PUVA Treatment Selectively Induces a Cell Cycle Block and Subsequent Apoptosis in Human T-Lymphocytes

Psoralen plus UVA (320–400 nm radiation; PUVA) is a highly effective therapy for cutaneous diseases caused by skin infiltration with normal or neoplastic T-lympho-cytes. In comparing the effects of pharmacologically relevant, low-dose PUVA treatment on growth of human keratinocytes, peripheral blood leukocytes (PBMC), and T-lymphocyte cell lines, we determined that PBMC or T-lymphocytes were >50-fold more sensitive to cytotoxic effects of PUVA, while antiproliferative effects were produced by similar PUVA levels in all cell types. Low doses of PUVA (10 ng/mL 8-methoxypsoralen and 1–2 J/cm²) were highly cytotoxic for phytohemagglutinin-activated normal lymphocytes or transformed T-lymphocytes as assessed by two viability assays and by flow cytofluo-rometry. Altered lymphocyte morphology, nuclear fragmentation, TUNEL+ nuclei or nuclear fragments, and the appearance of a sub-G, DNA peak indicated that cell death occurred by apoptosis, beginning about 1 day after PUVA treatment and continuing for several days thereafter. From assessment of cell cycle progression in mi-mosine-synchronized cells, PUVA treatment markedly slowed cell cycle progression, eventually producing cell cycle arrest and apoptotic entry. We propose that the probable basis for disease remissions (psoriasis, cutaneous T-cell lymphoma) produced by PUVA treatment is through selective cytotoxic effects on clonal T-lymphocyte populations that are concentrated in diseased skin.     

Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells

We studied the mechanism of the cytotoxic effects of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT; induction with 1 mM ALA for 4 h followed by a blue light dose of 18 J/cm(2)) on the human promyelocytic leukemia cell line HL60 using biochemical and electron microscopy methods. The disruption of mitochondrial membrane potential, deltapsi(m), was paralleled by a decrease in ATP level, unmasking of the mitochondrial antigen 7A6, release of cytochrome c into the cytoplasm, activation of caspases 9 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP). This was followed by DNA fragmentation. These data suggest that ALA-PDT activates the mitochondrial apoptotic pathway. The level of endoplasmic reticulum Ca(2+)-binding chaperones ERp57 and ERp72 and of anti-apoptotic proteins Bcl-2 and Bcl-x(L) was decreased whereas that of Ca(2+)-binding protein calmodulin and the stress protein HSP60 was elevated following ALA-PDT. Inhibition of the initiator caspase 9, execution caspase 3 and Ca(2+)-dependent protease m-calpain, did not prevent DNA fragmentation. We conclude that, in our in vitro model, ALA-based photodynamic treatment initiates several signaling processes in HL60 cells that lead to rapidly progressing apoptosis, which is followed by slow necrosis. Two apoptotic processes proceed in parallel, one representing the mitochondrial pathway, the other involving disruption of calcium homeostasis and activation of the endoplasmic reticulum stress-mediated pathway.     

Exploring the anticancer effects of tin oxide nanoparticles synthesized by pulsed laser ablation technique against breast cancer cell line through downregulation of PI3K/AKT/mTOR signaling pathway

Tin oxide nanoparticles (SnO₂ NPs) demonstrate potential anti-cancer functions. However, the anti-cancer mechanisms of SnO₂ NPs have not been explored in detail. In the present study, we synthesized SnO₂ NPs through laser ablation technique and examined their anticancer mechanisms and the probable involvement of the PI3K/AKT mediated pathways in human breast cancer cells (MCF-7) in vitro. The synthesized SnO₂ NPs were characterized by transmission electron microcopy (TEM), dynamic light scattering (DLS), and Fourier-transform infrared spectroscopy (FTIR) techniques. Afterwards, the breast cancer cells were incubated with increasing concentrations of SnO₂ NPs, and inhibition of cell proliferation was assessed by the viability assay. Furthermore, the quantification of reactive oxygen species (ROS) and apoptosis were examined by flow cytometry followed by superoxide dismutase (SOD) and catalase (CAT) activity as well as mitochondrial membrane potential assays. The expression levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), mechanistic target of rapamycin (mTOR), B-cell lymphoma 2 (Bcl-2), and Bax were also assessed by western blot and quantitative real time PCR (qRT-PCR). It was shown that SnO₂ NPs, 30 nm, with potential colloidal stability selectively prevented the proliferation of MCF-7 in comparison with MCF-10A cells and triggered ROS production, apoptosis, deactivation of SOD and CAT activity, and mitigation of mitochondrial membrane potential. Moreover, SnO₂ NPs stimulated mitochondrial-mediated apoptosis pathway by overexpression of Bax/Bcl-2 and downregulation of p-PI3K/p-AKT/p-mTOR signaling pathway. This data elucidates the possible mechanisms by which SnO₂ NPs may stimulate their anticancer effects.     

Association of Ceramide Accumulation with Photodynamic Treatment-Induced Cell Death

Abstract— Ceramide, a stress-induced second messenger, has been associated with apoptosis in several malignant and non-malignant cell lines. We have shown that photodynamic treatment (PDT), using the phthalocyanine photosensitiz-er Pc 4 (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂), causes increased ceramide generation and subsequent induction of apoptosis in L5178Y-R (LY-R) mouse lymphoma cells. To test further if ceramide generation accompanies photocytotoxicity, we treated various cell lines with a PDT dose producing a 99-99.9% loss of clonogenicity. Like LY-R cells, human leukemia (U937) cells underwent rapid DNA fragmentation initiating within 1 h after PDT. Similarly, Chinese hamster ovary (CHO) cells showed rapid DNA laddering, beginning 1 h following the treatment. In contrast, mouse radiation-induced fibrosarcoma (RIF-1) cells showed no apoptosis within 24 h post-PDT, as judged by the absence of 50 kbp and oligonucleosome-size DNA fragments, as well as no annexin V binding to cells with preserved membrane integrity. Using the same doses of PDT, we observed a time-dependent ceramide accumulation in all three cell lines. While a significant increase in ceramide levels was reached within 1 and 10 min in U937 and CHO cells, respectively, elevated ceramide production was measured only after 30 min in RIF-1 cells. In addition, exogenous N-acetyl-sphingosine was able to mimic PDT-induced apoptosis in U937 and CHO cells. We suggest that ceramide accumulation is associated with PDT-induced apoptosis and photocytotox-icity.     

Extracorporeal Photochemotherapy (Photopheresis) Induces Apoptosis in Lymphocytes: A Possible Mechanism of Action of PUVA Therapy

Abstract— The mechanism of action of psoralen plus UVA (PUVA) and photopheresis is not entirely understood. These therapies are assumed to be immunomodulating partly by gradually decreasing leukocyte viability. We investigated whether this delayed form of cell death was due to apoptosis. Untreated and treated (PUVA exposed) leukocytes obtained from six patients with systemic sclerosis and (untreated) leukocytes from healthy control individuals were studied. Qualitative gel electrophoresis and quantitative in situ nick translation analysis of DNA fragmentation was performed. Apoptosis of the treated cells did occur (gel electrophoresis) after 24 h. At t = 0 h, immediately after exposure to PUVA, there was no evidence of DNA fragmentation in the treated cells. The percentage of treated cells undergoing apoptosis was 20–55% at t = 24 h ( in situ nick translation). The untreated leukocytes of the patients and the healthy individuals showed no distinctive rise in apoptotic cells. Apoptosis of the leukocytes after PUVA or photopheresis treatment might be a mechanism of action and might explain the therapeutic response.     

Singlet oxygen-induced activation of Akt/protein kinase B is independent of growth factor receptors

Singlet oxygen (1O2)-induced cytotoxicity is believed to be responsible for responses to photodynamic therapy and for apoptosis of T helper cells after UV-A treatment. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. Both these oxidants stimulate the Akt/protein kinase B survival signaling pathway through activation of membrane tyrosine kinase growth factor receptors. We evaluated the ability of 1O2 to activate the Akt/protein kinase B pathway in NIH 3T3 cells and examined potential activation pathways. Exposure of fibroblasts to 1O2 elicited a strong and sustained phosphorylation of Akt, which occurred concurrently with phosphorylation of p38 kinase, a proapoptotic signal. Inhibition of phosphatidylinositol-3-OH kinase (PI3-K) completely blocked Akt phosphorylation. Significantly, cell death induced by 1O2 was enhanced by inhibition of PI3-K, suggesting that activation of Akt by 1O2 may contribute to fibroblast survival under this form of oxidative stress. 1O2 treatment did not induce phosphorylation of platelet-derived growth factor receptor (PDGFR) or activate SH-PTP2, a substrate of growth factor receptors, suggesting that PDGFR was not activated. In addition, specific inhibition of PDGFR did not affect Akt phosphorylation elicited by 1O2. Activation of neither focal adhesion kinase (FAK) nor Ras protein, both of which mediate responses to reactive oxygen species, appeared to be pathways for the 1O2-induced activation of the PI3-K-Akt survival pathway. Thus, activation of Akt by 1O2 is mediated by PI3-K and contributes to a survival response that counteracts cell death after 1O2-induced injury. However, unlike the response to other oxidants, activation of the PI3-K-Akt by 1O2 does not involve activation of growth factor receptors, FAK or Ras protein.     

Involvement of mitochondria-caspase pathway in Hemoporfin-mediated cell death

Hemoporfin is a novel second-generation porphyrin-related photosensitizer for ovarian cancer photodynamic treatment (PDT). The purpose of this study was to investigate the molecular mechanisms of Hemoporfin-mediated photocytotoxicity. Human epithelial ovarian cancer cell line 3AO was incubated with different concentrations of Hemoporfin, and phototoxic effects of Hemoporfin on cells were determined using a Cell Viability Analyzer. Apoptosis or necrosis was determined by flow cytometry analysis using the Annexin V-FITC apoptosis kit. Cellular caspase activation was determined using the fluorescent assay kit for caspase-3 and caspase-9. Rhodamine123 was used as a mitochondrial probe and Lucifer Yellow as a lysosomal probe to investigate the intracellular localization of Hemoporfin in 3AO cancer cells. We demonstrated that both high-dose (30 microg mL(-1)) and low-dose (3 microg mL(-1)) Hemoporfin significantly reduced the viability of ovarian cancer cell 3AO with light illumination, and the photocytotoxicity was dose-dependent (P < 0.01). Using a mitochondrial fluorescence probe, we demonstrated a distinct mitochondrial aggregation in 3AO cells with a low concentration of Hemoporfin. Loss of mitochondrial membrane potential was detected as early as 1 h after Hemoporfin-mediated PDT. PDT with low-dose Hemoporfin predominantly induced apoptosis but not necrosis, and both caspase-3 and caspase-9 were activated. Based on our results, mitochondria play an important role in the Hemoporfin-induced apoptosis, and mitochondria membrane potential loss initiated apoptosis via the activation of caspases. Understanding the mechanisms involved in PDT-mediated apoptosis may improve its therapeutic efficacy and facilitate its transition into the clinic.     

Molecular dynamics simulations reveal the allosteric effect of F1174C resistance mutation to ceritinib in ALK-associated lung cancer

Anaplastic lymphoma kinase (ALK) has become as an important target for the treatment of various human cancers, especially non-small-cell lung cancer. A mutation, F1174C, suited in the C-terminal helix αC of ALK and distal from the small-molecule inhibitor ceritinib bound to the ATP-binding site, causes the emergence of drug resistance to ceritinib. However, the detailed mechanism for the allosteric effect of F1174C resistance mutation to ceritinib remains unclear. Here, molecular dynamics (MD) simulations and binding free energy calculations [Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)] were carried out to explore the advent of drug resistance mutation in ALK. MD simulations observed that the exquisite aromatic-aromatic network formed by residues F1098, F1174, F1245, and F1271 in the wild-type ALK-ceritinib complex was disrupted by the F1174C mutation. The resulting mutation allosterically affected the conformational dynamic of P-loop and caused the upward movement of the P-loop from the ATP-binding site, thereby weakening the interaction between ceritinib and the P-loop. The subsequent MM/GBSA binding free energy calculations and decomposition analysis of binding free energy validated this prediction. This study provides mechanistic insight into the allosteric effect of F1174C resistance mutation to ceritinib in ALK and is expected to contribute to design the next-generation of ALK inhibitors.     

Anticancer Activity of Lesbicoumestan in Jurkat Cells via Inhibition of Oxidative Stress-Mediated Apoptosis and MALT1 Protease

This study explores the potential anticancer effects of lesbicoumestan from Lespedeza bicolor against human leukemia cancer cells. Flow cytometry and fluorescence microscopy were used to investigate antiproliferative effects. The degradation of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) was evaluated using immunoprecipitation, Western blotting, and confocal microscopy. Apoptosis was investigated using three-dimensional (3D) Jurkat cell resistance models. Lesbicoumestan induced potent mitochondrial depolarization on the Jurkat cells via upregulated expression levels of mitochondrial reactive oxygen species. Furthermore, the underlying apoptotic mechanisms of lesbicoumestan through the MALT1/NF-κB pathway were comprehensively elucidated. The analysis showed that lesbicoumestan significantly induced MALT1 degradation, which led to the inhibition of the NF-κB pathway. In addition, molecular docking results illustrate how lesbicoumestan could effectively bind with MALT1 protease at the latter’s active pocket. Similar to traditional 2D cultures, apoptosis was markedly induced upon lesbicoumestan treatment in 3D Jurkat cell resistance models. Our data support the hypothesis that lesbicoumestan is a novel inhibitor of MALT1, as it exhibited potent antiapoptotic effects in Jurkat cells.     

Discovery of Norbornene as a Novel Hydrophobic Tag Applied in Protein Degradation

Hydrophobic tagging (HyT) is a potential therapeutic strategy for targeted protein degradation (TPD). Norbornene was discovered as an unprecedented hydrophobic tag in this study and was used to degrade the anaplastic lymphoma kinase (ALK) fusion protein by linking it to ALK inhibitors. The most promising degrader, Hyt-9 , potently reduced ALK levels through Hsp70 and the ubiquitin−proteasome system (UPS) in vitro without compensatory upregulation of ALK. Furthermore, Hyt-9 exhibited a significant tumor-inhibiting effect in vivo with moderate oral bioavailability. More importantly, norbornene can also be used to degrade the intractable enhancer of zeste homolog 2 (EZH2) when tagged with the EZH2 inhibitor tazemetostat. Thus, the discovery of novel hydrophobic norbornene tags shows promise for the future development of TPD technology.     

QSAR‐based targeting anaplastic lymphoma kinase (ALK) variants with noncognate inhibitors in pediatric acute lymphoblastic leukemia

Human anaplastic lymphoma kinase (ALK) is a potential target for the treatment of pediatric acute lymphoblastic leukemia. However, a number of residue mutations in ALK kinase domain have been observed to cause drug resistance in pediatric acute lymphoblastic leukemia chemotherapy. Here, a chemometrics quantitative structure‐activity relationship predictor was developed using a structure‐based panel of kinase‐inhibitor activity data. The predictor was validated rigorously through internal cross‐validation and external blind test to ensure its statistical reliability, which was then used to computationally construct a systematic activity profile of 13 noncognate kinase inhibitors against both wild‐type ALK^wt and cancer‐related variants ALK^vt. It is revealed that most noncognate inhibitors exhibit weak potency on ALK^wt, but some of them are able to selectively target ALK^vt over ALK^wt. The chemometrics findings were then evaluated by using a kinase inhibition protocol; results showed that few noncognate inhibitors are 2‐ to 5‐fold higher potent against ALK variant than wild‐type kinase.     

Aqueous fraction of Nephelium ramboutan-ake rind induces mitochondrial-mediated apoptosis in HT-29 human colorectal adenocarcinoma cells

The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan) rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous) of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF) was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS) externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm) was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.     

The Anti-Tumor Effect and Underlying Apoptotic Mechanism of Ginsenoside Rk1 and Rg5 in Human Liver Cancer Cells

Ginsenoside Rk1 and Rg5 are minor ginseng saponins that have received more attention recently because of their high oral bioavailability. Each of them can effectively inhibit the survival and proliferation of human liver cancer cells, but the underlying mechanism remains largely unknown. Network pharmacology and bioinformatics analysis demonstrated that G-Rk1 and G-Rg5 yielded 142 potential targets, and shared 44 putative targets associated with hepatocellular carcinoma. Enrichment analysis of the overlapped genes showed that G-Rk1 and G-Rg5 may induce apoptosis of liver cancer cells through inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signal pathways. Methyl thiazolyl tetrazolium (MTT) assay was used to confirm the inhibition of cell viability with G-Rk1 or G-Rg5 in highly metastatic human cancer MHCC-97H cells. We evaluated the apoptosis of MHCC-97H cells by using flow cytometry and 4′,6-diamidino-2-phenylindole (DAPI) staining. The translocation of Bax/Bak led to the depolarization of mitochondrial membrane potential and release of cytochrome c and Smac. A sequential activation of caspase-9 and caspase-3 and the cleavage of poly(ADP-ribose) polymerase (PARP) were observed after that. The levels of anti-apoptotic proteins were decreased after treatment of G-Rk1 or G-Rg5 in MHCC-97H cells. Taken together, G-Rk1 and G-Rg5 promoted the endogenous apoptotic pathway in MHCC-97H cells by targeting and regulating some critical liver cancer related genes that are involved in the signal pathways associated with cell survival and proliferation.     

Betulinic Acid Restricts Human Bladder Cancer Cell Proliferation In Vitro by Inducing Caspase-Dependent Cell Death and Cell Cycle Arrest,and Decreasing Metastatic Potential

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid and generally found in the bark of birch trees (Betula sp.). Although several studies have been reported that BA has diverse biological activities, including anti-tumor effects, the underlying anti-cancer mechanism in bladder cancer cells is still lacking. Therefore, this study aims to investigate the anti-proliferative effect of BA in human bladder cancer cell lines T-24, UMUC-3, and 5637, and identify the underlying mechanism. Our results showed that BA induced cell death in bladder cancer cells and that are accompanied by apoptosis, necrosis, and cell cycle arrest. Furthermore, BA decreased the expression of cell cycle regulators, such as cyclin B1, cyclin A, cyclin-dependent kinase (Cdk) 2, cell division cycle (Cdc) 2, and Cdc25c. In addition, BA-induced apoptosis was associated with mitochondrial dysfunction that is caused by loss of mitochondrial membrane potential, which led to the activation of mitochondrial-mediated intrinsic pathway. BA up-regulated the expression of Bcl-2-accociated X protein (Bax) and cleaved poly-ADP ribose polymerase (PARP), and subsequently activated caspase-3, -8, and -9. However, pre-treatment of pan-caspase inhibitor markedly suppressed BA-induced apoptosis. Meanwhile, BA did not affect the levels of intracellular reactive oxygen species (ROS), indicating BA-mediated apoptosis was ROS-independent. Furthermore, we found that BA suppressed the wound healing and invasion ability, and decreased the expression of Snail and Slug in T24 and 5637 cells, and matrix metalloproteinase (MMP)-9 in UMUC-3 cells. Taken together, this is the first study showing that BA suppresses the proliferation of human bladder cancer cells, which is due to induction of apoptosis, necrosis, and cell cycle arrest, and decrease of migration and invasion. Furthermore, BA-induced apoptosis is regulated by caspase-dependent and ROS-independent pathways, and these results provide the underlying anti-proliferative molecular mechanism of BA in human bladder cancer cells.     

Chamaejasmine induces apoptosis in human lung adenocarcinoma A549 cells through a Ros-mediated mitochondrial pathway

In the present study, the anticancer activity of chamaejasmine towards A549 human lung adenocarcinoma cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of chamaejasmine, cell cycle distribution, ROS generation, mitochondrial membrane potential (Δψ(m)) disruption, and expression of cytochrome c, Bax, Bcl-2, caspase-3, caspase-9 and PARP were measured in A549 cells. Chamaejasmine inhibited the growth of A549 cells in a time and dose-dependent manner. The IC?? value was 7.72 μM after 72 h treatment. Chamaejasmine arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that chamaejasmine inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of chamaejasmine towards A549 in vitro.     

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidylcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.     

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.     

Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-XL involved in cardiotoxin III-induced apoptosis in K562 cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC(50) value of 1.7 microg/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-X(L). CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (DeltaPsim), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-XL, and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells.     

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.     

Mitochondrial-targeted fatty acid analog induces apoptosis with selective loss of mitochondrial glutathione in promyelocytic leukemia cells

Some fatty acids and derivatives are known to induce cell death in cancer cells. Mitochondria may have important roles in the death process. Therefore, we investigated the mitochondrial contribution in cell death induced by a modified fatty acid, tetradecylthioacetic acid (TTA), which cannot be beta-oxidized. TTA treatment induced apoptosis in IPC-81 leukemia cells via depolarization of the mitochondrial membrane potential (deltapsi) and early release of cytochrome c, accompanied by depletion of mitochondrial glutathione. Caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP) occurred at a late stage, but the broad-spectra caspase inhibitor zVAD-fmk did not block TTA-induced apoptosis. Overexpression of Bcl-2 partially prevented TTA-induced apoptosis, whereas cAMP-induced cell death was completely blocked. In conclusion, TTA seems to trigger apoptosis through mitochondrial-mediated mechanisms and selective modulation of the mitochondrial redox equilibrium.