p38 MAPK inhibition enhances PS-341 (bortezomib)-induced cytotoxicity against multiple myeloma cells

Although PS-341 (bortezomib) is a promising agent to improve multiple myeloma (MM) patient outcome, 65% of patients with relapsed and refractory disease do not respond. We have previously shown that heat shock protein (Hsp)27 is upregulated after PS-341 treatment, that overexpression of Hsp27 confers PS-341 resistance, and that inhibition of Hsp27 overcomes PS-341 resistance. Since Hsp27 is a downstream target of p38 mitogen-activated protein kinase (MAPK)/MAPK-mitogen-activated protein kinase-2 (MAPKAPK2), we hypothesized that inhibition of p38 MAPK activity could augment PS-341 cytotoxicity by downregulating Hsp27. Although p38 MAPK inhibitor SCIO-469 (Scios Inc, CA, USA) alone did not induce significant growth inhibition, it blocked baseline and PS-341-triggered phosphorylation of p38 MAPK as well as upregulation of Hsp27, associated with enhanced cytotoxicity in MM.1S cells. Importantly, SCIO-469 enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK) and augmented cleavage of caspase-8 and poly(ADP)-ribose polymerase. Moreover, SCIO-469 downregulated PS-341-induced increases in G2/M-phase cells, associated with downregulation of p21Cip1 expression. Importantly, SCIO-469 treatment augmented cytotoxicity of PS-341 even against PS-341-resistant cell lines and patient MM cells. These studies therefore provide the framework for clinical trials of SCIO-469 to enhance sensitivity and overcome resistance to PS-341, thereby improving patient outcome in MM.     

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of non-small cell lung cancer cells via the JNK/c-Jun/AP-1 signaling

Proteasome inhibitor PS-341 induces growth arrest and apoptosis of multiple myeloma (MM) cells via inactivation of NF-κB in vitro and has afforded some objective responses in individuals with relapsed, refractory MM. However, the activity of PS-341 against non-hematological malignancies remains to be fully elucidated. In this study, we found that PS-341 induced growth arrest and apoptosis of NCI-H520 and -H460 non-small cell lung cancer (NSCLC) cells in conjunction with markedly up-regulated levels of p21^waf1 and p53, and down-regulation of bcl-2 protein in these cells. Also, PS-341 caused phosphorylation of c-Jun NH₂-terminal kinase (JNK) and c-Jun, and enhanced AP-1/DNA binding activities in these cells as measured by western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Interestingly, when the JNK/ c-Jun/AP-1 signal pathway was disrupted by the JNK inhibitor SP600125, the ability of PS-341 to inhibit the growth of NSCLC cells and to up-regulate the levels of p21^waf1 in these cells was blunted, but the expression of p53 was sustained at a high level, suggesting that the JNK/c-Jun/AP-1 signal pathway might mediate the anti-lung cancer effects of PS-341, with p21^waf1 playing the central role. Thus, PS-341 might be useful for the treatment of individuals with NSCLC.     

The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells

Human multiple myeloma (MM) is a presently incurable hematological malignancy, and novel biologically based therapies are urgently needed. Proteasome inhibitors represent a novel potential anticancer therapy. In this study, we demonstrate that the proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis of human MM cell lines and freshly isolated patient MM cells; inhibits mitogen-activated protein kinase growth signaling in MM cells; induces apoptosis despite induction of p21 and p27 in both p53 wild-type and p53 mutant MM cells; overcomes drug resistance; adds to the anti-MM activity of dexamethasone; and overcomes the resistance to apoptosis in MM cells conferred by interleukin-6. PS-341 also inhibits the paracrine growth of human MM cells by decreasing their adherence to bone marrow stromal cells (BMSCs) and related nuclear factor kappaB-dependent induction of interleukin-6 secretion in BMSCs, as well as inhibiting proliferation and growth signaling of residual adherent MM cells. These data, therefore, demonstrate that PS-341 both acts directly on MM cells and alters cellular interactions and cytokine secretion in the BM millieu to inhibit tumor cell growth, induce apoptosis, and overcome drug resistance. Given the acceptable animal and human toxicity profile of PS-341, these studies provide the framework for clinical evaluation of PS-341 to improve outcome for patients with this universally fatal hematological malignancy.     

Efficacy and mechanism of action of the proteasome inhibitor PS-341 in T-cell lymphomas and HTLV-I associated adult T-cell leukemia/lymphoma

HTLV-I associated adult T-cell leukemia (ATL) and HTLV-I-negative peripheral T-cell lymphomas are associated with poor prognosis. Using pharmacological concentrations of the proteasome inhibitor PS-341, we demonstrate inhibition of cell proliferation and induction of apoptosis in fresh ATL cells, HTLV-I transformed and HTLV-I-negative malignant T cells, while normal resting or activated T lymphocytes were resistant. Combination of PS-341 and doxorubicin or etoposide resulted in an additive growth inhibition. In HTLV-I-negative malignant cells, PS-341 treatment significantly downregulated the antiapoptotic protein X-IAP and to a lesser extent c-IAP-1 and bcl-X(L) and resulted in caspase-dependent apoptosis. In HTLV-I transformed cells, the inhibition of the proteasomal degradation of Tax by PS-341 likely explains the relative protection of HTLV-I infected cells against caspase-dependent apoptosis. PS-341 treatment of these cells stabilized IkappaBalpha, IkappaBbeta, IkappaBvarepsilon, p21, p27 and p53 proteins and selectively inhibited Rel-A DNA binding NF-kappaB complexes. In both HTLV-I-positive and -negative cells, PS-341 treatment induced ceramide accumulation that correlated with apoptosis. We conclude that PS-341 affects multiple pathways critical for the survival of HTLV-I-positive and -negative malignant T cells supporting a potential therapeutic role for PS-341 in both ATL and HTLV-I-negative T-cell lymphomas, whether alone or in combination with chemotherapy.     

The proteasome inhibitor PS-341 (bortezomib) up-regulates DR5 expression leading to induction of apoptosis and enhancement of TRAIL-induced apoptosis despite up-regulation of c-FLIP and survivin expression in human NSCLC cells

The proteasome inhibitor PS-341 (bortezomib or Velcade), an approved drug for treatment of patients with multiple myeloma, is currently being tested in clinical trials against various malignancies, including lung cancer. Preclinical studies have shown that PS-341 induces apoptosis and enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells with undefined mechanisms. In the present study, we show that PS-341 induced caspase-8-dependent apoptosis, cooperated with TRAIL to induce apoptosis, and up-regulated death receptor 5 (DR5) expression in human non-small cell lung cancer (NSCLC) cells. DR5 induction correlated with the ability of PS-341 to induce apoptosis. Blockage of PS-341-induced DR5 up-regulation using DR5 small interfering RNA (siRNA) rendered cells less sensitive to apoptosis induced by either PS-341 or its combination with TRAIL, indicating that DR5 up-regulation mediates PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells. We exclude the involvement of c-FLIP and survivin in mediating these events because c-FLIP (i.e., FLIP(S)) and survivin protein levels were actually elevated on exposure to PS-341. Reduction of c-FLIP with c-FLIP siRNA sensitized cells to PS-341-induced apoptosis, suggesting that c-FLIP elevation protects cells from PS-341-induced apoptosis. Thus, the present study highlights the important role of DR5 up-regulation in PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells.     

Novel therapies targeting the myeloma cell and its bone marrow microenvironment.

Novel therapies in multiple myeloma (MM) target not only the tumor cell but also the bone marrow (BM) microenvironment. Thalidomide (Thal), as well as derivative immunomodulatory drugs (IMiDs), directly induce apoptosis or G1 growth arrest in MM cell lines and patient's MM cells which are resistant to melphalan (Mel), doxorubicin (Dox), and dexamethasone (Dex). Although Thal and IMiDs do not alter adhesion of MM cells to bone marrow stromal cells (BMSCs), they inhibit the upregulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion triggered by the binding of MM cells to BMSCs. Proteasome inhibitors represent another potential anticancer therapy targeting the MM cell and the BM microenvironment. The proteasome inhibitor PS-341 directly inhibits proliferation and induces apoptosis in both human MM cell lines and freshly isolated patient's MM cells which are resistant to Mel, Dox, and Dex. PS-341 inhibits p44/42 mitogen-activated protein kinase (MAPK) growth signaling triggered by IL-6 and induces apoptosis, despite induction of p21 and p27, in p53 wild-type and p53 mutant MM cells. PS-341 adds to the anti-MM activity of dexamethasone and overcomes IL-6-mediated protection against dexamethasone-induced apoptosis. PS-341 blocks the paracrine growth of human MM cells by decreasing their adherence to BMSCs and related NF-kappaB-dependent induction of IL-6 secretion in BMSCs. Moreover, proliferation and MAPK growth signaling of those residual adherent MM cells is also inhibited. Tumor necrosis factor-alpha (TNF-alpha), which is produced by some MM cells, induces only low-level MM proliferation and MAPK activation in MM cells, but markedly upregulates IL-6 secretion from BMSCs and upregulates expression of adhesion molecules (VLA-4 and LFA-1) on MM cells and their receptors (VCAM-1 and ICAM-1) on BMSCs, with resultant increased binding of MM cells to BMSCs. Inhibition of TNF-alpha-induced NF-kappaB activation with PS-341 inhibits both the upregulation of these molecules on MM cells and BMSCs and the resultant increased adhesion. Therefore, inhibiting TNF-alpha and its sequelae may be useful treatment strategies in MM. Our data show that VEGF causes proliferation and enhances migration of MM as well as plasma cell leukemia (PCL) cells. VEGF induced twofold activation of cell migration in MM cells and more than 100-fold activation of cell migration in PCL cells, suggesting an important role of VEGF in the progression of MM to PCL. These data indicate that VEGF plays a pivotal role not only in neoangiogenesis in MM BM but also in proliferation and migration of tumor cells.     

Overcoming drug resistance in multiple myeloma: the emergence of therapeutic approaches to induce apoptosis.

Drug resistance remains a major clinical challenge for cancer treatment. Early studies suggested that overexpression of P-glycoprotein was a major contributor to the chemotherapy resistance of myeloma cells and other tumor cells. Attempts in several clinical studies to reverse multidrug resistance protein (MDR) by using MDR modulators have not yet generated promising results. Recently, the emerging knowledge about the importance of overcoming antiapoptosis and drug resistance in treating a variety of malignancies, including multiple myeloma (MM), raises new hope of improving the treatment outcome for patients with cancer. The therapeutic value of targeting therapies that aim to reverse the antiapoptotic process in MM cells has been explored in a number of experimental systems, and the results have been promising. The proteasome inhibitor PS-341 is a new specifically targeted proapoptotic therapy that has been tested in clinical studies. The results indicate that PS-341 alone is an effective therapy for patients with MM who experience disease relapse. Recent in vitro data also demonstrate that PS-341 can markedly sensitize chemotherapy-resistant MM cells to various chemotherapeutic agents. On the basis of these encouraging results, clinical studies are underway to test the efficacy of PS-341 and chemotherapeutic agents as combination therapy in treating patients with refractory and relapsed MM.     

Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis

Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH(2)-terminal kinase (JNK)-dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca(2+) levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin.     

Blockade of Hsp27 overcomes Bortezomib/proteasome inhibitor PS-341 resistance in lymphoma cells

Bortezomib (PS-341), a selective inhibitor of proteasome, induces apoptosis in various tumor cells, but its mechanism of action is unclear. Treatment with PS-341 induces apoptosis in SUDHL6 (DHL6), but not SUDHL4 (DHL4), lymphoma cells. Microarray analysis shows high RNA levels of heat shock protein-27 (Hsp27) in DHL4 versus DHL6 cells, which correlates with Hsp27 protein expression. Blocking Hsp27 using an antisense strategy restores the apoptotic response to PS-341 in DHL4 cells; conversely, ectopic expression of wild-type Hsp27 renders PS-341-sensitive DHL6 cells resistant to PS-341. These findings provide the first evidence that Hsp27 confers PS-341 resistance.     

Biologic sequelae of c-Jun NH(2)-terminal kinase (JNK) activation in multiple myeloma cell lines

Although c-Jun NH(2)-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27(Kip1), without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-kappaB activation in a dose-dependent fashion, associated with phosphorylation of IkappaB kinase alpha (IKKalpha) and degradation of IkappaBalpha. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-kappaB activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-kappaB in MM cells.     

Long-term activation of SAPK/JNK, p38 kinase and fas-L expression by cisplatin is attenuated in human carcinoma cells that acquired drug resistance

Tumor cells chronically exposed to cisplatin (cDDP) acquire cDDP resistance that impacts tumor therapy. To elucidate the mechanism of acquired cDDP resistance (ACR), we compared HeLa cells that gained ACR upon chronic cDDP treatment with the parental strain. We show that ACR is due to a lower level of induced apoptosis. Further, upon cDDP treatment, the levels of Fas, Bax and Bid remained unchanged, whereas Bcl-2 and p-Bad were reduced at late times (120 hr) after treatment. At early times, Fas ligand (fas-L) expression was significantly enhanced in sensitive compared to resistant cells and remained upregulated up to the onset of apoptosis. Thus, activation of the Fas system is critical, which is in line with the finding that in sensitive cells, caspase-8 along with caspase-9 and -3 were activated by cDDP. cDDP provoked the activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 kinase dose-dependently, with significantly lower levels in ACR cells than in the sensitive parental line. cDDP induces c-Jun and AP-1 activity, as measured by a reporter gene assay, which was again attenuated in ACR cells. Time course analysis revealed that SAPK/JNK and p38 kinase activity was sustained upregulated (> 72 hr postexposure), which occurred at much higher level in sensitive than in ACR cells. Inhibition of either JNK or p38 kinase (by JNK inhibitor II and SB 203580, respectively) attenuated cDDP-induced apoptosis, supporting the role of JNK and p38 kinase in the cDDP response. Since several independently derived cDDP-resistant cell lines displayed attenuated MAPK signaling, sustained SAPK/JNK and p38 kinase activation may be a general mechanism of cDDP-induced cell death. ACR cells displayed a reduced level of DNA damage, indicating long-term stimulation of SAPK/JNK and p38 kinase is triggered by nonrepaired cDDP-induced DNA lesions.     

曲古菌素A和硼替佐米诱导卵巢癌细胞凋亡的协同作用

目的：探讨组蛋白去乙酰化酶抑制剂曲古菌素A与蛋白酶体抑制剂硼替佐米单独及联合应用对人卵巢癌细胞株SKOV3存活率和凋亡率的影响。初步探讨两种药物联合应用对诱导SKOV3细胞凋亡具有的协同作用。 方法曲古菌素A、硼替佐米单独或者联合应用于卵巢癌细胞后,用四甲基偶氮唑蓝（MTT）比色法测定细胞增殖活性,并计算细胞存活率,Annexin-V/PI法流式细胞仪检测细胞凋亡率,蛋白质免疫印迹法（Western blot）检测相关蛋白表达水平。通过检测Caspases-3的活性及其底物多聚ADP核糖聚合酶（PARP）的表达水平进一步说明不同用药组诱导细胞凋亡的情况。 结果联合用药组诱导的细胞凋亡率和单独用药组比较,差异有统计学意义（P<0.05）。几种凋亡相关蛋白Bcl-2、Mcl-1和Bcl-X_L, 在联合用药组的表达显著低于单独用药组。 在相同的时间点,联合用药组Caspase-3的活性和单独用药组比较,差异有统计学意义（P<0.001）。结论：低剂量的曲古菌素A和硼替佐米联合作用人卵巢癌细胞系能诱导凋亡,并且这种作用远远强于相同剂量单独用药引起的凋亡。这两种药物的联合应用可能成为人卵巢癌化疗中的新方案。     

A pivotal role for Mcl-1 in Bortezomib-induced apoptosis

Bortezomib is a proteasome inhibitor for the treatment of relapsed/refractory multiple myeloma (MM). Mechanisms of resistance to Bortezomib are undefined. Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic protein, which protects tumor cells against spontaneous and chemotherapy-induced apoptosis. In MM, specific downregulation of Mcl-1 induces apoptosis. Here, we examined the role of Mcl-1 in Bortezomib- and doxorubicin-induced apoptosis. We demonstrate that Bortezomib, but not doxorubicin, triggers caspase-dependent generation of a 28 kDa Mcl-1-fragment, in several MM cell lines, including MM.1S cells. Conversely, transient transfection of MM.1S cells with a previously reported 28 kDa Mcl-1(128-350) fragment, but not with the Mcl-1(1-127) fragment, induces apoptosis. Therefore, both downregulation of full-length antiapoptotic Mcl-1, as well as Bortezomib-induced generation of Mcl-1(128-350) cleaved protein, contribute to MM cell apoptosis. To verify further these findings, we next compared effects triggered by Bortezomib, doxorubicin and melphalan in Mcl-1(wt/wt) and Mcl-1(Delta/null) murine embryonic fibroblasts (MEFs). Our results show that Bortezomib, but not doxorubicin or melphalan, triggers Mcl-1 cleavage in Mcl-1(wt/wt), but not Mcl-1(Delta/null) MEFs and induces sub-G(1) phase cells; caspase-3 and -9, and PARP cleavage as well as morphological signs of apoptosis. Taken together, these results support an important role of Mcl-1 and a Mcl-1 fragment in Bortezomib-induced cell death in general, and in MM in particular. To prevent relapse of MM in patients treated with Bortezomib, we therefore recommend the combination of Bortezomib with agents that induce MM cell death independent of Mcl-1.     

The proteasome inhibitor PS-341 overcomes TRAIL resistance in Bax and caspase 9-negative or Bcl-xL overexpressing cells

We demonstrate that PS-341, a small molecule inhibitor of the proteasome, markedly sensitizes resistant prostate, colon, and bladder cancer cells to TNF-like apoptosis-inducing ligand (TRAIL)-induced apoptosis irrespective of Bcl-xL overexpression. PS-341 treatment by itself does not affect the levels of Bax, Bak, caspases 3 and 8, c-Flip or FADD, but elevates levels of TRAIL receptors DR4 and DR5. This increase in receptor protein levels is associated with the ubiquitination of the DR5 protein. When PS-341 is combined with TRAIL, the levels of activated caspase 8 and cleaved Bid are substantially increased. In Bax-negative TRAIL-resistant HC-4 colon cancer cells, the combination of PS-341 and TRAIL overcomes the block to activation of the mitochondrial pathway and causes SMAC and cytochrome c release followed by apoptosis. Similarly, murine embryonic fibroblasts lacking Bax undergo apoptosis when exposed to the combination of PS-341 and TRAIL; however, fibroblasts lacking Bak are significantly resistant. Taken together, these findings indicate that PS-341 enhances TRAIL-induced apoptosis by increasing the cleavage of caspase 8, causing Bak-dependent release of mitochondrial proapoptotic proteins.     

N-(4-hydroxyphenyl)retinamide-induced apoptosis triggered by reactive oxygen species is mediated by activation of MAPKs in head and neck squamous carcinoma cells

N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.     

Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341.

Almost all known conventional cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than in killing tumor cells grown as monolayer cell cultures. This "multicellular resistance" reflects the relative intrinsic drug-resistant phenotype of most solid tumors growing in vivo and is due to factors such as limited drug penetration or reduced fractions of proliferating cells. Proteasome inhibitors such as PS-341, a dipeptide boronic acid analogue, represent an interesting new class of potential anticancer drugs, which are entering early-phase clinical trials. PS-341 has been found to have good broad-spectrum cytotoxic activity in the 60-monolayer cell line National Cancer Institute screen. However, because its relative potency has not been tested in spheroid systems, we analyzed the activity of PS-341 in a spheroid/solid tumor context using four different human ovarian carcinoma cell lines and three prostate carcinoma cell lines, respectively. We found, with one exception, that PS-341 showed equal or greater activity in spheroids than in the respective monolayer cell cultures, even in a prostate cancer spheroid model with a very low growth fraction. PS-341 induced apoptotic cell death in carcinoma cells in both culture systems. We also noted a decrease in XIAP protein, a member of the inhibitor of apoptosis (IAP) family of apoptosis inhibitors, and phosphorylation of Bcl-XL in PS-341-treated ovarian carcinoma cells. Furthermore, DNA fragmentation, a hallmark of apoptosis (in this case, induced by PS-341), was completely inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). Taken together, the results indicate that unlike most other known anticancer cytotoxic drugs, PS-341 appears to be as effective in killing tumor cells grown in the form of multicell spheroids as in killing tumor cells grown in monolayer cell culture. Hence, this compound has the potential to circumvent multicellular drug resistance and, as such, may show promising activity against solid tumors with low growth fractions in vivo, which are frequently intrinsically resistant to conventional cytotoxic anticancer drugs.