Malignant Pleural Mesothelioma Lines Research Articles

Malignant pleural mesothelioma nodules remodel their surroundings to vascularize and grow.

BackgroundThe microanatomical steps of malignant pleural mesothelioma (MPM) vascularization and the resistance mechanisms to anti-angiogenic drugs in MPM are unclear.MethodsWe investigated the vascularization of intrapleurally implanted human P31 and SPC111 MPM cells. We also assessed MPM cell’s motility, invasion and interaction with endothelial cells in vitro.ResultsP31 cells exhibited significantly higher two-dimensional (2D) motility and three-dimensional (3D) invasion than SPC111 cells in vitro. In co-cultures of MPM and endothelial cells, P31 spheroids permitted endothelial sprouting (ES) with minimal spatial distortion, whereas SPC111 spheroids repealed endothelial sprouts. Both MPM lines induced the early onset of submesothelial microvascular plexuses covering large pleural areas including regions distant from tumor colonies. The development of these microvascular networks occurred due to both intussusceptive angiogenesis (IA) and ES and was accelerated by vascular endothelial growth factor A (VEGF-A)-overexpression. Notably, SPC111 colonies showed different behavior to P31 cells. P31 nodules incorporated tumor-induced capillary plexuses from the earliest stages of tumor formation. P31 cells deposited a collagenous matrix of human origin which provided “space” for further intratumoral angiogenesis. In contrast, SPC111 colonies pushed the capillary plexuses away and thus remained avascular for weeks. The key event in SPC111 vascularization was the development of a desmoplastic matrix of mouse origin. Continuously invaded by SPC111 cells, this matrix transformed into intratumoral connective tissue trunks, providing a route for ES from the diaphragm.ConclusionsHere, we report two distinct growth patterns of orthotopically implanted human MPM xenografts. In the invasive pattern, MPM cells invade and thus co-opt peritumoral capillary plexuses. In the pushing/desmoplastic pattern, MPM cells induce a desmoplastic response within the underlying tissue which allows the ingrowth of a nutritive vasculature from the pleura.

Picropodophyllin inhibits the growth of pemetrexed-resistant malignant pleural mesothelioma via microtubule inhibition and IGF-1R-, caspase-independent pathways.

BackgroundAcquired pemetrexed resistance leads to treatment interruption in patients with malignant pleural mesothelioma (MPM). Insulin-like growth factor-I receptor (IGF-1R) inhibitor is a candidate for treating pemetrexed-naïve MPM. However, the efficacy of cytotoxic and targeted drugs in acquired-pemetrexed resistant MPM is unclear. We explored anticancer drugs, including the IGF-1R inhibitor picropodophyllin, against acquired pemetrexed-resistant MPMs.MethodsAcquired pemetrexed-resistant human MPM cell lines, named as H2452/PEM and 211H/PEM after their parental lines H2452 and 211H, respectively, were established by exposure to pemetrexed in vitro. Picropodophyllin and siRNA for IGF-1R knockdown were used to evaluate the efficacy of IGF-1R inhibition. Immunofluorescence was used to evaluate microtubule localization. The efficacy of picropodophyllin was evaluated in 3-dimensional MPM models.ResultsThe acquired pemetrexed-resistant MPM lines retained their resistance after the removal of culture treatment. IGF-1R levels in H2452/PEM cells were higher than those in H2452 cells but not in 211H/PEM cells compared to the respective parental line. Picropodophyllin induced sub-G1 arrest in H2452/PEM cells but induced G2/M phase arrest in 211H/PEM cells, leading to caspase-independent cell death in the two acquired pemetrexed-resistant MPM lines. Although picropodophyllin inhibited phosphorylation of IGF-1R, specific inhibition of IGF-1R by RNA interference did not reduce the viability of pemetrexed-resistant MPM lines. Additionally, picropodophyllin reduced the viability of both IGF-1R knockdown pemetrexed-resistant MPM cells. Picropodophyllin was cytotoxic in acquired-pemetrexed-resistant MPM lines because of inhibition of microtubule formation and induction of aberrant mitosis. Moreover, combination treatment with picropodophyllin and vinorelbine synergistically affected the pemetrexed-resistant MPM lines but not the parental lines. Furthermore, we observed a similar efficacy of picropodophyllin in 3-dimensional pemetrexed-resistant MPM models.ConclusionsPicropodophyllin may offer novel therapeutic properties for treating acquired pemetrexed-resistant MPM. Targeting tubulin may be an important strategy in the treatment of MPM after the discontinuation of pemetrexed.

Identification of novel COX-2 / CYP19A1 axis involved in the mesothelioma pathogenesis opens new therapeutic opportunities

BackgroundBased on previous studies highlighting that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of malignant pleural mesothelioma (MPM), and that aromatase (CYP19A1), an enzyme that plays a key role in estrogen biosynthesis, along with estradiol (E2) were expressed in MPM, this study aimed to investigate the possible interplay between COX-2 and CYP19A1 in the pathogenesis of mesothelioma, as well as the underlying mechanism.MethodsThe interaction between COX-2 and CYP19A1 was first investigated on different MPM lines upon PGE2, and COX-2 inhibitor (rofecoxib) treatment by western blot, RT-PCR. The key regulatory pathways involved in the COX-2 and CYP19A1 axis were further studied in MPM cells, after rofecoxib and exemestane (CYP19A1 inhibitor) treatment in monotherapy and in combination, by cell cycle distribution, western blot, and combination index analysis. To explore the role of COX-2/CYP19A1 axis in 3D preclinical models of MPM cells, we analyzed the effect of combination of COX-2 and CYP19A1 inhibitors in mesosphere formation. Immunohistochemical analysis of MPM mesosphere and specimens was utilized to evaluate the involvement of COX-2 on the CYP19A1 activity and the relationship between E2 and COX-2.ResultsPGE2 or rofecoxib treatment caused in MPM cells an increased or decreased, respectively, CYP19A1 expression at mRNA and protein levels. The effect of rofecoxib and exemestane combination in MPM cell proliferation was synergistic. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for MPM cell lines. Increased expression levels of p53, p21, and p27, downregulation of cyclin D1 and inhibition of Akt activation (pAKT) were also found. The antagonistic effect of rofecoxib and exemestane combination found only in one cell line, was reverted by pretreatment with MK2206, a pAKT inhibitor, indicating pAKT as an actionable mediator in the COX-2-CYP19A1 axis. Reduction of size and sphere-forming efficiency in MPM spheres after treatment with both inhibitor and a decrease in COX-2 and E2 staining was found. Moreover, immunohistochemical analysis of 46 MPM samples showed a significant positive correlation between COX-2 and E2.ConclusionsCollectively, the results highlighted a novel COX-2/CYP19A1 axis in the pathogenesis of MPM that can be pharmacologically targeted, consequently opening up new therapeutic options.

Ponatinib is a potential therapeutic approach for malignant pleural mesothelioma

Purpose Malignant pleural mesothelioma (MPM) is a rare and deadly malignancy. Current MPM therapies remain inadequate, and outcomes are often disappointing. New meaningful therapeutic approaches are urgently needed. Accumulating evidence indicates that the cAbl pathway promotes various tumor-stimulating processes in MPM. In this study, we sought to determine ponatinib’s potential utility, a clinically approved and potent cAbl inhibitor, in MPM treatment. Material and methods Four MPM lines (MSTO211H, H28, H2452, H2052) were treated with ponatinib in vitro, and their growth was assessed. Scratch wound assay was used to investigate the ponatinib effect on cell migration. The expression levels of pAbl and its downstream effectors pCrkL, pAKT, and pSTAT5 were characterized. The in vivo ponatinib effect was evaluated in human MPM cells derived tumor model. Results In all four MPM lines, significant expression levels of phosphorylated cAbl/Arg and pCrkl were observed. Differentially but strongly, ponatinib inhibited the in vitro cell growth and migration of all four MPM line. Western blot analysis showed that the activation of Abl signaling was blocked in the ponatinib-treated MMP lines. In keeping, the cellular levels of pAbl and its downstream effector pCrkL, pAKT, and pSTAT5 were markedly decrease following ponatinib treatment. Moreover, ponatinib treatment amplified the levels of γH2AX in cells denoting increased double-strand DNA breaks levels. Notably, ponatinib treatment reduced in vivo tumor growth and reduced pCrkl and pSTAT5 levels in tumor samples. Conclusion Ponatinib may offer a new therapeutic strategy for MPM patients based on cAbl signaling pathway inhibition.

UHRF1 Is a Novel Druggable Epigenetic Target in Malignant Pleural Mesothelioma.

Ubiquitin-like with plant homeodomain and ring finger domains 1 (UHRF1) encodes a master regulator of DNA methylation that has emerged as an epigenetic driver in human cancers. To date, no studies have evaluated UHRF1 expression in malignant pleural mesothelioma (MPM). This study was undertaken to explore the therapeutic potential of targeting UHRF1 in MPM. Microarray, real-time quantitative reverse transcription-polymerase chain reaction, immunoblot, and immunohistochemistry techniques were used to evaluate UHRF1 expression in normal mesothelial cells (NMCs) cultured with or without asbestos, MPM lines, normal pleura, and primary MPM specimens. The impact of UHRF1 expression on MPM patient survival was evaluated using two independent databases. RNA-sequencing, proliferation, invasion, and colony formation assays, and murine xenograft experiments were performed to evaluate gene expression and growth of MPM cells after biochemical or pharmacologic inhibition of UHRF1 expression. UHRF1 expression was significantly higher in MPM lines and specimens relative to NMC and normal pleura. Asbestos induced UHRF1 expression in NMC. The overexpression of UHRF1 was associated with decreased overall survival in patients with MPM. UHRF1 knockdown reversed genomewide DNA hypomethylation, and inhibited proliferation, invasion, and clonogenicity of MPM cells, and growth of MPM xenografts. These effects were phenocopied by the repurposed chemotherapeutic agent, mithramycin. Biochemical or pharmacologic up-regulation of p53 significantly reduced UHRF1 expression in MPM cells. RNA-sequencing experiments exhibited the pleiotropic effects of UHRF1 down-regulation and identified novel, clinically relevant biomarkers of UHRF1 expression in MPM. UHRF1 is an epigenetic driver in MPM. These findings support the efforts to target UHRF1 expression or activity for mesothelioma therapy.

ES03.01 Targeting Altered microRNA Expression in Mesothelioma

ES03.01 Targeting Altered microRNA Expression in Mesothelioma

Proteasome stress sensitizes malignant pleural mesothelioma cells to bortezomib-induced apoptosis

Based on promising results in preclinical models, clinical trials have been performed to evaluate the efficacy of the first-in-class proteasome inhibitor bortezomib towards malignant pleural mesothelioma (MPM), an aggressive cancer arising from the mesothelium of the serous cavities following exposure to asbestos. Unexpectedly, only minimal therapeutic benefits were observed, thus implicating that MPM harbors inherent resistance mechanisms. Identifying the molecular bases of this primary resistance is crucial to develop novel pharmacologic strategies aimed at increasing the vulnerability of MPM to bortezomib. Therefore, we assessed a panel of four human MPM lines with different sensitivity to bortezomib, for functional proteasome activity and levels of free and polymerized ubiquitin. We found that highly sensitive MPM lines display lower proteasome activity than more bortezomib-resistant clones, suggesting that reduced proteasomal capacity might contribute to the intrinsic susceptibility of mesothelioma cells to proteasome inhibitors-induced apoptosis. Moreover, MPM equipped with fewer active proteasomes accumulated polyubiquitinated proteins, at the expense of free ubiquitin, a condition known as proteasome stress, which lowers the cellular apoptotic threshold and sensitizes mesothelioma cells to bortezomib-induced toxicity as shown herein. Taken together, our data suggest that an unfavorable load-versus-capacity balance represents a critical determinant of primary apoptotic sensitivity to bortezomib in MPM.

SFRP Tumour Suppressor Genes Are Potential Plasma-Based Epigenetic Biomarkers for Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is associated with asbestos exposure. Asbestos can induce chronic inflammation which in turn can lead to silencing of tumour suppressor genes. Wnt signaling pathway can be affected by chronic inflammation and is aberrantly activated in many cancers including colon and MPM. SFRP genes are antagonists of Wnt pathway, and SFRPs are potential tumour suppressors in colon, gastric, breast, ovarian, and lung cancers and mesothelioma. This study investigated the expression and DNA methylation of SFRP genes in MPM cells lines with and without demethylation treatment. Sixty-six patient FFPE samples were analysed and have showed methylation of SFRP2 (56%) and SFRP5 (70%) in MPM. SFRP2 and SFRP5 tumour-suppressive activity in eleven MPM lines was confirmed, and long-term asbestos exposure led to reduced expression of the SFRP1 and SFRP2 genes in the mesothelium (MeT-5A) via epigenetic alterations. Finally, DNA methylation of SFRPs is detectable in MPM patient plasma samples, with methylated SFRP2 and SFRP5 showing a tendency towards greater abundance in patients. These data suggested that SFRP genes have tumour-suppresive activity in MPM and that methylated DNA from SFRP gene promoters has the potential to serve as a biomarker for MPM patient plasma.

Targeting of the Hedgehog signal transduction pathway suppresses survival of malignant pleural mesothelioma cells in vitro

The present study sought to determine whether the Hedgehog (Hh) pathway is active and regulates the cell growth of cultured malignant pleural mesothelioma (MPM) cells and to evaluate the efficacy of pathway blockade using smoothened (SMO) antagonists (SMO inhibitor GDC-0449 or the antifungal drug itraconazole [ITRA]) or Gli inhibitors (GANT61 or the antileukemia drug arsenic trioxide [ATO]) in suppressing MPM viability. Selective knockdown of SMO to inhibit Hh signaling was achieved by small interfering RNA in 3 representative MPM cells. The growth inhibitory effect of GDC-0449, ITRA, GANT61, and ATO was evaluated in 8 MPM lines, with cell viability quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death was determined by annexinV/propidium iodide staining and flow cytometry. SMO small interfering RNA mediated a two- to more than fivefold reduction of SMO and Gli1 gene expression as determined by real-time quantitative reverse-transcriptase polymerase chain reaction, indicating significant Hh pathway blockade. This was associated with significantly reduced cell viability (34% ± 7% to 61% ± 14% of nontarget small interfering RNA controls; P=.0024 to P=.043). Treating MPM cells with Hh inhibitors resulted in a 1.5- to 4-fold reduction of Gli1 expression. These 4 Hh antagonists strongly suppressed MPM cell viability. More importantly, ITRA, ATO, GANT61 induced significant apoptosis in the representative MPM cells. Hh signaling is active in MPM and regulates cell viability. ATO and ITRA were as effective as the prototypic SMO inhibitor GDC-0449 and the Gli inhibitor GANT61 in suppressing Hh signaling in MPM cells. Pharmaceutical agents Food and Drug Administration-approved for other indications but recently found to haveanti-Hh activity, such as ATO or ITRA, could be repurposed to treat MPM.

Effect of the load versus capacity unbalance on the sensitivity of malignant pleural mesothelioma cells to proteasome inhibitors.

e18522 Background: Malignant Pleural Mesothelioma (MPM) is an aggressive cancer that originates from the mesothelium of the pleural cavities. Due to its resistance to all chemotherapies available, news therapies against MPM are urgently needed. Bortezomib is a potent Proteasome Inhibitors (PI) that induces apoptosis in different cancers and it was recently approved for treatment of Multiple Myeloma (MM). Since in MM an unbalance between a reduced proteasomal activity (capacity) and an increased proteins degradation (load) was shown to determine apoptotic sensitivity to PI, we challenged this paradigm also in MPM. Methods: Four different MPM (MM98, REN, MMB, MSTO) and a normal (TERT) pleural lines were treated with increasing doses (from 1 to 1000 nM) of Bortezomib and after 24h apoptosis was assessed by flow cytometric analysis of AnnexinV and propidium iodide positive cells. Levels of polyubiquitinated proteins were evaluated by immunofluorescence microscopy and western blotting with the specific monoclonal Ab Fk2 (BIOMOL International). The three main proteasome activities were measured in continuoin cellular extracts at 37°C with a Carry Eclipse spectrofluorometer (Varian, Palo Alto) by means of specific fluorogenic substrates (Suc-LLVY-amc, Bz-VGR-amc, Suc-YVAD-amc). Results: The sensitivity towards Bortezomib greatly differs between the 5 pleural lines analyzed. Specifically the EC50value was higher for the TERT line (130 nM) while for MPM lines ranged between 17 nM (MM98) and 71 nM (MSTO). Importantly, for all the 5 lines analyzed enhanced sensitivity to Bortezomib perfectly correlates with increased levels of ubiquitinated proteins and reduced proteasome specific activity. Conclusions: Our results prove that MPM lines are significantly more sensitive towards the pro-apoptotic effect of Bortezomib than normal pleural cells. Moreover, as in MM, also in MPM PI-sensitivity stems from an unbalance of the load/capacity ratio. Our data, therefore, establish a rationale for the use of Bortezomib (and new second generation PI) in MPM and indicate that combinatorial therapies with other drugs active on the proteostasis network (like HSP inhibitors) might be highly effective against this cancer.

Sensitivity of malignant mesothelioma lacking Merlin to the FAK inhibitor VS-6063: Evaluation of merlin/NF2 status in clinical samples.

e18541 Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung often resulting from prior exposure to asbestos. MPM patients are usually diagnosed at an advanced stage of disease and the prognosis is poor. Median survival after diagnosis is 9 to 12 months and standard-of-care agents such as cisplatin and pemetrexed have only a modest impact on median survival time for MPM patients. New therapeutic modalities are urgently needed to improve the prognosis of MPM patients. 40-50% of MPM patients exhibit homozygous disruption of the NF2 tumor suppressor gene by mutation and/or deletion resulting in lack of expression of functional merlin protein. Methods: Proliferation of drug-treated mesothelioma cell lines in 3-dimensional (3D) Matrigel culture was assessed by MTS, and MPM xenograft growth was measured in vehicle- vs. FAK inhibitor-treated SCID mice. Since absence of merlin expression can theoretically result from several mechanisms including NF2 mutation and chromosome 22 abnormalities, we assessed NF2 gene deletion by FISH and merlin protein levels by IHC in the same human mesothelioma tumor samples. Results: Among a panel of mesothelioma cell lines in 3D culture, MPM lines lacking expression of merlin protein were found to be especially sensitive to the selective FAK inhibitor VS-6063. In contrast, MPM cell lines with wildtype merlin were less sensitive with EC50 values greater than 1 μM. Accordingly, oral dosing with a FAK inhibitor induced significant tumor growth inhibition in a merlin-negative mesothelioma model in mice. To enable the planned stratification of MPM patients by merlin status, an immunohistochemistry (IHC) assay has been optimized to quantify merlin protein levels. A merlin IHC H-score below the defined cutoff was associated with loss of at least one copy of chromosome 22, indicating that chromosomal deletion is an important mechanism of merlin loss in mesothelioma patients. Conclusions: These data support the clinical development of VS-6063 for treatment of malignant pleural mesothelioma patients stratified by merlin/NF2 status. A potentially pivotal mesothelioma trial is set to initiate in 2013.

Sorafenib inhibits ERK1/2 and MCL-1L phosphorylation levels resulting in caspase-independent cell death in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive, rapidly progressive malignancy without effective therapy. We evaluate sorafenib efficacy and impact on the cellular pro-survival machinery in vitro, efficacy of Sorafenib as monotherapy and in combination with the naturally occurring death receptor agonist, TRAIL using human MPM cell lines, MSTO-211H, M30, REN, H28, H2052, and H2452. In vitro studies of the six MPM lines demonstrated remarkable single agent sensitivity to the multikinase inhibitor sorafenib and resistance to TRAIL. H28 and H2452 demonstrated augmented apoptosis with the addition of TRAIL to sorafenib in vitro. Treated cell lines demonstrated sorafenib- induced rapid dephosphorylation of AKT followed shortly by near complete dephosphorylation of the constitutively phosphorylated ERK1/2. Sorafenib therapy also decreased phosphorylation of B-raf and mTOR in several cell lines. Within 3 hr of sorafenib treatment, a number of known pro-survival molecules were dephosphorylated and/or downregulated in expression including MCL-1, c-FLIP, survivin, and cIAP1. These changes and eventual cell death did not elicit significant caspase-3 activation or PARP cleavage and pretreatment with the pan-caspase inhibitor, Z-VAD-FMK, did not block sorafenib efficacy but did block the effect of TRAIL monotherapy. Pre-treatment with Z-VAD-FMK did not block the synergistic effect of TRAIL and sorafenib in H28. In summary, single agent treatment with sorafenib results in widespread inhibition of the pro-survival machinery in vitro leading to cell death via a primarily caspase-independent mechanism. Combining sorafenib therapy with TRAIL, may be useful in order to provide a more efficient death signal and this synergistic effect appears to be caspase-independent. Pilot in vivo data demonstrates promising evidence of therapeutic efficacy in human tumor bearing xenograft nu/nu mice. We document single agent activity of sorafenib against MPM, unravel novel effects of sorafenib on anti-apoptotic signaling mediators, and suggest the combination of sorafenib plus TRAIL as possible therapy for clinical testing in MPM.

Antisense oligonucleotides directed at the bcl-xl gene product augment chemotherapy response in mesothelioma

Abstract Objective: Malignant pleural mesothelioma (MPM) is resistant to both conventional chemotherapy and apoptosis. The bcl-2 family proteins are major determinants of apoptotic homeostasis. MPM lines and tumors routinely overexpress the anti-apoptotic protein BCL-XL. We have previously shown that antisense inhibition of BCL-XL in MPM cells leads to apoptosis. We sought to determine whether antisense oligonucleotides directed at the bcl-xl gene product would augment response to a conventional chemotherapeutic agent in human mesothelioma cell lines. Methods: The human MPM cell lines REN and I-45 were exposed to two bcl-xl antisense oligonucleotides (15999, 16009) and one sense oligonucleotide (113529) construct at varying doses, followed by IC50 cisplatin. Cellular viability was assessed by a calorimetric assay, and apoptosis was evaluated by Hoechst staining, Annexin V staining, and sub-G1 fluorescence-activated cell sorter analysis. Western blot analysis of BCL-2 family proteins was performed following single agent and combined treatment. Isobologram mathematical analysis was used to determine whether or not combination therapies were additive or synergistic. Results: Cell viability was most affected with the 15999 antisense oligonucleotides plus IC50 cisplatin combination (70% of I-45 and 90% of REN cells killed), and apoptosis was markedly increased with this combination by all measures. Western blot demonstrated 15999 antisense oligonucleotides construct down-regulation of BCL-XL, but no further effect on expression of BCL-2 proteins with cisplatin. Isobologram analysis demonstrated 15999 + cisplatin synergistic effect. Conclusions: Exposure of human MPM cells to bcl-xl antisense oligonucleotides sensitizes human mesothelioma cells to the conventional chemotherapeutic agent cisplatin. Similar approaches using a combination of molecular and conventional treatment may have clinical utility for this tumor.

Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product.

Malignant pleural mesothelioma is resistant to conventional therapies and to apoptosis. The bcl-2 family genes are major determinants of apoptotic homeostasis. Malignant pleural mesothelioma lines and tumors rarely express the antiapoptotic Bcl-2 protein but routinely express the antiapoptotic protein Bcl-xl and the proapoptotic proteins Bax and Bak. We have previously shown pharmacologic inhibition of bcl-xl expression in malignant pleural mesothelioma can lead to apoptosis, so we sought to determine whether antisense oligonucleotides directed at bcl-xl messenger RNA would engender apoptosis, possibly through a "forced imbalance" of bcl-2 family proteins. Malignant pleural mesothelioma lines REN (epithelial) and I-45 (sarcomatous) were exposed to modified bcl-xl antissense oligonecleotides directed near the messenger RNA initiation sequence with and without a liposomal delivery system. Untreated cells and bcl-xl sense oligonucleotides were controls. Cell viability was measured by colorimetric assay, and apoptosis was evaluated with Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis. Bcl-xl protein expression after antisense oligonucleotides was downwardly regulated in both cell lines relative to sense oligonucleotides (>65%). Significant cellular killing in both the I-45 and REN cell lines was achieved with antisense oligonucleotides (compared with sense oligonucleotides) without (P =.003 and.006, respectively) and with (P =.006 and.0005, respectively) liposomal delivery. Hoechst staining and sub-G(1) fluorescence-activated cell sorter analysis demonstrated apoptosis to be the mechanism of cellular death. Use of a liposomal delivery system increased therapeutic effect and allowed lower doses of antisense oligonucleotides. Antisense oligonucleotides directed at the bcl-xl gene product engender apoptosis in mesothelioma cell lines. The therapeutic potential of inhibiting expression of this protein in mesothelioma should be evaluated.