Emodin upregulates urokinase plasminogen activator, plasminogen activator inhibitor-1 and promotes wound healing in human fibroblasts.

Urokinase plasminogen activator (uPA) system is important for several biological processes that call for extracellular proteolysis, fibrinolysis, cell migration, proliferation and angiogenesis. The current study highlights the fibrinolytic and wound healing potential of emodin, an anthraquinone, with relevance to the uPA system. Emodin increased the fibrinolytic activity of fibroblast cells in a dose-dependent manner. Zymography linked the activity to increased uPA activity. Subsequent RT-PCR and western analyses demonstrated uPA gene upregulation. Interestingly, PAI-1, the inhibitor of uPA was also upregulated. EMSA showed the upregulation occurred independent of emodin's effect on nuclear factor kappa B (NFkappaB). The effect on uPA system is supposedly via generation of reactive oxygen species (ROS) since cotreatment with ascorbic acid, an anti-oxidant, attenuated the activity. In addition to profibrinolytic potential, emodin also demonstrated wound healing activity in in vitro wound models. Presence of emodin in the medium enhanced the rate of migration of fibroblasts into the wounded region. These in vitro experiments reveal that emodin is a potent profibrinolytic and wound healing agent.

uPA dependent and independent mechanisms of wound healing by C-phycocyanin.

Wound repair requires both recruitment and well co-ordinated actions of many cell types including inflammatory cells, endothelial cells, epithelial cells and importantly fibroblast cells. Urokinase-type plasminogen activator (uPA) system plays a vital role in wound healing phenomenon. We have previously demonstrated that C-phycocyanin (C-pc), a biliprotein from blue-green algae, transcriptionally regulates uPA through cAMP-dependent protein kinase A (PKA) pathway. To date, a role for C-pc in wound-healing scenario is not elucidated. This study was designed to examine the wound-healing property of C-pc in relation to fibroblast proliferation and migration. C-pc increased fibroblast proliferation in a dose-dependent manner. It also enhanced G1 phase of cell cycle and increased the expressions of cyclin-dependent kinases 1 and 2, which facilitate cell cycle progression, in a uPA-independent manner. In vitro wound healing and migration assays revealed the pro-migratory properties of C-pc. Short-interference RNA studies demonstrated that uPA was necessary for C-pc-induced fibroblast migration. C-pc also significantly elevated the expressions of chemokines (MDC, RANTES, Eotaxin, GRO alpha, ENA78 and TARC) and Rho-GTPases (Cdc 42 and Rac 1) in a uPA-dependent manner. Pre-treatment of C-pc-stimulated cells with pharmacological inhibitor of PI-3K (LY294002) annulled the expression of GTPases implying that Rac 1 and Cdc 42 were induced through PI-3K pathway. C-pc-induced cellular migration towards wounded area was also negatively affected by PI-3K inhibition. In vivo wound-healing experiments in mice validated our finding that C-pc accelerates wound healing. Our data provides conclusive evidence of a novel therapeutic usage for C-pc as a wound-healing agent. C-pc is a food and drug administration (FDA)-approved health supplement. We believe this compound can also be beneficial in healing of internal wounds, such as ulcers.

Cell cycle regulation and induction of apoptosis by beta-carotene in U937 and HL-60 leukemia cells.

In this communication, we report the efficacy of beta-carotene towards differentiation and apoptosis of leukemia cells. Dose (20 microM) and time dependence (12 h) tests of beta- carotene showed a higher magnitude of decrease (significance p < 0.05) in cell numbers and cell viability in HL-60 cells than U937 cells but not normal cell like Peripheral blood mononuclear cell (PBMC). Microscopical observation of beta-carotene treated cells showed a distinct pattern of morphological abnormalities with inclusion of apoptotic bodies in both leukemia cell lines. When cells were treated with 20 microM of beta-carotene, total genomic DNA showed a fragmentation pattern and this pattern was clear in HL-60 than U937 cells. Both the cell lines, on treatment with beta- carotene, showed a clear shift in G(1) phase of the cell cycle. In addition the study also revealed anti-oxidant properties of beta-carotene since there was reduction in relative fluorescent when treated than the control at lower concentration. Collectively this study shows the dual phenomenon of apoptosis and differentiation of leukemia cells on treatment with beta-carotene.

C-phycocyanin transcriptionally regulates uPA mRNA through cAMP mediated PKA pathway in human fibroblast WI-38 cells.

We have previously demonstrated the efficacy of c-phycocyanin in up-regulation of urokinase-type plasminogen activator (uPA) in bovine endothelial cell line. However, the mechanism of action and pathway elucidation in uPA regulation is unclear. In experiments reported here, we have investigated the mechanism of action of c-phycocyanin (c-pc) induced uPA gene modulation in human fibroblast (WI-38) cell line. ELISA test confirmed that c-pc increased the uPA antigen whereas PAI-1 antigen level was unaffected. Treatment of cells with c-pc significantly (P<0.05) enhanced the uPA mRNA level in a dose (50 microg/ml) and time dependent (up to 4 h) manner. This effect of c-pc was abolished by treatment with dichloro-1-beta-D-ribofuranosyl benzamidazole (DRB) (10 microg/ml). Co-treatment of c-pc with 200 microg/ml cycloheximide (CHX), translation inhibitor, resulted in over accumulation of uPA mRNA. These results suggest that uPA induction by c-pc is transcriptionally regulated and does not require de novo protein synthesis. We also provide evidence that c-pc stimulates uPA gene through cAMP dependent pathway as adenylyl cyclase (AC) inhibitor, dideoxyadenosine (DDA) significantly inhibited the uPA mRNA expression and co-treatment with adenylyl cyclase analogue, dBcAMP recovered the effect of c-pc on gene activity. Furthermore, the present investigation provides evidence on the regulatory pathway involved in the c-pc stimulus. C-pc induced uPA expression was completely inhibited by PKA inhibitor (KT 5200), indicating the regulation is dependent on PKA pathway. Elimination of PKC pathway components by prolonged incubation with excess amount of phorbol 12-myristate 13-acetate (PMA) failed to abolish the c-pc effect on uPA expression indicating the regulation is independent of PKC pathway. Taken together, our data indicate that uPA gene regulation by c-pc is transcriptionally controlled through cAMP mediated PKA pathway.

Purification of c-phycocyanin from Spirulina fusiformis and its effect on the induction of urokinase-type plasminogen activator from calf pulmonary endothelial cells.

c-Phycocyanin (c-pc), a blue coloured, fluorescent protein was purified from blue-green alga, Spirulina fusiformis and its effect on fibrinolytic system in vascular endothelial cells was investigated. The c-pc consisted of two subunits, alpha and beta, whose molecular masses were 16 and 17 kDa, respectively. N-terminal sequences of both subunits were well conserved compared with other blue green algal phycobiliproteins. Fibrinolytic activity in the medium conditioned by calf pulmonary arterial endothelial cells was measured by the fibrin plate method. The c-pc increased the fibrinolytic activity in dose- and time-dependent manners. Fibrin zymographic studies indicated that c-pc-induced urokinase-type plasminogen activator in the cells. These in vitro results suggest that c-pc from S. fusiformis is a potent profibrinolytic protein in the vascular endothelial system.

Iron-mediated stability of PAI-1 mRNA in adenocarcinoma cells-involvement of a mRNA-binding nuclear protein.

This study reports the stability of mRNA of type-1 plasminogen activator inhibitor (PAI-1), the major physiologic inhibitor of plasminogen activation, by deferoxamine-aided iron deprivation, in PC3 adenocarcinoma cells. ELISA and Northern analyses studies revealed dose-dependent increase in PAI-1 expression by deferoxamine-treated cells. Co-treatment with ferric citrate quenched the effect of deferoxamine, confirming the role of iron in PAI-1 regulation. DRB-based RNA chase experiments suggested that post-transcriptional mechanism was involved in PAI-1 regulation. De-novo protein synthesis was necessary for this regulation. Electrophoretic mobility shift assay revealed the presence of a nuclear protein, binding to the 3'-UTR of PAI-1 mRNA in an iron-mediated manner. This is the first report of iron-mediated mRNA-protein interaction in PAI-1, involved in mRNA stability.

Post-transcriptional regulation of plasminogen activator inhibitor-1 by intracellular iron in cultured human lung fibroblasts--interaction of an 81-kDa nuclear protein with the 3'-UTR.

The proteinase inhibitor, type-1 plasminogen activator inhibitor (PAI-1), is a major regulator of the plasminogen activator system involved in plasmin formation and fibrinolysis. The present study explores the effects of intracellular iron on the expression of PAI-1 and associated cell-surface plasmin activity in human lung fibroblasts; and reports the presence of a novel iron-responsive protein. ELISA revealed a dose-dependent increase in PAI-1 antigen levels expressed in the conditioned medium of cells treated with deferoxamine, in the three cell lines studied. A concomitant increase in mRNA levels was also observed by Northern analyses. Presaturation with ferric citrate quenched the effect of deferoxamine. Experiments with transcription and translation inhibitors on TIG 3-20 cells demonstrated that intracellular iron modulated PAI-1 expression at the post-transcriptional level with the requirement of de-novo protein synthesis. Electrophoretic mobility shift assay and UV crosslinking assays revealed the presence of an approximately 81-kDa nuclear protein that interacted with the 3'-UTR of PAI-1 mRNA in an iron-sensitive manner. Finally, we demonstrated that the increased PAI-1 is functional in suppressing cell-surface plasmin activity, a process that can affect wound healing and tissue remodeling.

Growth and development of catla (Catla catla) fed with different levels of diet containing Spirogyra sp.

The effect of Spirogyra sp. incorporated into diet formulations on the growth and body composition of Indian major carp, catla (Catla catla) was investigated in a 45 days feeding trial. Spirogyra dry powder was mixed with different feed ingredients in different amounts (0%, 10%, 25%, 37% and 40% of the total feed). Carps fed with Spirogyra demonstrated higher feed conversion ratio. The study also revealed a direct relationship between the amount of Spirogyra in the diet, and muscle protein and fat contents in the fish. In general, this study demonstrated the benefits of incorporating Spirogyra into carp feeds.