Early apoptotic features of K562 cell death induced by 5-aminolaevulinic acid-based photodynamic therapy

5-Aminolaevulinic acid-based photodynamic therapy (ALA-PDT) is used to eliminate cancerous cells through photoactivation of endogenously formed protoporphyrin IX (PPIX) following the administration of PPIX precursor, 5-aminolaevulinic acid (ALA). We report on the kinetics of PPIX accumulation and the mechanism of cytotoxic effects of ALA-PDT studied in the chronic myelogenous leukaemia derived cell line K562. The PPIX distribution and, consequently, cytotoxic effects were found to be heterogenous. A subpopulation of K562 cells accumulating PPIX to a lesser extent exhibits only transient cell cycle arrest. A fraction of cells, probably those with higher PPIX accumulation, are irreversibly damaged by ALA-PDT. We detected several signs of an early apoptosis: lowering of Bcl-xL expression, decrease of the mitochondrial and plasma membrane potential, the cytochrome c release into the cytoplasm, and the unmasking of the mitochondrial antigen 7A6. However, late apoptotic events were lacking: neither caspase-3 activation nor DNA fragmentation occurred. Instead, rapidly progressing cell necrosis resulting from plasma membrane damage was observed. We suggest that the high level of the antiapoptotic heat-shock proteins HSP70 and HSP27 found by us in the K562 cells is responsible for the inhibition of the apoptotic process upstream of caspases activation.     

Biological evaluation of 2-methylpyrimidine derivatives as active pan Bcr-Abl inhibitors

We designed a series of 2-methylpyrimidine derivatives as new BCR-ABL inhibitors using scaffold-hopping strategy.These synthetic compounds exhibited significant inhibition against a broad spectrum of Bcr-Abl mutants including the gatekeeper T315I mutant.Compound 7u showed very potent kinase inhibitory activities against Bcr-Abl WT,Bcr-Abl E255K,Bcr-Abl Q252H,Bcr-Abl G250E and Bcr-Abl T315I,with IC50 values of 0.13 nM,0.17 nM,0.24 nM,0.19 nM and 0.65μM,respectively.This compound also displayed anti-proliferation activity against K562 cell line with an IC50 value of 1.1 nM,thus representing a new lead for further optimization.     

Selective destruction of leukaemic cells by photo-activation of 5-aminolaevulinic acid-induced protoporphyrin-IX

The effect of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) on the viability and proliferation of leukaemia/lymphoma cells as well as normal human lymphocytes has been investigated by flow cytometry-propidium iodide assay (FC-PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation and on clonogenic activity of normal human bone marrow progenitor cells by clonogenic methods. ALA-PDT (1 mM 5-ALA, 4 h, 18 J cm-2) reduces the number and/or suppressed proliferation of leukaemic cells of promyelocytic (HL60), B-cell-derived (DAUDI) and T-cell-derived (JURKAT) cell lines by 2 logs and that of the HEL erythroleukaemia cells by 77%. The effect of ALA-PDT on quiescent human lymphocytes is small (85% viable cells after ALA-PDT). The proliferation of lymphocytes subjected to ALA-PDT and induced with phytohaemagglutinin (PHA) decreases by 75% as compared to the untreated control. For normal human bone marrow progenitors, 58% of colony-forming units-granulocytes-macrophages (CFU-GM) and 55% burst-forming units-erythrocytes (BFU-E) activities are preserved.     

Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells

Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin β1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.     

硒化壳聚糖抗K562细胞的实验研究

用MTT法和AO/EB荧光染色法观察了硒化壳聚糖对K 562肿瘤细胞株生长的影响。结果发现,硒化壳聚糖可有效地抑制K 562细胞生长,并呈量效、时效关系。经硒化壳聚糖作用后的细胞可明显出现核固缩、碎裂等凋亡形态改变。硒化壳聚糖可诱导K 562细胞凋亡,抑制其生长。     

单独和合用硒化壳聚糖与阿霉素对K562细胞作用的研究

应用MTT法和AO／EB荧光染色法观察单独和合用硒化壳聚糖与阿霉素对K562细胞株的影响,结果发现硒化壳聚糖和阿霉素均可有效地抑制K562细胞生长。且两药联合使用效果更佳。硒化壳聚糖可诱导细胞凋亡。两药合用诱导细胞凋亡效果更好。     

Preferential cytotoxicity for multidrug-resistant K562 cells using the combination of a photosensitizer and a cyanine dye

The cyanine dye 1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide (HIDC) protects K562 leukemia cells from photodynamic membrane damage caused by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and 420 nm light. This wavelength of light is chosen because it is absorbed by TPPS2, but not by HIDC. The photodynamic system studied may be useful as a model for antineoplastic therapy. A subline of K562 leukemia (K562/DOX), expressing the multidrug-resistance (MDR) phenotype, is found to accumulate smaller amounts of HIDC than the parent cell line and thus has less photoprotection. In the absence of added HIDC, the K562/DOX cell line is more resistant to photodynamic cytotoxicity than the K562 cell line. The resistance of the K562/DOX cell line is not due to a smaller accumulation of TPPS2 than the K562 cell line. However, when both cell lines are incubated with HIDC and TPPS2, and then exposed to light, the K562/DOX cell line becomes more sensitive to photodynamic cell damage than the K562 cell line. The combination of a photosensitizer with a cationic or lysomorphotropic photoprotector represents a novel strategy for the eradication of malignant cells expressing the MDR phenotype.     

硒化壳聚糖对K 562和K 562/ADM作用的比较研究

应用MTT法和细胞集落形成率法观察了硒化壳聚糖对K562和K562／ADM肿瘤细胞株生长的影响，结果发现硒化壳聚糖可有效地抑制两种K562细胞生长，呈量效关系。同等剂量的硒化壳聚糖对K562细胞的作用强度高于对K562／ADM细胞。硒化壳聚糖对耐药的细胞株可产生抑制作用，若与化疗药合用则可减慢甚至部分逆转临床上K562细胞耐药性的发生。     

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.     

siRNA-mediated Knockdown of the Heme Synthesis and Degradation Pathways: Modulation of Treatment Effect of 5-Aminolevulinic Acid-based Photodynamic Therapy in Urothelial Cancer Cell Lines

Photodynamic therapy mediated by 5-aminolevulinic acid (ALA-PDT) has been developed as a therapeutic modality for refractory superficial bladder cancers. Here, in experiments using urothelial cancer cell lines, we investigated the effects of siRNA modulating heme-synthetic and degradation pathways for ALA-PDT. Targeted knockdown of ferrochelatase (FECH) suppressed heme synthesis and significantly increased intracellular protoporphyrin IX (PpIX) accumulation, leading to enhanced phototoxicity in four of five cell lines. Heme oxygenase-1 (HO-1) is recognized as important for cytoprotection against oxidative stress such as PDT. Targeted knockdown of HO-1 leads to decreased intracellular PpIX accumulation, resulting in a failure to enhance ALA-PDT effect in four cell lines. Knockdown of HO-1 caused marked growth inhibition in UM-UC-2 overexpressing HO-1, whereas no inhibitory effect was observed in UM-UC-3 lacking HO-1 expression. Moreover, HO-1 protein levels and (GT) _n repeat polymorphism of the HO-1 gene promoter region were examined with the implication that the constitutive expressions of HO-1 protein were associated with a shorter (GT) _n repeat. Our results suggested that (1) FECH siRNA improved the phototoxicity of ALA-PDT, (2) overexpression of HO-1 was associated with shorter (GT) _n repeat of the promoter region, and (3) siRNA-mediated knockdown of HO-1 could suppress the growth of bladder cancer cells overexpressing HO-1.     

Expression and Functional Characterization of Tumor-Targeted Fusion Protein Composed of NGR Peptide and 15-kDa Actin Fragment

To induce tumor cell apoptosis, a modified 15 kDa actin linked with a peptide NGR “homing” into tumor or tumor vessels was expressed in Escherichia coli. After refolding and purification, this fusion protein NGR-15actin was labeled with FITC to testify whether NGR-15actin could integrate into the cytoskeleton. It was found that this targeted peptide could induce HepG2 and HeLa cells apoptosis through its effect on the cytoskeleton function by binding to cytoskeleton protein. Thus, targeted NGR-15actin could be a candidate molecule for the therapy of cancer.     

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.     

Nanosecond pulsed electric field induced cytoskeleton, nuclear membrane and telomere damage adversely impact cell survival

We investigated the effects of nanosecond pulsed electric fields (nsPEF) on three human cell lines and demonstrated cell shrinkage, breakdown of the cytoskeleton, nuclear membrane and chromosomal telomere damage. There was a differential response between cell types coinciding with cell survival. Jurkat cells showed cytoskeleton, nuclear membrane and telomere damage that severely impacted cell survival compared to two adherent cell lines. Interestingly, disruption of the actin cytoskeleton in adherent cells prior to nsPEF exposure significantly reduced cell survival. We conclude that nsPEF applications are able to induce damage to the cytoskeleton and nuclear membrane. Telomere sequences, regions that tether and stabilize DNA to the nuclear membrane, are severely compromised as measured by a pan-telomere probe. Internal pore formation following nsPEF applications has been described as a factor in induced cell death. Here we suggest that nsPEF induced physical changes to the cell in addition to pore formation need to be considered as an alternative method of cell death. We suggest nsPEF electrochemical induced depolymerization of actin filaments may account for cytoskeleton and nuclear membrane anomalies leading to sensitization.     

A disposable impedance sensor for electrochemical study and monitoring of adhesion and proliferation of K562 leukaemia cells

A polyaniline-modified screen-printed carbon electrode (PANI/SPCE) was prepared by electropolymerization for the construction of a novel disposable cell impedance sensor. The conductive polymer improved greatly the electron transfer of SPCE and was very effective for cell immobilization. The adhesion of cells increased the electron transfer resistance (R_et) of redox probe on the PANI/SPCE surface, producing an impedance sensor for K562 leukaemia cells with a semilogarithm linear range from 10⁴ to 10⁷ cells ml⁻¹ and a limit of detection of 8.32 × 10³ cells ml⁻¹ at 10σ. The proliferation of cells on the conductive polymer increased the R_et, leading to a novel way to monitor the growth process of cells on the PANI/SPCE. The electrochemical monitoring indicated K562 leukaemia cells cultured in vitro on the PANI surface were viable for 60 h, consistent with the analysis from microscopic imaging and MTT assay. This method for monitoring the surface proliferation and detecting the number of viable cells was simple, low-cost and disposable, thus providing a convenient avenue for electrochemical study of cell immobilization, adhesion, proliferation and apoptosis.     

Protein changes in HL60 leukemia cells associated with 5-aminolevulinic acid-based photodynamic therapy. Early effects on endoplasmic reticulum chaperones

Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin precursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca(2+)-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.     

Antiproliferative effect of millimeter radiation on human erythromyeloid leukemia cell line K562 in culture: ultrastructural- and metabolic-induced changes

In the present study we compared the proliferation behavior, the ultrastructural morphology and the glycolitic metabolism of K562 cells irradiated by low-power wide-band millimeter waves, with those of sham-exposed K562 cells (control), maintained in the same culture conditions. The gigaHertz radiation treatments, performed between 53-78 10(9) Hz, induced a noticeable inhibition of the cell proliferation that could be related to relevant ultrastructural changes. Such effects brought the irradiated cell system to lose the homeostasis and to trigger defense/reparatory mechanisms in order to reestablish a new steady state. (13)C-Nuclear magnetic resonance data on the kinetic of glucose metabolism demonstrated that the irradiated cells enhanced the glycolitic aerobic pathway, indicating that such system need to produce an extra-bioenergy. Most of the ATP synthesized served probably to perform the above processes resulting in a significant decrease of the proliferation rate without significant cell death increment.     

Role of mTOR signaling in tumor cell motility, invasion and metastasis

Tumor cell migration and invasion play fundamental roles in cancer metastasis. The mammalian target of rapamycin (mTOR), a highly conserved and ubiquitously expressed serine/threonine (Ser/Thr) kinase, is a central regulator of cell growth, proliferation, differentiation and survival. Recent studies have shown that mTOR also plays a critical role in the regulation of tumor cell motility, invasion and cancer metastasis. Current knowledge indicates that mTOR functions as two distinct complexes, mTORC1 and mTORC2. mTORC1 phosphorylates p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), and regulates cell growth, proliferation, survival and motility. mTORC2 phosphorylates Akt, protein kinase C α (PKCα) and the focal adhesion proteins, and controls the activities of the small GTPases (RhoA, Cdc42 and Rac1), and regulates cell survival and the actin cytoskeleton. Here we briefly review recent knowledge of mTOR complexes and the role of mTOR signaling in tumor cell migration and invasion. We also discuss recent efforts about the mechanism by which rapamycin, a specific inhibitor of mTOR, inhibits cell migration, invasion and cancer metastasis.     

p53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin- damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced- tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA- PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.