Cyanine dyes as protectors of K562 cells from photosensitized cell damage

Several cyanine dyes were found to protect K562 leukemia cells against toxicity mediated by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and light. Most cyanine dyes derived from dimethylindole were better photoprotectors than cyanine dyes with other structures. This correlated with the fact that cyanine dyes derived from dimethylindole were predominately monomeric at millimolar concentrations within K562 cells, while other cyanine dyes formed aggregates. For cyanine dyes that are derived from dimethylindole and have absorption band wavelengths greater than 700 nm, fluorescence-energy transfer from TPPS2 to the cyanine dye was the most important mechanism for photoprotection. There was no spectroscopic evidence for complex formation between the cyanine dyes and TPPS2. The dimethylindole derivative, 1,1',3,3,3',3'-hexamethylindodicarbocyanine, was an excellent photoprotector, but a poor quencher of TPPS2 fluorescence and a relatively poor singlet-oxygen quencher. This cyanine dye may act by quenching excited triplet TPPS2. Singlet-oxygen quenching may contribute to the photoprotection provided by cyanine dyes not derived from dimethylindole. Differences in the subcellular distribution of the various cyanine dyes studied may have contributed to the different apparent mechanisms of photoprotection.     

5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells

To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells. Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells. A P-gp inhibitor could not increase PpIX accumulation. Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production. Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme. Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX. These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.     

Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells

Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is in clinical trials for the photodynamic therapy (PDT) of localized-stage cancer. The PDT susceptibility of cells expressing multidrug resistance (MDR) phenotype is an attractive possibility to overcome the resistance to cytotoxic drugs observed during cancer chemotherapy. The accumulation, photocytotoxicity and intracellular localization of mTHPC were examined using the doxorubicin selected MCF-7/DXR human breast cancer cells, expressing P-glycoprotein (P-gp), and the wild-type parental cell line, MCF-7. No significant difference in mTHPC accumulation was observed between the two cell lines up to 3 h contact. The photodynamic activity of mTHPC, measured 24 h after irradiation with red laser light (lambda=650 nm), was significantly greater in MCF-7/DXR as compared to MCF-7 cells. A light dose of 2.5 J cm(-2) inducing 50% of cytotoxicity in MCF-7, resulted in 85% cytotoxicity in MCF-7/DXR. The presence of P-gp inhibitors SDZ-PSC-833 and cyclosporin A did not modify the mTHPC-induced cytotoxicity. The difference in intracellular mTHPC distribution pattern between two cell lines may contribute to different photocytotoxicity. Our results indicate that mTHPC mediated PDT could be useful in killing cells expressing MDR phenotype.     

具有肿瘤多药耐药逆转作用的金雀异黄素衍生物的合成及生物活性研究

细胞膜P-糖蛋白(P-gp)介导的药物外流是肿瘤多药耐药(MDR)产生的重要机制,异黄酮类化合物可以通过抑制P-gp活性发挥MDR逆转作用.通过对P-gp抑制剂进行结构分析,以金雀异黄素为母体,在其7位、8位及4'位分别引进碱性边链,设计、合成了20个金雀异黄素衍生物(其中16个未见文献报道),并检测了其多药耐药逆转活性.结果表明,大多数目标化合物对人白血病耐药细胞株K562/A02具有不同程度的耐药逆转作用.其中目标化合物8a,8b,8d,8e逆转作用较强,逆转倍数分别为8.97,6.36,5.19和5.82.     

新型单取代两亲性酞菁锌的制备及其光动力活性研究

设计并制备了一种组成和结构单一的新型两亲性酞菁锌光敏剂. 采用固相合成法合成出2-单羧基取代酞菁锌, 并使之与五聚赖氨酸偶联成五聚赖氨酸2-羰基酞菁锌. 综合运用各种分析及光谱方法对所得化合物进行表征和确认. 研究了在光照下五聚赖氨酸2-羰基酞菁锌对3种肿瘤细胞(人源肝癌细胞Bel7402、人源胃癌细胞BGC823和人源白血病细胞K562) 与一种正常细胞(人源胚肺成纤维细胞HELF)的杀灭活性. 该光敏剂不仅克服了酞菁锌在水中溶解度低的问题, 且因所偶联的五聚赖氨酸对肿瘤细胞有靶向作用, 具有较高的杀灭肿瘤细胞活性.     

PORPHYRIN PHOTOSENSITIZATION OF MULTI-DRUG RESISTANT CELL TYPES

The P388 murine leukemia and P388/ADR, a subline expressing the multi-drug resistance (MDR) phenotype, were examined with regard to the role of MDR as a determinant of responsiveness to photodynamic therapy in vitro. Mesoporphyrin was used as a model substrate. We found no differences in porphyrin accumulation nor transport alterations associated with exposure of P388/ADR cells to the verapamil analog DMDP. There was a significant correlation between photodamage to mitochondria vs loss of cell viability in both cell lines, and LD50 sensitizer levels were not significantly different in P388 vs P388/ADR. P388/ADR cells were partly resistant to porphyrin-catalyzed photodamage to amino acid transport, but this result was not associated with differences in sensitizer localization, as indicated by fluorescence studies. Moreover, photodamage to membrane transport was not associated with loss of viability. These studies suggest that cells which express the MDR phenotype are unlikely to be cross-resistant to photodynamic therapy.     

Cytotoxic polyprenylated xanthones from the resin of Garcinia hanburyi

Thirteen xanthones (1-13) were isolated from the resin of Garcinia hanburyi. Among them, two new compounds (namely gaudichaudic acid, and isogambogenic acid, 1, 2), and one new natural product (deoxygaudichaudione A, 3) were identified on the basis of extensive spectral evidence including detailed 2D NMR data. Ten of these xanthones were tested for their cytotoxicities against human leukemia K562 (K562/S) and doxorubicin-resistant K562 (K562/R) cell lines, and showed similar inhibitory effects on both cell lines, suggesting that this group of polyprenylated xanthones might not be multidrug resistance (MDR) substrates.     

Inactivation of erythrocytic, lymphocytic and myelocytic leukemic cells by photoexcitation of endogenous porphyrins

The photodynamic sensitization of leukemic cells (erythrocytic, myelocytic and lymphocytic) via light activation of endogenous porphyrins is described. Human myelocytic-erythrocytic K562 cells and murine Friend erythroleukemia (FELC) and T-cell lymphoma Eb-Esb cells were stimulated to synthesize and accumulate porphyrins. K562 cells accumulated high amounts of protoporphyrin by stimulation with 5-aminolevulinic acid (ALA) plus sodium butyrate or hemin. For Friend and Eb-Ebs cells ALA was an adequate stimulator. The high-metastatic Esb lymphoma cells accumulated comparatively more porphyrin than the low-metastatic Eb cell line. Maximal porphyrin accumulation produced mortality rates of more than 99% after 10 min of photoactivation of the three leukemic lines. Thymidine incorporation was inhibited by the photodynamic effect depending on porphyrin concentration. These results confirm the photodynamic ability of endogenous porphyrins to inactivate cancer cells of different origins.     

Immunoassay of P-glycoprotein on single cell by capillary electrophoresis with laser induced fluorescence detection

The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5-10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.     

Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.     

CELLULAR UPTAKE AND RELATIVE EFFICIENCY IN CELL INACTIVATION BY PHOTO ACTIVATED SULFONATED meso-TETRAPHENYLPORPHINES

The cellular uptake, relative fluorescence quantum yields and photosensitizing efficiencies of meso-tetraphenylporphines sulfonated to different degrees (TPPSn) have been investigated using the human carcinoma cell line NHIK 3025. The efficiencies of these dyes in photoinactivation of cells were highly dependent on the number of sulfonate groups on the derivatives. These differences in phototoxicity were primarily due to different abilities to be taken up by cells, but were also dependent upon the cellular localization of the dyes. TPPS1 and TPPS2a were more efficiently taken up by the cells than TPPS2o and TPPS4. Plasma membrane associated TPPS4 was less efficient in cell inactivation per quantum of fluorescence emitted than intracellularly located dye. This was also to some extent the case for TPPS1 but not for TPPS2a and TPPS2o. The results presented here indicate that TPPS2a and TPPS1 are the most promising of the TPPSns for possible future use in photodynamic therapy.     

MEROCYANINE 540-SENSITIZED PHOTOINACTIVATION OF LEUKEMIA CELLS: EFFECTS OF DOSE FRACTIONATION

The differential sensitivity to merocyanine 540 (MC540)-sensitized photoirradiation of leukemia cells, selected solid tumor cells, and normal pluripotent hematopoietic stem cells has been successfully exploited for the extracorporeal purging of simulated autologous remission bone marrow grafts. In this communication, we compare the effects of fractionated vs continuous irradiation upon the MC540-sensitized photoinactivation of L1210 and K562 leukemia cells. Exposure to MC540 (15 micrograms/mL) and fractionated doses of white light inactivated fewer in vitro clonogenic cells than exposure to an equivalent dose of continuous irradiation, provided the irradiation doses were small (8.1-16.2 kJ/m2) and spaced 1-2 h apart. The dye-sensitized photoinactivation of leukemia cells was enhanced when cells were stored at 4 degrees C instead of 37 degrees C between irradiation periods, most likely in part because the cells were unable to repair sublethal photodynamic damages at the lower temperature. These data suggest that cells can recover from sublethal damage inflicted by the plasma membrane-active photosensitizer, MC540.     

Differences in Sensitivity to UVC, UVB and UVA Radiation of a Multidrug-Resistant Cell Line Overexpressing P-Glycoprotein

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best-studied mechanism involves the expression of a membrane protein that acts as an energy-dependent efflux pump, known as P-glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non-MDR) cell line.     

Mitochondrial localization and photodamage during photodynamic therapy with tetraphenylporphines

The subcellular localization sites of TPPS4 and TPPS1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS4 favors an aqueous environment, while TPPS1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS1 in the peri-nuclear region, while TPPS4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na(+)/K(+)-ATPase activity. As a result, cytosolic concentrations of Na(+) and Ca(2+) increased, and the level of K(+) decreased. In contrast, the lipophilic TPPS1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS1 has no effect on the mitochondria.     

Photodamage in a Mitochondrial Membrane Model Modulated by the Topology of Cationic and Anionic Meso‐Tetrakis Porphyrin Free Bases

The photodynamic effects of the cationic TMPyP (meso‐tetrakis [N‐methyl‐4‐pyridyl]porphyrin) and the anionic TPPS4 (meso‐tetrakis[4‐sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the influence of phorphyrin binding on lipid damage. Electronic absorption spectroscopy and zeta potential measurements demonstrated that only TMPyP binds to PC/CL large unilamellar vesicles (LUVs). The photodamage after irradiation with visible light was analyzed by dosages of lipid peroxides (LOOH) and thiobarbituric reactive substance and by a contrast phase image of the giant unilamellar vesicles (GUVs). Damage to LUVs and GUVs promoted by TMPyP and TPPS4 were qualitatively and quantitatively different. The cationic porphyrin promoted damage more extensive and faster. The increase in LOOH was higher in the presence of D₂O, and was impaired by sodium azide and sorbic acid. The effect of D₂O was higher for TPPS4 as the photosensitizer. The use of DCFH demonstrated that liposomes prevent the photobleaching of TMPyP. The results are consistent with a more stable TMPyP that generates long‐lived singlet oxygen preferentially partitioned in the bilayer. Conversely, TPPS4 generates singlet oxygen in the bulk whose lifetime is increased in D₂O. Therefore, the affinity of the porphyrin to the membrane modulates the rate, type and degree of lipid damage.     

A non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite in Bcr-Abl positive cells

Multidrug resistance (MDR) is often associated with overexpression of the P-glycoprotein (P-gp, ABCB1). It was demonstrated that the P-gp mediated efflux decreases the drug concentration in cancer cells which results in the failure of chemotherapy. However, the MDR phenotype in cancer cells obviously involves various mechanisms. Therefore, if we want to estimate a contribution of the P-gp expression to the MDR phenotype, a clear quantitative relationship between the intracellular drug level and cell sensitivity must be established. To achieve this goal, a sensitive and non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite N-desmethyl imatinib (CGP 74588) has been developed. The assay is based on an optimised extraction of cells with 4% formic acid after their separation from the growth medium by centrifugation through a layer of silicone oil. Cell extracts are subsequently analyzed by LC/MS/MS. Calibration curves were linear from 1 to 500 nmol/l for imatinib and from 2 to 500 nmol/l for CGP 74588, with correlation coefficients (r²) better than 0.998 and 0.996, respectively. The limit of quantitation (LOQ) was 1 nmol/l for imatinib and 2 nmol/l for CGP 74588. Our method has been successfully applied to the determination of intracellular levels of imatinib in sensitive K562 and their resistant variant, K562/Dox cells.     

Synthesis,dark toxicity and induction of in vitro DNA photodamage by a tetra(4-nido-carboranylphenyl)porphyrin

The total synthesis of tetra(4-carboranylphenyl)porphyrins 4 and 6 and their zinc(II) complexes 5 and 7 are described. These compounds were characterized by analytical and spectroscopic methods and, in the case of 5, by X-ray crystallography. The water-soluble nido-carboranylporphyrins 6 and 7 were found to have low dark toxicity towards V79 hamster lung fibroblast cells, using a clonogenic assay (50% colony survival, CS(50)>300 microM). Upon light activation nido-carboranylporphyrin 6 effectively induced DNA damage in vitro. Two different methods were used to assess the extent of DNA damage: the super-coiled to nicked DNA and the alkaline Comet assay using human leukemia K562 cells. Significant PDT-induced DNA damage was observed for porphyrin 6 using both assays, compared to light-only and porphyrin-only experiments. It is concluded that this type of nido-carboranylporphyrin is a promising sensitizer for both the boron neutron capture therapy and the photodynamic therapy of tumors.     

LIGHT INDUCED RELOCALIZATION OF SULFONATED meso-TETRAPHENYLPORPHINES IN NHIK 3025 CELLS AND EFFECTS OF DOSE FRACTIONATION

Human cervix carcinoma cells of the line NHIK 3025 were incubated for 18 h with sulfonated meso-tetraphenylporphines (TPPSn where n = 1, 2a, 2o or 4) followed by 1 h in sensitizer-free medium and then exposed to light. The fluorescing fraction of TPPS4, TPPS2o and TPPS2a has recently been shown to be located intracellularly in extracellular granules which are intracellularly localized in a similar pattern as acridine orange-stained granules, assumed to be endosomes and lysosomes (Berg, K., A. Western, J. Bommer and J. Moan. Photochem. Photobiol. 52, 481-487). Light exposure induced a relocalization of TPPS4 from its granular pattern to mainly the nuclear area while TPPS2o and TPPS2a relocalized mainly to cytoplasmic areas. After the light-induced relocalization TPPS4 became less efficient in sensitizing photoinactivation of cells as measured per fluorescing cellbound TPPS4 molecules while TPPS2a and TPPS2o became more efficient. These changes were independent of the extracellular concentration of TPPSn applied to the cells, except for cells incubated with 75 micrograms/mL TPPS4. These cells became more sensitive to light after a light exposure inactivating 20% of the cells. This increased photosensitivity seems to be related to a 2-2.5 fold increase in the amount of fluorescing cellbound TPPS4 induced by the first light exposure.     

一种液相色谱分离与质谱鉴定白血病K562细胞蛋白的方法

分别通过3种色谱模式:反相高效液相色谱(RPLC)、弱阴离子交换-反相高效液相色谱(WAX-RPLC)和排阻-反相高效液相色谱(SEC-RPLC)对K562细胞的蛋白质进行分离,收集的色谱馏分采用基质辅助电离解析时间飞行质谱(MALDI-TOF MS)进行鉴定后,比较所获得的蛋白质数据.结果显示:当选择SEC-RPLC模式构建K562细胞蛋白质图谱时,检测到的蛋白质数目最多,信息最为全面.此法的建立为白血病的临床研究提供了一种有效的手段.     

The cellular labeling and pH-sensitive responsive-drug release of celastrol in cancer cells based on Cys-CdTe QDs

As one of the active compounds derived from Traditional Chinese Medicine,Celastrol(CSL)had cytotoxicity for human leukemia cancer cells K562 and its multidrug-resistant cell line K562/A02.Here,we introduced cysteamine-modified CdTe QDs as the labeling and drug carrier into CSL research and found that the self-assembly and conjugation of anticancer molecular CSL with the Cys-CdTe QDs could significantly increase the drug’s cytotoxicity for K562 cells.More important,these CSL-Cys-CdTe nanocomposites could overcome the multidrug resistance of K562/A02 cells and efficiently inhibit the cancer cell proliferation by realizing the pH-sensitive responsive release of CSL to cancer cells.The enhanced cytotoxicity was caused by the increase of the G2/M phase arrest for K562/A02 cells as well as for K562 cells.Cys-CdTe QDs can readily bind on the cell plasma membranes and be internalized into cancer cells to trace and detect human leukemia cancer cells in real time.In addition,these Cys-CdTe QDs can facilitate the inhibition of the multidrug resistance of K562/A02 cells and readily induce apoptosis.As a good photosensitizer for the therapy,labeling,and tracing of cancer cells,the combination of CSL with Cys-CdTe QDs can optimize the use of and a new potential therapy method for CSL and yield new tools to explore the mechanisms of active compounds from Traditional Chinese Medicine.