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Identifier

000362713

Title

Διερεύνηση του μηχανισμού του κυτταρικού θανάτου που προκαλείται από τη συνδυασμένη δράση ταξανών και υπερθερμίας

Alternative Title

Investigation of the mechanism of cell death caused by the combined action of hyperthermia and taxanes

Author

Μιχαλάκης, Ιωάννης

Thesis advisor

Τσιφτσής, Δ.Δ

Reviewer

Γεωργούλιας, Β
Καστανάς, Η.
De Bree, E

Abstract

Background: Taxanes are plant alkaloids commonly used in the treatment of human carcinomas. They bind with high affinity to the β-subunit of tubulin, causing decreased dynamic instability and increased microtubule rigidity, leading to mitotic blockade. The cellular effects of the drug vary, depending on dose and treatment scheme. The cells usually die due to apoptosis. In vitro the cellular effect of hyperthermia depends on the duration and the level of temperature used. Apart from the temperature administered and the histological cellular characteristics of the tissue, the survival curve is influence by several factors including hypoxia, pH and blood flow. The influences of all these factors ultimately cause apoptosis or necrosis. Conflicting results have been reported from in vitro and in vivo studies on the combination of paclitaxel with hyperthermia. These studies varied in drug concentration, exposure time, degree and duration of hyperthermia and cell type studied. During Continuous Hyperthermic Peritoneal Perfusion Chemotherapy, (CHPPC) for primary or secondary peritoneal malignancies, tumour cells are exposed to high drug concentrations for a relatively short period of time. We investigated in vitro the effect of paclitaxel and hyperthermia on cell proliferation, cell cycle kinetics and cell death under conditions resembling those during CHPPC. Methods: Human breast MCF-7, ovarian SK-OV-3 and cervical HeLa cells were exposed to 10 and 20 μM paclitaxel at 37, 41.5 or 43C for 2 h. After the various treatments cell proliferation was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazol blue). Cell cycle analysis was done after the cells were treated in order to stain the DNA using the DNAPrep Coulter Reagent Kit (Beckman Coulter). Samples were subjected to FACS in a Coulter Epics Elite. The number of alive and dead cells was determined by incubating a fraction of total cells with Trypan Blue, placing the cell suspension in a Neubauer hemocytometer and counting the number of cells which incorporated (dead) or excluded (alive) the dye under a conventional microscope. Apoptosis was assayed by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. TUNEL assays were performed using an in situ cell death detection kit obtained from Boehringer Mannheim. All samples were analyzed by flow cytometry (FACS) in a Coulter Epics Elite. Results: Hyperthermia alone exerted a cytostatic effect to all cell lines (stable initial cell population) and at 430C a cytotoxic effect on MCF-7 and HeLa cells (a decrease of initial cell population at 30% and 60% respectively). Hyperthermia did not alter the cytotoxic effect of paclitaxel on HeLa cells. (50% decrease of the initial population). Hyperthermia also did not influence the mild cytostatic effect of paclitaxel on SK-OV-3 cells. Paclitaxel exerted a cytostatic effect on the MCF-7 cells at 370C, 41.50C and a cytotoxic at 43 0C (50% decrease of the initial population) At the first 24h hyperthermia showed an additive effect on cell necrosis at all cultures (up to 80% on HeLa cells). After 24h this effect was diminished. The MCF-7 cells were necrotic at 30% after 7 days, the SK-OV-3 at 70% after 7 days and the HeLa cells at 80% after 2 days. Apoptosis as a result of the treatments was minimal and did not affected by hyperthermia. HeLa cells were apoptotic at 18% after 3 days and SK-O-V3 cells at 20% after 5 days. The MCF-7 cells did not exert any apoptotic cells. MCF-7, HeLa and SK-OV-3 cells treated under normothermic conditions with paclitaxel were arrested at G2/M or M phase for a minimum 60% for at least 3 days. Hyperthermia at 41.50C altered cell cycle distribution and affected the paclitaxel-related effect on cell cycle kinetics of MCF-7 cells (43% of the cells at G2/M phase after 3 days). The subpopulation of G2/M cells consisted of mitotic and multinucleated cells. A direct (proportional) relationship between the decrease of the number of mitotic cells and the appearance of multinucleated cells was observed (except of the MCF-7 cells). When paclitaxel was combined with hyperthermia this balance between mitotic and multinucleated cells was disturbed, without stopping the formation of multinucleated cells. After 5 days the majority of the cells of the subpopulation G2/M were multinucleated at all cell lines. All the cells after exposure to paclitaxel showed nuclear envelope structural defects and abnormalities due to aberrant cytokinesis (gaps on the nuclear envelop integrity, nuclear pore clusters, multinucleated cells and asters of microtubules). The above effects did not influenced by the presence of hyperthermia. Conclusions: Hyperthermia did not inhibit the effect of paclitaxel on the studied carcinoma cell lines and the main cause of cellular death was necrosis. Given that hyperthermia has an additive effect on the cells studied and the advantages of the hyperthermia in vivo, we can say that the use of paclitaxel in the CHPPC is feasible.

Language

Greek

Subject

Apoptosis

Hyperthermia

Necrosis

Taxanes

Taxol

Νέκρωση

Issue date

2009-07-24

Collection