Proteasome Inhibitor PS-341 Research Articles

A phase I, open label, dose-escalating study of the proteasome inhibitor PS-341 (VELCADE¢) in combination with two schedules of trastuzumab, in patients with advanced breast cancer (ABC) that overexpresses HER-2

A phase I, open label, dose-escalating study of the proteasome inhibitor PS-341 (VELCADE¢) in combination with two schedules of trastuzumab, in patients with advanced breast cancer (ABC) that overexpresses HER-2

Sensitization of DNA damage–induced apoptosis by the proteasome inhibitor PS-341 is p53 dependent and involves target proteins 14-3-3σ and survivin

Proteasome inhibition following DNA damage results in the synergistic induction of apoptosis via a nuclear factor-kappaB-independent mechanism. In this study, we identify the role of p53 in mediating apoptosis by the sequence-specific treatment involving the DNA-damaging, topoisomerase I-targeting drug SN-38 followed by the proteasome inhibitor PS-341 (SN-38-->PS-341). The p53-dependent sensitization of DNA damage-induced apoptosis by PS-341 is accompanied by persistent inhibition of proteasome activity and increased cytosolic accumulation of p53, including higher molecular weight forms likely representing ubiquitinated species. In contrast, pretreatment with PS-341 followed by treatment with SN-38 (PS-341-->SN-38), which leads to an antagonistic interaction, results in transient inhibition of proteasome activity and accumulation of significantly lower levels of p53 localized primarily to the nucleus. Whereas cells treated with PS-341-->SN-38 undergo G2 + M cell cycle arrest, cells treated with SN-38-->PS-341 exhibit a decreased G2 + M block with a concomitant increase in the sub-G1 population. Decreased accumulation of cells in the G2 + M phase of the cell cycle in SN-38-->PS-341-treated cells compared with PS-341-->SN-38-treated cells correlates with enhanced apoptosis and reduced expression of two p53-modulated proteins, 14-3-3sigma and survivin, both of which play critical roles in regulating G2 + M progression and apoptosis. The functional role of 14-3-3sigma or survivin in regulating the divergent function of p53 in response to SN-38-->PS-341 and PS-341-->SN-38 treatment in inducing apoptosis versus G2 + M arrest/DNA repair, respectively, was confirmed by targeted down-regulation of these proteins. These results provide insights into the mechanisms by which inhibition of proteasome activity modulates DNA damage-induced apoptosis via a p53-dependent pathway.

Synergistic Activity of the Proteasome Inhibitor PS-341 with Non-Myeloablative 153-Sm-EDTMP in a Syngeneic, Orthotopic Model of Multiple Myeloma.

Multiple myeloma is a highly radiosensitive malignancy but, at the present time, radionuclide-based interventions have no proven place in disease management. Bone-seeking radionuclides such as 153-Sm-EDTMP and 166-Ho-DOTMP are promising agents for systemic delivery of ionizing radiation to sites of myeloma disease activity, but they are associated with significant myelosuppression at therapeutically effective doses and have therefore been used only in the context of myeloma stem cell transplantation protocols. We previously reported that the proteasome inhibitor PS-341 potently and selectively sensitizes myeloma cell lines and primary myeloma cells to the lethal effects of ionizing X-irradiation (Goel et al, Exp Hematol . 33, 784, 2005). To determine whether PS-341 is equally effective in sensitizing myeloma cells to ionizing beta-radiation and to extend our initial observations to an in vivo model, we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays were performed using a panel of myeloma cell lines and demonstrated synergistic killing following co-treatment with PS-341 and 153-Sm-EDTMP. Using the orthotopic, syngeneic 5TGM1 myeloma model, the median survivals of mice treated with saline, two doses of PS-341 (0.5 mg/kg), or a single non-myeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21, 22 and 28 days, respectively. In contrast, mice treated with combination therapy comprising two doses of PS-341 (0.5 mg/kg), one day prior to and one day following 153-Sm-EDTMP (22.5 MBq) showed a greatly prolonged median survival of 49 days. Correlative studies indicated that, compared to single-agent therapy, combination treatment with PS-341 and 153-Sm-EDTMP rapidly reduced the clonogenicity of bone-marrow resident 5TGM1 cells, slowed the elevation of serum myeloma-associated paraprotein levels, and was associated with longer term preservation of bone mineral density. The myelotoxicity of single agent and combination therapies was evaluated by comparing peripheral blood cell counts in each of the treatment groups, and by clonogenicity assays of hematopoietic progenitors isolated form normal mice receiving identical treatment regimens. Treatment with 153-Sm-EDTMP led to significant, but transient, myelosuppression which did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. In summary, PS-341 is a potent in vivo radiosensitizer that greatly enhances the therapeutic potency, without increasing myelotoxicity, of skeletal targeted radiotherapy in the syngeneic, orthotopic 5TGM1 myeloma model. Based on these findings, we propose to conduct a phase I clinical trial at Mayo Clinic to evaluate the combination of PS-341 plus non-myeloablative skeletal targeted radiotherapy (using 153-Sm-EDTMP) in patients with advanced or treatment-refractory multiple myeloma.

Accumulation of NFkB1 (p105) and NFkB2 (p100) Is Essential for Apoptosis Induced by Proteasome Inhibition in a Lymphoma Model.

The proteasome is a novel target in the therapeutic approach against different subtypes of lymphomas due to its role in the degradation of many proteins involved in cell cycle progression and apoptosis. This multi-enzyme complex controls gene expression by degrading transcription factors such as NF-kB, p53, c-Jun, c-Myc, HIFla, and MATa2. Among these, the best characterized is the NFkB family of transcription factors. Mammals express five NFkB proteins including REL-A (p65), c-REL, REL-B, p50 and p52. All NFkB proteins contain a highly conserved REL-homology domain (RHD), which is responsible for DNA binding, dimerization, nuclear translocation, and interaction with the IkB proteins. Active NFkB is present in the nucleus as heterodimers of p65 and p50 or p52 and REL-B subunits. In contrast, inactive NFkB dimers are sequestered in the cytoplasm due to their interaction with inhibitory proteins such as the IkB's or the large NFkB subunits p105 and p100. Activation of the NFkB pathway is mediated by proteasome processing of p105 and p100 to produce p50 and p52, respectively, and proteasome degradation of the IkB proteins. Therefore, the proteasome plays a central role in the equilibrium between anti-apoptotic signals derived from p50 and p52, and the NFkB inhibitory signals of p105 and p100. However, it is still undetermined whether modulation of the inhibitory signals or the anti-apoptotic signals by proteasome inhibition is more important for the induction of apoptosis in lymphomas. To address this question we tested the apoptotic effect of the proteasome inhibitor PS341 in two lymphoma cell lines (Daudi and SC) after knocking down p105 and/or p100, using lentivirus expressing siRNA's. After 24 hours of treatment with titration doses of PS341 (5, 10, 50, and 100nM), we measured apoptosis and NFkB inhibition by Annexin V-Alexa fluorescence and an NFkB luciferase reporter assay, respectively. Interestingly, co-expression of siRNA against both p100 and p105 rendered cells resistant to the induction of apoptosis at clinical doses of PS341 (5 and 10nM) compared to the controls (see table). Similar results were obtained after selective knock down of either p105 or p100. Western Blot analysis showed much lower accumulation of p105 and p100 in siRNA expressing cells than in control cells. However, IkB levels remain stable or increase during treatment in the p105 and/or p100 siRNA expressing cells, despite the observed reduction in apoptosis. These results suggest that the accumulation of p105 and p100, rather than IkB, contributes to the induction of apoptosis. In addition, the expression of NFkB anti-apoptotic signals, such as BCL-xL, increased in all cell lines, including controls. Therefore, the presence of anti-apoptotic signals has no impact on the apoptotic effect produced by PS341. In conclusion, the current study in our lymphoma model demonstrates that the accumulation of p105 and p100 is essential for the induction of apoptosis produced by proteasome inhibition.siRNA Against p100 and/or p105 Reduces PS341 Induction of Apoptosisps314(nM)51050100SCLuciferase siRNA61.2±3.270±3.672.5±3.679.5±5Both siRNA's23.5±1.533±2.959.2±3.972.1±5.7p100 siRNA27.2±0.429±0.556±2.766.3±6.3p105siRNA32.2±7.345±2.455.4±7.778.4±4.1DAUDILuciferase siRNA45.6±3.349.5±5.161.5±7.1 69.9±5Both siRNA's21.4±4.629.3±1.734.5±2.455.3±7.7p100 siRNA17.3±4.224.4±9.944.5±3.356.6±4.7p105 siRNA19.7±1.634.3±3.331.3±2.646.3±3.4

Sensitivity of human multiple myelomas and myeloid leukemias to the proteasome inhibitor I

The proteasome inhibitor PSI is potently cytotoxic in vitro against human chronic myeloid leukemia (CML) and acute myeloid leukemias (AML). Here, we have tested proteasome inhibitor I (PSI) in a panel of 11 human multiple myeloma (MM) cell lines and found that it has antiproliferative activity, with an IC50 between 4.5 and 557 nM at 48 h. PSI potentiated the toxicity of a number of chemotherapeutic agents in myeloid leukemia but not in MM cell lines, while in combination with therapeutic proteasome inhibitor PS-341 (Bortezomib) it had a synergistic effect. PSI suppressed the growth of AML cell lines more effectively than PS-341. CFU-GM colony assays revealed that CD34+ bone marrow progenitors from CML and AML patients were more sensitive to PSI than those from normal subjects (IC50: 5, 15 and 50 nM for AML, CML and normal, respectively). Moreover, the growth of normal primitive progenitors (LTC-IC) was unaffected by 15 nM PSI (P=0.576). PSI-induced cell death required RNA transcription and protein synthesis, but not DNA replication, was accompanied by the upregulation of Bcl-2 and modest reduction of Bax and Bcl-XL proteins, and involved the activation of caspases 2, 3, 7 and 8. These findings lend additional support to preclinical investigations with PSI.

Phosphorylation of AML1/RUNX1 Regulates Its Degradation and Nuclear Matrix Association

The acute myeloid leukemia 1 (AML1) transcription factors are key regulators of hematopoietic differentiation. Cellular AML1c protein is found in the nucleus and can be separated into two fractions, one soluble in buffers containing salt and nonionic detergent and the other insoluble and tightly bound to the nuclear matrix. We find that the AML1c protein is modified by both phosphorylation and ubiquitination. Our studies show that the majority of the ubiquitinated AML1c is associated with the insoluble nuclear matrix. Treatment of cells with the proteasome inhibitor PS341 (Velcade, Bortezomib) increases the levels of ubiquitinated AML1c. Mutation of the four phosphorylation sites necessary for transcriptional regulation (serine 276, serine 293, serine 303, and threonine 300) mimics the effects of the proteasome inhibitor, increasing the levels of ubiquitinated, matrix-bound AML1c. We find that the soluble and insoluble forms of AML1c are degraded at a similar rate. However, mutation of these four serine/threonine residues statistically increases the half-life of the matrix-associated AML1c. Thus, phosphorylation of AML1c on specific serine/threonine residues controls both transcriptional activity and rate of degradation.

The proteasome inhibitor PS-341 (bortezomib) in platinum (plat)-treated extensive-stage small cell lung cancer (E-SCLC): A SWOG (0327) phase II trial

7047 Background: The proteasome inhibitor PS-341 inhibits growth of SCLC cell lines through decreased bcl-2 via NFk-B. We conducted a phase II trial of PS-341 in plat-treated E-SCLC patients (pts) to determine the tumor response rate, toxicity and overall survival. Methods: : Pts with histologically-confirmed SCLC, measurable disease, Zubrod performance status 0–1 and prior plat-based therapy were eligible. Pts were stratified by platinum-sensitivity status: sensitive (progression >90 days after plat) or refractory (progression during or ≤90 days after plat). Pts were treated with PS-341 1.3mg/m2 IV on days 1, 4, 8 and 11 q 21 days. Results: Of 60 pts registered, 57 were evaluable for response. Seven pts discontinued treatment due to adverse events or side effects from therapy. Nine pts (16%) had grade (gr) 3/4 platelets. Eight (14%) pts had gr 3/4 fatigue. There was no gr 3/4 neuropathy. Conclusions: Although PS-341 induced a response in a plat-refractory pt, the efficacy is insufficient as a single agent in this heavily-pretreated population. Clinical testing of PS-341 in combination with an apoptotic-trigger such as cytotoxic chemotherapy, as supported by preclinical models, is warranted. A trial of topotecan plus PS-341 has been initiated to test this concept. (This study was supported by SWOG grant CA 30102) No significant financial relationships to disclose.

Pim-1 Kinase Stability Is Regulated by Heat Shock Proteins and the Ubiquitin-Proteasome Pathway

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.

APOPTOSIS AND CHEMOTHERAPY FOR BLADDER CANCER

We discuss the role of apoptosis, that is gene directed self-destruction of a cell, in the response of bladder transitional cell carcinoma cells to chemotherapy. A directed MEDLINE literature search of apoptosis, bladder cancer and chemotherapy was performed to extract relevant information for review. The characteristics of apoptotic cells were defined and the methods in common use to detect these traits is described. The role of the key mediators of the apoptotic process in bladder cancer is discussed in the context of chemosensitivity and disease stage. The importance of the apoptosis induction after chemotherapy is highlighted. On stimulus by appropriate external or internal signals a cell may alter the expression of genes encoding for proteins associated with the apoptotic process. The development of apoptosis depends on the balance between pro-apoptotic and anti-apoptotic proteins. Key alterations in genes and proteins related to apoptosis within bladder cancer result in a shift away from the default state of apoptosis toward a cell with increased survival properties that is chemoresistant. Much current research in bladder cancer is aimed at restoring chemosensitivity by shifting the cell toward a pro-apoptotic phenotype. Successful translation of this work into clinical practice may improve survival in patients in whom prognosis is currently poor.

Cellular Basis of Antiproliferative and Antitumor Activity of the Novel Camptothecin Derivative, Gimatecan, in Bladder Carcinoma Models

To investigate the cellular/molecular basis of the activity of a novel lipophilic camptothecin, gimatecan (ST1481), against slowly proliferating cells, we performed a comparative study of topotecan and gimatecan in human bladder cancer models (HT1376 and MCR). Gimatecan was significantly more effective than topotecan in inhibiting the growth of HT1376 tumor, thus reflecting anti proliferative potency. In both HT1376 and MCR cells, gimatecan caused a persistent S-phase arrest, indicating an efficient DNA damage checkpoint. This response was consistent with a cytostatic effect, because no evidence of apoptosis was detected. In contrast to gimatecan, topotecan at equitoxic concentrations caused an early and persistent downregulation of topoisomerase I. Modulation of protein level could not be solely ascribed to the proteasome-mediated degradation of the enzyme because the proteasome inhibitor PS341 sensitized MCR but not HT1376 cells to camptothecins, suggesting alternative mechanisms of drug-induced topoisomerase I downregulation. Indeed, the two camptothecins caused a differential inhibition of topoisomerase I transcription, which is more marked in topotecan-treated cells. The HT1376 model was more sensitive to this immediate decrease of mRNA level. Our data document a marked antitumor activity of gimatecan against a bladder carcinoma model. A limited downregulation of topoisomerase I by gimatecan provides additional insights into the cellular basis of drug potency.

Proteasome Inhibition Sensitizes Hepatocellular Carcinoma Cells, But Not Human Hepatocytes, to TRAIL *

TRAIL exhibits potent anti-tumor activity on systemic administration in mice. Because of its proven in vivo efficacy, TRAIL may serve as a novel anti-neoplastic drug. However, approximately half of the tumor cell lines tested so far are TRAIL resistant, and potential toxic side effects of certain recombinant forms of TRAIL on human hepatocytes have been described. Pretreatment with the proteasome inhibitor MG132 and PS-341 rendered TRAIL-resistant hepatocellular carcinoma (HCC) cell lines but not primary human hepatocytes sensitive for TRAIL-induced apoptosis. We investigated the different levels of possible MG132-induced interference with resistance to apoptotic signal transduction. Although proteasome inhibition efficiently suppressed nuclear factor-kappaB (NF-kappaB) activity, specific suppression of NF-kappaB by mutIkappaBalpha failed to sensitize TRAIL-resistant cell lines for TRAIL-induced apoptosis. In contrast to the previously reported mechanism of sensitization by 5-fluorouracil (5-FU), cellular FLICE-inhibitory protein (cFLIP)(L) and cFLIP(S) were markedly upregulated in the TRAIL death inducing signaling complex (DISC) by proteasome inhibitor pretreatment. Compared with 5-FU pretreatment, caspase-8 was more efficiently recruited to the DISC in MG132 pretreated cells despite the presence of fewer death receptors and more cFLIP in the DISC. But downregulation of cFLIP by short interference RNA (siRNA) further sensitized the HCC cell lines. In conclusion, these results show that otherwise chemotherapy-resistant tumor cells can be sensitized for TRAIL-induced apoptosis at the DISC level in the presence of high levels of cFLIP, which suggests the existence of an additional factor that modulates the interaction of FADD and the TRAIL death receptors. Of clinical relevance, proteasome inhibitors sensitize HCC cells but not primary human hepatocytes for TRAIL-induced apoptosis.

Avicins: A Novel Class of Anti-Myeloma Agents.

Multiple myeloma (MM) remains an incurable neoplasia, despite recent development of several novel therapies. As part of our efforts to identify new compounds with anti-MM activity, we evaluated the class of avicins, which are triterpenoid saponins that have been shown to induce apoptosis of neoplastic cells by affecting mitochondrial function independently of membrane-bound death receptors. Because we have previously shown that mitochondria constitute key regulators of MM cell responsiveness to diverse anti-tumor agents, (e.g. the proteasome inhibitor bortezomib), we evaluated the in vitro anti-MM effects of this class of compounds. Our vitro drug-sensitivity studies showed that Avicin D and Avicin G, the main members of this class of compounds, are active against a broad panel of MM cell lines and primary tumor cells, including tumor cells resistant to conventional (e.g. dexamethasone, alkylating agents, anthracyclines) or novel (e.g. thalidomide, immunomodulatory thalidomide derivatives, proteasome inhibitor PS-341[bortezomib], Apo2L/TRAIL) anti-MM agents. Using MTT survival assays, we confirmed that the IC50 values for both Avicins were highly concordant and were less than 250 nM for the overwhelming majority of MM cell lines tested. Importantly, this potent in vitro anti-MM activity was triggered by concentrations of Avicins which had minimal, if any, effect on the viability of normal hematopoietic cells or bone marrow stromal cells. Furthermore these IC50 values were comparable with the in vitro activity of this agent among the most Avicin-sensitive tumor models that have been previously tested. This potent anti-MM effect was not inhibited by transfection of MM cells with construct for constitutively active Akt. Although cytokine- or cell adhesion-mediated interactions of the local bone marrow (BM) microenvironment (e.g. BM stromal cells) protects MM cells from conventional therapies (e.g. dexamethasone or cytotoxic chemotherapy), avicins were able to overcome this protective effect in co-culture models of MM cells with BM stromal cells and sensitized MM cells to cytotoxic chemotherapy-induced cell death. Using hierarchical clustering analyses and relevance network algorithms, we compared the patterns of MM cell sensitivity to Avicin D vs Avicin G vs. other anti-cancer drugs and found that the pattern of dose-response relationships of the 2 main members of this class of compounds are very similar to each other, but clearly distinct from the patterns of sensitivity or resistance to other drugs, either conventional or investigational. This further supports the notion that the anti-MM properties of Avicins are mediated by molecular mechanisms distinct from those of currently available anti-MM drugs, and also suggests that Avicins may have anti-tumor activity even against subgroups of MM which may be resistant to other novel therapies that are currently in clinical development. These results have provided the framework for ongoing in vivo studies of anti-tumor activity of these agents, to evaluate the feasibility of future clinical trials of Avicins to improve patient outcome in MM.

Sigma 2-Agonist CB-64D Induces Apoptosis in Non Hodgkin's Lymphoma Cells: Association with Decreased Akt Expression.

Background and aim: Sigma (σ) receptors are expressed at high density in a variety of tumor cell types, with an increased expression of σ2-subtype in rapidly proliferating tumors. CB-64D is a recently developed sigma agonist, showing a higher affinity for σ2- receptor subtype. We evaluated the cytotoxic activity of CB-64D in the extensively characterized Non-Hodgkin's Lymphoma (NHL) cell lines DB, RL, and Ramos.Materials and methods: Human DB, RL (both derived from patients with diffuse large cell lymphoma), and Ramos (Burkitt's lymphoma) cells were exposed to increasing concentrations of CB-64D for various periods of time. Cells were harvested and stained with Annexin-V and PI and apoptosis was evaluated by flow cytometry. Expression of caspases, Akt and Bcl-2 was determined by Western blotting.Results: CB-64D induced apoptosis in all NHL cells tested in both a time- and dose- dependent manner. Apoptosis was detected as early as 3 hours (7.5%) and reached its maximum at 12 hours (75%). As little as 5 μM CB-64D was able to induce apoptosis and maximum effects were noted with 40 μM CB-64D. To investigate the mechanisms responsible for the observed apoptosis, expression of caspases, Bcl-2, and Akt was evaluated. Western blot experiments revealed that CB-64D induced the expression of activated caspases 3, 8, and 9. Inhibitors of both caspase 3 and caspase 8 significantly decreased CB-64D-induced apoptosis. The induction of apoptosis was not associated with changes in Bcl-2 levels. In contrast, CB-64D induced a major decrease of Akt as determined by Western blot. Northern blot analysis demonstrated that Akt mRNA was not affected by CB-64D suggesting that the decreased expression of Akt was controlled at the post-transcriptional level. Treatment with the proteasome inhibitor PS-341 failed to prevent CB-64D-induced Akt down-modulation suggesting that the decreased expression of Akt is not due to proteasome degradation.Conclusions: Our data demonstrate that the σ2-agonist CB-64D has a potent anti-lymphoma activity. CB-64D directly induces apoptosis through caspase-dependent mechanisms. Our data also show that CB-64D-induced apoptosis is associated with a decreased Akt expression that is controlled at the post-transcriptional level. The present results warrant the exploration of CB-64D in a pre-clinical model.

The Proteasome Inhibitor PS-341 Induces Early Apoptosis of CD14+ Dendritic Cell (DC) Precursors and of CD1a+ Immature DC.

DC play an essential role in the initiation of (allo)immune responses. Downregulation of NF-kB activity has been shown to prevent the differentiation and maturation of DC. PS-341 a novel antimyeloma terapeutic agent, promotes the degradation of NF-kB. In this study we have investigated whether PS-341 affects DC differentiation and/or maturation. DC were generated by culturing immunomagnetically purified CD14+ monocytes for 1–5 days with GM-CSF (50 ng/ml) and IL-4 (800 U/ml) in the presence or absence of PS-341 at 0.1 to 100 ng/ml. Addition of PS-341 at doses as low as 10 ng/ml significantly reduced the recovery of viable cells, as determined by trypan blue exclusion assay, both after 24h and 48h of culture (3±7% vs 35±25% in control cultures and 3±7% vs 32±25%, respectively, at 24 and 48 hours) (n=5 experiment, p<0.05). Doses higher than 10 ng/ml had a comparable effect. No viable cells were observed after 3 days of culture. Addition of 1 ng/ml PS-341 slightly reduced cell recovery both after 24h (22±26%) and 48h (25±28%) (p=n.s.), whereas cell recovery was not altered in the presence of 0.1 ng/ml PS-341. To test whether the reduced recovery of viable cells was due to apoptosis induction, the expression of annexin V was determined by flow cytometry. Upon addition of 10 ng/ml PS-341, annexin V+ cells increased both at 24h (87±1 vs 55±13% in control cultures) and 48h (93±4% vs 27±13%) (n=5 exps) (p<0.05), whereas no difference was observed with 1 ng/ml and 0.1 ng/ml PS-341. Induction of apoptosis was confirmed by microscopical analysis of cytospins, that showed nuclear fragmentation and massive cytoplasmic vacuolization, features suggestive of apoptosis. Differentiation of monocytes to dendritic cells, as measured by the progressive loss of CD14 and acquisition of CD1a expression, was not altered in cultures containing either 1 or 0.1 ng/ml PS-341. To test whether apoptosis induction by PS-341 required the presence of IL-4, that is necessary to DC differentiation, monocytes were cultured in the presence of GM-CSF only. 10 ng/ml PS-341 signifcantly reduced monocyte recovery after 48 hours of culture (4.5±3.1% vs 69±31% in control cultures, n=3 experiments, p<0.05), due to the induction of early apoptosis (75±4% annexin V+ cells vs 36±1% at 24 hours and 95±1% vs 26±19% at 48 hours in PS-341 and control cultures, respectively). To test whether PS-341 induced apoptosis of immature dendritic cells as well, monocytes were cultured for 6 days with GM-CSF and IL-4 then 1 ng/ml LPS was added for the last 48 hours with or without PS-341 at either 0.1, 1 or 10 ng/ml. Addition of PS-341 at 10 ng/ml strongly reduced day 8 cell recovery as compared to control cultures (3±1% vs 20±21%) (n=2 experiments). Following 48 hours of culture in the presence of 10 ng/ml PS-341, DC were mostly apoptotic (96±2% annexin V+ cells as compared to 28±5% in control cultures). However, PS-341 doses of either 0.1 or 1 ng/ml did not affect cell recovery as well as expression of CD40, CD80 and CD86 costimulatory molecules. Thus clinically relevant doses of PS-341 (in the range of 10−8 M) may effectively and rapidly induce apoptosis of CD14+ pre-DC as well as CD1a+ immature DC in vitro. Our data suggest that PS-341 might be tested in vivo in the prophylaxis of DC-triggered immunologic diseases, such as graft versus host disease.

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.

Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme (GBM)

The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of normal and tumor cells. PS-341, a novel boronic acid dipeptide that inhibits 26S proteasome activity, has prominent effects in vitro and in vivo against several solid tumors. We examined its antiproliferation, proapoptotic effects using three human glioblastoma multiforme (GBM) cell lines and five primary GBM explants. PS-341 markedly inhibited proliferation of GBM cell lines and explants in liquid and soft agar culture. These cells developed a G2/M cell cycle arrest with a concomitant decreased percentage of cells in S phase ( approximately 2-fold), associated with an increased expression of p21(WAF1), p27(KIP1), as well as cyclin B1 and decreased levels of CDK2, CDK4, and E2F4. About 35-40% of the cells became apoptotic when exposed to PS-341 (10(-7) M, 24-48 h) as shown by Annexin V analysis; in concert with these findings, immunobloting showed a C-terminal 85 kDa apoptotic fragment of poly ADP-ribose polymerase (PARP), and a decreased level of Bcl2 and Bcl-xl. PS-341 downregulated the expression of Bcl-2 and Bcl-xl in protein levels at an early time of treatment. These changes occurred irrespective of the p53 mutational status of the cells. PS-341 activated JNK/c-Jun signaling in GBM cells, and the JNK inhibitor SP600125 blocked the JNK signaling to reverse partially the PS-341 growth inhibition. PS-341 (10(-7) M, 24 h) decreased nuclear NF-kappaB levels as shown by Western blot, and reduced transcriptional activity of NF-kappaB as measured by reporter assays in these transformed cells. Also, PS-341 enhanced TRAIL (TNF-related apoptosis-inducing ligand) and TNFalpha (tumor necrosis factor alpha) induced cell death and apoptosis (two- to five-fold) in GBM cells. In summary, PS-341 has profound effects on growth and apoptosis of GBM cells, suggesting that PS-341 may be an effective therapy for patients with gliomas.

Proteasome Inhibitor PS-341 Induces Apoptosis through Induction of Endoplasmic Reticulum Stress-Reactive Oxygen Species in Head and Neck Squamous Cell Carcinoma Cells

PS-341, also known as Velcade or Bortezomib, represents a new class of anticancer drugs which has been shown to potently inhibit the growth and/or progression of human cancers, including head and neck squamous cell carcinoma (HNSCC). Although it has been logically hypothesized that NF-kappaB is a major target of PS-341, the underlying mechanism by which PS-341 inhibits tumor cell growth is unclear. Here we found that PS-341 potently activated the caspase cascade and induced apoptosis in human HNSCC cell lines. Although PS-341 could inhibit NF-kappaB activation, the inhibition of NF-kappaB was not sufficient to initiate apoptosis in HNSCC cells. Using biochemical and microarray approaches, we found that proteasome inhibition by PS-341 induced endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in HNSCC cells. The inhibition of ROS significantly suppressed caspase activation and apoptosis induced by PS-341. Consistently, PS-341 could not induce the ER stress-ROS in PS-341-resistant HNSCC cells. Taken together, our results suggest that in addition to the abolishment of the prosurvival NF-kappaB, PS-341 might directly induce apoptosis by activating proapoptotic ER stress-ROS signaling cascades in HNSCC cells, providing novel insights into the PS-341-mediated antitumor activity.

Effects of the proteasome inhibitor PS-341 on tumor growth in HTLV-1 Tax transgenic mice and Tax tumor transplants

AbstractRecent studies have shown that the transcription factor nuclear factor κB (NF-κB) regulates critical survival pathways in a variety of cancers, including human T-cell leukemia/lymphotrophic virus 1 (HTLV-1)–transformed CD4 T cells. The activation of NF-κB is controlled by proteasome-mediated degradation of the inhibitor of nuclear factor κBα (IκBα). We investigated the effects of PS-341, a peptide boronate inhibitor of the proteasome in HTLV-1 Tax transgenic tumors in vitro and in vivo. In Tax transgenic mice, PS-341 administered thrice weekly inhibited tumor-associated NF-κB activity. Quantitation of proliferation, apoptosis, and interleukin 6 (IL-6) and IL-10 secretion by tumor cells in culture revealed that the effects of PS-341 on cell growth largely correlated with inhibition of pathways mediated by NF-κB. However, the effect of PS-341 on the growth of tumors in Tax transgenic mice revealed heterogeneity in drug responsiveness. The tumor tissues treated with PS-341 show no consistent inhibition of NFκB activation in vivo. Annexin V staining indicated that PS-341 response in vivo correlated with sensitivity to apoptosis induced by γ irradiation. On the other hand, transplanted Tax tumors in Rag-1 mice showed consistent inhibition of tumor growth and prolonged survival in response to the same drug regimen. TUNEL staining indicated that PS-341 treatment sensitizes Tax tumors to DNA fragmentation.

A phase II study of the proteasome inhibitor PS-341 in patients (pts) with metastatic breast cancer (MBC)

546 Background: The ubiquitin-proteasome pathway plays an essential role in eukaryotic cellular homeostasis by regulating the degradation of vital intracellular regulatory proteins that govern cell cycle, transcription factor activation and apoptosis. Blocking this proteolytic pathway leads to the elevation of cell cycle inhibitory proteins, including IΚB, whose stabilization results in NF-KB inhibition and ultimately induction of apoptosis. PS-341 (bortezomib, Velcade) is a dipeptide boronic acid derivative that acts as a potent and highly specific inhibitor of the proteasome. PS-341 has demonstrated anti-tumor activity as a single agent in preclinical breast cancer models. Methods: The primary objective of this study was to determine the objective tumor response in pts with MBC receiving PS-341. Eligibility criteria included normal organ/marrow function, measurable disease, performance status (PS) 0–2, absence of brain metastases and ≥ 1 prior chemotherapy regimen for MBC. PS-341 was administered IV, 1.5 mg/m2 on days 1, 4, 8, 11 q 21 days. Dose escalation to 1.7 mg/m2 could be considered in the absence of significant toxicities. Re-evaluation was scheduled q 3 cycles. Results: 12 pts were enrolled for stage-one interim analysis. All 12 pts were evaluable for response. Pt characteristics: median age- 47 years (range 35–64); PS 0/1/2: 6/5/1. Toxicity: grade 4- none; grade 3- pulmonary (2); grade 2- thrombocytopenia (1), diarrhea (1), nausea (3), vomiting (1), pulmonary (4), pain (2), depression (1) and fatigue (2). Response: 1 pt had dose escalation to 1.7 mg/m2 for 1 cycle. Only 3/12 pts completed 3 cycles of therapy (median cycles/pt = 1) and 8/12 pts had disease progression after ≤ 1 cycle of therapy. No objective responses were observed. All 12 patients initially progressed while on therapy and the median time to disease progression was 25 days (range 10–62). At the time of this report, 6 patients have died with a median time to death of 136 days. Conclusions: Single-agent PS-341 does not appear to exhibit significant clinical activity in pts with MBC. This two-stage study was terminated after the first stage due to the absence of any objective response. No significant financial relationships to disclose.

A phase II study of the proteasome inhibitor PS-341 in patients (pts) with metastatic breast cancer (MBC)

A phase II study of the proteasome inhibitor PS-341 in patients (pts) with metastatic breast cancer (MBC)