Spin Trapping of Nitric Oxide by Hemoglobin and Ferrous Diethyldithiocarbamate in Model Tumors Differing in Vascularization.

Animal tumors serve as reasonable models for human cancers. Both human and animal tumors often reveal triplet EPR signals of nitrosylhemoglobin (HbNO) as an effect of nitric oxide formation in tumor tissue, where NO is complexed by Hb. In search of factors determining the appearance of nitrosylhemoglobin (HbNO) in solid tumors, we compared the intensities of electron paramagnetic resonance (EPR) signals of various iron-nitrosyl complexes detectable in tumor tissues, in the presence and absence of excess exogenous iron(II) and diethyldithiocarbamate (DETC). Three types of murine tumors, namely, L5178Y lymphoma, amelanotic Cloudman S91 melanoma, and Ehrlich carcinoma (EC) growing in DBA/2 or Swiss mice, were used. The results were analyzed in the context of vascularization determined histochemically using antibodies to CD31. Strong HbNO EPR signals were found in melanoma, i.e., in the tumor with a vast amount of a hemorrhagic necrosis core. Strong Fe(DETC)2NO signals could be induced in poorly vascularized EC. In L5178Y, there was a correlation between both types of signals, and in addition, Fe(RS)2(NO)2 signals of non-heme iron-nitrosyl complexes could be detected. We postulate that HbNO EPR signals appear during active destruction of well-vascularized tumor tissue due to hemorrhagic necrosis. The presence of iron-nitrosyl complexes in tumor tissue is biologically meaningful and defines the evolution of complicated tumor-host interactions.

EPR Monitoring of Oxygenation Levels in Tumors After Chlorophyllide-Based Photodynamic Therapy May Allow for Early Prediction of Treatment Outcome.

PURPOSE: Molecular oxygen, besides a photosensitizer and light of appropriate wavelength, is one of the three factors necessary for photodynamic therapy (PDT). In tumor tissue, PDT leads to the killing of tumor cells, destruction of endothelial cells and vasculature collapse, and the induction of strong immune responses. All these effects may influence the oxygenation levels, but it is the vasculature changes that have the main impact on pO2. The purpose of our study was to monitor changes in tumor oxygenation after PDT and explore its significance for predicting long-term treatment response. PROCEDURES: Electron paramagnetic resonance (EPR) spectroscopy enables direct, quantitative, and sequential measurements of partial pressure of oxygen (pO2) in the same animal. The levels of chlorophyll derived photosensitizers in tumor tissue were determined by transdermal emission measurements. RESULTS: The noninvasive monitoring of pO2 in the tumor tissue after PDT showed that the higher ΔpO2 (pO2 after PDT minus pO2 before PDT), the greater the inhibition of tumor growth. ΔpO2 also correlated with higher levels of the photosensitizers in the tumor and with the occurrence of a severe edema/erythema after PDT. CONCLUSION: Monitoring of PDT-induced changes in tumor oxygenation is a valuable prognostic factor and could be also used to identify potentially resistant tumors, which is important in predicting long-term treatment response.

The Blocking of Drug Resistance Channels by Selected Hydrophobic Statins in Chemoresistance Human Melanoma.

Despite the development of modern drugs, drug resistance in oncology remains the main factor limiting the curability of patients. This paper shows the use of a group of hydrophobic statins to inhibit drug resistance (Pgp protein). In a chemoresistance melanoma cell model, viability, necroptosis with DNA damage, the absorption of the applied pharmaceuticals, and the functional activity of the ABCB1 drug transporter after administration of docetaxel or docetaxel with a selected hydrophobic statin were studied. Taxol-resistant human melanoma cells from three stages of development were used as a model: both A375P and WM239A metastatic lines and radial growth phase WM35 cells. An animal model (Mus musculus SCID) was developed for the A375P cell line. The results show that hydrophobic statins administered with docetaxel increase the accumulation of the drug in the tumor cell a.o. by blocking the ABCB1 channel. They reduce taxol-induced drug resistance. The tumor size reduction was observed after the drug combination was administrated. It was shown that the structural similarity of statins is of secondary importance, e.g., pravastatin and simvastatin. Using cytostatics in the presence of hydrophobic statins increases their effectiveness while reducing their overall toxicity.

Silver Nanoparticles Induced Changes in DNA Methylation and Histone H3 Methylation in a Mouse Model of Breast Cancer.

The importance of epigenetic changes as a measurable endpoint in nanotoxicological studies is getting more and more appreciated. In the present work, we analyzed the epigenetic effects induced by citrate- and PEG-coated 20 nm silver nanoparticles (AgNPs) in a model consisting of 4T1 breast cancer tumors in mice. Animals were administered with AgNPs intragastrically (1 mg/kg b.w. daily-total dose 14 mg/kg b.w.) or intravenously (administration twice with 1 mg/kg b.w.-total dose 2 mg/kg b.w.). We observed a significant decrease in 5-methylcytosine (5-mC) level in tumors from mice treated with citrate-coated AgNPs regardless of the route of administration. For PEG-coated AgNPs, a significant decrease in DNA methylation was observed only after intravenous administration. Moreover, treatment of 4T1 tumor-bearing mice with AgNPs decreased histone H3 methylation in tumor tissue. This effect was the most pronounced for PEG-coated AgNPs administered intravenously. No changes in histone H3 Lys9 acetylation were observed. The decrease in methylation of DNA and histone H3 was accompanied by changes in expression of genes encoding chromatin-modifying enzymes (Setd4, Setdb1, Smyd3, Suv39h1, Suv420h1, Whsc1, Kdm1a, Kdm5b, Esco2, Hat1, Myst3, Hdac5, Dnmt1, Ube2b, and Usp22) and genes related to carcinogenesis (Akt1, Brca1, Brca2, Mlh1, Myb, Ccnd1, and Src). The significance of the observed changes and the mechanisms responsible for their development are unclear, and more research in this area is warranted. Nevertheless, the present work points to the epigenetic effects as an important level of interaction between nanomaterials and biological systems, which should always be taken into consideration during analysis of the biological activity of nanomaterials and development of nanopharmaceuticals.

Oxygen therapeutic window induced by myo-inositol trispyrophosphate (ITPP)-Local pO2 study in murine tumors.

Hypoxia, an inevitable feature of locally advanced solid tumors, has been known as an adverse prognostic factor, a driver of an aggressive phenotype, and an unfavorable factor in therapies. Myo-inositol trispyrophosphate (ITPP) is a hemoglobin modifier known to both increase O2 release and normalize microvasculature. Our goal was to measure the tumor oxygen partial pressure dynamic changes and timing of the therapeutic window after ITPP systemic administration. Two syngeneic tumor models in mice, B16 melanoma and 4T1 breast carcinoma, were used, with varying ITPP dose schedules. Tissue oxygenation level was measured over several days in situ in live animals by Electron Paramagnetic Resonance oximetry with implanted OxyChip used as a constant sensor of the local pO2 value. Both B16 and 4T1 tumors became more normoxic after ITPP treatment, with pO2 levels elevated by 10-20 mm Hg compared to the control. The increase in pO2 was either transient or sustained, and the underlying mechanism relied on shifting hypoxic tumor areas to normoxia. The effect depended on ITPP delivery intervals regarding the tumor type and growth rate. Moreover, hypoxic tumors before treatment responded better than normoxic ones. In conclusion, the ITPP-generated oxygen therapeutic window may be valuable for anti-tumor therapies requiring oxygen, such as radio-, photo- or immunotherapy. Furthermore, such a combinatory treatment can be especially beneficial for hypoxic tumors.

Silver Nanoparticles Inhibit Metastasis of 4T1 Tumor in Mice after Intragastric but Not Intravenous Administration.

The potential anticancer activity of different silver nanoformulations is increasingly recognized. In the present work, we use the model of 4T1 tumor in BALB/ccmdb immunocompetent mice to analyze the impact of citrate- and PEG-coated silver nanoparticles (AgNPs) on the development and metastatic potential of breast cancer. One group of mice was intragastrically administered with 1 mg/kg body weight (b.w.) of AgNPs daily from day 1 to day 14 after cancer cells implantation (total dose 14 mg/kg b.w.). The second group was intravenously administered twice with 1 or 5 mg/kg b.w. of AgNPs. A tendency for lowering tumor volume on day 21 (mean volumes 491.31, 428.88, and 386.83 mm3 for control, AgNPs-PEG, and AgNPs-citrate, respectively) and day 26 (mean volumes 903.20, 764.27, and 672.62 mm3 for control, AgNPs-PEG, and AgNPs-citrate, respectively) has been observed in mice treated intragastrically, but the effect did not reach the level of statistical significance. Interestingly, in mice treated intragastrically with citrate-coated AgNPs, the number of lung metastases was significantly lower, as compared to control mice (the mean number of metastases 18.89, 14.90, and 8.03 for control, AgNPs-PEG, and AgNPs-citrate, respectively). No effect of AgNPs treatment on the number of lung metastases was observed after intravenous administration (the mean number of metastases 12.44, 9.86, 12.88, 11.05, and 10.5 for control, AgNPs-PEG 1 mg/kg, AgNPs-PEG 5 mg/kg, AgNPs-citrate 1 mg/kg, and AgNPs-citrate 5 mg/kg, respectively). Surprisingly, inhibition of metastasis was not accompanied by changes in the expression of genes associated with epithelial-mesenchymal transition. Instead, changes in the expression of inflammation-related genes were observed. The presented results support the antitumor activity of AgNPs in vivo, but the effect was limited to the inhibition of metastasis. Moreover, our results clearly point to the importance of AgNPs coating and route of administration for its anticancer activity. Finally, our study supports the previous findings that antitumor AgNPs activity may depend on the activation of the immune system and not on the direct action of AgNPs on cancer cells.

Systemic Mobilization of Breast Cancer Resistance Protein in Response to Oncogenic Stress.

The breast cancer resistance protein (BCRP or ABCG2) involved in cancer multidrug resistance (MDR), transports many hydrophobic compounds, including a number of anti-cancer drugs. Our comprehensive study using a mouse model reveals that a subcutaneously growing tumor strongly affects the expression of BCRP in the host's normal organs on both the transcriptional and translational level. Additionally, the efflux of BCRP substrates is markedly enhanced. The levels of BCRP and its transcript in normal tissues distant from the tumor site correlate with tumor growth and the levels of cytokines in the peripheral blood. Thus, oncogenic stress causes transient systemic upregulation of BCRP in the host's normal tissues and organs, which is possibly mediated via cytokines. Because BCRP upregulation takes place in many organs as early as the initial stages of tumor development, it reveals a most basic mechanism that may be responsible for the induction of primary MDR. We hypothesize that such effects are not tumor-specific responses, but rather constitute a more universal defense strategy. The xenobiotic transporters are systemically mobilized due to various stresses, seemingly in a pre-emptive manner so that the body can be quickly and efficiently detoxified. Our findings shed new light on the biology of cancer and on the complexity of cancer-host interactions and are highly relevant to cancer therapies as well as to the design of new generations of therapeutics and personalized medicine.

Transient Vasodilation in Mouse 4T1 Tumors after Intragastric and Intravenous Administration of Gold Nanoparticles.

Gold nanoparticles (AuNPs) are foreseen as a promising tool in nanomedicine, both as drug carriers and radiosensitizers. They have been also proposed as a potential anticancer drug due to the anti-angiogenic effect in tumor tissue. In this work we investigated the effect of citrate-coated AuNPs of nominal diameter 20 nm on the growth and metastatic potential of 4T1 cells originated from a mouse mammary gland tumor inoculated into the mammary fat pad of Balb/ccmdb mice. To evaluate whether AuNPs can prevent the tumor growth, one group of inoculated mice was intragastrically (i.g.) administered with 1 mg/kg of AuNPs daily from day 1 to day 14 after cancer cell implantation. To evaluate whether AuNPs can attenuate the tumor growth, the second group was intravenously (i.v.) administered with 1 or 5 mg/kg of AuNPs, twice on day 5 and day 14 after inoculation. We did not observe any anticancer activity of i.v. nor i.g. administered AuNPs, as they did not affect neither the primary tumor growth rate nor the number of lung metastases. Unexpectedly, both AuNP treatment regimens caused a marked vasodilating effect in the tumor tissue. As no change of potential angiogenic genes (Fgf2, Vegfa) nor inducible nitric oxygenase (Nos2) was observed, we proposed that the vasodilation was caused by AuNP-dependent decomposition of nitrosothiols and direct release of nitric oxide in the tumor tissue.

CD44+ cells determine fenofibrate-induced microevolution of drug-resistance in prostate cancer cell populations.

Combinations of metabolic blockers (incl. fenofibrate) with chemotherapeutic drugs interfere with the drug-resistance of prostate cancer cells. However, their effect on cancer stem cells-dependent microevolution of prostate cancer malignancy remains unaddressed. Here, we hypothesize that the combined docetaxel/fenofibrate treatment prompts the selective expansion of cancer stem cells that affects the microevolution of their progenies. Accordingly, we adapted a combined in vitro/in vivo approach to identify biological and therapeutic consequences of this process. Minute subpopulations of docetaxel-resistant CD133high and/or CD44high cancer stem cell-like (SCL) cells were found in prostate cancer DU145 and PC3 cell populations. When pretreated with docetaxel, they readily differentiated into docetaxel-resistant CD44negative "bulk" cells, thus accounting for the microevolution of drug-resistant cell lineages. Combined docetaxel/fenofibrate treatment induced the generation of poly(morpho)nuclear giant cells and drug-resistant CD44high SCL cells. However, the CD44negative offspring of docetaxel- and docetaxel/fenofibrate-treated SCLs remained relatively sensitive to the combined treatment, while retaining enhanced resistance to docetaxel. Long-term propagation of drug-resistant SCL-derived lineages in the absence of docetaxel/fenofibrate resulted in their reverse microevolution toward the drug-sensitivity and invasive phenotype. Consequently, prostate tumors were able to recover from the combined docetaxel/fenofibrate stress after the initial arrest of their expansion in vivo. In conclusion, we have confirmed the potential of fenofibrate for the metronomic treatment of drug-resistant prostate tumors. However, docetaxel/fenofibrate-induced selective expansion of hyper-resistant CD44high SCL prostate cells and their "bulk" progenies prompts the microevolution of prostate tumor drug-resistance. This process can limit the implementation of metabolic chemotherapy in prostate cancer treatment.

Fenofibrate Augments the Sensitivity of Drug-Resistant Prostate Cancer Cells to Docetaxel.

Metronomic agents reduce the effective doses and adverse effects of cytostatics in cancer chemotherapy. Therefore, they can enhance the treatment efficiency of drug-resistant cancers. Cytostatic and anti-angiogenic effects of fenofibrate (FF) suggest that it can be used for the metronomic chemotherapy of drug-resistant prostate tumors. To estimate the effect of FF on the drug-resistance of prostate cancer cells, we compared the reactions of naïve and drug-resistant cells to the combined treatment with docetaxel (DCX)/mitoxantrone (MTX) and FF. FF sensitized drug-resistant DU145 and PC3 cells to DCX and MTX, as illustrated by their reduced viability and invasive potential observed in the presence of DCX/MTX and FF. The synergy of the cytostatic activities of both agents was accompanied by the inactivation of P-gp-dependent efflux, dysfunction of the microtubular system, and induction of polyploidy in DCX-resistant cells. Chemical inhibition of PPARα- and reactive oxygen species (ROS)-dependent pathways by GW6471 and N-acetyl-L-cysteine, respectively, had no effect on cell sensitivity to combined DCX/FF treatment. Instead, we observed the signs of adenosine triphosphate (ATP) deficit and autophagy in DCX/FF-treated drug-resistant cells. Furthermore, the cells that had been permanently propagated under DCX- and DCX/FF-induced stress did not acquire DCX/FF-resistance. Instead, relatively slow proliferation of DCX-resistant cells was efficiently inhibited by FF. Collectively, our observations show that FF reduces the effective doses of DCX by interfering with the drug resistance and energy metabolism of prostate cancer cells. Concomitantly, it impairs the chemotherapy-induced microevolution and expansion of DCX/FF-resistant cells. Therefore, FF can be applied as a metronomic agent to enhance the efficiency of palliative chemotherapy of prostate cancer.

Transplantable Melanomas in Hamsters and Gerbils as Models for Human Melanoma. Sensitization in Melanoma Radiotherapy-From Animal Models to Clinical Trials.

The focus of the present review is to investigate the role of melanin in the radioprotection of melanoma and attempts to sensitize tumors to radiation by inhibiting melanogenesis. Early studies showed radical scavenging, oxygen consumption and adsorption as mechanisms of melanin radioprotection. Experimental models of melanoma in hamsters and in gerbils are described as well as their use in biochemical and radiobiological studies, including a spontaneously metastasizing ocular model. Some results from in vitro studies on the inhibition of melanogenesis are presented as well as radio-chelation therapy in experimental and clinical settings. In contrast to cutaneous melanoma, uveal melanoma is very successfully treated with radiation, both using photon and proton beams. We point out that the presence or lack of melanin pigmentation should be considered, when choosing therapeutic options, and that both the experimental and clinical data suggest that melanin could be a target for radiosensitizing melanoma cells to increase efficacy of radiotherapy against melanoma.

Visualization and Quantitative 3D Analysis of Intraocular Melanoma and Its Vascularization in a Hamster Eye.

A tumor vasculature network undergoes intense growth and rebuilding during tumor growth. Traditionally, vascular networks are histologically examined using parameters such as vessel density determined from two-dimensional slices of the tumor. Two-dimensional probing of a complicated three-dimensional (3D) structure only provides partial information. Therefore, we propose the use of microcomputed tomography (micro-CT) imaging to analyze the evolution of a tumor vasculature in an experimental ocular tumor model. A Bomirski Hamster Melanoma was implanted in the anterior chamber of a hamster eye. Ultrasound (US) imaging of the same tumor was performed in vivo, and the vascular results obtained using the two methods were compared. Normal ocular tissues, a tumor, and a tumor vascular structure were revealed with high accuracy using micro-CT. The vessels that grew within the tumor were chaotic, leaky, and contained many convoluted micro-vessels and embolizations. They comprised 20-38% of the tumor mass. The blood flow in the larger functional vessels was in the range from 10 to 25 mm/s, as determined by in vivo Doppler US. The micro-CT imaging of the hamster eyeball enabled both qualitative and quantitative 3D analyses of the globe at a histological level. Although the presented images were obtained ex vivo, micro-CT noninvasive imaging is being developed intensively, and high-resolution in vivo imaging is feasible.

Optimization of Western blotting analysis for the isolation and detection of membrane xenobiotic transporter ABCG2.

All organisms are exposed to numerous stress factors, which include harmful xenobiotics. The diversity of these compounds is enormous, thus in the course of evolution diverse biological defense mechanisms at various levels of organization have developed. One of them engages an evolutionarily conserved family of transporters from the ABC superfamily, found in most species - from bacteria to humans. An important example of such a transporter is the breast cancer resistance protein (BCRP/ABCG2), a typical integral membrane protein. It plays a key role in the absorption, distribution and elimination of a wide variety of xenobiotics, including drugs used in chemotherapy, and is involved in multidrug resistance. It also protects against phototoxic chlorophyll derivatives of dietary origin. BCRP is a hemitransporter which consists of one transmembrane domain, made of six alpha-helices forming a characteristic pore structure, and one ATP-binding domain, which provides the energy from ATP hydrolysis, required for active transport of the substrates. The isolation of BCRP is still not an easy task, because its insolubility in water and the presence of membrane rafts pose serious methodological and technical challenges during the purification. The aim of this study was to optimize the methods for detection and isolation of BCRP-enriched fractions obtained from animal tissue samples. In this report we describe an optimization of isolation of a BCRP-enriched membrane fraction, which is suitable for further protein quantitative and qualitative analysis using the molecular biology tools.

Recent Progress in Chemical Modifications of Chlorophylls and Bacteriochlorophylls for the Applications in Photodynamic Therapy.

Since photodynamic therapy emerged as a promising cancer treatment, the development of photosensitizers has gained great interest. In this context, the photosynthetic pigments, chlorophylls and bacteriochlorophylls, as excellent natural photosensitizers, attracted much attention. In effect, several (bacterio) chlorophyll-based phototherapeutic agents have been developed and (or are about to) enter the clinics. The aim of this review article is to give a survey of the advances in the synthetic chemistry of these pigments which have been made over the last decade, and which are pertinent to the application of their derivatives as photosensitizers for photodynamic therapy (PDT). The review focuses on the synthetic strategies undertaken to obtain novel derivatives of (bacterio)chlorophylls with both enhanced photosensitizing and tumorlocalizing properties, and also improved photo- and chemical stability. These include modifications of the C- 17-ester moiety, the isocyclic ring, the central binding pocket, and the derivatization of peripheral functionalities at the C-3 and C-7 positions with carbohydrate-, peptide-, and nanoparticle moieties or other residues. The effects of these modifications on essential features of the pigments are discussed, such as the efficiency of reactive oxygen species generation, photostability, phototoxicity and interactions with living organisms. The review is divided into several sections. In the first part, the principles of PDT and photosensitizer action are briefly described. Then the relevant photophysical features of (bacterio)chlorophylls and earlier approaches to their modification are summarized. Next, a more detailed overview of the progress in synthetic methods is given, followed by a discussion of the effects of these modifications on the photophysics of the pigments and on their biological activity.

Determinants of the activity and substrate recognition of breast cancer resistance protein (ABCG2).

The xenobiotic transporters are among the most important constituents of detoxification system in living organisms. Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of xenobiotics. To understand its role in chemotherapeutic and multidrug resistance, it is crucial to establish the determinants of its substrate specificity, which obviously is of high relevance for successful therapy of many diseases. This article summarizes the current knowledge about the substrate preferences of BCRP. We overview the factors which determine its activity, inhibition and substrate recognition, focusing on the structural features of the transporter. BCRP substrate specificity is quite low as it interacts with a spectrum of substances with only a few common features: hydrophobic and aromatic regions, possibly a flat conformation and the metal ion-, oxygen- and nitrogen-containing functionalities, most of which may be the donors/acceptors of H-bonds. Several amino acid residues and structural motifs are responsible for BCRP activity and substrate recognition. Thus, the active form of BCRP, at least a dimer or a larger oligomer is maintained by intramolecular disulfide bridge that involves Cys(603) residues. The GXXXG motif in transmembrane helix 1, Cys residues, Arg(482) and Lys(86) are responsible for maintaining the protein structure, which confers transport activity, and the His(457) or Arg(456) residues are directly involved in substrate binding. Arg(482) does not directly bind substrates, but electrostatically interacts with charged molecules, which initiates the conformational changes that transmit the signal from the transmembrane regions to the ABC domain.

The role of strong hypoxia in tumors after treatment in the outcome of bacteriochlorin-based photodynamic therapy.

Blood flow and pO2 changes after vascular-targeted photodynamic therapy (V-PDT) or cellular-targeted PDT (C-PDT) using 5,10,15,20-tetrakis(2,6-difluoro-3-N-methylsulfamoylphenyl) bacteriochlorin (F2BMet) as photosensitizer were investigated in DBA/2 mice with S91 Cloudman mouse melanoma, and correlated with long-term tumor responses. F2BMet generates both singlet oxygen and hydroxyl radicals under near-infrared radiation, which consume oxygen. Partial oxygen pressure was lowered in PDT-treated tumors and this was ascribed both to oxygen consumption during PDT and to fluctuations in oxygen transport after PDT. Similarly, microcirculatory blood flow changed as a result of the disruption of blood vessels by the treatment. A novel noninvasive approach combining electron paramagnetic resonance oximetry and laser Doppler blood perfusion measurements allowed longitudinal monitoring of hypoxia and vascular function changes in the same animals, after PDT. C-PDT induced parallel changes in tumor pO2 and blood flow, i.e., an initial decrease immediately after treatment, followed by a slow increase. In contrast, V-PDT led to a strong and persistent depletion of pO2, although the microcirculatory blood flow increased. Strong hypoxia after V-PDT led to a slight increase in VEGF level 24h after treatment. C-PDT caused a ca. 5-day delay in tumor growth, whereas V-PDT was much more efficient and led to tumor growth inhibition in 90% of animals. The tumors of 44% of mice treated with V-PDT regressed completely and did not reappear for over 1 year. In conclusion, mild and transient hypoxia after C-PDT led to intense pO2 compensatory effects and modest tumor inhibition, but strong and persistent local hypoxia after V-PDT caused tumor growth inhibition.

Zinc-pheophorbide a-highly efficient low-cost photosensitizer against human adenocarcinoma in cellular and animal models.

BACKGROUND: Our previous study has shown a prolonged retention and accumulation of Zn-pheophorbide a, a water-soluble derivative of chlorophyll a, in tumor tissue (Szczygiel et al. [19]). This prompted us to further evaluate the phototherapeutic potential of this photosensitizer of excellent physicochemical properties. METHODS: Cellular uptake of Zn-pheophorbide, its localization in cells, cytotoxicity, phototoxicity and cell death mechanisms were studied in human adenocarcinoma cell lines: A549, MCF-7 and LoVo. The PDT efficacy was tested against A549 tumors growing in nude mice. RESULTS: Zn-pheophorbide a even at very low concentrations (∼1×10(-6)M) and at low light doses (5J/cm(2)) causes a strong photodynamic effect, leading to 100% cell mortality. Confocal microscopy showed that in contrast to most derivatives of chlorophyll, Zn-pheophorbide a does not localize to mitochondria. The photodynamic effects and the cell death mechanisms of Zn-pheophorbide a, its Mg analog (chlorophyllide a) and Photofrin were compared on the A549 cells. Zn-pheophorbide a showed the strongest photodynamic effect, at low dose killing all A549 cells via apoptosis and necrosis. The very high anti-cancer potential of Zn-pheophorbide was confirmed in a photodynamic treatment of the A549 tumors. They either regressed or were markedly inhibited for up to 4 months after the treatment, resulting, on average, in a 5-fold decrease in tumor volume. CONCLUSION: These results show that Zn-pheophorbide a is a very promising low-cost, synthetically easily accessible, second generation photosensitizer against human cancer.

Metastasis inhibition after proton beam, ß- and γ-irradiation of melanoma growing in the hamster eye.

Standard ocular tumor treatment includes brachytherapy, as well as proton therapy, particularly for large melanoma tumors. However, the effects of different radiation types on the metastatic spread is not clear. We aimed at comparing ruthenium ((106)Ru, emitting ß electrons) and iodine ((125)I, γ-radiation) brachytherapy and proton beam therapy of melanoma implanted into the hamster eye on development of spontaneous lung metastases. Tumors of Bomirski Hamster Melanoma (BHM) implanted into the anterior chamber of the hamster eye grew aggressively and completely filled the anterior chamber within 8-10 days. Metastases, mainly in the lung, were found in 100% of untreated animals 30 days after enucleation. Tumors were irradiated at a dose of 3-10 Gy with a (106)Ru plaque and at a dose of 6-14 Gy using a (125)I plaque. The protons were accelerated using the AIC-144 isochronous cyclotron operating at 60 MeV. BHM tumors located in the anterior chamber of the eye were irradiated with 10 Gy, for the depth of 3.88 mm. All radiation types caused inhibition of tumor growth by about 10 days. An increase in the number of metastases was observed for 3 Gy of ß-irradiation, whereas at 10 Gy an inhibition of metastasis was found. γ-radiation reduced the metastatic mass at all applied doses, and proton beam therapy at 10 Gy also inhibited the metastastic spread. These results are discussed in the context of recent clinical and molecular data on radiation effects on metastasis.

Central metal determines pharmacokinetics of chlorophyll-derived xenobiotics.

Chlorophyll derivatives are potentially dangerous xenobiotics of dietary origin. The interactions of water-soluble derivatives of chlorophyll a with the animal organism were investigated using chlorophyllide a and its Zn-substituted analogue as model xenobiotics. The chlorophyllides were administered to tumor-bearing mice and their uptake, distribution, and clearance were compared. The centrally bound metal determines important aspects of the in vivo behavior of metallochlorophyllides as xenobiotics. The uptake and clearance of chlorophyllide a were significantly faster than those of [Zn]-chlorophyllide a. Chlorophyllide a showed some tissue selectivity, while [Zn]-chlorophyllide a was uniformly distributed among tissues. Interestingly, the tissue levels of the latter compound were ten times higher than those of the Mg-derivative. These differences indicate that [Zn]-chlorophyllide a, in contrast to chlorophyllide a, is only weakly recognized by the system of active transport of xenobiotics and by enzymes involved in chlorophyll metabolism. The dependence of chlorophyllide pharmacokinetics on the central metal is of great relevance to chlorophyll-based phototherapy.

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.