INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Malignant mesotheliomas (MM) are primary tumors of the mesothelium that grow in the potential space of the pleural cavity. MM poses an important clinical problem for which currently available treatments are rarely effective. Unlike other pulmonary tumors, malignant mesotheliomas are exclusively localized to the surrounding tissues and seldom metastasize (1). Approximately 2,200 cases of MM are reported per year in the United States (2). Most patients with pleural mesothelioma die within a year from the onset of initial symptoms (3). Patients with mesothelioma generally die secondary to local disease extension and respiratory failure (4). Multimodiality approaches, including surgical excision, chemotherapy, and radiation therapy, have failed to show significant improvement or to prolong the survival of patients with mesothelioma except in stage IA mesothelioma (5, 6).

Patients at later stages of the disease are usually treated with palliative measures for their pleural effusion. A wide variety of sclerosing agents in various forms of solutions, slurries, and powders, including tetracycline, doxycycline, quinacrine, bleomycin, and talc, have been introduced into the pleural space with varying response rates (7, 8). Talc is considered to be the most effective sclerosing agent in patients with symptomatic malignant mesothelioma (9, 10). In Europe thoracoscopic talc poudrage is a widely used technique for pleural effusions of malignant etiology (11).

Apoptosis or programmed cell death is part of a natural mechanism that regulates cell populations by deletion of cells with DNA damage from the organism. The loss of a normal apoptotic response has been implicated in the development of malignancy and malignant transformation of cells (12). Knowledge of the biologic processes that promote and maintain the uncontrolled proliferation of mesothelioma cells in the pleural space and the mechanisms that they employ to evade the immune system of the host may provide a focus for tumor-specific therapeutic regimens. The fact that some therapeutic agents work by inducing apoptosis in tumor cells indicates that this form of cell death plays a major role in cancer (13). Talc is a relatively nontoxic chemical that has therapeutic potential as a sclerosing agent in patients with malignant mesothelioma. The present study has demonstrated that talc not only has prosclerotic properties but also induces apoptosis in mesothelioma cells in vitro and thus may result in containment of malignant mesothelioma.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Culture of Human Malignant Mesothelioma Cells (MMC)

The mesothelioma cell lines (CRL-2081, CRL-5820, and CRL-5915), were obtained from American Type Culture Collection (ATCC, Rockville, MD). These cell lines were established from the pleural effusion of patients with mesothelioma. The cell lines were cultured in RPMI-1640 culture medium (Sigma Chemical Co., St. Louis, MO), containing 10% FBS (Harlan Sprague Dawley Inc., Indianapolis, IN), 100 U/ml of penicillin, 100 µg/ml of streptomycin. The cells were plated in 75 cm² culture flasks (Corning Costar Corporation, Medfield, MA) and incubated overnight at 37° C in 5% CO₂ and 95% air. When the cells were confluent they were trypsinized and subcultured as required for different assays.

Isolation and Culture of Human Pleural Mesothelial Cells (PMC)

Pleural fluid was obtained via thoracentesis from patients with transudative pleural effusions secondary to congestive heart failure according to a protocol approved by the Indiana University Institutional Review Board. The majority of patients had intractable congestive heart failure and symptomatic pleural effusions. None of the subjects had evidence of an infectious etiology for the pleural effusion. The pleural fluid was removed into a heparinized container and centrifuged at 1,000 × g for 10 min and the supernatant was discarded. The cell pellet was briefly exposed to cold hypotonic solution to lyse the red blood cells. The cells were resuspended in Ham's 199 culture medium (Gibco Laboratories, Grand Island, NY) containing 15% FBS, 100 U/ ml of penicillin, 100 µg/ml of streptomycin. The cells were plated in 75 cm² culture flasks and incubated overnight at 37° C in 5% CO₂ and 95% air. The following day the medium was changed to remove nonadherent cells. The mesothelial cells were characterized by the presence of classic cobblestone morphology, absence of factor VIII antigen, and presence of cytokeratin (14). All cells were utilized between the second and fourth passages.

Talc Particle Preparation and Stimulation of MMC/PMC

Talc (3 MgO · 4 SiO₂ · H₂O; Humco Laboratory, Texarkana, TX) particles were suspended in endotoxin-free 0.89% normal saline at a concentration of 4.00 mg/ml. The particle size was 2.1 ± 0.89 µm as determined using a Sony CCD-IRIS/RGB video camera attached to an Olympus IMT-2 microscope interfaced with a DataStar 486-66 computer containing Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Glass (SiO₂) beads (Sigma Chemical) with a certified mean diameter of 2.1 ± 0.5 µm were processed in a manner similar to that of the talc particles. The particles were washed and then autoclave sterilized. The concentrated stock samples of talc and glass beads had undetectable levels of endotoxin as determined by limulus amebocyte lysate assay (Sigma Chemical).

Confluent PMC or MMC (CRL-2081, CRL-5820, and CRL-5915) were exposed to a range of (0, 3, 6, 12, and 24 µg/cm²) talc concentration in serum-free medium. Cell viability was tested at the end of 72 h by trypan blue dye exclusion. As the maximum time of talc exposure in the current study was 72 h, we evaluated the cell viability at the end of 72 h. On the basis of cell viability, equal numbers of MMC (CRL-2081, CRL-5820, and CRL-5915) and PMC were subsequently exposed to graded concentrations (0 to 24 µg/cm²) of talc in serum-free Ham's 199, and in serum-free RPMI-1640 medium, respectively, for 24 h in order to establish a dose-response curve of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL). On the basis of the dose-response curve, an optimal concentration of 6 µg/cm² of talc was selected. In order to establish the time-dependent response 6 µg/cm² of talc was applied to confluent mesothelial/mesothelioma cells for varying time intervals (24 to 72 h). To evaluate the specificity of the effect of talc, 6 µg/cm² of glass beads of a similar size were included as parallel controls. At this concentration of talc/glass beads there was no evidence of necrosis in PMC and MMC.

In Situ Detection of Apoptosis

The PMC and MMC (CRL-2081, CRL-5820, and CRL-5915) were cultured on chambered sides. TUNEL method was used for the determination of apoptosis visually in MMC and PMC. The in situ apoptag labeling (15) was performed according to the manufacturer's protocol (Oncor, Gaithersberg, MD). Briefly, the slides were covered with TUNEL equilibration buffer. After 10 min of incubation at room temperature (RT), terminal deoxinucleotidyle transferase (TdT) was added to the slide and reincubated in humidified chambers at 37° C for 1 h. The reaction was stopped by incubating slides with stop/wash buffer at 37° C for 30 min. After washing with PBS the slides were incubated with antidigoxigenin peroxidase for 10 min at RT. Slides were colorized with DAB and counterstained with methyl green. A positive control slide was prepared by nicking DNA with DNase I (16) and treated similarly to serve as positive controls (data not shown). Samples treated similarly without enzyme served as negative controls. The percent of positively stained nuclei to total nuclei (percent apoptotic cells) was determined by counting cells with a phase contrast microscope. A total of 1,000 cells were counted per slide and at least three slides per group were examined.

Determination of Apoptotic DNA (Ladder) by Electrophoresis

DNA extraction and electrophoresis were performed as described by Nicoletti and colleagues (17). In brief, 5 × 10⁶ cells were incubated at 37° C in medium alone, or medium containing glass beads (6 µg/cm²) or talc (6 µg/cm²) for 24, 48, and 72 h. The cells were washed, centrifuged at 200 × g for 10 min and lysed in hypotonic lysing buffer (100 mM NaCl, 10 mM EDTA, 1% SDS, 200 µg/ml proteinase K at pH 7.50). Samples were extracted once with phenol plus chloroform. RNAse-A (100 µg/ml) was added to each sample and DNA was extracted twice with phenol chloroform (1:1, wt/vol) and chloroform. DNA was recovered by centrifugation after overnight precipitation at 20° C in two volumes of ethanol in the presence of 0.3M sodium acetate. The DNA pellet was air-dried, dissolved in 10 mM TRIS-HCl, 1 mM EDTA (pH, 8.00) at 4° C, and the DNA concentration was determined by measuring absorbance at 260 nm with a spectrophotometer. Then 12 µg DNA along with DNA loading buffer (25% Ficoll 400, 0.25% bromophenol blue, and 0.25% xylene cyanol) was loaded onto a 1.6% agarose gel. Electrophoresis was carried out in TBE buffer (2 mM EDTA, 89 mM boric acid, 89 mM TRIS at pH 8.4) and the DNA was visualized by ethidium bromide staining.

Statistical Analysis

Data were analyzed by using the SigmaStat statistical software package (Apple Computer, Cupertino, CA) and expressed as mean ± SE. Data that appeared statistically significant were compared by analysis of variance (ANOVA) with the use of the Student-Newman-Keuls test for multiple comparisons and considered significant if p values were < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Quantitation of Apoptosis in MMC and PMC by TUNEL

During an average pleural sclerosing procedure an average of 2 to 4 g of talc are insufflated via a thoracoscope into the pleural space. In an average 70-kg man, the pleural surface is approximately 2,000 cm². This results in a talc dose of 1 to 2 mg/cm² surface area of pleura. The optimal concentration of talc used in the present study (6 µg/cm²) is several folds lower than that used in clinical practice. During talc insufflation, though attempts are made to spray talc uniformly over the pleural surface, it is possible that the distribution remains patchy and not necessarily uniform. In vitro, the MMC viability decreased with increasing concentration of talc (0 to 24 µg/cm²). At the highest concentration (24 µg/cm²) of talc, 93% viability was noticed in PMC (Table 1). Talc at 24 µg/cm² significantly affected the cell viability of MMC. Among the MMC, CRL-2081 is the most affected and CRL-5915 is least affected as tested by Trypan blue dye exclusion test. Talc induced apoptosis in MMC (CRL-2081, CRL-5820, CRL-5915) in a concentration-dependent manner (Figure 1). The apoptotic cells were characterized by chromatin condensation and fragmented nuclei. Talc at a lower (3 µg/cm²) concentration induced a marginal increase of apoptosis in MMC when compared with control. However, at 6 µg/cm² a significant (p < 0.001) increase in apoptosis was noticed, which plateaued thereafter. PMC did not show significant apoptosis to varying concentrations of talc. Apoptosis in MMC (CRL-2081, CRL-5820, and CRL-5915) and PMC as detected by in situ TUNEL labeling after exposing the cells to glass beads or talc is demonstrated in Figure 2.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Quantitation of apoptosis in MMC and PMC by TUNEL. Three MMC (CRL-2081, CRL-5820, and CRL-5915) and PMC were incubated either in the presence of SFM alone or SFM containing talc (3, 6, 12 µg/cm2) for 24 h. The data represent mean ± SE of three independent experiments; *p < 0.001 MMC versus PMC. SFM = serum-free medium.

View larger version (138K): [in this window] [in a new window]   Figure 2.   In situ detection of apoptosis in MMC and PMC by immunoperoxidase reaction. (A-C ) PMC in the presence of SFM, glass beads, and talc. (D-F ) MMC (CRL-2081) in the presence of SFM, glass beads, and talc. (G-I ) MMC (CRL-5820) in the presence of SFM, glass beads, and talc. ( J-L) MMC (CRL-5915) in presence of SFM, glass beads, and talc. The data shown are single representatives of three independent experiments. Bar represents 50 µm.

In MMC, talc induced apoptosis in a time-dependent manner (Figure 3). At 24 h, in talc-exposed MMC (CRL-2081, CRL-5820, and CRL-5915), 14.83 ± 1.51%, 10.60 ± 2.63%, and 7.98 ± 2.42 apoptosis were observed, respectively. After 48 h a maximum of 39.50 ± 2.55%, 31.86 ± 4.69%, and 15.10 ± 3.93% apoptotic cells were noted in MMC (CRL-2801, CRL-5820, and CRL-5915), and after 72 h, it was 36.83 ± 1.53%, 28.25 ± 3.33%, and 13.62 ± 2.39%, respectively. This is statistically significant (p < 0.001) when compared with untreated controls. The CRL-2081 cell line that had the most reduced viability upon talc exposure also demonstrated the most apoptosis when exposed to talc. Talc did not show significant apoptosis in PMC. The PMC were unaffected and looked healthy throughout the incubation periods. Glass beads did not affect MMC and PMC at any of the time points studied.

View larger version (25K): [in this window] [in a new window]   Figure 3.   Quantitation of apoptosis in MMC and PMC by TUNEL. The MMC (CRL-2081, CRL-5820, and CRL-5915) and PMC were incubated in presence of SFM alone, SFM containing glass beads, or talc for 24, 48, and 72 h. The data represent mean ± SE of four independent experiments. *p < 0.001 talc-treated versus untreated controls at each time point. SFM = serum-free medium.

Demonstration of Talc-induced Apoptotic DNA Ladder by Electrophoresis

Talc-induced apoptotic DNA fragmentation (DNA ladder) in MMC and in PMC are demonstrated in Figure 4. MMC (CRL-2081, CRL-5820, and CRL-5915) and PMC were treated either with talc (6 µg/cm²) or with glass beads (6 µg/cm²) for 24, 48, and 72 h and the DNA was extracted. The DNA obtained from MMC and PMC was subjected to DNA electrophoresis. The DNA stained with ethidium bromide in agarose gels demonstrated the characteristic laddering pattern in samples obtained from MMC but not from PMC. Among the MMC, the apoptotic DNA ladder intensity was higher in CRL-2081 than in CRL-5820 or CRL-5915 mesothelioma. Apoptosis was not detected in untreated controls. Glass beads did not show any significant effect in either MMC or PMC.

View larger version (68K): [in this window] [in a new window]   Figure 4.   The DNA gels stained with ethidium bromide demonstrated the laddering pattern. (Panel A) Lane 1: Commercial DNA ladder of 123 bp. Lanes 2-4: DNA from normal PMC cultured in presence of SFM, glass beads, and talc. Lanes 5-7: DNA from MMC (CRL-2081) cultured in presence of SFM, glass beads, and talc. Panels C and E are in the same order at 48 and 72 h, respectively. (Panel B) Lanes 1-3: DNA from MMC (CRL-5820) cultured in the presence of SFM, glass beads, and talc. Lanes 4-6: DNA from MMC (CRL-5915) cultured in presence of SFM, glass beads, and talc. Lane 7: Commercial DNA ladder of 123 bp. Panels D and F are in the same order at 48 and 72 h, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Malignant pleural mesotheliomas unlike other pulmonary tumors are exclusively localized to the pleura and cause pleural effusions. MM are unresponsive to standard medical and surgical therapies (1). A therapeutic agent that can inhibit the development of metastases and their subsequent growth on the pleura would be beneficial to patients with malignant mesothelioma by extending their survival time. Patients with mesothelioma who have successful pleurodesis with talc have decreased morbidity. In the present study we investigated the ability of talc to induce apoptosis in malignant mesothelioma cells and normal pleural mesothelial cells.

Talc is widely used as a sclerosing agent in patients with malignant mesothelioma (10, 11). There is no preceding literature available on the possible role of talc in the induction of apoptosis of malignant mesothelioma cells. Multiple approaches are necessary to convincingly demonstrate the mechanisms of action of a novel therapeutic agent. Therefore, we evaluated talc-mediated apoptosis in malignant mesothelioma cells and PMC by two different approaches. (1) In situ staining of fragmented DNA by TUNEL, which is an extremely sensitive technique. It labels 3'-OH ends in DNA fragments and is considered as a useful method of apoptotic cell quantitation (16). Staining of talc-exposed MM by TUNEL revealed the different steps of apoptotic process, namely, chromatin condensation, fragmented nuclei, and apoptotic bodies. (2) Apoptosis in MMC was also confirmed by a typical DNA ladder, which is the definitive biochemical feature of apoptosis (18). Both these methods convincingly demonstrate that talc at clinically achievable concentration (6 µg/cm²) induces significant apoptosis in a time- and concentration-dependent manner in MMC. Talc did not cause significant apoptosis in normal pleural mesothelial cells, and glass beads of similar size did not induce apoptosis in MMC and PMC.

Apoptosis is an intentional form of cell death that occurs in response to specific stimuli (19). Clinically, apoptosis has been found to be an important mechanism of the action of radiation and many chemotherapeutic drugs against malignant cells, including leukemia cells (20). A wide variety of chemotherapeutic agents work by initiating DNA damage, which results in apoptosis (21). Several theurapeutic agents such as doxorubicin, cisplatin, and cyclophosphamide were found to be promising against malignant mesotheliomas (22). However, the mechanisms whereby these drugs reduce the growth of mesothelioma are not clear. Evidence from the literature suggests doxorubicin (23), cisplatin, and cyclophosphamide are capable of inducing apoptosis (24, 25) in malignant cells. Lovastatin is known to induce apoptosis in malignant mesothelioma cells (26). Doxycycline, bleomycin, and talc are used as sclerosing agents against malignant meosotheliomas (9, 22). Among these sclerosing agents talc was found to be most effective. Doxycycline induced apoptosis in cultured human osteosarcoma cells (27) and also suppressed the growth of human prostate carcinoma cells (28). Bleomycin induced apoptosis in fibroblasts (29). Our findings demonstrate that talc induces apoptosis in MMC and may reduce the tumor burden after pleurodesis. Talc is more effective when pleurodesis is performed early in the history of malignant effusions (30). We speculate that this might occur because, at the early stages of malignant pleural disease, the tumor volume is small and talc covers a relatively larger tumor surface area. However, when the tumor mass is large, talc may not effectively reach the deeper areas of the tumor and thus induction of apoptosis is less likely in those tissues. An ideal locally acting chemotherapeutic agent would affect malignant cells and not harm the adjacent normal cells. Talc appears to fulfil this criterion since our studies demonstrate that normal pleural mesothelial cells do not undergo apoptosis when activated by talc.

Cell surface molecules are crucial in cell growth control, and they are associated with signal transmission essential for growth and homeostasis. The c-myc protoonocogene enhances the apoptosis (31). CRL-2081 mesothelioma cells are already characterized to constitively overexpress protooncogene c-myc, and are positive for H-ras, K-ras, and N-ras. Lovastatin enhance the apoptosis in fibroblasts constitutively expressing myc oncogene (32). The cell surface receptor Fas (33), and p⁵³ (34), are known to mediate apoptosis in several types of malignant cells. Melanoma cells express the Fas gene that causes apoptosis (35). Fas-mediated apoptosis was observed in lymphomas (36). Thus, several distinct cell surface molecules are involved in inducing apoptosis, and it can be speculated that talc may regulate the expression of these cell-surface molecules and thereby induce apoptosis in malignant mesothelioma cells. However, the mechanism whereby talc induces apoptosis of malignant mesothelioma cells needs to be further investigated.

In conclusion, the significance of this study is that talc induces apoptosis in MMC without affecting normal pleural mesothelial cells. These findings also demonstrate that talc, a palliative agent may have a therapeutic potential in decreasing tumor burden. Therefore, it may be construed that talc not only induces pleurodesis but also decreases the size and mass of tumor in patients with mesotheliomas.