Induction of Cell Cycle Arrest and Apoptosis by the Proteasome

Vol. 10, 3207–3215, May 1, 2004 Clinical Cancer Research 3207

Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-␬B Mutations or Activation of the CD30, CD40, and RANK Receptors

Bei Zheng, Georgios V. Georgakis, Yang Li, Conclusions: The in vitro activity of PS-341 against Alok Bharti, David McConkey, HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD. Bharat B. Aggarwal, and Anas Younes Departments of Lymphoma/Myeloma, Bioimmunotherapy, and Cancer Biology, M. D. Anderson Cancer Center, Houston, Texas INTRODUCTION Nuclear factor ␬B (NF-␬B) plays a central role in regulat- ABSTRACT ing the expression of various genes involved in cell survival, apoptosis, carcinogenesis, and inflammation, making it a poten- Purpose: The malignant Hodgkin and Reed-Sternberg tial therapeutic target (1, 2). The NF-␬B family of proteins cells of Hodgkin disease (HD) are known to constitutively (including p50/p105, p52/p100, p65, RelB, and c-Rel) exists in express high levels of activated nuclear factor ␬B (NF-␬B), homodimers and heterodimers. In mammalian cells, the domi- which plays an important role in their survival. The protea- nant responsive form is the p50/p65 heterodimer. In unstimu- some inhibitor PS-341 has been recently shown to modulate lated cells, NF-␬B is present in the cytoplasm in an inactive tumor cell proliferation and survival by inhibiting NF-␬B form, bound to inhibitors of NF-␬B(I␬B␣,I␬B␤, and I␬B⑀). On and modulating critical cellular regulatory proteins, but its activation, I␬B is rapidly phosphorylated and ubiquinated and activity in cells carrying I␬B␣ gene mutations has not been subsequently degraded by the proteasome pathway. Conse- reported previously. quently, the active p50/p65 heterodimer is translocated to the Experimental Design: The activity of PS-341 in four nucleus and binds to a specific DNA sequence to induce gene well-characterized, HD-derived cell lines. Cell proliferation transcription (3). and apoptosis were determined by the 3-(4,5-dimethylthiazol- The malignant Hodgkin/Reed-Sternberg (H/RS) cells of 2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetra- Hodgkin disease (HD) aberrantly express the activated p50/p65 zolium (MTS) and Annexin-V binding methods, respectively. (RelA) heterodimer form of NF-␬B (4–7). This expression is Cell cycle analysis was determined by flow cytometry. Intra- observed in HD cell lines and the vast majority of primary H/RS cellular protein levels were determined by Western blot. cells in HD lymph node sections (7). Several mechanisms have Results: PS-341 demonstrated a strong antiproliferative been implicated in the aberrant activation of NF-␬B in H/RS activity, which was irrespective of the status of mutations in cells, including genetic defects in the I␬B␣ gene, Epstein-Barr I␬B␣ and even the presence of CD30, CD40, or RANK viral infection, and stimulation by cytokines (8–13). Inhibition receptor activation. This effect was attributable to the in- of NF-␬B activation by a dominant-negative I␬B␣ in HD- duction of apoptosis and cell cycle arrest at the G -M phase. 2 derived cell lines has been shown to interfere with cell cycle PS-341 not only inhibited nuclear localization of NF-␬B but progression and inhibit proliferation in vitro and in severe also activated the caspase cascade, increased p21 and Bax combined immunodeficient mice (7). Thus, the pharmacological levels, and decreased Bcl-2 levels. Furthermore, PS-341 en- inhibition of this pathway may prove to be of clinical value for hanced the effect of gemcitabine chemotherapy and poten- the treatment of patients with HD. tiated the effect of tumor necrosis factor-related apoptosis- PS-341 (Velcade, bortezomib) is a small molecule inhibitor inducing ligand/APO2L and two agonistic antibodies to of the ubiquitin-proteasome pathway. Proteasome plays an es- tumor necrosis factor-related apoptosis-inducing ligand sential role in the degradation of most intracellular proteins, death receptors R1 and R2. including those that regulate the cell cycle, cell survival and apoptosis, cell adhesion and trafficking, and transcription factor activation (14). By inhibiting the degradation of cytoplasmic inhibitor of nuclear factor-␬B(I␬B␣), PS-341 inhibits the activation of NF-␬B. Furthermore, PS-341 has been reported to Received 10/29/03; revised 1/20/04; accepted 1/28/04. The costs of publication of this article were defrayed in part by the alter the levels of p21, p27, Bcl-2, Bax, XIAP, survivin, and payment of page charges. This article must therefore be hereby marked p53, leading to cell cycle arrest and apoptosis in several tumor advertisement in accordance with 18 U.S.C. Section 1734 solely to types (15). PS-341 has also been shown to enhance tumor cell indicate this fact. sensitivity to chemotherapy (16–18). Because of these favorable Requests for reprints: Anas Younes, the Department of Lymphoma/ Myeloma, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, antitumor properties, PS-341 was rapidly developed to treat Houston, TX 77030. Phone: (713) 792-2860; Fax: (713) 794-5656; E- human cancer (15, 19–21). mail: [email protected]. In this study, we investigated the activity of PS-341 in

HD-derived cell lines and determined the molecular mecha- CO2, and light absorbance of formazan was measured at 495 nm nisms for its biological activity on a ␮Quant plate reader equipped with KC4 software (Biotek Instruments, Winooski, VT). Each measurement was made in triplicate, and the mean value was determined. MATERIALS AND METHODS Flow Cytometry. Cell cycle fractions and apoptosis Cell Lines and Cell Culture. The human HD-derived were determined by staining with propidium iodide (Sigma- cell lines HD-MYZ, HD-LM2, L-428, and KM-H2 were ob- Aldrich) and Annexin-V-Fluos (Roche Molecular Biochemi- tained from the German Collection of Microorganisms and Cell cals, Indianapolis, IN), as described previously (24–26). Data Cultures, Department of Human and Animal Cell Cultures were collected on a Becton Dickinson FACScan flow cytometer (Braunschweig, Germany). Two cell lines are known to have using CellQuest software (BD Biosciences, San Jose, CA) and mutations in the I␬B␣ gene (L-428 and KMH-2). The pheno- analyzed using WinMDI 2.8 software (Joseph Trotter, Scripps types and genotypes of these cell lines have been published Research Institute, La Jolla, CA). previously (22). All cell lines were cultured in RPMI 1640 Western Blot Analysis. Whole-cell protein extract was supplemented with 10% heat-inactivated fetal bovine serum prepared by incubating the cells in lysis buffer (Cell Signaling (Life Technologies, Inc., Gaithersburg, MD), L-glutamine, and Technology) for 30 min at 4°C and then centrifuging to remove penicillin/streptomycin in a humid environment of 5% CO2 at cellular debris. The protein in the resulting supernatant was 37°C. quantified by the bicinchoninic acid method (Pierce Chemical Reagents, Antibodies, and Recombinant Proteins. PS- Co., Rockford, IL) according to the manufacturer’s instructions, 341 was kindly provided by Millennium Pharmaceuticals, Inc. diluted 1:1 in protein SDS loading buffer (Cell Signaling Tech- (Cambridge, MA). Recombinant human RANK ligand (RANKL, nology), and boiled for 30 min. A total of 30 ␮g of protein was TRANCE), CD40 ligand (CD40L, CD154), and CD30 ligand loaded onto 10 or 15% Tris-HCl SDS-PAGE Ready Gels (Bio- (CD30L, CD153) and the pan-caspase inhibitor Z-VAD-FMK Rad, Hercules, CA), transferred to a nitrocellulose transfer were obtained from Alexis Corp. (San Diego, CA). Recombi- membrane (Pierce Chemical), and detected using SuperSignal nant human tumor necrosis factor-related apoptosis-inducing West Dura Extended Duration Substrate (Pierce Chemical). ligand (TRAIL/APO2L) trimer was kindly provided by Dr. Avi Immunocytochemistry for NF-␬B p65 Localization. Ashkenazi (Genentech Corp., South San Francisco, CA). Ago- Cellular localization of the p65 subunit was performed as de- nistic monoclonal antibodies to the TRAIL death receptors R1 scribed previously (27). Briefly, HD cells treated with PS-341 (ETR1) and R2 (ETR2) were provided by Human Genome were plated on a glass slide by centrifugation at 750 rpm using Sciences, Inc. (Rockville, MD). Antibodies to extracellular a Cytospin 4 (Thermoshandon, Pittsburgh, PA), air dried for 1 h signal-regulated kinase (ERK)1/2 and phosphorylated ERK1/2 at room temperature, and fixed with cold acetone. After brief (Thr202, Tyr204) and antibodies to procaspases 7, 8, 9, and 10 washing in PBS, slides were blocked with 5% normal goat to cleaved caspase 3 and polyadenosine-5Ј-diphosphate-ribose serum for 1 h and then incubated with rabbit polyclonal antihu- polymerase were all from Cell Signaling Technology (Beverly, man p65 antibody (dilution 1:100). After overnight incubation, MA). A phospho p53 antibody sampler kit containing antibodies the slides were washed and then incubated with goat antirabbit to total p53 and phospho-specific antibodies to Ser6, Ser9, IgG (Alexa Fluor 594;1:100) for 1 h and counterstained for Ser15, Ser20, Ser37, Ser46, and Ser392 was also obtained from nuclei with Hoechst (50 ng/ml) for 5 min. Stained slides were Cell Signaling Technology. Antibodies to Mcl-1, Bcl-x, Bcl-2, mounted with mounting medium (Sigma Chemical) and ana- ARF WAF Kip1 Bax, Bak, cIAP2, p14 , p21 , and p27 were obtained lyzed under an epifluorescence microscope (Labophot-2; Nikon, from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies to Tokyo, Japan). Pictures were captured using Photometrics Cool- cFLIP, cIAP1, survivin, NAIP, and BID were from R&D Sys- snap CF color camera (Nikon, Lewisville, TX) and MetaMorph tems (Minneapolis, MN); antibodies to XIAP were obtained version 4.6.5 software (Universal Imaging Corp., Downing- from Transduction Laboratories (San Diego, CA); antibodies to town, PA). ␤-actin were obtained from Sigma Chemical Co. (St. Louis, Electrophoretic Mobility Shift Assay. Electrophoretic MO); antihuman p65 antibody was obtained from Santa Cruz mobility shift assay was performed to determine the activation and Biotechnology. Three NF-␬B inhibitors were prepared: (a) res- nuclear translocation of NF-␬B as described previously but using veratrol (Sigma Chemical) was prepared as a 100-mM stock minor modifications (28). Briefly, 4 ␮g of nuclear protein extract solution in DMSO; (b) Pyrrolidine dithiocarbamate (PDTC) were incubated with 16 fmol of a 32P-labeled, 45-mer double- (Alexis) was prepared as a 25 mM stock solution in PBS; and (c) stranded DNA oligonucleotide derived from the human immuno- N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL) (Sigma Chem- deficiency virus long terminal repeat (5Ј-TTGTTACAAG- ical) was prepared as a 25 mM stock solution in DMSO GGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3Ј; In Vitro Proliferation Assay. Cells were cultured in italicized areas indicate NF-␬B-binding sites) for 30 min at 37°C. 6 6-, 12-, and 24-well plates at a concentration of 0.5 ϫ 10 The resulting complex was resolved from free oligonucleotide cells/ml for all cell lines and assays. Cell viability was assessed using electrophoresis on 6.6% native polyacrylamide gels. with a nonradioactive cell proliferation 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay using CellTiter 96 Aqueous One Solution RESULTS Reagent (Promega, Madison, WI) as described previously (23). PS-341 Inhibits HD Cell Proliferation in Vitro Irrespec- Briefly, 80 ␮l of cell suspension were added to 20 ␮lofthe tive of I␬B␣ Gene Mutation. It is well established that H/RS reagent and incubated in 96-well plates for1hat37°Cin5% cells constitutively express activated NF-␬B (4, 6). On the basis

Fig. 1 Activity of PS-341 in Hodgkin disease (HD) cell lines is independent of inhibitor of nuclear factor-␬␣ mutation. In A, four HD cell lines (HD-MYZ and HD-LM2 have I␬-B␣ mutation) were incubated with increasing doses of PS-341 for 48 h, and the viable cell number was determined using an MTS assay. The dose-response curve shows the IC50 to be between 2.5 and 5 nM for the HDL-M2 cell line and between 10 and 20 nM for the other three cell lines. In B, HD cell lines were incubated with 20 nM PS-341 for 24, 48, and 72 h. PS-341 had an antiproliferative effect in all four cell lines. Each value represents a mean of three independent experiments performed in triplicate. There was no significant difference in the activity of PS-341 in cell lines that had inhibitor of nuclear factor-␬B␣ mutation (HD-MYZ and HD-LM2) and those that did not. In C, PS-341 maintained its antiproliferative activity in HD cells in the presence of 1 ␮g/ml CD30L, CD40L, or RANKL. L-428 cells were incubated with one of these ligands with or without PS-341 for 48 h, and viable cell numbers were determined using an MTS assay. This is a representative of two independent experiments performed in triplicate. Similar results were observed in the other three cell lines.

of the antiproliferative activity of PS-341 in other NF-␬B- proliferation in a time-dependent manner, with significant anti- expressing lymphoid malignancies, including multiple myeloma proliferative activity being observed as early as 24 h after (16–18), we first investigated whether PS-341 had a similar treatment and lasting for Ն72 h (Fig. 1B). Thus, PS-341 had antiproliferative effect on HD-derived cell lines. Results were antiproliferative activity in HD-derived cell lines irrespective of compared between cell lines that are known to have mutations in I␬B␣ mutations. the I␬B␣ gene (L-428 and KMH-2) and cell lines that do not PS-341 Activity Is Maintained in the Presence of CD30, have such mutations (HD-MYZ and HD-LM2; Ref. 10). For CD40, and RANK Receptor Activation. We and others have these experiments, HD cells were incubated with increasing reported previously that H/RS cells express CD30, CD40, and concentrations of PS-341 (0–50 nM) for 48 h, and the viable cell RANK receptors, all of which can activate NF-␬B (28–30). number was determined using an MTS assay. As shown in Fig. Because H/RS cells are frequently exposed to the ligands of 1A, PS-341 had antiproliferative activity in all four HD cell lines these receptors in vivo, we examined whether PS-341 can main- in a dose-dependent manner. The most sensitive cell line was the tain its antiproliferative activity in HD cells in the presence of

HD-LM2 line, with an IC50 between 2.5 and 5 nM. The other activating CD30L, CD40L, and RANKL. For these experi- three cell lines had an IC50 ranging between 10 and 20 nM. Thus, ments, HD cells were incubated with one of these ligands (1 a PS-341 concentration of 20 nM was used, unless otherwise ␮g/ml) in 10 or 0.5% fetal bovine serum for 48 h in the presence specified. Using this concentration, PS-341 inhibited HD cell or absence of PS-341 (20 nM). As shown in Fig. 1C, PS-341

Fig. 2 Effect of PS-341 on cell cycle. In A, HD cells were incubated with increasing concentrations of PS-341 (2.5–50 nM) for 48 h before cell cycle fractions were determined using the propidium iodide staining method. In the HD-LM2 cells, PS-341 induced cell death

(sub-G0 fraction) without G2-M arrest. In the other three cell lines, cell cycle arrest at the

G2-M phase was followed by cell death. Percentages in the left top quadrant, dead cells; percentage in the right top

quadrant, cells in the G2-M phase. In B, HD cells were incubated for 24 and 48 h with medium or 20 nM PS-341. Cell cycle fraction was determined using the propidium iodide staining method. PS-341 induced accumulation of the cells

in the G2-M phase in the L-428 and KM-H2 cells as early as 24 h after treatment.

maintained its antiproliferative effect in the presence of these The ability of PS-341 to induce cell death and apoptosis in activating ligands. the HD cells was further confirmed by Annexin-V staining. For PS-341 Induces Cell Cycle Arrest and Apoptosis in these experiments, cells were incubated with either medium or HD-Derived Cell Lines Irrespective of Ik-B Gene Mutation. PS-341 (20 nM) for 48 h. Cells were then double stained with To determine whether the antiproliferative activity induced by propidium iodide and Annexin-V before they were analyzed PS-341 was caused by cell cycle arrest or induction of apoptosis, using two-color flow cytometry. As shown in Fig. 3, PS-341 we examined the effect of PS-341 on cell cycle fractions. For increased Annexin binding in all four HD-derived cell lines, these experiments, HD-derived cells were incubated with in- indicative of the induction of apoptosis. creasing concentrations of PS-341 (2.5–50 nM) for 48 h. The PS-341 Inhibits NF-␬B Activation in HD-Derived Cell ␬ percentages of cells in the G0-G1, S, and G2-M phases were Lines. PS-341 has been reported to inhibit NF- B activation determined by flow cytometric analysis of propidium iodide- in several tumor types. Whether PS-341 could also inhibit stained cells as described previously (31). In addition, the per- NF-␬B activation in HD cell lines with normal or mutated I␬B␣

centage of dead cells in the sub-G0 fraction was also calculated. had not been determined. For these experiments, we examined ␬ PS-341 induced cell cycle arrest at the G2-M phase in three cell the effect of PS-341 on the nuclear localization of the NF- B lines, reaching a maximum at 10 nM (Fig. 2A). When higher p65 subunit in HD cells. HD cells were incubated with medium PS-341 concentrations were used, cell death rather than cell or PS-341 (20 nM) for 2–8 h. Cells were then immunostained cycle arrest predominated. In contrast, PS-341 induced cell with p65-specific immunofluorescent antibodies as described in

death in the HD-LM2 cells without significant G2-M cell cycle “Materials and Methods.” Nuclei were counterstained with arrest (Fig. 2A). The ability of PS-341 to induce cell cycle arrest Hoechst staining. Results were compared between cell lines that ␬ ␣ at the G2-M phase was observed as early as 24 h after treatment have I B mutations and those that do not. As shown in Fig. 4, in two cell lines (L-428 and KM-H2) and became more prom- unstimulated L-428 cells (I␬B␣ mutations) and HD-MYZ cells inent after 48 h, after which the cells died (Fig. 2B). (no I␬B␣ mutations) constitutively expressed active NF-␬B

these experiments, HD-derived cell lines were incubated with medium or PS-341 (20 nM) for 6, 24, or 48 h. Whole-cell lysates were then examined using Western blotting. PS-341 cleaved caspases 8, 9, and 3 within 48 h, with subsequent cleavage of polyadenosine-5Ј-diphosphate-ribose polymerase (Fig. 5A). In contrast, PS-341 had no effect on caspase 7, 10, or 6 (data not shown). In one cell line (HD-LM2), PS-341 decreased cFLIP levels. In two cell lines (HD-MYZ and HD-LM2), BID protein was detected in unstimulated cells. In these two cell lines, PS-341 reduced BID levels, indicative of BID cleavage (Fig. 5A). To determine whether caspase activation was the dominant mechanism for the induction of apoptosis in these cell lines, we incubated HD cells with PS-341 (20 nM) with or without the pan-caspase inhibitor Z-VAD-FMK (20 ␮M). After 48 h in culture, PS-341 reduced the viable cell number in all four HD cell lines. This effect was either partially or completely reversed by Z-VAD-FMK (Fig. 5B). Thus, caspase activation appeared to be a dominant mechanism for PS-341 activity in HD cells. PS-341 has been reported previously to regulate the level of several proteins that control the cell cycle. Here, we found that PS-341 increased p21WAF levels in all four HD cells lines without a significant effect on p27Kip1 (Fig. 6A). In addition, PS-341 increased cyclin D2 levels in the L-428 cell line and cyclin D1 levels in the HD-MYZ and L-428 cells (Fig. 6A). Furthermore, PS-341 did not change the total cellular content of p53 but increased its phosphorylation status at the serine 15 site in one cell line (KM-H2) and decreased the MDM2 protein level in two cell lines (HD-LM2 and KM-H2; Fig. 6A). Fig. 3 Induction of apoptosis by PS-341. Hodgkin disease (HD) cells The effect of PS-341 on members of the Bcl-2 family was were incubated with medium or PS-341 (20 nM) for 48 h before the also examined (Fig. 6B). In the HD-LM2 cells, the Bax level percentage of dead cells was determined using dual staining with An- nexin-V and propidium iodide. PS-341 induced apoptosis in all four HD significantly increased within 24 h and remained elevated for cell lines to a variable degree. Left bottom quadrant, viable cells. Ն48 h. In contrast, the Bcl-2 level decreased in the L-428 cells Percentages of dead cells in each quadrant are shown. without significant changes in the Bax protein levels. PS-341 had no significant effect on Bcl-XL or Mcl-1 levels in any of the cell lines. Finally, we examined the effect of PS-341 on several with localization of the p65 subunit in the nuclei and cytoplasm. members of the IAP family (Fig. 6C). All cell lines expressed When the L-428 and HD-MYZ cells were incubated with PS- detectable levels of XIAP, c-IAP1, and c-IAP2. PS-341 slightly 341 (20 nM), the active p65 subunit remained in the cytoplasm decreased c-IAP1 in one cell line (L-428) but had no significant without translocating into the nuclei, indicative of NF-␬B inac- effect on the level of other IAP family members in any of the tivation. This effect was observed as early as 2 h after treatment cell lines. and was maintained for Ն8 h. Thus, PS-341 inhibited NF-␬B PS-341 Enhances the Activity of Chemotherapy, activation even in cells that harbored I␬B␣ mutations. TRAIL/APO2L, and Agonistic Antibodies to TRAIL Death The constitutive expression of NF-␬B was inhibited in the Receptors R1 and R2. PS-341 has been reported to have HDLM-2 cell line by the antioxident PDTC (50 ␮M) but not by synergy with chemotherapy in several tumor models. To explore the proteasome inhibitor LLnL (6.25 ␮M) or antioxident res- whether a similar synergistic effect could be achieved in HD veratrol (50 ␮M; Fig. 4C). Although PDTC inhibited NF-␬B, it cells, we incubated HD cells with submaximal concentrations of had only a modest killing effect on HDLM-2 cells (20% cell PS-341 (5 nM), with or without submaximal concentrations of death within 48 h; Fig. 4C). In contrast, PS-341 was very gemcitabine (0.05 ␮M), for 72 h. The viable cells were counted effective in killing HD-LM2 cells (Fig. 4C). As expected, nei- using the MTS assay. A synergistic effect was observed only in ther resveratrol nor LLnL, which did not inhibit NF-␬B, was the HD-LM2 cells (Fig. 7A), whereas an additive effect was effective (data not shown). Collectively, these data suggest that, observed in the remaining cell lines (data not shown). under normal culture conditions, inhibition of NF-␬B activation We have reported previously that HD cell lines express the is not the predominant mechanism of PS341-induced cell death. TRAIL receptors R1 and R2 and that TRAIL/APO2L is mod- PS-341 Activates the Caspase Cascade and Alters the estly effective in inducing apoptosis in the cells (31). The Levels of Cell Cycle Regulatory Proteins. On the basis of activity of two agonistic antibodies to TRAIL receptors R1 the ability of PS-341 to induce apoptosis of HD cells, we (ETR1) and R2 (ETR2) has not been studied previously in HD investigated whether this process was caspase dependent. For cell lines. At equal concentrations of TRAIL/APO2L, ETR1,

Fig. 4 Effect of PS-341 on nuclear localization of nuclear factor ␬-B (NF-␬B) subunit p65. In A, L-428 cells, which are known to carry inhibitor of nuclear factor- ␬B␣ mutations, were incubated with 20 nM PS-341 for 2–8 h. P65 was visualized under a fluorescence microscope using Al- exa-Fluor 594 secondary antibody (top panel). The figure shows that PS-341 inhibited nuclear localization of p65 in L-428 cells within2hofincubation. Hoechst staining (bottom panel) shows that the nuclei were intact. In B, PS-341 shows a similar effect on the HD-MYZ cells, which carry wild-type I␬B␣.InC, inhibition of NF-␬B activation in the HD- LM2 cells was achieved by the antioxident PDTC but not resveratrol or LLnL (left panel). NF-␬B activity was determined after 48 h of incubation using electrophoretic mobility shift analysis as described in “Materials and Methods.” However, PDTC remained relatively ineffective compared with PS-341 (right panel).

and ETR2 (0.1 ␮g/ml), 40–50% of the HD-LM2 cells were resulting in a truncated, nonfunctional I␬B␣ protein (10). Thus, killed within 24–48 h. When HD-LM2 cells were incubated it would seem that proteasome inhibition might not be effective with a minimally toxic concentration of PS-341 (5 nM) with 0.1 in these cell lines. However, we found that I␬B␣ mutation had ␮g/ml TRAIL/APO2L, ETR1, or ETR2, a synergistic killing no effect on PS-341 activity, which suggests that I␬B␤ or other effect was observed within 24–48 h (Fig. 7B). This synergistic unknown inhibitors can substitute for the I␬B␣ protein. Further- effect was less prominent in the remaining HD cell lines (data more, some PS-341 activities may be mediated by NF-␬B- not shown). independent mechanisms. In any case, the presence of I␬B␣ mutations was therapeutically irrelevant. DISCUSSION An additional concern was whether PS-341 could maintain This is the first study demonstrating the activity of PS-341 its activity in the presence of activating cytokines (13). It is well in cell lines known to have I␬B␣ gene mutation. In this study, established that malignant H/RS cells express several receptors we found that PS-341 was effective as an antiproliferative agent that belong to the tumor necrosis factor family, including CD30, in all four HD-derived cell lines that were tested. Two of these CD40, and RANK (28, 32, 33). Activating these receptors by cell lines are known to have mutations in the I␬B␣ gene, their ligands activates several shared signaling pathways, in-

Fig. 5 PS-341-induced apoptosis in Hodgkin disease (HD) cells is caspase mediated. In A, HD cells were incubated with medium or 20 nM PS-341 for 6–48 h. Whole-cell lysates were examined by Western blot for changes in intracellular proteins. PS-341 cleaved caspases 8, 9, and 3 and poly(ADP-ribose) polymerase (PARP). Furthermore, PS-341 down-regulated c-FLIP in the HDL-M2 cells. In two cell lines that expressed BID (HD-MyZ and HD-LM2), PS-341 also decreased BID levels, suggesting its cleavage. In B, PS-341-induced cell death of HD cells was either partially or completely blocked by the pan-caspase inhibitor Z-VAD-FMK. HD cells were incubated with medium, Z-VAD-FMK (20 ␮M), PS-341 (20 nM), or a combination of PS-341 and Z-VAD-FMK. After 48 h in culture, the viable cells were counted using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H- tetrazolium (MTS) assay. Each value represents a mean of three independent experiments done in triplicate.

cluding ERK and NF-␬B (4, 31). We found that although PS-341 alters protein levels downstream from p-ERK that may PS-341 did not alter ERK levels or phosphorylation status, it be crucial for its function. maintained its activity in the presence of these ligands, suggest- In this study, PS-341 induced apoptosis and cell cycle ing that PS-341 would be active in vivo. This also suggests that arrest at the G2-M phase (16, 17, 34–36). Apoptosis was me-

Fig. 6 Effect of PS-341 on Bcl-2 family, IAPs, and cell cycle proteins. In A, PS-341 up- regulated p21 and increased p53 phosphorylation (serine 15) without inducing changes in total p53. Cyclins D2 and D1 also were each up-regulated in two cell lines. In B, PS-341 up- regulated Bax in the HD-LM2 cells and down-regulated Bcl-2 in the L-428 cells. PS-341 had little or no effect on Bcl-x or Mcl-1. In C, PS-341 had little or no effect on the IAP family.

increased in two cell lines (HD-MYZ and L-428), and the cyclin D2 level increased in one (L-428). PS-341 had no significant effect on total cellular content of wild-type p53, but it increased the phosphorylation level of p53 at serine 15 in one cell line (KM-H2). A similar observation was reported recently in multiple myeloma cells. In this study, we found that all HD lines produced members of the IAP family of proteins (XIAP, c-IAP1, and c-IAP2). PS-341 treatment either had no effect or only slightly decreased the level of one or more proteins in these cells, e.g., PS-341 decreased c-IAP1 in the L-428 cells. Interestingly, PS-341 reduced the XIAP level in the HD-LM2 cells within 6 h after treatment but lost this effect with extended incubation time. Using a submaximal concentration of gemcitabine or doxo- rubicin in combination with a submaximal concentration of PS-341 (5 nM), we found that PS-341 enhanced the activity of gemcitabine only in the HD-LM2 cells. However, this enhance- ment was modest. Similarly, PS-341 enhanced the activity of TRAIL/APO2L only in the HD-LM2 cells but had no significant effect in the other three cell lines. The ability of PS-341 to enhance TRAIL/APO2L and agonistic TRAIL death receptor antibodies in the HD-LM2 cells may have been related to its ability to down-regulate cFLIP levels (37). Fig. 7 PS-341 enhances the effect of chemotherapy and TRAIL in Collectively, these data show that PS-341 has significant Hodgkin disease cell lines. In A, Hodgkin disease-LM2 cells were antiproliferative activity against HD-derived cell lines in vitro, incubated with medium, PS-341 (5 nM), gemcitabine (0.01 nM), or a irrespective of I␬B␣ gene mutation and in the presence of combination of PS-341 and gemcitabine for 24–72 h. The viable cells activating cytokines. Evaluating PS-341 activity in vivo in pa- were counted using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay. 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Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Correction Research Correction: Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-kB Mutations or Activation of the CD30, CD40, and RANK Receptors

In this article (Clin Cancer Res 2004;10:3207–15), which was published in the May 1, 2004, issue of Clinical Cancer Research (1), Figure 5A contained duplicate panels for cleaved caspase-3 and cleaved PARP in the L-428 cells. Figure 6A contained duplicate panels for phospho-p53 and total p53 in the HD-Myz and HD-LM2 cells. These errors occurred during the assembly of the figures and have no bearing on the results or conclusions of the study. The corrected figures are shown below; the figure legends remain the same. Because this is an old study, some of the original blots could not be retrieved to generate complete, corrected figures. The authors regret these errors.

L-428 − + PS341

0 h 6 h 24 h 48 h 6 h 24 h 48 h Time (hrs)

Caspase 3

Cleaved PARP

Figure 5A.

HD-MYZ HD-LM2 L-428 KM-H2

− ++++− −−PS-341

0 6 24 48 6 24 48 0 6 24 48 6 24 48 0 6 24 48 6 24 48 0 6 24 48 6 24 48 Time (Hrs)

Phospho-p53 (ser15) Total p53

Figure 6A.

Reference 1. Zheng B, Georgakis GV, Li Y, Bharti A, McConkey D, Aggarwal BB, et al. Induction of cell cycle arrest and apoptosis by the proteasome inhibitor PS-341 in Hodgkin disease cell lines is independent of inhibitor of nuclear factor-kB mutations or activation of the CD30, CD40, and RANK receptors. Clin Cancer Res 2004;10:3207–15.

Published online May 30, 2014. doi: 10.1158/1078-0432.CCR-14-1017 2014 American Association for Cancer Research.

3044 Clin Cancer Res; 20(11) June 1, 2014 Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-κB Mutations or Activation of the CD30, CD40, and RANK Receptors

Bei Zheng, Georgios V. Georgakis, Yang Li, et al.

Clin Cancer Res 2004;10:3207-3215.

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