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Photochemically Generated Elemental Selenium Forms Conjugates with Serum Proteins That Are Preferentially Cytotoxic to Leukemia and Selected Solid Tumor Cells
Abstract
The objective of this study was to determine if and how photoproducts contribute to the antitumor effect of merocyanine-mediated PDT. A panel of barbituric, thiobarbituric and selenobarbituric acid analogues of Merocyanine 540 was photobleached, and the resulting photoproducts were characterized by absorption, fluorescence emission, mass, energy dispersive X-ray, and X-ray photoelectron spectroscopy and tested for cytotoxic activity against tumor cell lines and freshly explanted bone marrow cells. While all dyes were readily photobleached, only photoproducts of selone dyes showed cytotoxic activity. One-hour incubations with micromolar concentrations of selone-derived photoproducts were sufficient to reduce leukemia/lymphoma cells ≥10 000 fold, whereas preserving virtually all normal CD34-positive bone marrow cells. Of six multidrug-resistant tumor cell lines tested, five were as sensitive or more sensitive to photoproducts than the corresponding wild-type lines. Physicochemical characterizations of the cytotoxic activity indicated that it consisted of conjugates of subnano particles of elemental selenium and (lipo)proteins. The discovery of cytotoxic Se-protein conjugates provides a rare example of photoproducts contributing substantially to the antitumor effect of PDT and challenges the long-held view that Se in oxidation state zero is biologically inert. Agents modeled after our Se-protein conjugates may prove useful for the treatment of leukemia.
... An original approach to overcome drug resistance was utilized in a work that used selenobarbituric acid derivatives of merocyanine 540 (31 selected as representative example, Scheme 17) to be photobleached photochemically with a white fluorescent light, demonstrating a remarkable cytotoxicity towards 5 of the 6 drug-resistant cell lines evaluated (Daziano et al., 2012). Interestingly, the thiobarbituric and barbituric isosteres of these active selenobarbituric derivatives did not exhibit cytotoxicity. ...
... Interestingly, the thiobarbituric and barbituric isosteres of these active selenobarbituric derivatives did not exhibit cytotoxicity. The characterization of the photobleaching products revealed that this photochemical degradation formed within the subnanoparticles of elemental Se, which exerted their cytotoxicity through the formation of conjugates with proteins (Daziano et al., 2012). These compounds were synthesized according to a multi-step synthetic pathway described by Gunther et al., 1992. ...
Selenium is an essential trace element that is crucial for cellular antioxidant defence against reactive oxygen species (ROS). Recently, many selenium-containing compounds have exhibited a wide spectrum of biological activities that make them promising scaffolds in Medicinal Chemistry, and, in particular, in the search for novel compounds with anticancer activity. Similarly, certain tellurium-containing compounds have also exhibited substantial biological activities. Here we provide an overview of the biological activities of seleno- and tellurocompounds including chemopreventive activity, antioxidant or pro-oxidant activity, modulation of the inflammatory processes, induction of apoptosis, modulation of autophagy, inhibition of multidrug efflux pumps such as P-gp, inhibition of cancer metastasis, selective targeting of tumors and enhancement of the cytotoxic activity of chemotherapeutic drugs, as well as overcoming tumor drug resistance. A review of the chemistry of the most relevant seleno- or tellurocompounds with activity against resistant cancers is also presented, paying attention to the synthesis of these compounds and to the preparation of bioactive selenium or tellurium nanoparticles. Based on these data, the use of these seleno- and tellurocompounds is a promising approach in the development of strategies that can drive forward the search for novel therapies or adjuvants of current therapies against drug-resistant cancers.
... However, the SeNPs metabolism in human biological systems, the correlation with main selenoproteins, and their role in pharmacological protection should be studied more closely to understand their biological activities. Elemental selenium can form conjugate with proteins containing free thiol groups, which shows that elemental Se is not biologically inert and it may provide the basis of a new class of anticancer agents [104,105]. It would be interesting to find if selenium nanoparticles have a similar U-shape dose-depend association with a risk of DM. ...
Selenium is an important trace element for humans and animals as it plays a key role in several major metabolic pathways. Several studies were conducted to better understand the role of selenium against diabetes mellitus (DM), particularly type 2 (T2DM), but the obtained conclusions are contradictory. A simple linear relationship does not exist between the risk of T2DM and selenium levels but is best represented in a dose-dependent manner, getting often the U-graph. This relation also depends on selenium chemical forms that are present in a diet or supplements. Both too low and too high selenium intakes could increase the risk of diabetes. Moreover, the baseline status of Se should be taken into consideration to avoid over-supplementation. The focus of this brief overview is to report the recent updates concerning selenium participation in diabetes mellitus.
... Se(0) could then interact with suitable serum (lipo)proteins to generate cytotoxic conjugates. 60,61 2.7.2. Excretion. Two distinct pathways have been observed to be involved in the excretion of Se: the methylation pathway and the selenosugar pathway. ...
... However, the published data on photosensitizer distributions are limited for MC540 used in the blood tumors. Therefore, several researchers performed a basic inspection of the essence of MC540-mediated photodynamic damage for leukemia cells by examining the effects of MC540-sensitizer [47]. A recent study showed that the contribution of MC540 was useful for the treatment of leukemia. ...
This study aims to simulate a therapeutic plan for a normal human blood model under various patho-physiological conditions, such as the development of leukemia/blood diseases, by means of Monte Carlo multilayered simulation. The photosensitizing compound selectively accumulates in the target cells. A superficial treatment of a blood sample was performed at different ratios of oxygen saturation () under the concentration ( = 30 µM) effect of merocyanine 540 (MC540) in the blood irradiation. This was done under the application of visible light of wavelength ~ at an exposure time ~ 60 s. The dose of photodynamic therapy (PDT) was evaluated for the biological damage, leading to necrosis and blood damage during the treatment. In addition, the effect of PDT treatment response in the blood is related to hemoglobin oxygen saturation, resulting in an excellent relationship between the changes caused by the treatment in the blood at a peculiar oxygen saturation rate (for the highest response: 50%) and a light dose (LD) of 3.83 above the minimal toxicity of normal tissues. The photodynamic dose is related to the depth of necrosis and the time of treatment for the achievement of the LD delivery at the PDT of blood.
... As an example of this, studies on photoproducts of merocyanine analogues containing Se, such as selenomerocyanine dyes, showed potent cytotoxic activity against leukemia and other solid tumors, while being well tolerated by normal CD34-positive hematopoietic stem and progenitor cells (Sieber et al., 2005). This effect was reported to be the consequence of protein conjugate formation with serum albumin and lipoproteins by the elemental selenium generated during selenomerocyanine photobleaching, a response that was recently confirmed investigating other merocyanine analogues (Daziano et al., 2012). These conjugates are selectively cytotoxic to cancer cells in that these cells avidly take up serum albumin and lipoproteins, which thereby act as Trojan horses to deliver elemental selenium at cytotoxic levels to tumor cells. ...
Selenium (Se) is an essential micronutrient that functions as "redox gatekeeper" and homeostasis factor of normal and cancer cells. Epidemiology and experimental studies, in the last years suggested that both inorganic and organic forms of Se may have favorable health effects. In this regard, a protective action of Se on cellular systems that may help preventing cancer cell differentiation has been demonstrated, while the hypothesis that Se compounds may cure cancer and its metastatic diffusion appears speculative and is still a matter of investigation. Indeed, the overall actions of Se compounds in carcinogenesis are controversial. The recognition that cancer is a stem cell disease instigated major paradigm shifts in our basic understanding of cancer and attracted a great deal of interest. Although current treatment approaches in cancer are grounded in the need to kill the majority of cancer cells, targeting cancer stem cells (CSCs) may hold great potential in improving cancer treatment. In this respect, Se compounds have been demonstrated modulating numerous signaling pathways involved in CSC biology and these findings are now stimulating further research on optimal Se concentrations, most effective and cancer-specific Se compounds, and inherent pathways involved in redox and metabolic regulation of CSCs. In this review, we summarize the current knowledge about the effects of Se compounds on CSCs, by focusing on redox-dependent pathways and main gene regulation checkpoints that affect self-renewal, differentiation, and migration responses in this subpopulation of cancer cells.
... Protein binding of Se compounds also occurs at the extracellular level, and this may further help in developing selective drug delivery strategies for some tumors, such as leukaemia. Studies on photoproducts of merocyanine analogues containing Se, such as selenomerocyanine dyes, showed potent cytotoxic activity against leukemia and different solid tumors, while being well tolerated by normal CD34-positive hematopoietic stem and progenitor cells (Sieber et al., 2005); this effect was reported to be the consequence of protein conjugate formation by elemental selenium generated during selenomerocyanine photobleaching with serum albumin and lipoproteins, a response that was recently confirmed for other merocyanine analogues (Daziano et al., 2012). These conjugates are selectively cytotoxic to cancer cells because of selective uptake, which thereby act as Trojan horses serving to deliver elemental selenium at cytotoxic levels. ...
In vitro and in vivo experimental models clearly demonstrate the efficacy of Se compounds as anticancer agents, contingent upon chemical structures and concentrations of test molecules, as well as on the experimental model under investigation that together influence cellular availability of compounds, their molecular dynamics and mechanism of action. The latter includes direct and indirect redox effects on cellular targets by the activation and altered compartmentalization of molecular oxygen, and the interaction with protein thiols and Se proteins. As such, Se compounds interfere with the redox homeostasis and signaling of cancer cells to produce anticancer effects that include alterations in key regulatory elements of energy metabolism and cell cycle checkpoints that ultimately influence differentiation, proliferation, senescence, and death pathways.Cys-containing proteins and Se proteins involved in the response to Se compounds as sensors and transducers of anticancer signals, i.e., the pharmacoproteome of Se compounds, are described and include critical elements in the different phases of cancer onset and progression from initiation and escape of immune surveillance to tumor growth, angiogenesis, and metastasis. The efficacy and mode of action on these compounds vary depending on the inorganic and organic form of Se used as either supplement or pharmacological agent. In this regard, differences in experimental/clinical protocols provide options for either chemoprevention or therapy in different human cancers.
The diverse pharmacological activities of organoselenium compounds are closely correlated to their ability to scavenge and induce {AuQ: Edit OK?} reactive oxygen species (ROS), their intrinsic oxidative properties, and their Se(0) release property. The incorporation of selenium into small molecules, and particularly into heterocycles and natural products, has shown great potential in altering the potency and selectivity of these molecules. Therefore, selenium will play an important role in drug discovery in the near future. We summarize how different organoselenium species affect cellular oxidative stress levels, and try to correlate the structural properties of selenium-containing heterocycles and natural product derivatives to their biological activities and therapeutic applications. We also provide some information to guide the rational design of selenium-containing drugs.
Teaser: Incorporating selenium into heterocycles and natural products is an effective strategy in drug discovery because of the ability of selenium to fine tune the potency and selectivity of small molecules.
A series of novel thio- and seleno-barbituric acid derivatives were synthesized by varying the substituents at N1 and N3 (ethyl, methyl, allyl, and phenyl), and C5 tethered with dienyl and trienyl moieties attached to substituents such as phenyl, 2-furanyl, 2-thiophenyl, 1-naphthyl, and 3-pyridyl. The cytotoxic potential of these derivatives was evaluated by using MTT assay against melanoma cell lines expressing either wild-type (CHL-1) or mutant (UACC 903) BRAF gene. Among all, 2b and 8b were identified as the most potent compounds. Both 2b and 8b inhibited viability of various melanoma cells and induced cell death as evidenced by Live and Dead assay. Western blot analysis showed that they induce PARP cleavage and inhibit anti-apoptotic Bcl-2, Bcl-xL and Survivin in a dose-dependent manner within 24 h of the treatment. Novel thiobarbituric acid analogs also inhibited viability of various other solid tumor cell lines, such as pancreatic, breast, and colon. Overall, 2b, 2d, and 8b emerged as the most effective compounds and make good leads for the development of future therapeutic agents.
Objective: This study aims to evaluate the efficacy of carcinophotorin (PSD-007) photosensitization on the apoptosis-induced response in human breast cancer cells and analyze the mechanisms of PSD-007 involved in this process. Methods: Methyl thiazolyl tetrazolium (MTT) assay and in situ labeling were performed to examine the effects of the photodynamic therapy (PDT) on the proliferation and apoptosis of the cancer cells MDA-MB-231, respectively. Changes in cellular morphology were assessed using an optical microscope. Real-time polymerase chain reaction and Western blot analysis were conducted to clarify the underlying mechanisms. Results: The MTT assay revealed that at a concentration of 10 μg/mL and in combination with 9.0 J/cm2 laser radiation power 24 h after cell culture, PSD-007 markedly inhibited the proliferation of breast tumor cells, with an inhibition rate of 97.01%." Under a fluorescent microscope, apoptotic cells in the treatment groups with 5 and 10 μg/mL PSD-007-PDT were observed to have dramatically outnumbered the control groups. The dead cells after PSD-007-PDT mainly consisted of necrotic and late apoptotic cells. Caspase-3, caspase-8, P65, and P53 expression was upregulated in the treatment groups compared with the control group, with 5 and 10 μg/ml PSD-007 therapies, whereas no significant alteration in Bcl-2 and Bcl-x was found. Conclusion: PDT inhibits the proliferation and induces the apoptosis of cancer cells MDA-MB-231 by upregulating the caspase-3, caspase-8, P53, and NF-KB pathways, indicating a new strategy for treating breast cancer in the future.
The biological activity of selenium is dependent upon its speciation. We aim to integrate selenium speciation and metabolism into a discussion of the mechanisms by which selenium exerts its biological activity. First, we present the current status of selenium in the prevention of cancer, cardiovascular and neurodegenerative diseases with particular attention paid to the results of major chemoprevention trials involving selenium supplementation. A comprehensive review of the current understanding of the metabolism of common dietary selenium compounds - selenite, selenomethionine, methylselenocysteine and selenocystine - is presented, with discussion of the evidence for the various metabolic pathways and their products. The antioxidant, prooxidant and other mechanisms of the dietary selenium compounds have been linked to their disease prevention and treatment properties. The evidence for these various mechanisms - in vitro, in cells and in vivo - is evaluated with emphasis on the selenium metabolites involved. We conclude that dietary selenium compounds should be considered prodrugs, whose biological activity will depend on the activity of the various metabolic pathways in, and the redox status of, cells and tissues. These factors should be considered in future laboratory research and in selecting selenium compounds for trials of disease prevention and treatment by selenium supplementation.
Many Type II photosensitizers are substrates of the singlet oxygen they
generate. They are, therefore, converted to photoproducts when exposed
to light in the presence of oxygen. While most photoproducts appear to
be biologically inert, one of the photoproducts generated by
selenomerocyanine photosensitizers is a form of elemental selenium that
is surprisingly toxic to tumor cells if allowed to form conjugates with
serum albumin or lipoproteins. Albumin and lipoproteins act as delivery
vehicles for the cytotoxic entity, exploiting the fact that many tumor
cells have an increased capacity to bind and internalize albumin and/or
lipoproteins. The cytotoxic mechanism of Se(0)-protein conjugates is not
yet fully understood but is obviously quite different from the singlet
oxygen-mediated mechanism of merocyanine-mediated photodynamic therapy
(PDT). Whereas merocyanine-PDT is directed against the plasma membrane,
is more effective at 4 °C than at room temperature, and is inhibited
by excess albumin and lipoproteins, selenomerocyanine-derived cytotoxic
photoproducts act on intracellular targets, are ineffective at low
temperatures, and require albumin or lipoproteins as carrier molecules.
The discovery of cytotoxic Se(0)-protein conjugates provides a rare
example of photoproducts contributing substantially to the cytotoxic
effect of PDT and challenges the widely held notion that elemental
selenium is biologically inert.
IMPORTANCE IN THE FIELD: Activated cells metabolize albumin to cover their increased need for amino acids and energy. In inflamed, diseased and malignant tissue, extravasation of macromolecules into the tissue is upregulated. Drug carriers such as albumin have been used to target specifically diseased and malignant cells, resulting in higher efficacy of treatment and reduced side effects.
Owing to its advantageous biochemical and pharmacological properties, albumin has been regarded as an interesting candidate as a drug carrier. Covalent coupling to albumin carries drugs specifically to tumors and sites of inflammation, leading to reduced side effects as long as the native structure of albumin is unchanged. In this review, the means of coupling drugs to native albumin as well as exemplary studies for the use of albumin as drug carrier are summarized and discussed.
An overview of the state-of-the-art using albumin as drug carrier for specific accumulation in tumors and inflammatory cells using the advantageous properties of native albumin is given in this review.
Native albumin is an effective drug carrier to sites of inflammation or malignancy.
The amphipathic dye, merocyanine 540 (MC540), which preferentially photosensitizes enveloped viruses and virus-infected cells, is currently being evaluated in preclinical models as a blood sterilizing agent. In this communication, we report on an initial analysis of the site and nature of MC540-mediated photodynamic damages to human herpes simplex virus type 1 and human cytomegalovirus. The comigration of dye molecules and virions on a gel filtration column, the red-shift of the fluorescence emission spectrum of virus-containing fractions, and the distribution of MC540-treated virions in an aqueous two-phase partition system were indicative of MC540 binding to the enveloped viruses and localizing in a lipophilic environment (most likely the viral envelope). Fluorescence quenching and fluorescence resonance energy transfer experiments suggested that both dye monomers and dimers were capable of partitioning into the lipid bilayer of the viral envelope. Adsorption and penetration assays and immunohistochemical analyses of viral antigen expression showed that MC540-sensitized irradiation interfered with early phases of the infectious process, the adhesion to the host cell, the penetration of the host cell, and the translocation of the virus into the nucleus of the host cell. The inactivation of viruses was inhibited if oxygen in the medium was displaced by argon, enhanced if air was displaced by pure oxygen or if water was replaced by deuterium oxide. This suggested that the MC540-sensitized photoinactivation of enveloped viruses is an oxygen-dependent process and that singlet oxygen is one but not necessarily the only mediator of the antiviral effects of MC540.
We have established cis-diamminedichloroplatinum(II) (cisplatin) resistant human small cell lung cancer cell lines, H69/CDDP0.2 and H69/CDDP, to investigate the mechanism of acquired resistance to cisplatin. H69/CDDP0.2 and H69/CDDP were 6- and 11-fold resistant to cisplatin compared with the H69 parental cell line. H69/CDDP was also resistant to cadmium chloride (2-fold), cis-diammine(glycolato)platinum (4-fold), 4-hydroperoxycyclophosphamide (3-fold) and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosour ea (4-fold) if the drug concentrations that inhibit cell growth by 50% from growth inhibition assay were compared. There was no significant difference in the cisplatin accumulation among these cell lines. Although DNA interstrand cross-link formations, determined by filter elution assay in H69/CDDP0.2 and H69/CDDP, was decreased to 20 to 30% of that in H69 parental cells, the repair capacity of DNA interstrand cross-links was equivalent in all three cell lines. Intracellular glutathione content was equal in all cell lines. H69/CDDP had the highest glutathione S-transferase activity (H69, 11 nmol/min/mg protein, H69/CDDP0.2, 12 nmol/min/mg protein; H69/CDDP, 74 nmol/min/mg protein, respectively) and an overexpression of glutathione S-transferase pi mRNA. The drug concentrations that inhibit cell growth by 50% for cisplatin in all cell lines were decreased by treatment with ethacrynic acid, an inhibitor of glutathione S-transferase pi, but this did not alter the relative degree of resistance. Intracellular metallothionein content (H69, 14 pmol/mg protein, H69/CDDP0.2, 22 pmol/mg protein; H69/CDDP, 33 pmol/mg protein, respectively) and expression of metallothionein mRNA were correlated with the drug concentrations that inhibit cell growth by 50% of the three cell lines for cisplatin and cadmium chloride. The present study suggested the importance of metallothionein in the mechanisms of cisplatin resistance.
Albumin content has been reported to be significantly different in cytosols from benign and malignant breast tumors, with a higher level in benign lesions. Low albumin content is suggested to be associated with a higher tendency to axillary nodal involvement in breast cancer patients. Albumin contributes greatly to the total amount of protein in tumor cytosol, and is easily measured. Albumin was measured in cytosols from 382 patients with breast cancer stage I and II, to evaluate correlations to other tumor variables and to investigate whether it may add information as a prognostic factor. The albumin content was expressed in percentages of total cytosol protein, with a median value of 18.5% for the study population. It was found to be significantly inversely correlated to estrogen receptor (ER) content. Cytosol protein content was inversely correlated to albumin. In addition to tumor size and axillary nodal involvement, albumin content was found to be an independent prognostic factor for relapse-free survival in an analysis of different prognostic variables in patients not given adjuvant endocrine treatment. Low albumin content (less than median) seems to predict effect of adjuvant tamoxifen treatment.
The success of cancer chemotherapy is dependent on the possibility to utilize biological differences between malignant and normal cells to selectively destroy the tumor cells. One such difference may be that of receptor-mediated cellular uptake of low density lipoproteins (LDLs). Previous studies have shown that leukemic cells from patients with acute myelogenous leukemia have elevated receptor-mediated uptake and degradation rates of plasma LDL in vitro compared to normal white blood and bone marrow cells, and that plasma cholesterol levels at diagnosis are inversely correlated with the LDL receptor activity of the malignant cells. An important question is whether the uptake of LDL by the leukemic cells is also increased in vivo. To evaluate the in vivo uptake of LDL, 11 adult patients with newly diagnosed acute myelogenous leukemia received an i.v. injection of [14C]-sucrose-labeled LDL. On degradation of [14C]sucrose-LDL, the radiolabeled sucrose moiety is known to remain trapped in the lysosomal compartment of the cells. After injection, radioactivity accumulated progressively for at least 12 hr in the leukemic cells. The uptake of radioactivity in vivo correlated with the rate of receptor-mediated degradation of 125I-labeled LDL by the leukemic cells assayed in vitro (r = +0.88, P less than 0.001). An inverse correlation between plasma LDL cholesterol concentrations and the in vivo cellular uptake of [14C]sucrose-LDL in whole blood (r = -0.76, P less than 0.01) indicates that the hypocholesterolemia is due to elevated LDL uptake by the leukemic cells. Postmortem biopsies from virtually all tissues were obtained from one patient, and the distribution of radioactivity revealed that the liver and bone marrow had accumulated most radioactivity; the adrenals had the highest uptake of label per gram of tissue weight. The results indicate that LDL may be used as a carrier targeting lipophilic cytotoxic drugs to leukemic cells.
The photochemistry of merocyanine 540 (MC 540), a sensitizing dye that binds preferentially to leukemia and electrically excitable cells, has been investigated. MC 540-mediated photooxidation of histidine, arachidonate, and unsaturated phospholipid vesicles was assessed by spin label oximetry and shown to involve type II (singlet oxygen) chemistry. The dye was also shown to be a potent sensitizer of lipid peroxidation in a natural cell membrane, the erythrocyte ghost. Inhibition by azide, stimulation by 2H2O, and identification of the cholesterol product 5 alpha-cholest-6-ene-3 beta,5-diol in this system, all were consistent with singlet oxygen intermediacy. Finally, MC 540 was found to be considerably more phototoxic to K-562 leukemia cells in 2H2O than in H2O. We conclude that singlet oxygen plays a major role in the phototherapeutic effects of this dye.
To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.
Cells from three different human neuroblastoma cell lines and normal human bone marrow cells were exposed to the lipophilic fluorescent dye, merocyanine 540 (MC 540), and white light. In vitro clonogenic tumor cells were inactivated up to 25,000 times more rapidly than multipotent hematopoietic progenitor cells (CFU-GEMM). It is conceivable that this pronounced difference in sensitivity to MC 540-mediated photolysis can be exploited for the selective killing of residual neuroblastoma cells in autologous remission marrow grafts.
Murine L1210 leukemia cells resistant to the antineoplastic agent L-phenylalanine mustard have a 1.5-2.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of L-[U-14C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of gamma-glutamylcysteine, GSH, and GSSG is elevated 1.5-2.0-fold in cell-free preparations of the resistant tumor. This increased synthesis of GSH is attributed to increased cellular content (1.6-fold) of gamma-glutamylcysteine synthetase. GSH synthetase activity is equivalent in both drug-sensitive and -resistant cells. Investigation into the hydrolysis of selected peptides by cell-free preparations of both sensitive and resistant tumors suggest that aminopeptidase M participates in the formation of L-cysteine from L-Cys-Gly. This is supported by the observation that these preparations readily degrade L-Leu-p-nitroanilide and L-Ala-L-Ala-L-Ala, known substrates for aminopeptidase M, but not dipeptidase. The failure of the tumors to degrade Gly-D-Ala, a dipeptidase substrate, and the marked inhibition of L-Ala-Gly, L-Cys-Gly, and L-Ala-L-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L-cysteine from L-Cys-Gly. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both gamma-glutamyl transpeptidase-initiated catabolism of GSH to cysteine and its reutilization by gamma-glutamylcysteine synthetase.
Various chemical compounds have been described to induce photosensitization of tumor cells resulting in cell death. We studied the effect of merocyanine-540 (MC-540) on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) and common acute lymphoblastic leukemia antigen-positive non-T, non-B acute lymphoblastic leukemia (Reh) cell lines were incubated with MC-540 and simultaneously exposed to white light. Normal human BM and mixtures of leukemic cells with BM cells were treated under similar conditions. At constant illumination rates of 50,000 lx, significant (at least 4 to 5 logs) tumor cell destruction was obtained with MC-540 concentrations of 20 micrograms/ml or more for HL-60, and 10 micrograms/ml or more for Reh cells. Incubation of BM under equivalent conditions preserved 18.0% of granulocyte-macrophage colony-forming units and 14.2% of erythroid burst-forming units. Similar results were obtained when tumor cells were mixed with irradiated BM and then treated with MC-540. In summary, cell photosensitization with MC-540 has a selective cytotoxic effect towards leukemic cells and therefore may be useful for purging tumor cells from autologous BM.
In vitro incubation of leukemic bone marrow with the lipophilic fluorescent dye merocyanine 540 and simultaneous exposure to light reduced the concentration of L1210 leukemia cells by about 5 orders of magnitude but spared enough normal pluripotent hematopoietic stem cells to allow hematopoietic reconstitution of lethally irradiated syngeneic mice. This simple and rapid procedure may find an application in the purging of tumor cells from autologous bone marrow grafts.
Elemental selenium generated by the photobleaching of selenomerocyanine dyes forms conjugates with serum albumin and serum lipoproteins that are toxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic stem and progenitor cells. Serum albumin and lipoproteins act as Trojan horses that deliver the cytotoxic entity (elemental selenium) to tumor cells as part of a physiological process. They exploit the fact that many tumors have an increased demand for albumin and/or low-density lipoprotein. Se(0)-protein conjugates are more toxic than selenium dioxide, sodium selenite, selenomethionine, or selenocystine. They are only minimally affected by drug resistance mechanism, and they potentiate the cytotoxic effect of ionizing radiation and several standard chemotherapeutic agents. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood. Currently available data are consistent with the notion that Se(0)-protein conjugates act as air oxidation catalysts that cause a rapid depletion of intracellular glutathione and induce apoptosis. Drugs modeled after our Se(0)-protein conjugates may prove useful for the local and/or systemic therapy of cancer.
Methotrexate covalently bound to human serum albumin in a 1:1 molar ratio (MTX‐HSA) is a new macromolecular drug which is currently being studied in phase I clinical trials by the German Association for Medical Oncology (AIO) Phase I/II study group. Previous studies have shown that MTX‐HSA differs favorably from unbound MTX in terms of plasma half‐life time, tumor accumulation of albumin and uptake mechanisms into cancer cells. To achieve optimal drug efficacy, repeated treatment cycles were necessary. To evaluate the anti‐tumor activity of MTX‐HSA and MTX in pre‐clinical in vivo models, we selected 7 solid human tumor xenografts growing s.c. in nude mice and administered drug either i.p. or i.v. weekly for 3 weeks. The maximal tolerated dose (MTD) of MTX‐HSA in nude mice was 12.5 mg/kg given i.p. on days 1, 8 and 15, whereas the MTD for free MTX was 100 mg/kg given i.v. MTX‐HSA was significantly more active (p > 0.01) than MTX in 3 models. In the soft tissue sarcoma SXF 1301, MTX‐HSA effected complete remission/cure after a single injection, whereas free MTX resulted in short‐lasting, partial tumor regression. In the prostate‐cancer model PRXF PC3M, MTX‐HSA produced growth inhibition of 92.8% of control or an optimal test/control (T/C) of 7.2% compared to a T/C of 20.8% for MTX (p = 0.05). In the osteosarcoma model SXF 1410, optimal T/C values were 10.2% and 14.5%, respectively (p = 0.025). In lung cancers LXFE 409 and LXFL 529, bladder cancer BXF 1258 and breast cancer MAXF 449, both compounds were inactive. The improved therapeutic effects seen in 3 xenograft models under MTX‐HSA treatment are promising and might be due to specific accumulation of the compound in solid tumors owing to their enhanced permeability and retention effect. Thus, clinical development of MTX‐HSA will continue and sarcomas as well as prostate cancers will be included as potential target tumors for upcoming clinical phase II trials. © 2001 Wiley‐Liss, Inc.
Cells from three different human neuroblastoma cell lines and normal human bone marrow cells were exposed to the lipophilic fluorescent dye, merocyanine 540 (MC 540), and white light. In vitro clonogenic tumor cells were inactivated up to 25,000 times more rapidly than multipotent hematopoietic progenitor cells (CFU-GEMM). It is conceivable that this pronounced difference in sensitivity to MC 540-mediated photolysis can be exploited for the selective killing of residual neuroblastoma cells in autologous remission marrow grafts.
The amphipathic dye, merocyanine 540 (MC540), which preferentially photosensitizes enveloped viruses and virus-infected cells, is currently being evaluated in preclinical models as a blood sterilizing agent. In this communication, we report on an initial analysis of the site and nature of MC540-mediated photodynamic damages to human herpes simplex virus type 1 and human cytomegalovirus. The comigration of dye molecules and virions on a gel filtration column, the red-shift of the fluorescence emission spectrum of virus-containing fractions, and the distribution of MC540-treated virions in an aqueous two-phase partition system were indicative of MC540 binding to the enveloped viruses and localizing in a lipophilic environment (most likely the viral envelope). Fluorescence quenching and fluorescence resonance energy transfer experiments suggested that both dye monomers and dimers were capable of partitioning into the lipid bilayer of the viral envelope. Adsorption and penetration assays and immunohistochemical analyses of viral antigen expression showed that MC540-sensitized irradiation interfered with early phases of the infectious process, the adhesion to the host cell, the penetration of the host cell, and the translocation of the virus into the nucleus of the host cell. The inactivation of viruses was inhibited if oxygen in the medium was displaced by argon, enhanced if air was displaced by pure oxygen or if water was replaced by deuterium oxide. This suggested that the MC540-sensitized photoinactivation of enveloped viruses is an oxygen-dependent process and that singlet oxygen is one but not necessarily the only mediator of the antiviral effects of MC540.
To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6- contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.
Structural analogs of Merocyanine 540 (MC540) were synthesized to probe biological mechanisms of action and to enhance viral inactivation. Novel pyrimidine-2- selone analogs of known and modified merocyanine sensitizers are described. A surprising result of placing selenium at the barbiturate site was the 100-fold enhancement of photogeneration efficiency for singlet oxygen (1O2). Conversely, selenium directly attached to the chromophore π-system was much less efficacious. The production of more 1O2 is reflected in greatly improved biological effectiveness of the new dyes. A puzzling high to low bioactivity isomer effect among molecules containing naphthoxazole or naphthothiazole components is shown by both thione and selone dyes.
Fifteen unsymmetrical alkyl methyl and alkyl phenyl diselenides were prepared by reacting (1) symmetrical dialkyl diselenides with benzeneselenol, (2) benzeneselenenyl chloride with an alkaneselenol, (3) dimethyl diselenide with an alkaneselenol, (4) lithium alkylselenenylselenolate with an alkyl iodide, and (5) alkaneselenenyl halide with sodium alkaneselenolate. The easily prepared unsymmetrical diselenides are thermally labile species undergoing disproportionation to the symmetrical diselenides upon distillation with the single exception of ethyl methyl diselenide that distills as a mixture, thermally stable enough to fully characterize with GC/mass spectroscopy. Phenyl isopropyl diselenide, the only compound to display evidence of dynamical chemical exchange in the NMR spectra (13C and 77Se), was investigated by a 77Se DANTE selective magnetization transfer experiment. Chemical exchange was suggested to occur via scission of selenium-selenium bonds and not carbon-selenium bonds and was found to stop (or slow extensively) upon subsequent aqueous workup. The 77Se chemical shifts, spanning a range of 406 ppm, were found to correlate well with the number of α minus β protons, suggesting additivity of the 77Se chemical shifts similar to that of 13C chemical shifts, β and γ 77Se chemical shift additivity parameters were determined for α-methyl substitution in alkyl methyl diselenides. Correction factors for branching at the remote selenium were found necessary to compute the 77Se chemical shifts in contrast with 13C chemical shifts that only require correction factors for adjacent branching. The one-bond 77Se-77Se spin-spin coupling constant 1JSeSe varies in magnitude from 36.3 Hz for n-butyl methyl diselenide to ±2.7 Hz for tert-butyl methyl diselenide. A linear correlation of 1JSeSe with the sum of the α minus β protons for both substituents tentatively suggests that a change in sign of the coupling constant may occur for tert-butyl methyl diselenide and with phenyl isopropyl and tert-butyl phenyl diselenide. A new preparation of di-tert-butyl selenide and di-tert-butyl diselenide is described along with their characterization.
Abstract— Merocyanine dyes with an oxygen in the electron donor heterocycle were rapidly degraded by plasma, serum and serum components. Replacement of the oxygen by a sulfur or selenium atom rendered the dyes refractory to degradation. The degradation of labile merocyanine dyes was temperature dependent and oxygen independent. The plasma component that was responsible for the degradation of merocyanine dyes was sensitive to heat and detergent, suggesting an enzymatic process. The identification of the structural requirements for sensitivity/resistance to degradation provides the experimenter with a simple means to manipulate the stability of mer-ocyanines in high serum or plasma environments and may expand the clinical utility of merocyanine photosen-sitizers beyond their traditional role in the extracorporeal purging of bone marrow grafts.
Purpose:
The non-ionic detergent Tween-80, a surface-active agent, has been shown to modulate the cytocidal effect of certain antitumor agents. In the present study, we sought to determine whether or not Tween-80 could enhance the antitumor effect of etoposide (VP16) in human lung cancer cells in vitro.
Methods:
Survival fractions were measured by growth inhibiton assays of PC14, H69, KB, and PC14/CDDP (the corresponding cisplatin-resistant subline of PC14) cells. An in vitro clonogenic assay of PC14 and PC14/CDDP cells was undertaken after incubation for 10-12 days in RPMI-1640 medium with 20% fetal calf serum and 1.72% methyl cellulose, plus continuous exposure to VP16 with Tween-80. We also investigated the direct toxicity of Tween-80 to PC14 and PC14/CDDP cells using a clonal assay. The intracellular accumulation of VP16 was further analyzed using [3H]VP16 in PC14, PC14/CDDP, A549, KB and H69 cells, and compared with that of daunorubicin (DNR), a hydrophilic anti-cancer agent, using [3H]DNR in PC14, A549 and KB cells.
Results:
It was found that PC14/CDDP had collateral sensitivity to VP16 and Tween-80 markedly enhanced the killing effect of VP16 not only of PC14 cells but also of PC14/CDDP cells while exerting little cytotoxic effect. Moreover, Tween-80 increased the intracellular accumulation of VP16 in PC14, PC14/CDDP and A549 cells, and not in KB and H69 cells. Tween-80 did not increase the intracellular DNR levels in PC14, A549 and KB cells.
Conclusions:
Tween-80 was shown to potentiate the cytotoxicity of VP16 against several human lung adenocarcinoma cells by increasing the accumulation of VP16 in vitro. Tween-80-mediated sensitization of lung adenocarcinoma cells to VP16 is considered to be related to both the characteristics of the cell membrane in adenocarcinoma cells and the lipotropic properties of VP16. These results suggest that this combination might have the potential to improve the therapeutic index of VP16 in human lung adenocarcinoma.
Action spectra of the antileukemic and antiviral activities of merocyanine 540 (MC540) were determined using L1210 leukemia cells and human Herpes simplex virus type 1. The major peak of both action spectra aligned closely with the absorption spectrum of membrane-bound dye monomer, and by implication, the action spectrum of 1O2 generation. These results are compatible with the notion that the antileukemic and antiviral activities of MC540 are primarily attributable to membrane-bound monomer and at least in part mediated by 1O2.
Dry or wet heat, solvents, and detergents combined with ultraviolet irradiation provide effective means of sterilizing soluble blood products such as albumin or factor VIII. For obvious reasons, these procedures are not applicable to cellular blood components. We have recently shown that simultaneous exposure to the photosensitizer, merocyanine 540 (MC 540) and white light rapidly inactivates the Friend erythroleukemia virus complex and Friend virus-transformed cells, but causes relatively little damage to pluripotent hematopoietic stem cells. In this communication, we show that several lipid-enveloped human pathogenic viruses are also highly susceptible to MC 540-sensitized photoirradiation, and we report on an initial evaluation of the ability of MC 540-sensitized photoirradiation to sterilize blood products.
Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. In this paper we present evidence that the MC 540-sensitized photoinactivation of leukemia cells is an oxygen-dependent process and that unsaturated plasma membrane lipids are substrates for singlet oxygen and/or other activated oxygen species generated by photoirradiated MC 540. A comparison of the inhibition of clonal growth, the inhibition of mitochondrial respiration, and the exclusion of trypan blue by the plasma membrane after exposure to MC 540 and graded doses of light showed that mitochondrial respiration is compromised relatively early in the course of the dye-mediated photoinactivation of cells, well before the plasma membrane loses its capacity to exclude trypan blue. It also showed that trypan blue exclusion assays can greatly underestimate the cytotoxic effects of MC 540-sensitized photoirradiation.
The efficacy of photosensitization by merocyanine 540 (MC540), a lipophilic fluorescent dye, was investigated in the murine B cell leukemia (BCL1). Normal BALB/c mice were injected with BCL1 cells exposed to MC540, followed by photosensitization with white light for 15 min to 2 h. Mice injected with BCL1 cells exposed for 1 or 2 h showed no sign of leukemia. Lethally irradiated mice were successfully reconstituted with mixtures of syngeneic bone marrow (BM) and BCL1 cells treated with MC540 following exposure to white light. Exposure of BM/BCL1 mixtures for 2 h proved to be effective in purging all BCL1 cells without affecting BM viability, as documented by normal hemopoietic reconstitution of all recipients surviving without evidence of leukemia. All recipients of untreated BM/BCL1 cell mixtures developed leukemia within 42 days. Adoptive transfer of 10(6) spleen cells obtained from treated mice into secondary naive syngeneic recipients was carried out in order to test for dormant BCL1 cells in treated recipients. No leukemia developed in any of the secondary recipients. Previous studies indicate that as few as 10, or possibly less, BCL1 cells are sufficient to cause lethal disease in BALB/c recipients. Our results suggest that MC540 may be an extremely potent tool for in vitro elimination of residual tumor cells while leaving uncommitted progenitor hemopoietic cells intact for hemopoietic reconstitution following lethal marrow ablation.
The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.
The purpose of this study was to determine the sensitivity to merocyanine 540 (MC 540)-mediated photolysis of normal human hematopoietic progenitor cells and four leukemia cell lines (Daudi, Raji, K562 and HL-60). Late erythroid progenitors were the most sensitive normal cells. Early erythroid progenitors were of intermediate sensitivity. Granulocyte/macrophage progenitors and multipotent progenitors were the least sensitive normal marrow cells. A combination of dye concentration, serum concentration, and illumination that eliminated 50% of multipotent progenitor cells reduced the concentration of leukemic cells by greater than or equal to 4.5 log. It is conceivable that this difference in photosensitivity can be exploited for the extracorporeal purging of autologous remission marrow grafts.
HL60 cells isolated for resistance to Adriamycin (HL60/ADR) overexpress a 190-kDa ATP binding protein which has a minor sequence homology with P-glycoprotein. It has also been observed that HL60/ADR overexpress the MRP gene which was first identified as a component of a non-P-glycoprotein mediated multidrug resistance of H69/ADR cells [Cole et al., Science (Washington DC), 258: 1650, 1992]. A complementary DNA of MRP has been cloned and based on the deduced sequence encodes a member of the superfamily of proteins which bind ATP and function in various transport processes [Cole et al., Science (Washington DC), 258: 1650, 1992]. In view of this it was of interest to identify the protein encoded by MRP and determine if it may be related to p190. In the present study we have prepared antisera against three synthetic peptides which correspond to the deduced sequence of the MRP protein. Proteins reactive with the antisera have been examined in HL60/ADR cells using Western blot analysis. All antisera react with a 190 kDa protein contained in membranes of resistant but not sensitive cells. One antiserum used for further studies is not reactive with P-glycoprotein contained in membranes of HL60 cells isolated for resistance to vincristine. Analysis of subcellular fractions demonstrates that p190 is present primarily in the endoplasmic reticulum with lower levels also present in plasma membranes. Treatment of HL60/ADR cells with tunicamycin results in the appearance of a 165-kDa resistance associated protein which reacts with the antipeptide serum. The results of this study therefore demonstrate that the MRP gene encodes a 190-kDa membrane bound glycoprotein.
Since there is strong evidence of a preferential LDL accumulation in tumor cells, LDL might be of interest for tumor imaging. We have tested the ability of 99mTc-LDL in tumor imaging with B16-melanoma-bearing mice as a model for further applications in human studies. The LDL fixation rate was higher with 99mTc-labeled LDL than with 125I labeled LDL. Since technetium-99m remains trapped in the cells, 99mTc-LDL is a well-adapted radioligand because of information given by this radiotracer on the receptor metabolism. We observed that, at early growth stages, the tumor took up the LDL at a maximal rate, suggesting differences in cholesterol metabolism as a function of tumor growth. Accumulation of label in the tumor area was perfectly observable in tumor-bearing mice on scintigraphic images. Computerized quantification of the regions of interest (as well as biodistribution studies including killing of the animals) showed a 1.81-fold increase in uptake by the tumor as compared to the liver and a 28-fold increase as compared with corresponding normal tissue (muscle of the left leg) at day 8 of tumor growth. These data give strong support to the value of this non-invasive method in visualizing and quantifying the tissue LDL uptake in vivo, including the precise information provided by nuclear scintigraphy on the distribution of the radiolabeled LDL in the different tissues. 99mTc-LDL could be an efficient tool for further diagnostic or therapeutic exploration in cancer patients.
The molecular basis of the differential sensitivity of normal hematopoietic stem cells and of leukemia, lymphoma, and neuroblastoma cells to merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) is not yet completely understood. While the capacity to bind dye molecules appears to be the major determinant of a cell's susceptibility of MC540-mediated PDT, we here present evidence that under certain experimental conditions a cell's capacity to repair MC540-mediated photodynamic damage is also an important factor. Two parameters, temperature and intracellular glutathione (GSH) content, were varied to investigate the role of cellular defense mechanisms in the dye-sensitized photoinactivation of normal murine granulocyte/macrophage progenitors (CFU-GM) and K562, L1210, and melphalan-resistant L1210/L-PAM1 leukemia cells. When exposed to MC540 and light at room temperature, the three leukemia cell lines bound similar amounts of dye and accumulated similar amounts of lipid hydroperoxide (LOOH) but differed markedly in their sensitivity to MC540-mediated PDT. Performing MC540-mediated PDT at 4 degrees C instead of at room temperature reduced dye binding and LOOH generation and enhanced cytotoxicity in some but not all cell lines. A brief (< or = 120 minutes) incubation at 37 degrees C immediately following MC540-mediated PDT accelerated the decay of LOOH in all leukemic cell lines and reduced cell kill by about 2 log in both CFU-GM and leukemia cells. The effect of post-PDT incubation at 37 degrees C on LOOH decay was most pronounced in K562 and least pronounced in L1210/L-PAM1 cells, whereas its effect on cell survival was less pronounced in L1210 cells than in the remaining cell types. L1210/L-PAM1 cells whose GSH content had been reduced from 8.2 to 1.6 micrograms/mg protein by incubation with buthionine sulfoximine recovered from potentially lethal photodynamic damage as rapidly as untreated L1210/L-PAM1 cells and more rapidly than wild-type L1210 cells with a GSH content of 4.5 micrograms/mg protein. Thus, with regard to capacity of L1210/L-PAM1 cells to recover from photodynamic damage, the cells' enhanced capacity to synthesize GSH appeared more decisive than intracellular GSH levels per se. Taken together, these data suggest that temperature-dependent cellular defense mechanisms are significant determinants of a cell's susceptibility to MC540-mediated PDT. The data emphasize the need for temperature control during and immediately after the photochemical purging of autologous bone marrow or peripheral blood stem cells.
We have recently reported that albumin accumulates in solid tumors and serves there as a source of nitrogen and energy. Methotrexate-albumin conjugates [MTX(I)-RSA] derivatized at a molar ratio of 1:1 differ favorably from original MTX in terms of plasma presence and tumor uptake. The purpose of this study was to evaluate the therapeutic efficacy of these novel conjugates in a comparative study with low m.w. MTX is Sprague-Dawley rats bearing a Walker-256 carcinoma. The maximum tolerated dose (MTD) for MTX and MTX(I)-RSA was determined (2 mg/kg based on MTX injected on days 1, 3 and 8). The tumor-bearing rats received injections of either the MTD or MTD/2 of MTX, MTX-albumin or mixtures containing the MTD/2 or MTD/4 of both MTX and MTX-albumin. No toxic side effects were observed. Cure rate and tumor growth retardation were slightly better for the conjugate compared with MTX alone in the MTD group (16 complete remissions vs. 14 of 20 rats). The best results were achieved for the combination treatment with MTX and MTX-albumin, with complete remission in all 20 rats. In conclusion, MTX-albumin conjugates show therapeutic activity in vivo without toxic side effects. Additive effects were observed for a combination of MTX-albumin and MTX. These effects might be caused by the much longer tumor exposition time of the conjugate in conjunction with a different route of uptake (pinocytosis for MTX-albumin vs. folate receptors for MTX).
Based on the rationale of a preferred albumin uptake by tumors, conjugates comprising of rat serum albumin (RSA) as a drug carrier and of methotrexate (MTX) as chemotherapeutic drug were prepared. For a comparative study of MTX-RSA and MTX we chose a slow growing Dunning R3327 HI prostate cancer model. In a radiopharmacologic study blood kinetics and the tumor and organ distribution pattern of residualizingly labeled MTX-RSA were determined, and were found to be similar to that of residualizingly labeled RSA. The MTD was established for Copenhagen rats at a total four injections of 2 mg/kg MTX or MTX-RSA administered at days 0, 4, 8 and 12. Tumor volume measurements and tumor removal showed a small non-significant growth delay in the MTX treatment group, suggesting MTX resistance for the Dunning R3327 HI prostate carcinoma. In contrast, treatment with MTX-RSA resulted in a significant (50%) growth inhibition of the Dunning R3327 HI tumor. The cellular mechanisms responsible for MTX resistance in Dunning HI tumor cells is not known. The improved therapeutic effects seen during MTX-RSA treatment in this slow growing adenocarcinoma might be a result of prolonged tumor exposure time and an altered cellular uptake by a lysosomal route. MTX-albumin conjugates have shown antitumor activity exceeding that of MTX in several tumor xenografts in nude mice, including human prostate cancer. The recently initiated clinical development of MTX-human serum albumin will be continued and cancer of the prostate will be included as a potential target tumor during further clinical phase II testing.
To delineate the tumor margins of malignant gliomas laser-induced fluorescence detection technique was applied using 5-aminofluorescein-albumin as the fluorescent dye. The 5-aminofluorescein was linked to serum albumin (= AFlc-SA) as a cumulative protein label using residualizing markers. In a C6-glioma model the biodistribution and pharmacokinetics of the injected dye were investigated by labeling the protein conjugate with 111In-DTPA. Twenty-four hours after intravenous injection of the dye, fluorescence was activated by an argon laser and inspected in the C6-gliomas. Histological examinations were performed to compare the microscopic margins of the fluorescence-stained tumors with hematoxylin/eosin. The tumor uptake 24 h after dye injection was 23-fold higher than in the surrounding brain. Fluorescence inspection under laser activation demonstrated clearly stained and sharply demarcated tumors. The microscopic borders of the tumors corresponded exactly with the fluorescence, also demonstrating intracellular tumor uptake of the dye. In a preliminary study, three patients with malignant gliomas were operated using laser-induced fluorescence detection technique after injection of AFlc-SA. In all patients, the borders of the malignant gliomas were clearly stained by AFlc-SA during surgery. Laser-induced fluorescence imaging using the albumin conjugate AFlc-SA may be a promising method for delineating tumor margins which are hard to detect under the operating microscope alone.
Methotrexate covalently bound to human serum albumin in a 1:1 molar ratio (MTX-HSA) is a new macromolecular drug which is currently being studied in phase I clinical trials by the German Association for Medical Oncology (AIO) Phase I/II study group. Previous studies have shown that MTX-HSA differs favorably from unbound MTX in terms of plasma half-life time, tumor accumulation of albumin and uptake mechanisms into cancer cells. To achieve optimal drug efficacy, repeated treatment cycles were necessary. To evaluate the anti-tumor activity of MTX-HSA and MTX in pre-clinical in vivo models, we selected 7 solid human tumor xenografts growing s.c. in nude mice and administered drug either i.p. or i.v. weekly for 3 weeks. The maximal tolerated dose (MTD) of MTX-HSA in nude mice was 12.5 mg/kg given i.p. on days 1, 8 and 15, whereas the MTD for free MTX was 100 mg/kg given i.v. MTX-HSA was significantly more active (p > 0.01) than MTX in 3 models. In the soft tissue sarcoma SXF 1301, MTX-HSA effected complete remission/cure after a single injection, whereas free MTX resulted in short-lasting, partial tumor regression. In the prostate-cancer model PRXF PC3M, MTX-HSA produced growth inhibition of 92.8% of control or an optimal test/control (T/C) of 7.2% compared to a T/C of 20.8% for MTX (p = 0.05). In the osteosarcoma model SXF 1410, optimal T/C values were 10.2% and 14.5%, respectively (p = 0.025). In lung cancers LXFE 409 and LXFL 529, bladder cancer BXF 1258 and breast cancer MAXF 449, both compounds were inactive. The improved therapeutic effects seen in 3 xenograft models under MTX-HSA treatment are promising and might be due to specific accumulation of the compound in solid tumors owing to their enhanced permeability and retention effect. Thus, clinical development of MTX-HSA will continue and sarcomas as well as prostate cancers will be included as potential target tumors for upcoming clinical phase II trials.
Renal cell carcinoma (RCC) has a poor prognosis when metastasized to distant sites, although immunotherapy may offer a prolongation of survival in selected patient groups. Unfortunately, no treatment options remain when immunotherapy fails. In this phase IIa trial the tolerability and efficacy of the antifolate drug methotrexate-human serum albumin (MTX-HSA) were evaluated in patients with metastatic RCC who progressed after first-line immunotherapy.
A total of 17 patients started treatment, and 14 (12 men, 2 women) were evaluable for response according to the phase IIa Gehan design. Patients had had prior tumor nephrectomy, were in relatively good general condition, had no impairment of renal, liver or bone marrow function, and had progressive metastatic disease after treatment with interferon-alpha (IFN-alpha) with or without cis-retinoic acid (EORTC protocols 30951 and 30947). MTX-HSA was given once a week intravenously on an outpatient basis at a dose of 50 mg/m(2). The treatment interval was prolonged in those patients who had not yet recovered from previous toxicities.
Toxicity was manageable, relatively mild to moderate and reversible in most cases. Grade 2/3 mucositis (10/17) and grade 3 elevated transaminase levels (4/17) were most frequent, and in only one patient was a grade 4 thrombocytopenia reported. Of three inevaluable patients, one discontinued treatment due to drug-related toxicities. The mean administration interval was 12.1 days, and 7 of 14 evaluable patients had treatment intervals of 1 or 2 weeks. No objective responses were seen, although eight patients had stable disease (stabilization >2 months) for up to 8 months (median 121 days).
MTX-HSA was generally well tolerated and can be given on an outpatient basis, but no objective responses were seen in patients with metastatic RCC who had progressed after previous immunotherapy.
Paediatric solid tumours exhibit steep dose-response curves to alkylating agents and are therefore considered candidates for high-dose chemotherapy and autologous stem cell support. There is growing evidence that autologous stem cell grafts from patients with solid tumours are frequently contaminated with live tumour cells. The objective of this study was to perform, in a preclinical purging model, an initial assessment of the safety and efficacy of a two-step purging procedure that combined Merocyanine 540-mediated photodynamic therapy (MC540-PDT) with a brief exposure to the alkyl-lysophospholipid, Edelfosine. Human and murine bone marrow cells and Neuro-2a murine neuroblastoma, SK-N-SH human neuroblastoma, SK-ES-1 and U-2 OS human osteosarcoma, G-401 and SK-NEP-1 human Wilms' tumour, and A-204 human rhabdomyosarcoma cells were exposed to a fixed dose of MC540-PDT followed by a brief incubation with graded concentrations of Edelfosine. Survival was subsequently assessed by in vitro clonal assay or, in the case of CD34-positive haematopoietic stem cells, by an immunohistochemical method. Combination purging with MC540-PDT and Edelfosine depleted all tumour cells by >4 log while preserving at least 15% of murine granulocyte/macrophage progenitors (CFU-GM), 34% of human CFU-GM, and 31% of human CD34-positive cells. The data suggest that combination purging with MC540-PDT and Edelfosine may be useful for the ex vivo purging of autologous stem cell grafts from patients with paediatric solid tumours.
The purpose of this study was to determine in a preclinical purging model, how effective crystal violet-mediated photodynamic therapy (CV-PDT) is against solid tumor and drug-resistant mutant tumor cells, and if certain limitations of CV-PDT can be overcome by using crystal violet (CV) in combination with the membrane-active photosensitizer, Merocyanine 540 (MC540). When used under conditions that preserved an adequate fraction of normal human granulocyte/macrophage progenitors (CFU-GM), CV-PDT failed to achieve meaningful reductions of DU145 prostate, H69 small cell lung cancer, and MDA-MB-435S breast cancer cells. Melphalan-resistant L1210/L-PAM1, adriamycin-resistant P388/ADR, and adriamycin-resistant HL-60/ADR leukemia cells were markedly less sensitive to CV-PDT than their wild-type counterparts, whereas cisplatin-resistant H69/CDDP cells were more sensitive than wild-type H69 cells. Sequential exposure to MC540- and CV-PDT under conditions that preserved an adequate fraction (73% and 29%, respectively) of normal CD34-positive hematopoietic stem cells and granulocyte/macrophage progenitors was highly effective against H69 (99.997% reduction) and H69/CDDP (99.999% reduction) cells, but ineffective against HL-60/ADR, MDA-MB-435S, and DU145 cells. CV thus shows only limited promise as a single-modality purging agent. However, in certain situations, clinically meaningful tumor cell depletions can be obtained by using CV in combination with a second photosensitizer such as MC540.
In this study, we showed that smaller size particles of Nano-Se have better scavenging effects on the following free radicals: carbon-centered free radicals (R*) generated from 2,2'-azo-bis-(2-amidinopropane) hydrochloride (AAPH), the relatively stable free radical 1,1-diphenyl-2-picryhydrazyl (DPPH), the superoxide anion (O2*-) generated from the xanthine/xanthine oxidase (X/XO) system, singlet oxygen (1O2) generated by irradiated hemoporphyrin. Furthermore, the three sizes of Nano-Se studied also show protective effects against the oxidation of DNA. The three samples all have potential size-dependent characteristics on scavenging the free radicals. Although in this study we regarded Nano-Se as a whole without considering interactions between BSA and the red selenium nano-particles, there is the possibility that the apparent free radical scavenging effects may be partially contributed by such interactions.
All about Albumin—Biochemistry
- Peters Jr
Peters Jr., T. (1996) All about Albumin—Biochemistry, Genetics, and Medical Applications, pp. 1–432.