Ioannis I. Verginadis

ATF4-dependent induction of heme oxygenase 1 prevents anoikis and promotes metastasis

The integrated stress response (ISR) is a critical mediator of cancer cell survival, and targeting the ISR inhibits tumor progression. Here, we have shown that activating transcription factor 4 (ATF4), a master transcriptional effector of the ISR, protects transformed cells against anoikis - a specialized form of apoptosis - following matrix detachment and also contributes to tumor metastatic properties. Upon loss of attachment, ATF4 activated a coordinated program of cytoprotective autophagy and antioxidant responses, including induced expression of the major antioxidant enzyme heme oxygenase 1 (HO-1). HO-1 upregulation was the result of simultaneous activation of ATF4 and the transcription factor NRF2, which converged on the HO1 promoter. Increased levels of HO-1 ameliorated oxidative stress and cell death. ATF4-deficient human fibrosarcoma cells were unable to colonize the lungs in a murine model, and reconstitution of ATF4 or HO-1 expression in ATF4-deficient cells blocked anoikis and rescued tumor lung colonization. HO-1 expression was higher in human primary and metastatic tumors compared with noncancerous tissue. Moreover, HO-1 expression correlated with reduced overall survival of patients with lung adenocarcinoma and glioblastoma. These results establish HO-1 as a mediator of ATF4-dependent anoikis resistance and tumor metastasis and suggest ATF4 and HO-1 as potential targets for therapeutic intervention in solid tumors.

Synthesis, structural characterization and biological studies of the triphenyltin(IV) complex with 2-thiobarbituric acid

The reaction between 2-thiobarbituric acid (H(2)TBA), which was treated with an equimolar amount of potassium hydroxide, in a water with triphenytin chloride in methanol, results in the formation of the {[Ph(3)Sn(O-HTBA)]}(n) (1) complex. Crystals of the hydrated 1 with formula {[Ph(3)Sn(O-HTBA)]·0.7(H(2)O)}(n) were growth from methanol/acetonitrile solution, of the white precipitation, filtered off, from the reaction. The crystal structure of complex 1 has been determined by X-ray diffraction at 120 K. Complex 1 is polymeric. The geometry around the tin(IV) ions is trigonal bi-pyramidal with coordination to three C atoms from phenyl groups and one O atom from a de-protonated HTBA ligand. Complex 1 and the already known [(n-Bu)(3)Sn(O-HTBA)·H(2)O] (2) were evaluated for their in vitro cytotoxic activity (cell viability) against human cancer cell lines: HeLa (cervical), OAW-42 (ovarian), MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER negative), A549 (lung), Caki-1 (renal) and additionally, the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells) and normal immortalized human mammary gland epithelial cell line MTSV17 with a Trypan Blue assay. Moreover complex 1 was evaluated for its in vitro cell growth proliferation activity against leiomyosarcoma cells (LMS), MCF-7 and MRC-5 cells with a Thiazolyl Blue Tetrazolium Bromide (MTT) assay. The type of cell death caused by complexes 1 and 2 was also evaluated by use of flow cytometry assay. The results showed that these compounds mediate a strong cytotoxic response to normal and cancer cell lines tested through apoptosis and induce cell cycle arrest in S phase of the cell cycle, suggesting DNA intercalation (direct or indirect) with the complexes. Finally, the influence of these complexes 1 and 2 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied.

Cell intrinsic and extrinsic activators of the unfolded protein response in cancer: Mechanisms and targets for therapy

A variety of cell intrinsic or extrinsic stresses evoke perturbations in the folding environment of the endoplasmic reticulum (ER), collectively known as ER stress. Adaptation to stress and re-establishment of ER homeostasis is achieved by activation of an integrated signal transduction pathway called the unfolded protein response (UPR). Both ER stress and UPR activation have been implicated in a variety of human cancers. Although at early stages or physiological conditions of ER stress, the UPR generally promotes survival, when the stress becomes more stringent or prolonged, its role can switch to a pro-cell death one. Here, we discuss historical and recent evidence supporting an involvement of the UPR in malignancy, describe the main mechanisms by which tumor cells overcome ER stress to promote their survival, tumor progression and metastasis and discuss the current state of efforts to develop therapeutic approaches of targeting the UPR.

Anticancer and cytotoxic effects of a triorganotin compound with 2-mercapto-nicotinic acid in malignant cell lines and tumor bearing Wistar rats.

Nowadays, investigation for possible therapeutic applications of various metal-based drugs attracts the scientific interest worldwide. The triorganotin compound bis[triphenyltin(IV)](3-carboxy-pyridine-2-thionato) (SnMNA), was tested for its anti-proliferative and antitumor activities. Cytotoxic activity was assessed by Trypan blue and 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide assay (MTT). SnMNA exhibited potent cytotoxic effects against leiomyosarcoma cells (LMS) and human breast adenocarcinoma cells (MCF-7), which is 200 times stronger than that of cisplatin. Moreover, SnMNA induced significant apoptosis in LMS and MCF-7 cells characterized by flow cytometry analysis and DNA fragmentation. Acute and chronic toxicity studies on Wistar rats caused kidney and lung toxicity at a single dose of 80mg/kgBody Weight (BW) or four repeated doses of 8mg/kgBW once per week. Furthermore, antitumor activity studies on sarcoma bearing Wistar rats revealed that SnMNA complex at four repeated doses of 5.4mg/kgBW every three days prolonged mean survival time of the animal at 200% and decreased mean tumor growth rate (MTGR) compared to the control group (p<0.05). It is noteworthy to mention that the 30% (3 out of 10) of the bearing animals were totally cured. These findings indicate that SnMNA might be a promising new antitumor agent.

A simple procedure for estimation of total body surface area and determination of a new value of Meeh's constant in rats

Precise calculation of total body surface area (TBSA) or premarked surface areas (P-MSAs) is of great importance in many biomedical applications. The aim of the paper was to present a simple procedure of measurement of P-MSAs in small animals and to determine a more accurate Meehs constant (k), for a commonly used weight range of laboratory rats. A series of 30 Wistar rats, weighing 195–240 g, were anaesthetized and weighted. The TBSA of each animal was measured using a clear pocket and a planimeter. The data obtained were entered into the Meehs formula (TBSA = kW 2/3), the most commonly used for small experimental animals, so that a k value for each animal as well as a mean k value (9.83) were obtained. The TBSA of the animals was also calculated using the aforementioned mean k value and compared with that obtained using k values reported in previous studies. According to our findings, the new mean k value, determined with the use of our procedure of surface area measurement, ensured greater accuracy in the determination of the TBSA of experimental rats of a specific weight range. We also suggest a new procedure of surface area measurement which is easy, accurate and does not require animal sacrifice.

Oxidative stress in patients treated with continuous ambulatory peritoneal dialysis (CAPD) and the significant role of vitamin C and E supplementation

PurposeChronic renal failure patients undergoing peritoneal dialysis (PD) are characterized by increased oxidative stress (OS), which is associated with enhanced cardiovascular risk. Moreover, oxidative stress also contributes to peritoneal membrane changes and ultrafiltration failure. The aim of this study was to evaluate OS in PD patients and the effect of treatment with ascorbic acid and α-tocopherol.MethodsPlasma, erythrocyte, urine, and peritoneal effluent samples from 20 patients on PD were evaluated for glutathione peroxidase and superoxide dismutase activity, total antioxidant capacity (TAC) and malondialdehyde (MDA) levels, as well as protein carbonyl formation, before and after administration of vitamin C, alone or in combination with vitamin E, in comparison with 10 apparently healthy control individuals.ResultsAll studied markers showed enhanced OS in the PD group, compared to controls. The supplementation of vitamin C and E resulted in improvements of all the OS markers, as indicated by increased erythrocyte antioxidant enzymes activity and TAC levels, as well as decreased MDA concentration and carbonyl compound formation.ConclusionsThe oral supplementation of antioxidant vitamins C and E, in combination, can lead to decreased OS, thus providing a useful and cost-effective therapeutic option in PD patients.

Effects of catechin and epicatechin on superoxide dismutase and glutathione peroxidase activity, in vivo

Abstract Objectives The objective of this study was to investigate the effects of catechin and epicatechin on the activity of the endogenous antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) (as well as the total antioxidant capacity (TAC)) of rats after intra-peritoneal (i.p.) administration. Methods Twenty-four Wistar rats were randomly divided into two groups: the experimental group which was administered daily with a 1:1 mixture of epicatechin and catechin at a concentration of 23 mg/kg body weight for 10 days and the control group which was injected daily with an equal amount of saline. Blood and urine samples were collected before and after the administration period, as well as 10 days after (follow-up). Results Intra-peritoneal administration of catechins led to a potent decrease in GPx levels and a significant increase in SOD levels. TAC was significantly increased in plasma and urine. Malonaldehyde levels in urine remained stable. In the animals treated with catechins, SOD activity showed a moderate negative correlation with GPx activity. Discussion Boosting the activity of the antioxidant enzymes could be a potential adjuvant approach for the treatment of the oxidative stress-related diseases.

Cytotoxic and anticancer effects of the triorganotin compound [(C6H5)3Sn(cmbzt)]: An in vitro, ex vivo and in vivo study

Since the initial success of cisplatin, metal complexes and organometallic compounds have been gaining growing interest in cancer therapy. It is well known that organotin(IV) compounds display strong biological activity. The triorganotin compound [(C(6)H(5))(3)Sn(cmbzt)] (cmbzt=5-chloro-2-mercaptobenzothiazole) (SnCMB), was tested for its antiproliferative and antitumour activities. Two sets of experimental procedures were followed: (1) In vitro and ex vivo procedures included the study of the cytotoxic activity of the complex against leiomyosarcoma cells (LMS) and on a normal human fibroblast line (MRC5) by the MTT assay (cell proliferation), colony formation efficiency and flow cytometric analysis with Annexin V-FITC. The anticoagulation properties of the complex were also studied. (2) In vivo procedures included acute toxicity studies and finally administration of the complex to tumour bearing Wistar rats. The results showed that the complex exhibited potent cytotoxic activity (LMS IC(50)=155 nM) and induced significant apoptosis against LMS cells. Acute toxicity studies on Wistar rats presented kidney and liver toxicity at a single dose of 40 mg/kg body wt. Furthermore, antitumour activity studies on sarcoma bearing Wistar rats revealed that SnCMB complex, administrated in two different therapeutic schemes (treated with 4 × 2 mg/kg body wt every 5 days and 3 × 2.67 mg/kg body wt every 10 days of SnCMB complex), prolonged mean survival time (by 50% and 70% respectively), but failed to decrease the mean tumour growth rate (MTGR) compared to the control group (p<0.01). In conclusion, the organic complex SnCMB possess potent cytotoxic and antimetastatic effects, and low toxicity introducing it as possible successor of organometallic compounds used nowadays in chemotherapy.

Intra-peritoneal application of catechins and EGCG as in vivo inhibitors of ozone-induced oxidative stress.

Oxidative stress is considered as a prominent feature of many acute and chronic diseases as well as of the normal aging process. We examined the effects of intra-peritoneal administration of catechins and EGCG as in vivo inhibitors of oxidative stress induced by ozone administration in two groups of Wistar rats. The first group was treated by intra-peritoneal administration of catechins and EGCG after the administration of ozone and the second group was pretreated by intra-peritoneal administration of catechins and EGCG prior to ozone administration. We determined in blood the activity of the enzymes superoxide dismutase and glutathione peroxidase, total antioxidant capacity, levels of copper and zinc and in urine malonaldehyde contents. Ozone administration resulted in significant reduction of glutathione peroxidase activity, plasma zinc levels and plasma and Red Blood Cells antioxidant capacity. Catechins and EGCG upregulate superoxide dismutase activity and maintain plasma and Red Blood Cells antioxidant capacity. Malonaldehyde levels at the end of the study were significantly increased only in the first group. Our data demonstrate that treatment with catechins and EGCG cannot reverse or prevent the effects of oxidative stress although some modulation occurs.

A Novel Mouse Model to Study Image-Guided, Radiation-Induced Intestinal Injury and Preclinical Screening of Radioprotectors

Radiation is an important treatment modality for gastrointestinal tumors, but intestinal injury is a common side effect. Here we describe a physiologically relevant model for studying the molecular determinants of radiation-induced intestinal damage and testing novel radioprotectors. The model employs a radiopaque marker implanted into the surface of the mouse jejunum, serving as a fiducial marker for precise radiation targeting. Mice were imaged with Cone-Beam CT (CBCT) and irradiated (IR) to the marked area using the Small Animal Radiation Research Platform (SARRP). IR-induced damage was acute but reversible and largely restricted to the area of the marker, leaving the surrounding tissues intact. Although whole gut irradiation with these doses caused lethal GI syndrome, focal (5 mm) radiation of the intestine did not cause any weight loss or lethality. However, fibrosis and collagen deposition 4 months post-IR indicated chronic intestinal damage. A separate cohort of mice was treated daily with curcumin, a clinically tested radioprotector, prior to and post-IR. Curcumin-treated mice showed significant decreases in both local and systemic inflammatory cytokine levels and in fibrosis, suggesting it is an effective radioprotector of the intestine. Our results indicate that this model, which emulates clinically relevant intestinal radiation-induced injury, can be used to assess radioprotectors prior to testing in the clinic. Cancer Res; 77(4); 908-17. ©2016 AACR.