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10.1245/s10434-006-9305-4
Annals of Surgical Oncology 14:1220-1228 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Short-Term Exposure of Cancer Cells to Micromolar Doses of Paclitaxel, with or without Hyperthermia, Induces Long-Term Inhibition of Cell Proliferation and Cell Death In Vitro

John Michalakis1,2, Spyros D. Georgatos3, Eelco de Bree2, Hara Polioudaki1, John Romanos2, Vassilis Georgoulias4, Dimitris D. Tsiftsis2 and Panayiotis A. Theodoropoulos1

1 Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, 71003, Heraklion, Greece
2 Department of Surgical Oncology, School of Medicine, University of Crete, 71003, Heraklion, Greece
3 Laboratory of Biology, School of Medicine, University of Ioannina, 45110, Ioannina, Greece
4 Department of Medical Oncology, School of Medicine, University of Crete, 71003, Heraklion, Greece

Correspondence: Address correspondence and reprint requests to: Panayiotis A. Theodoropoulos; E-mail: takis{at}med.uoc.gr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: During intraoperative hyperthermic intraperitoneal chemotherapy for primary or secondary peritoneal malignancies, tumor 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 intraoperative hyperthermic intraperitoneal chemotherapy.

Methods: Human breast MCF-7, ovarian SKOV-3 and hepatocarcinoma HEpG2 cells were exposed to 10 and 20 µM paclitaxel at 37, 41.5 or 43°C for 2 h. Cell proliferation, cell cycle kinetics, necrosis and apoptosis were evaluated.

Results: Hyperthermia exerted a cytostatic effect to all cell lines and at 43°C a cytotoxic effect on MCF-7 cells. MCF-7 and SKOV-3 cells treated under normothermic conditions with paclitaxel were arrested at G2/M or M phase for at least 3 days. Most of MCF-7 cells and approximately half of SKOV-3 cells were in interphase and became multinucleated without properly completing cytokinesis. Hyperthermia at 41.5°C altered cell cycle distribution and affected the paclitaxel-related effect on cell cycle kinetics of MCF-7 and SKOV-3 cells. Analysis of the mode of cell death showed that cell necrosis prevailed over apoptosis. Hyperthermia at 43°C increased paclitaxel-mediated cytotoxicity in MCF-7 cells and to a lesser extent in SKOV-3 and HEpG2 cells.

Conclusions: Short-time treatment of carcinoma cells with high (micromolar) concentrations of paclitaxel in normothermic and hyperthermic conditions is highly efficient for cell growth arrest and could be of clinical relevance in locoregional chemotherapy.

Key Words: Paclitaxel • Intraperitoneal chemotherapy • Hyperthermia • Cell cycle • Cell necrosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the last decades, primary and secondary peritoneal malignancies have been treated by a combination of cytoreductive surgery and intraperitoneal chemotherapy with encouraging results, especially in advanced ovarian cancer.1,2 Intraperitoneal drug administration results in high locoregional drug concentrations, increased exposure of drug to the microscopic or small macroscopic peritoneal disease and high peritoneal tumor concentrations, while systemic toxicity is limited. Paclitaxel has a highly favorable pharmacokinetic profile, because of slow clearance from the peritoneal cavity due to its large molecular weight and its significant first pass effect in the liver.3,4 Because of its apparent dose–effect relation, this pharmacokinetic advantage may lead to increased efficacy. Because of its above mentioned properties and its high activity in various malignancies, intraperitoneal chemotherapy with paclitaxel is an attractive treatment option in peritoneal carcinomatosis from ovarian or gastric origin and malignant mesothelioma.3–7

Intraoperative intraperitoneal chemotherapy allows better exposure of the entire seroperitoneal surface to chemotherapeutic agents, minimizes the number of viable exfoliate tumor cells after resection and avoids complications related to intra-abdominal catheters. Moreover, intraoperatively intraperitoneal chemotherapy may be combined with hyperthermia.8–11 Hyperthermia enhances the cytotoxicity of some chemotherapeutic agents.10,11 Conflicting results have been reported from in vitro and in vivo studies on the combination of paclitaxel with hyperthermia.11–19 These studies varied in drug concentration, exposure time, degree and duration of hyperthermia and cell type studied. In intraperitoneal chemotherapy cancer cells are exposed to significantly increased drug concentrations. While in systemic chemotherapy paclitaxel concentrations are in the nanomolar range, micromolar concentrations of the drug are achieved in the peritoneal cavity after its intraperitoneal administration.20 During intraoperative hyperthermic intraperitoneal chemotherapy this high intracavitary drug concentration lasts, however, for a much shorter time than during intraperitoneal installation chemotherapy (1.5–2 h versus 24 h).

To evaluate the efficacy of the use of paclitaxel for this treatment modality in vitro, we studied paclitaxel-mediated cytotoxicity, cell cycle kinetics and cell death in ovarian and breast human cell lines after 2-h treatment with high dose micromolar drug concentrations under normothermic and hyperthermic conditions, circumstances mimicking those in intraoperative hyperthermic intraperitoneal chemotherapy.21,22


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Culture
Cell lines from mammary (MCF-7), ovary (SKOV-3) adenocarcinoma and hepatocarcinoma (HEpG2) obtained from American Type Tissue Culture Collection (Manassas, VA) were maintained at 37°C in a humidified atmosphere containing 5% CO2. MCF-7 and HEpG2 cells were cultured in DMEM/HAM’S F-12 plus 10 µg/ml insulin and SKOV-3 cells in Mc Coy’s 5A modified medium. All media were purchased from Biochrom (Berlin, Germany) and were supplemented with 10% heat-inactivated fetal bovine serum, penicillin and streptomycin.

Cell Treatments
Upon reaching 50–60% confluence, cells grown either on culture dishes or on coverslips were incubated with 10 or 20 µM paclitaxel (Taxol®, Bristol-Meyer Squibb, NJ, USA) diluted in culture medium, at 37, 41.5 or 43°C for 2 h, washed with fresh medium and incubated in drug-free medium at 37°C for up to 7 days. Hyperthermic treatment was performed by washing and incubating the cell cultures with medium at the appropriate temperature and by placing culture dishes in a CO2 incubator regulated at 41.5 or 43°C.

Cell Proliferation and Cell Death Assays
Cells were grown on 24 well plates and after the various treatments cell proliferation was determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazol blue), as specified by the supplier (Sigma Chemical Co, St Louis, MO). The percentages of viable cells for each treatment were determined by measurement of MTT absorbance, relative to the initial cell population before any treatment. All cell proliferation data are the means of eight measurements from three independent experiments.

The number of alive and dead cells was determined by incubating a fraction of total cells (adherent and in suspension) with Trypan blue for 5 min, 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, according to the manufacturer’s instructions. All samples were analyzed by flow cytometry (FACS) in a Coulter Epics Elite.

Cell Cycle Analysis
Adherent cells were trypsinized, and added to the supernatants which contained the nonadherent cells. The whole cell population was washed with PBS and treated in order to stain the DNA using the DNA-Prep Coulter Reagent Kit (Beckman Coulter), as specified by the supplier. Samples were subjected to FACS in a Coulter Epics Elite.

Examination of Nuclear Morphology
The mitotic index and the morphology of inter-phase nuclei were assessed using total cells (adherent and in suspension), after staining with the DNA-binding dye, DAPI. At least 1,000 nuclei per sample were scored using a conventional fluorescence microscope, as mitotic (cells in prophase, metaphase, anaphase or telophase), normal-shaped or multinucleated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of Cell Proliferation
Assessment of viable MCF-7 and SKOV-3 cells 3 days after their treatment showed that both cell lines exhibited retarded proliferation (Fig. 1Go, panels A and B). To differentiate between cytostatic and cytotoxic effects, we compared the percentage of viable cells counted before any treatment (initial population, white bars) with the fraction of viable cells after treatment. Percentages of viable cells after treatment that were higher than that of the initial population denoted a cytostatic effect, whereas those below the initial level showed cytotoxicity. As shown in Fig. 1Go (panels A and B), the type of the anti-proliferating effect depended on cell type and treatment. Hyperthermic treatment at 41.5°C exerted a cytostatic effect to both cell lines, whereas raising the temperature to 43°C was cytotoxic only for MCF-7 cells. No substantial differences in the proliferation of SKOV-3 cells were noted after paclitaxel treatments under normal and hyperthermic conditions. However, a substantial enhancement of paclitaxel cytotoxicity was observed when MCF-7 cells were treated with paclitaxel at 43°C.


Figure 1
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  		FIG. 1. Effect of paclitaxel and hyperthermia on cell proliferation. An initial population (white bars) of MCF-7 (A, C) and SKOV-3 cells (B, D) was treated with 0, 10 or 20 µM of paclitaxel for 2 h at 37, 41.5 or 43°C and then cultured in drug-free medium at 37°C for 3 (A, B) and 7 (C, D) days. Cell proliferation was estimated using the MTT assay, as described in Methods. Results are expressed as percentages of viable cells by comparison to the maximal (100%) cell proliferation of normal cultures (no paclitaxel, 37°C). Values are means ± SE of eight measurements in three separate experiments.

 
The extent and type of the anti-proliferating effect changed when treated cells were cultured for longer periods (7 days, Fig. 1C and DGo). Hyperthermia no longer had an antiproliferating action on SKOV–3 cells, whereas a cytostatic effect was still observed in MCF-7 cells. The minor cytotoxic effect of paclitaxel at 37°C and 41.5°C on MCF-7 cells which has been noted on the third day of culture was no more apparent on the seventh day, although the cytotoxic effect of paclitaxel at 43°C was still observed (compare panels A and C in Fig. 1Go). The inhibitory effect of paclitaxel on SKOV-3 cell proliferation was more pronounced when compared to that described above for shorter time intervals. Specifically, after 7 days of culture we found a more potent inhibition of cell proliferation at normothermic and mild hyperthermic conditions as well as a minor cytotoxic additive effect when SKOV-3 cells were treated with 20 µM paclitaxel at 43°C (compare panels B and D in Fig. 1Go).

We further studied the effect of hyperthermia and paclitaxel on the proliferation of a hepatocarcinoma cell line, HEpG2. Although hyperthermia alone inhibited partially cell proliferation for up to 7 days, the cytostatic/cytotoxic effects of paclitaxel with or without hyperthermia on HEpG2 cells were very similar to that reported above for SKOV-3 cells (not shown).

Effects on Cell Cycle Progression
To examine the parameters that contribute to the anti-proliferative effect of 10 µM paclitaxel and hyperthermia at 41.5°C, we first studied cell cycle kinetics by FACS analysis (Fig. 2Go). We decided to treat cells with 10 µM and mild hyperthermia, because of our results from proliferation assays, demonstrating no differences between SKOV-cells treated with either 10 or 20 µM combined or not with hyperthermia at 41.5 or 43°C (Fig. 1Go). Hyperthermia, when applied alone, induced a disturbance on cell cycle kinetics (Fig. 2Go, panel Hyperthermia). More specifically, immediately after treatment (point "0") a decrease in the G1 for MCF-7 cells and the S sub-population for SKOV-3 cells with a parallel increase of the G2/M population was noticed. Two days after treatment, the percentages of the G1 and S phase SKOV-3 cells became similar to those in controls (Fig. 2Go, panel Hyperthermia/SKOV-3), whereas the effect on MCF-7 cells persisted for at least 3 days (Fig. 2Go, panel Hyperthermia/MCF-7).


Figure 2
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  		FIG. 2. Cell cycle analysis. The distribution of cells in the different phases, G1 (black bars), S (white bars) and G2/M (grey bars), of the cell cycle was estimated in cultures growing under normal conditions (c) and in cultures treated for 2 h with hyperthermia at 41.5°C (Hyperthermia), paclitaxel 10 µM (Paclitaxel) and both paclitaxel and hyperthermia (Paclitaxel with Hyperthermia). Cell cycle analysis was performed immediately after treatment (0), and 1, 2 and 3 days after cells been cultured under normal conditions. Total (adherent and no-adherent) cells were collected, stained with propidium iodide and analyzed by flow cytometry as described in Methods. Results are expressed as the percentage of each sub-population and are representative of three independent experiments.

 
Independently of the cell line used, more than 60% of the cells treated with paclitaxel remained arrested in the G2/M phase of the cell cycle for at least 3 days after release from the drug, indicating a sustained accumulation of mitotic or interphase tetraploid cells (Fig. 2Go, panel Paclitaxel).

When paclitaxel was combined with hyperthermia (Fig. 2Go, panel Paclitaxel with Hyperthermia), the percentages of the G2/M subpopulation of SKOV-3 cells were similar to those observed for cells treated with paclitaxel alone (Fig. 2Go panels, Paclitaxel and Paclitaxel with Hyperthermia). On the contrary, the effect of paclitaxel on the cell cycle of MCF-7 cells was completely abolished by hyperthermia for a period of at least 1 day post-treatment (Fig. 2Go, panels Paclitaxel and Paclitaxel with Hyperthermia). Thereafter, the G2/M subpopulation increased reaching 48% of the total cell population after 3 days (Fig. 2Go, panel Paclitaxel with Hyperthermia/MCF-7).

Analysis of the G2/M Subpopulation
In order to distinguish between interphase and mitotic cells in the G2/M subpopulation, we counted mitotic and interphase figures after DAPI staining. Examination of more than a thousand cells for each treatment protocol and cell line used revealed that almost all mitotic cells were in metaphase or a "metaphase like" state. No anaphase and very few of aberrant cytokinesis figures were detected, even after 3 days in culture. The proportion of mitotic cells varied and depended on cell line and treatment (Fig. 3Go, Paclitaxel 37°C). One day after paclitaxel treatment, 52% of SKOV-3 cells were blocked in metaphase, whereas this percentage was only 17% for MCF-7 cells.


Figure 3
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  		FIG. 3. Mitotic arrest and the nuclear morphology of treated cells. Cells grown on 6-well plates were treated with paclitaxel 10 µM for 2 h at 37 or 41.5°C and then cultured under normal conditions for up to 5 days. Total (adherent and in suspension) MCF-7 (open squares) and SKOV-3 (solid triangles) cells were stained with DAPI and more than 1,000 specimens from each treatment were microscopically examined in order to determine the number of mitotic cells and cells with multinucleated morphology. Results are expressed as a percentage of total cells. Values are means ± SE of three measurements in three separate experiments.

 
A decrease in the number of mitotic cells by more than 90% for MCF-7 and SKOV-3 cells was observed when mild hyperthermia at 41.5°C was combined to paclitaxel (Fig. 3Go, Paclitaxel 41.5°C). In fact, 1 day after combined treatment, the percentages of mitotic MCF-7 and SKOV-3 cells became similar to those of control cultures. The number of MCF-7 mitotic cells remained unchanged (between 1 and 7% of total cells) during a period of 5 days after treatment, whereas the percentage of SKOV-3 mitotic cells increased to 37% 2 days after treatment and then decreased as cells were cultured for longer periods.

The obtained results strongly suggest that the mode and the extent of responsiveness to paclitaxel and hyperthermic treatment depend on cell type. Most of MCF-7 cells arrested at G2/M phase 1 day after treatment with paclitaxel were in interphase. The nuclear morphology of those cells was not altered: they possessed normally shaped nuclei, even after been cultured in drug-free medium for 2 days (Fig. 3Go, Paclitaxel 37°C). The number of multinucleated cells increased rapidly from the third until the fifth day of culture. In contrast, the G2/M subpopulation of SKOV-3 cells differed significantly from that of MCF-7 cells and included an appreciable population of mitotic cells (52%) 1 day after their treatment with paclitaxel. Thereafter, as cells were cultured in drug-free medium, a direct (proportional) relationship between the decrease of the number of mitotic cells and the appearance of multinucleated cells was observed (Fig. 3Go, Paclitaxel 37°C). When paclitaxel was combined with hyperthermia this balance between mitotic and multinucleated SKOV-3 cells was disturbed for 24 h in the beginning of cell culture (Fig. 3Go, Paclitaxel 41.5°C).

Determination of the Mode of Cell Death
The results presented above indicated that the cytotoxicity of hyperthermia, paclitaxel or the combination of both, is due to both inhibition of cell cycle progression and cell death. In order to define the relative contribution of necrosis and apoptosis on cell death, the total cell population (adherent and non-adherent cells) was analyzed using Trypan blue, DAPI staining and TUNEL assays.

Paclitaxel’s cytotoxicity was dependent on the cell type. Treatment of SKOV-3 cells with paclitaxel at 37 and 41.5°C resulted in 28 and 41% of dead cells, respectively, after 5 days of cell culture in drug-free medium (Fig. 4Go). At that time, the number of apoptotic cells was lower and represented 17–23% of the total cells. In contrast, MCF-7 cells were less sensitive, as more than 80% of the total population remained alive and less than 5% became apoptotic 5 days after treatment with paclitaxel, with or without hyperthermia (Fig. 4Go). Hyperthermia had only a minor effect on both apoptotic and necrotic cell death in both cell lines (not shown).


Figure 4
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  		FIG. 4. Effect of paclitaxel and hyperthermia on cell necrosis and apoptosis. MCF-7 and SKOV-3 cells were treated with paclitaxel 10 µM for 2 h at 37 (dashed lines) or 41.5°C (solid lines) and then cultured under normal conditions for up to 5 days. Total (adherent and no-adherent) cells were collected and stained either with Try-pan blue for the determination of necrotic cells (circles) or with TUNEL for the measurement of apoptotic cells (triangles) using flow cytometry as described in Methods. Results are expressed as a percentage of total cells and are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Paclitaxel is a plant alkaloid commonly used in the treatment of human carcinomas.23 It binds with high affinity to the ß-subunit of tubulin and results in decreased dynamic instability and increased micro-tubule rigidity.24,25 The cellular effects of the drug vary depending on dose and treatment scheme. In the range of nanomolar concentrations, it induces sustained mitotic arrest,26 inhibits protein prenylation,27 affects nuclear envelope organization, alters macro-molecular trafficking through the nuclear pore complex28 and triggers apoptosis.28,29 At micromolar doses paclitaxel exerts other effects in addition to mitotic arrest, which occur almost immediately: it promotes synthesis and release of cytokines30–32 and induces "early response" genes, including those that encode tumor suppressors.33

Paclitaxel-treated cells have been observed to develop multiple or lobulated nuclei. Ishikawa cells treated with nanomolar concentrations of paclitaxel and allowed to complete mitosis, re-enter the cell cycle, effectively complete mitosis and cross the G1 checkpoint.28 Roughly half of the interphase cells have atypical nuclear shapes and died by apoptosis 3 days after removal of the drug.28 Alternatively, cells that fail to undergo cytokinesis after mitotic arrest caused by either DNA or spindle damage, become multinucleated and undergo a nonapoptotic form of cell death known as ‘mitotic catastrophe’.34,35 The observation that paclitaxel leads to the formation of multinucleated cells would, therefore, be consistent with the possibility that cell death could be due to mitotic catastrophe.

This study showed that short-term incubation of MCF-7, SKOV-3 and HEpG2 cells with micromolar concentrations of paclitaxel results on sustained inhibition of cell proliferation. This effect could be, in part, explained by the finding that both cell types remained arrested at the G2/M phase of the cell cycle. Further analysis of the G2/M sub-population, revealed that the number of cells in metaphase varied significantly among the cell lines. Moreover, the nuclear morphology (normal shaped or multinucleated) of the interphase tetraploid cells was also dependent on the cell line at early time points of culture. However, multinucleated cells became the predominant phenotype for both cell types as culture progressed indicating that the tetraploid multinucleated state is partially related to the inhibition of cell proliferation.

Some investigators have suggested that the multinucleated phenotype plays a role in paclitaxel resistance36,37 while others have concluded that multinucleation reflects drug sensitivity.29,38,39 The results of the present study taken together with our previous findings on HeLa cells14 strongly suggest that there is no direct relationship between multinucleation and cell death as most of the cells, independently of cell type, became multinucleated but did not die to the same extent. It seems, however, that cells arrested in metaphase before they became multinucleated, i.e. SKOV-3 and HeLa cells, are more sensitive to cell death than MCF-7 cells which became multinucleated without been arrested at the meta-phase/anaphase transition.

The mode of cell death after paclitaxel treatment seems to be dependent on drug concentration, duration of treatment and cell line. Generally, in vitro studies on paclitaxel cytotoxicity have been performed using low (nanomolar concentrations) and high (micromolar concentrations) drug doses during prolonged periods ranging from 20 to 72 h.28,39,40 It is generally considered that paclitaxel’s cytotoxicity results from apoptotic cell death. However in one experimental study, cell necrosis was observed at micromolar doses and apoptosis at nanomolar doses in various breast cancer cell lines.40 In other cell lines such as A549 cells, no signal for apoptosis has been found after treatment with paclitaxel concentrations that triggered G2/M arrest.41 Moreover, it has been reported that nontransformed 3T3.A31 cells do not develop apoptosis, although they become micronucleated after treatment with a fluorescent taxoid.42 We have previously shown that treatment of Ishikawa cells for 20 h with low concentrations of paclitaxel is sufficient to inhibit cell proliferation and to block more than 80% of cells at the metaphase/anaphase transition. Cells released from the drug reentered cell cycle but presented structural and functional defects of the nuclear envelope, multinucleation and died by apoptosis.28 When HeLa cells were treated with high concentrations of paclitaxel for 2 h, similar effects on cell proliferation and on cell cycle were observed.14 However, cells did not reenter cell cycle, presented structural defects of the nuclear envelope, multinucleation and died mostly by necrosis. In this study, using the same short period and high dose treatment scheme, we show that SKOV-3 and MCF-7 cells become multinucleated, after been partially (50 and 18%, respectively) arrested in the metaphase/anaphase transition. Although the extent of cell death was dependent on cell line, the proportion of necrotic cells was constantly higher than that of apoptotic cells.

Thermal enhancement would alter membrane fluidity. As a consequence, spatial rearrangement of membrane components and increase of drug entry into cells could be expected. In yeast, movement and clustering of the nuclear pores have been reported when cells were shifted to growth at 37 instead of 23°C.43 We have previously shown that treatment of human cell lines with paclitaxel alter nuclear pore organization and function28 and the extent of such alterations remained unchanged when hyperthermia was combined with paclitaxel.14 No changes in nuclear pore organization and nuclear envelope morphology have been noted when HeLa,14 SKOV-3 and MCF-7 cells (this report, not shown) have been treated with hyperthermia. However, hyperthermia inhibited proliferation of MCF-7 cells and hepatocarcinoma HEpG2 cells and this effect persisted for at least 7 days.

When combined to paclitaxel treatment, hyperthermia at 41.5°C could not significantly decrease the number of viable cells even in the presence of high paclitaxel concentrations. Nevertheless, cell death mechanisms seem to be different under hyperthermic compared with normothermic conditions, because cell necrosis was significantly higher in hyperthermic than normothermic conditions in SKOV-3 cells. Despite the increased cell necrosis of SKOV-3 cells observed under hyperthermic conditions (Fig. 4Go), the number of viable cells remained unchanged (Fig. 1Go). A possible explanation could be differences in doubling time of the cells at normothermic and hyperthermic conditions as well as the slight decrease in cell apoptosis observed at 41.5°C (Fig. 4Go). Paclitaxel-mediated cytotoxicity has been significantly increased in MCF-7 cells but only slightly in SKOV-3 and HEpG2 cells when hyperthermia at 43°C was combined to high concentrations of paclitaxel.

Finally, our findings may be of importance in vivo because hyperthermia is expected to enhance vascular permeability of the tumor and consequently to exert its cytostatic or cytotoxic action in no-cycling and otherwise nonaccessible cancer cells. Moreover, the fact that most cells died by necrosis, which is a process that in vivo increases local inflammation and recruits the resources of the immune system, supports the clinical relevance of the combination of paclitaxel and hyperthermia for the treatment of locoregional disease.

In conclusion, proliferation of MCF-7 (breast cancer), SKOV-3 (ovarian cancer) and HEpG2 (hepatocarcinoma) cells is inhibited after short time exposure of cells to high, micromolar, concentrations of paclitaxel, conditions encountered during intraoperative hyperthermic intraperitoneal chemotherapy. The inhibitory effect is due both to cell cycle arrest and cell death. Cell death mechanisms seem to be different under hyperthermic compared with normothermic conditions, although necrosis prevailed over apoptosis in both conditions. In this study, hyperthermia at 43°C increased paclitaxel cytotoxicity significantly in MCF- 7 cells and slightly in SKOV-3 and HEpG2 cells.


   ACKNOWLEDGMENTS
 
This work was supported partly by PENED 2001, 01E{Delta}376 from the Greek Secretariat of Research and Technology (PAT) and the Cretan Association for Biomedical Research (PAT and SDG).


   FOOTNOTES
 
Authors Contribution: JM: Acquisition, analysis and interpretation of data, drafting the manuscript; SDG: Design of the study, interpretation of data and revising the manuscript; EdB: Interpretation of data, drafting and revising the manuscript; HP: Acquisition of data and drafting the manuscript; JR and DDT: Conception of the study and revising the manuscript; VG: Interpretation of data and revising the manuscript; PAT: Conception and design of the study, analysis and interpretation of data, drafting and revising the manuscript. All authors have given final approval of the version to be published.

Received for publication January 10, 2006. Accepted for publication August 8, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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