comparative investigations of cytotoxic action of …ﬂuence of both drugs to the change in number...

CARYOLOGIA Vol. 61, no. 1: 26-44, 2008

INTRODUCTION

Taxol (paclitaxel) isolated from Pacific yew tree Taxus brevifolia (WANI et al. 1971) and its semisynthetic Taxotere® (docetaxel) presently be-long to the most effective anticancer drugs used in therapy of many cancerous diseases, including mainly breast cancer (LAVELLE et al. 1995) and in monotherapy of non-small lung cancer (PICCART 1998), therapy of ovarian cancer and, in lower de-gree, of soft tissue sarcomas, malignant melano-ma, non-Hodgkin lymphoma or alimentary canal cancers.

Both drugs belong to diterpene pseudoalka-loids. However, Taxotere shows higher anticancer activity than Taxol and is easier soluble in water,

which is important in clinical tests and anticancer therapy (GUERITTE – VOEGELEIN et al. 1991).

Reports indicate that both Taxol and Taxotere inhibit cell divisions due to their ability to stabilize microtubules, preventing tubulin depolymeriza-tion (SCHIFF et al. 1979), (MANFREDI and HORWITZ 1984). However, these works do not describe the details of the process following Taxotere treat-ment, and there are no data regarding changes of the phase index or induction of chromosome aberrations and micronuclei. The mechanism of action of both drugs is believed to consist in their binding to microtubules, in particular with b-tubulin, which enhances formation of micro-tubules and leads to decrease of the amount of free tubulin dimmers in cytoplasm (HAMEL et al. 1981), (SCHIFF and HORWITZ 1981). This inhibits cell divisions due to abnormal functioning of the mitotic spindle, as microtubules do not undergo depolymerization and they cannot transport chro-matids to the spindle poles (SCHIFF and HORWITZ 1980). As a consequence, cells are arrested at G2/

Comparative investigations of cytotoxic action of anticancer drugs Taxol and Taxotere on meristematic cells of Allium cepaMajewska1* Agnieszka, Elwira Sliwinska2, Małgorzata Wierzbicka1, Mieczysław Kuras1

1 Department of Ecotoxicology, Warsaw University, 1 Miecznikowa Str, 02-096 Warsaw, Poland.2 Department of Genetics and Plant Breeding, University of Technology and Agriculture, 85-796 Bydgoszcz, Poland.

Abstract — Taxotere and Taxol are anticancer drugs used mainly in breast cancer therapy. Although both of them act by stabilizing microtubules, reports indicate differences in their action, mainly connected with their binding affinity to tubulin, sensitivity of cancer lines to the drugs, gene expression induction, which may indicate a different mechanism of action of both drugs. The present work was focused on specific character and differences in action of Taxotere and Taxol on subsequent mitotic phases, amount of chromosome aberration and micronuclei, and on ultrastructure of meristematic cells of Allium cepa.Analysis of the mitotic index following treatment with Taxol and Taxotere revealed differences in their effect on mitotic activity of cells and changes in chromosome morphology. Taxol inhibited cell divisions and caused changes in chromosome morphology, having however no mutagenic character. Treatment with Taxotere did not inhibit cell divisions, but it caused accumulation of a large number of chromosome aberrations and micronuclei. The drugs showed similar effectiveness in arresting cells at G2/M phase and led to higher heterochromatinization of cell nu-clei, partial degradation of mitochondria, which may largely affect the energetic state of cells.

Keywords: Allium cepa, cell cycle, chromosomal aberrations, degradation of mitochondria, micronuclei, Taxol, Taxotere.

* Corresponding author: phone + 48-22-55 42 001; fax + 48-22-55 42 022; e-mail: [email protected]

comparative investigations of cytotoxic action of taxol and taxotere 27

M phase (SCHIFF and HORWITZ 1980), (FUCHS an JOHNSON 1978) and enter apoptosis (HALDAR et al. 1997).

Although both Taxol and Taxotere inhibit cell divisions by stabilization of microtubules, many works indicate differences in their action. They pertain to the capacity of both compounds to transform protofilaments into microtubules. Tax-otere proves to be a twice stronger inhibitor of microtubule depolymerization (GELMON 1994), it shows higher affinity to binding to tubulin and it induces higher MT polymerization than paclitaxel (RINGEL and HORWITZ 1991). Under some condi-tions, Taxol and Taxotere also cause different in-duction of gene expression (BURKHART et al. 1994). In macrophage RAW 264.7 line, it is Taxol and not Taxotere that induces overexpression of the TNF-a gene and the expression of prostaglandin synthetase-2 gene (MOOS et al. 1999); moreover, it shows differences in induction of other genes of interleukin 1-b, cyclooxygenase-2, transcription agents ATF-4, Crg-2, Krox-24 and others (MOOS and FITZPATRIK 1998). Different induction of gene expression leads to presumption that, in at least several cell lines, taxanes act independently from microtubules, and that the mechanism of Taxol action may be slightly different from the one of Taxotere.

Moreover, there are no explicit results on ul-trastructural changes following administration of Taxotere and Taxol. As has been shown so far, stabilization of MTs causes translocation of some organelles, such as centrosomes, Golgi structures and lysosomes, and their chaotic topography (e.g., lack of centrosomes at the nuclear envelope), and blocking organizational ability of microtubule organization center (MtOC) by reduction of the critical concentration of tubuline (BRABANDER et al. 1981). Additionally, disturbances in microtu-bule system induced by paclitaxel may influence the transport inside and between cells and the se-cretion of some compounds (RAINEY et al. 1985). Paclitaxel disturbs functioning of Golgi apparatus and causes changes in endosomes and lysosomes motion (MOORTELE et al. 1992). However, the in-fluence of both drugs to the change in number and area of mitochondria in cell, and their ener-getic state, is yet unknown.

High demand for anticancer drugs is often a reason for their introduction to the phase I of clinical studies following a series of investigations focused mainly on their antiproliferative action connected with inhibition of tumor growth. The precise mechanism of their anticancer action is of-ten elucidated only after their admission to thera-

peutic use. This is the case with taxanes, whose mechanism of action is not fully known, and re-ports are often contradictory and disputable. It appears that the basic mechanism of action of both drugs is similar; however, the vulnerability of cancers and cancer lines is largely varied. This leads to a hypothesis that the observed differences in therapeutic efficacy of these drugs can result from differences in their mechanism of action.

Both drugs (and particularly Taxotere) cause severe side effects (ROWINSKY et al. 1993); how-ever, the reasons for their toxicity still remain un-clear. The side effects reduce the effectiveness of anticancer therapy. The drugs are often used in combination with other cytostatics in a so-called combination therapy, in order to enhance the an-tineoplastic effect and reduce the side effects of taxane therapy. Although many of those combina-tions are more effective, their side effects are not reduced, and may be even stronger. High toxicity of taxanes gives rise to a question on their influ-ence on the mitotic process and the amount of chromosome aberrations. Apart of tests on Dro-sphila melanogaster (CUNHA et al. 2001), no in-vestigations on mutagenicity of those compounds have been carried out so far.

In 1982 BAJER et al. investigated the influence of Taxol on mitosis and karyokinetic spindle of Haemanthus katherinae endosperm cells (BAJER et al. 1982). They showed that Taxol, in a wide range of concentrations (5x10-5, 5x10-6, 10-6, 10-7, 10-8), caused prolongation of mitosis, but it did not totally inhibit cell divisions. BASKIN et al. car-ried out investigations on MT organization and morphology in roots of Arabidopsis thaliana and the polarity of their growth following treatment with 1 and 10mM Taxol (BASKIN et al. 1994). The investigations showed that Taxol enhances the ra-diant growth of roots in their elongation area, but it has no effect on the meristematic area. BALUŠKA et al. in their tests of maize root meristematic cells showed that a 24h treatment with 30-100mM Tax-ol led to enlargement of cell nuclei and deconden-sation of chromatin (BALUŠKA et al. 1997). These cells were unable to divide and their nuclei got fragmented due to previous disturbances in the process of cell divisions.

Our previous investigations (MAJEWSKA et al. 2000; MAJEWSKA et al. 2002) regarding action of extract from yew tree Taxus baccata var. Elegan-tissima with paclitaxel content of 81, 6mg/g fresh mass on meristematic cells of Allium cepa L. root tips showed that, in the initial phase of incuba-tion, cells in changed prophases and C-meta-phases were accumulated, which was followed by

majewska, sliwinska, wierzbicka, kuras 28

inhibition of cell divisions due to arrest of the cell cycle at G2/M phase. The applied Allium test has been long used for investigations of genotoxicity of many compounds (ATEEQ et al. 2002, KHORA et al. 1997). Its usefulness in examining mutagenic-ity of different compounds is undisputable. It is a perfect tool for a safe, quick, easy and reliable evaluation of disturbances in the process of cell divisions and has been used by, inter alia, (AN-TOSIEWICZ 1990; PIETROSIUK et al. 1999; MAJEW-SKA et al. 2000; MAJEWSKA et al. 2002; ATEEQ et al. 2002). Moreover, its substantial value is sup-ported by the fact that effects of mutagenic action of many compounds on the Allium test cells and mammalian cells are very similar (FISKESJÖ 1971; FISKESJÖ 1979).

The present studies were aimed at description of specific character and differences in action of Taxotere and Taxol with respect to the phase of mitosis, and investigation of the proportion of chromosome aberrations and micronuclei in the Allium test, also known as Levan’s test, and addi-tionally, examination of effects of the toxic action of both drugs on ultrastructure of meristematic cells, including morphometric measurements.

MATERIAL AND METHODS

Drugs tested – Taxotere® produced by Rhone Poulenc Rorer (France) and Taxol (Bristol Mey-ers Squibb) were dissolved in 100% DMSO (di-methyl sulphoxide), and their final concentra-tions – Taxotere: 26, 160, 640mg/ml, and Taxol: 16 and 26mg/ml, were obtained by adding distilled water. DMSO concentration in final solutions of the drugs did not exceed 0.5%. According to re-sults obtained both for plant cells (MAJEWSKA et al. 2000; MAJEWSKA et al. 2002) and HL-60 cells (Majewska et al. 2006), 0.5% DMSO is neutral for cells, i.e., it does not induce structural chang-es. This DMSO concentration was the control in the present studies.

Plant Material – The study was carried out on adventitious root tips of onion (Allium cepa L.), grown according to the method described by MAJEWSKA et al. 2000 and 2002. The onions were obtained from the Plant Genetic Resourc-es Laboratory, Research Institute of Vegetable Crops (Skierniewice, Poland). When the roots were approximately 4cm (±1cm) long, they were transferred into the Taxol and Taxotere solutions for the period of incubation. Control roots were

grown in 0.5% dimethyl sulphoxide (DMSO) solution. In the course of incubation, 2mm long root tips were collected to study mitotic, phase and micronuclei indices and morphological and ultrastructure changes. Only meristematic cells of the cortex were investigated.

Microscopic preparations for mitotic, phase, micro-nuclei indices and morphological changes of chro-mosomes – The material for analysis of mitotic and phase indices, collected after 3, 6, 12, 24h of incu-bation in drug solutions was prepared according to the method used in the work of MAJEWSKA et al. 2000; (MAJEWSKA et al. 2002) i.e., by fixation in a Carnoy fixer (CARNOY 1886), and further mac-eration and staining in 2% acetoorceine. On the basis of squash preparations, mitotic (IM), aber-rations, micronuclei and phase indices (IP) were counted according to the method of LOPEZ-SAEZ and FERNANDEZ-GOMEZ (1965). 4 root apices of 3 different onions were collected for every variant. Calculations and microphotographs were made using light microscope (Nikon).

Flow cytometry – Root tips about 2mm long were cut off after 6, 12 and 24h of incubation in 26mg/ml of Taxotere and 26mg/ml of Taxol. In this ex-periment, three onions were used for each treat-ment. For cytometric analysis, a two-step proce-dure was used (OTTO 1990). Three root tips from each onion were chopped into small pieces in a Petri dish with 250ml of pretreatment solution (0.1M citric acid monohydrate, 0.5% Tween 20) and incubated for 3min. for the isolation of nu-clei. Next the suspension was passed through a 50mm mesh nylon filter, and 1ml of staining solu-tion (0,4M Na2HPO4×12 H2O, 2 mg/ml 4’6- dia-midino-2-phenylindole; DAPI) was added. For each sample, DNA content of 8000-10000 nu-clei was measured with a Partec CCA (Münster, Germany) flow cytometer. From obtained histo-grams, the average G2/G1 ratio was calculated. The results were presented in charts as 100% of control.

Ultrastructural studies – 2mm root tips, excised after 6, 12 and 24h of incubation in 26mg/ml of Taxol and Taxotere, were fixed according to the procedure used by MAJEWSKA (MAJEWSKA et al. 2000; MAJEWSKA et al. 2002), i.e., by fixation for 2hs in 2.5% glutaraldehyde at pH 7.2 (cacodyl-ate buffer), and then in 1% OsO4, dehydration in ethanol, and by propylene oxide, embedding in the Epon-Spurr mixture of epoxide resins. Ultra-thin sections 60-80nm) were cut with a ultrami-


crotome produced by LKB (Sweden). They were contrasted with uranyl acetate and lead citrate according to REYNOLDS (1983). Observations and electronograms of the ultrastructure were taken with a JEOL JEM 100C transmission electron mi-croscope (TEM).

Additionally, morphometric investigations were carried out. In cell sections, the mean number of mitochondria, plastids and their area per 1 cell was counted using Lucia Measurement Program (Nikon). The results were presented in charts as 100% of control. In all investigations, 3 root tips from each of 3 onions for each time pe-riod were collected. In each tip, 20 meristematic cortical cells were tested.

All data were analyzed by one-way analysis of variance (ANOVA), followed by the least-signifi-cant Student-Newman-Kelus test, if significance was indicated by the ANOVA.

RESULTS

Mitotic index after treatment of Taxol and Taxoter – Treatment with Taxol, in concentrations of 16 and 26mg/ml, resulted with marked and propor-

tional decrease in mitotic activity of cells (Fig. 1). The lower concentration of the drug (16mg/ml) led to reduction of the mitotic index (IM) by 50% in comparison with the control as soon as after 3h of incubation (Fig. 1). Starting from 6h of incubation, IM gradually increased until 24h of incubation, when it exceeded the control value. Treatment with the higher concentration of Taxol (26mg/ml) caused gradual reduction of the IM during 24h incubation, reaching its lowest values (2.8%) after 12 and 24h (Fig. 1).

Unlike Taxol, even a very high concentration of Taxotere (640mg/ml) did not inhibit the IM (Fig. 1). Moreover, after 6h incubation, in almost all tested concentrations of the drug, IM showed significant statistical increase above the control level. That increase, however, was not correlated with Taxotere concentrations. After 12h incuba-tion in most of the investigated concentrations, the index was significantly slightly lower than the control level, and almost equal to it after 24h.

Phase indices and changes in morphology of chro-mosomes after treatment of Taxol – Treatment with Taxol resulted both in changes of frequency of particular mitotic phases and in distinct percent

Fig. 1 — Changes of the mitotic index of the Allium test cells during 24 h treatment with different concentrations of Taxol and Taxotere.


increase of abnormal mitoses (Fig. 2 and Fig. 4). Changes, described as turbagenic, consisted main-ly in thickening and shortening of prophase and metaphase chromosomes, occurrence of C-met-aphases (with chromosomes chaotically distribut-ed within cells instead of forming the metaphase plate). Scarce clastogenic changes, connected with chromosome thickening during divisions, were observed (according to GRANT 1978 and GRANT 1982). Micronuclei were not observed.

After 3 h incubation in Taxol at concentration of 16mg/ml, a significant increase of the prophase index was noted (Fig. 2A). Among mitotic phases, half of prophases and most of metaphases were changed. Following 6h incubation, the prophase index started to decrease, but the metaphase in-dex and percentage of abnormal mitoses vs. nor-mal mitoses increased. After 12h, proportions of particular mitotic phases were similar to the con-trol ones (Fig. 2A).

Fig. 2 — Changes of the phase index of the Allium test cells following Taxol treatment at the concentrations of 16 µg/ml (A) and 26 µg/ml (B), including abnormal phases. * – statistically significant change of the prophase or metaphase index as compared to the control.


26mg/ml Taxol, after 3 and 6h incubation, in-duced a marked increase of the prophase index in comparison to the control (Fig. 2B). The amount of prophase turbagenic changes was higher than under the lower concentration (16mg/ml). Meta-phase chromosomes were also largely changed and slightly more clastogenic changes were ob-served. Following 12h incubation, the prophase index was markedly lower, but telophase index was elevated, which indicated unblocking of the accumulated changed mitoses and their progress to the final karyokinesis stages.

Phase index and morphological changes in chromo-somes following Taxotere treatment – Application of all tested Taxotere concentrations resulted in very strong disturbances of the cell division proc-ess and significant changes of the phase index, mainly of the metaphase index (Fig 3 and Fig. 4).

The percentage of changed prophases, during the whole experiment and in all Taxotere concen-trations, was significantly lower than in the case of Taxol (Fig. 3). Unlike Taxol, however, incuba-tion in Taxotere resulted in marked increase of the metaphase index (Fig. 3A- C), with was observed even after 3h incubation in all tested concentra-tions. Most of metaphases were abnormal: C-met-aphases (Fig. 4H, L, Ł) or ones showing metaphase turbagenic (Fig. 4J- K) or, more commonly, met-aphase clastogenic changes (Fig. 4G-L). The per-centage of clastogenic changes rose proportionally to the concentration (Fig. 3A-C), while turbagenic changes were fewer. Additionally, morphological changes of anaphase and telophase chromosomes occurred. Anaphase chromosomes often showed features of clastogenic anaphases (with one or more daughter chromosomes outside the mitotic spindle) (Fig. 4M). This resulted in abnormalities at the telophase stage: appearance of telophase bridges (Fig. 4N), uneven formation of phragmo-plast between several nuclei of various sizes (Fig. 4R). That in turn led to abnormal separation of chromosomes and accumulation of numerous mi-cronuclei (Fig. 4O-T). In post-telophase cells con-taining micronuclei, separating fragments of cells with micronuclei were often seen (Fig. 4T), which was a result of the previous formation of phrag-moplast between micronuclei and the nucleus. Bi-nuclear cells occurred as well (Fig. 4 P and T).

Index of micronuclei following treatment with Taxotere – Unlike Taxol treatment, during which almost no micronuclei appear, treatment with Taxotere caused formation and accumulation of a large number of micronuclei (Fig. 5). Their in-

dex increased starting with 3h incubation, and its percentage was dependent on the drug concentra-tion. The highest values of the index were noted following 24h incubation (Fig. 5).

Changes in percentage of cells at G1, S and G2 phases of the cell cycle following Taxol and Taxo-tere treatment – Histograms obtained from cyto-metric analysis of the Allium tests revealed, in all cases, presence of two classes of nuclei, presented as peaks for the contents of 2oC and 4oC DNA and being at phase G0/G1 or G2/M (Fig. 6). A longer incubation in Taxol and Taxotere caused gradual increase of the proportion of G2/M phase cells, and decrease in number of G0/G1 phase cells (Fig. 6). This is a symptom of accumulation of cells with doubled DNA content (Fig. 4C).

In control cells, the G2/G1 ratio was av. 0.5. Both drugs caused its marked, statistically sig-nificant increase (Fig. 7). After Taxol treatment at concentration of 26mg/ml, the ratio increased starting with 3h of incubation, reaching its peak values after 24h (Fig. 7). Treatment with Tax-otere, at the same concentration, caused a sig-nificant elevation of the G2/G1 ratio above the control value only after 12h, with its peak value after 24h.

Ultrastructural changes in cells after Taxol and Taxotere treatment – Changes in cell ultrastruc-ture following treatment with Taxol (26mg/ml) and Taxotere (26mg/ml) were similar, and the dif-ferences pertained mainly to their intensity. Most of the changes after treatment with both drugs ap-peared after a longer incubation period, i.e., 12h and 24h, only after Taxotere treatment after short time of incubation – 3h.

A typical change caused by both drugs was a marked heterochromatinization of cell nuclei, which was particularly distinct after 12 and 24h of incubation (Fig. 8B). Cytoplasm of cells after treatment with Taxol and Taxotere was rarefied as compared to the control. Also a higher vacuoliza-tion of cells than in the control was noted (Fig 8B, Fig.9 A, Fig. 10A and C). Vacuoles were often accompanied by concentrations of very active dic-tyosomes with large vesicles (Fig. 10C) and short, swollen and budding cisterns of smooth and rough endoplasmic reticulum (Fig. 9B).

Both Taxol and Taxotere induced typical changes in ultrastructure of mitochondria. In the control, mitochondria were usually elliptic, with distinct cristae (Fig. 8D). After 6h incubation in Taxol or 3h treatment with Taxotere, they became spherical, with visible clear spaces in their matrix;


Fig. 3 — Changes of the phase index of the Allium test cells following Taxotere treatment at the concentrations of 16 µg/ml (A), 26 µg/ml (B) and 160 µg/ml (C), including abnormal phases. * - statistically significant change of the prophase or metaphase index as compared to the control.


Fig. 4 — Subsequent mitotic changes in control Allium test cells, and following Taxol and Taxotere treatment at the concentration of 26 µg/ml. Preparations squeezed in acetoorceine (light microscope). Bar A-D and F-T 40 µm; Bar E 60 µm. A. Prophase. B. Metaphase. C. Anaphase. D. Telophase in control cells. E. Taxol (26 µg/ml), 6 h of incubation. Changed prophases with shortened and thickened chromosomes, with light patches of karyolymph be-tween them (arrow). F. Taxotere (26 µg/ml), 6 h of incubation. Changed prometaphase with chaotically distributed chromosomes. G-L. Taxotere (26 µg/ml), 6 of incubation. Changed metaphases /C-metaphases with shortened and often thickened chromosomes. Some chromosomes located out of the metaphase plate (arrows). Ł. Taxol (26 µg/ml), 24 h of incubation. A C-metaphase with shortened and often thickened chromosomes (arrow). M. Taxotere (26 µg/ml), 24 h of incubation. Irregular movement of chromosomes in anaphase. Two of them are “lost” (asterisk). N. Taxotere (26 µg/ml), 24 h of incubation. Telophase with chromosome bridge. O. Taxotere (26 µg/ml), 36 h of incubation. Post-telophase following abnormal chromosome segregation; micronuclei. P. Taxotere (26 µg/ml), 48 h of incubation. Binuclear post-telophase cell with a micronucleus. R-S. Taxotere (26 µg/ml), 48 of incubation. Accumulation of micronuclei in cells. T. A post-telophase cell showing asymmetric division of cytoplasm between two nuclei and a micronucleus (arrow) separated during cytokinesis (72 h of incubation).


Fig. 5 — Percentage of cells with micronuclei in the Allium test following treatment with different concentrations of Taxotere.

Fig. 6 — Histograms showing changes of nuclear DNA level in control cells of the Allium test and following 6, 12 and 24 h of treatment with Taxol at the concentration of 26 µg/ml.


the number of cristae was significantly reduced (Fig. 9A-B). These changes were observed until 24h of incubation. A result of Taxotere treatment was appearance of another type of mitochondria, very long and often with a thread-like narrowing in the middle and swollen at their ends (Fig. 9C and D), occurring beside spherical mitochondria. Such modifications were most frequent after a short incubation (3h)in Taxotere.

Similar changes also occurred in ultrastructure of plastids, although less commonly. The plastids were either very electron-dense (Fig. 10A and B), or, mainly after Taxotere treatment, had typically changed shapes (similar to the shape of mitochon-dria) – narrowed in the middle and swollen at their ends (Fig. 10A and B).

Treatment with both drugs resulted in local uneven thickenings of the cell wall. These places often contained concentrations of microtubules (Fig. 10D).

Changes in number and area of chosen organelles – Changes in ultrastructure of mitochondria and plastids were connected with modifications in their number and area. Treatment with both Taxol (at concentration of 26mg/ml) and Taxotere (26mg/ml) resulted in basically similar changes in number and area of chosen cell organelles (Fig. 11-12).

During the incubation, the number of mi-tochondria and plastids gradually decreased, reaching its lowest values after 24h incubation

in treatment with both drugs; however, this ef-fect was much stronger in the case of Taxol (Fig. 11A). A 24h treatment with this compound re-duced the number of mitochondria and plastids almost by half as compared to the control (Fig. 11A), whereas treatment with Taxotere during the same period resulted only in a 20% decrease 20% (Fig. 12A). The number of dictyosomes fol-lowing Taxol treatment oscillated at the control level until 12h, increasing by 20% after 24h (Fig. 11A), while treatment with Taxotere reduced that number particularly after 6 and 24h (Fig. 12A).

The amount of vacuoles changed during incu-bation in Taxol and Taxotere in a different way, depending on the drug used (Fig. 11A and Fig. 12A). In Taxol, that number markedly increased after 12 and 24h incubation (Fig. 11A), while the same period of incubation in Taxotere resulted in its reduction (Fig. 12A).

As the number of mitochondria and plastids decreased after treatment with both drugs, their area enlarged. That increase was much higher fol-lowing treatment with Taxol, which caused the mitochondrial area increase starting from 6h in-cubation and stay at the high level until the end of the experiment, exceeding almost twice the size of control mitochondria (Fig. 11B). In treatment with Taxotere, the mitochondrial area increased as soon as after 3 h, when it reached its peak val-ues (Fig. 12B), but it shrunk further on during the experiment to reach control values. The area of

Fig. 7 — Changes of the G2/G1 ratio during incubation of Allium test cells in Taxol and Taxotere.


Fig. 8 — Ultrastructure of control cells of the Allium test and following treatment with Taxol and Taxotere at the concentration of 26 µg/ml. A. Meristematic cells of the root tip – general view. B. Taxol 26 µg/ml; note the marked heterochromatinization of nuclei; prominent changes in mitochondria, including lucent spaces in matrix; increased vacuolation of cells. C. A control root tip cell; note regular of cortical MTs near the cell wall. D. Control cell – a por-tion of nucleus with moderate chromatin dispersion; electron dense plastids, spherical mitochondria with distinct cristae and dictyosomes; cell wall evenly thickened at its whole length.


Fig. 9 — Ultrastructure of Allium test cells following treatment with Taxol and Taxotere at the concentration of 26 µg/ml. The material was fixed and embedded according to standard methods used in electron microscopy (see Fig. 8). A. Taxol 26 µg/ml; 6 h of incubation. Mitochondria with very large lucent spaces in matrix. Cristae almost absent; electron-dense plastids; large vacuole with membranous structures inside. B. Taxotere 26 µg/ml; 3 h of incubation. Accumulation of spherical nuclei with lucent spaces in matrix and markedly reduced number of cristae; small electron-dense plastids; numerous short and swollen ER cisterns (arrows) and active dictyosomes. C. Taxotere 26 µg/ml; 3 h of incubation. A long mitochondrion with a thread-like narrowing and swellings at the ends (arrows); a portion of nucleus and nucleolus. D. Taxotere 26µg/ml, 3 h of incubation. A very long and narrow mitochondrion with normal structure of cristae; marked vacuolization of the cell.


Fig. 10 — Ultrastructure of Allium test cells following treatment with Taxol and Taxotere at the concentration of 26 µg/ml. The material was fixed and embedded according to standard methods used in electron microscopy (see Fig. 8). A and B. Taxotere 26µg/ml, 12 h of incubation. Changed plastids of shapes not met in the control, with narrow-ing in the middle and swellings at the ends (arrows); spherical mitochondria with marked lucent spaces in matrix. C. Taxol (26µg/ml), 24 h of incubation. Accumulation of very active dictyosomes with numerous vesicles (arrows). D. Taxotere (26µg/ml), 24 h of incubation. Chaotic array of cortical MTs, arranged slantly or perpendicularly to the cell wall; the MTs are crossed in some places (arrow).


Fig. 11 — Changes of the mean number (A) and area (B) (% of control) of chosen organelles of Allium test cells following treatment with Taxol at the concentration of 26 µg/ml. Bars marked with different letters show statisti-cally different values.

plastids reached its highest level after 12 and 21h in Taxol (Fig. 12A), and after 6 and 12h in Taxo-tere.

During incubation in Taxol and Taxotere, the area of vacuoles was enlarged to a similar degree, especially after 24h. It often exceeded the control vacuole area 2-4 times (Fig. 12A-B).

DISCUSSION

Clinical tests and scientific reports indicate ex-istence of significant differences in the efficacy of therapeutic action of Taxol and Taxotere. It is also known that both drugs, and particularly Taxol, cause severe side effects. The aim of the present


study was to find cytological and ultrastructural symptoms of varied therapeutic effect of both drugs. Existing data suggest that the basic mecha-nism of action of both Taxol and Taxotere is simi-lar; however, there are marked differences in sus-ceptibility of tumors and cancer lines. This allows speculating that the observed differences in their therapeutic efficacy may be due to different mech-anisms of their action. Both Taxol and Taxotere therapy results in severe side effects, the origin of which remains unclear. For this reason, further studies are needed, concentrated on a detailed analysis of the mechanism of action of both drugs on one hand, and the possibilities of enhancing

their efficacy with simultaneous reduction of their side effects on the other.

Analysis of changes in the mitotic and phase in-dices and chromosome aberrations – The analysis of the mitotic index under treatment with Taxol and Taxotere showed that Taxol, depending on its concentration, reduced the mitotic activity or inhibited it completely, which was correlated with changes in the phase index. During incuba-tion, accumulation of prophases occurred, most of which were abnormal. Changes in chromo-some morphology were also commonly observed in metaphase cells. Most of them were turbagenic

Fig. 12 — Changes of average number (A) and area (B) (% of control) of chosen organelles in Allium test cells following treatment with Taxotere at the concentration of 26 µg/ml. Bars marked with different letters show sta-tistically different values.


changes, whereas clastogenic ones were scarce. Such changes could not result in formation of mi-cronuclei. So it can be assumed that they were not aberrations, i.e. of mutagenic character. These ob-servations are supported in existing results con-cerning animal cells (SCHIFF and HORWITZ 1981; BRABANDER et al. 1981; WATERS et al. 1996; RIEDER et al. 1994). Treatment with high concentrations of Taxol causes accumulation of cells at the bor-der of metaphase and anaphase stage, preventing them from entering the anaphase stage, which leads to inhibition of divisions. This is a result of stabilization of MTs by paclitaxel. It is known that the MTs in treatment with Taxol do not bind to chromosome kinetochores, which causes chaotic distribution of chromosomes all over the cell in-stead of formation of the equatorial plate (BRA-BANDER et al. 1981; WATERS et al. 1998). Prevent-ing depolymerization of kinetochore microtubules makes the progress of cells to anaphase A and B stage impossible (WATERS et al. 1996). According to RIEDER et al. (1994), lack of normal binding of chromosomes to the karyokinetic spincle before anaphase is a signal for delaying the anaphase un-til the proper binding with the microtubules oc-curs. This way, preventing by paclitaxel of bind-ing the kinetochores with the microtubules may block the further process of mitosis (WATERS et al. 1998).

Treatment with Taxotere resulted in wholly different changes of the mitotic and phase indices. This may be due to its different mechanism of ac-tion. Within 24 hours, none of the concentrations used (even very high ones) resulted in inhibition of cell divisions, but in their peculiar accumulation. This phenomenon was correlated with a marked increase of metaphase index, most of which were abnormal metaphases, mainly clastogenic ones. The mechanism of action of Taxotere seems to be connected with accumulation of cells at the met-aphase stage, showing mainly clastogenic features, which results in formation of numerous micronu-clei during further incubation. Continuation of cell divisions during incubation may result from the fact that some cells accumulated at the met-aphase stage managed to progress through further stages of karyokinesis. Investigations carried out on human cancer lines (JORDAN et al. 1993) show that, in some cases, a prolonged exposition to tax-anes results in breaking the mitotic blockade and progress through further mitotic phases; however, cytokinesis is abnormal, leading to formation of multinuclear cells, unable to live. Such changes were also observed in the present investigations on Allium test cells treated with Taxotere.

Blocking the cell cycle in the M phase was also evidenced by cytometric tests. In cells accumulated at the prophase stage during treatment with Taxol and at metaphase in the case of Taxotere, which did not progress to anaphase, despiralization of chromosomes and formation of interphase nuclei at 4C DNA level occurred. Changes of the G2/M ratio indicate that Taxol causes earlier blockade of the cycle at the G2/M phase, as it increased as ear-ly as after 3 and 6h of incubation, when the most intensive accumulation of the abnormal prophases was observed. Instead, Taxotere induced an in-crease of the G2/G1 ratio above the control level only after 12h, which was correlated with accumu-lation of metaphase cells at that time.

These results correspond with existing reports of investigations of the cell cycle following treat-ment with taxanes, carried out on animal and human cells, where blockade of the cell cycle at the border of G2 and M phase and changes in ploidy of cells occur as well (SCHIFF and HORWITZ 1981).

Taxotere concentrations, giving similar effects in the present work, were much higher than the concentration of Taxol. However, the existing data (LAVELLE et al. 1995; PICCART 1998) indicate that Taxotere is a much more efficient cytostatic than Taxol. Its twice lower doses are able to inhibit proliferation of cancer cells. This made us expect a similar result in the present investigations. How-ever, even high doses of Taxotere did not inhibit the cell divisions in the Allium test within 24h of incubation. This indicates species-connected dif-ferences in the reaction to Taxotere, and possibly a different mechanism of action of Taxotere than the one of Taxol.

The influence of Taxotere on the Allium test cells caused their accumulation at metaphase and induced a large number of clastogenic changes in chromosomes of that phase. This led to appearance of numerous micronuclei. Unlike Taxol, the action of Taxotere is strongly mutagenic. Few studies on genotoxicity of Taxol (TINWELL and ASHBY 1994) and mutagenic effect of Taxotere on Drospohila melanogaster (CUNHA et al. 2001) have been published so far. These works evidence that the micronuclei are associated with previous disorganization of the karyokinetic spindle and the clastogenic action of Taxotere, leading to its aneugenic effect (CUNHA et al. 2001), which was also supported by our results presented herein.

Analysis of changes in cell ultrastructure and of morphometric investigations – The most typical ul-trastructural changes observed in the present stud-


ies as a result of treatment with Taxol and Taxotere consisted in thinning of cytoplasm by accumula-tion of numerous dictyosomal vesicles, budding short ER cisterns, higher vacuolation. Addition-ally, cell walls were irregularly thickened with MTs depositing at those places, the number, area and ultrastructure of mitochondria was changed, and changes were observed in the number of plastids and condensation of chromatin.

During incubation of the Allium test cells in Taxol and Taxotere, the number of mitochondria and plastids was reduced. This probably resulted from degradation of these organelles and simul-taneous inhibition of their divisions. Also the marked increase of the mean area of mitochon-dria and plastids during the incubation may be due to inhibition of their divisions, which is cor-related with inhibition of cell divisions during the incubation, especially in Taxol.

Degradation of mitochondria and plastids was reflected by their ultrastructure: reduction of number of their inner membranous structures (cristae) and lucent spaces in their matrix. Such changes were often observed as a result of toxic substances (LEMASTERS et al. 1997). A similar deg-radation of mitochondria was observed inter alia by LUX (1984) in development of xylem in barley roots, and by FARBOS et al. (2001) in flower mer-istem of male-sterile plants of Nicotiana tabacum. Moreover, abnormalities in ultrastructure and functioning of mitochondria, consisting in the decrease of their metabolic activity, might be an indirect consequence of condensation of nuclear chromatin. It is known that more than 95% of mitochondrial proteins (responsible for, among others, transport of electrons, ATP synthesis and expression of mitochondrial genome) are coded by the nuclear genome (DE PAEPE et al. 1990; PASCHEN and NEUPERT 2001). Degradation of a larger number of mitochondria during incubation might in turn result in lower cell metabolism, by reduction of the cell ATP level (BERGMAN et al. 2000).

Apart from appearance of mitochondria with reduced membranous structures and lucent spaces in their matrix, treatment with Taxotere resulted in formation of very long mitochondria and ones having a distinct narrowing in their central parts and swollen at the ends. The amount of membra-nous structures in such mitochondria was similar as in the control. It can be speculated that the oc-currence of such mitochondria, by their larger res-piration area, was to compensate energetic losses resulting from degradation of other mitochondria. Mitochondrial degradation could lead to hypox-

ion, and further appearance of mitochondria of very oblong shape. Such ultrastructure of mito-chondria was often observed under stress of hy-poxion (LIU and DU 2002; LORENTE et al. 2002).

Another typical change resulting from the treatment was the increased vacuolation of cells. Additionally, a large number of very active dictyo-somes with abundant vesicles was observed. Those changes could be connected with active removal of harmful substances by the cells – “detoxification” process, or could result from autolysis of organelles degraded during the incubation. Simultaneous changes in the ER activity (swollen and budding cisterns of smooth and rough endoplasmic reticu-lum) could be connected with synthesis by these organelles of vacuolar or secretion proteins, hav-ing lytic function or connected with defense reac-tion of cells. This may evidence intensive transport connected with secretion and excretion. Existing reports notice that disturbances in the microtu-bule system caused by paclitaxel may influence the transport inside and between cells and the secre-tory activity of cells (RAINEY et al. 1985); these re-sults correspond with my observations presented here. By its affecting the interphase cytoskeleton, paclitaxel may disturb the normal function of the plasmalemma, as it impacts the transmembrane signalization and intercellular transport and loco-motion (LONG and FAIRCHILD 1994).

An especially typical feature of cell ultrastruc-ture following Taxol and Taxotere treatment was inhomogeneous distribution of MTs in the cyto-plasm, particularly at the cell wall. It caused irreg-ular its thickening at the sites of MT aggregations. These changes resulted from MT stabilization and their tendency to form larger aggregates, which disturbed the normal orientation of cellulose mi-crofibrils in the cell wall (BASKIN 2001).

The results of the present studies indicate that the mechanism of action of Taxol on plant cells is identical as in the case of animal cells. By changes in the cytoskeleton (MT stabilization) (MAJEWSKA and KURAs in press), the drug causes accumulation of cells at the G2/M phase of the cell cycle, thus inhibiting cell divisions. The results of the inves-tigations showed as well that the mechanism of Taxol antimitotic action is different than that of Taxotere. Taxotere did not block mitotic divisions during 24 h of incubation, but, by causing numer-ous clastogenic aberrations, it made the progress through subsequent mitotic phases impossible. Its result was formation of cells with micronuclei, unable to further divide. The significant amount of chromosome aberrations caused by Taxotere indicates its high genotoxicity.


The present studies also revealed that the cy-tostatic action of Taxol and Taxotere is reflected by plant cell ultrastructure; inter alia, a partial degradation of mitochondria and plastids occurs, which may affect the energetic state of the cell.

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Received November 21th 2007; accepted March 20th 2008

comparative investigations of cytotoxic action of …ﬂuence of both drugs to the change in number...

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