The synergistic effect of PDT and oxacillin on clinical isolates of Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a major pathogen in clinical microbiology. It is known to cause infections at various body sites and can be life-threatening. The development of resistance to many well-established antibiotic treatments and the prevalence of methicillin-resistant S. aureus (MRAS) among hospital patients and the general community pose challenges in treating the pathogen. The antimicrobial effect of photodynamic therapy (PDT) has been a subject of study for a long time and can offer new strategies for dealing with resistant strains. OBJECTIVE: In our study, we searched for a positive synergistic relationship between PDT and the standard antibiotics used to treat S. aureus and MRSA infections. MATERIALS AND METHODS: The phototoxic profile of deuteroporphyrin (DP) in both resistant and susceptible clinical strains of S. aureus was determined by plating of treated and untreated broth cultures. Electron microscopy imaging was done to explore possible sites of damage and free-radical accumulation in the cells during DP-PDT. Minimal inhibitory concentration (MIC) of oxacillin, gentamicin, vancomycin, rifampin, and fusidic acid was determined using the broth dilution method, and the checkerboard method was used to detect and evaluate the synergistic potential of DP-PDT and antibiotic combinations. A synergistic combination was further characterized using broth cultures and plating. RESULTS: DP-PDT using a light dose of 15 J/cm2 showed a bactericidal effect even with a small concentration of 17 µM DP. Transmission electron microscopy indicated profound damage in the cell wall and cell membrane, and the appearance of mesosome-like structures. Free radicals tend to localize in the cell membrane and inside the mesosome. No synergistic effect was detected by combining PDT with gentamicin, vancomycin, rifampin, and fusidic acid treatments. A positive synergistic effect was observed only in DP-PDT-oxacillin combined treatment using the checkerboard method. The effect was observed in clinical antibiotic-resistant isolates after DP-PDT using a light dose of 46 J/cm2 and small concentrations of DP. Oxacillin MIC decreased below 2 µg/ml in resistant strains under such conditions. Cultures which did not undergo new cycles of DP-PDT recovered their original oxacillin resistance after a few generations. CONCLUSIONS: PDT with porphyrins shows possible new therapeutic options in treating drug-resistant S. aureus at body sites suitable for irradiation. The synergistic effect of DP-PDT with oxacillin on clinical strains illustrates the potential of PDT to augment traditional antibiotic treatment based on cell wall inhibitors. Lasers Surg. Med. 50:535-551, 2018. © 2018 Wiley Periodicals, Inc.

Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

Sperm interaction with bacteria induces the spontaneous acrosome reaction.

Bacterial contamination in the semen deteriorates spermatozoa function. One mechanism through which this may occur is by inducing a premature form of the acrosome reaction (spontaneous acrosome reaction (sAR)) which has been shown to abrogate fertilization. To understand the mechanism by which bacteria affect sperm functions, we determined the effects of bacteria on sperm sAR and on other parameters involved in sperm capacitation. Sperm cells undergo biochemical changes in the female reproductive tract collectively called capacitation. Only capacitated sperm can undergo the physiological acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize the egg. Bovine sperm incubated with the bacteria Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) or Pseudomonas aeruginosa (P. aeruginosa), revealed a sperm-bacteria interaction, however only E. coli and P. aeruginosa caused an increase in sperm sAR. This effect was seen only when the bacteria were present with the sperm during the full incubation under capacitation conditions but not when the bacteria were added to capacitated sperm. These results indicate that bacteria affect sperm during capacitation and not at the AR step. In addition, Ca2+ influx, protein kinase A, and protein tyrosine phosphorylation activities, three essential processes that promote capacitation, were inhibited by the bacteria. Moreover, increasing intracellular cAMP, which also occur during sperm capacitation, caused significant reverse of sAR induced by the bacteria.

Multifunctional 5-aminolevulinic acid prodrugs activating diverse cell-death pathways.

Herein we describe a series of multifunctional 5-aminolevulinic-acid (ALA) prodrugs for photodynamic dependent and independent cancer therapy (PDT). We studied the cell-death mechanisms in glioblastoma U251 cells treated with four ALA-prodrugs: (1) AlaAcBu, that releases ALA, acetaldehyde, and butyric acid; (2) AlaFaBu, that releases ALA, formaldehyde, and butyric acid; (3) AlaFaPi, that releases ALA, formaldehyde and pivalic acid (4) AlaAcPi that releases ALA, acetaldehyde and pivalic acid. We examined the light-activated and dark cell-death mechanisms of the active metabolites released from the prodrugs by unspecific cellular hydrolases. The active moieties accelerated biosynthesis of protoporphyrin IX (PpIX) due to upregulated porphobilinogen deaminase (PBGD) activity. AlaAcBu was found to be the superior prodrug for PDT due to its ability to induce the highest PpIX synthesis. Photo-irradiation of AlaAcBu-treated cells led to dissipation of the mitochondrial membrane potential and reduction in the mitochondria metabolic activities; apoptosis and necrosis. Electron microscopy analyses of these cells revealed mitochondrial and endoplasmic reticulum swelling, membrane blebbing, apoptotic bodies and necrotic cell rupture. The formaldehyde-releasing prodrugs AlaFaBu and AlaFaPi induced low PDT efficacy, moreover sequestering the formaldehyde with semicarbazide resulted in high PpIX synthesis, suggesting that formaldehyde inhibited its synthesis. ALA and AlaAcBu phototherapy resulted in a dramatic accumulation of ubiquitinated proteins due to reduced proteasome activity and expression. In conclusion, the PDT potency of the prodrugs was in the order: AlaAcBu, AlaAcPi > AlaFaBu ≥ ALA > AlaFaPi, and the superiority of AlaAcBu stems from lower molar concentrations of AlaAcBu and lower light intensity needed to activate cell death following PDT.

The centrality of PBGD expression levels on ALA-PDT efficacy.

Successful 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is dependent on efficient porphyrin synthesis in the inflicted cancer tissue, which is regulated by several enzymes. Irradiation of the tumor excites the light-sensitive porphyrins and results in ROS production and cell death. In this study we investigated the effect of the expression levels of two main enzymes in heme biosynthesis, ALA dehydratase (ALAD) and porphobilinogen deaminase (PBGD), on the capacity of K562 cells to undergo cell death following ALA-PDT. We manipulated PBGD and ALAD expression levels by shRNAs and PBGD overexpressing plasmid. PBGD down-regulation induced an elevation in ALAD activity, while overexpression of PBGD reduced ALAD activity, indicating a novel regulation feedback of PBGD on ALAD activity. This feedback mechanism enabled partial PpIX synthesis under PBGD silencing, whereas ALAD silencing reduced PpIX production to a minimum. ALA-PDT efficacy was directly correlated to PpIX levels. Thus, only ALAD-silenced cells were not affected by ALA+ irradiation, while following PBGD silencing, the accumulated PpIX, though decreased, was sufficient for successful ALA-PDT. The alterations in ALAD activity level initiated by changes in PBGD expression indicates PBGD's central role in heme synthesis. This enables efficient ALA-PDT, even when PBGD is not fully active. Conversely, ALAD loss resulted in reduced PpIX synthesis and consequently failure in ALA-PDT, due to the absence of compensation mechanism for ALAD.

Modulating ALA-PDT efficacy of mutlidrug resistant MCF-7 breast cancer cells using ALA prodrug.

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.

Silencing of ALA dehydratase affects ALA-photodynamic therapy efficacy in K562 erythroleukemic cells.

Synthesis of protoporphyrin IX (PpIX) by malignant cells is essential for the success of ALA-based photodynamic therapy (PDT). Two key enzymes that were described as affecting PpIX accumulation during ALA treatment are porphobilinogen deaminase (PBGD) and ferrochelatase. Here, we show that down regulation of ALA dehydratase (ALAD) expression and activity by specific shRNA induced a marked decrease in PpIX synthesis in K562 erythroleukemic cells. Photo-inactivation efficacy following ALA-PDT was directly correlated with ALAD-silencing and cellular levels of PpIX. MTT metabolism following ALA-PDT was shown to be 60% higher in ALAD-silenced cells in comparison to control cells, indicating that mitochondria were protected in the silenced cells. Morphological analysis by scanning electron microscopy (SEM) of cells treated by ALA-PDT showed no morphological changes in ALAD-silenced cells, in contrast to controls exhibiting cell deformations and lysis. Membrane integrity following ALA-PDT was kept intact and undamaged in ALAD-silenced cells as examined by Annexin V-FITC/PI staining and LDH-L leakage. We conclude that ALAD, although it is present in the cell at abundant levels, has a major and limiting role in regulating PpIX synthesis and ALA-PDT outcome.

Accelerated proteasomal activity induced by Pb2+, Ga3+, or Cu2+ exposure does not induce degradation of alpha-synuclein.

The involvement of environmental heavy metals in Parkinson's disease (PD) has been suggested by epidemiologic studies; however, the mechanism of this effect is unknown. PD is characterized by the aggregation of alpha-synuclein in Lewy bodies. We previously showed that Pb2+ accelerates proteasomal activity. Therefore, we examined the effect of Pb2+, Ga3+, and Cu2+ on alpha-synuclein in human SH-SY5Y cells. The heavy metals induced an increase in heme-oxygenase-1 levels without significant cell death or ROS generation. The metals inhibited ALA-dehydratase, which is the inhibitory subunit of the proteasome, thereby accelerating proteasomal activity and decreasing protein levels of CDK-1 and PBGD. However, alpha-synuclein protein levels increased after exposure to metals, similar to the effect obtained with the proteasome inhibitor, hemin, suggesting that alpha-synuclein is inaccessible to proteasomal degradation. Indeed, electron microscopy revealed the formation of aggresomes in Pb2+- or hemin-treated cells. Thus, although heavy metals enhance proteasomal activity, alpha-synuclein is protected from degradation, and its protein levels and aggregation are increased.

Proteasomal degradation regulates expression of porphobilinogen deaminase (PBGD) mutants of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a neuropathic disease caused by a dominant inherited deficiency in porphobilinogen deaminase (PBGD). We investigated the expression and the degradation of the human PBGD-mutations G748A, G748C and 887insA following transfection into human SH-SY5Y neuroblastoma cells. Mutant proteins exhibited reduced protein expression compared to transfected wild-type (wt) PBGD as revealed by Western blotting. The transcription levels assessed by real-time PCR of these mutant species were identical to those of the wild type. Immuno-fluorescence microscopy revealed reduced cellular distribution of the mutated PBGDs in the cytosol and the nucleus in comparison to the wild-type PBGD. Enhanced cellular accumulation of the mutated and wild-type PBGDs was detected following inhibition of the proteasome by the inhibitors CLBL and hemin. Elevated expression of wt and mutated PBGD protein levels was either achieved by hemin or heme-arginate treatment. On the other hand, enhanced PBGD degradation was achieved by lead poisoning of ALAD in the SH-SY5Y cells concomitant with acceleration of proteasomal activity, most probably by ALAD participation in proteasomal regulation [G.G. Guo, M. Gu, J.D. Etlinger, 240-kDa proteasome inhibitor (CF-2) is identical to delta-aminolevulinic acid dehydratase. J Biol Chem 1994; 269:12399-402.] Our results suggest that the difference in expression between the wild-type and mutant proteins appears to be regulated on the level of protein degradation. In conclusion, we demonstrate that the PBGD cellular pool is controlled by the proteasome activity, which in turn is down regulated by hemin or up-regulated by Pb-ALAD.

The anticancer prodrugs of butyric acid AN-7 and AN-9, possess antiangiogenic properties.

The antiangiogenic and antineoplastic activities of the butyric acid prodrugs AN-7 and AN-9 were demonstrated in vitro with HUVEC by inhibition of proliferation and vascular tubes formation, enhanced apoptosis, and inhibition of 22Rv-1 cells migration. In the sc implanted human prostate tumors (22Rv-1) in nude mice, AN-7 significantly inhibited Ki-67, HIF-1alpha, HER-2/neu, bFGF and increased PTEN level. AN-7 and AN-9 reduced hemoglobin accumulation in matrigel plugs implanted sc in Balb-c mice. Herein, we show that the anticancer activity of AN-7 and AN-9 can be attributed in part to their antiangiogenic activities suggesting potential therapeutic benefits for prostate cancer patients.

In vitro and in vivo photosensitization by protoporphyrins possessing different lipophilicities and vertical localization in the membrane.

Photodynamic therapy (PDT) is being evaluated in clinical trials for treatment of various oncologic and ophthalmic diseases. The main cause for cell inactivation and retardation of tumor growth after photoactivation of sensitizers is very short-lived singlet oxygen molecules that are produced and have limited diffusion distances. In this paper we show that the extent of biological damage can be modulated by using protoporphyrin, which was modified to increase its lipophilicity, and which also places the tetrapyrrole core deeper within the membrane by the carboxylate groups being anchored at the lipid:water interface. The uptake of the parent molecule (PPIX) and its diheptanoic acid analogue (PPIXC6) by WiDR and CT26 cells was investigated by fluorescence microscopy and by fluorescence intensity from the cells. The uptake of PPIXC6 increased almost linearly with incubation length for over 24 h, whereas for PPIX only 1 h was needed to reach maximal intracellular concentration. Fluorescence microscopy of both cell lines indicated that both drugs were distributed diffusely in the plasma membrane and cytoplasm, but remained outside the nucleus. The efficiency of in vitro inactivation of WiDr and CT26 cells increased with the length of the alkylcarboxylic chain. Tumors in mice that were treated with PPIX-PDT grew more slowly than control tumors. However, tumors that were given PPIXC6 followed by light exposure showed a significant delay in their growth.

Regulation of porphyrin synthesis and photodynamic therapy in heavy metal intoxication.

Protoporphyrin IX (PpIX) synthesis by malignant cells is successfully exploited for photodynamic therapy (PDT) following administration of 5-aminolevulinic acid (ALA) and light irradiation. The influence of two environmental heavy metal poisons, lead and gallium, on PpIX-synthesis and ALA-PDT was studied in two neu-ronal cell lines, SH-SY5Y neuroblastoma and PC12 pheochromocytoma. The heavy metal intoxication affected two of the heme-synthesis enzymes, ALA-dehydratase (ALAD) and porphobilinogen deaminase (PBGD). The present results show that lead poisoning significantly decreased the PBGD cellular level and inhibited its enzymatic activity, whereas the effects of gallium were less prominent. Although, the protein levels were reduced, the mRNA levels of PBGD remained unchanged during metal intoxication. These findings show additional inhibitory activity of lead on top of its classical effect on ALAD. Proteasome activity was enhanced during lead treatment, as measured by the AMC fluorigenic proteasome assay. The reduction in PBGD levels was not a consequence of PBGD mRNA reduced synthesis, which remained unchanged as shown by RT-PCR analysis. As a result of the lead poisoning, marked alterations in the cell cycle were observed, including a decreased G1 phase and an increased number of S phase cells. The efficacy of ALA-PDT was reduced in correlation with decreased activities of the enzymes during lead intoxication. We may conclude that lead poisoning adversely affects the outcome of ALA photodynamic therapy of cancer.

Bi-functional prodrugs of 5-aminolevulinic acid and butyric acid increase erythropoiesis in anemic mice in an erythropoietin-independent manner.

Anemia is a major cause of morbidity and mortality worldwide resulting from a wide variety of pathological conditions. In severe cases it is treated by blood transfusions or injection of erythroid stimulating agents, e.g., erythropoietin (Epo), which can be associated with serious adverse effects. Therefore, there is a need to develop new treatment modalities. We recently reported that treatment of erythroleukemic cells with the novel the bi-functional prodrugs of 5-aminolevulinic acid (ALA) and butyric acid (BA), AN233 and AN908, enhanced hemoglobin (Hb) synthesis to a substantially higher level than did ALA and BA individually or their mixture. Herein, we describe that these prodrugs when given orally to mice induced histone deacetylase inhibition in the kidneys, bone marrow and spleen, thus, indicating good penetrability to the tissues. In mice where anemia was chemically induced, treatment with the prodrugs increased the Hb, the number of red blood cells (RBCs) and the percentage of reticulocytes to normal levels. The prodrugs had no adverse effects even after repeated treatment at 100-200mg/kg for 50days. The lack of increased levels of Epo in the blood of mice that were treated with the prodrugs suggests that AN233 and AN908 affected the Hb and RBC levels in an Epo-independent manner. Taken together with our previous studies, we propose that the prodrugs increase globin expression by BA inhibition of histone deacetylase and elevation heme synthesis by ALA. These results support an Epo-independent approach for treating anemia with these prodrugs.

A porphobilinogen deaminase (PBGD) Ran-binding protein interaction is implicated in nuclear trafficking of PBGD in differentiating glioma cells.

Porphobilinogen deaminase (PBGD) is a rate-limiting enzyme of the heme biosynthesis pathway, whose level is elevated in various human tumors. PBGD was observed in both nuclear and cytoplasmic fractions of C6 glioma cells by immunostaining. During mitosis, chromatids were intensely stained for PBGD in comparison to the interphase chromatin. Using the yeast two-hybrid system, we identified RanBPM, the nuclear Ran-binding protein, as an interacting partner of PBGD. During butyrate-induced differentiation of C6, both nuclear and cytoplasmic PBGD levels declined as did Ran protein and its nucleotide exchange factor RCC1. N,N'-hexamethylene bis-acetamide-dependent differentiation resulted in an increase of the cytoplasmic PBGD, whereas nuclear PBGD, Ran protein and RCC1 remained unchanged. mRNA levels of PBGD remained unchanged during stimulation with both butyrate and N,N'-hexamethylene bis-acetamide. The enzymatic activity of PBGD and protoporphyrin IX synthesis in C6 cells were dependent on the differentiation induction agent. We conclude that PBGD possibly has a nuclear role in addition to its cytosolic enzymatic activity required for heme synthesis, which is related to cell transformation and differentiation.

Spectrally resolved microscopy of GFP trafficking.

Folding and chromophore cyclization-oxidation processes of green and cyan fluorescent fusion proteins (GFP and CFP) in subcellular microenvironments of transfected C6 glioma cells were studied by multipixel spectrally resolved microscopy (SRM). Discrete time-dependent spectral transitions were characterized during protein folding and chromophore maturation in the cytosol, nucleus, mitochondria, endoplasmic reticulum (ER), and Golgi. Spectral similarity mapping of fluorophore transition phases demarcated spatio-temporal fluorescence correlation at a subcellular level. Folding stages were characterized by a transition from red-shifted spectral populations in the time interval of 7-10 hr after transfection to a fully matured fluorophore emitting typical GFP or CFP fluorescence after 10-15 hr. The nascent protein revealed an initial focal accumulation in cytosol emitting in the range of 580-680 nm. After 10 hr, mixed pixel population spectra were measured and at 15 hr GFP was visualized in the cytoplasm by its specific spectral fingerprints with maxima at 545 nm. For nucleus- and mitochondrion-targeted CFPs, the mature conformer was discovered only in its final destination, whereas intermediate steps of fluorophore synthesis (at 10 hr) were found in the cytoplasm. Enhanced fluorescence maturation was manifested only by the ER-Golgi-targeted CFP after 10 hr post transfection by spectral imaging. Moreover, only remnants of initial intermediate fluorescent pixels were localized externally to the Golgi framework at 15 hr. SRM assessed the competence of ER-Golgi to maintain efficient CFP folding in comparison to the rest of the cellular compartments.

Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light.

Propionibacterium acnes is a Gram-positive, microaerophilic bacterium that causes skin wounds. It is known to naturally produce high amounts of intracellular porphyrins. The results of the present study confirm that the investigated strain of P. acnes is capable of producing endogenic porphyrins with no need for any trigger molecules. Extracts from growing cultures have demonstrated emission peaks around 612 nm when excited at 405 nm, which are characteristic for porphyrins. Endogenic porphyrins were determined and quantified after their extraction from the bacterial cells by fluorescence intensity and by elution retention time on high-performance liquid chromatography (HPLC). The porphyrins produced by P. acnes are mostly coproporphyrin, as shown by the HPLC elution patterns. Addition of delta-aminolevulinic acid (ALA) enhanced intracellular porphyrin synthesis and higher amounts of coproporphyrin have been found. Eradication of P. acnes by its endogenic porphyrins was examined after illumination with intense blue light at 407-420 nm. The viability of 24 h cultures grown anaerobically in liquid medium was reduced by less than two orders of magnitude when illuminated once with a light dose of 75 J cm(-2). Better photodynamic effects were obtained when cultures were illuminated twice or three times consecutively with a light dose of 75 J cm(-2) and an interval of 24 h between illuminations. The viability of the culture under these conditions decreased by four orders of magnitude after two illuminations and by five orders of magnitude after three illuminations. When ALA-triggered cultures were illuminated with intense blue light at a light dose of 75 J cm(-2) the viability of the treated cultures decreased by seven orders of magnitude. This decrease in viability can occur even after a single exposure of illumination for the indicated light intensity. X-ray microanalysis and transmission electron microscopy revealed structural damages to membranes in the illuminated P. acnes. Illumination of the endogenous coproporphyrin with blue light (407-420 nm) apparently plays a major role in P. acnes photoinactivation. A treatment protocol with a series of several illuminations or illumination after application of ALA may be suitable for curing acne. Treatment by both pathways may overcome the resistance of P. acnes to antibiotic treatment.

Regulation and gene expression of heme synthesis under heavy metal exposure--review.

Environmental pollution of heavy metals is very abundant nowadays from industry, chemicals, old paints, and pipes or resulting from previous contaminants accumulating in the food chain. Most of the iron demands of the body are needed for heme synthesis and assembly, but iron is also required for Fe-S cluster proteins and other redox enzymes. Heme is an essential, iron-binding molecule used as a prosthetic group of hemoproteins or as a regulator in multiple cellular pathways. In this review, we focused on the effect of exposure to heavy metals, such as Pb, Ga, Cu, Kd, Hg and Al, on heme synthesis as the main iron-sequestering process of the human body. These metals compete with iron on transporters, reduce the cellular iron pool and moreover, bind to proteins, and cause physical and mental disturbances. Heavy metals mainly impair various aspects of the heme synthesis pathway: gene expression, enzyme activity, and iron integration into protoporphyrin IX. Main risk factors are described as well as effects on iron dependent processes in order to increase public awareness to the distribution of heavy metals in our close environment and the harsh consequences of exposure, even in low doses.

Regulation of heme synthesis and proteasomal activity by copper: possible implications for Wilson's disease.

Wilson's disease (Wd) is a genetic disorder resulting in Cu2+ accumulation, and is caused by mutations in the ATP7B gene, the copper transporter. In vivo studies show a correlation between Cu2+ accumulation and malfunction of the heme biosynthesis pathway. In this study, we describe multiple effects of Cu2+ accumulation on heme synthesis, which, in turn, affect proteasomal activity. Cu2+ toxicity was examined in two hepatocellular carcinoma cell lines, HepG2 and Hep3B, with Hep3B cells containing an integrated hepatitis B virus genome. Exposure of HepG2 and Hep3B cells to Cu2+ inhibited the enzymes PBGD and ALAD of the heme synthesis pathway and, in parallel, upregulated heme oxygenase-1 (HO-1). Proto-porphyrin IX (PpIX) and the heme pool were reduced as a result of these processes. PpIX synthesis was found to be lower in cells expressing the mutant ATP7B (P1134P), compared to those expressing the WT enzyme. Proteasomal activity was inhibited under Cu2+ treatment in HepG2 cells; however, Cu2+ induced marked proteosomal acceleration in Hep3B cells. Under these conditions, Ub-conjugated proteins were gradually accumulated, whereas treatment with bathocuproine disulfonic acid (BCS), a Cu2+ chelator, reversed this effect. In conclusion, our data suggest that copper downregulates the heme synthesis pathway in hepatocellular cells and further reduces it in the presence of mutated ATP7B.

Novel multifunctional acyloxyalkyl ester prodrugs of 5-aminolevulinic acid display improved anticancer activity independent and dependent on photoactivation.

Multifunctional acyloxyalkyl ester prodrugs of 5-aminolevulinic acid in cancer cell lines inhibited the proteasome and induced apoptosis and heme synthesis. The most potent prodrug was butyryloxymethyl 5-amino-4-oxopentanoate (1a). The metabolically released formaldehyde from the prodrugs was the dominant factor affecting cell viability by a ROS-dependent mechanism and was responsible for rapid phosphorylation of H2AX, suppression of the cell survival protein c-myc, and transient elevation in the expression of p21. 1a, which differs from 2a by releasing butyric instead of pivalic acid, was a more potent inducer of heme and acetylated H4 expression and induced apoptosis through activation of caspase 9. 1a and 1b specifically increased the level of the photosensitizer protoporphyrin 9, leading to enhancement of cell death by photodynamic therapy (PDT). The advantage of these multifunctional prodrugs over 5-ALA is their greater potency in the non-PDT mechanism of cancer cell killing and their ability to also augment PDT.

In vivo and in vitro antitumor activity of butyroyloxymethyl-diethyl phosphate (AN-7), a histone deacetylase inhibitor, in human prostate cancer.

AN-7, a prodrug of butyric acid, induced histone hyperacetylation and differentiation and inhibited proliferation of human prostate 22Rv1 cancer cells in vitro and in vivo. In nude mice implanted with these cells, 50 mg/kg AN-7 given orally thrice a week led to inhibition of tumor growth and metastasis, tumor regression in >25% of animals and increased survival. Median time to the experimental end point (tumor volume 2 cm3 or death) in the untreated was 52 days, and average tumor volume was 0.8 +/- 0.18 cm3. At the same time, 94.4% of AN-7-treated mice survived and had average tumor volumes of 0.37 +/- 0.1 cm3. PSA expression was a useful marker for 22Rv1 lung metastasis detection. Sizeable metastases positively stained for PSA and limited air gaps were found in lungs of untreated mice. In animals treated with AN-7, lung morphology appeared normal. Primary tumors of treated animals were highly positive for PSA and had an elevated level of p21 and the proapoptotic protein Bax. Sections taken from AN-7-treated animals, examined under an electron microscope, exhibited condensed chromatin and apoptotic bodies. PSA serum levels were higher in untreated compared to treated animals and correlated with tumor volume. Since prolonged oral administration with 50 mg/kg or a single oral dose of 1.2 g/kg AN-7 did not cause adverse effects and the former exhibited significant anticancer activity, AN-7 is likely to display a high therapeutic index and may be beneficial for prostate cancer patients.