硼替佐米体外抑制耐全反式维甲酸白血病细胞株NB_4-R2的增殖

  目的　探讨硼替佐米（Bortezomib）在体外对耐全反式维甲酸（ATRA）的急性早幼粒细胞白血病（APL）细胞株NB4-R2增殖、凋亡的影响,为用Bortezomib治疗耐ATRA的APL提供新的理论依据。方法　体外用不同浓度的Bortezomib处理NB4-R2细胞株48 h后,用四甲基偶氮唑蓝染色法（MTT）观察细胞增殖活力、Hoechst33342染色法观察细胞凋亡形态、流式细胞术检测细胞凋亡百分率以及Western blotting蛋白印迹检测剪切多聚ADP-核糖聚合酶（cleaved PARP）和Caspase-3凋亡蛋白的表达水平。结果　体外Bortezomib能明显抑制NB4-R2细胞增殖,在质量浓度1～5 μg／L的范围内,细胞的抑制作用和Bortezomib浓度呈剂量依赖关系,当质量浓度为5 μg／L时,细胞增殖抑制率可达74.9 %;在质量浓度1～5 μg／L的Bortezomib范围内,NB4-R2细胞增殖受抑主要表现为细胞凋亡增加,凋亡百分率可达78.7 %,且以晚期凋亡为主;Western blotting证实Bortezomib处理后的NB4-R2细胞中,cleaved PARP和Caspase-3蛋白表达明显增强。结论　体外Bortezomib可通过诱导细胞凋亡来抑制NB4-R2细胞的增殖。     

Inhibition of autophagy enhances the anticancer activity of bortezomib in B-cell acute lymphoblastic leukemia cells

B-cell acute lymphoblastic leukemia (B-ALL) remains a challenging disease to treat in adults because of the high rates of relapse and refractory. Bortezomib, as a proteasome inhibitor, exerts obvious cytotoxicity against ALL cells and increases the sensitivity of ALL cells to conventional chemotherapeutic agents. We observed that bortezomib inhibited proliferation, induced apoptosis, arrested the cell cycle and induced autophagy in the Nalm-6 cell line and CD34⁺ primary cells. Additionally, we demonstrated that bortezomib promoted the disruption of the Bcl-2/Beclin-1 complex and increased the formation of the Beclin-1/PI3KC3 complex, leading to the initiation of autophagy. Autophagy inhibitors were employed in this study, and we found that autophagy inhibitors enhanced the anti-ALL activity of bortezomib. Taken together, these results revealed that autophagy protected B-ALL cells against the cytotoxicity of bortezomib and, in combination with autophagy inhibitors, can enhance the anticancer effects of bortezomib.     

Effects of the proteasome inhibitor bortezomib alone and in combination with chemotherapeutic agents in gastric cancer cell lines

The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of tumor cells. Bortezomib is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome and has prominent effects in vitro and in vivo against several solid tumors. We examined the anti-proliferative and apoptotic effects of bortezomib in three gastric cancer cell lines (SNU638, MUGC-3 and MKN-28), along with its antitumor combination effects with other chemotherapeutic agents. Tumor cell growth inhibition and apoptosis was measured by MTT assay and FACS analysis, respectively. Apoptosis- and cell cycle-associated protein expression levels were measured by Western blot assay. Bortezomib induced the suppression of tumor cell growth and apoptosis in a dose-dependent manner with an inhibitory dose (ID)50 of approximately 0.5 microg/ml in all gastric cancer cell lines tested. Further combination treatment with cisplatin and docetaxel, in particular with docetaxel displayed dramatically increased tumor cell growth suppression in all three gastric cancer cell lines, as compared to single drug treatment alone. This was concomitant with the induction patterns of apoptotic cells. Bortezomib treatment increased the Bax protein expression. Moreover, combination treatment of bortezomib plus docetaxel resulted in a dramatic increase in the Bax expression. In contrast, Bcl-2 expression was decreased by combination treatment with bortezomib plus docetaxel in SNU638 cells. Finally, bortezomib, docetaxel and to a greater degree bortezomib plus docetaxel increased the expression levels of p27 proteins even without influencing p53 expression levels. Bortezomib has profound effects on tumor cell growth inhibition and induction of apoptosis in human gastric cancer cells, suggesting that bortezomib may be an effective therapeutic drug for patients with gastric cancer. Further combination studies with other chemotherapeutic drugs, in particular docetaxel showing more tumor cell growth inhibition and apoptosis suggest that combining bortezomib with docetaxel might be more effective for displaying tumor cell growth inhibitory effects in gastric cancer cells through regulation of Bcl-2, Bax and p27 proteins in vitro.     

1，6二磷酸果糖增强蛋白酶体抑制剂对人乳腺癌细胞MCF-7的抑瘤效应

目的探讨抑制蛋白酶体活性是否可以诱导人乳腺癌细胞MCF-7的自我吞噬,1,6二磷酸果糖对自我吞噬的影响及自我吞噬对细胞增殖的作用。方法采用MTT法检测细胞增殖,蛋白质印迹检测自我吞噬相关蛋白LC3的表达。结果蛋白酶体抑制剂硼替佐米以剂量依赖方式抑制MCF-7细胞的增殖并诱导细胞的自我吞噬,但是,当1,6二磷酸果糖与硼替佐米联合应用后,可逆转硼替佐米诱导的自我吞噬并增强硼替佐米对MCF-7细胞的增殖抑制。结论蛋白酶体活性的抑制可诱导人乳腺癌细胞MCF-7的自我吞噬代偿性激活,1,6二磷酸果糖可抑制激活的自我吞噬,其与硼替佐米的联合应用可增强其抑瘤效应。     

WEB-2086 and WEB-2170 trigger apoptosis in both ATRA-sensitive and -resistant promyelocytic leukemia cells and greatly enhance ATRA differentiation potential.

PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation in murine and human leukemia cells. The present study describes the apoptotic-differentiative effect of WEBs in all-trans-retinoic acid (ATRA)-sensitive (NB4) and -resistant (NB4-007-6 and NB4-MR4) acute promyelocytic leukemia (APL) cell lines as well as blasts from patients with t(15;17) APL. NB4 cells exposed to 0.5-1 mM WEBs underwent striking growth arrest and massive apoptosis without appreciable differentiation; IC50 values after 3-day treatment of NB4 were 0.4 and 0.25 mM for WEB-2086 and WEB-2170, respectively. WEBs induced apoptosis also in the two ATRA-resistant NB4-007-6 and NB4-MR4 cell lines and in blasts from patients with t(15;17) APL. Moreover, subapoptotic WEBs acted synergistically with low-dose (0.025-0.05 microM) ATRA; this allowed to increase ATRA differentiation potential up to 40-fold and to improve both number and intensity of NBT-positive NB4 cells at definitely higher levels than with 1 muM ATRA alone. The powerful antiproliferative-apoptotic activities of WEBs in vitro on ATRA-sensitive, ATRA-resistant APL cells and blasts from patients with APL as well as drug capabilities to enhance ATRA differentiation potential suggested that these agents also due to their recognized tolerability in vivo might improve, alone or in combination, clinical treatment of APL.     

Bortezomib induces AMPK-dependent autophagosome formation uncoupled from apoptosis in drug resistant cells

The proteasome inhibitor bortezomib is an effective anti-cancer agent for the plasma cell malignancy multiple myeloma but clinical response is hindered by the emergence of drug resistance through unknown mechanisms. Drug sensitive myeloma cells were exposed to bortezomib to generate drug resistant cells that displayed a significant increase in subunits of the energy sensor AMP-activated protein kinase (AMPK). AMPK activity in resistant cells was increased and bortezomib resistant cells contained a ~4-fold greater level of autophagosomes than drug sensitive cells. Real-time measurements indicated that bortezomib reduced the oxygen consumption rate in drug sensitive cells more readily than in resistant cells. Genetic ablation of AMPK activity reduced the bortezomib effect on autophagy. The autophagy-related gene (Atg)5 is required for autophagosome formation and enhances cellular susceptibility to apoptotic stimuli. Atg5 knockout eliminated bortezomib-induced autophagosome formation and reduced susceptibility to bortezomib. Bortezomib treatment of myeloma cells lead to ATG5 cleavage through a calpain-dependent manner while calpain inhibition or a calpain-insensitive Atg5 mutant promoted bortezomib-resistance. In contrast, AICAR, an AMPK activator, enhanced bortezomib-induced cleavage of ATG5 and increased bortezomib-induced killing. Taken together, the results demonstrate that ATG5 cleavage provokes apoptosis and represents a molecular link between autophagy and apoptosis with therapeutic implications.     

mTHPC-based photodynamic therapy induction of autophagy and apoptosis in cultured cells in relation to mitochondria and endoplasmic reticulum stress

Photodynamic therapy (PDT), an approved anticancer treatment, is reported as a potent inducer of programmed cell death (PCD) by both apoptosis and autophagy. The present study investigated the kinetics of both autophagy and caspase activation in MCF-7 cells submitted to mTHPC-PDT upon condition of treatment promoting ER accumulation of mTHPC. Fluence-dependent immediate cytochrome c (cyt C) release followed by caspase-9 and -7 activation at 1 h post-PDT evidenced a mitochondrial oxidative stress triggered by high light doses leading to >90% of cell death. ER oxidative stress was monitored by the induction of the glucose-related protein chaperone GRP78. From 6 h post-PDT, GRP78 induction was accompanied by the conversion of LC3-I into LC3-II, the hallmark of autophagosome formation. The formation of acid vesicles evidenced by fluorescence microscopy was obvious from 22 h post-PDT. Twenty-four hours post-PDT, cyt C release decreased and caspase-9 cleavage disappeared, while the expression of cleaved caspase-7 remained significant. At the same time, the profiles of GRP78, cleaved caspase-7 and LC3-II expression were similar irrespective of light doses. In contrast to an inhibitor of caspase activation Z-VAD-FMK, the use of autophagy inhibitor, Wortmannin, impaired cytotoxicity along with an increase in caspase-7 activation. These results demonstrate a valuable contribution of autophagy to cell death in mTHPC-photosensitized MCF-7 cells.     

Effect of inhibition of the Ubiquitin-Proteasome System and Hsp90 on growth and survival of Rhabdomyosarcoma cells in vitro

ABSTRACT: BACKGROUND: The ubiquitin-proteasome system (UPS) and the heat shock response (HSR) are two critical regulators of cell homeostasis, as their inhibition affects growth and survival of normal cells, as well as stress response and invasiveness of cancer cells. We evaluated the effects of the proteasome inhibitor Bortezomib and of 17-DMAG, a competitive inhibitor of Hsp90, in rhabdomyosarcoma (RMS) cells, and analyzed the efficacy of single-agent exposures with combination treatments. METHODS: To assess cytotoxicity induced by Bortezomib and 17-DMAG in RMS cells, viability was measured by MTT assay after 24, 48 and 72 hours. Western blotting and immunofluorescence analyses were carried out to elucidate the mechanisms of action. Apoptosis was measured by FACS with Annexin-V-FITC and Propidium Iodide. RESULTS: Bortezomib and 17-DMAG, when combined at single low-toxic concentrations, enhanced growth inhibition of RMS cells, with signs of autophagy that included intensive cytoplasmic vacuolization and conversion of cytosolic LC3-I protein to its autophagosome-associated form. Treatment with lysosomal inhibitor chloroquine facilitates apoptosis, whereas stimulation of autophagy by rapamycin prevents LC3-I conversion and cell death, suggesting that autophagy is a resistance mechanism in RMS cells exposed to proteotoxic drugs. However, combination treatment also causes caspase-dependent apoptosis, PARP cleavage and Annexin V staining, as simultaneous inhibition of both UPS and HSR systems limits cytoprotective autophagy, exacerbating stress resulting from accumulation of misfolded proteins. CONCLUSION: The combination of proteasome inhibitor Bortezomib with Hsp90 inhibitor 17-DMAG, appears to have important therapeutic advantages in the treatment of RMS cells compared with single-agent exposure, because compensatory survival mechanisms that occur as side effects of treatment may be prevented.     

Bortezomib induces apoptosis and growth suppression in human medulloblastoma cells, associated with inhibition of AKT and NF-ĸB signaling, and synergizes with an ERK inhibitor

Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.     

A metal chelator,diphenylthiocarbazone, induces apoptosis in acute promyelocytic leukemia (APL) cells mediated by a caspase-dependent pathway without a modulation of retinoic acid signaling pathways

A metal chelator, diphenylthiocarbazone (dithizone), has been reported to induce differentiation and apoptosis of the human myeloid leukemia cell line HL-60, however, very little is known about the mechanism of dithizone-induced apoptosis. Here, we report for the first time that dithizone can induce inhibition of cellular growth of retinoic acid (RA)-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis but not differentiation. Treatment of NB4 cells with dithizone markedly-induced apoptosis, which was associated with the loss of mitochondrial transmembrane potentials (Delta Psi(m)) and activation of caspase-3 and -9. Further investigation of the RA-resistant UF-1 APL cells showed that dithizone-induced apoptosis to a lesser extent. However, neither dithizone alone nor in combination with all-trans RA induced the expression of myeloid differentiation antigen CD11b. Concomitantly, the degradation of PML/RARalpha fusion protein was not observed after treatment with dithizone alone, and the degradation was not enhanced by the combination of dithizone and all-trans RA. We conclude that dithizone, a metal chelator, induced apoptosis without differentiation in APL cells in association with Delta Psi(m) collapse and caspase-3 and -9 activation.     

硼替佐米对白血病细胞株HEL抑制作用的初步探讨

 目的　探讨硼替佐米单独或联合化疗药物对急性白血病细胞株HEL生长的影响及其机制。方法　MTT法检测药物对HEL的生长抑制效应,流式细胞术检测细胞周期变化及凋亡,Western blotting检测凋亡及细胞周期相关蛋白表达。结果　硼替佐米对HEL细胞的生长抑制作用呈浓度依赖关系,其半数抑制浓度（IC50）为7.15 nmol／L;硼替佐米联合化疗药物明显增强抑制HEL细胞生长;流式细胞术检测可见时间依赖性的G2／M周期阻滞;Western blotting检测显示bcl-2表达下降,bax、p27表达增高,联合用药时具有相加效应,但不管是单独或联合用药,p53蛋白的表达均无明显改变。结论　硼替佐米可能是一种有效治疗急性白血病的药物,与柔红霉素联合应用有更强的抑制肿瘤细胞生长和诱导其凋亡的作用,其机制可能与bcl-2,bax及p27蛋白的调节有关,是非p53依赖性的。     

Inhibition of autophagy overcomes glucocorticoid resistance in lymphoid malignant cells

Glucocorticoid (GC) resistance remains a major obstacle to successful treatment of lymphoid malignancies. Till now, the precise mechanism of GC resistance remains unclear. In the present study, dexamethasone (Dex) inhibited cell proliferation, arrested cell cycle in G0/G1-phase, and induced apoptosis in Dex-sensitive acute lymphoblastic leukemia cells. However, Dex failed to cause cell death in Dex-resistant lymphoid malignant cells. Intriguingly, we found that autophagy was induced by Dex in resistant cells, as indicated by autophagosomes formation, LC3-I to LC3-II conversion, p62 degradation, and formation of acidic autophagic vacuoles. Moreover, the results showed that Dex reduced the activity of mTOR pathway, as determined by decreased phosphorylation levels of mTOR, Akt, P70S6K and 4E-BP1 in resistant cells. Inhibition of autophagy by either chloroquine (CQ) or 3-methyladenine (3-MA) overcame Dex-resistance in lymphoid malignant cells by increasing apoptotic cell death in vitro. Consistently, inhibition of autophagy by stably knockdown of Beclin1 sensitized Dex-resistant lymphoid malignant cells to induction of apoptosis in vivo. Thus, inhibition of autophagy has the potential to improve lymphoid malignancy treatment by overcoming GC resistance.     

Bortezomib induces autophagy in head and neck squamous cell carcinoma cells via JNK activation

The molecular mechanism of autophagy induction following proteasome inhibition is not fully understood. We report that the proteasome inhibitor bortezomib potently induces autophagy in head and neck squamous cell carcinoma (HNSCC) cells, as demonstrated by autophagosome formation and induction of complete autophagic flux. Bortezomib treatment led to phosphorylation/activation of jun N-terminal kinase (JNK) enzymes and JNK-dependent phosphorylation of Bcl-2 on serine 70. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited bortezomib induction of autophagy regulatory proteins and autophagosome formation. These results demonstrate a key requirement for JNK signaling in the activation of autophagy by bortezomib.     

The Notch ligand, Delta-1, alters retinoic acid (RA)-induced neutrophilic differentiation into monocytic and reduces RA-induced apoptosis in NB4 cells

Effects of Notch activation on retinoic acid (RA)-induced differentiation and apoptosis were investigated. NB4, an acute promyelocytic leukemia (APL) cell line, undergoes neutrophilic differentiation and apoptosis by RA. Notch activation induced by a recombinant Notch ligand, Delta-1, did not affect the growth by itself. Treatment with RA plus Delta-1 made part of NB4 cells monocyte-like shaped and reduced the apoptosis. Similar phenomenon was also observed in primary APL cells. RA treatment induced cleavage of caspase-8 and PARP in NB4. Delta-1 suppressed the RA-induced cleavage of them, which may be a possible mechanism through which Delta-1 suppressed the RA-induced apoptosis.     

Apoptotic effect of oridonin on NB4 cells and its mechanism

The anti-proliferation effects of oridonin on acute promyelocytic leukemia (APL) cells and its mechanisms were studied in vitro. NB4 cells as well as fresh leukemia cells obtained from APL patients in culture medium were treated with different concentrations of oridonin. Cell growth inhibition, apoptosis and related pathways were assessed by MTT assay as well as flow cytometry (FCM) and western blot analysis. The data revealed that oridonin (over 16 micromol/L) could inhibit the growth of NB4 cells by induction of apoptosis. Marked changes of cell apoptosis were observed very clearly by using electron microscopy and DNA fragmentation analysis after the cells exposed to oridonin for 48 h; Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit as well as a cleaved 89-kDa fragment of 116-kDa PARP when apoptosis occurred. The expression of Bcl-2 was down-regulated remarkably accompanied by the disruption of the mitochondrial membrane potential (delta(psi)m). The anti-proliferative and apoptosis-inducing effects by oridonin in fresh APL cells were also found remarkably using Trypan Blue dye exclusion method and Wright's staining. We concluded that oridoning has significant anti-proliferative and apoptosis-inducing effects on NB4 cells by activation of caspase-3 and cleavage of PARP as well as by down regulation of Bcl-2 and disruption of the delta(psi)m. Furthermore, oridonin demonstrated apparent cell growth inhibition effects on fresh APL cells in vitro. The results indicated that oridonin may serve as a potential anti-leukemia reagent.     

Arsenic enhances the activation of Stat1 by interferon gamma leading to synergistic expression of IRF-1

Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia (APL), including those who have relapsed after treatment with all-trans-retinoic acid (RA). In vitro studies with the APL-derived NB4 cell line showed that As2O3 exerts a dose-dependent dual effect, which induces apoptosis at 1 microM, whereas at a lower concentration of 0.1 microM, a partial differentiation of APL is observed. In non-APL cells, interferon (IFN) alpha and 1 microM As2O3 act synergistically to induce apoptosis. In this report, we show that in NB4 cells and in two RA-resistant NB4-derived cell lines, NB4-R1 and NB4-R2, IFNalpha or IFNgamma combined with 0.1 microM As2O3 lead to an increased maturation effect. Moreover, IFNgamma alone is able to differentiate RA-sensitive and -resistant cells with a higher maturation effect on NB4-R2 cells. In contrast, all these cells underwent apoptosis in the presence of the cytokine and a higher concentration of As2O3. IFNgamma boosted As2O3-induced apoptosis in APL cells as tested by TUNEL, Annexin V staining and activation of caspase 3. As2O3 differently altered IFN-induced gene products; it downregulated PML/RARalpha and PML, did not alter PKR and Stat1, and upregulated interferon regulatory family (IRF)-1. Synergism by IFNgamma and arsenic on IRF-1 expression is mediated by a composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a nonconsensus site for nuclear factor kappa B (NFkappaB). Arsenic has no effect on NFkappaB, whereas it enhances the activation of Stat1 by IFNgamma in NB4 cells leading to an increase in IRF-1 expression.     

Ursolic acid promotes cancer cell death by inducing Atg5‐dependent autophagy

Ursolic acid (UA) has been reported to possess anticancer activities. Although some of the anticancer activities of UA have been explained by its apoptosis‐inducing properties, the mechanisms underlying its anticancer actions are largely unknown. We have found that UA‐activated autophagy induced cytotoxicity and reduced tumor growth of cervical cancer cells TC‐1 in a concentration‐dependent manner. UA did not induce apoptosis of TC‐1 cells in vitro as determined by annexin V/propidium iodide staining, DNA fragmentation, and Western blot analysis of the apoptosis‐related proteins. We found that UA increased punctate staining of light chain 3 (LC3), which is an autophagy marker. LC3II, the processed form of LC3I which is formed during the formation of double membranes, was induced by UA treatment. These results were further confirmed by transmission electron microscopy. Wortmannin, an inhibitor of autophagy, and a small interfering RNA (siRNA) for autophagy‐related genes (Atg5) reduced LC3II and simultaneously increased the survival of TC‐1 cells treated with UA. We also found that LC3II was significantly reduced and that survival was increased in Atg5?/? mouse embryonic fibroblast (MEF) cells compared to Atg5+/+ MEF cells under UA treatment. However, silencing BECN1 by siRNA affected neither the expression of LC3II nor the survival of TC‐1 cells under UA treatment. These results suggest that autophagy is a major mechanism by which UA kills TC‐1 cells. It is Atg5 rather than BECN1 that plays a crucial role in UA‐induced autophagic cell death in TC‐1 cells. The activation of autophagy by UA may become a potential cancer therapeutic strategy complementing the apoptosis‐based therapies. Furthermore, regulation of Atg5 may improve the efficacy of UA in cancer treatment.     

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.