may be responsible for the failure of BG to induce prolonged inhibition of direct repair in TMZ co-treated GBM cells with mt p53 status.

Bortezomib downregulates MGMT expression in T98G glioblastoma cells.

The efficacy of treatment for glioblastoma multiforme is currently limited by the development of resistance, particularly, but not exclusively, due to the expression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) in a significant proportion of astrocytic tumors. MGMT is post-translationally regulated by the 26S proteasome, a multi-subunit organelle responsible for degradation of misfolded cellular proteins. The boronic acid dipeptide bortezomib is the first and only proteasome inhibitor in clinical use so far, and has been reported as a strategy to restrict growth and promote apoptosis of glioblastoma cells. In this study we investigated the effect of bortezomib on MGMT expression in T98G cells, looking for an effect on the nuclear factor kappa B (NFκB) pathway, which is a major player in MGMT regulation and is also under tight control by the ubiquitin-proteasome system. Administration of bortezomib led to a significant reduction of T98G cell viability and induction of DNA fragmentation. These effects coincided with reduced expression of MGMT transcript levels, and a decrease in cellular amount and IκBα-mediated, proteasomal activity-dependent nuclear translocation of NFκB. In addition, bortezomib-induced phosphorylation of the translation initiation factor 2alpha (eIF2α) was in parallel with translational repression of MGMT. Taken together, these results suggest a novel role for bortezomib as a potent MGMT inhibitor and support its ongoing testing as a chemosensitizer in glioblastoma.

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells.

BACKGROUND: Upregulation of nuclear factor kappa B (NFκB) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NFκB, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro. METHODS: We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment. RESULTS: Neuropeptide (NP) stimulation induced nuclear translocation of NFκB in a dose-dependent manner in AI cells, also evident as reduced total inhibitor κB (IκB) levels and increased DNA binding in EMSA. These effects were preceded by increased 20 S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NFκB, IκB kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA. CONCLUSIONS: Our results support evidence for a direct mechanistic connection between the NPs and NFκB/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

OBJECTIVES: Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. METHODS: Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. RESULTS: Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. CONCLUSIONS: These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Bortezomib represses HIF-1α protein expression and nuclear accumulation by inhibiting both PI3K/Akt/TOR and MAPK pathways in prostate cancer cells.

Bortezomib represents the first proteasome inhibitor (PI) with demonstrated antitumor activity in the clinical setting, particularly for treatment of hematological malignancies. At the preclinical level, its action is shown to be mediated by induction of growth arrest and apoptosis in many tumor types, including androgen-dependent (AD) and androgen-independent (AI) prostate cancer (PCa) cells. Hypoxia-inducible factor-1α (HIF-1α), which is directly involved in tumor growth, is one of the most studied and promising molecular targets for anti-cancer therapy and is often overexpressed in PCa. Bortezomib has been reported to impair tumor growth by also inhibiting HIF-1α. In this study, we investigated the effect of bortezomib on the expression, activity and localization of HIF-1α in LNCaP (AD) and PC3 (AI) PCa cells. First, we show that hypoxic upregulation of HIF-1α protein levels and activity involves both the PI3K/Akt/mTOR and p44/42 MAPK pathways. Second, bortezomib inhibits expression of HIF-1α protein under both normoxic and hypoxic conditions, represses HIF-1 transcriptional activity and attenuates the release of vascular endothelial growth factor. These effects correlate with the ability of bortezomib to cause dephosphorylation of phospho-Akt, phospho-p70S6K, and phospho-S6RP, thus inactivating a pathway known to be required for HIF-1α protein expression at the translational level. Furthermore, bortezomib also abrogates p44/42 MAPK phosphorylation, which results to reduced nuclear translocation of HIF-1α. Taken together, these results suggest that bortezomib inhibits HIF-1α protein synthesis and its nuclear targeting through suppression of PI3K/Akt/mTOR and MAPK pathways, respectively, in both AD and AI PCa cells.

Evidence of association between methylenetetrahydrofolate reductase gene and susceptibility to breast cancer: a candidate-gene association study in a South-eastern European population.

The methylenetetrahydrofolate reductase (MTHFR) gene has been proposed as a candidate gene for breast cancer (BC). However, the specific role of MTHFR polymorphisms and haplotypes has not been fully clarified and replicated. We examined the association of two common MTHFR polymorphisms (C677T and A1298C) and their haplotypes in a candidate-gene association study, involving 300 female patients with BC and 283 healthy women. The single locus analysis for the two polymorphisms revealed an association only for the C677T polymorphism [odds ratio (95% confidence interval), OR=2.05 (1.21-3.48)], but adjustment for age diminished this association [OR=1.76 (0.92-3.42)]. The menopausal status showed no significant effect in the association between the MTHFR polymorphisms and BC. The analysis of haplotypes showed an association for the C677-A1298 haplotypes (p=0.04). The available evidence from our study may support a contributory role of MTHFR polymorphisms in BC development. Future larger studies may help in elucidating the genetics of BC further.

Inverse baseline expression pattern of the NEP/neuropeptides and NFκB/proteasome pathways in androgen-dependent and androgen-independent prostate cancer cells.

BACKGROUND: Castration-resistance in prostate cancer (PC) is a critical event hallmarking a switch to a more aggressive phenotype. Neuroendocrine differentiation and upregulation of NFκB transcriptional activity are two mechanisms that have been independently linked to this process. METHODS: We investigated these two pathways together using in vitro models of androgen-dependent (AD) and androgen-independent (AI) PC. We measured cellular levels, activity and surface expression of Neutral Endopeptidase (NEP), levels of secreted Endothelin-1 (ET-1), levels, sub-cellular localisation and DNA binding ability of NFκB, and proteasomal activity in human native PC cell lines (LnCaP and PC-3) modelling AD and AI states. RESULTS: At baseline, AD cells were found to have high NEP expression and activity and low secreted ET-1. In contrast, they exhibited a low-level activation of the NFκB pathway associated with comparatively low 20S proteasome activity. The AI cells showed the exact mirror image, namely increased proteasomal activity resulting in a canonical pathway-mediated NFκB activation, and minimal NEP activity with increased levels of secreted ET-1. CONCLUSIONS: Our results seem to support evidence for divergent patterns of expression of the NFκB/proteasome pathway with relation to components of the NEP/neuropeptide axis in PC cells of different level of androgen dependence. NEP and ET-1 are inversely and directly related to an activated state of the NFκB/proteasome pathway, respectively. A combination therapy targeting both pathways may ultimately prove to be of benefit in clinical practice.

Impaired phospholipases A2production by stimulated macrophages from patients with acute respiratory distress syndrome.

The aim of this study was to investigate whether early phase of acute respiratory distress syndrome (ARDS) is associated with changes in immune response, either systemic or localized to the lung. ARDS and control mechanically ventilated patients, as well as healthy volunteers were studied. Alveolar macrophages (AMΦ) and blood monocytes (BM) were treated ex vivo with lipopolysaccharide (LPS), interferon-γ (IFNγ), and surfactant. Phospholipase A2 (PLA2) activity and TLR4 expression were evaluated as markers of cell response. AMΦ from ARDS patients did not respond upon treatment with either LPS or IFN-γ by inducing PLA2 production. On the contrary, upon stimulation, in control patients the intracellular PLA2, (mainly cPLA2) levels were increased, but secretion of PLA2 (mainly sPLA2-IIA) was observed only after treatment with LPS. Surfactant suppressed PLA2 production in cells from both groups of patients. Increased relative changes of total PLA2 activity and an upregulation of TLR4 expression upon stimulation was observed in BM from primary ARDS, control patients and healthy volunteers. In BM from secondary ARDS patients, however, no PLA2 induction was observed, with a concomitant down-regulation of TLR4 expression. Cytosolic PLA2, its activated form, p-cPLA2, and sPLA2-IIA were the predominant PLA2 types within the cells, while extracellularly only sPLA2-IIA was identified. These results support the concept of down-regulated innate immunity in early ARDS that is compartmentalized in primary and systemic in secondary ARDS. PLA2 isoforms could serve as markers of the immunity status in ARDS. Finally, our data highlight the role of surfactant in controlling inflammation.

Additive inhibition of colorectal cancer cell lines by aspirin and bortezomib.

PURPOSE: To investigate the effect of cyclooxygenase-2 (Cox-2) inhibitor aspirin (acetylsalicylic acid, ASA) and proteasome inhibitor bortezomib in the proliferation and apoptosis of colorectal cancer cell lines. METHODS: MTT assay, trypan blue exclusion and DNA fragmentation have been used to investigate cell proliferation and apoptosis in the presence of drugs. For the determination of Cox activity a colorimetric method was used. Western blotting was used for the measurement of the effect of the drugs in different proteins expression. RESULTS: Bortezomib together with aspirin inhibit the growth of colorectal cancer cell lines HCT116, HT-29, and CaCo2 more than each drug alone. In the first two cell lines ASA inhibitory effects are Cox-2 independent because HCT116 cells do not express the enzyme while in HT-29 cells, Cox-2 has no activity as shown by a Cox activity assay. In CaCo2 cells that express enzymatically active Cox-2 this activity is inhibited by ASA. ASA is also able to suppress the increase in Cox-2 activity induced by bortezomib in these cells. Cell cycle inhibitors p21 and p27 are induced in the three cell lines by bortezomib and the combination treatment. Akt1 kinase is down-regulated in all three lines by the same treatments. Transcription factor NF-kappaB is retained in the cytoplasm by drug treatment in cell lines HCT116 and HT-29, a fact that may play a role in their pro-apoptotic activity. Pro-apoptotic bcl-2 family member, bad is down-regulated in cell lines HCT116 and CaCo2 by bortezomib treatment, a neoplasia-promoting event that is reversed by combination treatment. CONCLUSION: The combination of bortezomib and ASA cooperates to decrease proliferation and induce apoptosis in three human colorectal cell lines with different genetic lesions. These effects are at least in some cases Cox-2 independent and involve common and diverse mechanisms in the three lines.

Phospholipases A2 and platelet-activating-factor acetylhydrolase in patients with acute respiratory distress syndrome.

OBJECTIVE: Phospholipases A2 (PLA2) comprise a family of enzymes probably implicated in the development of acute respiratory distress syndrome (ARDS). The aim was to investigate PLA2 activities and characteristics in bronchoalveolar lavage (BAL) fluid, BAL cells, and plasma from patients with ARDS by a fluorometric method. DESIGN: Prospective, controlled study. SETTING: Fourteen-bed polyvalent intensive care unit in a university hospital. PATIENTS: A total of 31 mechanically ventilated patients, 20 with and 11 without ARDS, were studied. INTERVENTION: BAL was performed by fiberoptic bronchoscopy in mechanically ventilated patients with a controlled mechanical ventilation mode. MEASUREMENTS: PLA2 and platelet-activating-factor acetylhydrolase were determined in BAL fluid, cells, and plasma. For the classification of PLA2-specific inhibitors, Western blot analysis and their biochemical characteristics were used. RESULTS: In ARDS patients, increased PLA2 levels were detected in BAL fluid, BAL cells, and plasma compared with the control patients. PLA2 in BAL fluid was mainly type IIA secretory and cytosolic types. In plasma, type IIA secretory and cytosolic and a Ca-independent PLA2 were found. In BAL cells, a cytosolic form, probably a Ca-independent intracellular form, and a low activity of type IIA secretory PLA2 was also observed. Total PLA2 activity correlated inversely with Pao2/Fio2 ratio and positively with the mortality rate. Patients with direct ARDS exhibited higher PLA2 activity compared with patients with indirect ARDS. Platelet-activating-factor acetylhydrolase activity was higher in BAL fluid and plasma, but it was lower in BAL cells. CONCLUSION: Ca-dependent, secretory, cytosolic, and Ca-independent forms of PLA2 and platelet-activating-factor acetylhydrolase could play important roles in the development or down-regulation of inflammation in ARDS, respectively.