A role for manganese superoxide dismutase in apoptosis after photosensitization

The oxidative stress triggered by photodynamic therapy (PDT) involves generation of cytotoxic reactive oxygen species, including superoxide radical, accumulation of de novo-generated ceramide, and induction of apoptosis. Since PDT with the photosensitizer phthalocyanine Pc 4 induces mitochondrial damage and the superoxide scavenger manganese superoxide dismutase (MnSOD) is localized to mitochondria, here we tested genetically the role of MnSOD in apoptosis and ceramide accumulation after photosensitization with Pc 4. Jurkat cells overexpressing wild-type MnSOD were protected from Pc 4-PDT-initiated apoptosis, but not from increased ceramide response to Pc 4-PDT. In Jurkat cells overexpressing mutant MnSOD, however, DEVDase activation and ceramide formation were promoted post-Pc 4-PDT. Similarly, in MnSOD-null cells, Pc 4-PDT-induced apoptosis, as well as ceramide accumulation, were enhanced compared to their normal counterparts. The data show that MnSOD affects sensitivity of cells to Pc 4-PDT-initiated apoptosis, and partly ceramide accumulation, suggesting that the processes are superoxide-mediated.     

A role for the de novo sphingolipids in apoptosis of photosensitized cells

Sphingolipids have been implicated in apoptosis after various stress inducers. To assess the involvement of the de novo sphingolipid pathway in apoptosis, photodynamic therapy (PDT) with the photosensitizer Pc 4 was used as a novel stress inducer. Here we provide biochemical and genetic evidence of the role of the de novo sphingolipids in apoptosis post-Pc 4-PDT. In Jurkat cells PDT-induced intracellular sphinganine accumulation, DEVDase activation, PARP cleavage, and apoptosis were suppressed by the de novo sphingolipid synthesis inhibitor ISP-1 (Myriocin). Coincubation with sphinganine, sphingosine, or C16-ceramide specifically reversed the antiapoptotic actions of ISP-1 or the singlet oxygen scavenger L-histidine. PDT-induced cytochrome c release from mitochondria into the cytosol was inhibited by L-histidine, but not by ISP-1. Cotreatment with sphinganine did not reverse the inhibitory effect of L-histidine. In addition, PDT-induced sphinganine accumulation and apoptosis were ISP-1-sensitive in A431 human epidermoid and HT29 human carcinoma cells. Furthermore, in LY-B cells, CHO-derived mutants deficient in the de novo sphingolipid synthesis enzyme serine palmitoyltransferase (SPT) activity, DEVDase activation and apoptosis were delayed and suppressed post-PDT. Hence, the data are consistent with the partial involvement of the de novo sphingolipid pathway in apoptosis via DEVDase activation downstream of mitochondrial cytochrome c release post-Pc 4-PDT.     

Ceramide response post-photodamage is absent after treatment with HA14-1

The oxidative stress induced by photodynamic therapy using the phthalocyanine Pc 4 (PDT) can lead to apoptosis, and is accompanied by photodamage to Bcl-2 and accumulation of de novo ceramide. Similar to PDT, the oxidative stress inducer and Bcl-2 inhibitor HA14-1 triggers apoptosis. To test the specificity of the ceramide response, Jurkat cells were exposed to an equitoxic dose of HA14-1. Unlike PDT, HA14-1 did not induce accumulation of de novo ceramide, although levels of sphingomyelin, phosphatidylserine and phosphatidylethanolamine were below control values after either treatment. In contrast to PDT, (i) the transient inhibition of serine palmitoyltransferase induced by HA14-1 was associated with the initial decrease in de novo ceramide, and (ii) HA14-1-initiated inhibition of sphingomyelin synthase and glucosylceramide synthase did not result in accumulation of de novo ceramide. These results show that the ceramide response to PDT is not induced by another pro-apoptotic stimulus, and may be unique to PDT as described here.     

Reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells

Our recent studies have shown that the de novo sphingolipids play a role in apoptosis of photosensitized cells. To elucidate the involvement of the de novo sphingolipids in reactive oxygen species (ROS) production and mitochondrial depolarization during apoptosis, the stress inducer photodynamic therapy (PDT) with the photosensitizer Pc 4 was used. In Jurkat cells PDT-triggered ROS production or mitochondrial membrane potential (deltapsi(m)) loss was not prevented by the de novo sphingolipid synthesis inhibitor ISP-1. However, PDT + C16-ceramide led to enhanced mitochondrial depolarization and DEVDase activation. The superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected Jurkat cells from ROS generation and apoptosis, but not from deltapsi(m) reduction. Sphinganine or C16-ceramide counteracted MnTBAP-induced protection from apoptosis in Jurkat, as well as CHO cells. In LY-B cells, CHO-derived mutants deficient in serine palmitoyltransferase (SPT) activity and the de novo sphingolipid synthesis, mitochondrial depolarization, but not ROS generation, was suppressed post-PDT. In LY-B cells transfected with the SPT component LCB1, deltapsi(m) collapse post-PDT was restored. The data support the following hypotheses: MnTBAP protects against apoptosis via steps downstream of deltapsi(m) loss; de novo sphingolipids are not required for ROS generation, but can play a role in deltapsi(m) dissipation in photosensitized apoptotic cells.     

FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4

Oxidative stress, such as photodynamic therapy with the silicon phthalocyanine Pc 4 (Pc 4-PDT), can induce apoptosis and tumor necrosis factor alpha (TNF) production. TNF receptors, as well as other death receptors, have been implicated in stress-induced apoptosis. To assess directly the role of FADD, a death receptor-associated protein, in induction of apoptosis post-Pc 4-PDT, embryonic fibroblasts from FADD knock out (k/o) and wild-type (wt) mice were used. Pc 4-PDT induced casp-3 activation and apoptosis in both cell types. In the presence of zVAD, a pancaspase inhibitor, Pc 4-PDT-induced apoptosis was abrogated in both cell lines. Fumonisin B1 (FB), an inhibitor of ceramide synthase, had no effect on apoptosis after Pc 4-PDT in either cell line. Similar to Pc 4-PDT, exogenous C6-ceramide bypassed FADD deficiency and induced zVAD-sensitive apoptosis. In contrast to Pc 4 photosensitization, TNF did not induce either apoptosis or ceramide accumulation in FADD k/o cells. In the absence of FADD deficiency, TNF-induced apoptosis was zVAD-sensitive and FB-insensitive. Induced ceramide levels remained elevated after cotreatment with TNF and zVAD in FADD wt cells. Taken together, these data provide genetic evidence for a lack of FADD requirement in Pc 4-PDT- or C6-ceramide-induced apoptosis. FB-sensitive ceramide production accompanies, but does not suffice, for apoptosis after Pc 4 photosensitization or TNF.     

Rapid induction of apoptosis in human keratinocytes with the photosensitizer QLT0074 via a direct mitochondrial action

QLT0074 is a newly introduced, porphyrin-derivative for use in photodynamic therapy (PDT). In the current study, the intracellular distribution of QLT0074 and the mode of cell death induced by photosensitization with this compound in vitro were assessed for transformed human HaCaT keratinocytes. Fluorescence microscopy studies indicated a distribution of the drug to the cytoplasm, nuclear membrane and mitochondria of these cells. In the absence of light, QLT0074 produced no evidence of apoptosis-related biochemical changes or affected cell viability. When combined with blue light exposure, cytotoxicity was exerted in a QLT0074- and light-dose-related manner. Appearance of the mitochondrial protein cytochrome c in the cytosolic fraction and expression of the apoptosis-associated mitochondrial 7A6 antigen were demonstrable following photosensitization at nano-molar levels of QLT0074. Evidence of processing of the apoptosis-effector molecules caspase-3, -6, -7, -8 and -9 as well as cleavage of the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) were demonstrable subsequent to cytochrome c release after PDT. Treatment with the anti-oxidant pyrrolidine dithiocarbamate (PDTC) inhibited cytochrome c release, caspase-3 activation and PARP cleavage associated with PDT thereby supporting the contention that QLT0074 induces apoptosis through the generation of reactive oxygen species upon light activation. QLT0074 is a potent photosensitizer with the capacity to directly initiate apoptosis by acting upon mitochondria.     

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.     

Photodynamic therapy-induced death of MCF-7 human breast cancer cells: a role for caspase-3 in the late steps of apoptosis but not for the critical lethal event

Photodynamic therapy (PDT) causes mitochondrial damage and induces apoptosis through release of cytochrome c and activation of caspase-3. To test whether caspase 3 is the sole executioner of apoptosis and its role in overall cell lethality, we compared the response of MCF-7c3 cells that express a stably transfected CASP-3 gene to that of parental MCF-7:SW8 cells transfected with vector alone (MCF-7v). Following photosensitization with the phthalocyanine Pc 4 and red light, cytochrome c was released from the mitochondria to equivalent extents in the two cell lines. However, the appearance of apoptotic indicators, such as active caspase-3 (DEVDase), cleavage of poly(ADP-ribose) polymerase, and oligonucleosomal DNA fragmentation, was observed only in MCF-7c3 cells during the first 6 h after photosensitization. Although production of 50-kb DNA fragments and chromatin condensation were found in PDT-treated MCF-7v cells by 20-24 h posttreatment, the rate and extent of apoptosis were much less than in MCF-7c3 cells. MCF-7c3 cells were more sensitive to photosensitization than were MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to photodynamic killing when evaluated by a clonogenic assay. These results show (a) the importance of assessing overall cell death by clonogenic assay; (b) that the critical lethal event is independent of caspase-3, perhaps at or near the release of cytochrome c from mitochondria; and (c) that the caspase-3-mediated events appear to be irrelevant in determining overall killing of cells.     

Suppression of sphingomyelin synthase 1 by small interference RNA is associated with enhanced ceramide production and apoptosis after photodamage

We have shown that overexpression of SMS1, an enzyme that converts de novo ceramide into sphingomyelin, is accompanied by attenuated ceramide response and apoptotic resistance after photodamage with the photosensitizer Pc 4 (photodynamic therapy; PDT). To test whether SMS1 overexpression-related effects after PDT can be reversed, in this study SMS1 was downregulated in Jurkat T lymphoma/leukemia cells using small inhibitory RNA (siRNA) for SMS1. Compared to scrambled (control) siRNA-transfectants, in SMS1 siRNA-transfected cells the activity of SMS at rest was downregulated with concomitant decrease in sphingomyelin mass. In SMS1 siRNA-transfected cells increases in ceramides were higher than in control siRNA-transfectants after PDT. Similar findings were obtained for dihydroceramides suggesting the involvement of de novo ceramide pathway. PDT-induced DEVDase (caspase-3-like) activation was enhanced in SMS1 siRNA-transfected cells compared to their control counterparts. The data show that RNA interference-dependent downregulation of SMS1 is associated with increased accumulation of ceramide and dihydroceramide with concomitant sensitization of cells to apoptosis after photodamage. Similarly, in SMS2 siRNA-transfected cells, downregulation of SMS activity was accompanied by potentiated DEVDase activation post-photodamage. These findings suggest that SMS is a potential novel molecular target that can augment therapeutic efficacy of PDT.     

Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax^+/−) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax^+/− cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax^+/− cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax^+/− and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.     

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation

L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9 dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40-60%, and via Complex III by 50-90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumption via electron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.     

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.     

Niemann-Pick human lymphoblasts are resistant to phthalocyanine 4-photodynamic therapy-induced apoptosis.

Stress-induced activation of sphingomyelinase (SMase) leading to generation of ceramide, a lipid mediator, has been associated with apoptosis in several malignant and nonmalignant cell lines. Photodynamic therapy (PDT), with the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], is an oxidative stress associated with increased ceramide generation and subsequent induction of apoptosis in various cell types. We assessed the role of SMase in photocytotoxicity. Normal human lymphoblasts accumulated ceramide and underwent apoptosis after Pc 4-PDT. In contrast, Niemann-Pick disease (NPD) lymphoblasts, which are deficient in acid sphingomyelinase (ASMase) activity, failed to respond to Pc 4-PDT with ceramide accumulation and apoptosis, suggesting that ASMase may be a Pc 4-PDT target. NPD lymphoblasts were exposed to exogenous bacterial sphingomyelinase (bSMase) to test whether these defects are reversible. Treatment of NPD cells with bSMase itself led to elevated ceramide formation, which did not translate into induction of apoptosis. However, a combination of Pc 4-PDT + bSMase induced a significant apoptotic response. Thus, the combined treatment of Pc 4-PDT + bSMase, rather than bSMase alone, was required to restore apoptosis in NPD cells. These data support the hypothesis that SMase is a proapoptotic factor determining responsiveness of cells to Pc 4-PDT.     

Photodynamic therapy induces caspase-3 activation in HL-60 cells

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.     

Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.     

Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis

Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.     

Caspases-3 and -7 are activated in goniothalamin-induced apoptosis in human Jurkat T-cells.

Goniothalamin, a plant styrylpyrone derivative isolated from Goniothalamus andersonii, induced apoptosis in Jurkat T-cells as assessed by the externalisation of phosphatidylserine. Immunoblotting showed processing of caspases-3 and -7 with the appearance of their catalytically active large subunits of 17 and 19 kDa, respectively. Activation of these caspases was further evidenced by detection of poly(ADP-ribose) polymerase cleavage (PARP). Pre-treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked apoptosis and the resultant cleavage of these caspases and PARP. Our results demonstrate that activation of at least two effector caspases is a key feature of goniothalamin-induced apoptosis in Jurkat T-cells.     

Disparate cleavage of poly-(ADP-ribose)-polymerase (PARP) and a synthetic tetrapeptide, DEVD, by apoptotic cells

In the present investigations, we have shown differential cleavage of cellular PARP and a caspase 3-selective synthetic tetrapeptide substrate, Z-DEVD-AFC or Ac-DEVD-AMC using a T lymphoblastoid cell line Jurkat, and its variant clone E6.1(J-E6). Anti-Fas antibody-mediated apoptosis resulted in DNA fragmentation and PARP cleavage in both Jurkat and J-E6 cells. However, unlike Jurkat, J-E6 cells did not cleave a synthetic tetrapeptide substrate efficiently. The failure to cleave the DEVD tetrapeptide by apoptotic J-E6 cells was not due to insufficient expression or processing of caspase 3 in J-E6 cells. Interestingly, when the J-E6 cells were transiently transfected with a cDNA encoding caspase 3, efficient cleavage of Z-DEVD-AFC was achieved. The observations that apoptotic J-E6 cells barely cleaved a synthetic DEVD tetrapeptide, but efficiently cleaved endogenous PARP, potentially at the most preferred DEVD site, suggest that active caspases may have disparate characteristics to recognize substrates presented in different context.     

Unveiling Ga(III) phthalocyanine—a different photosensitizer in neuroblastoma cellular model

Phthalocyanines (Pc) and their metallated derivatives are strongly considered for photodynamic therapy (PDT) possessing unique properties as possible new photosensitizers (PS). We have used toxicological assessments, real‐time monitoring of cellular impedance, and imagistic measurements for assessing the in vitro dark toxicity and PDT efficacy of Ga(III)‐Pc in SHSy5Y neuroblastoma cells. We have established the non‐toxic concentration range of Ga(III)‐Pc, a compound which shows a high intracellular accumulation, with perinuclear distribution in confocal microscopy. By choosing Ga(III)Pc non‐toxic dose, we performed in vitro experimental PDT hampering cellular proliferation. Our proposed Ga(III)‐Pc could complete a future PS panel for neuroblastoma alternate therapy.