Molecular Dynamics of Cobalt Protoporphyrin Antagonism of the Cancer Suppressor REV-ERBß.

Nuclear receptor REV-ERBß is an overexpressed oncoprotein that has been used as a target for cancer treatment. The metal-complex nature of its ligand, iron protoporphyrin IX (Heme), enables the REV-ERBß to be used for multiple therapeutic modalities as a photonuclease, a photosensitizer, or a fluorescence imaging agent. The replacement of iron with cobalt as the metal center of protoporphyrin IX changes the ligand from an agonist to an antagonist of REV-ERBß. The mechanism behind that phenomenon is still unclear, despite the availability of crystal structures of REV-ERBß in complex with Heme and cobalt protoporphyrin IX (CoPP). This study used molecular dynamic simulations to compare the effects of REV-ERBß binding to Heme and CoPP, respectively. The initial poses of Heme and CoPP in complex with agonist and antagonist forms of REV-ERBß were predicted using molecular docking. The binding energies of each ligand were calculated using the MM/PBSA method. The computed binding affinity of Heme to REV-ERBß was stronger than that of CoPP, in agreement with experimental results. CoPP altered the conformation of the ligand-binding site of REV-ERBß, disrupting the binding site for nuclear receptor corepressor, which is required for REV-ERBß to regulate the transcription of downstream target genes. Those results suggest that a subtle change in the metal center of porphyrin can change the behavior of porphyrin in cancer cell signaling. Therefore, modification of porphyrin-based agents for cancer therapy should be conducted carefully to avoid triggering unfavorable effects.

Schisandrin C enhances odontoblastic differentiation through autophagy and mitochondrial biogenesis in human dental pulp cells.

OBJECTIVE: To investigate the role of Schisandrin C in odontoblastic differentiation, and its relations between autophagy and mitochondrial biogenesis in human dental pulp cells (HPDCs). DESIGN: Fresh third molars were used, and cultured for HDPCs. Western blotting technique, Alizarin red S staining, alkaline phosphatase (ALP) activity, and confocal microscopy were used to detect autophagy, mitochondrial biogenesis, and odontoblastic differentiation. To understand the mechanism of Schisandrin C, the HDPCs were treated with lipopolysaccharide (LPS), autophagy and heme oxygenase-1 (HO-1) inhibitors: 3-Methyladenine (3-MA) and Zinc protoporphyrin IX (ZnPP), respectively. RESULTS: LPS decreased the expression of autophagy molecules [autophagy protein 5 (ATG-5), beclin-1, and microtubule-associated protein 1A/1B light chain 3 (LC3-I/II)] and mitochondrial biogenesis molecules [heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)], and disrupted odontoblastic differentiation. The down-regulation of autophagy and mitochondrial biogenesis with 3-MA and ZnPP inhibited odontoblastic differentiation. However, Schisandrin C restored the expression of all the above molecules, even with LPS and inhibitor treatment. This result demonstrates that autophagy and mitochondrial biogenesis plays an essential role in odontoblastic differentiation, and Schisandrin C activates these systems to promote odontoblastic differentiation of HDPCs. CONCLUSION: Schisandrin C has potential characters to regulate odontoblastic differentiation, and may be recommended for use as a compound for pulp homeostasis.

Cimetidine/lactulose therapy ameliorates erythropoietic protoporphyria-related liver injury.

A 21-year-old Japanese man was admitted to our hospital because of severe abdominal pain and jaundice. He had been suffering from abdominal pain attacks and liver dysfunction since 18 years of age. Liver histology showed amorphous brown deposits in the sinusoidal space and significant periportal fibrosis without apparent hepatitis. Increased protoporphyrin in serum and feces and ferrochelatase gene mutation confirmed the final diagnosis of erythropoietic protoporphyria (EPP). Since ursodeoxycholic acid (UDCA) intake and glucose infusion are insufficient to ameliorate jaundice and abdominal attacks, cimetidine and lactulose were added in order to suppress hepatic delta-aminolevulinic acid synthase and limit re-absorption of protoporphyrin, respectively. Afterwards, the jaundice, liver dysfunction and abdominal symptoms improved and UDCA, cimetidine, and lactulose administration was continued. A repeat biopsy at 1.5 years after adding cimetidine/lactulose revealed marked attenuation of periportal fibrosis and protoporphyrin deposits. As far as we know, this is the first demonstration of histological improvement of EPP-induced liver abnormalities due to persistent cimetidine/lactulose administration. These treatments may be useful for EPP-related liver injury.

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Cytotoxic effects of metal protoporphyrins in glioblastoma cells: roles of albumin, reactive oxygen species, and heme oxygenase-1.

We investigate the cytotoxic effect of metal protoporphyrins including ferric protoporphyrin (FePP; hemin), cobalt protoporphyrin (CoPP), and tin protoporphyrin (SnPP) in glioblastoma cells C6 and GBM8401. Data of MTT assay show that FePP and CoPP, but not SnPP, significantly reduce the viability of glioma cells C6 and GBM8401 in the absence of serum. In the condition with fetal bovine serum (FBS) or bovine serum albumin (BSA), the cytotoxic effect of FePP and CoPP was completely inhibited. Binding of FePP, CoPP, and SnPP with BSA was examined via spectrophotometer analysis, and the protective effect of serum against FePP and CoPP-induced cell death was abolished by BSA depletion. A loss in the integrity of DNA with an occurrence of apoptotic events including DNA ladders, caspase 3 and PARP protein cleavage, and chromatin-condensed cells is observed in FePP-treated or CoPP-treated C6 cells. An increase in intracellular peroxide level was examined in FePP, but not CoPP, -treated C6 cells, and N-acetyl-l-cysteine (NAC) addition significantly protected C6 cells from FePP, but not CoPP, -induced cell death with reducing FePP-stimulated reactive oxygen species (ROS) production. Activation of extracellular regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs) with an increase in the heme oxygenase-1 (HO-1) protein was observed in FePP-treated or CoPP-treated C6 cells in the absence of FBS or BSA, and adding JNKs inhibitor SP600125 (SP), but not ERKs inhibitor PD98059 (PD), significantly attenuated FePP-induced or CoPP-induced HO-1 protein expression in accordance with reducing JNKs protein phosphorylation. However, PD98059, SP600125, or transfection of C6 cells with antisense HO-1 oligonucleotides show no effect on the cytotoxicity elicited by FePP and CoPP in C6 cells. Effect of serum and BSA on the cytotoxicity of metal protoporphyrins in glioma cells is first demonstrated in the present study, and the roles of ROS, MAPKs, and HO-1 were elucidated.

Characterization of heme as activator of Toll-like receptor 4.

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.

Co-administration of melatonin reverses the tin-protoporphyrin (SnPP) induced decline of cytochrome P450 content in vivo in rats.

Melatonin (N-acetyl-5 methoxytryptamine) is a low molecular weight antioxidant and is an endogeneous defense system against the deleterious actions of the extremely reactive hydroxyl radical. Among the enzymes that participate in the antioxidant functions is cytochrome P-450, a stalwart of the detoxification system in the body. Our results revealed that tin-protoporphyrin administration brought about a marked decline in cytochrome P-450 levels. This decline was, however, reversed by the coadministration of the antioxidant, melatonin. Thus, the enhanced antioxidant status in melatonin-treated rats may act as a protective mediator of various pharmacological functions altered during tin-protoporphyrin (an antihyperbilirubemenic agent) administration to Wistar rats.

Inhibition of substance P-induced histamine release from rat peritoneal mast cells by low doses of protoporphyrin plus long-wave ultraviolet light irradiation: decreased intracellular calcium as a possible mechanism.

BACKGROUND: A considerable amount of recent interest has been devoted to the down-regulatory effects of photosensitizers plus long-wave ultraviolet light (UVA) irradiation on multiple biologic systems. However, these effects on mast cells are controversial. OBJECTIVE: We have investigated the effect of low doses of protoporphyrin (PP) plus UVA irradiation (PP/UVA) on substance P (SP)-induced histamine release from rat mast cells. METHODS: Rat peritoneal mast cells purified on a Percoll gradient were treated with 3 ng/mL PP and/or UVA, and challenged with SP. In some experiments, IgE-sensitized mast cells were stimulated by antirat IgE in the presence of phosphatidylserine. Histamine released from mast cells and intracellular calcium concentrations ([Ca2+]i) were measured, respectively. RESULTS: SP at a concentration of 10(-5) mol/L caused a significant histamine release with the increase in [Ca2+]i. PP or UVA irradiation alone at doses used in the present study induced no histamine release from mast cells and had no significant effects on SP-induced histamine release from the cells. On the other hand, PP/UVA inhibited SP-induced histamine release in a dose-dependent manner of UVA with the reduction of SP-induced maximal increases in [Ca2+]i. Comparing with the inhibitory effects of PP/UVA on anti-IgE-induced histamine release from IgE-sensitized mast cells and maximal increases in [Ca2+]i in the cells, the inhibitory effects of PP/UVA on the findings in SP-stimulated mast cells were less. CONCLUSION: These data suggest that low doses of PP/UVA inhibits histamine release from SP-activated rat peritoneal mast cells through the suppression of [Ca2+]i.

The effect of protoporphyrin on the susceptibility of human erythrocytes to oxidative stress: exposure to hydrogen peroxide.

Binding of protoporphyrin caused a perturbation of the erythrocyte membrane, as reflected by a change in cell shape from discoid to echinocyte, and a concomitant increase in mean cellular volume and K(+)-loss. Protoporphyrin-induced changes could be prevented by the presence of BaCl2, whereas binding of protoporphyrin was not affected. Exposure of erythrocytes to hydrogen peroxide leads to K(+)-leakage and lipid peroxidation. In de presence of protoporphyrin, H2O2-induced K(+)-leakage was enhanced, whereas lipid peroxidation was inhibited. The increase in H2O2-induced K(+)-leakage by protoporphyrin was not affected by diamide or various K+ channel blockers, but could be prevented by the addition of BaCl2. The inhibition of lipid peroxidation, on the other hand, was not affected by BaCl2. These results indicate that the enhancement of H2O2-induced K(+)-leakage was most likely caused by the change in cell shape. Addition of chlorpromazine and promethazine, positively charged molecules that induce stomatocytosis, did not cause an enhancement of H2O2-induced K(+)-leakage.