|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
It has been shown that mesothelioma expresses the antiapoptotic protein BCL-XL, but not BCL-2, rendering bcl-xl gene expression a potential therapeutic target. Sodium butyrate (NaB) is a histone deacetylase inhibitor capable of alteration of bcl-2 family protein expression in other tumor types. Mesothelioma cell lines (REN, I-45) were exposed to NaB, and viability (colorimetric assay) and apoptosis (TUNEL, Hoescht staining, flow cytometry) were evaluated. Effects on bcl-2 family protein, fas-fas ligand, and caspases were examined by Western blot analysis and functional assay. An RNase assay evaluated bcl-2 family messenger RNA (mRNA) expression. Overexpressing BCL-XL mesothelioma clones were created by plasmid transfer. Cells were sensitive to NaB at low IC50 (REN, 0.3 mM; I-45, 1 mM) and demonstrated apoptosis (percentage of cells below G1 phase by flow cytometry [sub-G1]: REN, 38.5%; I-45, 30.9%). A significant decrease in BCL-XL protein expression was noted with BAK, BAX, and BCL-2 unchanged, and this was corroborated at the transcriptional level with selectively decreased bcl-xl mRNA production after sodium butyrate exposure. Fas expression and fas-fas ligand sensitivity were unchanged. Caspases demonstrated low-level activation. Stable overexpressing BCL-XL clones were proportionally resistant to the NaB effect. This study suggests that mesothelioma cells are sensitive to the induction of apoptosis related to the attenuation of antiapoptotic bcl-xl gene and protein expression. Additional study of the therapeutic benefit of targeting bcl-xl gene expression in mesothelioma is warranted.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Malignant pleural mesothelioma is a tumor that continues to be a difficult clinical problem. Although advances in staging and evaluation have identified subgroups that may benefit from aggressive therapy, the prognosis for patients remains poor (1). This tumor is clinically resistant to conventional chemotherapeutic agents, with no single or combination regimen consistently demonstrating response rates of greater than 20%. Accordingly, the majority of all published chemotherapy trials have not demonstrated a significant benefit (2). Inadequacy of conventional treatments has led to a great deal of interest in novel approaches to treatment such as prodrug gene therapy using the herpes simplex virus thymidine kinase/ganciclovir system and immunotherapy (5, 6).
The unresponsiveness of this tumor to most conventional agents may in part be explained by a resistance to
the induction of programmed cell death or apoptosis. Although the discrete mechanism of action varies, many conventional treatments depend on an ability to engender
apoptosis as a final common pathway. Narasimhan and coworkers (7) demonstrated that mesothelioma cells are
very resistant to oxidant (asbestos, H2O2) and nonoxidant
(calcium ionophore) stimulators of apoptosis. Other investigators have noted that p53 gene mutations are infrequent
in mesothelioma, rendering the possibility of targeting of
this gene less attractive (8). Of interest, however, is the
finding that BCL-XL expression is relatively uniform for
this tumor, with BCL-2 expression rare (9). The finding
that expression of only one of these proteins is common
may indicate a potential therapeutic target
i.e., downregulation of expression of BCL-XL with resultant "unopposed" proapoptotic protein expression.
Sodium butyrate (NaB) is a four-carbon, fatty acid histone deacetylase inhibitor that is physiologically present in the gut lumen as a byproduct of digestion of complex carbohydrates. It is thought to be important in the maintenance of healthy gut epithelium (10). The bulk of previous experience with the effects of NaB on neoplastic cells has been garnered in colonic carcinoma, but its effect on prostate, breast, hepatocellular carcinoma, and many others has also been reported (11). In addition to differentiation, induction of apoptosis has been noted in many tumor cell types after NaB exposure, but a unifying mechanism has not been described. Rather, a number of disparate explanations have been suggested, including alterations in bcl-2 family protein expression, increased caspase activity, sensitivity to fas-fas ligand interaction and effects on mitochondrial membrane function (12, 15). Novel protein/ DNA interactions after histone deacetylase inhibition and changes in the expression of a myriad of cellular genes, including c-myc, K-ras, and others, have also been suggested as causative (19). Cogent to this study evaluating effect in mesothelioma, inhibition of BCL-XL expression has been noted by investigators evaluating the effects of NaB on colonic and hepatic tumor cell lines as well as neonatal fibroblasts (11, 13, 20).
This study evaluates the effect of alteration of BCL-XL
protein expression on cell lines derived from human malignant mesotheliomaa tumor type thought resistant to
apoptotic cell death by both clinical and experimental behavior. We sought to examine whether NaB could alter
bcl-2 family protein expression in this tumor, with a specific goal of BCl-XL downregulation. We show that exposure to NaB leads to differentiation as well as apoptotic
cell death in two well-characterized mesothelioma cell lines
and suggest that a decrease in antiapoptotic bcl-xl gene
and protein expression may be responsible. Other experiments using a stable overexpressing BCL-XL mesothelioma
clone demonstrate the potential importance of the relationship of bcl-xl gene expression to apoptotic homeostasis in
this tumor cell type.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Cell Lines, Culture Conditions, and Growth
All cells were maintained in RPMI medium supplemented with 10% fetal bovine serum and 100 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY) in an atmosphere of 5% CO2 at 37°C. Both REN and I-45 are human mesothelioma cell lines originally derived from patients with pleural tumors. The REN (inflammatory epithelial subtype, p53 mutant) cell line was developed by the primary author (W.R.S.) and has been previously described (5). The I-45 (sarcomatous subtype, p53 wild-type) cell line was originally provided by Dr. Joe Testa of the Fox Chase Cancer Center in Philadelphia, PA.
Alkaline Phosphatase Activity Assay
Each cell line was seeded onto a six-well plate with 5.0 × 105
cells/well. Cells were incubated for 24 h to allow adherence and
then 3 mM of NaB (Sigma Chemical Co., St. Louis, MO) were added to the study wells and the cells were reincubated for 3 h.
Medium was then aspirated from the wells, and the cells were washed with phosphate-buffered saline (PBS). After aspirating the PBS, cells were then placed into a 
70°C freezer. At the time of cell lysis, the plates were removed from the 70°C freezer and
100 µl of PBS were added to each well. The cells underwent three
thaw-freeze cycles from 70° to +37°C. Finally, the lysed cells
were removed with a cell scraper for quantitative protein analysis. Alkaline phosphatase substrate (Sigma Chemical Co.) was
added to the cell lysis extract and then incubated at 37°C for 1 h.
Alkaline phosphatase activity was measured using a microplate reader at a wavelength of 405 nm (Dynatech Laboratories, Chantilly, VA).
Cell Viability Assay
Cell viability was tested using the XTT assay (Sigma Chemical Co.). After REN and I-45 exposure to NaB at varying doses from 0.1 to 10 mM, the assay evaluated surviving cells at 48 h after dose. The labeling reagent was added to the electron-coupling reagent (Roche, Mannheim, Germany) in a 1:50 ratio. A total of 50 µl of the XTT mixture was added to each well in a 96-well plate. Plates were then incubated at 37°C for 4 h. The plates were then analyzed using a colorimetric microplate reader at a wavelength of 450 nm (Dynatech Laboratories).
Western Blot Analysis
After mesothelioma cell exposure to 3 mM NaB and culture for 24, 48, or 72 h, total cell lysates were prepared by lysing plated cell monolayers with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The protein content of the lysates was then determined by BCA protein assay (Pierce, Rockford, IL). Each lane on an SDS-polyacrylamide (12%) gel was loaded with 20 µg of cell lysate and electrophoresed to separate proteins under reducing conditions for the protein of interest. After electrophoresis at 20 mA for 2 h, the proteins were transferred to high-bond enhanced chemiluminescence (ECL) membranes (Amersham Corp., Arlington Heights, IL). The membranes were then incubated with the primary and secondary antibodies, and developed according to the Amersham ECL protocol. Actin was used as a control. Actin, fas, BCL-XL, BAK, and BAX antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). BCL-2 antibody was obtained from Dako Corporation (Carpenteria, CA).
DNA Content Analysis for Apoptosis
Apoptotic cell death was evaluated by changes in cell morphology and flow cytometry. Fluorescence-activated cell sorter analysis was performed as follows: after 3-mM NaB exposure and 48-h culture, cells were trypsinized, collected by centrifugation, resuspended in PBS, and fixed in 70% ethanol at 4°C for 1 h. After centrifugation, the cells were washed in PBS and resuspended in phosphatidylinositol staining solution (Boehringer Mannheim Co., Indianapolis, IN). Specimens were incubated in the dark for 30 min at 37°C; the specimens were analyzed with the use of an EPICS Profile II flow cytometer (Coulter Corp., Hialeah, FL). An analysis region was set based on the negative controls, and the percentage of sub-G1 cells was calculated from this region.
TUNEL Assay and Hoescht Staining
For terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) staining, cells were plated on slides after 24 h of 3-mM NaB exposure and then fixed in ice-cold paraformaldehyde (Sigma Chemical Co.) for 5 min. Next, a methanol/3% H2O2 (Sigma Chemical Co.) wash was applied for 10 min. After incubation, the slides were washed three times with PBS for 2 min each time. The slides were then reincubated at 37°C for 2 h in terminal deoxynucleotidyl transferase and biotin-16-uridine triphosphate enzyme mix (Vector Labs, Burlingame, CA). Afterward, the slides were soaked in TB buffer (30 mM NaCl 30 mM sodium citrate) for 10 min and washed with PBS for 2 min. Slides were then soaked for 30 min in diluted blocking serum (1 ml horse serum in 3.3 ml PBS/0.1% bovine serum albumin [BSA]) and washed twice in PBS for 2 min each time). Avidin-biotin complex reagent (1:50 dilution, 60 µL of A and 60 µL of B into 3 mL of PBS/0.1%BSA) (Vector Labs) was used to soak the slides. Slides were rinsed in PBS three times for 2 minutes. Slides were then exposed to diaminobenzidine tetrahydrochloride/H2O2 (Vector Labs) for 1 to 2 min and then washed with running water for 2 min. Slides were counterstained with 0.4% methyl green solution for 10 s. Counterstained slides were rinsed again under running water for 2 min. Slides were then mounted with cytoseal after being air dried.
For Hoescht staining, cells were plated in chamber slides using a concentration of 5,000 cells in 500 µL of medium. After 24 h, the cells were exposed to 3 mM NaB. After 72 h, cells were washed with PBS and then fixed with acetone/acetic acid (3:1). After fixation, Hoechst nuclear staining solution (0.1 µg/ml) was added. Nuclei were then examined using a fluorescent microscope (Nikon Diaphou; Nikon Inc., Melville, NY). Apoptosis was characterized as cells with large segmented nuclei.
Fas-Fas Ligand Assay
Three thousand cells per well were plated on a 96-well plate and incubated. After 24 h cells were treated with NaB at different concentrations ranging from 0.1 to 6 mM. At the same time, Fas active antibody (Upstate Biotechnology, Lake Placid, NY) was added in varying doses from 0.3 to 30 ng/µl. The cells were then incubated for 72 h. After the incubation, an XTT assay was performed on the plates. From the XTT data, cell viability curves were created.
Caspase-3 Functional Assay
Three million cells were plated on a 10-cm dish and incubated for 24 h. After incubation, the cells were treated with 3 mM NaB. Cells were incubated again for 24 h. Next, cells were washed with PBS and lysed using 10 ml of cell lysis buffer (Pharmingen, San Diego, CA), and then transferred in 100-µl aliquots to 1.5-ml Eppendorf tubes. For each reaction, 10 µl of the substrate Ac-DEVD-AMC (Pharmingen) and 1 ml of Hepes buffer (Pharmingen) were added. The reaction mixtures were incubated at 37°C for 1 h. The reaction mixture was then transferred to a 96-well plate. After this, the fluorescent 7-amino-4-methylcoumarin (AMC) liberated from the reaction was measured using a spectrofluorometer (Dynatech Labs) at an excitation wavelength of 380 nm and an emission wavelength of 440 nm.
An inhibitory reaction was set up using the four sets of cell lysates as described previously. Ac-DEVD-AMC (10 µL) was combined with 10 µl of the inhibitory Ac-DEVD-CHO (Pharmingen) and then added to 1 ml of Hepes buffer, making a new reaction buffer. This new reaction mixture was then combined with 100 µl of each cell lysate in a 1.5-ml Eppendorf tube. Fluorescent activity was measured as previously described.
Overexpressing BCL-XL Mesothelioma Clones
Plasmid pcDNA3.1/BCL-XL was constructed by inserting human bcl-xl complementary DNA into the EcoR1 site of a pcDNA 3.1 vector (Invitrogen, Carlsbad, CA). The plasmid pd2EGFP-N1 vector was purchased from (Clontech, Palo Alto, CA). The I-45 mesothelioma cell line was used for transfection experiments. The I-45/GFP and I-45/BCL-XL were generated by transfecting parental I-45 cells with pcDNA3.1/BCL-XL and pd2EGFP-N1 using FUGENE-6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN). Cells were then selected with 400 µg/ml G418 medium. Single cell clones were isolated and further cultured in 400 µg/ml G418 medium. Established cell clones confirmed their BCL-XL expression by Western blot analysis. The overexpressing BCL-XL I-45 clones and I-45 were cultured in RPMI 1640 with 10% fetal bovine serum (FBA) lacking G418. Cells were plated into a 96-well plate and treated with varying doses of NaB. After 72 h, the number of viable cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.
RNase Protection Assay-bcl-xl Messenger RNA Quantification
RNase protection assays were performed using an RNase protection assay kit (Pharmingen). In brief, total RNA was isolated from cells with Trizol (Life Technologies, Inc). A multiprobe set, hapo-2c, which contains the probes for bcl-x, bad, bax, bak, mcl-1 and the housekeeping genes L-32 and glyceraldehyde-3-phosphate dehydrogenase (GADPH), was labeled with [32P]adenosine triphosphate using T7 RNA polymerase. Total RNA and probes were hybridized at 56°C overnight, and these hybrids were treated with RNase cocktail. Protected hybrids were resolved on a denaturing polyacrylamide sequencing gel and exposed to radiographic film overnight. The band density was quantitated with digital scanning and OPTIMAS software (Media Cybernetics, Silver Spring, MD).
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
NaB Leads to Differentiation and Tumor Cell Death in a Dose-Dependent Fashion in Human Mesothelioma Cell Lines
The histone deacetylase inhibitor NaB has been classified as a differentiating agent capable of converting tumor cells to a more mature phenotype. Tumor cells undergoing this redifferentiation process would be expected to exhibit a more mature morphology, different growth patterns, and possibly altered protein expression. The level of expression of the enzyme alkaline phosphatase has been correlated with a more mature differentiation state in a number of tumor cell types treated with NaB (21, 22). After a 3-h exposure to 3 mM NaB, REN and I-45 cells demonstrated increased contact inhibition and more mature morphology with increased cytoplasm and fewer nucleoli. Alkaline phosphatase activity was evaluated after this same exposure by a substrate colorimetric assay. Production of the enzyme was markedly increased after NaB (Figure 1). The alkaline phosphatase level increased approximately 2.5-fold in the I-45 line at 24 h and approximately sevenfold in REN at 48 h after NaB exposure.
|
0.01).
In addition to differentiation and decreased rate of growth, mesothelioma cells were noted to be dying after NaB exposure. A cell viability assay was performed to quantify the actual number of cells surviving in a dose- response fashion. After exposure to varying doses of NaB (0 to 30 mM), cell viability at 48 h after the dose was assayed using the XTT colorimetric method. The NaB IC50 (concentration of drug required to kill 50% of the cells) was approximately 0.3 mM for the REN cell line and 1 mM for the I-45 cell line, respectively (Figure 2). Fewer cells survived at higher doses of NaB, but the effect was attenuated above 1 mM concentration.
|
Apoptosis Is the Mechanism for Tumor Cell Death after NaB Exposure
By TUNEL and Hoescht staining, a significant population of mesothelioma cells exposed to NaB demonstrated nuclear condensation and fragmentation consistent with apoptosis (Figure 3). In an effort to quantify apoptosis, fluorescence-activated cell sorter (FACS) cell cycle analysis was performed, evaluating the percentage of cells in the sub-G1 phase after a 3-mM NaB exposure. Both lines demonstrated in excess of 30% of cells in the sub-G1 phase after NaB exposure (REN, 38.5%; I-45, 30.9%), with minimal sub-G1 component noted in the control cells (Figure 4).
|
|
NaB Does Not Lead to Significant Caspase Activation or Increased Fas-Fas Ligand-Mediated Apoptosis
Direct or indirect activation of caspases has been implicated in the induction of apoptosis in cells treated with NaB (18). Western blot analysis was performed in an effort to identify caspase cleavage products after NaB exposure in mesothelioma cells. Although only very faint cleavage bands were noted for caspases 3, 8, and 9 after NaB exposure (data not shown), a small increase in caspase 3 activity was noted by substrate assay. This increase in activity was ameliorated by the inhibitor Ac-DEVD-CHO (Figure 5).
|
Sensitization to fas-fas ligand-mediated apoptosis has been demonstrated after exposure of colon cancer cell lines to NaB (18, 23). We first evaluated cell-surface fas expression after a 3-mM NaB exposure by Western blot analysis and noted no change in the amount of protein produced at 24 to 72 h. We then evaluated this system by exposure of mesothelioma cells to fas-active antibody (ligand) at varying concentrations. No increase in cellular death was noted above the baseline after antibody and NaB exposure (data not shown).
BCL-XL Protein Expression Is Altered by NaB Exposure
A number of investigators have demonstrated alterations in bcl-2 family protein expression in tumor cell lines after NaB exposure. We evaluated expression of the proapoptotic proteins bax and bak together with the antiapoptotic proteins BCL-2 and BCL-XL after varying doses of NaB and at varying time points after exposure by Western blot analysis. As demonstrated in Figure 6, a marked decrease in BCL-XL protein expression was noted in both cell lines after 3-mM NaB exposure. BCL-2 protein expression, very low at baseline, was not appreciably altered, nor was expression of the proapoptotic bcl-2 family members bak and bax. The relative level of actin protein expression was preserved at all time points.
|
bcl-xl Messenger RNA Level Is Altered by NaB Exposure
To determine if the alteration in BCL-XL protein expression noted previously was related to changes in messenger RNA (mRNA), an RNase protection assay was performed on REN and I-45 cells, evaluating for a panel of apoptosis-related genes. Both NaB-treated and control cells were evaluated for mRNA expression and the results are depicted in Figure 7. Band densities were compared using digital scanning software, and values are expressed relative to the mRNA expression level of housekeeping gene expression GAPDH in each group. A significant decrease of bcl-xl mRNA level is noted in both cell lines after NaB exposure. In addition, the level of mcl-1 mRNA is significantly reduced after NaB treatment in the REN cell line.
|
Stable Overexpression of BCL-XL Correlates with Lowered Sensitivity to NaB-Induced Apoptosis
To test further the hypothesis that BCL-XL protein expression is an important determinant of apoptotic death in mesothelioma cells, stable overexpressing BCL-XL clones of the I-45 mesothelioma cell line were created using plasmid transfer techniques. Several clones were generated with varying increased levels of BCL-XL protein expression compared with the parent I-45 cell line (clones I, Q, 47, S; Figure 8). Figure 8 also demonstrates that sensitivity to the induction of cellular death with NaB exposure was inversely proportional to the level of BCL-XL protein expression (ratios of BCL-XL expression based on parent line set at 1, ratios of cell death based on highest expressing clone set at 1).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Malignant pleural mesothelioma, albeit relatively uncommon when compared with other intrathoracic malignancies such as nonsmall cell lung carcinoma, remains a frustratingly difficult clinical problem with a case:fatality ratio approaching one. Local control has improved with aggressive surgical therapy; however, recurrences are common, and metastatic disease, once thought unlikely, is becoming more recognized as a result (1, 24, 25). The relative unresponsiveness of this tumor to conventional chemotherapeutic agents is unfortunately well known (2). In large part owing to dissatisfaction with available systemic treatment options, a number of novel therapies, including adenoviral-mediated gene therapy using both immune and prodrug suicide gene transfer, are currently being investigated (26, 27). The artificial induction of programmed cell death, or apoptosis, is one of the more promising novel approaches to treatment of malignant disease (28). In this study, we were able to readily induce apoptosis in two well-characterized human mesothelioma cell lines after exposure to NaB, with a lower IC50 than has been noted with many epithelial tumor types. We also show that a reduction in BCL-XL protein expression is associated with cell death and apoptosis induced by NaB and that this seems to be related to events at the level of gene transcription as mRNA levels are reduced as well by RNase assay.
Published literature regarding apoptosis and mesothelioma has been limited. p53 mutations are uncommon in this tumor, suggesting that alterations in the expression of this gene are unlikely to be responsible for either creation of a malignant phenotype or for insensitivity to apoptotic induction (8). Crocidolite, a type of asbestos fiber commonly associated with the development of mesothelioma, has been shown to paradoxically induce apoptosis via reactive oxygen species (29). It has been proposed that mesothelioma might be relatively resistant to apoptosis once established, and this may in part explain the tumor's insensitivity to conventional therapies. Narasimhan and coworkers (7) demonstrated the resistance of mesothelioma cells to fairly rigorous challenge with both oxidant and nonoxidant stimulators of in vitro apoptosis such as hydrogen peroxide and calcium ionophore. This group demonstrated that the antiapoptotic protein BCL-2 was infrequently expressed in mesothelioma cell lines and that expression of the proapoptotic gene bax was common. Other investigators have also demonstrated bcl-2 gene product expression to be rare in both mesothelioma tumors as well as cell lines, but bcl-xl and bax gene product expression are common and pronounced (9, 30). In this study, we found both REN and I-45 mesothelioma cell lines to be positive at baseline for strong BCL-XL, BAX, and BAK protein expression with a very low-level BCL-2 protein expression.
Depending on the study reviewed, virtually every mechanism thus far characterized has been proposed for the ability of NaB to engender apoptosis in malignant cells. It seems likely on the basis of myriad reports that the effects of NaB are variable and cell-type specific. Sensitization to fas-fas ligand interaction, reported elsewhere, was not noted in this study (18, 22). We demonstrated that mesothelioma bcl-xl gene and protein expression were markedly attenuated by NaB exposure. The literature on discrete NaB effect on bcl-xl expression in other tumor types is conflicting. Bonnotte and colleagues (18) did not note changes in BCL-XL or BCL-2 expression in rat or human colon cancer cells after NaB exposure. Alternatively, Wang and associates (13) noted significant decreases in BCL-XL expression in two different hepatic carcinoma cell lines after NaB exposure, and Litvak and coworkers (11) showed that bcl-2 as well as bcl-xl mRNA expression were markedly downregulated in human colon cancer cell lines by ribonuclease protection assay. As bax and bak protein expression were not altered by NaB exposure in our study, it is possible that by virtue of markedly lowered BCL-XL expression, either a lack of Apaf-1 binding or an alteration in the proapoptotic:antiapoptotic protein ratio led to apoptosis in this study. Caspase activity was increased slightly by a functional assay in mesothelioma cells after NaB exposure, but significant caspase cleavage bands on Western blot analysis were not noted. This raises the possibility that a caspase-independent mechanism such as has been noted in yeast (overexpression of proapoptotic proteins) or that a combination of both caspase-dependent and caspase-independent processes is responsible. Although it is certainly possible that expression of a number of cellular genes in mesothelioma is effected by NaB, we further demonstrated that BCL-XL overexpression in mesothelioma cells is sufficient to reverse the effects, namely apoptotic cell death, seen with NaB exposure and bcl-xl downregulation in control cells.
Although other antiapoptotic proteins have been described in cells, BCL-XL and BCL-2 are among the more potent thus far identified in many cell lines and tissues, and when overexpressed demonstrate an equivalent ability to inhibit apoptosis (31). Additional support for an important role for bcl-xl expression in prevention of apoptosis in mesothelioma is provided by antisense studies in nonsmall cell lung carcinoma. In a report evaluating bcl-xl antisense therapy in lung carcinoma cells, it was noted that apoptosis occurred in those expressing BCL-XL at high levels and little or no BCL-2. In cell lines with comparable levels of BCL-2 and BCL-XL, no effect was noted in one study and another study suggested that cell death could not be engendered if both proteins were expressed unless an antisense oligonucleotide with specificity for both BCL-2 and BCL-XL was used (32). In the REN cell line, the level of mcl-1 mRNA is also reduced by NaB treatment. Although not as well studied as other members of the bcl-2 family, mcl-1 is known to be an important inhibitor of apoptosis in some tissues, such as the ovary (33). The fact that BCL-XL is downregulated in both these cell lines with little difference in cell death or apoptosis noted between the two argues for a lesser role for the mcl-1 protein compared with BCL-XL. However, this observation will require further investigation as malignant mesothelioma does exhibit some other interesting molecular correlates with ovarian tissue such as overexpression of the WT-1 gene (34).
Additional study of the constituents of this bcl-xl gene expression-dependent apoptotic pathway in mesothelioma at both the transcriptional and translational level may be informative and allow for the future development of more effective therapeutic strategies for this and other neoplasms exhibiting similar patterns of bcl-2 family protein expression.
| |
Footnotes |
|---|
Address correspondence to: W. Roy Smythe, Dept. of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer
Center, 1515 Holcombe Blvd.Box 109, Houston, TX 77030. E-mail:
rsmythe{at}mdanderson.org
(Received in original form February 21, 2001 and in revised form May 30, 2001).
Abbreviations: glyceraldehyde-3-phosphate dehydrogenase, GAPDH; concentration of drug required to kill 50% of the cells, IC50; messenger RNA, mRNA; sodium butyrate, NaB; phosphate-buffered saline, PBS.Acknowledgments: This study was supported in part by the University of Texas M.D. Anderson Physician-Scientist Award (W.R.S.) and by the University of Texas M.D. Anderson-W. M. Keck Center for Cancer therapy.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
1.
Sugarbaker, D. J.,
R. M. Flores,
M. T. Jaklitsch,
W. G. Richards,
G. M. Strauss,
J. M. Corson,
M. M. DeCamp,
S. J. Swanson,
R. Bueno,
J. M. Lukanich,
E. H. Baldini, and
S. J. Mentzner.
1999.
Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: results in
183 patients.
J. Thorac. Cardiovasc. Surg.
117:
54-65
2.
Ong, T., and
N. J. Vogelzang.
1996.
Chemotherapy in malignant mesothelioma: a review.
J. Clin. Oncol.
14:
1007-1017
3. Sahmoud, T., P. E. Postmus, C. van Pottelsberghe, K. Mattson, L. Tammilehto, T. A. W. Splinter, A. S. T. Planting, T. Sutedja, J. van Pawel, N. van Zandwijk, P. Baas, K. J. Roozendaal, M. Schrijver, A. Kirkpatrick, M. V. van Glabbake, A. Ardizzoni, and G. Giaccone. 1997. Etoposide in malignant pleural mesothelioma: two phase II trials of the EORTC Lung Cancer Cooperative Group. Eur. J. Cancer 33: 2211-2215 .
4. van Meerbeeck, J., C. Debruyne, N. van Zandwijk, P. E. Postmus, M. C. Pennucci, F. van Breukelen, D. Galdermans, H. Groen, P. Pinson, M. van Glabbeke, E. van Marck, and G. Giaccone. 1996. Paclitaxel for malignant pleural mesothelioma: a phase II study of the EORTC Lung Cancer Cooperative Group. Br. J. Cancer 76: 961-963 .
5.
Smythe, W. R.,
H. C. Hwang,
K. M. Amin,
S. L. Eck,
B. L. Davidson,
J. M. Wilson,
L. R. Kaiser, and
S. M. Albelda.
1994.
Use of recombinant adenovirus to transfer the herpes simplex virus thymidine kinase (HSVtk) gene
to thoracic neoplasms: an effective in vitro drug sensitization system.
Cancer Res.
54:
2055-2059
6.
Pass, H. I.,
B. W. Robinson,
J. R. Testa, and
M. Carbone.
1999.
Emerging
translational therapies for mesothelioma.
Chest
116:
455S-460S
7.
Narasimhan, S. R.,
L. Yang,
B. I. Gerwin, and
V. C. Broaddus.
1998.
Resistance of pleural mesothelioma cell lines to apoptosis: relation to expression of bcl-2 and bax.
Am. J. Physiol.
275:
L165-L171
8. Mor, O., P. Yaron, M. Huszar, A. Yellin, O. Jakobovitz, F. Brok-Simoni, G. Rechavi, and N. Reichert. 1997. Absence of p53 mutations in malignant mesotheliomas. Am. J. Respir. Cell Mol. Biol. 16: 9-13 [Abstract].
9.
Soini, Y.,
V. Kinnula,
W. R. Kaarteenhaho,
E. Kurttil,
K. Linnainmaa, and
P. Paako.
1999.
Apoptosis and expression of apoptosis regulating proteins
bcl-2, mcl-1, bcl-X and bax in malignant mesothelioma.
Clinical Cancer
Research
5:
3508-3515
10.
Cummings, J. H..
1981.
Short chain fatty acids in the human colon.
Gut
22:
763-779
11. Litvak, D. A., B. M. Evers, K. O. Hwang, M. R. Hellmich, T. C. Ko, and C. M. Townsend. 1998. Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21 WAF1/Cip1 and p27Kip1. Surgery 124: 161-170 [Medline].
12. Mandel, M., and R. Kumar. 1996. Bcl-2 expression regulates sodium butyrate-induced apoptosis in human MCF-7 breast cancer cells. Cell Growth Differ. 7: 311-318 [Abstract].
13. Wang, X.-M., X. Wang, J. Li, and B. M. Evers. 1998. Effects of 5-azacytidine and butyrate on differentiation and apoptosis of hepatic cancer cell lines. Ann. Surg. 227: 922-931 [Medline].
14. Ellerhorst, J., T. Nguyen, D. N. W. Cooper, Y. Estrov, D. Lotan, and R. Lotan. 1999. Induction of differentiation and apoptosis in the prostate cancer cell line LNCaP by sodium butyrate and galectin-1. Int. J. Oncol. 14: 225-232 . [Medline]
15. Hague, A., G. D. Diaz, D. J. Hicks, S. Krajewski, J. C. Reed, and C. Paraskeva. 1997. bcl-2 and bak may play a pivotal role in sodium butyrate-induced apoptosis in colonic epithelial cells; however, overexpression of bcl-2 does not protect against bak-mediated apoptosis. Int. J. Cancer 72: 898-905 [Medline].
16. Heerdt, B. G., M. A.. Houston, and L. H. Augenlicht. 1997. Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function. Cell Growth and Differ. 8:523-532.
17.
Medina, V.,
B. Edmonds,
G. Y. Young,
R. James,
S. Appleton, and
P. D. Zalewski.
1997.
Induction of caspase-3 protease activity and apoptosis by
butyrate and trichostatin A. (inhibitors of histone deacetylase): dependence on protein synthesis and synergy with a mitochondrial.cytochrome
c-dependent pathway.
Cancer Res.
57:
3697-3707
18. Bonnotte, B., N. Favre, S. Reveneau, O. Micheau, N. Drion, C. Garrido, A. Fontana, B. Chauffert, E. Solary, and F. Martin. 1998. Cancer cell sensitization to Fas-mediated apoptosis by sodium butyrate. Cell Growth Differ. 5: 480-487 .
19. Herold, K., and P. Rothberg. 1998. Evidence for a labile intermediate in the butyrate induced reduction of the level of c-myc RNA in SW837 rectal carcinoma cells. Oncogene 4: 423-428 .
20. Chung, D. H, F. Zhang, F. Chen, W. P. McLaughlin, and M. Ljungman. 1998. Butyrate attenuates BCLX(L) expression in human fibroblasts and acts in synergy with ionizing radiation to induce apoptosis. Radiat. Res. 149: 187-194 [Medline].
21.
Gum, J.,
W. Kam,
J. Byrd,
J. Hicks,
M. Sleisenger, and
Y. Kim.
1987.
Effects
of sodium butyrate on human colonic adenocarcinoma cells.
J. Biol. Chem.
262:
1092-1097
22. Barnard, J., and G. Warwick. 1993. Butyrate rapidly induces growth inhibition and differentiation in HT-29 cells. Cell Growth Differ. 4: 495-501 [Abstract].
23.
Giardina, C.,
H. Boulares, and
M. S. Inan.
1999.
NSAIDS and butyrate sensitize a human colorectal cancer cell line to TNF
and fas ligation: the role
of reactive oxygen species.
Biochim. Biophys. Acta
1448:
425-438
[Medline].
24.
Baldini, E. H.,
A. Recht,
G. M. Strauss,
M. M. DeCamp Jr,
S. J. Swanson,
M. J. Liptay,
S. J. Mentzer, and
D. J. Sugarbaker.
1997.
Patterns of failure
after trimodality therapy for malignant pleural mesothelioma.
Ann. Thorac. Surg.
63:
334-348
25. Kawai, A., Y. Nagasaka, M. Muraki, M. Fukuoka, T. Satou, M. Kimura, and S. Hashimoto. 1997. Brain metastasis in malignant pleural mesothelioma. Intern. Med. 36: 591-594 [Medline].
26. Schwarzenberger, P.. 1998. The treatment of malignant mesothelioma with a gene modified cancer cell line: a phase I study. Hum. Gene Ther. 9: 2641-2649 [Medline].
27. Sterman, D. H., J. Treat, L. A. Litzky, K. M. Amin, L. Coonrod, K. Molnar-Kimber, A. Recio, L. Knox, J. M. Wilson, S. M. Albelda, and L. R. Kaiser. 1998. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with low malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum. Gene. Ther. 9: 1083-1092 [Medline].
28. Korsmeyer, S. J.. 1999. BCL2 gene family and the regulation of programmed cell death. Cancer Res. 59: 1693-1700 .
29. Broaddus, V. C., L. Yang, L. M. Scavo, J. D. Ernst, and A. M. Boylan. 1996. Asbestos induces apoptosis of human and rabbit pleural mesothelial cells via reactive oxygen species. J. Clin. Invest. 98: 2050-2059 [Medline].
30. Segers, K., M. Ramael, S. K. Singh, J. Weyler, J. Van Meerbeeck, P. Vermeire, and E. Van Marck. 1994. Immunoreactivity for bcl-2 protein in malignant mesothelioma and non-neoplastic mesothelium. Virchows Arch. 424: 631-634 [Medline].
31. Huang, D. C. S., S. Cory, and A. Strasser. 1997. Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. Oncogene. 14: 405-414 [Medline].
32. Leech, S. H., R. A. Olie, O. Gautschi, A. P. Simoes-Wust, S. Tschopp, R. Haner, J. Hail, R. A. Stahel, and U. Zangemeister-Wittke. 2000. Induction of apoptosis in lung cancer cells following bclXL antisense treatment. Int. J. Cancer 86: 570-576 [Medline].
33.
Hsu, S. Y., and
A. J. Hsueh.
2000.
Tissue-specific Bcl-2 protein partners in
apoptosis: An ovarian paradigm.
Physiol. Rev.
80:
593-614
34. Amin, K. M, L. A. Litzky, W. R. Smythe, and et al. 1995. Wilms' tumor 1 susceptibility (WT1) gene products are selectively expressed in malignant mesothelioma. Am. J. Pathol. 146: 344-356 [Abstract].
This article has been cited by other articles:
 |
|
 |
|
  S. K. Kulp, C.-S. Chen, D.-S. Wang, C.-Y. Chen, and C.-S. Chen Antitumor Effects of a Novel Phenylbutyrate-Based Histone Deacetylase Inhibitor, (S)-HDAC-42, in Prostate Cancer Clin. Cancer Res., September 1, 2006; 12(17): 5199 - 5206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Scherpereel, B. Grigoriu, M. Conti, T. Gey, M. Gregoire, M.-C. Copin, P. Devos, B. Chahine, H. Porte, and P. Lassalle Soluble Mesothelin-related Peptides in the Diagnosis of Malignant Pleural Mesothelioma Am. J. Respir. Crit. Care Med., May 15, 2006; 173(10): 1155 - 1160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Rascoe, X. Cao, J. C. Daniel, S. D. Miller, and W. R. Smythe Receptor tyrosine kinase and phosphoinositide-3 kinase signaling in malignant mesothelioma J. Thorac. Cardiovasc. Surg., August 1, 2005; 130(2): 393 - 400. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Munshi, J. F. Kurland, T. Nishikawa, T. Tanaka, M. L. Hobbs, S. L. Tucker, S. Ismail, C. Stevens, and R. E. Meyn Histone Deacetylase Inhibitors Radiosensitize Human Melanoma Cells by Suppressing DNA Repair Activity Clin. Cancer Res., July 1, 2005; 11(13): 4912 - 4922. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. K. Kelly, O. A. O'Connor, L. M. Krug, J. H. Chiao, M. Heaney, T. Curley, B. MacGregore-Cortelli, W. Tong, J. P. Secrist, L. Schwartz, et al. Phase I Study of an Oral Histone Deacetylase Inhibitor, Suberoylanilide Hydroxamic Acid, in Patients With Advanced Cancer J. Clin. Oncol., June 10, 2005; 23(17): 3923 - 3931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Doi, H. Soda, M. Oka, J. Tsurutani, T. Kitazaki, Y. Nakamura, M. Fukuda, Y. Yamada, S. Kamihira, and S. Kohno The histone deacetylase inhibitor FR901228 induces caspase-dependent apoptosis via the mitochondrial pathway in small cell lung cancer cells Mol. Cancer Ther., November 1, 2004; 3(11): 1397 - 1402. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Ozvaran, X. X. Cao, S. D. Miller, B. A. Monia, W. K. Hong, and W. R. Smythe Antisense oligonucleotides directed at the bcl-xl gene product augment chemotherapy response in mesothelioma Mol. Cancer Ther., May 1, 2004; 3(5): 545 - 550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. D. Zhang, S. K. Gillespie, J. M. Borrow, and P. Hersey The histone deacetylase inhibitor suberic bishydroxamate regulates the expression of multiple apoptotic mediators and induces mitochondria-dependent apoptosis of melanoma cells Mol. Cancer Ther., April 1, 2004; 3(4): 425 - 435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Terui, K. Murakami, R. Takimoto, M. Takahashi, K. Takada, T. Murakami, S. Minami, T. Matsunaga, T. Takayama, J. Kato, et al. Induction of PIG3 and NOXA through Acetylation of p53 at 320 and 373 Lysine Residues as a Mechanism for Apoptotic Cell Death by Histone Deacetylase Inhibitors Cancer Res., December 15, 2003; 63(24): 8948 - 8954. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Singhal, R. Wiewrodt, L. D. Malden, K. M. Amin, K. Matzie, J. Friedberg, J. C. Kucharczuk, L. A. Litzky, S. W. Johnson, L. R. Kaiser, et al. Gene Expression Profiling of Malignant Mesothelioma Clin. Cancer Res., August 1, 2003; 9(8): 3080 - 3097. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Crit. Care Med. |