Chandra Sekhar Ravuri

The Role of Reactive Oxygen Species in Integrin and Matrix Metalloproteinase Expression and Function

Cell adhesion and migration is largely dependent on integrin binding to extracellular matrix, and several signalling pathways involved in these processes have been shown to be modified by reactive oxygen species (ROS). In fact, integrin activation is linked to increased ROS production by NADPH-oxidases, 5-lipoxygenase, and release from mitochondria. Cell migration is intimately linked to degradation of the extracellular matrix, and activated matrix metalloproteinases (MMPs) are a prerequisite for cancer cell invasion and metastasis. In this minireview, we focus on the interplay between integrin-mediated ROS production and MMP expression as well as its biological and pathobiological significance.

Seasonal appetite regulation in the anadromous Arctic charr: Evidence for a role of adiposity in the regulation of appetite but not for leptin in signalling adiposity

The aim of this study was to investigate whether the seasonal feeding cycle of the anadromous Arctic charr (Salvelinus alpinus) is regulated by a lipostatic mechanism and if leptin (Lep) might act as an endocrine signal of adiposity. Offspring of anadromous Arctic charr with a body mass of 121 g were divided into two treatment groups; one was given feed in excess from March to November, and the other was fasted between April and early June and fed in excess thereafter. In the continuously fed group there was an 8-fold increase in body mass, and a doubling of percentage body fat, from March to August, after which there was no further increase. Fish in the other group lost weight and body fat during fasting, but grew rapidly on being fed, and had partially compensated for their deficit in body mass by August. Differences in percentage body fat between treatment groups were eliminated by August, providing evidence for a lipostatic regulation of feeding and energy homeostasis in Arctic charr. Neither liver total LepA gene expression nor plasma Lep concentrations correlated positively with fish adiposity, so there was no evidence that Lep acts as a signal of adiposity in this species. On the other hand, there was a strong increase in liver LepA1 gene expression at the end of the fasting period, concomitant with fat mobilization and increased plasma glucose, indicating that LepA1 may play a role in regulating metabolic processes associated with fasting.

Long-term fasting in the anadromous Arctic charr is associated with downregulation of metabolic enzyme activity and upregulation of leptin A1 and SOCS expression in the liver

SUMMARY The life strategy of the anadromous Arctic charr (Salvelinus alpinus) includes several months of voluntary fasting during overwintering in freshwater, leading to emaciation prior to seawater migration in spring. In this study we compared changes in condition, substrate utilization and liver metabolism between captive anadromous charr subjected to food deprivation during late winter and spring, and conspecifics fed in excess. In March, nine out of the 10 sampled fed fish had not eaten, indicating that they were in a voluntary anorexic state. In June, the fed fish were eating and all had higher body mass, condition factor and adiposity than in March. In fasted fish there were only small decreases in body mass, condition factor and adiposity between March and May, but all these parameters decreased markedly from May to June. The fasted fish were depleted in fat and glycogen in June, had suppressed activity of hepatic enzymes involved in lipid metabolism (G6PDH and HOAD) and seemed to rely on protein-derived glucose as a major energy source. This was associated with upregulated liver gene expression of leptin A1, leptin A2, SOCS1, SOCS2 and SOCS3, and reduced IGF-I expression. In an in vitro study with liver slices it was shown that recombinant rainbow trout leptin stimulated SOCS1 and SOCS3 expression, but not SOCS2, IGF-I or genes of enzymes involved in lipid (G6PDH) and amino acid (AspAT) metabolism. It is concluded that liver leptin interacts with SOCS in a paracrine fashion to suppress lipolytic pathways and depress metabolism when fat stores are depleted.

Complete regression and systemic protective immune responses obtained in B16 melanomas after treatment with LTX‑315

Malignant melanoma is the most aggressive and deadliest form of skin cancer due to its highly metastatic potential, which calls for new and improved therapies. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line of defense against pathogens, and several CAPs have shown promising potential as novel anticancer agents. Structure–activity relationship studies on the CAP bovine lactoferricin allowed us to de novo design short chemically modified lytic anticancer peptides. In the present study, we investigated the in vivo antitumor effects of LTX-315 against intradermally established B16 melanomas in syngeneic mice. Intratumoral administration of LTX-315 resulted in tumor necrosis and the infiltration of immune cells into the tumor parenchyma followed by complete regression of the tumor in the majority of the animals. LTX-315 induced the release of danger-associated molecular pattern molecules such as the high mobility group box-1 protein in vitro and the subsequent upregulation of proinflammatory cytokines such as interleukin (IL) 1β, IL6 and IL18 in vivo. Animals cured by LTX-315 treatment were protected against a re-challenge with live B16 tumor cells both intradermally and intravenously. Together, our data indicate that intratumoral treatment with LTX-315 can provide local tumor control followed by protective immune responses and has potential as a new immunotherapeutic agent.

Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of γ-glutamyltransferase expression

Abstract γ-Glutamyltransferase (GGT) plays a significant role in antioxidant defence and participates in the metabolism of glutathione (GSH). The enzyme is up-regulated after acute oxidative stress and during pro-oxidant periods, but the underlying regulatory mechanisms are not well known. The present investigation studied whether the endogenous reactive oxygen species (ROS) level was a determinant for GGT expression. A substantial amount of ROS is produced through the NADPH oxidase (NOX) system and knockdown of p22phox, a sub-unit of NOX1-4, resulted not only in reduced ROS levels but also in reduced GGT expression in human endometrial carcinoma cells. Phorbol-12-myristate-13-acetate (PMA) is an activator of NOX and it was found that PMA treatment of human colon carcinoma cells both increased cellular ROS levels and subsequently up-regulated GGT expression. On the other hand, the NOX inhibitor apocynin reduced ROS levels as well as GGT expression. The GGT mRNA sub-type A was increased after PMA-induced NOX activation. These results demonstrate that ROS generated from NOX enzymes are a significant determinant for GGT expression and activity.

Organ specific regulation of tumour invasiveness and gelatinolytic activity at the invasive front

Proteolytic enzymes play a complex role in tumour growth and invasion. To explore the impact of tumour stroma on invasiveness and expression of proteolytic enzymes, we used a xenograft mouse model where tumours in tongue and skin were established from various human cancer cell lines. Gelatinolytic activity in the tumours was investigated by a novel in situ zymography technique which enables high image resolution. In vivo and in vitro expression of various proteolytic enzymes were analysed at transcriptional and protein level using RT-qPCR, immunohistochemistry and SDS-PAGE substrate zymography. At the mRNA level all cell lines were found to express MMP-2, -7, -14, uPA and uPAR. In addition, two out of three cell lines expressed MMP-9. Histological analyses revealed that tongue tumours had an invasive growth pattern, associated with reduced E-cadherin expression. In contrast, the skin tumours established from the same cell lines were non-invasive. Tongue tumours of all cell lines showed strong gelatinolytic activity especially at the invasive front, which was not seen in the non-invasive skin tumours. Our results show a close relationship between tumour invasiveness and gelatinolytic activity at the tumour front. Furthermore, in our model, both invasiveness and activity of tumour-associated proteolytic enzymes were more dependent on the tumour microenvironment than on inherent properties of the cancer cells.

Differential regulation of γ-glutamyltransferase and glutamate cysteine ligase expression after mitochondrial uncoupling: γ-glutamyltransferase is regulated in an Nrf2- and NFκB-independent manner

Abstract The enzymes γ-glutamyltransferase (GGT) and glutamate cysteine ligase (GCL) have important roles in glutathione (GSH) homeostasis, and both are frequently upregulated after acute oxidative stress. Mitochondria are major producers of ROS, and incubating the colorectal adenocarcinoma cell line HT-29 cells with mitochondrial uncouplers significantly increased endogenous ROS as well as mRNA for both GGT and GCLC (the catalytic subunit of GCL). However, no elevation in GGT protein or activity was detected, in contrast to the increased levels of GCLC protein found. The uncouplers initiated endoplasmic reticulum (ER) stress, as demonstrated by highly increased levels of CHOP and GRP78 mRNA. Using inhibitors of proteasomes and ER-associated degradation (ERAD) together with a mitochondrial uncoupler, increased GGT protein and activity levels were obtained indicating that GGT may be a substrate for ERAD. Uncoupling increased the mRNA levels of the two redox-regulated transcription factors Nrf2 and NFκB. Using siRNA to suppress Nrf2 and NFκB expression, downregulation of GCLC expression both at the basal level and after mitochondrial uncoupling was achieved. In contrast, the expression level of GGT was not affected by this treatment. These data strongly indicate a discrepancy between the regulation of GCLC and of GGT following the oxidative stress situation due to mitochondrial uncoupling. Both the enzymes are considered to be part of the cellular antioxidant system; however, the role of GGT as a consistent oxidative response parameter needs to be reevaluated.

Seasonal Differences in Relative Gene Expression of Putative Central Appetite Regulators in Arctic Charr (Salvelinus alpinus) Do Not Reflect Its Annual Feeding Cycle

The highly seasonal anadromous Arctic charr (Salvelinus alpinus) was used to investigate the possible involvement of altered gene expression of brain neuropeptides in seasonal appetite regulation. Pro-opiomelanocortin (POMCA1, POMCA2), Cocaine and amphetamine regulated transcript (CART), Agouti related Peptide (AgRP), Neuropeptide Y (NPY) and Melanocortin Receptor 4 (MC4-R) genes were examined. The function of centrally expressed Leptin (Lep) in fish remains unclear, so Lep (LepA1, LepA2) and Leptin Receptor (LepR) genes were included in the investigation. In a ten months study gene expression was analysed in hypothalamus, mesencephalon and telencephalon of immature charr held under natural photoperiod (69°38’N) and ambient temperature and given excess feed. From April to the beginning of June the charr did not feed and lost weight, during July and August they were feeding and had a marked increase in weight and condition factor, and from November until the end of the study the charr lost appetite and decreased in weight and condition factor. Brain compartments were sampled from non-feeding charr (May), feeding charr (July), and non-feeding charr (January). Reverse transcription real-time quantitative PCR revealed temporal patterns of gene expression that differed across brain compartments. The non-feeding charr (May, January) had a lower expression of the anorexigenic LepA1, MC4-R and LepR in hypothalamus and a higher expression of the orexigenic NPY and AgRP in mesencephalon, than the feeding charr (July). In the telencephalon, LepR was more highly expressed in January and May than in July. These results do not indicate that changes in central gene expression of the neuropeptides investigated here directly induce seasonal changes in feeding in Arctic charr.

The proteasome inhibitor lactacystin enhances GSH synthesis capacity by increased expression of antioxidant components in an Nrf2-independent, but p38 MAPK-dependent manner in rat colorectal carcinoma cells.

ABSTRACT Proteasome inhibitors may induce ER stress and oxidative stress, disrupt signaling pathways, and trigger apoptosis in several cancer cells. However, they are also reported to increase glutathione (GSH) synthesis and protect cells from oxidative stress. In the present study, we showed that the proteasome inhibitor lactacystin increased reactive oxygen species (ROS) and GSH levels after the treatment of HT-29 colorectal cancer cells. The increased GSH depended upon the activity of glutamate cysteine ligase (GCL), uptake of cystine/cysteine via the cystine/glutamate transporter , and the activity of γ-glutamyltransferase (GGT). Increased transcription levels of the catalytic subunit of glutamate cysteine ligase (GCLC), the catalytic subunit xCT of , and GGT were induced by lactacystin, although with different kinetics and stoichiometry. Lactacystin treatment also augmented protein levels of GCLC, xCT, and GGT, but significant levels were not detected until 48 h after initiation of lactacystin treatment. These increases in protein levels were dependent on the p38 MAPK pathway. Studies in cells transfected with siRNA against the transcription factor Nrf2 demonstrated that the promoter activities of xCT and GCLC, but not of GGT, depended on Nrf2. However, depletion of Nrf2 had no effect on lactacystin-induced upregulation of the GGT, GCLC, and xCT mRNA levels. Taken together, our results suggest that oxidative stress provoked by proteasomal inhibition results in the elevation of cellular GSH levels due to increased synthesis of GSH and uptake of cystine/cysteine. Following treatment with lactacystin, enhanced expression of antioxidant components involved in GSH homeostasis is p38 MAPK-dependent, but Nrf2-independent, resulting in increased GSH synthesis capacity.

Abstract 2584: Complete regression and protective immune responses obtained in B16 melanomas after treatment with LTX-315 (Oncopore®)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Malignant melanoma is the most aggressive and deadliest form of skin cancer, with a high mortality rate among late-stage melanoma patients. This calls for new and improved therapies within the field. Cationic antimicrobial peptides (CAPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line of defense against pathogens, and several CAPs have shown promising anticancer activity. Structure-activity relationship studies on the CAP bovine lactoferricin has resulted in the design of a short chemically modified lytic anticancer peptide, LTX-315, which has potential as a new anticancer drug. Previous animal studies conducted with LTX-315 have demonstrated that intratumoral (i.t.) treatment of syngeneic murine A20 B-cell lymphomas and CT26WT colon carcinomas resulted in complete tumor regression. In the present study, we investigated the antitumor effects of LTX-315 against highly aggressive B16 melanomas in syngeneic mice. Methods: The cytotoxicity of LTX-315 was tested against a series of cell lines in vitro using the MTT assay. B16F1 melanoma cells (5 x 104) were intradermally inoculated into the abdomen of syngeneic C57BL/6N mice and established tumors treated i.t. with LTX-315. Tumor tissue and blood samples were taken for analysis to elaborate the mechanism of action of LTX-315 and for potential immune modulatory effects following i.t. treatment. Blood plasma was analyzed for IL 1β and IL 6 content and tumor tissue was analyzed for infiltrating immune cells and expression of pro-inflammatory cytokines using immunohistochemistry and real time qPCR, respectively. Results: LTX-315 rapidly kills B16 melanoma cells and induces the release of Danger-Associated Molecular Pattern molecules such as the High Mobility Group Box-1 protein in vitro. Intratumoral (i.t.) administration of LTX-315 resulted in tumor necrosis and the infiltration of immune cells into the tumor parenchyma followed by a complete regression of the tumor in the majority of the animals. Animals cured by LTX-315 treatment were protected against a re-challenge with live B16 tumor cells. Conclusions: Our results demonstrate that LTX-315 i.t. treatments with LTX-315 induces complete regression of solid B16F1 melanoma tumors, thus leading to local tumor control, followed by protective immune responses. Thus, LTX-315 has potential as a novel immunotherapeutic agent. Citation Format: Ketil Andre Camilio, Gerd Berge, Chandra Sekhar Ravuri, Oystein Rekdal, Baldur Sveinbjornsson. Complete regression and protective immune responses obtained in B16 melanomas after treatment with LTX-315 (Oncopore®). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2584. doi:10.1158/1538-7445.AM2014-2584