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The aim of this study was to examine the effects of 5‑fluorouracil (5‑FU), anti‑epidermal growth factor receptor (EGFR) antibody and aspirin (ASA) on the characteristics of two CRC cell lines, HCT116 and HT29, maintained in a spherical culture system. We observed that the morphology of both the HCT116 and HT29 cell‑derived spheres was significantly impaired and the size of the colonospheres was markedly reduced following treatment with the aforementioned three drugs. In contrast to adherent cultures, the spherical cultures were more resistant to the tested drugs, as was reflected by their capacity to re‑create the colonospheres when sustained in serum‑free medium. Flow cytometric analysis of the drug‑treated HCT116 cell‑derived spheres revealed changes in the fraction of cells expressing markers of cancer stem cells (CSCs), whereas the CSC phenotype of HT29 cell‑derived colonospheres was affected to a lesser extent. All reagents enhanced the percentage of non‑viable cells in the colonospheres despite the diminished fraction of active caspase‑3‑positive cells following treatment of the HT29 cell‑derived spheres with anti‑EGFR antibody. Increased autophagy, assessed by acridine orange staining, was noted following the incubation of the HT29‑colonospheres with ASA and 5‑FU in comparison to the control. Notably, the percentage of cyclooxygenase (COX)‑2‑positive cells was not affected by ASA, although its activity was markedly elevated in the colonospheres incubated with anti‑EGFR antibody. On the whole, the findings of this study indicate that all the tested drugs were involved in different cellular processes, which suggests that they should be considered for the combined therapeutic treatment of CRC, particularly for targeting the population of CSC‑like cells. Thus, cancer cell‑derived spheres may be used as a preferable model for in vitro anticancer drug testing.

Whereas cancer immunotherapy with cytokines in recent research was demonstrated effective in activating immune response against tumor cells, one major obstacle with the use of these cytokines is their severe side effects when delivered systemically at high doses. Another challenge is that advanced tumor cells often evade immunosurveillance of the immune system as well as of the Fas-mediated apoptosis by various mechanisms. We report the design and preliminary evaluation of the antitumor activity of a novel fusion protein-mIL-12/FasTI, consisting of mouse interleukin-12 and the transmembrane and intracellular domains of mouse Fas. The fusion construct (pmIL-12/FasTI) was transfected into mouse lung carcinoma cell line TC-1. Stable cell clones expressing the fusion protein were established as assayed by RT-PCR and immunohistochemistry. ELISA and cell proliferation analyses demonstrated that NK cells were effectively activated by the fusion protein with increased IFN-γ production and cytotoxicity. Enhanced caspase-3 activity of the clones when co-cultured with NK cells indicated that apoptosis was induced through Fas/FasL signaling pathway. The preliminary results suggest a synergized anticancer activity of the fusion protein. It may represent a promising therapeutic agent for cancer treatment.

Carcinoma cells that have acquired drug resistance often exhibit cross-resistance to various other cytotoxic stimuli. Here, we investigated the effects of ursodeoxycholic acid (UDCA), a gastrointestinal tumor-suppressor, on a cisplatin‑resistant SNU601 gastric cancer subline (SNU601/R). While other anticancer drugs, including L-OHP, etoposide, and death ligand TRAIL, had minimal effects on the viability of these resistant cells, they were sensitive to UDCA. The UDCA‑induced reduction in the viability of the SNU601/R cells was accomplished through autophagy while the primary means of cell death in the parental SNU601 cells (SNU601/WT) was apoptosis. Previously, we demonstrated that the UDCA-triggered apoptosis of gastric cancer cells was regulated by a cell surface death receptor, TRAIL-R2/DR5, which was upregulated and re-distributed on lipid rafts. The UDCA stimulation of TRAIL-R2/DR5 also occurred in the SNU601/R cells despite the lack of apoptosis. In the present study, we found that CD95/Fas, another cell surface death receptor, was also translocated into lipid rafts in response to UDCA although it was not involved in the decrease in cell viability. Specifically, raft relocalization of CD95/Fas was triggered by UDCA in the SNU601/WT cells in which apoptosis occurred, but not in the SNU601/R cells where autophagic death occurred. Notably, UDCA reduced ATG5 levels, an essential component of autophagy, in the SNU601/WT, but not in the SNU601/R cell line. Moreover, in CD95/Fas-silenced SNU601/WT cells, UDCA did not decrease ATG5 levels and induced autophagic cell death rather than apoptosis. These results imply that raft‑distributed CD95/Fas may support UDCA-induced apoptosis via downregulation of ATG5 levels, preventing the autophagic pathway. Taken together, these results suggest that UDCA induces both apoptotic and autophagic cell death depending on the intracellular signaling environment, thereby conferring the advantage to overcome drug resistance through apoptotic defects.

Swainsonine, an extract from Astragalus membranaceus, exhibits broad inhibition of growth and pro-apoptotic activity in a number of tumor types. However, the underlying mechanism involved remains unclear. To investigate the effects and mechanisms of swainsonine on hepatocellular carcinoma (HCC), we performed experiments on HepG2, SMCC7721, Huh7 and MHCC97-H human hepatoma and HL-7702 human hepatocyte cells. We observed that swainsonine significantly inhibited the viability of human hepatoma cells in a dose- and time-dependent manner, but did not affect human hepatocytes. Due to their highly proliferative and tumorigenic nature, we selected MHCC97-H cells as a model system to examine. Swainsonine significantly inhibited MHCC97-H cell growth by causing cell cycle arrest at the G0/G1 phase and the induction of apoptosis. Blockage of G0/G1 phase was accompanied by a decrease in cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4) and an increase in the Cdk inhibitors p21 and p27. Furthermore, swainsonine enhanced the apoptosis of MHCC97-H cells with the induction of the upregulation of Bax and the downregulation of Bcl-2, whereas the expressionof Fas and Fas-L remained almost unchanged. These changes were accompanied by the enhanced cytoplasmic accumulation of nuclear factor κB (NF-κB) with a concomitant decrease in the nuclear fraction. Importantly, swainsonine also potentiated the cytotoxic effects of paclitaxel in vitro and in vivo, in part, by restricting the paclitaxel-induced nuclear accumulation of NF-κB. Taken together, these results suggest that swainsonine may be an important agent against HCC via directly inhibiting HCC cell growth and enhancing the responsiveness of HCC cells to paclitaxel.

Norcantharidin (NCTD) is one of the ingredients of blister beetles which have been used in Chinese medicine for a long time. The purpose of this study was to investigate the inhibitory effects of NCTD on TSGH 8301 human bladder cancer cells in vitro and the mechanisms through which it exerts its anticancer action. Cell morphological analysis was performed using a phase-contrast microscope. The percentage of viable cells, cell cycle distribution, sub-G1 phase (apoptosis), reactive oxygen species (ROS) production and the levels of mitochondrial membrane potential (∆Ψ(m)) were analyzed by flow cytometry. DNA condensation and damage were determined by DAPI staining and comet assay. Apoptosis-associated protein level changes in TSGH 8301 cells following exposure to NCTD were examined, measured and determined by western blotting. Analysis of protein translocation was conducted by immunostaining and confocal laser microscopy. The results indicated that NCTD promoted cytotoxic effects, including the induction of cell morphological changes and the decrease in the percentage of viability, the induction of S-phase arrest as well as sub-G1 phase (apoptosis) in TSGH 8301 cells. The activities of caspase-3 and -9 were upregulated following NCTD treatment. Western blotting indicated that NCTD upregulated Fas, FasL, Bax, Bid, cytochrome c, caspase-3, -8 and -9 that led to the induction of apoptosis through the Fas extrinsic pathway. Furthermore, NCTD induced AIF and Endo G that were released from mitochondria to induce apoptosis through the mitochondrial-independent pathway. NCTD upregulated ROS production, downregulated ∆Ψ(m) and ERK, JNK, p38 protein kinases in TSGH 8301 cells. These findings suggest that NCTD triggers apoptosis in TSGH 8301 human bladder cancer cells via the Fas receptor, activation of the caspse-8, -9 and -3, mitochondrial-dependent and -independent pathways. NCTD may be useful for developing new therapeutic regimens for the treatment of bladder cancer.

Glioblastoma is the most common primary brain tumor, characterized by its resistance to treatments. To define efficient therapy, the origin of tumor-forming cells needs to be elucidated in order to search for new therapeutic pathways. The objective of this study was to determine the different cell populations constituting a human glioblastoma cell line, U-87 MG and their sensitivity to apoptosis induced through the activation of Fas, a membranous death receptor. By a cell sorting method, the sedimentation field flow fractionation, two major cell subpopulations were identified, a most differentiated cell fraction, containing large and adherent cells, sensitive to Fas-induced apoptosis and another one, characterized by small cells forming aggregates, expressing CD133, a marker of stem cells and more resistant to Fas-activated apoptosis. By using a selective method of culture, adapted for neural stem cell cultures, we have verified that the U-87 MG cell line contained cancer stem cells similar to the immature ones obtained by the cell sorting method. Interestingly, while these tumor stem cells, expressing CD133, were resistant to Fas-induced apoptosis, monomeric form of Fas protein was detected predominantly in these cells. In contrast, the most mature cells, responsive to Fas-activated apoptosis, collected in another cell fraction, contained oligomeric aggregates of Fas protein, a pre-signalling form of the Fas receptor, essential for the initiation of apoptosis through its activation. These results suggest that these immature stem cells in glioma could be an important factor of resistance to chemotherapy requiring apoptosis through Fas signalling system. Indeed, future strategies of treatment, inducing differentiation of these stem cells need to be considered to enhance therapeutic efficiency.

Apoptosis is a physiological mechanism responsible for a wide range of cellular processes during growth, development, carcinogenesis, and inflammation. In this study, we determined whether MUC1 affects Fas ligand (FasL)-induced apoptosis using MUC1 expressing (MUC1(+)) and MUC1(-) cells. Following treatment with 50 nM FasL, apoptosis, caspase-8 activity, and cell surface Fas receptor were measured by cytosolic nucleosome ELISA, colorimetric enzyme assay, and immunofluorescence analysis, respectively. Our results showed that (i) treatment with FasL increased caspase-8 activity (maximum at 4 h) and apoptosis (maximum at 8 h) in both MUC1(+) and MUC1(-) cells, (ii) FasL-induced caspase-8 activity and apoptosis were significantly greater in MUC1(+) cells compared with MUC1(-) cells, (iii) FasL treatment increased cell surface expression of Fas receptor in MUC1(+) cells to a greater extent compared with MUC1(-) cells, (iv) increased cell surface expression of Fas in MUC1(+) cells was not blocked by an inhibitor of protein synthesis (cycloheximide), but was completely abrogated by brefeldin A, an inhibitor of post-translational protein trafficking to the cell surface, and (v) brefeldin A inhibited the increased sensitivity of MUC1(+) cells to FasL-induced apoptosis. We conclude that MUC1(+) cells were more sensitive to FasL-induced apoptosis compared with MUC1(-) cells due to up-regulation of Fas receptor on the cell surface by a mechanism involving increased intracellular trafficking.

Drug resistance in ovarian cancer treatment urges the exploration of new targets for drugs against this malignancy. Fas is a cell membrane receptor which, after engagement with Fas ligand (FasL), triggers apoptotic death. In this study Fas and FasL levels in cyst fluids and sera of patients with benign, borderline and malignant ovarian tumors and in corresponding tumors are determined. Fas and FasL were determinded by ELISA and immunohistochemistry in 30 patients with benign, 5 patients with borderline and 24 patients with malignant epithelial ovarian tumors. In serum there were no differences in median Fas levels, while median FasL levels were higher in healthy women (p=0.02). In malignant cyst fluids, median Fas levels where higher compared to benign cyst fluids (p<0.01). FasL immunostaining was more frequent in malignant ovarian tumors (p=0.002). In conclusion, serum Fas or FasL levels do not seem useful markers. Elevated Fas and equal FasL levels in malignant cyst fluids, suggest an increased production of Fas, and not of FasL by malignant cells. High expression of both Fas and FasL, in malignant ovarian tumors present Fas/FasL as an interesting route to explore for innovative cancer therapy.

Adenoviral p53 gene transfer (Ad-p53) induces apoptosis in glioma cells expressing mutant p53, but fails in cells with wild-type p53. Endogenously, gliomas express varied levels of Fas/CD95, yet constitutively high levels of Fas/CD95 ligand. Because the mechanism behind the differential apoptotic response to Ad-p53 infection remains elusive, we examined how the Fas/CD95 pathway is involved in U87MG (wt-p53), D54 (wt-p53), U251MG (mutant-p53), and U373MG (mutant-p53) glioma cell lines. Ad-p53 infection did not alter the levels of Fas/CD95 ligand in either wild-type or mutant p53-expressing cell lines. In contrast, Ad-p53 infection led to an approximately 3-fold increase in Fas/CD95 mRNA expression in mutant p53-bearing cell lines but not in their wild-type (wt) counterparts, as assessed in an RNase protection assay. Fas/CD95 mRNA induction appeared to be regulated at the transcriptional level because Ad-p53 infection resulted in up to a 4-fold increase in Fas/CD95 promoter reporter activity. Subsequently, flow cytometric analysis revealed a 2- to 4-fold increase in surface Fas/CD95 expression following Ad-p53 infection in mutant-p53-containing cell lines. Use of the protein transport inhibitor Brefeldin A significantly inhibited Ad-p53-induced surface Fas/CD95 expression, but only partially inhibited apoptosis in mutant-p53 cell lines. These results suggest that p53 regulates Fas/CD95 expression at the transcriptional level and through protein trafficking in mutant-p53 cell lines. Fluorogenic activity assays demonstrated that induction of caspase-8 activity following Ad-p53 infection correlated with increases in Fas/CD95 expression. Incubating cells with a caspase-8-specific inhibitor Ac-IETD-CHO prior to Ad-p53 infection inhibited caspase-8 activity and apoptosis. Together, our results suggest that regulation of the Fas/CD95 pathway is partly responsible for Ad-p53-induced apoptosis in glioma cells, which depends on the p53 status of the involved cells. Additionally, the inability of Ad-p53 to activate the Fas/CD95 pathway in wt-p53 glioma cells coincides with their apoptotic-resistant phenotype. Further elucidation of the nature of this resistance could ultimately augment the efficacy of Ad-p53 gene therapy.

The widespread expression of CD40, a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily, is likely to account for the central role of CD40 in the regulation of humoral immunity and host defense. Interestingly, the expression of the CD40 in various types of carcinoma cells was often observed and conveys signals regulating diverse cellular responses, ranging from proliferation to growth suppression. Thus, the biologic role of the CD40-CD40L interaction in solid tumors is still controversial. In this study, we investigated the expression and function of the CD40 in gastric carcinoma cells. In 3-4,5 dimethylthiozol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) assay and Annexin V/propidium iodide staining, CD40 stimulation using a soluble form of CD40 ligand did not affect cell viability, but significantly inhibited Fas-mediated or chemotherapy-mediated apoptosis in three CD40-positive gastric cancer cell lines. Moreover, in migration assay, CD40 stimulation induced an elevation of cell motility in CD40-positive gastric carcinoma cells. Our results show that the CD40 expression on gastric carcinoma makes cells less vulnerable to apoptosis induced by Fas or chemotherapy. These results suggest that the CD40 expression on gastric carcinoma may be associated with cell survival and elevation of cell motility.

Apoptosis is a crucial event for anticancer drug efficacy. The signal pathways activated by anticancer drugs are classified as death receptor (DR) -dependent or -independent. The mechanisms by which anticancer drugs induce apoptosis via DR-dependent pathways are not fully understood. In the present study, we assessed differential activation of signal transduction pathways leading to apoptosis by anticancer drugs in breast cancer cell lines. Three breast cancer cell lines, MDA-MB-231, MCF-7, and MCF-7ADM, which is drug-resistant, were used. Drug sensitivity and apoptosis were assessed by MTT assay and TUNEL, respectively. Expression of apoptosis-related protein was assessed by Western blotting and RT-PCR. The sensitivities of MDA-MB-231 and MCF-7 cells to mitomycin C (MMC) and adriamycin (ADM) were similar. In contrast, sensitivity of MDA-MB-231 cells to paclitaxel (TXL) was 30-fold greater than that of MCF-7 cells, 0.03 micro M in MDA-MB-231 and 0.9 micro M in MCF-7 cells, respectively. Treatment with MMC increased expression of DR4 and Fas in a time-dependent manner in MDA-MB-231 cells. Treatment with ADM increased expression of DR4 and DR5 but not Fas, whereas treatment with TXL increased expression of Fas but not DR4 and DR5 in MDA-MB-231 cells. Although treatment of MCF-7 cells with ADM increased expression of DR4 and DR5 but not Fas, expression of DR4, DR5, and Fas by the drug-resistant cells did not change following treatment with ADM. Activation of Fas, DR4, and DR5 in drug-sensitive cells in response to anticancer drugs is dependent on the cytotoxic effect of each drug. In drug-resistant cells, apoptosis is induced via DR-independent pathways mediated by mitochondrial dysfunction.

Menstrual cycle-dependent expressions of activin A in normal human endometrial tissues have been reported. Expression of activin receptor mRNAs and increased activin A production were also observed in human endometrial adenocarcinoma tissues, suggesting that activin A might enhance cell proliferation and inhibit apoptotic signaling in endometrial cancer cells. In this study, we have examined the effects of activin A on cell proliferation, anticancer drug-induced apoptosis and Fas-mediated apoptosis in 3 differentiated human endometrial adenocarcinoma cell lines, namely HEC-1, HHUA and Ishikawa. Flow cytometric analyses revealed moderate expressions of all 4 types of activin receptor subunits on the cell surfaces of the 3 cell lines. The proliferations of the 3 endometrial cancer cells were completely unaffected by activin A, whereas it suppressed the cell proliferation of a human ovarian endometrioid adenocarcinoma cell line, OVK-18, in a dose-dependent manner. Moreover, activin A did not affect the apoptotic changes in the 3 endometrial adenocarcinoma cells treated with 4 different anticancer drugs, namely CDDP, paclitaxel, etoposide and SN38. The apoptotic changes in HHUA cells treated with anti-Fas IgM were also unaffected by activin A. These results indicate that the increased activin A production in human endometrial adenocarcinoma tissues in vivo may not stimulate carcinoma cell proliferation or inhibit apoptotic signaling in carcinoma cells. Insensitivity to the usual growth suppression signals induced by activin A might be one of the mechanisms of immortality of human endometrial adenocarcinoma cells.

The aim of this study was to evaluate the possible role of apoptosis-related proteins (ARP: Fas and Fas ligand, bcl-2, p53) in the progress of tumorigenesis in breast cancer. Epithelial tumor cells and lymphocytes were analyzed immunohistochemically for the rate of lymphoid infiltration and presence of ARP in 50 human breast tumors of different types. The tumors included: i) fibrocystic disease (n=12); ii) benign fibroadenoma (n=11); iii) carcinoma in situ (n=8); iv) invasive ductal carcinoma with high lymphoid infiltration (n=12); and v) invasive ductal carcinoma with lymphoid depletion (n=7). Both fibrocystic disease and fibroadenomas had low amounts of lymphocytes and very little lymphoid infiltration. In cancer in situ, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a rise in the total number of lymphocytes, reflecting intensification of the immune response. In carcinomas with high lymphoid infiltration, a significant increase in the number of Fas and FasL and p53-positive cells was found. The number of bcl-2-positive tumor cells in these tumors was not changed, whereas the number of bcl-2-positive lymphocytes decreased. In carcinomas with lymphoid depletion, the opposite picture was found as a result of deep subcompensation of the lymph system. ARP are present in a significantly higher number of lymphocytes than of the epithelial tumor cells. This indicates that the cells initiating apoptosis in tumors are themselves damaged by the process. The intense apoptosis in lymphocytes in malignant tumors may be one of the reasons for the progress of breast tumors.

Keratinocyte injury in drug-induced toxic epidermal necrolysis (TEN) is attributed to a dysregulation in the complex apoptotic machinery. This study was performed to investigate some aspects of the apoptotic pathomechanism involving calprotectin and the CD95 receptor (CD95R, FasR) in TEN. The expression of these two molecules corresponds to calcium-dependent and calcium-independent processes, respectively. Biopsies were collected from blistering skin in 21 TEN patients and from clinically uninvolved skin in 16 of these patients. Immunohistochemistry was performed using specific antibodies directed to CD95R and calprotectin. Half (8/16) of the biopsy specimens taken from clinically uninvolved sites showed the expression of calprotectin throughout the epidermis or restricted to the suprabasal layers. In these samples, a strong CD95R immunoreactivity was often restricted to the basal layer (8/16). Calprotectin was present in all biopsies of bullous skin, especially in suprabasal layers (15/21) or throughout the epidermis (6/21). By contrast, the prominent expression of CD95R was confined almost exclusively to the basal layer (15/21), and more rarely throughout the epidermis (2/21), and it remained sometimes unexpressed (4/21). A clear-cut boundary was often present between the areas labeled by the two antibodies with exceptional overlap between them. The simultaneous expression of calprotectin and CD95R in TEN at distinct levels of the epidermis indicates that the pathomechanism leading to keratinocyte death does not belong to a single process in TEN. Their expression in clinically uninvolved skin suggests that these processes are early and widespread events in TEN.

Nasopharyngeal carcinoma (NPC) is a malignant disease of the head/neck region with a 5-year survival level of approximately 65%. To explore the novel therapeutic strategies in the management of this disease, the potential effects of photodynamic therapy (PDT) in NPC cells were investigated. PDT, a new mode of treatment, is based on the combined use of light-absorbing compounds and light irradiation. Two human NPC cells such as, poorly differentiated (NPC/CNE2) and moderately differentiated (NPC/TW0-1) and other types of tumor cells like colon (CCL-220.1) and bladder (SD) undergo rapid apoptosis when treated with PDT sensitized with hypericin (HY). It has been shown that this compound has a strong photodynamic effect on tumors and viruses. However, the initiating events of PDT sensitized HY-induced apoptosis are not identified completely. In this study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HY-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases 8 and 3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photosensitization of HY enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/CD95L expression appeared within 2 h following light irradiation and appeared to be a principal event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm within 2-3 h post PDT is a secondary event following the activation of initiator caspase-8 preceding Apaf-1, caspase-9 and caspase-3 activation, cleavage of PARP and DNA fragmentation.

The therapeutic efficacy of low dose administration of 5-fluorouracil (5-FU) and cisplatin (CDDP) (low dose FP) has been reported in patients with advanced and recurrent gastric carcinoma. Mechanism(s) by which low dose FP exerts antitumor effect is not entirely clear. We investigated mechanism(s) of the therapeutic efficacy in combination with 5-FU and CDDP in terms of signal transduction pathways leading to apoptosis. Using two human gastric carcinoma cell lines, MKN28 and MKN45, antitumor effect in combination treatment with 5-FU and CDDP was assessed by MTT 5-day assay. The significant antitumor effect was determined with more than 50% growth inhibition compared to control cells. Enhancement of antitumor effect in the combination treatment was analyzed using isobologram. Apoptotic cell death was assessed by DNA ladder formation assay, and expression of apoptosis-related genes was detected by Western blotting. Concentration of free platinum and 5-FU was measured by high-pressure liquid chromatography (HPLC), and dihydropyrimidine dehydrogenase (DPD) activity and total folate levels were assessed by enzyme immunoassays. Antitumor effect in single treatment with 5-FU was not observed significantly with the concentration from 1 to 5 microM in vitro. In contrast, antitumor effect in combination treatment with 5-FU and CDDP showed a synergism with the concentration of CDDP from 1.5 to 3 microM. Single treatment with CDDP also did not show significant antitumor effect with the concentration from 1.5 to 3 microM. The enhancement in the synergistic effect by CDDP was dose-dependent. Any free platinum treated with low dose CDDP was not detected into gastric carcinoma cells, however, treatment with CDDP induced a receptor signaling pathway, that is mediated by Fas but not DR4. It may directly activate caspase 3 leading to apoptosis. Although the receptor signaling pathway in apoptosis was not observed by 5-FU, Bax-induced cytochrome c and caspase 3 was also observed in a receptor-independent pathway by 5-FU and CDDP. Total folate levels by cotreatment with CDDP was increased to 1.5-fold compared to 5-FU alone, whereas DPD activity and 5-FU concentration were not changed by cotreatment of CDDP in vivo. The enhancement of antitumor effect by low dose FP can be explained as follows: i) low dose treatment with CDDP induces apoptotic cell death through a receptor signaling pathway even in absence of free platinum into cells; ii) increased folate level by CDDP and a non-receptor signaling pathways by 5-FU contribute to apoptotic cell death in gastric carcinoma.

In head and neck clinical oncology, recurrent cancer after initial irradiation therapy is no longer sensitive to irradiation. To explore the irradiation resistance in head and neck squamous cell carcinoma, a human cell line, KB, derived from the floor of the oral cavity was used. The participation of the Fas-mediated apoptotic pathway was suggested by the upregulation of the surface Fas molecule, the reduction of the apoptotic cell fraction after inhibition of caspase 8 which is a Fas-related initiator caspase, and the changes in Fas-related genes after irradiation. Therefore, it is suggested that disruption of the Fas-mediated apoptotic pathway participates in the acquisition of irradiation-resistance in HNSCC.

The effect of extremely large hepatomas on splenic lymphoid elements and apoptosis-related proteins in rats were studied. Hepatoma cells were inoculated subcutaneously into 6-week-old rats, and 4 months later the quantities of T and B cells, macrophages, and cells positive for Fas, Fas ligand (FasL) and interleukin-2 (IL-2) were immunohistochemically evaluated in spleens. Grafting of hepatoma cells caused hyperplasia of the spleen and development of giant tumors that could reach one-third of the rat's body weight. A 7-fold increase in the weight of the spleen was mainly due to proliferation of B lymphocytes and macrophages in the red pulp, while the relative quantity of CD4+ and CD8+ T cells decreased. Extremely small amount of Fas+ and FasL+ lymphocytes were present in the marginal zone, the follicles, red pulp, and occasionally in the PALS. All the splenic zones were abundant with IL-2+ cells, while macrophages and siderophages were present mainly in the red pulp and in the marginal zone of the white pulp. We suggest that all these changes are compensatory processes of the host's lymphatic system.

Glial fibrillary acidic protein (GFAP)-v-src transgenic mice develop spontaneous gliomas with a high incidence of malignant progression. We characterize the first glial cell line derived from v-src transgenic mice, Tu-2449 in comparison with a virally induced murine glioma, SRB-10, and a spontaneous murine glioma, P497. Doubling times were lowest, as clonogenicity in soft agar was highest for Tu-2449, and to a lesser degree, Tu-2449 cells formed spheroids and showed migratory behaviour and invaded fetal rat brain aggregates. BCL-2 and BAX expression were lower in Tu-2449 and P497 than in SRB-10 cells. Only Tu-2449 cells accumulated p53 protein in response to genotoxic stress. Tu-2449 and SRB-10 cells that showed low CD95 expression were resistant to CD95 ligand (CD95L)-induced apoptosis unless coexposed to CD95L and inhibitors of RNA or protein synthesis. A chemosensitivity profile revealed Tu-2449 to be rather chemoresistant. Tu-2449 thus displays growth characteristics and patterns of resistance to apoptosis similar to those of other mouse and human glioma cell lines and may therefore become a valuable tool to evaluate new therapies for malignant gliomas in vitro and in vivo.

To establish a new clinical index for immunological abnormalities occurring in silicosis, several clinical parameters related to Fas-mediated apoptosis; i.e., membrane Fas expression on peripheral blood lymphocytes (mFas), serum soluble Fas levels (sFas), serum soluble Fas ligand levels (sFasL), and soluble/membrane Fas mRNA expression ratios (s/mFas ExR) in peripheral blood mononuclear cells (PBMC) were investigated. Fifty-eight silicosis patients with no clinical symptoms of autoimmune diseases were the subjects of this study. Factor analysis was performed using 12 clinical parameters including four parameters related to Fas-mediated apoptosis. Two common factors were identified. Factor 1 which consisted of the following parameters; duration of exposure, symptomatic dyspnea, PO2, PCO2, and A-aDO2, should be designated as the respiratory factor for cases with silicosis. The parameters of factor 2 were serum IgG, sFas with high factor loading, titer of ANA, sFasL, and s/mFas ExR. These parameters of factor 2 are indicative of the immunological disorders occurring in silicosis cases. Some cases exhibited abnormalities in parameters of factor 2 but not factor 1. The factor analysis clearly demonstrated that the parameters related to Fas-mediated apoptosis should be the most beneficial for predicting the pre-clinical status of complicated autoimmune diseases in silicosis.

Fas ligand (FasL), a cell surface molecule belonging to the tumor necrosis factor family, binds to its receptor Fas, thus inducing apoptosis of Fas bearing cells. In the present study we assessed the expression of Fas, activation molecule interleukin (IL)-2 receptor alpha chain (CD25) and an index of functional activity such as thymidine uptake under mitogen stimulation of tumor associated lymphomonocytes (TALM) from 7 neoplastic effusions of advanced cancer patients. The same parameters were studied in peripheral blood mononuclear cells (PBMC) of 7 patients with cancer of different sites and in 7 normal subjects. The proliferative response to phytohemagglutinin (PHA), measured as [3H]-thymidine uptake, of TALM was significantly lower than that of PBMC of cancer patients. The expression of CD25 on unstimulated fresh TALM was slightly higher than that of PBMC from normal subjects: after 24 h of PHA stimulation the CD25 was expressed both on TALM and PBMC from normal subjects. The expression of Fas was assessed on unstimulated TALM, PBMC from cancer patients and normal subjects immediately after (by 2 h, t0) the cell separation, and at different times (24 h and 48 h) thereafter, and on PHA-stimulated TALM, PBMC from cancer patients and normal subjects after 24 h and 48 h of culture (in RPMI 1640). At all times (t0, 24 h and 48 h) the Fas expression by unstimulated TALM was significantly higher than that of PBMC from normal subjects: the Fas expression by PBMC from cancer patients was roughly in the same range as PBMC from normal subjects. At 24 h the Fas expression by PHA-stimulated TALM was significantly higher than that of PBMC from normal subjects, whereas at 48 h the difference was not significant. The TALM studied by us showed to be functionally defective and expressing relatively high levels of Fas showing the characteristics to be considered as a target for FasL expressing tumor cells, which in this way may escape immune control.

Fas antigen, also termed APO-1 or CD95, is a transmembrane protein and a member of the tumor necrosis factor receptor/nerve growth factor receptor superfamily which mediates apoptosis upon oligomerization. The Fas/Fas ligand system is considered to be a key regulator of apoptosis. Recently, we have demonstrated that Fas antigen expression is induced by low-dose irradiation of some types of lymphomas, and we also demonstrated that irradiation-induced Fas antigen expression increased with the passage of time until peaking at 48 h after irradiation in CML-C1, CML-C2, DL-40, and DL-95 cell lines. In this study, we also examined the potential cytotoxicity of Fas ligand peptide against several types of lymphoma/leukemia cell lines that showed induction of Fas antigen expression under irradiation. Flow cytometry analysis was performed at 6, 24 and 48 h after irradiation. Samples (1 x10(6) cells/ml) from irradiated and non-irradiated cells of each cell line were incubated with or without 5 microg/ml of Fas ligand peptide for 2 h at 37 degrees C in a humidified atmosphere of 5% carbon dioxide (CO2) in air. The killing effect of Fas ligand against cell lines of CML-C1, DL-40, and DL-95 were clearly identified as the percentage of cells with Fas antigen expression induced by irradiation. Concerning HD-70 cell line, for which soluble Fas antigen has been identified, the killing effects were clearly observed in samples pre-treated with PBS washings. To our knowledge, this is the first report describing a possible application of the Fas/Fas ligand system in treatment of certain types of malignancies in which Fas antigen is inducible by irradiation.

The mechanism by which radiation induces human peripheral T cell apoptosis is not known. We examined sequential changes in post-irradiated peripheral blood mononuclear cells (PBMC(S)) taken from normal volunteers, by using flow-cytometer and an anti-CD3 monoclonal antibody, annexin V, propidium iodide, anti-Fas antibody, and anti-Fas ligand antibody. After 5 or 10 Gy of irradiation with a 60Co radiation therapy unit, most of the human peripheral T cells showed positivity against annexin V in 15 h, and positivity against propidium iodide in 23 h after irradiation. On a microscopy-video system, approximately 80% of mononuclear cells revealed apoptotic changes in 24 h after irradiation. Because of its proposed role in activation-induced cytotoxicity, we also examined the Fas (CD95/Apo-1) pathway in killing T cells by irradiation. Irradiated PBMC, displayed no increase in surface Fas expression and caspase-3 activity relative to non-irradiated cells. In addition, the anti-Fas ligand failed to eliminate the apoptotic death of PBMC, after irradiation. These results suggest that irradiation induces direct apoptosis of T cells by a Fas-independent mechanism.

Fas antigen belongs to the tumor necrosis factor receptor family and is known to induce apoptosis. This protein is abundantly expressed in hepatocytes, especially in acute and chronic hepatitis. To elucidate the clinical significance of Fas in hepatocellular carcinoma (HCC), we investigated its expression in 50 HCC having various characteristics. Fas was comprehensively expressed in non-cancerous hepatocytes as well as bile ducts. It was moderately expressed in histiocytes in the stroma rather than in infiltrating lymphocytes. In HCC, Fas expression was significantly reduced when there was poor differentiation (p<0.001), portal tumor thrombus and extracapsular invasion (p<0.05) according to univariate analysis. Multivariate analysis revealed statistical significance between Fas expression and the degree of differentiation (p=0.0002). These results suggest that HCC, like non-cancerous hepatocytes, express Fas antigen, when they are well or moderately differentiated but lose this ability when they become poorly differentiated as the carcinoma advances.