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Experimental Therapeutics |
Departments of Radiation Oncology [K. Z., S. R., H. R., C. G., S. B., M. P.] and Neuropathology [I. H.], University Hospital Zurich, CH-8091 Zurich; Department of Biochemistry, ETH Zurich, CH-8091 Zurich [S. R.]; and Novartis Pharma Inc. Department of Oncology Research, CH-4002 Basel [D. F.], Switzerland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Besides DNA damage, IR induces different cytoplasmic signal transduction cascades that are part of the cellular stress response, activating growth-promoting, and growth-inhibiting, pro- and antiapoptotic pathways (3) . The transduction of mitogenic and stress-related stimuli is governed by a network of multiple kinase cascades from the plasma membrane to the nucleus that serve as suitable targets for the development of antineoplastic agents. Members of the PKC family function as transducers for various lipid second messengers in the regulation, transduction, and propagation of cell proliferative stimuli and, thus, are interesting targets for antiproliferative cancer treatments (4 , 5) . Previous studies have indicated that these serine/threonine kinases are interesting targets not only for a single treatment modality but also in combination with additional chemotherapeutic agents and IR. PKC-inhibitors are potent inducers of apoptosis but also sensitize tumor cells to antimetabolites or cytotoxic and DNA-damaging agents. On the other hand, PKC stimulation by phorbol esters can rescue different cell types from glucocorticoid- and growth factor withdrawal-induced cell death (6, 7, 8) .
Several types of antineoplastic PKC-inhibitors are well investigated.
The two staurosporine-related drugs UCN-01 (7-hydroxystaurosporine)
and PKC412 [N-benzoyl staurosporine (formerly called
CGP-41251)] specifically inhibit the conventional calcium- and
diacylglycerol-stimulated PKC isoforms 
, ß, and 
and are
currently under clinical investigations for their potential anticancer
activity. Despite their similar and high specificity against these PKC
isoforms, these compounds also display a dissimilar inhibitory spectrum
against other targets that might be coresponsible for their
antiproliferative activity (9, 10, 11, 12)
.
The state of the tumor suppressor p53 is pivotal for the response of tumor cells to irradiation. Mutations in the p53 gene are involved in acquired and intrinsic treatment resistance in human tumors and render tumor cells refractory to many anticancer therapies (13 , 14) . After irradiation, p53 is activated and induces a crucial block to cell cycle progression providing enough time for sufficient DNA repair prior to deleterious DNA replication in S phase. On the other hand, apoptosis may arise through p53-mediated signal transduction cascades leading to the activation of the apoptotic machinery. Radioresistance of tumor cells devoid of p53 may be a consequence of a diminished ability to undergo apoptosis in vitro and in vivo (15 , 16) . Thus, chemotherapeutic agents that alone or in combination with additional treatment modalities bypass the p53-dependent death pathway and induce p53-independent cell killing are interesting compounds for cancer treatment.
In this study, we have investigated in vitro and in
vivo the potency of the novel, clinically relevant
,ß,-subtype-specific PKC-inhibitor PKC412 to sensitize p53
wild-type and p53-deficient tumor cells for IR. Furthermore, we
demonstrate that in p53+/+ tumor cells, combined treatment with PKC412
and IR drastically induces apoptotic cell death, whereas a
non-apoptosis-related mechanism of radiosensitization occurs in
p53-deficient cells determined in vitro and in histological
sections from p53-deficient tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Irradiation of cell cultures was carried out at room temperature in tissue culture dishes (100 x 100 mm) with a 6-MV linear accelerator at a dose rate of 2 Gy/min or in 96-well plates using a Pantak Therapax 300 kV X-ray unit at 0.7 Gy/min.
Cell Proliferation and Clonogenic Assay.
Tumor cell proliferation was assessed during 4 consecutive days after
treatment by the colorimetric alamarBlue assay that is based on
detection of metabolite activity according to the protocol of the
manufacturer (Biosource International, Camarillo, CA). Absorption was
measured at 570 and 600 nm using a Dynatech MR5000 spectrophotometer.
To determine clonogenic survival, the number of singular seeded cells
was adjusted to obtain 
100 colonies per dish with a given treatment.
After exposure to the different regimens, cells were maintained at
37°C in a humidified atmosphere containing 5%
CO2. Cells were then allowed to grow for 8–10
days before fixation in methanol/acetic acid (75%/25%) and staining
with crystal violet. Only colonies with more than 50 cells/colony were
counted. The plating efficiency (PE) of untreated cells was
determined and calculated by PE (%) = (scored colonies/number of plated cells) x 100. The
surviving fraction (SF) with a given treatment was
determined by SF = (scored colonies)/(number of plated
cells x PE/100). All of the
proliferation and clonogenic assays (in triplicate) were repeated as
independent experiments at least twice, and a representative experiment
is shown. For combined treatment modalities, cells were preincubated
with PKC412 1 h prior to irradiation. For in vitro
experiments, PKC412 was dissolved in DMSO (10 mM
stock solution) and further diluted with media in the presence of 10%
FCS.
Tumor Xenografts in Nude Mice and Administration of Chemotherapy
and Irradiation.
p53-/- and p53+/+ fibrosarcoma cells and human colon carcinoma cells
(SW480) were injected s.c. (4 x 106cells) on the back of 4–8-weeks-old athymic
nude mice. Tumor volumes were determined from caliper measurements of
tumor length (L) and width (l) according
to the formula (L x l2)/2. Tumors were allowed to expand
to a volume of at least 0.175 cm3 (±25%) before
treatment. Using a shielding device mice were given a locoregional
applied body dose of 4 x 3 Gy using a Pantak Therapax
300-kV X-ray unit at 0.7 Gy/min. PKC412 (dissolved in 5% DMSO, 0.5%
Tween 80, and 94.5% H2O) was applied p.o. 4 h prior to irradiation at the indicated dosage. Statistical analysis
was performed with the Mann-Whitney U test. The tumor AGD
was defined as the time for tumor volume in the treated groups to
triple the initial treatment size minus the time in the untreated
control group to reach the same size (17)
.
Histology of Xenografts.
Detection of apoptosis was performed by the use of the dUTP
TUNEL assay (Boehringer Mannheim, Indianapolis, IN).
Ten-µm sections from selected formalin-fixed, paraffin-embedded
tissue blocks were placed on coated slides. Briefly, tissue sections
were dewaxed and rehydrated routinely. After rehydration, the slides
were incubated with proteinase K (20 µg/ml; Sigma, St. Louis, MO) in
10 mmol of Tris-HCl (pH 8) at 37°C for 15 min, according to the
manufacturer’s protocol. The sections were washed in PBS for 10 min,
covered by TdT-FITC-dUTP enzyme-labeling solution, and incubated
at 37°C in a humidified incubator for 1.5 h. The slides were
rinsed for 10 min in PBS and covered with alkaline phosphatase
converter solution. After 1-h incubation, the slides were washed twice
in PBS for 10 min, and BM purple substrate (BCIP/NBT) was
added. The dark-purple color was visible in 2–5 min. Slides were
washed, and a coverslip was placed on mounting media. The nuclear
staining was evaluated under a light microscope. Identical slides were
also stained with H&E to evaluate tissue structure. For negative
controls, deionized water was used instead of TdT.
Cell Cycle Analysis.
Cells were prepared for cell cycle analysis using flow cytometry.
Twenty-four h after the different treatment regimens, both adherent and
floating cells were collected, washed with ice-cold sample buffer
(0.1% glucose in PBS) for 10 min by centrifugation at
400 x g, fixed in 70% ethanol overnight at
4°C; and stained with propidium iodide in presence of the
RNase (1 mg/ml). FACS analysis was performed on a FACScan and data were
analyzed using Multi Cycle software (Becton Dickinson). At least two
independent experiments, each in duplicate, were performed for
each set of data. For cell cycle quantification, the amount of
apoptotic cells were subtracted from the total cell population, and the
amount of surviving cells are used as 100%. Statistical
analysis was performed with a Student t test.
Preparation of Cell Fractions.
Cells were harvested by centrifugation at 1800 x g for 10 min at 4°C and washed with ice-cold PBS. The cell
pellet was suspended in five volumes of ice-cold buffer A [20
mM HEPES-KOH (pH 7.5), 10
mM KCl, 1.5 mM MgCl2, 1
mM sodium EDTA, 1 mM sodium
EGTA, 1 mM DTT, 250 mM
sucrose, and 0.1 mM phenylmethylsulfonyl
fluoride], supplemented with protease inhibitors (5 µg/ml pepstatin
A, 10 µg/ml leupeptin, and 2 µg/ml aprotinin). After sitting on ice
for 15 min, the cells were disrupted by douncing 15 times in a dounce
homogenizer. Cell lysates were centrifuged at 1,000 x g for 10 min at 4°C (crude nuclear pellet), and the
supernatant was further centrifuged at 100,000 x g for 1 h. The resulting supernatant (S-100 fraction)
and pellet (mitochondrial fraction) was stored at -80°C.
In Vitro Caspase 3-like Activity Assay.
Protein (50–80 µg) of S-100 fraction was incubated at 37°C in the
presence of the colorimetric caspase-3 substrate Ac-DEVD-pNA (100
µM; Calbiochem) and 1 mM dATP in a final
volume of 100 µl. Cleavage was monitored at 405 nm using a Dynatech
MR5000 spectrophotometer. Horse cytochrome c (Sigma) and
caspase-3 inhibitor Ac-DEVD-CHO (5 nM) were added
to the reaction mixture as indicated in the text.
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RESULTS |
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Clonogenic survival of p53-wild-type and p53-deficient tumor cells was
reduced on treatment with increasing concentrations of PKC412 alone,
but p53-wild-type tumor cells were more sensitive to PKC412 than were
p53-deficient cells at all of the concentrations tested (Fig. 1, A and B)
. Staurosporine was also more toxic
against the p53+/+ tumor cells than against the p53-/- tumor cells
and in a dose range 10 times lower than the benzoylated staurosporine
derivative PKC412. The difference in the antiproliferative potency
between staurosporine and PKC412 obtained with these cell lines is in a
similar range as observed against other tumor cell lines
(9)
.
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. The cooperative effect of the
PKC-inhibitor and IR was further enhanced when concentrations of PKC412
and irradiation doses were applied that massively reduced clonogenic
survival as a single agent. Similarly, a supra-additive reduction of
clonogenic survival was observed when tumor cells were treated with IR
in combination with staurosporine (Fig. 2, C and D)
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Radiosensitizing Effect of N-Benzoylstaurosporine on
Tumor Xenografts.
PKC412 is less toxic than staurosporine and applicable to in
vivo studies. Thus, combined treatment with PKC412 and IR was
tested in vivo against tumors derived from the p53-/-
deficient fibrosarcoma cells s.c. injected into the back of nude mice.
Treatment was started when tumors reached a minimal size of 165
mm3 ± 10% (days 12–17 after cell
injection).
In vivo studies were performed with locoregional application of IR using a shielding device and a fractionated single dose of 3 Gy. This daily dose is applied when fractionated radiotherapy is used for the treatment of human malignancies. For practical reasons, only four fractions were chosen as the treatment regimen, but the response to such a regimen was useful for treatment evaluation.
Fig. 3A
summarizes the effect of tumor treatment with the
PKC-inhibitor PKC412 alone (4 x 100 mg/kg), IR alone
(4 x 3 Gy), and in combination (4 x 100
mg/kg combined with 4 x 3 Gy) applied on 4 consecutive
days, in comparison with an untreated control group. Treatments were
started with a minimal tumor size of 150–300 mm3
and each curve represents the mean tumor volume per group
(n = quantity of animals per group).
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Mice-borne tumors that were derived from the radioresponsive p53+/+ fibrosarcoma cells almost completely regressed on treatment with irradiation (4 x 3 Gy) alone, and, thus, almost no enhanced response was observed when IR was combined with the PKC-inhibitor using this fractionated treatment regimen (data not shown).
In addition, combined treatment was tested in vivo against
tumors derived from the p53-mutated, human colon adenocarcinoma cell
line SW480 (Fig. 3B)
. Although no tumor-growth-delaying
effect was observed on treatment with PKC412 alone, combined treatment
with PKC412 increased the antitumor effect of IR, which resulted in
extended tumor growth delay. (P = 0.009,
RT versus combined treatment). In this human colon
tumor-xenograft, a significant AGD tripling the tumor volume was
observed on combined treatment in comparison with the AGD on treatment
with IR or PKC412 alone (16 days versus 4 or 1 day,
respectively), which resulted in extended tumor growth control.
Differential Mechanism of Radiosensitization.
The p53-deficient E1A/ras-transformed tumor cell
line does not undergo apoptosis as part of the therapeutic response to
different DNA-damaging and cytotoxic agents (16
, 18
, 19)
.
To evaluate the extent of apoptosis after combined treatment with
PKC412, tumor histology from p53+/+ and p53-/- tumors were compared 4
days after treatment start. Both tumor types contained some necrotic
zones, and combined treatment induced massive DNA fragmentation in
p53+/+ tumors detected with the TUNEL assay using biotin-labeled
dUTP, that was not observed in the p53-/- tumors (Fig. 4)
. The amount of TUNEL-positive cells was also increased and
interspersed throughout the p53+/+ tumor on treatment with IR alone but
to a smaller extent than after combined treatment (data not shown).
These data indicate that the induction of apoptosis is responsible for
tumor regression in the p53+/+ tumors but not for the extended growth
control in the p53-/- tumors on combined treatment.
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. These data clearly support the
histological observations that the response of the p53+/+ but not of
the p53-/- tumors to combined treatment is attributable to
apoptosis and that PKC412 sensitizes p53+/+ tumor cells for the
activation of the apoptotic machinery.
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In accordance, a decreasing fraction of cells in
G1 and S phase was identified. Interestingly,
treatment of the p53+/+ tumor cells with IR alone or in combination
with PKC412 resulted in a diminished fraction of cells in
G1, most probably because of the p53-dependent
induction of apoptosis. |
DISCUSSION |
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In the p53-wild-type tumor cells, induction of apoptosis on combined treatment was observed both on the biochemical and cellular level (caspase-3 activity and FACS analysis, respectively) and correlated with the strong TUNEL-positive, immunohistochemical analysis of p53+/+-tumor xenografts after treatment with PKC412 and IR. Already after a singular treatment application, initial signs of apoptosis were detectable (not shown), but apoptosis on the biochemical, cellular, and histochemical level was much more extended after 2 or 4 daily treatment fractions that also more closely paralleled the treatment regimen used for the in vivo tumor growth measurement. Quantitative analysis of cell death by flow cytometry indicated that a large portion of these cells die via apoptosis by irradiation alone, which has already been suggested for this tumor cell (16) . Treatment in combination with PKC412 even potentiated this mode of cell death.
No signs of apoptosis were observed when p53-/- tumor cells, treated
with single or combined treatment modalities after one or after
multiple fractions, were analyzed for these different parameters.
Nevertheless, irradiation in combination with PKC412 revealed a
supra-additive, antitumoral effect both on clonogenic survival and on
in vivo tumor growth delay against these treatment-resistant
p53-deficient fibrosarcoma tumor cells. Combined treatment with PKC412
and IR also resulted in decreased clonogenic survival and an extended
growth delay against xenografts derived from the human colon
adenocarcinoma cell line SW480, which is p53 mutated, and does not
undergo IR-induced apoptosis (Fig. 3
and unpublished
results).5
Using this treatment regimen, we did not observe any short- or
long-term unspecific toxicities in control mice. Thus, it would be
interesting to test an extended combined-treatment regimen against
these tumor xenografts, in particular because the dosages of the IR and
PKC412 applied in our regimen are well below the maximal daily
tolerated dose of PKC412 or locally applied IR (20)
.
Nevertheless, already this minimal combined treatment schedule in
vivo indicates both a significant supra-additive, antitumoral
effect and a potentially broad therapeutic window.
On the basis of our cellular and histological results, combined treatment with PKC412 and IR may induce different mechanisms of cell death. The in vivo tumor growth delay on combined treatment in the p53-/- tumors might be attributable to the enhanced G2-M arrest as observed on the cellular level by FACS measurement. This (most probably) permanent cell cycle arrest also explicates the effect on clonogenicity of the p53-/- tumor cells on combined treatment. On the other hand, combined treatment drastically potentiated apoptosis in tumor cells with p53-positive background. There is an ongoing discussion on the relevance of apoptosis in the treatment response of tumor cells against pharmacological agents and in particular in response to IR (21) . In our tumor model system, we observe a striking difference in the mode of cell death as part of the treatment response that is depending on the p53 status of the tumor cells. Both single treatment with IR or PKC412 alone and combined treatment have a more drastic effect on the clonogenic survival in the p53+/+ tumor cells. This suggests that the difference in the survival rate between the two cell lines is attributable to the remaining capacity of the treatment-sensitive p53+/+ tumor cells to undergo apoptosis. However, it is not possible to quantitatively discriminate which proportion of cells did undergo p53-mediated apoptosis or other forms of clonogenic cell death and thereby explicating the different treatment sensitivity of the p53 +/+ and p53 -/- tumor cells observed with the clonogenic survival assay.
The tumor system used for this study consists of E1A/ras-transformed MEFs. We cannot exclude the possibility that transformation of these cells might lead to subsequent genetic alterations. But such changes could also reflect the process in carcinogenesis. Nevertheless, the significant difference in the mode of cell death induced by PKC412 and IR is still primarily dependent on the p53 status. More important, combined treatment with PKC412 and IR resulted in a significant tumor growth control against tumors derived from the otherwise-resistant p53-/- murine fibrosarcoma cells as well as against tumors derived from human p53-mutated, colon adenocarcinoma tumor cells.
Staurosporine and its derivatives used as single agents are well-known inducers of apoptosis but at doses 25–100 times higher as applied in this report in combination with irradiation (22 , 23) . Interestingly, radiosensitization with specific PKC inhibitors like chelerythrine at low concentrations was mechanistically linked to the generation of the apoptotic second-messenger ceramide leading to apoptosis in specific tumor cells that still have a remaining capacity to undergo apoptosis even in the absence of functional p53. A decrease in an apoptotic threshold was achieved in in vitro and in vivo experiments by combined treatment with IR and the PKC inhibitor chelerythrine, presumably through the activation of sphingomyelinase leading to elevated ceramide concentrations (24, 25, 26) . We previously demonstrated that the p53-wild-type tumor cells used in this study undergo ceramide-induced apoptosis, whereas these treatment-resistant p53-deficient tumor cells are refractory to ceramide (18) . These responses to ceramide parallel the different responses to treatment with PKC412 in combination with IR in our tumor cell system.
The effects of staurosporine and its derivatives on cell cycle progression is complex and concentration dependent and might be attributable to the multiple interference with the cyclin-dependent kinase system (27) . Inhibition of CDC2 and CDK2 kinase activity by high concentrations of PKC412 alone (1 µM), induced a substantial increase of glioblastoma cells into the particularly radiosensitive G2-M phase of the cell cycle (12) . We did not observe any increase of a G2-M cell population in the p53-/- tumor cells at the low concentrations of PKC412 (0.2 µM) used, but only when combined with IR. Thus, a possible antitumoral synergism might have resulted in the concurrent accumulation into the IR-most-sensitive G2-M phase of cells in the course of the fractionated combined radiochemotherapy with PKC412 and IR.
On the other hand, low concentrations of 7-hydroxy-staurosporine (UCN-01) inhibit the critical cell cycle kinase chk1, thereby suppressing a DNA-damage-induced G2 arrest by G2 checkpoint abrogation (10) . On the basis of this G2-M-modulatory effect, UCN-01 was also tested in vitro and in vivo and showed a synergistic activity in combination with DNA-damaging agents such as cis-DDP and multiple fractions of high doses of IR (10 Gy) against rather small murine fibrosarcomas (28) . Hence, a thus-far-unknown radiosensitizing mechanism and different molecular target must be responsible for the radiosensitizing effect of PKC412 in the p53-/- tumor cells, because the N-benzoylated staurosporine derivative PKC412 does not abrogate a G2 checkpoint but rather enhances a G2 arrest in combination with IR. Nevertheless, we must consider the possibility that the observed increase in G2-M cell population on combined treatment is only an unrelated consequence and not the initial cause for increased radiosensitivity.
The protein kinase inhibitor PKC412 is currently in phase I/II trials
for treatment of advanced cancer and was preclinically tested as
antitumor agent alone and in combination with chemotherapeutica. PKC412
displayed potent antitumor activity against various tumor types when
used alone, but more important also significantly enhanced the
antitumoral activity of 5-FU, cis- and carboplatinum,
doxorubicin, vinblastine and Taxol against solid tumors that do not
show a response against single treatment with PKC412. PKC412 is known
to have a strong affinity to the human -1 acidic glycoprotein (AAG)
present in human plasma. Nevertheless, a major PKC412 metabolite has an
even higher affinity to AAG and will keep the remaining free PKC412
concentration sufficiently high to still effect radiosensitization
(20)
.
In this report, we have presented for the first time complementary in vitro and in vivo data on PKC412 in combination with irradiation. In tumor cells with an intact apoptotic program, combined treatment drastically enhanced the apoptotic response but still induced substantial growth control against tumors with a nonfunctional apoptotic machinery. PKC412 alone had only minimal antitumoral activity when tested in vivo against the radio- and chemoresistant p53-deficient murine fibrosarcoma and human colon adenocarcinoma tumor cell xenografts, but it significantly enhanced tumor growth control when used in combination with clinically relevant daily fractions of IR.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from the Zurich and
Aargau Cancer League (to K. Z.), the Baugarten Foundations (to
S. R.), and the University of Zurich (to S. B.). 
2 Present address: Department of Radiation
Oncology, Kantonsspital St.Gallen, CH-9007 St. Gallen,
Switzerland.
3 To whom requests for reprints should be
addressed, at Laboratory for Molecular Radiobiology, Department of
Radiation Oncology, University Hospital Zurich, CH-8091 Zurich,
Switzerland. Phone: 41-1-255-8549; Fax: 41-1-255-44-35; E-mail: pum{at}dmr.usz.ch
4 The abbreviations used are: IR, ionizing
radiation; MEF, mouse embryo fibroblast; AGD, absolute growth delay;
PKC, protein kinase C; RT, irradiation; TdT, terminal deoxynucleotidyl
transferase; TUNEL, TdT-mediated nick end labeling.
5 S. Rocha and M. Pruschy, unpublished results.
Received 7/ 5/00. Accepted 11/ 9/00.
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down-regulation. J. Biol. Chem., 272: 23481-23484, 1997.This article has been cited by other articles:
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