Mitochondrial targeting of TR3 is involved in TPA induced apoptosis in breast cancer cells.

TPA is considered to be a tumor promoting molecule that induces the expression of COX-2 protein. However, it is contradictory to find that TPA can induce tumor cell apoptosis and exert antitumor activity. Therefore, the role of TPA in tumorigenesis and development has not yet been elucidated. Here we show that TPA can promote the apoptosis of breast cancer cells and increase the ratio of Bax/Bcl-2. It is suggested that TPA may induce apoptosis of breast cancer cells through mitochondrial apoptosis pathway. Further studies showed that TPA could cause mitochondrial dysfunction and trigger mitochondrial apoptotic pathway. In mechanism, the mitochondrial targeting of TR3 is involved in TPA induced apoptosis in breast cancer cells. In conclusion, our findings suggest that TPA can play a role in inhibiting cancer by inducing apoptosis and TR3 is expected to be a new target for cancer treatment.

Association Between PAI-1 Activity Levels and t-PA Antigen with Glycemic Status in Prediabetic Population.

AIM: to evaluate an association between fibrinolysis defect and glycemic status in prediabetic population by assessing the levels of t-PA antigen and PAI-1 activity. METHODS: it was an observational study with cross-sectional approach. There were 72 subjects aged 30-50 years who had met the inclusion criteria. The diagnosis of diabetes mellitus (DM) and glycemic index were determined based on the American Diabetes Association (ADA) criteria. The PAI-1 and t-PA antigen levels were measured quantitatively using enzyme-linked immunosorbent assay (ELISA). Analysis between the levels of t-PA antigen and PAI-1 activity was performed using ANOVA. RESULTS: the t-PA antigen level was significantly higher in subjects with impaired glucose tolerance (IGT) and impaired fasting blood glucose (IFBG) as well as subject with impaired fasting blood glucose (IFBG) than those with normal glucose tolerance (NGT) (p=0.047). The PAI-1 activity was significantly higher in subjects with IGT, IFBG and subjects with IFBG than NGT (p=0.024). There was a significant association between glycemic status in prediabetic subjects and PAI-1 activity (p=0.04). CONCLUSION: the level of t-PA antigen and PAI-1 activity were significantly higher in prediabetic subjects than those with NGT; and there was a significant association between glycemic status in prediabetic subjects and PAI-1 activity.

Loss of CRABP-II Characterizes Human Skin Poorly Differentiated Squamous Cell Carcinomas and Favors DMBA/TPA-Induced Carcinogenesis.

Retinol and its derivatives play an important role in epidermal growth and differentiation and represent chemopreventive agents in nonmelanoma skin cancer. Retinoic acid binding protein II (CRABP-II) is a cytoplasmic receptor that critically regulates all-trans-retinoic acid (ATRA) trafficking. We documented the marked reduced expression of CRABP-II and its promoter methylation in human poorly differentiated squamous cell carcinomas. To investigate the role of CRABP-II in skin carcinogenesis we used skin lesion induction by dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate in CRABP-II-knockout C57BL/6 mice. We observed earlier and more diffuse epidermal dysplasia, greater incidence and severity of tumors, reduced expression of cytokeratin 1/cytokeratin 10 and involucrin, increased proliferation, and impaired ATRA inhibition of tumor promotion compared with wild-type animals. CRABP-II-transfected HaCaT, FaDu, and A431 cells showed expression of differentiation markers, retinoic acid receptor-ß/-γ signaling, ATRA sensitivity, and suppression of EGFR/v-akt murine thymoma viral oncogene homolog 1 (AKT) pathways in a fatty acid binding protein 5/peroxisome proliferator-activated receptor-ß/-δ-independent manner. The opposite was true in keratinocytes isolated from CRABP-II-knockout mice. Finally, CRABP-II accumulation induced ubiquitination-associated reduction of EGFR. Our results showed reduced CRABP-II expression in human poorly differentiated squamous cell carcinomas, and its gene deletion favored experimental skin carcinogenesis and impaired ATRA antitumor efficacy, likely modulating EGFR/AKT pathways and retinoic acid receptor-ß/-γ signaling. Therapeutic interventions aimed at restoring CRABP-II-mediated signaling may amplify therapeutic retinoid efficacy in nonmelanoma skin cancer.

EGFR mutations predict a favorable outcome for malignant pleural effusion of lung adenocarcinoma with Tarceva therapy.

The aim of the present study was to evaluate the therapeutic effects and adverse reactions of Tarceva treatment for malignant pleural effusion (MPE) caused by metastatic lung adenocarcinomas. One hundred and twenty-eight patients who failed first-line chemotherapy drug treatment were divided into a mutation and a non-mutation group according to the presence or absence of epidermal growth factor receptor (EGFR) mutations. Each patient received closed drainage combined with simple negative pressure suction after thoracoscopic talc poudrage pleurodesis and oral Tarceva treatment. Short-term and long-term clinical therapeutic effects of Tarceva were evaluated. The EGFR mutation rate in pleural metastatic tissues of lung adenocarcinoma acquired through video-assisted thoracoscopic surgery was higher compared to that in surgical resection specimens, plasma specimens and pleural effusion specimens compared to previously reported results. There were significant statistical differences in the average extubation time (p<0.01), drainage volume of pleural effusion (p<0.05), Karnofsky score and formation of encapsulated pleural effusion 4 weeks after surgery (p<0.05) between these two groups. The number of patients with mild pleural hypertrophy in the mutation group was significantly higher compared to the non-mutation group (p<0.01), while the number of patients with severe pleural hypertrophy was significantly reduced (p<0.05). There was significant statistical discrepancy between these two groups in terms of improvement of peripheral blood carcinoembryonic antigen and tissue polypeptide antigen after 4 weeks of therapy. The complete remission rate and the efficacy rate were higher in the mutation group compared to that in the non-mutation group (p<0.05). There was a longer overall survival time after Tarceva treatment in patients with EGFR mutations than those without EGFR mutation. EGFR mutations predict a favorable outcome for malignant pleural effusion of lung adenocarcinoma with Tarceva therapy. Detection of EGFR mutations may determine the responsiveness of malignant pleural effusion to Tarceva treatment.

The peritoneal fibrinolytic response to conventional and laparoscopic colonic surgery.

BACKGROUND: Laparoscopic surgery is considered to induce less peritoneal trauma than conventional surgery. The peritoneal plasmin system is important in the processes of peritoneal healing and adhesion formation. The present study assessed the peritoneal fibrinolytic response to laparoscopic and conventional colonic surgery. METHODS: Twenty-four patients scheduled for a right colonic resection were enrolled in the trial. Twelve underwent conventional surgery and 12 were operated laparoscopically. Biopsies of the parietal peritoneum were taken at standardized moments during the procedure. Tissue concentrations of tissue-type plasminogen activator (tPA) and its specific activity (tPA-activity), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor type 1 (PAI-1) were measured, using commercial assays. RESULTS: After mobilization of the colon, peritoneal levels of tPA antigen and activity were significantly higher in the laparoscopic group (p < 0.005) due to a decrease in the conventional group (p < 0.05). At the end of the procedure, the concentrations of tPA antigen and activity significantly (p < 0.05) decreased in the laparoscopic group to levels comparable with the conventional group. Neither uPA antigen nor PAI-1 antigen changed throughout the procedures. CONCLUSIONS: Both conventional and laparoscopic surgery inflict a decrease in tPA antigen and its specific activity. Peritoneal hypofibrinolysis initiates more rapidly during conventional, compared to laparoscopic, surgery, but at the conclusion of the surgery, the effect was the same.

Heating of carbon dioxide during insufflation alters the peritoneal fibrinolytic response to laparoscopic surgery : A clinical trial.

BACKGROUND: Laparoscopic surgery is evolving rapidly. It involves the creation of a pneumoperitoneum, mostly using carbon dioxide. Cooling of the peritoneum, due to insufflation, might traumatize the peritoneum and disturb peritoneal fibrinolysis, important in peritoneal healing processes. The current study was performed to elucidate the effects of the temperature of insufflation gas on the peritoneal fibrinolytic response to laparoscopic surgery. METHODS: Thirty patients scheduled for laparoscopic cholecystectomy were randomized in two groups: one group in which the pneumoperitoneum was created with carbon dioxide at room temperature, and one wherein carbon dioxide at body temperature was used. Peritoneal biopsies were taken at the start and at the end of surgery. Tissue concentrations of tPA antigen, tPA activity, uPA antigen, and PAI-1 antigen were measured using ELISA techniques. RESULTS: Peritoneal PAI-1 antigen levels were significantly higher at the end of the procedure in patients operated with carbon dioxide at room temperature (p < .05). A slight, but not significant, decrease in tPA antigen and activity was observed in both groups during the procedure. Peritoneal concentrations of uPa antigen did not change during the procedure. CONCLUSIONS: The temperature of carbon dioxide used for insufflation of the abdominal cavity affects peritoneal biology. Cooling of the peritoneum by unheated carbon dioxide causes increased peritoneal PAI-1 levels, important in peritoneal healing processes.

The bHLH protein MyoR inhibits the differentiation of early embryonic endoderm.

MyoR is a bHLH protein whose expression was reported to be almost exclusively restricted to the precursors of the skeletal muscle lineage where it was postulated to function as a transcriptional repressor of myogenesis. However, previous studies in our laboratory suggested a much broader role for MyoR in embryonic cell differentiation. We demonstrated that, besides being expressed in several adult tissues of non-muscle lineage, MyoR was expressed at a much earlier stage in mammalian development than had previously been reported, that is, as early as the blastocyst stage, well before skeletal muscle specification. We also found that, as in skeletal muscle precursor cells, MyoR expression is inversely correlated with the cellular differentiative state of ectodermal, non-muscle embryonal carcinoma (EC) cells. Retinoic acid (RA) treatment of ectodermal EC or embryonal stem (ES) cells promotes their differentiation into primitive endoderm. However, in the present study, we show that the RA-induced expression of endodermal markers such as EndoA, collagen IV, and t-PA are inhibited by exogenous MyoR expression and that the level of inhibition of these markers correlates with the level of MyoR expressed. Conversely, knock-down of MyoR expression via RNA interference enhances RA-induced differentiation of EC cells, promoting earlier and much higher expression of the above-mentioned endodermal markers following RA treatment. Finally, we have narrowed the period of exogenous MyoR-induced embryonic lethality to between 3.5 and 5.5 days post-coitum (dpc), the period during which embryonic endoderm differentiates from the embryonic ectoderm. Our results suggest, therefore, that inhibition of endodermal differentiation between 3.5 and 5.5 dpc contributes to the embryonic death of mouse embryos overexpressing exogenous MyoR and consequently that MyoR may serve as a repressor of embryonal endoderm differentiation.

Cytoplasmic c-Fos induced by the YXXQ-derived STAT3 signal requires the co-operative MEK/ERK signal for its nuclear translocation.

A STAT3 (signal transducer and activator of transcription 3)- and a MEK/Erk-mediated signal can be activated by cytokines, including IL-6 (interleukin-6), PDGF, and EGF. Recently, STAT3 and an ERK-signal were shown to co-operatively activate the c-fos gene. Activation of a truncated form of the IL-6 receptor subunit, gp130, that had only one YXXQ motif, induced both c-Fos and JunB in NIH3T3 cells through STAT3 without an apparent increase in the AP-1 (activator protein-1) activity. In contrast, concomitant stimulation of the STAT3 signal and a MEK/Erk-signal markedly increased AP-1 activity with enhanced c-Fos expression. Surprisingly, the c-Fos induced by the YXXQ-signal alone was localized to the cytoplasm, from which it translocated into the nucleus following TPA (12-O-tetradecanoyl-phorbol 13-acetate) treatment in a MEK/Erk-dependent manner. c-Fos that was expressed from a constitutive promoter localized to the nucleus and did not move into the cytoplasm in response to the YXXQ-signal. Rather, the YXXQ-signal was required during c-Fos production for it to be retained in the cytoplasm. Thus, the YXXQ-signal induces c-Fos expression through STAT3 and anchors the new c-Fos in the cytoplasm. In addition, the YXXQ-signal and an Erk signal co-operatively cause c-Fos activation in the nucleus.

Evaluation of different markers in non-small cell lung cancer: prognostic value of clinical staging, tumour cell detection and tumour marker analysis for tumour progression and overall survival.

We compared the prognostic value of routine pathology, cytokeratin-positive (CK+) cells in the bone marrow (BM) and serum tumour markers (TM) in patients with non-small cell lung cancer (NSCLC) at the time of diagnosis with regard to overall survival (OS) and time to progression (TTP). Eighty patients with NSCLC, staged as T2-4, N0-3, M0 (n=52), M1 (n=27), (Mx = 1) were evaluated. Treatment included chemo-radiotherapy with cisplatin/etoposide and subsequent radical surgical resection. There were 23 complete responders, 50 non-responders and 7 patients who died of non-lung cancer causes. The median follow-up was 12 months (range 1-44 months). Besides routine pathology for tissue and BM, CK+ BM cells were detected by immunocytochemistry (IC) and 4 different tumour markers as well as the shedded domain of the oncoprotein Her-2/neu and urokinase plasminogen activator uPA were determined by radio- or enzyme-immunoassay. Patients classified as stage IV and patients with metastases had a significantly lower TTP and OS. No significant correlation was demonstrated for grading, tumour size or number of involved lymph nodes. The tumour marker tissue polypeptide antigen (TPA) and Cyfra 21-1 were the only marker which significantly correlated with OS. Interestingly, routine pathology could not detect minimal residual BM involvement as IC was able to (p=0.0004) and the presence of even a few CK+ cells significantly correlated with reduced OS. Thus, we conclude that the detection of CK+ cells should be added to routine pathology and for tumour marker determination, studies should focus on Cyfra 21-1 and TPA.

Tissue concentrations of tissue polypeptide antigen (TPA) and prostatic specific antigen (PSA) in 42 patients with prostatic carcinoma.

BACKGROUND: Following development of methods to quantitate biochemical markers in aspiration biopsies we showed that tissue concentration of prostate specific antigen (T-PSA) decreased with increasing malignancy while serum PSA increased. We also found that T-PSA predicts the clinical outcome better than earlier used prognostic markers. METHODS: In order to further study biochemical markers in prostatic cancer a membrane protein, tissue polypeptide antigen (TPA), which is a complex of polypeptide fragments of cytokeratins 8, 18, and 19, was quantitated in 42 patients with newly diagnosed carcinoma of the prostate. The samples had previously been analyzed for T-PSA. RESULTS: Correlation to TGM classification showed that higher malignancy is correlated to lower tissue TPA values. There is a significant positive correlation (r(s) = 0.49, P < 0.01) between T-TPA and T-PSA. Pretreatment values of T-PSA, but not T-TPA, had association to time to progression or time to death. CONCLUSIONS: Increasing prostatic malignancy is correlated to decreasing values of T-TPA. This indicates that the concentrations of membrane and secretory proteins are changed in the same direction in tissue during cancer development. Tissue TPA seem to have no prognostic value in endocrine treatment of prostatic carcinoma.

Quantitative measurement of soluble cytokeratin fragments in tissue cytosol of 599 node negative breast cancer patients: a prognostic marker possibly associated with apoptosis.

Apoptosis is associated with caspase-mediated proteolysis of Type I (K18 and K19) cytokeratins. We previously showed a positive association between the levels of tissue polypeptide antigen (TPA), that recognizes cytokeratins K8, K18, and K19 fragments, and induced apoptosis in breast cancer cell lines. The aim of the present study was to evaluate the interrelationships between TPA, steroid receptors, and p53, and their joint prognostic role in node-negative breast cancer patients not treated with adjuvant therapies. Age and pT were also considered since they are known prognostic factors. Five hundred and ninety-nine cases with N- breast cancer were evaluated (median follow-up: 60 months). TPA was measured by an immunoradiometric assay and p53 by an immunochemiluminescent assay in tumor cytosol. Multiple correspondence analysis was used to study the associations among variables. Their prognostic role (univariate analysis) and their joint effect (multivariate analysis) on RFS were investigated with Cox regression models. TPA showed a direct association with ER and PgR. Higher p53 values were weakly associated to low values of ER, PgR, and TPA. Younger age was related to low and intermediate values of ER and PgR and to low p53 values, while older age was related to high values of ER. Multivariate analysis showed a significant prognostic impact for pT, age, ER, and TPA. Among the interactions considered clinically relevant, only that between ER and age was found. RFS estimated values were poorer in cases with lower than in those with higher TPA values, both in patients expected to have a poor (pT2, young age, low ER) and a better prognosis (pT1, older age, high ER). From the findings of the present study we can draw the following conclusions: The relationship of TPA with prognosis gives an additional contribution to pT, age, and steroid receptors in N- breast cancer; TPA may be considered the first marker of apoptosis measured with a fully standardized quantitative method in tumor cytosol and could be evaluated in prognostic indexes including markers related to different biological mechanisms.

The effect of dexamethasone on tissue fibrinolytic system in male and female rats.

BACKGROUND: Dexamethasone in low and high doses affects blood fibrinolytic activity both in animals and humans. In this study the effect of a high dose of dexamethasone on plasminogen activator activity (PAA), t-PA antigen level, plasminogen activator inhibition (PAI) and plasmin inhibition (Pl) in rat heart, brain, liver, lungs and kidneys was investigated in both sexes. MATERIALS AND METHODS: Twenty male and twenty female adult Wistar rats were used. Dexamethasone was administered as a single intraperitoneal injection (3mg/kg/day) in rats, once daily, for a period of 5 consecutive days. t-PA antigen level was assayed by an enzyme-linked immunoabsorbant assay method. PAA, PAI and Pl were determined by spectrophotometric methods. The plasminogen used was isolated from rat plasma. RESULTS: Dexamethasone induced variable changes in the fibrinolytic parameters in rat heart, brain and liver of both sexes; in lungs and kidneys dexamethasone had no effect. CONCLUSION: These changes of PAA, PAI and t-PA antigen level in heart, brain and liver induced by dexamethasone might be of importance regarding the involvement of glucocorticoids and plasminogen activators/plasmin in many pathophysiological conditions.

Tissue polypeptide antigen and carcinoembryonic antigen lack diagnostic accuracy in urothelial carcinoma.

Carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA) levels in serum and urine from 25 patients with bladder cancer and 42 patients with cancer of the renal pelvis/ureter have been evaluated as an aid for clinical diagnosis of urothelial cancer. The tumour CEA content varied markedly, from values obtained in normal urothelium up to 822 and 7306 ng/g wet tissue in cancer of the renal pelvis/ureter and bladder cancer, respectively. Serum and urine CEA levels were found not to correlate with the tumour CEA content. Serum CEA levels were found increased over 5 microg/L in up to 16% of bladder cancer patients, but only in 4.8% in renal pelvis/ureter cancer. Urine of cancer patients contained usually normal CEA levels. Increased serum TPA levels were found in 48% and 35.7% of patients with bladder cancer and cancer of renal pelvis/ureter, respectively. Urine TPA levels were significantly increased in both, patients with bladder cancer (p<0.001) and cancer of renal pelvis/ureter (p<0.01). The median values of urine TPA were 59, 1095 and 1325 U/L, in controls, patients with bladder cancer and cancer of renal pelvis/ureter, respectively. However, considering previously described increase of TPA in inflammatory diseases of urinary tract and in renal failure patients, results of urinary TPA obtained in the diagnostic workup of urothelial cancer should be cautiously interpreted. This study shows that serum and urine levels of CEA and TPA have no diagnostic accuracy required for clinical diagnosis of urothelial cancer.

Establishment and characterization of a new human extrahepatic bile duct carcinoma cell line (ICBD-1).

A new human extrahepatic bile duct carcinoma cell line (ICBD-1) was established from surgically resected tumor of a 71-year-old Japanese male patient. ICBD-1 cells proliferate in a layer with a population doubling time of 31.5 h and secrete tissue polypeptide antigen. ICBD-1 cells have a tetraploid pattern with a DNA index of 1.83 and chromosome counts showed equally distribution in a range from 65 to 69. IC50 values for ICBD-1 cells were 200 ng/ml for adriamycin, 400 ng/ml for mitomycin C, 2 microg/ml for cisplatin and 300 ng/ml for 5-fluorouracil. ICBD-1 cells were successfully transplanted to male nude mice, inducing progressive tumor growth. Histologically, nude mouse tumors were less differentiated than the original human tumor. Tumor cells showed alveolar structures with thin fibrous stroma, classified as poorly-differentiated adenocarcinoma. ICBD-1 is the fourth established cell line that originate from extrahepatic bile duct carcinoma and it will be applicable for the experimental studies of this disease.

Differential responsiveness of proliferation and cytokeratin release to stripped serum and oestrogen in the human breast cancer cell line, MCF-7.

In vitro research into hormone sensitivity and the relation to proliferation of cytokeratin release from cancer cells is scarce. Therefore, we examined the stimulation of proliferation and the release of cytokeratins in a breast cancer cell culture model. Cell growth was stimulated by 17 beta-oestradiol (10(-11) M), stripped serum (10%) and by the two together. Cytokeratin release was stimulated only by stripped serum, oestradiol having no effect. After long incubation periods (> 12 h), cytokeratin release also commenced in the control and oestradiol treatments. Release rate versus time analysis suggested that there are two different release processes. Cytokeratin release was first stimulated at a stripped serum concentration approximately 100 times lower than that which initiated proliferation. Pharmacological alteration of proliferation with cordyceptin resulted in growth changes without alterations in cytokeratin release. We conclude that cytokeratin release in these cells is unrelated to proliferation, independent of oestrogen action and probably in some way related to growth factor receptor function.

Tissue polypeptide antigen production in a uterine carcinosarcoma cell line in a serum-free culture.

Carcinosarcomas (malignant mixed Mullerian tumors, MMMT) of the female genital tract are uncommon neoplasms, most of which show a highly aggressive behavior. However, the biological characteristics of MMMT, especially the producibility of tumor-related substances in vitro, have yet to be clarified. Using a serum-free culture model system which was newly established from an FU-MMT-2 cell line originating from an MMMT of the human uterus, the biological and immunocytochemical characteristics of MMMT cells were examined in vitro. As a control model, the uterine fibroblasts were also cultured in the same conditions. Moreover, several tumor markers were also measured by a radioimmunoassay in the serum of five patients with MMMT. FU-MMT-2 produced and secreted tissue polypeptide antigen (TPA), a tumor marker closely related to the keratin family, under serum-free culture conditions. Moreover, TPA was also immunocytochemically demonstrated in carcinoma cells as well as sarcoma cells in FU-MMT-2. On the other hand, the uterine fibroblasts showed no detectable producibility of the substances examined in vitro. An elevation of the serum TPA levels was also detected in four of five (80%) patients with MMMT. This is the first serum-free culture study of MMMT, which appears to provide a useful model for further studies of the tumor biology in MMMT of the female genital tract. An evaluation of the serum TPA level thus appears to be useful in determining the optimum management of patients with MMMT. In addition, the demonstration of TPA in sarcoma cells of FU-MMT-2 in vitro supports both the theory of a single cell origin and the current concept of MMMT as a "metaplastic carcinoma," as substantiated in our previous study.

Synthetic wound dressings--evaluation of interactions with epithelial and dermal cells in vitro.

Comparative testing of seven wound dressings (WD) has been performed with human HaCaT keratinocyte and mouse 3T3 fibroblast cultures. To assess biocompatibility, morphologic examinations were combined with cell counting. Supernatants were subjected to measurements of tissue peptide antigen (TPS), soluble intercellular adhesion molecule 1 (ICAM-1), and interleukins (IL-1 alpha, -1 beta, -6). Furthermore, monoxygenation, the reduced glutathione/oxidized gluthathione (GSH/GSSG) ratio and lipid peroxides were determined. Initial morphologic events were noted within the first day of exposure to WD. After 72 h, inhibition of cell growth was observed in the presence of hydrocolloids and hydrogels. The cytochrome-P-450-dependent ethoxyresorufin 0-deethylation rate and the GSH/GSSG ratio were not altered by WD in HaCaT cells. Lipid peroxide generation, IL-1 and ICAM-1 were scarcely detectable. TPS and IL-6 release indicate the presence of an 'activated stage' of keratinocytes and fibroblasts exposed to WD. Peptide release in vivo may contribute to the beneficial effects of modern dressings in the treatment of superficial cutaneous wounds.