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.

Knocking out the Vitamin D Receptor Enhances Malignancy and Decreases Responsiveness to Vitamin D3 Hydroxyderivatives in Human Melanoma Cells.

Vitamin D3 is not only involved in calcium and phosphate metabolism in humans, but it can also affect proliferation and differentiation of normal and cancer cells, including melanoma. The mechanism of the anti-cancer action of vitamin D3 is not fully understood. The nuclear vitamin D receptor (VDR) is crucial for the phenotypic effects of vitamin D hydroxyderivatives. VDR expression shows an inverse correlation with melanoma progression and poor outcome of the disease. In this study we knocked out the VDR in a human melanoma cell line using CRISPR methodology. This enhanced the proliferation of melanoma cells grown in monolayer culture, spheroids or colonies and their migration. Activated forms of vitamin D, including classical 1,25(OH)2D3, 20(OH)D3 and 1,20(OH)2D3, inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while VDR KO cells showed a degree of resistance to their action. These results indicate that expression of VDR is important for the inhibition of melanoma growth induced by activated forms of vitamin D. In conclusion, based on our previous clinicopathological analyses and the current study, we suggest that the VDR can function as a melanoma tumor suppressor gene.

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.

Melanin presence inhibits melanoma cell spread in mice in a unique mechanical fashion.

Melanoma is a highly aggressive cancer that exhibits metastasis to various critical organs. Unlike any other cancer cells, melanoma cells can synthesize melanin in large amounts, becoming heavily pigmented. Until now the role of melanin in melanoma, particularly the effect of melanin presence on the abilities of melanoma cells to spread and metastasize remains unknown. Recently, we have shown that melanin dramatically modified elastic properties of melanoma cells and inhibited the cells invasive abilities in vitro. Here, we inoculated human melanoma cells with different melanin content into nude mice and tested the hypothesis that cell elasticity is an important property of cancer cells for their efficient spread in vivo. The obtained results clearly showed that cells containing melanin were less capable to spread in mice than cells without the pigment. Our findings indicate that the presence of melanin inhibits melanoma metastasis, emphasizing possible clinical implications of such an inhibitory effect.

Vitamin D receptors (VDR), hydroxylases CYP27B1 and CYP24A1 and retinoid-related orphan receptors (ROR) level in human uveal tract and ocular melanoma with different melanization levels.

In recent years, a significant number of studies have investigated the preventive role of vitamin D in a number of different neoplasms. In this study, we analyze various components of the vitamin D signaling pathways in the human uveal tract and uveal melanoma, including analysis of the expression of vitamin D receptors (VDR), the activating and inactivating hydroxylases, respectively, CYP27B1 and CYP24A1, and the retinoic acid-related orphan receptors (ROR) α (RORα) and γ (RORγ) in these tissues. We further analyzed the expression of VDR, CYP27B1, CYP24A1, and ROR in relation to melanin levels, clinical stage and prognosis. Our study indicated that the uveal melanoma melanin level inversely correlated with VDR expression. We further showed that vitamin D is metabolized in uveal melanoma. This is significant because until now there has been no paper published, that would describe presence of VDR, hydroxylases CYP27B1 and CYP24A1, and RORα and RORγ in the human uveal tract and uveal melanomas. The outcomes of our research can contribute to the development of new diagnostic and therapeutic methods in uveal tract disorders, especially in uveal melanoma. The presented associations between vitamin D signaling elements and uveal melanoma in comparison to uveal tract encourage future clinical research with larger patients' population.

Increased elasticity of melanoma cells after low-LET proton beam due to actin cytoskeleton rearrangements.

Cellular response to non-lethal radiation stress include perturbations in DNA repair, angiogenesis, migration, and adhesion, among others. Low-LET proton beam radiation has been shown to induce somewhat different biological response than photon radiation. For example, we have shown that non-lethal doses of proton beam radiation inhibited migration of cells and that this effect persisted long-term. Here, we have examined cellular elasticity and actin cytoskeleton organization in BLM cutaneous melanoma and Mel270 uveal melanoma cells. Proton beam radiation increased cellular elasticity to a greater extent than X-rays and both types of radiation induced changes in actin cytoskeleton organization. Vimentin level increased in BLM cells after both types of radiation. Our data show that cell elasticity increased substantially after low-LET proton beam and persisted long after radiation. This may have significant consequences for the migratory properties of melanoma cells, as well as for the cell susceptibility to therapy.

Calcitriol and Calcidiol Can Sensitize Melanoma Cells to Low⁻LET Proton Beam Irradiation.

Proton beam irradiation promises therapeutic utility in the management of uveal melanoma. Calcitriol (1,25(OH)2D3)-the biologically active metabolite of vitamin D3-and its precursor, calcidiol (25(OH)D3), exert pleiotropic effects on melanoma cells. The aim of the study was to evaluate the effect of both calcitriol and calcidiol on melanoma cell proliferation and their response to proton beam irradiation. Three melanoma cell lines (human SKMEL-188 and hamster BHM Ma and BHM Ab), pre-treated with 1,25(OH)2D3 or 25(OH)D3 at graded concentrations (0, 10, 100 nM), were irradiated with 0⁻5 Gy and then cultured in vitro. Growth curves were determined by counting the cell number every 24 h up to 120 h, which was used to calculate surviving fractions. The obtained survival curves were analysed using two standard models: linear-quadratic and multi-target single hit. Calcitriol inhibited human melanoma proliferation at 10 nM, while only calcidiol inhibited proliferation of hamster lines at 10 and 100 nM doses. Treatment with either 1,25(OH)2D3 or 25(OH)D3 radio sensitized melanoma cells to low doses of proton beam radiation. The strength of the effect increased with the concentration of vitamin D3. Our data suggest that vitamin D3 may be an adjuvant that modifies proton beam efficiency during melanoma therapy.

Acute hepatologic and nephrologic effects of calcitriol in Syrian golden hamster (Mesocricetus auratus).

Although vitamin D is included in the group of fat-soluble vitamins, it must be considered as a prohormone. Its active forms, including calcitriol, have pleiotropic effects and play an important role in the regulation of cell proliferation, differentiation and apoptosis, as well as in hormone secretion, and they demonstrate anti-cancer properties. Since calcitriol delivery can be beneficial for the organism, and Syrian golden hamsters represent a unique experimental model, we decided to investigate its toxicity in this species. In this study, we injected calcitriol intraperitoneally at doses 0 (control), 0.180±0.009 µg/kg and 0.717±0.032 µg/kg. Animal behavior was observed for 72 hrs after injection, and afterwards blood, liver and kidneys were collected for post-mortem examination, electron microscopy, and hematology analyses. The highest dose of calcitriol induced a change in animal behavior from calm to aggressive, and the liver surface showed morphological signs of damage. Following injection of calcitriol, ultrastructural changes were also observed in the liver and kidneys, e.g. vacuolization and increased number of mitochondria. There was also a trend for increased serum levels of aspartate aminotransferase (AST), but not of alanine aminotransferase (ALT) or GGTP (gamma-glutamyl transpeptidase). There was no change in Ca, Mg and P levels, as well as in blood morphology between experimental and control groups. These results indicate that calcitriol at 0.717, but not at 0.180 µg/kg, may induce acute damage to the liver and kidneys, without inducing calcemia. We propose that the hepatotoxic effect of calcitriol in hamster constitutes the primary cause of behavioral changes.

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.

Nanomechanical Phenotype of Melanoma Cells Depends Solely on the Amount of Endogenous Pigment in the Cells.

Cancer cells have unique nanomechanical properties, i.e., they behave as if they were elastic. This property of cancer cells is believed to be one of the main reasons for their facilitated ability to spread and metastasize. Thus, the so-called nanomechanical phenotype of cancer cells is viewed as an important indicator of the cells' metastatic behavior. One of the most highly metastatic cancer cells are melanoma cells, which have a very unusual property: they can synthesize the pigment melanin in large amounts, becoming heavily pigmented. So far, the role of melanin in melanoma remains unclear, particularly the impact of the pigment on metastatic behavior of melanoma cells. Importantly, until recently the potential mechanical role of melanin in melanoma metastasis was completely ignored. In this work, we examined melanoma cells isolated from hamster tumors containing endogenous melanin pigment. Applying an array of advanced microscopy and spectroscopy techniques, we determined that melanin is the dominating factor responsible for the mechanical properties of melanoma cells. Our results indicate that the nanomechanical phenotype of melanoma cells may be a reliable marker of the cells' metastatic behavior and point to the important mechanical role of melanin in the process of metastasis of 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.

Proton beam irradiation inhibits the migration of melanoma cells.

PURPOSE: In recent years experimental data have indicated that low-energy proton beam radiation might induce a difference in cellular migration in comparison to photons. We therefore set out to compare the effect of proton beam irradiation and X-rays on the survival and long-term migratory properties of two cell lines: uveal melanoma Mel270 and skin melanoma BLM. MATERIALS AND METHODS: Cells treated with either proton beam or X-rays were analyzed for their survival using clonogenic assay and MTT test. Long-term migratory properties were assessed with time-lapse monitoring of individual cell movements, wound test and transpore migration, while the expression of the related proteins was measured with western blot. RESULTS: Exposure to proton beam and X-rays led to similar survival but the quality of the cell colonies was markedly different. More paraclones with a low proliferative activity and fewer highly-proliferative holoclones were found after proton beam irradiation in comparison to X-rays. At 20 or 40 days post-irradiation, migratory capacity was decreased more by proton beam than by X-rays. The beta-1-integrin level was decreased in Mel270 cells after both types of radiation, while vimentin, a marker of EMT, was increased in BLM cells only. CONCLUSIONS: We conclude that proton beam irradiation induced long-term inhibition of cellular motility, as well as changes in the level of beta-1 integrin and vimentin. If confirmed, the change in the quality, but not in the number of colonies after proton beam irradiation might favor tumor growth inhibition after fractionated proton therapy.

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.

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.

Photodynamic therapy efficacy enhanced by dynamics: the role of charge transfer and photostability in the selection of photosensitizers.

Progress in the photodynamic therapy (PDT) of cancer should benefit from a rationale to predict the most efficient of a series of photosensitizers that strongly absorb light in the phototherapeutic window (650-800 nm) and efficiently generate reactive oxygen species (ROS = singlet oxygen and oxygen-centered radicals). We show that the ratios between the triplet photosensitizer-O2 interaction rate constant (kD) and the photosensitizer decomposition rate constant (kd), kD/kd, determine the relative photodynamic activities of photosensitizers against various cancer cells. The same efficacy trend is observed in vivo with DBA/2 mice bearing S91 melanoma tumors. The PDT efficacy intimately depends on the dynamics of photosensitizer-oxygen interactions: charge transfer to molecular oxygen with generation of both singlet oxygen and superoxide ion (high kD) must be tempered by photostability (low kd). These properties depend on the oxidation potential of the photosensitizer and are suitably combined in a new fluorinated sulfonamide bacteriochlorin, motivated by the rationale.

Proteomic analysis of proton beam irradiated human melanoma cells.

Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy) of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times) change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i) DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH), (ii) cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70), (iii) cell metabolism (TIM, GAPDH, VCP), and (iv) cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B). A substantial decrease (2.3 x) was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma.

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.

Nitrosylhemoglobin in photodynamically stressed human tumors growing in nude mice.

The role of nitric oxide in human tumor biology and therapy has been the subject of extensive studies. However, there is only limited knowledge about the mechanisms of NO production and its metabolism, and about the role NO can play in modern therapeutic procedures, such as photodynamic therapy. Here, for the first time, we report the presence of nitrosylhemoglobin, a stable complex of NO, in human lung adenocarcinoma A549 tumors growing in situ in nude mice. Using electron paramagnetic resonance spectroscopy we show that the level of nitrosylhemoglobin increases in the course of photodynamic therapy and that the phenomenon is local. Even the destruction of strongly vascularized normal liver tissue did not induce the paramagnetic signal, despite bringing about tissue necrosis. We conclude that photodynamic stress substantiates NO production and blood extravasation in situ, both processes on-going even in non-treated tumors, although at a lower intensity.