Fibroblast activation protein as a potential theranostic target in brain metastases of diverse solid tumours.

Brain metastases are a very common and serious complication of oncological diseases. Despite the vast progress in multimodality treatment, brain metastases significantly decrease the quality of life and prognosis of patients. Therefore, identifying new targets in the microenvironment of brain metastases is desirable. Fibroblast activation protein (FAP) is a transmembrane serine protease typically expressed in tumour-associated stromal cells. Due to its characteristic presence in the tumour microenvironment, FAP represents an attractive theranostic target in oncology. However, there is little information on FAP expression in brain metastases. In this study, we quantified FAP expression in samples of brain metastases of various primary origin and characterised FAP-expressing cells. We have shown that FAP expression is significantly higher in brain metastases in comparison to non-tumorous brain tissues, both at the protein and enzymatic activity levels. FAP immunopositivity was localised in regions rich in collagen and containing blood vessels. We have further shown that FAP is predominantly confined to stromal cells expressing markers typical of cancer-associated fibroblasts (CAFs). We have also observed FAP immunopositivity on tumour cells in a portion of brain metastases, mainly originating from melanoma, lung, breast, and renal cancer, and sarcoma. There were no significant differences in the quantity of FAP protein, enzymatic activity, and FAP+ stromal cells among brain metastasis samples of various origins, suggesting that there is no association of FAP expression and/or presence of FAP+ stromal cells with the histological type of brain metastases. In summary, we are the first to establish the expression of FAP and characterise FAP-expressing cells in the microenvironment of brain metastases. The frequent upregulation of FAP and its presence on both stromal and tumour cells support the use of FAP as a promising theranostic target in brain metastases.

Regulation of Fibroblast Activation Protein by Transforming Growth Factor Beta-1 in Glioblastoma Microenvironment.

The proline-specific serine protease fibroblast activation protein (FAP) can participate in the progression of malignant tumors and represents a potential diagnostic and therapeutic target. Recently, we demonstrated an increased expression of FAP in glioblastomas, particularly those of the mesenchymal subtype. Factors controlling FAP expression in glioblastomas are unknown, but evidence suggests that transforming growth factor beta (TGFbeta) can trigger mesenchymal changes in these tumors. Here, we investigated whether TGFbeta promotes FAP expression in transformed and stromal cells constituting the glioblastoma microenvironment. We found that both FAP and TGFbeta-1 are upregulated in glioblastomas and display a significant positive correlation. We detected TGFbeta-1 immunopositivity broadly in glioblastoma tissues, including tumor parenchyma regions in the immediate vicinity of FAP-immunopositive perivascular stromal cells. Wedemonstrate for the first time that TGFbeta-1 induces expression of FAP in non-stem glioma cells, pericytes, and glioblastoma-derived endothelial and FAP+ mesenchymal cells, but not in glioma stem-like cells. In glioma cells, this effect is mediated by the TGFbeta type I receptor and canonical Smad signaling and involves activation of FAP gene transcription. We further present evidence of FAP regulation by TGFbeta-1 secreted by glioma cells. Our results provide insight into the previously unrecognized regulation of FAP expression by autocrine and paracrine TGFbeta-1 signaling in a broad spectrum of cell types present in the glioblastoma microenvironment.

BOK displays cell death-independent tumor suppressor activity in non-small-cell lung carcinoma.

As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFß-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients.

Species-specific real-time RT-PCR analysis of expression of stromal cell genes in a tumor xenotransplantation model in mice.

Human tumor xenografts in mice together with the species-specific analysis of expressed genes allow to study the molecular processes driving tumor growth and progression in vivo and help to develop and evaluate anticancer therapies. In the present work, we designed and validated species-specific real-time RT-PCR assays for discrimination and quantitation of expression of human and mouse transcripts in cancer and stromal cells including dipeptidyl peptidase (DPP) 4, DPP8, DPP9, fibroblast activation protein (FAP) and CXC chemokine receptor 4 in mixed human-mouse biological samples. Using single species RNA samples and mixed human-mouse RNA samples, we formulated and characterized two-step real-time RT-PCR assays to quantitate expression of the indicated transcripts and described analytical performance of the assays. We also demonstrated the applicability of these assays for species-specific quantitation of transcriptional expression of mouse stromal cell genes including Dpp4, Dpp8, Dpp9, Fap and Cxcr4 in mixed human-mouse RNA samples from human glioma cell-derived tumor xenografts growing in mouse brain.

Fibroblast activation protein alpha is expressed by transformed and stromal cells and is associated with mesenchymal features in glioblastoma.

Glioblastomas are deadly neoplasms resistant to current treatment modalities. Fibroblast activation protein (FAP) is a protease which is not expressed in most of the normal adult tissues but is characteristically present in the stroma of extracranial malignancies. FAP is considered a potential therapeutic target and is associated with a worse patient outcome in some cancers. The FAP localization in the glioma microenvironment and its relation to patient survival are unknown. By analyzing 56 gliomas and 15 non-tumorous brain samples, we demonstrate increased FAP expression in a subgroup of high-grade gliomas, in particular on the protein level. FAP expression was most elevated in the mesenchymal subtype of glioblastoma. It was neither associated with glioblastoma patient survival in our patient cohort nor in publicly available datasets. FAP was expressed in both transformed and stromal cells; the latter were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. In a mouse xenotransplantation model, FAP was expressed in glioma cells in a subgroup of tumors that typically did not express the astrocytic marker GFAP. Endogenous FAP was frequently upregulated and part of the FAP+ host cells coexpressed the CXCR4 chemokine receptor. In summary, FAP is expressed by several constituents of the glioblastoma microenvironment, including stromal non-malignant mesenchymal cells recruited to and/or activated in response to glioma growth. The limited expression of FAP in healthy tissues together with its presence in both transformed and stromal cells suggests that FAP may be a candidate target for specific delivery of therapeutic agents in glioblastoma.

Differential sensitivity to apoptosome apparatus activation in non-small cell lung carcinoma and the lung.

The intrinsic apoptosis pathway represents an important mechanism of stress-induced death of cancer cells. To gain insight into the functional status of the apoptosome apparatus in non-small cell lung carcinoma (NSCLC), we studied its sensitivity to activation, the assembly of apoptosome complexes and stability of their precursors, and the importance of X-linked inhibitor of apoptosis (XIAP) in the regulation of apoptosome activity, using cell-free cytosols from NSCLC cell lines and NSCLC tumours and lungs from 62 surgically treated patients. Treatment of cytosol samples with cytochrome c (cyt-c) and dATP induced proteolytic processing of procaspase-9 to caspase-9, which was followed by procaspase-3 processing to caspase-3, and by generation of caspase-3-like activity in 5 of 7 studied NSCLC cell lines. Further analysis demonstrated formation of high-Mr Apaf-1 complexes associated with cleaved caspase-9 in the (cyt-c + dATP)-responsive COLO-699 and CALU-1 cells. By contrast, in A549 cells, Apaf-1 and procaspase-9 co-eluted in the high-Mr fractions, indicating formation of an apoptosome complex unable of procaspase-9 processing. Thermal pre-treatment of cell-free cytosols in the absence of exogenous cyt-c and dATP lead to formation of Apaf-1 aggregates, unable to recruit and activate procaspase-9 in the presence of cyt-c and dATP, and to generate caspase­3­like activity. Further studies showed that the treatment with cyt-c and dATP induced a substantially higher increase of caspase-3-like activity in cytosol samples from NSCLC tumours compared to matched lungs. Tumour histology, grade and stage had no significant impact on the endogenous and the (cyt-c + dATP)-induced caspase-3-like activity. Upon addition into the cytosol, the XIAP-neutralizing peptides AVPIAQK and ATPFQEG only moderately heightened the (cyt-c + dATP)-induced caspase­3­like activity in some NSCLC tumours. Taken together, the present study provides evidence that the apoptosome apparatus is functional in the majority of NSCLCs and that its sensitivity to the (cyt-c + dATP)-mediated activation is often enhanced in NSCLCs compared to lungs. They also indicate that XIAP does not frequently and effectively suppress the activity of apoptosome apparatus in NSCLCs.

Down-regulated expression of apoptosis-associated genes APIP and UACA in non-small cell lung carcinoma.

The Apaf-1 interacting protein (APIP) and the uveal autoantigen with coiled coil domains and ankyrin repeats (UACA) belong to endogenous regulators of the apoptosome apparatus, but their role in tumourigenesis and progression of non-small cell lung carcinoma (NSCLC) is not known. Previous studies demonstrated that APIP inhibits the apoptosome-mediated procaspase-9 activation while UACA induces translocation of Apaf-1 from the cytoplasm into the nucleus. Here, we report for the first time that the expression of APIP and UACA genes is down-regulated on the level of both mRNA and protein in NSCLC cells and tumours. In particular, the expression of APIP protein was strikingly decreased and the expression of UACA mRNA and protein was frequently down-regulated in NSCLC tumours of different histopathological types. Moreover, stage IA NSCLC tumours showed significantly lower expression of UACA mRNA compared to higher stage tumours. The weak increase of both APIP and UACA mRNA levels in the 5-aza-2'-deoxycytidine-treated NSCLC cells indicates that mechanisms other than DNA methylation are involved in the regulation of APIP and UACA gene expression in these cancer cells. Taken together, the down-regulation of APIP and UACA expression suggests that the threshold to activate the apoptosome apparatus may be decreased in NSCLC cells due to the lack of APIP-mediated suppression and UACA-assisted Apaf-1 nuclear entry. Moreover, the loss of UACA-assisted Apaf-1 nuclear translocation may underlie the failure of DNA damage checkpoint activation in NSCLC cells leading to their genomic instability.

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity.

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.

Coupled expression of dipeptidyl peptidase-IV and fibroblast activation protein-α in transformed astrocytic cells.

Dipeptidyl peptidase-IV (DPP-IV) and fibroblast activation protein-α (FAP) are speculated to participate in the regulation of multiple biological processes, because of their unique enzymatic activity, as well as by non-hydrolytic molecular interactions. At present, the role of DPP-IV and FAP in the development and progression of various types of tumors, including glioblastoma, is intensively studied, and their functional crosstalk is hypothesized. In this article, we describe the correlative expression of DPP-IV and FAP mRNA in primary cell cultures derived from human glioblastoma and associated expression dynamics of both molecules in astrocytoma cell lines depending on culture conditions. Although the molecular mechanisms of DPP-IV and FAP co-regulations remain unclear, uncoupled expression of transgenic DPP-IV and the endogenous FAP suggests that it occurs rather at the transcriptional than at the posttranscriptional level. Understanding of the expressional and functional coordinations of DPP-IV and FAP may help clarify the mechanisms of biological roles of both molecules in transformed astrocytic cells.

Granzyme B-induced apoptosis in cancer cells and its regulation (review).

The granzyme B-induced cell death has been traditionally viewed as a primary mechanism that is used by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells to eliminate harmful target cells including allogeneic, virally infected and tumour cells. Granzyme B (GrB) is the most abundant serine protease which is stored in secretory granules of CTLs and NK cells. After recognition of the target cell, the engaged CTLs and NK cells vectorially secrete GrB along with other granule proteins including perforin into the immunological synapse. From this submicroscopic intercellular cleft GrB translocates into the cytoplasm of the target cell. Although several models have been proposed to explain the GrB delivery mechanism, conclusive understanding of this process remains still elusive. Once in the cytoplasm, GrB cleaves and activates, or inactivates, multiple protein substrates, resulting eventually into apoptotic demise of the target cell. This review is focused on the gene structure and expression of GrB, its biosynthesis and activation, delivery mechanisms into the target cell cytoplasm, direct proteolytic involvement in activation of several pro-apoptotic pathways, and on regulation of its activity in cancer cells. Moreover, emphasis is given to the GrB-mediated anticancer effects and future clinical applications of the GrB-based and tumour-targeted recombinant fusion constructs.

Expression of dipeptidyl peptidase-IV activity and/or structure homologs in human meningiomas.

Meningiomas are tumors derived from arachnoid cap cells that represent approximately 30% of all intracranial tumors. In this study, we investigated 22 human meningiomas for the expression of dipeptidyl peptidase (DPP)-IV activity and/or structure homologs (DASH), including canonical DPP-IV/CD26, fibroblast activation protein-alpha (FAPalpha), DPP8 and DPP9. DPP-IV-like enzymatic activity, including all enzymatically-active DASH molecules, was found in all 18 benign meningiomas WHO grade I and IV atypical meningiomas WHO grade II by continuous rate fluorimetric assay in tissue homogenates and catalytic enzyme histochemistry in situ. In atypical meningiomas, this activity was significantly higher and was associated with higher cell proliferation as detected by Ki67 antigen immunohistochemistry. The expression of DPP-IV/CD26 and FAPalpha demonstrated by real-time RT-PCR and immunohistochemistry was low. As shown histochemically, it occurred most often on the surface of fibrous bundles and whorls rich in extracellular matrix. Compared to DPP-IV/CD26 and FAPalpha, the expression of DPP8 and DPP9 was higher and, in addition, it was present also in the cells inside these structures. Expression of CXCR4, the receptor of pro-proliferative chemokine stromal cell-derived factor-1alpha (SDF-1alpha), DPP-IV substrate, was found in all tumors, suggesting higher values in atypical grade II samples. This is the first report on the expression status of dipeptidyl peptidase-IV and related molecules in meningiomas. It shows that DPP8 and DPP9 prevail over canonical DPP-IV/CD26 and FAPalpha in all examined patients. In addition, the study suggests an increase of DPP-IV-like enzymatic activity in these tumors of WHO grade II.

Expression of proteinase inhibitor-9/serpinB9 in non-small cell lung carcinoma cells and tissues.

Human proteinase inhibitor-9 (PI-9)/serpinB9 is an intracellular ovalbumin-family serpin with nucleocytoplasmic distribution which is expressed in certain normal cell types and cancer cells of different origin. Due to binding and inactivating of granzyme B (GrB), PI-9 can protect the cells from GrB-mediated apoptosis. High levels of PI-9 expression in certain cancer cells may contribute to their resistance against the immune mediated killing. So far, it is not known whether non-small cell lung cardinomas (NSCLCs) express PI-9 mRNA and a functional PI-9 protein. Herein we report for the first time that NSCLC cells express both PI-9 mRNA and protein and that there is a subset of NSCLC cells with upregulated PI-9 mRNA and protein expression. Futhermore, our work revealed that the PI-9 protein expressed in NSCLC cells can inhibit the active GrB. Finally, analysis of PI-9 mRNA expression in NSCLC tumours from surgically treated patients showed that the expression of this transcript is upregulated in the less-differentiated lung adenocarcinomas. We suggest, that the upregulated expression of PI-9 in NSCLC cells may serve to protect them from apoptosis induced by GrB.

Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma.

Members of the inhibitor of apoptosis protein (IAP) family, survivin and X-chromosome-linked IAP (XIAP), contribute to apoptosis resistance of cancer cells, and an increase in their expression may elevate the apoptotic threshold of malignant tumours during their growth and progression. In the present study, we investigated the expression status of survivin and its interactants hepatitis B X-interacting protein (HBXIP) and XIAP in non-small cell lung carcinoma (NSCLC) cell lines and NSCLC tumours and matched lungs from surgically treated patients in relation to their clinicopathological data. The expression of survivin, HBXIP and XIAP mRNAs was quantitated by real-time RT-PCR. The expression of survivin and XIAP proteins was analysed by Western blotting and ELISA. Survivin mRNA and protein levels were highly upregulated in NSCLC cells and tissues as compared to the lungs. In fact, the levels of survivin mRNA and protein in the tumours were more than 10-fold higher in 96 (64%) and 72 (82%) of the 150 and 88 examined NSCLC patients, respectively. The expression of survivin mRNA was higher in squamous cell lung carcinomas than in lung adenocarcinomas (LACs; P=0.003) and in less-differentiated tumours than in well-differentiated ones (P=0.007). The level of survivin protein was higher in stage IB and stage II+III tumours (P=0.049 and P=0.044), than in stage IA tumours. The BIRC5 promoter polymorphism at nucleotide -31 did not influence the expression of survivin mRNA and protein in NSCLC cells and tumours. HBXIP mRNA was abundantly expressed in NSCLC cell lines and NSCLC tumours and lungs, while its level was comparable in the tumours and lungs. The expression of XIAP mRNA in NSCLC cell lines and NSCLC tumours and lungs was not significantly different. However, the expression of XIAP protein was higher in NSCLC tumours, particularly in LACs, as compared to the lungs (P=0.017 and P=0.004). In conclusion, the overexpression of survivin in the majority of NSCLCs together with the abundant or upregulated expression of HBXIP and XIAP suggest that tumours are endowed with resistance against a variety of apoptosis-inducing conditions.

Expression and enzymatic activity of dipeptidyl peptidase-IV in human astrocytic tumours are associated with tumour grade.

Alterations in dipeptidyl peptidase-IV (DPP-IV) enzymatic activity are characteristic of malignant transformation. Through its well-characterized functionality in regulating the activity of bioactive peptides by removal of the N-terminal dipeptide, DPP-IV activity may have profound effects upon metastatic potential and cell growth. Although DPP-IV/CD26 (EC 3.4.14.5) is the canonical representative of the group, a number of other proteins including DPP-7, 8, 9, and seprase/fibroblast activation protein-alpha (FAP-alpha) have been shown to have similar enzymatic activity. This study was set up to address the relative representation and enzymatic activity of plasma membrane localized DPP-IV/CD26 and FAP-alpha in human brain and astrocytic tumours. In parallel, expression of CXCR4, receptor for glioma cell growth stimulator chemokine SDF-1alpha known to be a DPP-IV substrate, was investigated. This is the first report showing that non-malignant brain tissue contains a DPP-IV-like enzymatic activity attributable mostly to DPP-8/9, while the substantial part of the activity in glioma is due to increased DPP-IV/CD26, localized in both the vascular and parenchymal compartments. DPP-IV enzymatic activity increased dramatically with tumour grade severity. A grade-related increase in CXCR4 receptor paralleled the rise in DPP-IV expression and activity. These data might support a role for DPP-IV regulation of the CXCR4-SDF-1alpha axis in glioma development.

Expression of apoptosome pathway-related transcripts in non-small cell lung cancer.

PURPOSE: Tumour cells killing by cytotoxic therapies largely depends on triggering the intrinsic apoptosome-mediated caspase activation pathway but it had never been evaluated whether the expression of transcripts encoding the core components of apoptosome pathway is altered in non-small cell lung carcinoma (NSCLC). METHODS: We investigated the expression status of several apoptosome pathway-related transcripts including Apaf-1, procaspase-9, -3, -6, -7 and Smac in tumour and lung tissue samples from 65 surgically treated NSCLC patients and in 10 NSCLC cell lines with using real time RT-PCR. RESULTS: NSCLC tissues and cell lines showed significantly increased expression of procaspase-9, -3, -6 and Smac mRNAs as compared to the lungs and expression of these transcripts was simultaneously upregulated in a subset of NSCLCs belonging to different histopathological type, grade and stage categories. The expression of procaspase-7 mRNA in NSCLC tissues and cell lines and lungs was not significantly different. By contrast, the expression of Apaf-1 mRNA was frequently downregulated in the tumours as compared to matched lungs. Nevertheless, the examined NSCLC cell lines showed significantly higher expression of Apaf-1 mRNA than the lungs. The expression of Apaf-1, procaspase-9 and -6 mRNAs was higher in lung adenocarcinomas as compared to squamous cell lung carcinomas but the expression levels of the studied apoptosome pathway-related transcripts in the tumours were independent of tumour's grade and stage. CONCLUSIONS: The results of the present study suggest that there is a subgroup of NSCLCs, which may be intrinsically primed for apoptosis through upregulated expression of transcripts encoding the apoptosome pathway components.

Lung cancer in women.

Lung cancer is one of the most important avoidable causes of death around the world, it is the most widespread carcinoma with a very poor prognosis, and is the leading cause of cancer death in both developed and developing countries. At present more men than women die each year from lung cancer, but in recent years a rapid increase in lung cancer mortality has been observed among women in developed countries, contrasting with a levelling off or decrease among men. The rising trend in female lung cancer mortality has been observed to parallel with the past and current prevalence of cigarette smoking among women in the United States and elsewhere. An important role of other factors acting either as independent risk factors or interacting with the effect of smoking has been suggested by some studies among women, among them genetic, biologic and hormonal factors, and probably some factors related to the environment and lifestyle. There is a controversy concerning the claim that women have a different susceptibility to tobacco carcinogens, which might or might not be greater than men do. Since tobacco is far and away the strongest epidemiological risk factor for the development of lung cancer, comprehensive smoking control efforts are the priority in the prevention of lung cancer among women.

Concurrent versus sequential chemoradiotherapy with cisplatin and vinorelbine in locally advanced non-small cell lung cancer: a randomized study.

PURPOSE: The superiority of chemoradiotherapy (CRT) over radiation alone in locally advanced non-small cell lung cancer (NSCLC) has been proven, but the relative merits of a concurrent schedule versus their sequential administration are less clear. This study compared the safety and efficacy of concurrent and sequential CRT, with chemotherapy (CT) consisting of a cisplatin and vinorelbine regimen, in patients with locally advanced NSCLC. PATIENTS AND METHODS: One hundred and two previously untreated patients (aged 42-75 years) with locally advanced, stage IIIA (n = 15) or stage IIIB (n = 87) NSCLC were entered into the study. The CT schedule consisted of up to four cycles of cisplatin 80 mg/m(2) on day 1, and vinorelbine 25 mg/m(2) at the first and fourth cycles (12.5 mg/m(2) during the 2nd/3rd cycles) on days 1, 8, 15 of a 28-day cycle. Radiotherapy (RT) was prescribed at a dose of 60 Gy/30 fractions, given as five fractions per week for 6 weeks. In the concurrent arm (arm A), RT was started on day 4 of cycle 2; whilst in the sequential arm (arm B), RT started within 2 weeks after completion of CT. Fifty-two patients were randomized to concurrent treatment and 50 to the sequential schedule. RESULTS: Overall survival was significantly longer in arm A (median survival 16.6 months) versus arm B (median survival 12.9 months) (P = 0.023 by means of log-rank test; hazard ratio HR = 0.61, 95% CI of HR (0.39-0.93)), and time to progression (TTP) was also significantly longer in arm A (median time to progression 11.9 months) versus arm B (median time to progression 8.5 months) (P = 0.024 by means of log-rank test; HR = 0.62, 95% CI of HR (0.38-0.93)). Ninety-eight patients were evaluable for response and 101 for toxicity. The overall response rate was significantly higher in arm A, 80% (with 21% complete response (CR)) compared with 47% (with 17% CR) in arm B (P = 0.001 by means of chi(2)-test). WHO grade 3 or 4 toxicity was more frequent in arm A than in arm B, with a significantly greater incidence of leucopenia (53% versus 19%, P = 0.009 by means of chi(2) test) and nausea/vomiting (39% versus 15%, P = 0.044 by means of chi(2) test). There were no treatment related deaths. CONCLUSION: In this study population, concurrent CRT demonstrated significant benefit in terms of response rate, overall survival and time to progression over sequential CRT. The concurrent CRT schedule was associated with higher toxicity; however, the adverse event profile was acceptable in both arms.

Increased expression of Apaf-1 and procaspase-3 and the functionality of intrinsic apoptosis apparatus in non-small cell lung carcinoma.

The intrinsic apoptosis apparatus plays a significant role in generating and amplifying cell death signals. In this study we examined whether there are differences in the expression of its components and in its functioning in non-small cell lung carcinoma (NSCLC) and the lung. We show that NSCLC cell lines express Apaf-1 and procaspase-9 and -3 proteins and that the expression of Apaf-1 and procaspase-3, but not of procaspase-9 and -7, is frequently up-regulated in NSCLC tissues as compared to the lung. NSCLC tissues and lungs and some NSCLC cell lines expressed also caspase-9S(b) and displayed a high caspase-9S(b)/procaspase-9 expression ratio. Procaspase-3 from NSCLCs and lungs was readily processed to caspase-3 by granzyme B or caspase-8, and the granzyme B-generated caspase-3-like activity was significantly higher in tumor tissues and cells than in lungs. By contrast, cytochrome c plus dATP could induce a significant increase of caspase-3-like activity in cytosol only in some NSCLC cell lines and in subsets of studied NSCLC tissues and lungs, while procaspase-3 and -7 were detectably processed only in NSCLC tissues which showed a high (cytochrome c+dATP)-induced caspase-3-like activity. Taken together, the present study provides evidence that the expression of Apaf-1 and procaspase-3 is up-regulated in NSCLCs and indicates that the tumors have a capability to suppress the apoptosome-driven caspase activation in their cytosol.