Signaling pathways involved in apoptosis induced by novel angucycline antibiotic landomycin E in Jurkat T-leukemia cells

Aim. To study the molecular mechanisms of action of novel anticancer antibiotic landomycin E (LE). Methods. Annexin V/propidium iodide, DAPI (4',6-diamidino-2-phenylindole) staining, Western-blot analysis. Results. LE applied in 2 μg/ml dose (IC50), induced reactive oxygen species (ROS)-dependent splitting of poly [ADP-ribose] polymerase 1 (PARP-1) and DNA Fragmentation Factor 45 (DFF45) proteins involved in DNA reparation. This effect was observed 6 h after the start of treatment and it positively correlated with phosphatidyl serine externalization (early morphological marker of apoptosis). We suggest that cleavage of PARP-1 and DFF45 was mediated by active caspase-7 which is a key effector caspase in the LE-induced apoptosis in leukemia cells. We found that activation of initiator procaspase-10 (involved in receptormediated apoptosis) was the earliest detected event in LE-induced apoptotic signaling pathways; however, this activation was shown to be ROS-independent. We also demonstrated that the induction of apoptosis by LE is accompanied by activation of apoptosis-inducing factor (AIF) in mitochondria. Conclusions. Our data suggest that LE-induced cascade of apoptotic events is started by the initiator caspase-10 which leads to activation of the effector caspase-7 and AIF that is known to induce caspase-independent apoptosis involving ROS generation.


Introduction.
Streptomyces species play a significant role in the production of bioactive natural products, many of which are polyketides, such as anthracyclines and tetracylines [1].The angucyclines are the largest group of polycyclic aromatic polyketides with more than 120 members that is constantly growing [2,3].The most characteristic feature of angucyclines is their uniquely shaped benz [a]anthracene tetracyclic framework with an angularly condensed ring [4].The group is rich in chemical scaffolds and various biological ac-tivities, predominantly antitumor and antibacterial.Yet, none of these compounds have been developed to clinically applied drugs usually due to toxicity or solubility issues [1][2][3].Landomycins are the most perspective group of angucyclines possessing strong antineoplastic potential.
All natural landomycins identified to date share the same aglycon (landomycinone) and vary only in their oligosaccharide chain, a linear glycosidic chain containing only di-and trideoxysugars (b-D-olivose and a-L-rhodinose) [5].They show broad activity against many cancer cell lines with the general tendency that compounds with longer saccharide chains show higher activity [6,7].
The main compound, landomycin A, containing a hexasaccharide side chain, has so far been shown to be the most potent congener and was extensively tested by the National Cancer Institute (USA) towards 60 selected human cancer cell line panel and particularly towards prostate cancer lines [8,9].
Flow cytometry experiments showed that landomycin A specifically blocked cell cycle progression from G1 phase to S phase (DNA synthesis) [8].Structurally unrelated anticancer drugs (e. g. bleomycin, mitomycin C, and neocarzinostatin) possess a similar pattern of cell cycle inhibition.However, in contrast to many clinically useful drugs of a similar structure, like the anthracyclines and chromomycins, landomycins do not bind directly to DNA [4,13,14].Landomycin E whose glycosidic chain contains only 3 deoxysugars, does not possess cell cycle specificity [14], but it strongly impairs mitochondria functions leading to the generation of reactive oxygen species (ROS).Nevertheless, role of mitochondria and ROS in LE-mediated apoptosis remains poorly understood.It needs to be explained whether the LE-induced early generation of ROS in mitochondria can trigger caspase activation, or it is only a supplementary step in general scheme of apoptosis induced by this drug.
The main goal of present study was to investigate in more detail specific apoptotic signaling pathways which are induced by the landomycin E in leukemia cells.This might allow to identify potential molecular targets of LE action in tumor cells.For experiments, cells were seeded into 24-well tissue culture plates («Greiner Bio-one», Germany).The cytotoxic effect of antitumor drugs was studied under the inverted microscope («Biolam-P1», «LOMO», Russian Federation) after cell staining with trypan blue dye (0.1 %).N-acetylcysteine («Sigma», USA) was dissolved in 1 ´ phosphate buffered saline (PBS) and added to cell culture 30 min before addition of anticancer drugs (final concentration 1 mM).Broad caspase inhibitor z-VAD-fmk («BD Pharmingen», USA), 20 mM stock solution in DMSO was dissolved in cell culture medium prior to addition to cell culture to achieve final concentration 100 µM.
FITC-conjugated annexin V («BD Pharmingen», USA) and propidum iodide («Sigma») double staining was performed to detect early apoptotic events under treatment of Jurkat cels by LE.At 1, 3, 6, 12, 24 h after addition of LE Jurkat cells were centrifuged at 2000 rpm, washed twice with 1 ´ PBS, and incubated for 15 min in annexin V binding buffer («BD Pharmingen») containing 1/20 volume of FITC-conjugated annexin V solution and PI (20 µg/ml).10 µl of cell suspension was placed on slides.
DAPI staining was performed to study chromatin condensation in Jurkat cells at various timepoints of LE treatment.1, 3, 6, 12, and 24 h after the addition of LE, Jurkat cells were centrifuged at 2,000 rpm, washed twice with 1 ´ PBS, fixed in 4 % paraformaldehyde so-lution for 15 min at room temperature, and then permeabilized by 0.1 % Triton X-100 solution in PBS for 3 min.After that, cells were incubated with 1 µg/ml DAPI solution (4',6-diamidino-2-phenylindole) («Sigma») for 5 min, washed twice with PBS, 10 µl of cell suspension was added to slides and cover glasses placed.Cytomorphological investigations were performed on Zeiss AxioImager A1 fluorescent microscope.
Results and discussion.LE was shown to be highly active for all studied tumor cell lines of both epithelial and mesenchymal origin, and its effect was comparable with that of doxorubicin and vincristin.Leukemia cells were most sensitive to LE action [11].That is why we used human T-leukemia Jurkat cells in our further studies.
Phosphatidylserine translocation to the external layer of the cell membrane of dying cells measured by annexin V test is considered to be one of the earliest hallmarks of apoptosis [16].Landomycin E (2 µg/ml) induced early phosphatidylserine translocation already at 6 h after treatment of Jurkat cells, while doxorubicin in the same concentration caused PS exposure only at 12 h after the treatment start (Fig. 1, A, see inset).Massive DNA fragmentation was measured by cytomorphological evaluation of apoptotic bodies and hypercondensed chromatine in Jurkat cells using DAPI staining.Such fragmentation has appeared only at 12 h both after LE and Dx treatment (Fig. 1, B, see inset).Thus, LE induces more rapid apoptosis, than Dx, in spi-te of LE's lower cytotoxic activity [11,13,14].Doxorubicin is classified as a topoisomerase II poison [17], although other mechanisms of its action have been also proposed: ROS generation, DNA intercalation, inhibition of nucleic acid synthesis [18,19].Thus, an induction of apoptosis by Dx can be explained by its DNAdamaging nature, while LE may use other pathways taking place more rapidly than at DNA damaging in p53dependent apoptosis.
Mitochondria are perfect candidate for LE targeting.ROS are overproduced by mitochondria under pathological conditions, such as ionizing radiation, heat shock or anticancer drug action [20].ROS scavenger N-acetylcysteine (NAC) was used to study in detail ROS involvement in LE and Dx apoptotic signaling in Jurkat cells.
Pre-treatment of Jurkat cells for 30 min with 1 mM NAC restored number of live cells to control levels at 6 h and 12 h after LE treatment (P < 0,01), while at 24 h no significant effect of NAC at IC 50 concentration of LE (2 µg/ml) could be observed (Fig. 2).It is known, that Dx anticancer activity also involves ROS [18], but in our case, no statistically significant differences between Dx-treated and Dx + NAC-pre-treated cells were found (Fig. 2).
Caspase-dependent signaling pathways were studied by Western-blot analysis.
Activation of key effector caspases -caspase-3 which cleaves enzymes PARP-1 and DFF45, as well as activation of caspase-6 which cleaves nuclear lamina, take place only at 24 h after LE treatment of Jurkat cells (Fig. 3).This was also confirmed by a study of expression of procaspase-3 that was cleaved only at 24 h, while at all time points from 1h till 12 h no signs of its cleavage were detected.However, slight activation of other key effector caspase-7 was observed already at 6 h after LE treatment of Jurkat cells, which perfectly correlated with time-dependent cleavage of PARP-1 and DFF45 (Fig. 3).
In contrast to LE, doxorubicin (2 µg/ml) induced simultaneous activation of effector caspases-3 and -7 at 12 h, while active form of effector caspase-6 appeared only at 24 h after treatment (Fig. 4).We suggest that caspase-7 plays a key role in late stages of LE-induced apoptosis in leukemia cells, while caspase-3 and caspase-6 are not so important.
Pre-treatment of Jurkat cells with 1 mM NAC for 30 min led to inhibition of production of reactive oxygen species under LE treatment and also completely blocked activation of caspase-7 and cleavage of DFF45 and PARP-1 by this enzyme at 6, 12 h, and partly at 24 h (Fig. 3).
However, there were no significant changes in the levels of proapoptotic protein Bax involved in mitochondria-induced apoptosis (Fig. 5).Thus, one can see that ROS do not activate Bax and, thus, their role in LE-induced apoptosis is supplementary, but not initiative one.This can be also confirmed by absence of changes in expression of antiapoptotic proteins of Bcl-2 family -Bcl-X L and Bcl-2 itself (Fig. 5), which clearly indicates that small mitochondrial proteins are not involved in LE-induced apoptosis.NAC pre-treatment also had no effect on their levels in target cells.Also in case of LE cleavage of caspase-9 (key initiator caspase, involved in mitochondria-mediated apoptosis) takes place only at 24 h after treatment (Fig. 5) and cytochrome C release from mitochondria -only at 12 h (data not shown), while effector caspase-7 was activated already at 6 h after LE treatment.This again indicates important, but not initiatory role of mitochondria in these processes.
That is why FAS/CD95 receptor in plasma membrane of cells can be another potential target for this drug [21].Since all landomycins have linear glycosidic chain, they can interact in some way with extracellular part of CD95 receptor or cell glycocalyx and induce receptor-mediated apoptosis via caspase-8 or caspase-10.This suggestion can be indirectly supported by the results of National Cancer Institute testing of landomycin A, landomycin E and landomycin D, where landomycins with longer glycosidic chain had significantly higher antineoplastic activity (see www.dtp.nci.nih.gov).To study this hypothesis, Western-blot analysis was performed with antibodies against initiator caspases-2, -8, -10.
It was found that LE (2 µg/ml, 24 h) induced cleavage of all tested initiator caspases, but at early stages (6 h), caspase-10 was the only active enzyme, and its activation was not blocked by NAC (Fig. 5).Thus, caspase-10 is the only initiator enzyme, which is activated at earliest timepoint by LE, and its activation is not ROS-mediated.So, caspase-10 seems to act upstream of mitochondria in the LE-induced apoptosis.
Doxorubicin is also known to induce ROS production in target cells [18], but this effect was much weaker than that of LE (see Fig. 2).NAC pre-treatment when it is absent (as in case with Jurkat cells where p53 is inactivated) via cleavage of small mitochondrial Bid protein via caspase-8 [22], which was also observed in our case (Fig. 5).Landomycin E led to Bid cleavage only in 24 h which excludes caspase-8-Bid bridge in apoptosis induction.This suggests principal difference of the mechanisms of LE-induced cell death from Dxinduced ones.Most anticancer drugs induce classical caspase-dependent apoptosis.However, several chemotherapeutic drugs of clinical use and some natural agents with anti-tumorigenic properties can also lead to caspase-independent type of cell death potentiated by specific «backup death pathways» [23].Broad caspase inhibitor z-VAD-fmk was used to find out what death pathway is preferentially stimulated by LE in Jurkat cells.Pretreatment of Jurkat cells for 1 h with 100 µM of z-VADfmk attenuated Dx-induced (2 µg/ml) apoptosis for 24 h, but failed to stop LE-induced (2 µg/ml) apoptosis measured by counting annexin V + /PI-cells (Fig. 6).
Apoptosis-inducing factor (AIF) whose level was significantly increased during LE-induced apoptosis (Fig. 5), can be another candidate for key apoptotic mediator.
AIF was the first mitochondrial protein shown to mediate caspase-independent cell death [24].It was initially characterized as a protein confined within the mitochondrial intermembrane space of healthy cells.During apoptosis AIF is released from mitochondria and translocates to the nucleus, where it mediates nuclear features of apoptosis such as chromatin condensation and large-scale (~50 kb) DNA degradation [25].In healthy cells, AIF plays role in oxidative phosphorylation and redox control [26].Thus, AIF can generate ROS playing role downstream in the LE-induced signaling apoptotic pathways.We found a significant increase in AIF level in Jurkat cells treated with LE (Fig. 4), which confirms our suggestion of its role in LE action.
Thus, it is AIF, but not caspase-7, which seems to play a major role at terminal stages of LE-induced apoptosis.This might explain why NAC, even used for 24 h, does not stop death of LE-treated cells.NAC can block activation of effector caspases, but not of AIF, which itself is a source of ROS, and thus NAC pretreatment cannot stop AIF's proapoptotic action.

Fig. 6 .
Fig. 6.Effect of broad caspase inhibitor z-VAD-fmk on survival of Jurkat T-leukemia cells under treatment with landomycin E and doxorubicin (2 µg/ml, 24 h): 1 -no z-VAD-fmk; 2 -+ 100 mM z-VAD-fmk Materials and methods.Landomycin E (95 % purity, according to TLC data) was prepared in the laboratory of Dr. B. Matselyukh at D. K. Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine.Doxorubicin produced by «Ebeve» (Austria) was bought at the local pharmacy.