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Experimental Therapeutics, Preclinical Pharmacology |
Biological Sciences Program, Cancer Biology Research, Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, M4N 3M5 Canada [A. F., S. M., R. S. K.]; Toronto-Sunnybrook Regional Cancer Centre [R. S. K.], Department of Medical Biophysics [R. S. K.], and Laboratory Medicine and Pathobiology [A. F., R. S. K.], University of Toronto, Toronto, Ontario, Canada; and Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139 [P. E., J. A.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By growing cells in culture as multicellular tumor spheroids, it is possible to mimic some of the in vivo microenvironmental and behavioral characteristics of solid tumors, including hypoxia and intrinsic resistance to chemotherapeutic agents (4, 5, 6) . For example, significant reductions in the proportion of proliferating tumor cells can result in spheroids with values that are more akin to the cell cycle kinetics of most solid tumors in vivo (4, 5, 6) . Because the majority of conventional cytotoxic anticancer drugs preferentially kill cycling cells, this feature of solid tumors can severely limit the efficacy of such drugs (7 , 8) . Moreover, some of these drugs may also have a diminished capability to diffuse into the inner layers of multicell spheroids or the tumor parenchyma of a solid tumor in vivo (9) . Limited penetration can also be a factor in reducing the effectiveness of various immunotherapeutic strategies in the treatment of multicellular spheroids and solid tumors in vivo (10, 11, 12) . Furthermore, hypoxia can also result in spheroids expressing resistance to ionizing radiation (4 , 5) .
We have been using tumor spheroids as a means of evaluating the properties and relative potential of new anticancer drugs in the treatment of solid tumors. The rationale is that if a drug demonstrates antitumor activity in a spheroid model or screen comparable to or greater than that seen in a conventional monolayer system, it could indicate more promising efficacy for the treatment of solid tumors in vivo. In other words, the results of the spheroid assay may be of greater predictive value than those of conventional monolayer cell cultures, at least as far as the response of solid tumors is concerned.
With these considerations in mind, we decided to evaluate the relative
effects of a new class of anticancer drug, proteasome inhibitors, in
monolayer versus spheroid cultures. The proteasome
represents the cell’s major nonlysosomal tool to degrade or process
proteins by ATP/ubiquitin-dependent proteolysis (13, 14, 15)
.
In higher eukaryotic cells, the proteasome is involved in the
degradation of most of the cytosolic proteins, in particular, the
short-lived ones, many of which are critical for cell proliferation and
cell cycle regulation, as well as apoptosis. Examples include the tumor
suppressor protein p53 (16)
, various cyclins
(17)
, and the cyclin-dependent kinase inhibitor
p27Kip1 (18
, 19)
. The ordered and
temporal degradation of these numerous key proteins is required for
cell cycle progression and mitosis; thus, the ubiquitin-proteasome
pathway plays a significant role in neoplastic growth, survival, and
metastasis. The proteasome is also required for the activation of
NF-
B3
by
degradation of its inhibitory protein IB. NF-B is required to
maintain cell viability through the transcription of inhibitors of
apoptosis in response to environmental stress or cytotoxic agents.
Stabilization of the IB protein and blockade of NF-B activity
have been demonstrated to render cells more sensitive to apoptosis
(19, 20, 21, 22)
.
Recently, a unique series of proteasome inhibitors that are potent, selective, and reversible in their action have been described by Adams et al. (23 , 24) . These compounds are dipeptide boronic acid analogues (23) that inhibit the chymotryptic activity of the proteasome and, as a result, suppress or block activity of the enzyme (23 , 24) . One such drug, PS-341, has been found to have an excellent cytotoxic profile and activity when tested on the National Cancer Institute screen or panel of 60 different tumor cell lines, all grown as monolayer cell cultures (24) . However, PS-341 has not yet been evaluated in a tumor spheroid context. The results of such studies could be informative with respect to the use and mechanisms of proteasome inhibitors as anticancer agents for solid tumors. To this end, we report here a series of experiments designed to evaluate the cytotoxic (proapoptotic) activity of PS-341 using a panel of seven different human carcinomas (four ovarian and three prostate carcinomas). The results indicate PS-341 has activity against spheroids that is comparable or even superior to that observed in monolayer cell culture systems.
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MATERIALS AND METHODS |
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Multicellular spheroids of the various cell lines were prepared by the liquid overlay technique, as described previously (25) . In brief, 24-well or 96-well tissue culture plates (Nunc) were coated with 0.25 ml or 60 µl, respectively, of 1% Seaplaque-agarose (FMC, Bioproducts, Rockland, ME) prepared from a 4% stock solution. For the MTS and [3 H]thymidine incorporation assays with the prostate cell lines, an alternate protocol for spheroid culture was used, i.e., 2% poly(2-hydroxyethylmethacrylate) or "polyhema" (Sigma-Aldrich) was used instead to coat U-bottomed 96-well polystyrene multiwell plates (Nunc) as described previously (26) . One ml of 105 cells or 100 µl of 104 cells were added to to each well of the 24-well or 96-well plates, respectively. To allow the cells to come in close contact with one another, the 24-well plates were placed on an orbital shaker and rotated at 250 rpm for 5 min, and the 96-well plates were centrifuged at 1000 rpm for 5 min.
Monolayer culture conditions for the MTS and [3 H]thymidine assays were the same as those for spheroid cultures, except that uncoated 96-well tissue culture plates were used, and the plates were not rotated or centrifuged. For all other assays, cells were grown in 100-mm-diameter tissue culture dishes (Nunc) and harvested when approximately 75% confluent.
MTS Staining.
Cell viability was assessed essentially as described previously
(27)
. This assay is composed of two solutions:
(a) a tetrazolium compound (MTS); and (b) an
electron-coupling reagent (PMS). MTS was purchased from Promega and
Fisher Scientific, and PMS was purchased from Sigma-Aldrich. Briefly,
cells were cultured in 100 µl of media in 96-well plates for 48 h, and then drugs/compounds were added and incubated for certain time
intervals, as indicated. After incubation, 20 µl of the MTS/PMS
solution were added to each well and incubated at 37°C for 6 h.
MTS is bioreduced by cells into a formazan that is soluble in tissue
culture medium. The relative cell viability was obtained by measuring
the absorbance of formazan at 490 nm on the Bio-Rad 3550 microplate
reader.
[3
H]Thymidine Incorporation.
The conditions for this assay were essentially the same as those for
the MTS assay, except that the cells were pulsed with 50 µl of
[3
H]thymidine (Amersham Pharmacia) at 40
µCi/ml for 6 h at 37°C. Proliferating cells incorporated the
[3
H]thymidine into their DNA, which was
harvested and collected onto a filtermat using the Titertek Cell
Harvester and counted on the Wallac 1205 BetaPlate scintillation
counter. Proliferation is expressed as a percentage of the maximum
incorporation by the control.
S-phase Analysis by BrdUrd Incorporation.
The procedure used in this assay has been described previously
(28)
. Cells grown in monolayer or spheroid culture were
pulsed with 10 µM BrdUrd (Sigma-Aldrich) for 3 h.
Cells were then rinsed with PBS, trypsinized, rinsed again, and fixed
with cold 70% ethanol for at least 1 h on ice. Cells were washed
in PBS, resuspended in 1 ml of cold 0.1 N HCl/0.7% Triton
X-100, and left on ice for 10 min. The cells were then washed with PBS,
resuspended in 0.5 ml of distilled water, and transferred to a 0.5-ml
microcentrifuge tube already containing 16 µl of 0.1 N
HCl. The tube was then heated in a PCR machine for 8 min at 95°C and
placed immediately on ice. Cells were transferred to a 5-ml tube,
rinsed twice with HNFN buffer containing 0.5% Tween 20, and rinsed
once with HNFN alone. Pellets were then resuspended in a 1:40 dilution
of FITC-conjugated anti-BrdUrd antibody (Cedarlane Laboratories) and
incubated on ice in the dark for 45 min. Stained cells were rinsed
twice with HNFN/Tween 20, rinsed once with HNFN, pelleted, resuspended
in 1 ml of PI solution (50 µg/ml PI and 10 µg/ml RNase A in PBS),
and incubated for 1 h at 4°C in the dark before analyzing on a
Becton Dickinson FAScan flow cytometer.
Flow Cytometry for Detection of Apoptosis.
The proteasome inhibitor PS-341 was synthesized by Millennium
Pharmaceuticals, Inc. (23
, 24)
. Briefly, cells grown in
monolayer culture or in spheroid culture were incubated with varying
concentrations of PS-341 for 48 h. Cells were collected, fixed
with 1% paraformaldehyde for 15 min, and then fixed with 70% ethanol
for 1 h. The Boehringer Mannheim in situ cell death
detection kit was then used to study apoptosis. The assay is based on
terminal deoxynucleotide transferase labeling of DNA strand breaks by
the addition of fluorescein dUTP to free 3'-OH DNA ends. The cells were
then analyzed by flow cytometry for detection of shifts in fluorescence
that indicated increased DNA fragmentation.
Western Blot Analysis.
Cells grown in monolayer or spheroid culture were incubated with 5
µM PS-341 and harvested at various time points. Cells
were then lysed with lysis buffer supplemented with protease inhibitors
(1 mM sodium orthovanadate and 2 mM
phenylmethylsulfonyl fluoride). The lysate was then centrifuged at
14,000 rpm for 15 min, and postnuclear supernatant was harvested and
sampled for quantitation of protein concentration, using the Bradford
dye. The lysate (30 µg) was then mixed with 5x SDS-PAGE sample
buffer, boiled for 5 min, and subjected to electrophoresis in 12% SDS
gels under reducing conditions. The separated proteins were then
electrophoretically transferred to Immobilon-P membranes (Millipore,
Bedford, MA). After blocking by TBS-T (5% nonfat dried milk in
Tris-buffered saline with 0.125% Tween 20) for 1 h at room
temperature, the membranes were blotted with an anti-p21 antibody
(Santa Cruz Biotechnology) at a concentration of 0.1 µg/ml, an
anti-Bcl-x antibody (Transduction Laboratories) at a concentration of
0.25 µg/ml, an anti-Bax antibody (Santa Cruz Biotechnology) at a
concentration of 0.25 µg/ml, and an anti-XIAP antibody
(Apoptogen, Inc.) at a 1:2000 dilution. After washing in TBS-T, the
membrane was incubated with antirabbit immunoglobulin-horseradish
peroxidase (1:1000 dilution) for 1 h. Protein bands were detected
by the enhanced chemiluminescence detection system (Amersham Pharmacia
Biotech).
Cell Death Detection ELISA.
The kit for this assay was purchased from Roche Molecular Biochemicals.
This is a photometric enzyme-immunoassay for the qualitative and
quantitative determination of cytoplasmic histone-associated DNA
fragments (mono- and oligonucleosomes) after induced cell death.
Briefly, SKOV3 cells were plated in either monolayer or spheroid
cultures at 1 x 105 cells/well in a 24-well
dish (Nunc). Forty-eight h later, cells were pretreated overnight with
100 µM Z-VAD (Calbiochem), a caspase inhibitor,
and then treated with 5 µM PS-341 in combination with 100
µM Z-VAD for an additional 24 h. Duplicate samples
were then collected from the dishes, lysed, and placed into a
streptavidin-coated 96-well plate. Subsequently, a mixture of
anti-histone-biotin and anti-DNA-peroxidase-conjugated antibodies were
added and incubated for 2 h, both of which bind to histone-DNA
complexes in the nucleosomes. After removal of unbound
antibodies by a washing step, the amount of nucleosomes was quantified
by the peroxidase retained in the immunocomplex. Peroxidase was
determined photometrically with ABTS
(2,2'-azino-di[3-ethylbenzthiazolin-sulfonat] as substrate.
The absorbance reading at 405 nm was corrected to a negative
control and expressed as an enrichment factor (or fold increase in DNA
fragmentation), according to the manufacturer’s instructions.
Drug Sensitivity.
The sensitivity of DU-145, PC3, and LNCaP cells to
4-hydroperoxycyclophosphamide (a metabolically active form of
cyclosphamide; Nova Pharmaceutical Corp.), Taxol (paclitaxel;
Bristol-Myers Squibb), CDDP (Sigma-Aldrich), Adriamycin
(doxorubicin hydrochloride; Pharmacia, Inc.), or PS-341 was assessed by
the MTS and/or [3
H]thymidine assay. The cells
were plated at 104 cells/well in 100 µl of
complete medium and incubated for 48 h, after which 100 µl of
the drugs at various concentrations were added, and the plates were
incubated for an additional 12 h before pulsing with MTS or
[3
H]thymidine as described above.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows OVCA 429 cells grown
as a multicellular tumor spheroid without the addition of PS-341,
whereas Fig. 1B
shows the effect of 300
nM PS-341 on a previously intact spheroid. A
significant disruption of cellular adhesion in the treated cells was
observed. Similar effects were observed with three other human ovarian
cancer cell lines, namely SKOV3, HEY, and A2780 (data not shown).
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demonstrates the effect of
PS-341 on the viability and tritiated thymidine incorporation of OVCA
429 and SKOV3 ovarian carcinoma cell lines plated in both monolayer and
spheroid culture treated over a range of concentrations for 48 h.
The viability curves are superimposable when comparing the effects of
PS-341 on monolayer versus spheroid cultures in the OVCA 429
cell line (Fig. 2A)
. Comparable with the OVCA 429 cell line,
the viability curves of the spheroid and monolayer cultures of the
SKOV3 cells treated with PS-341 are similar at low nanomolar
concentrations of the drug, i.e., at 5
nM PS-341, 88.2% of the spheroid and 86.8% of
the monolayer cultured cells remain viable. However, at concentrations
from 300 nM to 5 µM
PS-341, the cell viability of the monolayer cultured cells is actually
greater than the viability of the cells grown as multicellular tumor
spheroids, i.e., at 300 nM PS-341,
75.8% of the cells remain viable in the monolayer cultures compared
with 63.8% of the spheroid cultured cells.
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To confirm the MTS results and rule out a possible problem with
penetration of the MTS dye into the interior of the spheroid, tritiated
thymidine incorporation was measured in both the spheroid and monolayer
cultures after PS-341 treatment. As can be observed in Fig. 2, C and D
, there are no significant differences in
cell proliferation (as measured by the tritiated thymidine uptake) in
the two cell culture systems in either the OVCA 429 cells or the SKOV3
cells.
PS-341 Induces a Comparable Level of Apoptosis in Both Culture
Systems.
SKOV3 cells were plated in either monolayer or spheroid cultures and
exposed 48 h later to varying concentrations of PS-341 for 48 h. In the monolayer culture (Fig. 3A)
, treatment with 5
nM PS-341 induced a shift to the right in the
graph, indicative of apoptosis. At this low concentration, no induction
of apoptosis was observed in the spheroid culture (Fig. 3B)
.
However, at 50, 250, and 500 nM concentrations of
the drug, similar levels of apoptosis were observed in both cultures.
At 50 nM PS-341, 40.42% of the monolayer
cultured cells had undergone apoptosis compared with 43.47% of the
spheroid cultured cells. There were comparable increases in the
percentage of apoptotic cells with increasing concentrations of PS-341
in both culture systems. Similar results were observed with the HEY and
OVCA 429 cell lines (data not shown). To further validate the induction
of apoptosis by PS-341 in ovarian carcinoma cells, treatment with the
compound was able to cleave PARP into its
Mr 89,000 fragment, which is
known to serve as an early specific marker of apoptosis (Fig. 3C)
.
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that were comparable
in monolayer and spheroid cultures. After 6 h in both culture
systems, the levels began to increase significantly, and by 12 h,
there was a strong up-regulation of the protein. The pattern of
up-regulation in spheroid culture mirrors that of the monolayer
culture. The upper band of the bcl-XL protein,
which represents the hyperphosphorylated form of the protein, becomes
more significant after 12 h of treatment with PS-341 in the
monolayer culture system and as early as 6 h in the spheroid
culture, implying that this antiapoptotic protein becomes inactivated
after treatment with the proteasome inhibitor. The levels of the
apoptotic protein, bax, did not change, even after a 48 h
treatment with PS-341. The level of XIAP, a member of the inhibitor of
apoptosis (IAP) family of antiapoptotic proteins, was down-regulated
24 h after PS-341 treatment in both culture systems, and after
48 h, the protein was barely detectable.
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. Cells were treated with 100
µM Z-VAD and treated 24 h later with 5
µM PS-341 in combination with Z-VAD to ensure
the irreversibility of the caspase inhibitor. The increase in
absorbance was indicative of an increase in oligonucleosomal-sized
fragments of DNA in the cytoplasm, which is a hallmark of DNA
fragmentation. This was measured by a cell death detection ELISA.
Z-VAD, on its own, had a protective effect over the cells. There was a
3.4-fold decrease in the level of DNA fragmentation in the monolayer
culture and a 2.2-fold decrease in the spheroid culture. PS-341, on its
own, induced DNA fragmentation in both culture systems. In the
monolayer culture, there was a 2.9-fold increase in DNA fragmentation
as compared with the control monolayer cultured cells after PS-341
treatment and a 5.1-fold induction of DNA fragmentation in the spheroid
culture. Pretreatment with Z-VAD resulted in complete protection of the
cells, indicating that PS-341 does not induce DNA fragmentation in the
apparent absence of caspase activation.
Effects of PS-341 on Human Prostate Carcinoma Cell Lines.
We attempted to grow the PC3, LNCaP, and DU-145 human prostate cancer
cell lines as multicellular spheroids. Only DU-145 cells were competent
to form a highly compact or cohesive spheroid, as shown in Fig. 5A
. When S-phase analysis was
undertaken with DU-145 cells grown in subconfluent or confluent
monolayer cell culture, 34.34% and 39.26%, respectively, were found
to be BrdUrd positive (Fig. 5B)
. This may help explain the
results presented in Fig. 6
, which show
the relative effects on thymidine incorporation of Taxol, CDDP,
4-hydroperoxycyclophosphamide, or Adriamycin on monolayer cultures
(high growth fraction) versus spheroids (low growth
fractions) of DU-145 cells. Monolayer cell cultures were more sensitive
to these conventional cytotoxic drugs, which are known in general to
target rapidly dividing cells. These results may help resolve the
paradox of Teicher et al. (30)
, who showed that
solid tumors derived from the injection of human prostate cancer cell
lines into nude mice tend to be resistant to chemotherapy, whereas the
same cell lines grown as monolayer cell cultures were found to be
relatively drug sensitive (30)
. The solid tumors in
vivo would typically have much lower growth fractions than
monolayer cell cultures. Spheroids of DU-145 cells, on the other hand,
appear to mimic the low growth fractions of metastatic human prostate
cancer (8)
. In light of these results, it is of interest
to note the different activity profile that is obtained when PS-341 is
used to treat prostate cancer spheroids and monolayer cell cultures. As
shown in Fig. 7
, PS-341 was actually more
effective at the higher doses against DU-145 spheroids than against the
corresponding monolayer cell cultures.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results indicate that unlike the majority of anticancer chemotherapeutic drugs, the proteasome inhibitor PS-341 appears to be as effective in killing tumor cells exposed in the form of dense multicellular spheroids as in killing those in subconfluent monolayer cell cultures. Of particular interest was the observation that in DU-145 prostate cancer cells in which the S-phase growth fraction (approximately 2.5%) was more than 1 order of magnitude lower in spheroids than in monolayer cell cultures of the same tumor, the drug was actually more effective against spheroids. This suggests that the activity of PS-341 may not be compromised by the relatively low growth fractions characteristic of many types of solid tumor such as prostate cancer, a common feature of many solid tumors that is known to limit the initial effectiveness of many conventional cell cycle-dependent chemotherapeutic agents (4 , 5 , 7) . This feature of PS-341, along with its impressive broad spectrum antitumor activity and its steep dose-response effects, as assessed in the National Cancer Institute 60-cell line screen (24) , may bode well for PS-341 and possibly for other proteasome inhibitors as anticancer drugs for the treatment of solid tumors, provided that such drugs have a good therapeutic index, i.e., the toxic effects to normal tissues of such drugs are not too severe. This is currently being tested in Phase I clinical trials.
Our results suggest that PS-341 treatment induces tumor cell apoptosis
and is able to do so equally well in tumor spheroids and monolayer cell
cultures. For example, according to the flow cytometry results
measuring apoptosis, there are comparable increases in the percentage
of apoptotic cells with increasing concentrations of PS-341. To further
implicate this apparent induction of apoptosis, treatment with PS-341
gave a similar pattern of PARP cleavage in both culture systems. In
addition, phosphorylation of the Bcl-xL protein
is observed in the cells grown as spheroids as well as monolayers,
suggesting that this antiapoptotic protein may become similarly
inactivated in both culture systems after treatment with PS-341.
Furthermore, the level of the antiapoptotic protein XIAP was found to
be down-regulated in both culture systems after treatment with PS-341.
It is of considerable interest to note that XIAP represents one of the
NF-B-regulated genes (37)
. Thus, proteasome inhibition
resulting in the stabilization of IB, NF-B’s inhibitory
"partner" protein, suggests that the PS-341-induced down-regulation
of XIAP may be due to inhibition of NF-B activation. However,
additional experiments to ascertain the direct involvement of NF-B
are clearly required. Finally, we also observed that pretreatment with
the caspase inhibitor Z-VAD protects both the spheroid and monolayer
cultures, implying that caspase activation may be critical for
PS-341-induced apoptosis. These results do not indicate whether the
particular regulators of apoptosis we examined are actually directly
involved in mediating the cytotoxic (proapoptotic) effects of PS-341
treatment that we have observed.
These results may seem in contrast to several reports in the literature suggesting that proteasome inhibitors are cytotoxic to proliferating cells (38, 39, 40) . However, in addition to cell cycle proteins, the ubiquitin-proteasome system degrades a number of cell survival proteins, such as Bcl-2 (41) and other members of the Bcl-2 family of apoptotic and antiapoptotic proteins including Bid (42) and Bax (43) . Other proteins degraded by this system include STAT3 (44) , topoisomerase II (45) , and MHC-1 (46) restricted antigens. Moreover, Teicher et al. (47) and Adams et al. (24) tested the in vivo effects of PS-341 in the EMT-6 mouse mammary carcinoma and PC-3 human prostate carcinoma systems, respectively. Teicher et al. (47) demonstrated a 90% tumor cell kill by i.p. injection of a single dose of PS-341 in EMT-6 tumors growing s.c. Similarly, Adams et al. (24) reported that direct injection of PS-341 into the tumor growing s.c. resulted in a 70% decrease in tumor volume. The proportion of proliferating cells in a spheroid is highly representative of the cell cycle kinetics of most solid tumors in vivo (4, 5, 6) . Therefore, these in vivo therapy studies would appear to support out spheroid results.
In summary, our results add to a small but growing body of evidence that indicates that the proteasome may be a novel target for anticancer therapy (48) . In addition to their potential as anticancer agents on their own (24 , 49) , proteasome inhibitors can also be used in a combination-type therapy as chemosensitization agents to increase the apoptotic sensitivity of tumor cells to other drugs such as tumor necrosis factor (50) . As such, it will be of considerable interest to evaluate the effects of PS-341 on the efficacy of chemotherapy and/or radiation therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the National Cancer
Institute of Canada and the CaPCURE Foundation of the United
States (to R. S. K.). A. F. is a Medical Research Council of Canada
student awardee. 
2 To whom requests for reprints should be
addressed, at Division of Cancer Biology Research, S-218, Research
Building, Sunnybrook and Women’s College Health Sciences Centre, 2075
Bayview Avenue, Toronto, Ontario, M4N 3M5 Canada. Phone:
(416) 480-5711; Fax: (416) 480-5703; E-mail: kerbel{at}srcl.sunnybrook.utoronto.ca
3 The abbreviations used are: NF, nuclear factor;
FBS, fetal bovine serum; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy
methoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium; PARP, poly(ADP-ribose)
polymerase; PMS, phenazine methosulfate; BrdUrd, bromodeoxyuridine;
HNFN, 10 mM HEPES (pH 7.4), 150 mM NaCl, 4%
FBS, and 0.1% NaN2; PI, propidium iodide; CDDP, cisplatin;
Z-VAD, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.
Received 4/ 6/00; revised 6/28/00; accepted 6/28/00.
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 G. Francia, S. K. Green, G. Bocci, S. Man, U. Emmenegger, J. M.L. Ebos, A. Weinerman, Y. Shaked, and R. S. Kerbel Down-regulation of DNA mismatch repair proteins in human and murine tumor spheroids: implications for multicellular resistance to alkylating agents Mol. Cancer Ther., October 1, 2005; 4(10): 1484 - 1494. [Abstract] [Full Text] [PDF]
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A. Mani and E. P. Gelmann The Ubiquitin-Proteasome Pathway and Its Role in Cancer J. Clin. Oncol., July 20, 2005; 23(21): 4776 - 4789. [Abstract] [Full Text] [PDF]
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Y. Fernandez, M. Verhaegen, T. P. Miller, J. L. Rush, P. Steiner, A. W. Opipari Jr., S. W. Lowe, and M. S. Soengas Differential Regulation of Noxa in Normal Melanocytes and Melanoma Cells by Proteasome Inhibition: Therapeutic Implications Cancer Res., July 15, 2005; 65(14): 6294 - 6304. [Abstract] [Full Text] [PDF]
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 G. Francia, S. Green, and R. S. Kerbel Epigenetic Aspects of Environmentally Mediated "Multicellular" Drug Resistance Am. Assoc. Cancer Res. Educ. Book, April 1, 2005; 2005(1): 114 - 118. [Full Text] [PDF]
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O. A. O'Connor, J. Wright, C. Moskowitz, J. Muzzy, B. MacGregor-Cortelli, M. Stubblefield, D. Straus, C. Portlock, P. Hamlin, E. Choi, et al. Phase II Clinical Experience With the Novel Proteasome Inhibitor Bortezomib in Patients With Indolent Non-Hodgkin's Lymphoma and Mantle Cell Lymphoma J. Clin. Oncol., February 1, 2005; 23(4): 676 - 684. [Abstract] [Full Text] [PDF]
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S. V. Rajkumar, P. G. Richardson, T. Hideshima, and K. C. Anderson Proteasome Inhibition As a Novel Therapeutic Target in Human Cancer J. Clin. Oncol., January 20, 2005; 23(3): 630 - 639. [Abstract] [Full Text] [PDF]
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 A. Fribley, Q. Zeng, and C.-Y. Wang Proteasome Inhibitor PS-341 Induces Apoptosis through Induction |
