Masaya Ueno

Redox control of cell death.

Cellular redox is controlled by the thioredoxin (Trx) and glutathione (GSH) systems that scavenge harmful intracellular reactive oxygen species (ROS). Oxidative stress also evokes many intracellular events including apoptosis. There are two major pathways through which apoptosis is induced; one involves death receptors and is exemplified by Fas-mediated caspase-8 activation, and another is the stress- or mitochondria-mediated caspase-9 activation pathway. Both pathways converge on caspase-3 activation, resulting in nuclear degradation and cellular morphological change. Oxidative stress induces cytochrome c release from mitochondria and activation of caspases, p53, and kinases, including apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase. Trx inhibits apoptosis signaling not only by scavenging intracellular ROS in cooperation with the GSH system, but also by inhibiting the activity of ASK1 and p38. Mitochondria-specific thioredoxin (Trx-2) and Trx peroxidases (peroxiredoxins) are suggested to regulate cytochrome c release from mitochondria, which is a critical early step in the apoptotis-signaling pathway. dATP/ATP and reducing factors including Trx determine the manifestation of cell death, apoptosis or necrosis, by regulating the activation process and the activity of redox-sensitive caspases. As mitochondria are the most redox-active organelle and indispensable for cells to initiate or inhibit the apoptosis process, the regulation of mitochondrial function is the central focus in the research field of apoptosis and redox.

Thioredoxin-dependent Redox Regulation of p53-mediated p21 Activation

Thioredoxin (TRX) is a dithiol-reducing enzyme that is induced by various oxidative stresses. TRX regulates the activity of DNA-binding proteins, including Jun/Fos and nuclear factor-κB. TRX also interacts with an intranuclear reducing molecule redox factor 1 (Ref-1), which enhances the activity of Jun/Fos. Here, we have investigated the role of TRX in the regulation of p53 activity. Electrophoretic mobility shift assay showed that TRX augmented the DNA binding activity of p53 and also further potentiated Ref-1-enhanced p53 activity. Luciferase assay revealed that transfection of TRX enhanced p53-dependent expression of p21 and further intensified Ref-1-mediated p53 activation. Furthermore, Western blot analysis revealed that p53-dependent induction of p21 protein was also facilitated by transfection with TRX. Overexpression of transdominant negative mutant TRX (mTRX) suppressed the effects of TRX or Ref-1, showing a functional interaction between TRX and Ref-1.cis-Diamminedichloroplatinum (II) (CDDP) induced p53 activation and p21 transactivation. The p53-dependent p21 transactivation induced by CDDP was inhibited by mTRX overexpression, suggesting that TRX-dependent redox regulation is physiologically involved in p53 regulation. CDDP also stimulated translocation of TRX from the cytosol into the nucleus. Hence, TRX-dependent redox regulation of p53 activity indicates coupling of the oxidative stress response and p53-dependent repair mechanism.

Possible association of thioredoxin and p53 in breast cancer

Expression of thioredoxin (TRX), a dithiol-reducing enzyme, and mutations of p53 have been detected in various cancer tissues. We recently reported that TRX-dependent redox regulation plays a crucial role in DNA binding activity of p53. In this study, we investigated the possibility of functional association between TRX and p53 in breast cancer. First, we examined the expression of TRX and mutated p53 in 100 primary breast cancer tissues by immunohistochemistry. Expression of TRX was detected in cases of 84/100 (84%) and expression of p53, which means existence of mutated p53, in cases of 63/100 (63%). TRX positive cases was 89% (56/63) in mutant p53 positive cases. Next, we examined the expression of TRX and p53 in breast cancer cell line MCF-7 cells after CDDP treatment or irradiation. CDDP treatment or irradiation augmented expression of TRX and p53 in MCF-7 cells by western blotting. Immunofluorescence cell analysis by confocal microscopy showed that CDDP treatment induced translocation of TRX into nuclei. These results suggest the possible association of TRX with p53-dependent function including DNA repair in breast cancer.

The reversal of recurrence hazard rate between ER positive and negative breast cancer patients with axillary lymph node dissection (pathological stage I-III) 3 years after surgery

BackgroundsPrognostic factors are defined as biological or clinical measurement associated with overall survival and/or disease-free survival. Previous studies have shown that patients with estrogen receptor (ER) positive cancers have a better prognosis than patients whose cancers do not have these receptors.MethodsThis study investigated the assessment of variables in defining prognosis of 742 breast cancer women with pathological stage (pTNM) I-III diagnosed between 1980 and 2005 at the Kyoto University Hospital in Japan, by age, clinical stage (cTNM), pTNM, the numbers of positive lymph nodes (pN), and ER status.ResultsMultivariate analysis demonstrated that pTNM and ER status were the independent prognostic factors for overall survival, and that pTNM and pN were the independent prognostic factors for disease-free survival. For the 0- to 2-year interval, the hazard of recurrence was higher for the ER-negative patients than the ER-positive patients, and beyond 3 years the hazard was higher for ER-positive patients.ConclusionThe present study confirmed the previous reports which showed favorable prognosis of the patients with lesser pTNM or positive ER status. A reversal of recurrence hazard rate between ER positive and negative breast cancer patients beyond 3 years after operation was detected. The fact may indicate the importance of long term adjuvant hormone therapy for ER positive cancer patients.

Prognostic factors for survival after first recurrence in breast cancer: a retrospective analysis of 252 recurrent cases at a single institution.

IntroductionPrevious studies have shown that primary breast cancer patients with estrogen receptor (ER)-positive status have better outcomes in terms of both overall survival and disease-free intervals (DFI). However, 25.2xa0% of our ER-positive patients experienced recurrence. This study aimed to define factors potentially predicting survival after first recurrence in surgically treated patients with stage I–III breast cancer.MethodsWe retrospectively analyzed 252 females with recurrent breast cancer who had undergone surgery and been followed at Kyoto University Hospital in Japan. Age, clinical stage, pathological stage, axillary lymph node involvement, ER status at the time of diagnosis, progesterone receptor status, human epidermal growth factor receptor 2 status, operative method, adjuvant chemotherapy, adjuvant endocrine therapy, use of trastuzumab after recurrence, site of recurrence, DFI, and time of recurrence were examined for possible influences on survival after the first recurrence.ResultsPositive ER status and positive PR status at the time of diagnosis were significantly favorable factors of survival after first recurrence for patients with recurrence, pxa0<xa00.001 and pxa0=xa00.021, respectively. More than two sites of recurrence (pxa0<xa00.001) were associated with shorter survival time after the first recurrence on multivariate analysis. Survival of patients with recurrent breast cancer steadily improved from 1980–1994 to 1995–2008, significantly in ER-negative subgroups.ConclusionsPositive ER status at the time of diagnosis is a powerful predictor for favorable survival after first recurrence. Survival time after first recurrence of breast cancer has steadily increased in recent decades. Advances in treatments and attitudes about breast cancer have contributed to this improvement in survival after first recurrence.

Efficient proliferation and adipose differentiation of human adipose tissue-derived vascular stromal cells transfected with basic fibroblast growth factor gene.

Human vascular stromal (VS) cells obtained from mature adipose tissue were transfected with an adenovirus vector carrying the basic fibroblast growth factor (bFGF) gene. bFGF protein was observed in VS cell nuclei 24 h after transfection and in the cytoplasm and extracellular space 72 h after transfection. Naive VS cells were almost static in vitro and proliferated in a dose-dependent manner on stimulation with recombinant bFGF (rbFGF). However, bFGF-transfected VS cells proliferated spontaneously to the same extent as naive VS cells when stimulated with rbFGF at 100 ng/ml. The former cells started to proliferate on day 3 after transfection and the proliferation pattern was similar to that of the latter cells, although only a slight amount of bFGF protein was detected in the culture medium when the bFGF-transfected cells started to proliferate. The proliferation of bFGF-transfected VS cells was completely inhibited by bFGF neutralizing antibody, which also completely inhibited the proliferation of naive VS cells stimulated with rbFGF. Under conditions favoring differentiation to adipocytes, bFGF-transfected VS cells stopped proliferating and started to accumulate lipid in the cytoplasm. bFGF-transfected VS cells, which spontaneously and efficiently proliferate while preserving their ability to differentiate into adipocytes, may be an adequate cell source for human adipose tissue regeneration.

13 – Role of Thioredoxin and Redox Regulation in Oxidative Stress Response and Signaling

This chapter discusses the role thioredoxin (TRX) and redox regulation in oxidative stress response as well as signaling. Oxidative stress evokes various cellular events, including activation of transcription factors, apoptosis, and cell cycle arrest. The TRX system, composed of several related molecules, protects cells against oxidative stress. Redox regulation is deeply involved in the cell death mechanism as is p53 transcriptional function. Thus, in addition to a function of scavenging Reactive Oxygen Species (ROS), TRX appears to be a regulator/modulator involved in cellular signaling against oxidative stress. Enhancement of the thioredoxin system is beneficial against oxidative stress-induced pathological conditions. Elucidation of oxidative stress signaling and response also provide a new therapeutic approach by the redox regulation of oxidative stress-mediating target molecules.