Pemetrexed in the treatment of advanced non-small-cell lung cancer: a review of the clinical data

Pemetrexed is a novel multitargeting antimetabolite that has first-line and second-line activity against non-small cell lung cancer (NSCLC). Phase II studies have shown significant efficacy and a favorable toxicity profile of the combination of pemetrexed plus platinum as first-line therapy for NSCLC. Second-line activity against NSCLC was demonstrated in a phase III trial comparing single-agent pemetrexed with docetaxel; in that trial, survival was comparable between these agents but side effects were significantly less for patients who received pemetrexed. Pemetrexed is also an active agent against mesothelioma. A phase III trial comparing pemetrexed plus cisplatin with cisplatin alone showed for the first time a regimen that improves survival in this disease and led to FDA approval of pemetrexed in combination with cisplatin for mesothelioma. As a radiosensitizer, pemetrexed has been well-tolerated when given concurrent with chest radiation, and a phase I study is under way assessing its tolerability in combination with carboplatin in this setting. Pemetrexed is clearly a useful agent in the treatment of thoracic malignancies, and is worthy of further study in combination with other drugs having novel mechanisms of action.     

Schedule-dependent interactions between pemetrexed and cisplatin in human carcinoma cell lines in vitro

The combination of pemetrexed and cisplatin shows good clinical activity against mesothelioma and lung cancer. In order to study the potential cellular basis for this, and provide leads as to how to optimize the combination, we studied the schedule-dependent cytotoxic effects of pemetrexed and cisplatin against four human cancer cell lines in vitro. Tumor cells were incubated with pemetrexed and cisplatin for 24 h at various schedules. The combination effects after 5 days were analyzed by the isobologram method. Both simultaneous exposure to pemetrexed and cisplatin for 24 h and sequential exposure to cisplatin for 24 h followed by pemetrexed for 24 h produced antagonistic effects in human lung cancer A549, breast cancer MCF7, and ovarian cancer PA1 cells and additive effects in colon cancer WiDr cells. Pemetrexed for 24 h followed by cisplatin for 24 h produced synergistic effects in MCF7 cells, additive/synergistic effects in A549 and PA1 cells, and additive effects in WiDr cells. Cell cycle analysis of MCF7 and PA1 cells supported these findings. Our results suggest that the simultaneous clinical administration of pemetrexed and cisplatin may be suboptimal. The optimal schedule of pemetrexed in combination with cisplatin at the cellular level is the sequential administration of pemetrexed followed by cisplatin and this schedule is worthy of clinical investigations.     

Interaction and cross-resistance of cisplatin and pemetrexed in malignant pleural mesothelioma cell lines

Although cisplatin and pemetrexed are key drugs in the treatment of malignant pleural mesothelioma, their drug-drug interactions, cross-resistance and resistance mechanisms in malignant pleural mesothelioma are not well understood. In the present study, the interaction of these 2 agents was determined by clonogenic assays followed by isobologram analysis of 4 human malignant pleural mesothelioma cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase mRNA expression was evaluated in the drug-resistant sublines. As a consequence, cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar pemetrexed sensitivity in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High thymidylate synthase expression did not correlate with natural pemetrexed resistance. Elevated thymidylate synthase expression correlated with acquired pemetrexed resistance in 2 sublines. In conclusion, cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 malignant pleural mesothelioma cell lines, suggesting the clinical relevance of their combination in chemotherapy. Thymidylate synthase expression did not necessarily correlate with pemetrexed resistance. The information together with the experimental model presented here would be useful for further investigating therapeutic targets of malignant mesothelioma.     

Upregulated p53 expression activates apoptotic pathways in wild-type p53-bearing mesothelioma and enhances cytotoxicity of cisplatin and pemetrexed

The majority of malignant mesothelioma possesses the wild-type p53 gene with a homologous deletion of the INK4A/ARF locus containing the p14(ARF) and the p16(INK4A) genes. We examined whether forced expression of p53 inhibited growth of mesothelioma cells and produced anti-tumor effects by a combination of cisplatin (CDDP) or pemetrexed (PEM), the first-line drugs for mesothelioma treatments. Transduction of mesothelioma cells with adenoviruses bearing the p53 gene (Ad-p53) induced phosphorylation of p53, upregulated Mdm2 and p21 expression levels and decreased phosphorylation of pRb. The transduction generated cleavage of caspase-8 and -3, but not caspase-9. Cell cycle analysis showed increased G0/G1- or G2/M-phase populations and subsequently sub-G1 fractions, depending on cell types and Ad-p53 doses. Transduction with Ad-p53 suppressed viability of mesothelioma cells and augmented the growth inhibition by CDDP or PEM mostly in a synergistic manner. Intrapleural injection of Ad-p53 and systemic administration of CDDP produced anti-tumor effects in an orthotopic animal model. These data collectively suggest that Ad-p53 is a possible agent for mesothelioma in combination with the first-line chemotherapeutics.     

SOCS‐1 gene delivery cooperates with cisplatin plus pemetrexed to exhibit preclinical antitumor activity against malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS‐1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of MPM. We infected MPM cells (MESO‐4, H28 and H226) with adenovirus‐expressing SOCS‐1 vector to examine the effect of SOCS‐1 overexpression on MPM cells. We evaluated the antitumor effect of SOCS‐1 gene delivery combined with cisplatin plus pemetrexed by cell proliferation, apoptosis and invasion assay. We also investigated the regulation of NF‐κB and STAT3 signaling related to apoptotic pathways. Furthermore, we evaluated the inhibition of tumor growth by SOCS‐1 gene delivery combined with cisplatin plus pemetrexed in vivo. SOCS‐1 gene delivery cooperated with cisplatin plus pemetrexed to inhibit cell proliferation, invasiveness and induction of apoptosis in MPM cells. SOCS‐1 regulated NF‐κB and STAT3 signaling to induce apoptosis in MESO‐4 and H226 cells. Furthermore, SOCS‐1 gene delivery cooperated with cisplatin plus pemetrexed to regulate NF‐κB signaling and significantly inhibit tumor growth of MPM in vivo. These results suggest that SOCS‐1 gene delivery has a potent antitumor effect against MPM and a potential for clinical use in combination with cisplatin plus pemetrexed.     

Cytotoxicity of cisplatin and cisdiammine-1,1-cyclobutane dicarboxylate in MGH-U1 cells grown as monolayers, spheroids, and xenografts

The cytotoxicity of cisplatin and cisdiammine-1,1-cyclobutane dicarboxylate (CBDCA) was examined using the MGH-U1 human bladder carcinoma cell line, grown as monolayer cultures, multicellular tumor spheroid(s) (MTS), and xenografts in immune-deprived CBA/CaJ mice. The cell survival of exponentially growing monolayers and MTS treated with cisplatin declined in a monoexponential fashion with a concentration of drug resulting in 10% colony survival (D10) of 7.75 micrograms/ml and 9.5 micrograms/ml, respectively. MTS growth delay determination demonstrated a drug concentration-dependent increase in growth delay and a correlation between decreasing surviving fraction and increasing growth delay. In vivo treatment of MGH-U1 xenografts with cisplatin caused a modest decrease in surviving fraction although the xenografted cells treated in vitro demonstrated the same sensitivity to cisplatin as those cells maintained continuously in vitro. The D10 for CBDCA treatment was 246 micrograms/ml for exponentially growing monolayer cells and 196 micrograms/ml for MTS. Growth delay studies with CBDCA showed a concentration-dependent increase in spheroid growth delay and a correlation between decreasing surviving fraction and growth delay similar to cisplatin. The conclusions were that: 1) cisplatin and CBDCA did not have any difficulty penetrating into spheroids, 2) both agents appeared to be active against the noncycling poorly nourished cells found near the necrotic center of spheroids, 3) both cisplatin and CBDCA were cytotoxic toward MGH-U1 cells but cisplatin was 20-30 times more effective, and 4) the limited cytotoxic effect of cisplatin in vivo may be due to the low area under the concentration times the time curve achieved in vivo and not due to intrinsic cell resistance.     

Evaluation of drug efficacy in vitro using human small cell carcinoma of the lung spheroids

Five human small cell carcinoma of the lung (SCCL) cell lines selected from 25 established cultures were grown as three-dimensional spheroid tumor models in either spinner culture or in static, agar-coated multiwells. Volume doubling times for the cell lines were approximately 4.5 days. Decreases in spheroid volumes after exposure to a variety of chemotherapeutic agents were used as indicators of drug activity. To further quantify cell killing in SCCL spheroids by chemotherapeutic agents 24 hours after exposure to drugs, a technique was employed that measured maximum levels of incorporation of 125IUdR after continuous labeling for 48 hours. The results of the use of this assay report for SCCL spheroid responses to various concentrations of doxorubicin hydrochloride, cytosine arabinoside, mechlorethamine hydrochloride, cisplatin, or etoposide. Some evidence for an intertumor heterogeneous response to chemotherapy is presented for some of the drugs tested. This assay was also used to characterize a potentiated cell kill when etoposide is combined with cisplatin and to identify activity by a new compound, diazoacetylcholine iodide (DACI), which was synthesized as an agent targeted for SCCL cells.     

Radiosensitivity of different human tumor cells lines grown as multicellular spheroids determined from growth curves and survival data

Five human tumor cell lines were grown as multicellular tumor spheroids (MTS) to determine whether multicellular tumor spheroids derived from different types of tumors would show tumor-type dependent differences in response to single-dose irradiation, and whether these differences paralleled clinical behavior. Multicellular tumor spheroids of two neuroblastoma, one lung adenocarcinoma, one melanoma, and a squamous cell carcinoma of the oral tongue, were studied in terms of growth delay, calculated cell survival, and spheroid control dose50 (SCD50). Growth delay and cell survival analysis for the tumor cell lines showed sensitivities that correlated well with clinical behavior of the tumor types of origin. Similar to other studies on melanoma multicellular tumor spheroids our spheroid control dose50 results for the melanoma cell line deviated from the general pattern of sensitivity. This might be due to the location of surviving cells, which prohibits proliferation of surviving cells and hence growth of melanoma multicellular tumor spheroids. This study demonstrates that radiosensitivity of human tumor cell lines can be evaluated in terms of growth delay, calculated cell survival, and spheroid control dose50 when grown as multicellular tumor spheroids. The sensitivity established from these evaluations parallels clinical behavior, thus offering a unique tool for the in vitro analysis of human tumor radiosensitivity.     

Proliferation,migration and invasion of human glioma cells exposed to antifolate drugs

The present study describes the effects of 2 folate antagonists, methotrexate (MTX) and the lipophilic antifolate trimetrexate (TMX) on 2 permanent human glioma cell lines (GaMg and D-54Mg) grown as monolayers and as multicellular tumor spheroids. In addition, the effects of drug exposure on tumor cell invasion was studied using a three-dimensional organ co-culture system. In monolayer cultures, TMX was a more potent inhibitor of cell growth than MTX, especially towards the GaMg cell line. The 2 drugs, however, showed similar cytotoxicity as assessed by the plating efficiency assay. Reduced ability of directional migration of cells on a plastic surface was seen by either antifolate usually at concentrations to 10-fold higher than those exerting a cytotoxic effect in the plating efficiency assay. TMX was somewhat more potent than MTX as an inhibitor of spheroid growth. When tumor spheroids were exposed to MTX or TMX at concentrations that caused 65 to 70% inhibition of cell migration, there was a latent period of 4 to 5 days before inhibition of spheroid growth ensued. Invasion was investigated in a co-culture system, where tumor spheroids were confronted with fetal rat brain cell aggregates. Neither drug reduced tumor cell invasion, although histological examination revealed toxic effects both in GaMg and in D-54Mg spheroids. We conclude that spheroids from human glioma cells were less sensitive to the antifolates than monolayers. For both drugs a latency period was observed before inhibition of spheroid growth. The spheroids retained their ability to invade normal brain tissue when exposed to levels of folate antagonists inhibiting spheroid growth.     

Imatinib mesylate enhances therapeutic effects of gemcitabine in human malignant mesothelioma xenografts.

PURPOSE: Platelet-derived growth factor receptor beta (PDGFRbeta), frequently activated in malignant mesothelioma, is a promising cancer therapeutic target. Imatinib mesylate (STI571; Glivec) is a selective inhibitor of tyrosine kinases as bcr-abl, c-kit, c-fms, and PDGFRbeta and enhances tumor drug uptake by reducing the interstitial fluid pressure. We previously showed that imatinib mesylate synergizes with gemcitabine and pemetrexed in PDGFRbeta-positive mesothelioma cells. Here, we aimed at investigating these combined treatments in a novel mesothelioma model. EXPERIMENTAL DESIGN: REN mesothelioma cells, infected with a lentiviral vector carrying the luciferase gene, were injected in the peritoneum of severe combined immunodeficient mice. This model allowed imaging of live animals treated with pemetrexed or gemcitabine chemotherapeutics, or with imatinib mesylate alone, as well as with a combination of gemcitabine and imatinib mesylate. RESULTS: We show here that, consistent with our previous in vitro studies, gemcitabine inhibited tumor growth, whereas pemetrexed was ineffective, even at the highest dosage tested. Compared with monotreatment, the combination of gemcitabine with imatinib mesylate led to a further tumor growth inhibition and improved mice survival, by a decrease rate of tumor cell proliferation and an increase in number of apoptotic tumor cells. CONCLUSIONS: Imatinib mesylate enhances the therapeutic response to gemcitabine, in accordance with our previous in vitro data. These in vivo results validate imatinib mesylate and gemcitabine as a combination treatment of malignant mesothelioma, also in view of its known positive effects on tumor drug uptake. These evidences provide the rationale for the currently ongoing clinical trials.     

Recombinant erythropoietin differently affects proliferation of mesothelioma cells but not sensitivity to cisplatin and pemetrexed

The combination of cisplatin and pemetrexed represents the newly established standard of care for patients with unresectable malignant mesothelioma (MM). However, this chemotherapy regimen appears to be associated with an increased prevalence of higher grade anemia as compared to treatment with cisplatin alone. Human recombinant erythropoietin (rHuEpo) is currently used for the treatment of anemia in cancer patients. Still, following the finding that the erythropoietin receptor (EpoR) is expressed by several tumor cells types and after the trials reporting that the recombinant cytokine can adversely affect tumor progression and patient survival, the clinical safety of rHuEpo administration to neoplastic patients has recently been questioned. The observation that the expression of EpoR, variably associated with the expression of the cognate ligand, is a common feature of MM cells prompted us to investigate whether treatment with rHuEpo could elicit proliferative and cytoprotective signals in EpoR-positive MM cell lines. Biochemical responsiveness of MM cells to rHuEpo was demonstrated by the time-course activation of both ERK1/2 and AKT following treatment with the recombinant cytokine. A moderately increased mitogenic activity was observed in two out of five MM cell lines treated with pharmacologically relevant concentrations of rHuEpo. On the other hand, the recombinant cytokine, administered either before or after cisplatin and pemetrexed, failed to interfere with the cytotoxic effects exerted by the chemotherapeutic drugs on the five MM cell lines. According to the presented findings, rHuEpo appears to have an overall limited impact on cell growth and no effect on MM sensitivity to chemotherapy.     

Current data with pemetrexed (Alimta) in non-small-cell lung cancer

Pemetrexed (Alimta ) is a novel multitargeted antifolate that inhibits 3 enzymes involved in folate metabolism and purine and pyrimidine synthesis. These enzymes are thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. This agent has broad antitumor activity in phase II trials in a wide variety of solid tumors. In non-small-cell lung cancer (NSCLC), single-agent activity has been documented in the first- and second-line settings. Promising activity has also been demonstrated when pemetrexed is combined with cisplatin and gemcitabine. A pivotal phase III study in mesothelioma has been presented, indicating the superiority of pemetrexed in combination with cisplatin versus cisplatin alone in this disease. Approval for pemetrexed in combination with cisplatin in advanced mesothelioma is expected within the next 12 months. This review discusses the activity of pemetrexed in NSCLC.     

Evaluation of adenoviral oncolytic effect on glioma spheroids by 18F-DG positron-emission tomography

Multicellular tumor spheroids are used as a model to assess the efficacy of replicating oncolytic adenoviruses. As most assays used to assess cellular viability are unsuitable for oncolytic viruses because of ongoing viral replication, we have used positron emission tomography (PET) to sequentially determine the incorporation of 18F-labeled deoxyglucose (18F-DG) as a measure of viability and compared the results to more commonly used assays for measuring the effect of oncolytic therapy. Glioma monolayer cultures and spheroids were infected with wild-type replicating adenovirus and viability was measured by 18F-DG incorporation, WST-1 assay, crystal violet assay, and spheroid volume 2 to 10 days following infection. Results show that volume measurements in adenovirus-infected spheroids are confounded by the cytopathic effect occurring in infected cells. 18F-DG PET provides a useful method to assess small differences in cell number and viability following oncolytic viral therapy in glioma monolayer cultures and spheroids without the need for disintegration of these cultures. Moreover, using 18F-DG PET, repeated sequential measurements of spheroid viability can be made, decreasing the required number of spheroids per experiment. This is a valuable feature when using spheroids derived from limited amounts of patient material.     

Multi-targeted-Enzyminhibition in der Tumortherapie

Pemetrexed is a newly developed therapeutic agent which inhibits several key enzymes in the folate metabolic pathway. In phase I trials, this novel multitargeted antifolate showed a broad antitumor activity as a single agent and in combination chemotherapy. Based on these findings, phase III studies have been conducted including patients with malignant pleural mesothelioma (MPM), non-small-cell lung cancer (NSCLC), and colorectal cancer. In a recent phase III trial in MPM, the combination of pemetrexed and cisplatin was significantly more efficacious than cisplatin alone. In addition, vitamin supplementation reduced treatment-associated toxicities with no apparent affect on activity. In patients with NSCLC, a phase III trial showed clinically equivalent efficacy of pemetrexed and docetaxel, but pemetrexed was associated with significantly fewer toxicities in second-line therapy. This review summarizes preclinical and clinical data to define the future role of pemetrexed in the treatment of tumor patients.     

Therapy of advanced non-small-cell lung cancer with irinotecan and gemcitabine in combination

Malignant mesothelioma is a refractory malignancy. Treatment for unresectable disease may provide a palliative benefit, but survival duration is impacted only minimally, if at all. Several newer agents, including difluorodeoxycytidine (gemcitabine) and pemetrexed disodium (LY231514, Alimta) appear to have activity against this neoplasm. Phase II data for combination regimens of gemcitabine and a platinum in mesothelioma patients have been encouraging. However, no phase III data are available to place these phase II results in true perspective. In phase I studies of a cisplatin/pemetrexed combination, objective responses occurred in several mesothelioma patients. This led to a phase II trial of pemetrexed alone in untreated mesothelioma patients and a randomized phase III trial of cisplatin alone versus pemetrexed/cisplatin. Phase II activity (15% partial response) was seen with single-agent pemetrexed. The phase III trial accrued over 450 patients. Primary analysis of the phase III data set has been completed and the results will be presented at the American Society of Clinical Oncology Meeting in May 2002.     

Potential of a Cetuximab‐based radioimmunotherapy combined with external irradiation manifests in a 3‐D cell assay

Targeting epidermal growth factor receptor (EGFR)‐overexpressing tumors with radiolabeled anti‐EGFR antibodies is a promising strategy for combination with external radiotherapy. In this study, we evaluated the potential of external plus internal irradiation by [⁹⁰Y]Y‐CHX‐A″‐DTPA‐C225 (Y‐90‐C225) in a 3‐D environment using FaDu and SAS head and neck squamous cell carcinoma (HNSCC) spheroid models and clinically relevant endpoints such as spheroid control probability (SCP) and spheroid control dose 50% (SCD₅₀, external irradiation dose inducing 50% loss of spheroid regrowth). Spheroids were cultured using a standardized platform. Therapy response after treatment with C225, CHX‐A″‐DTPA‐C225 (DTPA‐C225), [⁹⁰Y]Y‐CHX‐A″‐DTPA (Y‐90‐DTPA) and Y‐90‐C225 alone or in combination with X‐ray was evaluated by long‐term monitoring (60 days) of spheroid integrity and volume growth. Penetration kinetics into spheroids and EGFR binding capacities on spheroid cells were identical for unconjugated C225 and Y‐90‐C225. Spheroid‐associated radioactivity upon exposure to the antibody‐free control conjugate Y‐90‐DTPA was negligible. Determination of the SCD₅₀ demonstrated higher intrinsic radiosensitivity of FaDu as compared with SAS spheroids. Treatment with unconjugated C225 alone did not affect spheroid growth and cell viability. Also, C225 treatment after external irradiation showed no additive effect. However, the combination of external irradiation with Y‐90‐C225 (1 µg/ml, 24 hr) resulted in a considerable benefit as reflected by a pronounced reduction of the SCD₅₀ from 16 Gy to 9 Gy for SAS spheroids and a complete loss of regrowth for FaDu spheroids due to the pronounced accumulation of internal dose caused by the continuous exposure to cell‐bound radionuclide upon Y‐90‐C225‐EGFR interaction.     

Alteration of pemetrexed excretion in the presence of acute renal failure and effusions: presentation of a case and review of the literature

Pemetrexed is a multitargeted antifolate approved by the Food and Drug Administration for patients with mesothelioma and non small cell lung cancer. As this agent is almost exclusively cleared by renal excretion, trials leading to its approval were conducted in patients with normal renal function. However, this cycle active agent often is administered with cisplatin to patients who may have pleural effusions and ascites. As a result, the potential for drug accumulation in effusions and for accompanying renal insufficiency is real. Recommendations for the management of pemetrexed toxicity in the presence of renal failure have not been established. A case of treatment-related acute renal failure following chemotherapy with cisplatin and pemetrexed in a patient with advanced mesothelioma and ascites is presented. Pharmacologic studies in this patient revealed persistent pemetrexed levels in ascites and plasma. This is the first time that significant accumulation of pemetrexed in ascetic fluid has been reported in the literature. Treatment with leucovorin, folate, and continuous veno-venous hemodialysis was initiated. Thymidine and carboxypeptidase were not available. Dialysis was unsuccessful in removing pemetrexed. Theoretical and practical approaches to management of similar cases are presented.     

Modeling Selective Elimination of Quiescent Cancer Cells from Bone Marrow

Patients with many types of malignancy commonly harbor quiescent disseminated tumor cells in bone marrow. These cells frequently resist chemotherapy and may persist for years before proliferating as recurrent metastases. To test for compounds that eliminate quiescent cancer cells, we established a new 384-well 3D spheroid model in which small numbers of cancer cells reversibly arrest in G1/G0 phase of the cell cycle when cultured with bone marrow stromal cells. Using dual-color bioluminescence imaging to selectively quantify viability of cancer and stromal cells in the same spheroid, we identified single compounds and combination treatments that preferentially eliminated quiescent breast cancer cells but not stromal cells. A treatment combination effective against malignant cells in spheroids also eliminated breast cancer cells from bone marrow in a mouse xenograft model. This research establishes a novel screening platform for therapies that selectively target quiescent tumor cells, facilitating identification of new drugs to prevent recurrent cancer.     

Preclinical studies of the proteasome inhibitor bortezomib in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm that is resistant to chemotherapy. Bortezomib is an FDA-approved proteasome inhibitor that is currently under clinical investigation in multiple neoplasms but has not been studied extensively in MPM. In this report, we determine the biological and molecular response of cultured MPM cells to bortezomib alone and in combination with cisplatin or pemetrexed. We used four MPM cell lines (MS589, H28, H2052, JMN), a normal mesothelial cell line (HM3), and a lung cancer cell line (H23) in survival studies utilizing bortezomib, cisplatin, and pemetrexed alone and in combination by administering concurrently or by varying the order of administration. We determined the effect of bortezomib on the cell cycle, apoptosis, and on the expression of cell cycle proteins p21/WAF1 and p27/KIP1 and on apoptosis-related proteins IAP-1, IAP-2, survivin, and XIAP. Bortezomib was highly cytotoxic to MPM cells and induced both G₂/M and G₁/S cell cycle arrest. Apoptosis increased in a concentration- and time-dependent manner in 3 of 4 MPM cell lines. Bortezomib stabilized or increased protein levels of p21/WAF1 and IAP-1 and to a lesser degree p27/KIP1, IAP-2, XIAP, and survivin. In combination studies with cisplatin, bortezomib was generally synergistic at high concentrations and antagonistic at low concentrations. Bortezomib increased the cytotoxicity of cisplatin and pemetrexed in a concentration-dependent manner when administered prior to either. Bortezomib may improve outcome in MPM patients alone or in combination with standard chemotherapy but the order of administration is likely to be important. This study justifies further evaluation of bortezomib in MPM.