Cytotoxic and antiproliferative effects of heptaacetyltiliroside on human leukemic cell lines

Leukemia Research 23 (1999) 1021 – 1033 www.elsevier.com/locate/leukres

Cytotoxic and antiproliferative effects of heptaacetyltiliroside on human leukemic cell lines Kostas Dimas a, Costas Demetzos b, Basilios Vaos c, Marios Marselos d, Dimitrios Kokkinopoulos a,* b

a Department of Immunology, Hellenic Anticancer Institute, Athens GR-115 22, Greece School of Pharmacy, Department of Pharmacognosy, Uni6ersity of Athens, Athens, GR-157 71, Greece c Clinical Laboratory, Kesarias 2, Athens, Greece d Department of Pharmacology, Medical School, Uni6ersity of Ioannina, Ioannina GR-451 10, Greece

Received 9 December 1998; accepted 16 May 1999

Abstract The peracetylated derivative of kaempferol-3-O-b-D-(6¦-E-p-coumaroyl) glycopyranoside (tiliroside) (1a) was tested for its cytotoxic and cytostatic activity against several human leukemic cell lines. The significant cytotoxic activity of this derivative, prompted to an additional examination on some of the cell lines used. The effect on the uptake of [3H]thymidine as a marker of DNA synthesis and on the cell proliferation, was investigated as well as the morphology of the cells and the kind of death induced, using the Wright-Giemsa dye and horizontal agarose-gel electrophoresis. Flow cytometric experiments of 1a on some leukemic cell lines was also performed. Compound 1a showed a significant antiproliferative effect as soon as 1 h of continuous incubation at all cell lines tested. Cells were killed, through the process of apoptosis and the appearance of the apoptotic signs was time and dose-dependent, while from the flow cytometric experiments, a synchronisation (through a delay probably in the G0/1 phase) of the cells seems to take place. © 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Tiliroside; Human leukemic cell lines; Apoptosis; Cytotoxic/cytostatic effect

1. Introduction Flavonoids are one of the classes of natural products, widely distributed in plants. It has been found that components of this kind have many pharmacological functions such as antimicrobial, antitumor, antiviral, central vascular system and enzyme inhibiting activities [1 – 3]. Kaempferol-3-O-b-D-(6¦-E-p-coumaroyl) glycopyranoside (tiliroside) (1) isolated from natural sources [4–6], is known to be inactive against a panel of human cell lines [4,7]. Incubation of 1 with Aspergillus nidulans produced 7-methylether tiliroside (2), which was also inactive against human leukemic cells lines [8]. The peracetylated derivative of 2, exhibited better cytotoxic activity and arrested DNA synthesis. Thus taking

into account that improvement, compound 1 was acetylated and yielded the heptaacetyltiliroside (1a), in order to study its effects on the same panel of human leukemic cell lines [8]. Furthermore the morphology of the cells and the kind of death induced, was investigated and a flow cytometric study of its action is presented.

Abbre6iations: CPM, counts per minute; DAPI, 4%,6%-diamidin-2phenylindol dihydrochloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; PBML, peripheral blood mononuclear leukocytes. * Corresponding author. Present address: G. Papandreou str. 110, Zografou 15773, Athens, Greece. Tel.: +30-1-7487233; fax: +30-17487233. E-mail address: [email protected] (D. Kokkinopoulos) 0145-2126/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 9 9 ) 0 0 1 2 4 - 1

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PBML, these were treated with 10 mg/ml of PHA-P (Sigma Chemical Co.) for 3 days.

2. Materials and methods

2.1. Synthesis of 1a

2.3. Determination of toxicity

Compound 1, was dissolved in Ac2O-Py and left for 48 h at room temperature to yield heptaacetyltiliroside [4].

2.2. Cell cultures Compound 1a, was tested for its cytotoxic activity on several human leukemic cell lines. The following cell lines have been used: CCRF-CEM [9], MOLT3 [10], H33AJ-JA13 [11], HUT78 [12], H9 [13],(T cells), KM3 [14], NAMALWA [15], DAUDI [16], SDK [17], JIYOYE [18], CCRF-SB [19] (B cell lines), HL60 [20] (promyelocytic cell line), K562 [21] (proerythrocytes), U937 [22] (monocytes). All cell lines were maintained as exponentially proliferating suspension cultures in RPMI-1640 medium (Gibco Europe Ltd., Scotland), supplemented with 10% heat inactivated fetal calf serum (Myoclone Gibco), 2 mM L-glutamine (Gibco) and 50 mg/ml gentamycin and incubated at 37°C, in a humidified atmosphere with 5% CO2. All subsequent incubations were carried out under these conditions. Peripheral blood mononuclear cells (PBML), were obtained from the peripheral blood of normal volunteers after Ficoll-Hypaque centrifugation [23]. For activating

Table 1 In vitro cytotoxicity of acetylated tiliroside on leukemic cell linesa Cell lines

IC50 (mg/ml)

T cell lines CCRF-CEM MOLT3 H33AJ-JA13 HUT 78 H9

12.6 8.9 16.1 7.4 6.5

B cell lines KM3 NAMALWA JIYOYE DAUDI CCRF-SB SDK

6.9 37.4 11.4 8.4 12.5 44.4

Granulocytic K562

48.1

Promyelocytic HL60

11.0

Monocytic U937

13.9

a Vincristine was used as control and exhibited an IC50B1 mg/ml in cell lines tested.

To determine the induced cytotoxicity, exponentially growing cells from each cell line, and resting or activated PBML (1× 106 cells/ml), were incubated with compound 1a for 48 h, in 96-well flat-bottomed microplates. Cultures used as controls contained an equivalent amount of DMSO (negative) or vincristine (Vincristine sulphate, Pharmachemie B.V., Haarlem Holland) (positive). The IC50 for each cell line was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)2,5 diphenyltetrazolium bromide) method [24,25]. The optical density was measured with an ANTHOS HT II Microelisa reader, using a test wavelength of 550 nm. Viability of the cells was assessed by trypan blue dye exclusion, at the beginning of the incubation time and was always greater than 98%.

2.4. Trypan blue exclusion Cell death, due to drug was determined by the Trypan blue dye exclusion test. Cells were incubated with 20, 10, or 2 mg/ml of the compound for 48 h and trypan blue-excluding cells were counted by hematocytometer, on cell aliquots removed from cultures at 1, 4, 24, 48 hours after the addition of the compound 1a. Viability of untreated and DMSO treated cells was also assessed and was always greater than 95%. Vincristine at two concentrations (5 and 1 mg/ml), was used as positive control.

2.5. Cell proliferation All tests on the samples were evaluated at three concentrations 2, 10 and 20 mg/ml after 1, 4, 24 and 48 h. Cells were incubated with 10 mCi of [3H]thymidine (Amersham, UK), added 1 h before the end of each interval and harvested in an automatic cell harvester. The amount of radioactivity incorporated into macromolecules was measured in a liquid scintillation counter (Packard IL) and expressed as counts per minute (CPM). The same controls as for trypan blue assay were used. In both trypan blue test and cell proliferation assay, data represent the mean of experiments done in triplicates analyzed by a two-tailed Student’s t-test. PB 0.05 was considered significant.

2.6. Light microscopy and electrophoresis Exponentially growing cells (5× 105 cells/ml) from MOLT3, H33AJ-JA13 and HL60 cell lines were incubated with 20 or 10 mg/ml of 1a. Control cultures with

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Fig. 1. Effect of 1a on viability (A, C) and DNA synthesis (B, D) of MOLT3 and H33AJ-JA13 cell lines. The cells were incubated for 1, 4, 24 and 48 h, in the presence of 1a. Viability and DNA synthesis were assayed as described in Section 2. The values represent means 9S.D.

DMSO (negative control) or etoposide (Vepesid, Bristol-Myers SQUIBB, Germany) treated cells (positive control [26–28]) were also tested in parallel. After 8 and 24 h of incubation aliquots from each culture were removed, fixed with cytospin and 70% methanol onto microscopic slides, stained with Wright-Giemsa dye and observed under a light microscope (1000 × magnification). DNA from the above cell lines was analysed for endonucleolytic DNA damage, using horizontal agarose gel electrophoresis. At 8 and 24 h, cell aliquots (2 × 106 cells) were collected, washed and the cells were lysed with TNE buffer (50 mM Tris – HCl, 0.15 M NaCl, 5 mM EDTA and 0.5% SDS; pH 8). DNA was extracted, purified as described by Maniatis et al. [29] and analyzed on 1.2% horizontal agarose gels in TBE

buffer (89 mM Tris, 89 mM borate, 0.25 mM EDTA; pH 8.0). Electrophoresis performed at 2.5 V/cm and DNA was stained with ethidium bromide (Sigma) and visualized under UV.

2.7. Flow cytometric study Cells from MOLT3 and H33AJ-JA13 were incubated with 20 and 10 mg/ml of 1a for 4, 8, 24,and 32 h extended to 48 and 56 h for the concentration of 10 mg/ml. DMSO or 10 mg/ml etoposide was added, were used as controls. At the given times aliquots (1×106 cells) were removed and the cells harvested by centrifugation, resuspended in PBS, washed and finally resuspended in ice-cold 70% ethanol. Afterwards DAPI (4%,

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6%-diamidin-2-phenylindoldihydroclorid) (Boehringer Mannheim, Germany) at a final concentration of 1.0 mg/ml was added and cells were analyzed for DNA content by quantitation of green fluorescence in a Partec PAS III i flow cytometry system (Partec GmbH, Mu´nster, Germany). At least 10 000 events for H33AJJA13 and 16 000 for MOLT3 were counted. Single parameter histograms were analyzed using the program supplied from the manufacturer.

stimulated PBML from normal donors, where exhibited an IC50 at 20 mg/m and 19, respectively, after 48 h of continuous incubation. The results of the activity of the flavonoid on the leukemia cell lines are summarized in Table 1. Most cell lines tested at this phase exhibited IC50s lower than 20 mg/ml after the same incubation period. The most resistant was the cell line K562 (granulocytic, highly undifferentiated cells), while the most sensitive were the mature T-cell line H9 (T-ALL, single positive, CD4+ cells) and the pre-B cell line KM3 (c-ALL) with IC50 s 6.5 and 6.9 mg/ml, respectively.

3. Results

3.2. Effect on cell growth and DNA synthesis 3.1. Cytotoxic acti6ity on human leukemic cell lines Compound 1a was tested first on resting and PHA-P

Figs. 1 and 2 show the effects of 1a on cell growth and DNA synthesis of the two T and two B cell lines,

Fig. 2. Effect of 1a on viability (A, C) and DNA synthesis (B, D) of JIYOYE and DAUDI cell lines. The cells were incubated for 1, 4, 24 and 48 h, in the presence of 1a. Viability and DNA synthesis were assayed also as described in Section 2. The values represent means 9S.D.

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Fig. 3. Light microscopy examination of H33JA-AJ13 exposed to 1a and etoposide (see Section 2) (1000 × ). (A) DMSO treated cells (had no difference with untreated cells-not shown). (B) Etoposide treated cells after 8 h. (C) Cells treated with 20 mg/ml of 1a after 8 h and (D) after 24 h. (E) Cells treated with 10 mg/ml of 1a for 8 h and (F): for 24 h.

used for this kind of experiment. Viability of all cell lines declined at levels lower than 60% of the control, after a 24-h continuous incubation with 20 mg/ml of the compound, and even below 20% after 48 h (Figs. 1 and 2A, C). Cells were more resistant at 10 mg/ml, where, however, viability was lower than 80% in all cell lines tested. Cell survival rate was unaffected after 48 h treatment with 2 mg/ml of the compound. On the contrary DNA synthesis, was strongly affected at all doses tested. DNA synthesis fell at extremely low levels, as soon as 1 h after the addition either 20 or 10 mg/ml of the compound and to undetectable levels after 24 h, even at the more resistant H33AJ-JA13 cells (Fig. 1B, D and 2B, D). At 2 mg/ml, DNA synthesis declined more slowly and fell at 80% in H33AJ-JA13 and below

60% of the control in the rest of cell lines at the end of the incubation period.

3.3. Morphological changes and assessment of DNA clea6age The morphological examination of the cells from three leukemic cell lines, two of the T-lineage (H33AJJA13 and MOLT3) and one from the promyelocytic (HL60) revealed severe morphological changes (Figs. 3–5). Reduction in cell volume, chromatin condensation and fragmentation of the nuclei — a morphology consistent with apoptosis — were evident as early as 8 h of incubation with 20 mg/ml of the flavonoid (Figs. 3–5C).The phenomenon was getting more intense after

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a 24-h incubation with the same concentration (Figs. 3 – 5D). Such morphological changes were also observed in etoposide treated cells (Figs. 3 – 5B) but not in DMSO-treated cells (Figs. 3 – 5A). Apoptotic signs were also obvious at 10 mg/ml-treated cells (Figs. 3 – 5E, F). DNA extracted from H33AJ-JA13 cells treated with 20 mg/ml of compound, exhibited an intense ladder pattern, with integer multiples of roughly 180 base pairs (nucleosome-like fragments), as soon as 8 h after the start of treatment (Fig. 6C; lanes 4, 6). The same pattern exhibited also, although not so intense, when the cells treated with 10 mg/ml for the same period (Fig. 6C; lanes 5, 7). Nucleosome-like DNA cleavage induced also in HL60 cells treated with both concentrations (Fig. 6B). Additionally the intense of this nucleosomal ladder was increased in a dose- and time-dependent manner. Similar endoucleolytic damage was also induced in DNA extracted from H33AJ-JA13 and HL60 cells treated with 20 mg/ml of etoposide for 8 h (Fig.

6B, C; lane 3), but not in DMSO-treated cells (Fig. 6B, C; lane 3). No DNA ladder was observed in MOLT3 cells treated with either the flavonoid or with etoposide (Fig. 6A).

3.4. Flow cytometric study Figs. 7 and 8 represent the data obtained from the flow cytometer for H33AJ-JA13 and MOLT3, respectively. Most obviously a subdiploid population appeared, as soon as 4 h after the start of the incubation in both cell lines and doses tested. The population with sub G0/1 DNA content was well separated from the G0/1 phase in H33AJ-JA13 (Fig. 7), but on the contrary it was to close in G0/1 peak in MOLT3, suggesting that in this cell line there is indeed DNA cleavage, which however does not follow the oligonucleosomal pattern. That for, probably, we did not observe a ladder pattern in DNA electrophoresis. MOLT3 cells was also more

Fig. 4. Light microscopy examination of MOLT3 exposed to 1a and etoposide (see Section 2) (1000 ×). (A – F as in Fig. 3).

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Fig. 5. Light microscopy examination of HL60 exposed to 1a and etoposide (see Section 2) (1000 ×) (A – F as in Figs. 3 and 4).

sensitive to the action of the acetylated tiliroside, undergoing massive degeneration after 32 h of treatment with 10 mg/ml (Fig. 8D). In H33AJ-JA13, which were more resistant, the treatment with 10 mg/ml, revealed however that a synchronisation effect took also place. After a perturbance of DNA content at the first 4 h of incubation, the sub G0/1 peak was clearly separated. The remaining viable cells were accumulated at G0/1 phase until 24 h (56.3 against 49.0% of the controls), while 8 h latter (at 32 h incubation with that concentration) a release occured and an increase of the G2/M phase begun (20.9% at 48 h against 14.5% of the controls). At 56 h G2/M begun to decrease slowly and G0/1 to slightly increase. Taking into account that G2/M was not largely affected, we can assume that the apoptotic cells most probably originated from the other two cell cycle phases. The huge apopptosis however (the apopptotic peak was increased at 40.2% at the end of

the incubation period) was not allowed to us a further evaluation of the cell cycle analysis data. 4. Discussion Tiliroside (1) is known to be an inactive product against a panel of human cell lines [4,7]. In a previous study however we observed that the acetylation of 7-methylether tiliroside (2) (a biotransformation product of tiliroside) produced the derivative 2a, which showed a better cytotoxic activity. Further examination revealed that the compound 2a exhibited significant antiproliferative effects. That observation led us to test the peracetylated derivative of tiliroside. From the results it is obvious that the acetylation produced a compound by far more potent than the parent compound. Compound 1a exhibited a high antiproliferative activity, which observed 1 h after the incubation

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started. Moreover 1a suppressed DNA synthesis in a dose and time dependent manner. There was not observed however, a cell lineage or maturity phase specifi-

city. The dramatic change in the action of 1a compared to tiliroside (1), led us to investigate furthermore the kind of death compound induced. The morphological

Fig. 6. (A) Agarose gel analysis of 1a induced DNA cleavage in MOLT3; (lanes 1, 2) DNA extracted from untreated and DMSO treated cells respectively; (lane 3) DNA extracted from cells treated with 20 mg/ml of etoposide after 8 h; (lane 4, 6) DNA from cells treated with 20 mg/ml of 1a after 8 and 24 h of incubation respectively; (lane 5, 7) DNA after treatment with 10 mg/ml for 8 and 24 h, respectively; lane 8: Hind iii standards. (B) Agarose gel analysis of 1a induced DNA cleavage in HL60: (lanes 1 – 7) as in 6A; (lane 8) DNA from H33JA-AJ13 treated with 20 mg/ml of etoposide for 8 h. (C) Agarose gel analysis of 1a induced DNA cleavage in H33JA-AJ13 (lanes 1 – 8 as in 6A).

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Fig. 7. DNA histogramms of H33JA-A13 cells: (A, B): histograms of untreated and DMSO treated cells. (C) Cells treated with 10 mg/ml of etoposide after 8 and 24 h (D) histograms of cells treated with 20 mg/ml of 1a and (E) with 10 mg/ml at different time intervals. (for details see Section 2). Arrows show the cells with sub G0/1 DNA content.

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Fig. 7. (continued)

examination of the cells and the DNA electrophoresis revealed, that 1a killed cells activating the apoptotic machinery of cells, in a dose and time dependent man-ner. The flow cytometric analysis revealed also a population with sub G0/1 DNA content, as early as after 4 h of incubation. However at that time, the trypan blue test, revealed that more than 95% of the cells, treated with both concentrations, excluded the dye. Thus plasma membrane integrity was intact at a time when the genome had already sustained serious damage, which is indicative of death through apoptosis. Addi-tionally, a synchronisation of cells was observed in H33AJ-JA13, but the extensive apoptosis did not allowed a clear cell cycle phase specificity to be established. Unlikely many other flavonoids [2,3,30 – 32], derivatives of kaempferol are very poorly studied. Thus the

amount of data concerning the activity of that flavonoids (and especially of their glucosides), is too small for establishing a clear explanation either for their mechanism of action or for why acetylation changes so dramatically the action of studied flavonoid glucosides. However other relative flavonoids, such as quercetin [30,31] and genistein [32] has been already observed to affect cell cycle progression and growth of cells. A number of enzymes have been proposed as the target molecules (such as p34cdc2 and cyclins [30–32], protein kinace C [33], tyrosinespecific protein kinases [34]), which could very possibly be also targets for kaempferol derivatives. Continuing experiments on that group of flavonoids in our laboratories, we hope to clarify as more as possible aspects of their largely unknown mechanism of action.

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Fig. 8. DNA histograms of MOLT3 cells: (A, B): histograms of untreated and DMSO treated cells. (C) Cells treated with 10 mg/ml of etoposide after 8 and 24 h. (D) Histograms of cells treated with 20 mg/ml of 1a and (E): with 10 mg/ml at different time intervals (for details see Section 2). Arrows show the suspected apoptotic cell population.

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Fig. 8. (continued)

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