Molecular Cancer Therapeutics
Inhibition of constitutive signal transducer and activator of
transcription 3 activation by novel platinum complexes
with potent antitumor activity
James Turkson,1,4 Shumin Zhang,1,4
Jay Palmer,3,7,8 Heidi Kay,8 Joseph Stanko,3,7
Linda B. Mora,1,4 Said Sebti,3,4,5 Hua Yu,2,4
and Richard Jove1,4,5,6
pathways, such as Stat3, may be one of the key
molecular mechanisms for the antitumor effects of
platinum (IV) – containing complexes. [Mol Cancer Ther
2004;3(12):1533– 42]
1
Molecular Oncology, 2Immunology and 3Drug Discovery
Programs, H. Lee Moffitt Cancer Center & Research Institute;
and Departments of 4Interdisciplinary Oncology, 5Biochemistry
and Molecular Biology, 6Pathology, 7Chemistry, and 8College of
Public Health, University of South Florida, Tampa, Florida
Introduction
Abstract
DNA-alkylating agents that are platinum complexes induce
apoptotic responses and have wide application in cancer
therapy. The potential for platinum compounds to modulate signal transduction events that contribute to their
therapeutic outcome has not been extensively examined.
Among the signal transducer and activator of transcription
(STAT) proteins, Stat3 activity is frequently up-regulated
in many human tumors. Various lines of evidence have
established a causal role for aberrant Stat3 activity in
malignant transformation and provided validation for its
targeting in the development of small-molecule inhibitors
as novel cancer therapeutics. We report here that
platinum-containing compounds disrupt Stat3 signaling
and suppress its biological functions. The novel platinum
(IV) compounds, CPA-1, CPA-7, and platinum (IV) tetrachloride block Stat3 activity in vitro at low micromolar
concentrations. In malignant cells that harbor constitutively activated Stat3, CPA-1, CPA-7, and platinum (IV)
tetrachloride inhibit cell growth and induce apoptosis in a
manner that reflects the attenuation of persistent Stat3
activity. By contrast, cells that do not contain persistent
Stat3 activity are marginally affected or are not affected
by these compounds. Moreover, CPA-7 induces the regression of mouse CT26 colon tumor, which correlates
with the abrogation of persistent Stat3 activity in tumors.
Thus, the modulation of oncogenic signal transduction
Received 4/29/04; revised 9/27/04; accepted 10/15/04.
Grant support: National Cancer Institute grants CA78038, CA55652,
and CA82533 from the NIH.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: James Turkson, Molecular Oncology Program,
H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia
Drive, SRB 22214, Tampa, FL 33612. Phone: 813-745-6725;
Fax: 813-632-1436. E-mail: [email protected]
Copyright C 2004 American Association for Cancer Research.
Cellular responses to growth factors and cytokines are
characterized by the activation of the signal transducer and
activator of transcription (STAT) family of cytoplasmic
transcription factors (1 – 10). STATs are activated by
tyrosine phosphorylation, which is induced by protein
tyrosine kinases of growth factor receptors and receptorassociated cytoplasmic kinases, such as the Janus-activated
kinase ( Jak) or Src kinase families. This in turn allows
phosphotyrosine (pTyr)-SH2 interactions between two
STAT monomers, and the formation of dimers, which then
translocate to the nucleus. In the nucleus, active STATs
bind to specific DNA response elements and regulate the
expression of genes essential for cell proliferation, differentiation, development, and survival.
Normal physiologic STAT activation is tightly regulated
and has a short duration, which is in keeping with the
cellular requirements for mounting a response to external
stimuli. However, persistent activation of specific STAT
proteins, particularly Stat3 and Stat5, occurs with high frequency in a wide range of tumors (11 – 21). Furthermore,
compelling evidence supports persistently active Stat3 as
having a causal role in malignant transformation by promoting growth and survival of cells. This has been observed in solid and hematologic cancers, including breast
and prostate cancers, head and neck squamous carcinoma,
lymphomas and leukemias [reviewed in (refs. 22 – 29)].
Of clinical importance is the observation that the blockade
of aberrant Stat3 signaling induces tumor cell apoptosis
and tumor regression (15, 18, 21, 30), which provides the
rationale for developing small-molecule Stat3 inhibitors
as anticancer drugs [31, 32, for reviews see (refs. 25, 26,
29, 33)].
Platinum complexes, the prototype of which is cisplatin,
are widely used as active anticancer agents (34, 35) in a
variety of human tumors, including testicular, ovarian and
bladder carcinomas, head and neck squamous cell carcinoma, and non – small cell lung cancers. The biological
outcome of cisplatin and other platinum-containing complexes is strongly linked to their alkylating effects on
DNA. The effects on DNA, as well as possible interactions
with proteins, may be part of a series of molecular events
induced by platinum complexes (36) that influence their
overall therapeutic outcome. In this context, earlier reports
show that cisplatin induces the activation of members of
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
1533
1534 Stat3 Inhibition by Novel Platinum Compounds
the mitogen-activated protein kinase (MAPK) family (37,
38). Thus, understanding the interactions of platinum
compounds with signal transduction pathways may provide new approaches to enhance the therapeutic benefits of
platinum complexes.
We determined the effects of platinum-containing compounds on persistently active Stat3 signaling and biological functions in the context of malignant transformation.
Evaluation of a series of novel platinum complexes identified two, CPA-1 and CPA-7, which interfere with Stat3
and disrupt its ability to bind to DNA in vitro. Treatment
with CPA-1 or CPA-7 of v-Src – transformed mouse fibroblasts, human breast and prostate cancer cells, as well as
mouse melanoma and colon cancer cells blocks constitutive
Stat3 activity that is harbored in these cells, and induces
growth inhibition and apoptosis. Along with CPA-1 and
CPA-7, both of which are platinum (IV) complexes,
platinum (IV) tetrachloride (Pt(IV)Cl4) induces similar
effects in the same cellular background. Moreover, the
administration of CPA-7 in a mouse tumor model of colon
cancer attenuates aberrant Stat3 activity in this tumor, and
induces tumor regression. These platinum (IV) compounds
may represent a new group of agents that inhibit Stat3
activation.
Materials and Methods
Cells, Plasmids, and Other Reagents
v-Src – transformed (NIH3T3/v-Src) mouse fibroblasts
and their counterparts stably expressing Stat3 reporter,
pLucTKS3 (NIH3T3/v-Src/pLucTKS3) or Stat3-independent plasmid, pRLSRE (NIH3T3/v-Src/pRLSRE), and Rastransformed (NIH3T3/v-Ras) mouse fibroblasts, human
breast carcinoma MDA-MB-231, MDA-MB-435, MDA-MB453, MCF-7, and MDA-MB-468 cells, mouse melanoma B16
and M2 cells, human prostate cancer DU145 cells, human
lung carcinoma A459 cells, as well as colon cancer cells
CT26 (mouse) have been previously described (21, 31, 39 – 41).
HCT116p53 / cell line was generously provided by Dr.
B. Vogelstein (Johns Hopkins University, Baltimore, MD).
Plasmids pLucTKS3 (driving expression of the firefly
luciferase gene) and pRLSRE (driving renilla luciferase
gene expression) have been previously described (31, 42).
Recombinant human epidermal growth factor (EGF) was
obtained from Invitrogen (Carlsbad, CA), recombinant
murine interferon gamma (IFN-g) from Research Diagnostics, Inc. (Flanders, NJ), and the Src tyrosine kinase
inhibitor, SU6656 from SUGEN (South San Francisco,
CA). The EGF and IFN-g were used at 9 ng/AL and
10 units/mL, respectively. Cells were grown in DMEM
containing 5% iron-supplemented bovine calf serum, with
or without G418. Cisplatin and (Pt(IV)Cl4) were purchased from Sigma-Aldrich (Milwaukee, WI). Novel
platinum (II) and (IV) complexes, CPA-3, or CPA-1, and
CPA-7 were synthesized according to previously published procedures (43, 44).
Cytosolic Extract Preparation and Luciferase Assays
Cytosolic lysate preparation from fibroblasts for
luciferase assays or from baculovirus-infected Sf-9 insect
cells have been previously described (12, 31, 42, 45).
Luciferase assays were done as outlined in the supplier’s
(Promega, Madison, WI) manual and measured with a
luminometer.
Nuclear Extract Preparation and Gel Shift Assays
Nuclear extracts were prepared from cell lines and used
for electrophoretic mobility shift assay (EMSA) as previously described (12, 40, 46). In some cases, cells were
pretreated with platinum complexes for the indicated times
prior to harvesting. Where cells were stimulated with EGF
(9 ng/AL) or IFN-g (10 units/mL), duration of treatment
was 15 to 30 minutes. Nuclear extracts (normalized for
equal amounts of total protein) were preincubated with
compounds for 30 minutes at room temperature prior to
incubation with radiolabeled probe. The 32P-labeled oligonucleotide probes used are high-affinity sis-inducible
element (hSIE, m67 variant, 5V-AGCTTCATTTCCCGTAAATCCCTA), which binds both Stat1 and Stat3 (46,
47), mammary gland factor element (MGFe) from the
bovine h-casein gene promoter, 5V-AGATTTCTAGGAATTCAA, which binds Stat1 and Stat5 (48, 49), the NF-B –
binding oligo (5V-TCGACAGAGGGGACTTTCCGAGAGGC-3V), and the 90-bp oligonucleotide sequence from
the dihydrofolate reductase promoter that binds E2F1 (50).
Western Blot Analyses
Whole-cell lysates were prepared in boiling SDS sampleloading buffer to extract total proteins from the cytoplasm
and nucleus as well as preserve the in vivo phosphorylation states. Equivalent amounts of total cellular protein
were electrophoresed on a SDS-10% polyacrylamide gel
and transferred to nitrocellulose membranes. Probing of
nitrocellulose membranes with primary antibodies and
detection of horseradish peroxidase-conjugated secondary
antibodies by enhanced chemiluminescence (Amersham,
Piscataway, NJ) were done as previously described (12, 42,
45). The probes used were anti-Stat3 (Santa Cruz, Santa
Cruz, CA), anti-pTyr705Stat3 (Cell Signaling, Beverly, MA),
antiactive (phosphorylated) JNK, p38mapk, Erks, or pAkt,
and pJak1, as well as anti-JNK, p38, Erks, Akt and Jaks
(Cell Signaling), and antiphosphotyrosine, clone 4G10
(Upstate Biotechnology, Lake Placid, NY).
Cell Proliferation and TUNEL Staining
Proliferating cells were counted by phase-contrast
microscopy for viable cells (using trypan blue exclusion).
Thymidine incorporation assays were done in triplicate
with 5 103 cells per well exposed to the indicated
concentrations of compounds for 30 hours. Cells were
pulsed with [3H]-thymidine (0.5 ACi/well) for 6 hours of
culture, then transferred onto glass fiber filters to determine
the [3H]-thymidine incorporated using liquid scintillation
counter. Terminal nucleotidyl transferase-mediated nick
end labeling (TUNEL) staining was carried out according
to supplier’s (Roche, Indianapolis, IN) instructions to detect
apoptotic cells. Cells were first treated with or without
compounds for 24 to 48 hours prior to staining.
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
Figure 1. Structural formulas of novel platinum complexes. Cisplatin
analogues of novel platinum (II) complex, CPA-3, and platinum (IV)
complexes, CPA-1, and CPA-7, as well as (Pt(IV)Cl4).
Mice and In vivo Tumor Studies
Six-week-old female Balb/C mice were purchased from
the National Cancer Institute (Frederick, MD) and maintained in the institutional animal facilities approved by the
American Association for Accreditation of Laboratory
Animal Care. Balb/C mice were shaved in the left flank
area and injected s.c. with 2 105 colon carcinoma CT26
cells in 100 AL of PBS. After 5 to 10 days, tumors with a
diameter of 3 to 6 mm were established. Animals were
stratified so that the mean tumor sizes in all treatment
groups were nearly identical. Tumor volume was calculated
according to the formula V = 0.52 a 2 b, where a, smallest
superficial diameter; b, largest superficial diameter.
Results
Inhibition of In vitro Stat3 DNA-Binding Activity
A panel of novel platinum complexes was evaluated for
inhibitory effects against STAT activity in vitro, measured
in terms of the level of DNA-binding activity associated
with nuclear extracts prepared from EGF-stimulated mouse
(NIH3T3/hEGFR) fibroblasts. Of the group of novel plati-
num complexes, the three (CPA-1, CPA-3, and CPA-7) that
showed inhibitory effects on Stat3 DNA-binding activity
in vitro are presented in Fig. 1. The structures in Fig. 1
were confirmed by various analytic approaches, including
nuclear magnetic resonance, infrared, X-ray diffraction, and
elemental analysis (data not shown). Nuclear extracts
containing activated Stat1, Stat3, and Stat5 were preincubated with different concentrations of platinum complexes
for 30 minutes prior to incubation with radiolabeled hSIE
probe that binds Stat1 and Stat3 and subjected to EMSA
analysis. Preincubation of nuclear extracts with CPA-1,
CPA-3, or CPA-7 results in a dose-dependent reduction
in the level of DNA-binding activity of Stat3 and Stat1
(Fig. 2A), with compounds approximately two to four
times more potent against Stat3 over Stat1 homodimers
(IC50 values in the low micromolar range shown in Table 1).
In contrast, similar treatment of nuclear extracts with
platinum compounds showed that they have a much
reduced inhibitory effect on Stat5 DNA-binding activity
using the MGFe probe (Fig. 2B). This suggests that CPA-1,
CPA-3, and CPA-7 preferentially disrupt Stat3 and Stat1
activity in vitro. Supershift analyses in the presence of
antibodies were done, which identify the hSIE-bound
complexes as Stat3:Stat3 (top band), Stat1:Stat3 (intermediate
band) and Stat1:Stat1 (lower band; Fig. 2A, second and third
lanes from the right), and the MGFe-bound complexes as
Stat5:Stat5 (top band) and Stat1:Stat1 (lower band; Fig. 2B, the
first and third lanes from the right).
Because both CPA-1 and CPA-7 are platinum (IV)
complexes, we determined whether the observed effects
are associated with platinum (IV) by evaluating (Pt(IV)Cl4)
Figure 2. EMSA analyses of DNAbinding activities and effects of platinum complexes. Nuclear extracts
containing activated Stat1, Stat3,
Stat5, E2F1, or NF-nB were treated
with the indicated concentrations of
platinum complexes CPA-1, CPA-3,
CPA-7, or Pt(IV)Cl4 for 30 minutes at
room temperature prior to incubation
with radiolabeled oligonucleotide
probes. A, Stat1 and Stat3 binding
activities to hSIE probe; B, Stat1 and
Stat5 binding activities to MGFe
probe; C, the binding of E2F1 to
dihydrofolate reductase sequence as
probe; and D, the binding of NF-nB to
binding sequence in a-2 macroglobulin promoter as probe. Positions of
complexes of DNA with STATs, E2F1,
or NF-nB (p65, p50) in gel are labeled.
In (A) and (B), the last four lanes from
the right represent supershift analysis
in the presence of antibodies against
Stat1 (a-St1 Ab), Stat3 (a-St3 Ab), or
Stat5 (a-St5 Ab); *, supershifted
DNA-STAT complex; Control lanes ,
nuclear extracts that are not preincubated with platinum complexes.
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
1535
1536 Stat3 Inhibition by Novel Platinum Compounds
Table 1. IC50 values for disruption of STAT DNA-binding activity
in vitro (Amol/L)
CPA-1
CPA-3
CPA-7
Stat3:Stat3
Stat1:Stat3
Stat1:Stat1
5.0
5.8
1.5
9.3
27.0
3.5
20.0
8.3
4.0
in similar assays (Fig. 1). By EMSA analysis, we show that
preincubation of nuclear extracts with (Pt(IV)Cl4) disrupts
DNA-binding activity of Stat3 and Stat1, but not Stat5, as
similarly observed for CPA-1 and CPA-7 (Fig. 2A and B).
To determine the selectivity of platinum complexes for
Stat3, we investigated effects on the binding of E2F1 and
NF-nB to their cognate DNA-binding sequences. Analysis
by EMSA shows that the DNA-binding activities of the
two non – STAT-related transcription factors are not
significantly altered by these platinum compounds except
at the very highest concentration (Fig. 2C and D).
Abrogation of Constitutive Stat3 Signaling in Malignant Cells by Platinum (IV) ^ Containing Compounds
To extend our findings to in vivo conditions, we
investigated the effects of platinum compounds on Stat3
signaling in malignant cells that contain aberrant Stat3
activation. Platinum complexes were first evaluated to
determine their ability to inhibit the induction of Stat3
transcriptional activity using v-Src – transformed mouse
fibroblasts, NIH3T3/v-Src/pLucTKS3, and NIH3T3/
v-Src/pRLSRE, which stably express Stat3-dependent
and Stat3-independent luciferase reporters, respectively.
Results show that CPA-7 significantly suppresses the
expression of the Stat3-dependent luciferase reporter
pLucTKS3 (Fig. 3A), with little effect on induction of the
Stat3-independent luciferase reporter pRLSRE (Fig. 3B).
Although CPA-1 inhibited Stat3-dependent luciferase
reporter induction, this effect was weaker than observed
for CPA-7. In contrast, little inhibition of Stat3 transcriptional activity by cisplatin and platinum analogue CPA-3
was observed (Fig. 3A and B).
We then determined the effects of platinum compounds
on constitutive activation of Stat3 in v-Src – transformed
mouse fibroblasts (NIH 3T3/v-Src), human breast carcinoma cells (MDA-MB-231, MDA-MB-435, and MDA-MB-468),
colon carcinoma (CT26), and melanoma cells (M2 and Cl10).
Cells were treated with or without compounds for 24 to 48
hours prior to preparing nuclear extracts for DNA-binding
assay. EMSA analysis shows strong inhibition by CPA-1 and
CPA-7 of constitutive Stat3 activation (Fig. 4A and C, (iii, iv)),
in contrast to cisplatin and CPA-3 that show minimal or no
effect on Stat3 activation (Fig. 4A). Altogether, these
findings indicate that both CPA-1 and CPA-7 are strong
inhibitors of Stat3 activation in intact cells.
The kinetics of inhibition of constitutive Stat3 activation
by platinum complexes were determined in MDA-MB-435.
Analysis by EMSA of nuclear extracts prepared from
treated cells shows that CPA-1, CPA-7, and Pt(IV)Cl4
significantly inhibit Stat3 activation and DNA-binding
activity by as early as 6 hours, with no recovery for up to
48 hours (Fig. 4B, (i)). To determine if this inhibition was
accompanied by changes in Stat3 tyrosine phosphorylation,
whole-cell lysates were prepared and analyzed by SDSPAGE and Western blot. Except for treatment with CPA-7
for 12 hours, treatment with platinum complexes for short
durations of 12 hours or less induced weak or no significant effect on pTyrStat3, whereas prolonged treatments
(24 – 48 h) resulted in relatively stronger or complete reduction in pTyrStat3 (Fig. 4B, (ii)), without altering total Stat3
protein levels, suggesting that platinum (IV) compounds
disrupt phosphotyrosine levels of Stat3 on prolonged treatment. Altogether, our findings suggest two mechanisms by
which platinum compounds disrupt Stat3 DNA-binding
activity in cells. One mechanism occurs early, which is
consistent with the effects of these compounds on Stat3
DNA-binding activity in vitro (Fig. 2), and a second mechanism involves a reduction in the phosphotyrosine levels
of Stat3. In the context of both modes of disruption, results
show that CPA-7 is the most potent.
Because platinum compounds were observed to disrupt
Stat1 DNA-binding activity in vitro (Fig. 1A and B), we
examined whether Stat1 activation in cells could be affected
by compounds. Normal NIH3T3 or NIH3T3/hEGFR
fibroblasts were first treated with platinum compounds
Figure 3. Inhibition of Stat3-mediated gene expression in intact cells by
platinum complexes. v-Src – transformed mouse fibroblasts that stably
express Stat3-dependent (NIH3T3/v-Src/pLucTKS3) and Stat3-independent (NIH3T3/v-Src/pRLSRE) luciferase reporters were treated with
platinum complexes for the indicated times. Cytosolic extracts were then
prepared from cells for (A) Stat3-dependent firefly luciferase activity and
(B) Stat3-independent renilla luciferase measurements. Columns , means
of five independent assays; bars , SE.
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
Figure 4. Evaluation of effects of
platinum complexes on Stat3 activation in cells analyzed by EMSA and
Western blot. Nuclear extracts and
whole-cell lysates were prepared from
ligand-stimulated normal cells or malignant cells that contain constitutively
activated Stat3 and were treated with
or without platinum complexes for the
indicated times. A and B (i), EMSA
analysis of in vitro DNA-binding activity
of Stat3 that is associated with extracts
prepared from v-Src – transformed
NIH3T3/v-Src, human breast carcinoma
MDA-MB-231, MDA-MB-435, and
MDA-MB-468; (ii) , SDS-PAGE and
Western blot analysis of whole-cell
lysates from MDA-MB-435 that probes
for pTyrStat3, and total Stat3. C,
EMSA analysis of in vitro DNA-binding
activity of Stat3 that is associated with
extracts prepared from NIH3T3 treated
with IFN-g, NIH3T3/hEGFR treated with
EGF, mouse colon carcinoma CT26,
and mouse melanoma Cl10 and M2 cell
lines. Positions of STAT-DNA complex,
pTyrStat3, and total Stat3 are shown.
h-Actin is shown for normalization of
total protein.
for 24 hours, stimulated with IFN-g or EGF, and then
nuclear extracts prepared for DNA-binding assay and
EMSA analysis. Stimulation of cells by IFN-g induces
predominantly Stat1:Stat1 (lower band) and to a lesser
extent, Stat1:Stat3 (intermediate band), and Stat3:Stat3 (Fig.
4C, (i); top band), whereas treatment with EGF induces all
three complexes (Fig. 4C, (ii)). Pretreatment of cells with
CPA-1, CPA-7, or Pt(IV)Cl4 has no significant effect on the
activation of Stat1:Stat1 DNA-binding activity by IFN-g or
EGF (Fig. 4C, (i) and (ii); lower band). In contrast, Stat1:Stat3
(intermediate band) is partially blocked, consistent with
strong inhibition of Stat3:Stat3 (top band) in both cell lines
(Fig. 4C, (i) and (ii)), and in mouse colon, CT26, or
melanoma, Cl10, and M2 cell lines that harbor constitutively active Stat3 (Fig. 4C, (iii) and (iv)). Inhibition is
stronger with CPA-7 than CPA-1 or Pt(IV)Cl4. Moreover,
constitutively active Stat3 levels are lower in the melanoma
cell line, Cl10, compared with M2 cells (Fig. 4C, (iv)).
Together, these findings indicate that CPA-1, CPA-7 and
Pt(IV)Cl4 selectively inhibit Stat3 activation in cells and
have minimal effect on Stat1 activity. This may reflect
differences in activities of platinum compounds in cells
compared with the cell-free system.
CPA-1, CPA-7, and (Pt(IV)Cl4) Are Selective Inhibitors
of Stat3 Signal Transduction
To determine the possibility of widespread nonspecific
effects in vivo, we studied changes in other signaling
pathways that could be induced by platinum complexes,
including changes in MAPK family, Erks, p38mapk and
JNK, as well as phosphatidylinositol 3-kinase/Akt, and
Jaks. Viral Src-transformed fibroblasts (NIH 3T3/v-Src)
were used in these studies to take advantage of the
extensive signal transduction pathways that are induced
due to the presence of the Src oncoprotein. In NIH3T3/vSrc or NIH3T3/hEGFR cells stimulated with EGF, pretreatment with CPA-1, CPA-7, or Pt(IV)Cl4 does not significantly alter the levels of total cellular tyrosine phosphorylated
proteins, as analyzed by Western blot using antiphosphotyrosine antibody, clone 4G10 (Fig. 5A). As a positive
control, treatment with the Src kinase inhibitor SU6656 (51)
suppressed phosphotyrosine levels in the NIH3T3/v-Src
cells (Fig. 5A). Consistent with no change in tyrosine
phosphorylated proteins (Fig. 5A), the induction by v-Src of
p38mapk (Fig. 5B; ref. 42), Akt (Fig. 5C; ref. 52), JNK
(Fig. 5D; ref. 42), and Jak1 (Fig. 5E; ref. 45) was not affected
by treatment with platinum compounds. Similarly, the
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
1537
1538 Stat3 Inhibition by Novel Platinum Compounds
Figure 5. Effects of platinum complexes on signal molecules. Fibroblasts over-expressing EGF receptor (NIH3T3/hEGFR) and their v-Src –
transformed counterparts (NIH3T3/v-Src) were treated with platinum
complexes, cisplatin, CPA-1, CPA-7, and Pt(IV)Cl4 (20 Amol/L), or Src
inhibitor, SU6656 (10 Amol/L) for 24 hours and stimulated with or without
EGF for 5 minutes. Whole-cell lysates were prepared and analyzed on 5%
SDS-PAGE and transferred to a nitrocellulose membrane and probed by
Western blot using (A) antiphosphotyrosine antibody (4G10) or antibodies
against activated forms of (B) p38mapk, (C) Akt, (D) JNK, (E) Jak1, and
(F) Erk1 and Erk2.
induction by EGF of Erk1 and Erk2 was not significantly
altered by the same treatment (Fig. 5F). These findings
together indicate that CPA-1, CPA-7, and Pt(IV)Cl4 are
selective and potent inhibitors of constitutive activation of
Stat3 DNA-binding and Stat3-mediated gene expression in
malignant cells.
Inhibition of Cell Proliferation by Platinum Complexes
Based on previous studies that Stat3 has a key role in cell
growth (11 – 17), we investigated whether platinum complexes have effects on cell proliferation, which were
measured by trypan blue counting of viable cells and
[3H]-thymidine incorporation. Treatment with CPA-1,
CPA-7, or Pt(IV)Cl4 of human breast cancer cell lines
(MDA-MB-231 and MDA-MB-435), human prostate cancer
cell line (DU145), human non – small cell lung cancer line
(A549), mouse colon cancer cell line (CT26), mouse
melanoma cell line (M2), or v-Src – transformed mouse
fibroblasts, all of which harbor persistently activated Stat3,
strongly inhibited growth as measured by trypan blue
exclusion (Fig. 6, and data not shown) and [3H]-thymidine
incorporation (Fig. 7, and data not shown). In the tumor cell
lines, HCT116p53 / (human colon), and Cl10 (mouse
melanoma), which have low levels of constitutively active
Stat3 (Fig. 4C, (iv), and data not shown), treatment with
platinum compounds weakly diminished viability (Fig. 6).
Similarly, normal or tumor cell lines harboring no
constitutive Stat3 activity (normal mouse fibroblasts,
NIH3T3, and human breast carcinoma, MCF-7 or MDAMB-453) are less sensitive to these platinum complexes and
are partially inhibited in their growth (Figs. 6 and 7, and
data not shown). Moreover, treatment of mouse NIH3T3
and NIH3T3/v-Src fibroblasts with cisplatin has no effect
on growth of these cells (data not shown). Together, these
data suggest that malignant cells that contain constitutively activated Stat3 signaling are more sensitive to
platinum complexes in terms of cell proliferation.
Induction of Apoptosis by Platinum Complexes
Numerous studies have established that constitutive
Stat3 signaling is critical for the enhanced survival of
malignant cells (15, 19, 20, 51, 53, 54). We therefore
evaluated the effects of platinum complexes on the
viability and survival of malignant cells that harbor
constitutive Stat3 activity compared with those that do
not. Normal mouse (NIH3T3) and viral Src-transformed
(NIH3T3/v-Src) fibroblasts, as well as human breast
cancer cell lines and mouse colon, were treated with the
platinum compounds and subjected to TUNEL analysis
for evidence of DNA damage and apoptosis. Compared to
vehicle (DMSO) – treated cells, there is significant apoptosis observed in NIH3T3/v-Src, MDA-MB-435, and CT26
cell lines that harbor high constitutively active Stat3 (12,
21, 40, 41) and that are treated with CPA-1, CPA-7, or
Pt(IV)Cl4 (Fig. 8). In contrast, minimal to no significant
apoptosis was detected in similarly treated normal mouse
fibroblasts (NIH3T3), human breast cancer cells (MDAMB-453 and MCF-7) that lack activated Stat3. Similarly, in
the human colon cancer cell line, HCT116p53 / harboring
Figure 6. Effects of platinum complexes on cell viability. Human breast
carcinoma MDA-MB-453, MDA-MB-435, colon carcinoma HCT116p53 /
(human), and CT26 (murine), and mouse melanoma Cl10 and M2 cell lines
in culture were treated with different concentrations of platinum
complexes for the indicated times. The number of viable cells were then
enumerated by visualization under microscope and using trypan blue
exclusion. Values are the means of four independent determinations.
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
Figure 7.
Effects of platinum
complexes on cell proliferation. Normal and Src-transformed mouse fibroblasts, human tumor cells of the
breast (MDA-MB-231, MDA-MB-435,
MDA-MB-453, and MCF-7), lung
(A549) and prostate (DU145) were
treated with or without platinum complexes for 48 hours and analyzed for
extent of [3H]thymidine incorporation. Columns , means of three independent determinations; bars , SE.
low constitutively active Stat3, which is insensitive to Stat3
inhibition by dominant-negative Stat3h,9 no significant
apoptosis is observed. These findings parallel the minimal
effects of platinum compounds on proliferation of MCF-7
cells (Fig. 6), and together show that CPA-1, CPA-7, and
Pt(IV)Cl4 preferentially inhibit the survival of malignant
cells that are dependent on constitutively active Stat3. Our
findings have strong implications for treatment of tumors
derived from malignant cells that harbor constitutively
active Stat3, suggesting that such tumors might have
greater sensitivity to platinum (IV) compounds due to
blockade of aberrant Stat3 activity.
Platinum (IV) ^ Containing Compounds Induce Regression of ColonTumors in a Mouse Model
We extended our studies to evaluate the antitumor
efficacy of CPA-7 using mouse model colon tumors
harboring persistently active Stat3. Mouse colon tumor –
bearing mice were given i.v. injection with CPA-7 on days
1, 4, 7, 10, and 12 following implantation of tumor, and
tumor sizes measured every 3 days. Compared to control
(vehicle-treated) tumors, we observed strong or complete
regression in colon CT26 tumors following the i.v.
administration of CPA-7 (Fig. 9, top). Furthermore, significant to complete inhibition of constitutively active Stat3 is
observed in the majority of treated tumors (Fig. 9, bottom),
which correlates with tumor regression. In the tumor that
only partially regressed following treatment, there was
significant Stat3 activity, suggesting that failure to regress
is likely due to lack of inhibition of Stat3. These findings
indicate that platinum (IV) complexes induce the regression of colon tumors, accompanied by inhibition of Stat3
activity. Together with cell-based studies in Figs. 6, 7, and
9
G. Niu, et al. Role of Stat3 in regulating p53 expression and function, submitted
manuscript, 2004.
8, the findings here provide support for platinum (IV)
compound – induced tumor regression in part due to loss of
viability and apoptosis of tumor cells through inhibition
of constitutively active Stat3.
Discussion
Current application of cisplatin and its analogues as
anticancer agents is predominantly based on their DNAdamaging effects which contribute to cell death. Although
initial evidence indicates that cisplatin modulates MAPK
signaling pathway (36, 37), the effects of platinum
complexes on signal transduction events have not been
extensively studied. We provide evidence that novel
platinum (IV) compounds, CPA-1, CPA-7, and (Pt(IV)Cl4)
are potent disruptors of STAT activity. Of the three STAT
family members (Stat1, Stat3, and Stat5) examined here,
Stat3 is preferentially disrupted. In addition, CPA-7 is the
most potent compound in vitro, in whole cells and in
animal models of cancer. The disruption of STAT activity
by platinum compounds in part depends on their interaction with the proteins, as suggested by the direct inhibition
of DNA-binding activity in vitro, and by effects on cellular
phosphotyrosine levels of Stat3. It remains to be determined how prolonged treatment of cells with platinum
compounds alters phosphotyrosine Stat3 levels without
changes in the levels of Stat3 protein.
The biological effects of CPA-1, CPA-7, and Pt(IV)Cl4 in
our study include growth inhibition and apoptosis of
malignant cells that harbor constitutively active Stat3.
These effects are stronger and more selective against
malignant cells that contain persistently active Stat3 than
those that do not, reflecting in part the inhibition of
constitutive activation of Stat3 signaling, and providing
further support for a critical role for abnormal Stat3 activity
in the growth control and survival of transformed cells
(22 – 29). In contrast, the absence of consistent cellular
effects of cisplatin or CPA-3 in malignant cells harboring
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
1539
1540 Stat3 Inhibition by Novel Platinum Compounds
Figure 8. Induction of apoptosis by platinum complexes. Normal NIH3T3 fibroblasts and their v-Src – transformed counterparts, human breast carcinoma
cells (MDA-MB-453, MCF-7, and MDA-MB-435), as well as colon tumor cell lines HCT116p53 / (human) and CT26 (mouse) were treated with platinum
complexes for 48 hours and analyzed for evidence of DNA damage using TUNEL staining kit.
persistent Stat3 signaling parallels their lack of ability to
significantly inhibit Stat3 activity at the concentrations
used, which raises the possibility of different modes of
action for platinum complexes inside cells. Moreover, CPA-1
and CPA-7 are platinum (IV) complexes, in contrast to
cisplatin and CPA-3, which are platinum (II) – containing
complexes. It remains to be determined how inhibitory
effect against Stat3 activity and biological functions are
influenced by the oxidative state of platinum in CPA-1 and
CPA-7, an issue that requires evaluation of a series of novel
platinum (IV) complexes to resolve.
Although current studies indicate that disruption of
constitutive Stat3 signaling might not be an important
factor in the induction of biological effects to
cisplatin and CPA-3, they as well as others (36, 37)
strongly support modulation of signal transduction pathways as one of the key mechanisms underlying the
biological effects of platinum-containing complexes. Thus,
in addition to alkylation of DNA, the effects on signal
transduction pathways owing to cisplatin (36, 37), carboplatin, and other platinum complexes may be part of a
general molecular mechanism for their antitumor efficacy.
On the basis of the present data, we can conclude that
inhibition of constitutive Stat3 signaling is a key component
of the mechanism whereby the biological effects of the three
platinum (IV) complexes, CPA-1, CPA-7, and Pt(IV)Cl4, are
manifested.
Our studies also provide the rationale for evaluating
the antitumor effects of these platinum (IV) – containing
complexes in patients with tumors harboring aberrant Stat3
Figure 9. Tumor regression induced by platinum complex. Mouse colon
CT26 tumor – bearing mice were given CPA-7 (5 mg/kg) i.v. on days 1, 4,
9, 12, and 15. Tumor sizes were monitored every 3 to 4 days and plotted
(top ). Extracted tumor tissues following treatment were investigated for
Stat3 activity in in vitro DNA-binding assays with EMSA analysis (bottom).
Broken lines, control tumors (50% DMSO); solid lines, treated tumors.
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
signaling. Compared to cells that do not contain aberrant
Stat3 activity, malignant cells harboring constitutively
active Stat3 are more sensitive to the platinum IV
complexes, CPA-1, CPA-7, and Pt(IV)Cl4. This observation
indicates that tumors showing evidence of persistently
active Stat3 are more likely to be targeted by these
platinum (IV) compounds. Indeed, our initial observation
of the regression of mouse colon tumors harboring
constitutively active Stat3 upon platinum (IV) compound
administration provides a proof-of-concept for their
antitumor efficacy that is based on anti-Stat3 activity, as
supported by the abrogation of constitutive Stat3 activity
in the regressed tumors. These studies have to be
extended to other tumor types that harbor constitutively
active Stat3 for evaluation of sensitivity to novel platinum
(IV) complexes.
We do not exclude other modes of action for platinum
(IV) complexes, including possible interactions with other
signaling proteins, which might contribute to their antitumor cell activity. Contrary to other reports on cisplatin
(37, 38, 55 – 58), no significant effects of these novel
platinum (IV) complexes are observed on ERK1/2, p38,
JNK, Akt and Jaks, suggesting that direct modulation of
these signaling proteins may not contribute to the
biological effects of CPA-1, CPA-7, and Pt(IV)Cl4. A parallel
and independent study by our collaborators also provides
evidence for intracellular release of nitric oxide in the
presence of CPA-1 or CPA-7 but not CPA-3 or cisplatin.10
This raises the possibility that nitric oxide release might
represent one of the mechanisms for the antitumor cell
activity that is associated with these compounds. This other
study is in part consistent with our findings reported here
on the biological effects of CPA-1 and CPA-7, including the
induction of tumor regression.10 We also note the absence
of gross toxicity of compounds in cell types that do not
harbor aberrant Stat3 signaling. Except for the reduction in
their proliferation rate, cells with no evidence of constitutively active Stat3 are not affected. Thus, overall, the
biological actions of the novel platinum (IV) compounds
reflect their specificity towards tumor cells with abnormal
Stat3 activation.
In summary, our findings reported here strongly
support the potential of platinum compounds to
modulate signaling proteins, and the importance of
these interactions for their biological properties, including their overall therapeutic outcomes. Modulation of
Stat3 signaling pathway by certain platinum (IV)
compounds might be part of their general mode of
action. These findings provide the rationale for further
evaluation of the therapeutic potential of platinum (IV) –
containing compounds as inhibitors of Stat3 with
antitumor activity.
Acknowledgments
We thank members of the laboratory for stimulating discussions and the
Moffitt Cancer Center’s Molecular Imaging Core Facilities for imaging
support.
References
1. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling
proteins. Science 1994;264:1415 – 21.
2. Schindler C, Darnell JE Jr. Transcriptional responses to polypeptide
ligands: the JAK-STAT pathway. Annu Rev Biochem 1995;64:621 – 51.
3. Bromberg JF, Horvath CM, Wen Z, Schreiber RD, Darnell JE Jr.
Transcriptionally active Stat1 is required for the antiproliferative effects of
both interferon alpha and interferon gamma. Proc Natl Acad Sci U S A
1996;93:7673 – 8.
4. Fukada T, Hibi M, Yamanaka Y, et al. Two signals are necessary for cell
proliferation induced by a cytokine receptor gp130: involvement of signal
transducers and activators of transcription 3 in anti-apoptosis. Immunity
1996;5:449 – 60.
5. Darnell JE Jr. STATs and gene regulation. Science 1997;277:1630 – 5.
6. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells
respond to interferons. Annu Rev Biochem 1998;67:227 – 64.
7. Smithgall TE, Briggs SD, Schreiner S, Lerner EC, Cheng H, Wilson MB.
Control of myeloid differentiation and survival by stats. Oncogene
2000;19:2612 – 8.
8. Akira S. Roles of signal transducers and activators of transcription 3
defined by tissue-specific gene targeting. Oncogene 2000;19:2607 – 11.
9. Hirano T, Ishihara K, Hibi M. Roles of signal transducers and activators
of transcription 3 in mediating the cell growth, differentiation and survival
signals relayed through the IL-6 family of cytokine receptors. Oncogene
2000;19:2548 – 56.
10. Kotenko SV, Pestka S. Jak-Stat signal transduction pathway through
the eyes of cytokine class II receptor complexes. Oncogene 2000;
19:2557 – 65.
11. Nielsen M, Kaltoft K, Nordahl M, et al. Constitutive activation of a
slowly migrating isoform of Stat3 in mycosis fungoides: tyrphostin AG490
inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines.
Proc Natl Acad Sci U S A 1997;94:6764 – 9.
12. Turkson J, Bowman T, Garcia R, Caldenhoven E, De Groot RP, Jove
R. Stat3 activation by Src induces specific gene regulation and is required
for cell transformation. Mol Cell Biol 1998;18:2545 – 52.
13. Bromberg JF, Horvath CM, Besser D, Lathem WW, Darnell JE Jr.
Stat3 activation is required for cellular transformation by v-src. Mol Cell
Biol 1998;18:2553 – 8.
14. Grandis JR, Drenning SD, Chakraborty A, et al. Requirement of Stat3
but not Stat1 activation for epidermal growth factor receptor-mediated
cell growth in vitro . J Clin Invest 1998;102:1385 – 92.
15. Catlett-Falcone R, Landowski TH, Oshiro MM, et al. Constitutive
activation of Stat3 signaling confers resistance to apoptosis in human
U266 myeloma cells. Immunity 1999;10:105 – 15.
16. Bromberg JF, Wrzeszczynska MH, Devgan G, et al. Stat3 as an
oncogene. Cell 1999;98:295 – 303.
17. Nielsen M, Kaestel CG, Eriksen KW, et al. Inhibition of constitutively
activated Stat3 correlates with altered Bcl-2/Bax expression and induction
of apoptosis in mycosis fungoides tumor cells. Leukemia 1999;13:735 – 8.
18. Niu G, Heller R, Catlett-Falcone R, et al. Gene therapy with dominantnegative Stat3 suppresses growth of the murine melanoma B16 tumor
in vivo. Cancer Res 1999;59:5059 – 63.
19. Grandis JR, Drenning SD, Zeng Q, et al. Constitutive activation of
Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis
in vivo. Proc Natl Acad Sci U S A 2000;97:4227 – 32.
20. Epling-Burnette PK, Lui JH, Catlette-Falcone R, et al. Inhibition of
STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression. J Clin Invest 2001;107:351 – 62.
21. Garcia R, Bowman TL, Niu G, et al. Constitutive activation of Stat3
by the Src and JAK tyrosine kinases participates in growth regulation
of human breast carcinoma cells. Oncogene 2001;20:2499 – 513.
10
M.A. Blaskovich, et al. Submitted manuscript, 2004.
22. Garcia R, Jove R. Activation of STAT transcription factors in
oncogenic tyrosine kinase signaling. J Biomed Sci 1998;5:79 – 85.
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
1541
1542 Stat3 Inhibition by Novel Platinum Compounds
23. Catlett-Falcone R, Dalton WS, Jove R. STAT proteins as novel targets
for cancer therapy. Signal transducer an activator of transcription. Curr
Opin Oncol 1999;11:490 – 6.
24. Bowman T, Garcia R, Turkson J, Jove R. STATs in oncogenesis.
Oncogene 2000;19:2474 – 88.
25. Turkson J, Jove R. STAT proteins: novel molecular targets for cancer
drug discovery. Oncogene 2000;19:6613 – 26.
26. Song JI, Grandis JR. STAT signaling in head and neck cancer.
Oncogene 2000;19:2489 – 95.
27. Coffer PJ, Koenderman L, de Groot RP. The role of STATs in myeloid
differentiation and leukemia. Oncogene 2000;19:2511 – 22.
28. Lin TS, Mahajan S, Frank DA. STAT signaling in the pathogenesis
and treatment of leukemias. Oncogene 2000;19:2496 – 504.
29. Yu H, Jove R. The STATs of cancer: new molecular targets come
of age. Nat Rev Cancer 2004;4:97 – 105.
30. Niu G, Shain K, Huang M, et al. Overexpression of a dominantnegative signal transducer and activator of transcription 3 variant in tumor
cells leads to production of soluble factors that induce apoptosis and cell
cycle arrest. Cancer Res 2001;61:3276 – 80.
31. Turkson J, Ryan D, Kim JS, et al. Phosphotyrosyl peptides block
Stat3-mediated DNA-binding activity, gene regulation and cell transformation. J Biol Chem 2001;276:45443 – 55.
32. Turkson J, Kim JS, Zhang S, et al. Novel peptidomimetic inhibitors
of signal transducer and activator of transcription 3 dimerization and
biological activity. Mol Cancer Ther 2004;3:261 – 9.
33. Buettner R, Mora LB, Jove R. Activated STAT signaling in human
tumors provides novel molecular targets for therapeutic intervention. Clin
Cancer Res 2002;8:945 – 54.
34. Ardizzoni A, Antonelli G, Grossi F, Tixi L, Cafferata M, Rosso R. The
combination of etoposide and cisplatin in non-small-cell lung cancer
(NSCLC). Ann Oncol 1999;10:S13 – 7.
35. Nitiss JL. A copper connection to the uptake of platinum anticancer
drugs. Proc Natl Acad Sci U S A 2002;99:13963 – 5.
36. Bose RN. Biomolecular targets for platinum antitumor drugs. Mini Rev
Med Chem 2002;2:103 – 11.
37. Persons DL, Yazlovitskaya EM, Cui W, Pelling JC. Cisplatin-induced
activation of mitogen-activated protein kinases in ovarian carcinoma cells:
inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin. Clin Cancer Res 1999;5:1007 – 14.
38. Sanchez-Perez I, Murguia JR, Perona R. Cisplatin induces a persistent
activation of JNK that is related to cell death. Oncogene 1998;16:533 – 40.
43. Burdge J, Stanko J, Palmer J. Oxidation of platinum (II) bis(ethylenediamine) complexes with the oxides of nitrogen, NO and NO2: a
model for the synthesis of platinum (IV) nitro compounds as potential
antitumor agents. Florida Scientist 1995;58:274 – 85.
44. Palmer J, Burdge J, Stanko J. Oxidation of platinum (II) mono(ethylenediamine) complexes with the oxides of nitrogen, NO and NO2:
possible antitumor agents. Florida Scientist 1995;58:359 – 65.
45. Zhang Y, Turkson J, Carter-Su C, et al. Activation of Stat3 in v-Src
transformed fibroblasts requires cooperation of Jak1 kinase activity. J Biol
Chem 2000;275:24935 – 44.
46. Garcia R, Yu CL, Hudnall A, et al. Constitutive activation of Stat3 in
fibroblasts transformed by diverse oncoproteins and in breast carcinoma
cells. Cell Growth Differ 1997;8:1267 – 76.
47. Wagner BJ, Hayes TE, Hoban CJ, Cochran BH. The SIF binding
element confers sis /PDGF inducibility onto the c-fos promoter. EMBO J
1990;9:4477 – 84.
48. Gouilleux F, Moritz D, Humar M, Moriggl R, Berchtold S, Groner B.
Prolactin and interleukin-2 receptors in T lymphocytes signal through
a MGF-STAT5-like transcription factor. Endocrinology 1995;136:
5700 – 8.
49. Seidel HM, Milocco LH, Lamb P, Darnell JE Jr., Stein RB, Rosen J.
Spacing of palindromic half sites as a determinant of selective STAT
(signal transducers and activators of transcription) DNA binding and
transcriptional activity. Proc Natl Acad Sci U S A 1995;92:3041 – 5.
50. Croxton RL, Ma Y, Cress WD. Differences in DNA binding properties
between E2F1 and E2F4 specify repression of the Mcl-1 promoter.
Oncogene 2002;21:1563 – 70.
51. Bowman T, Broome M, Sinibaldi N, et al. Stat3-mediated Myc
expression is required for Src oncogenesis and PDGF-induced mitogenesis.
Proc Natl Acad Sci U S A 2000;98:7319 – 24.
52. Liu AX, Testa JR, Hamilton TC, Jove R, Nicosia SV, Cheng JQ. AKT2,
a member of the protein kinase B family, is activated by growth factors,
v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human
ovarian epithelial cancer cells. Cancer Res 1998;58:2973 – 7.
53. Grandis JR, Zeng Q, Drenning SD. Epidermal growth factor receptormediated stat3 signaling blocks apoptosis in head and neck cancer.
Laryngoscope 2000;110:868 – 74.
54. Horiguchi A, Oya M, Marumo K, Murai M. STAT3, but not ERKs,
mediates the IL-6-induced proliferation of renal cancer cells. Kidney Int
2002;61:926 – 38.
39. Johnson PJ, Coussens PM, Danko AV, Shalloway D. Overexpressed
pp60c-src can induce focus formation without complete transformation
of NIH 3T3 cells. Mol Cell Biol 1985;5:1073 – 83.
55. Mansouri A, Ridgeway LD, Korapati AL, et al. Sustained activation of
JNK/p38 MAPK pathways in response to cisplatin leads to Fas ligand
induction and cell death in ovarian carcinoma cells. J Biol Chem
2003;278:19245 – 56.
40. Yu CL, Meyer DJ, Campbell GS, et al. Enhanced DNA-binding activity
of a Stat3-related protein in cells transformed by the Src oncoprotein.
Science 1995;269:81 – 3.
56. Zanke BW, Boudreau K, Rubie E, et al. The stress-activated protein
kinase pathway mediates cell death following injury induced by cis platinum, UV irradiation or heat. Curr Biol 1996;6:606 – 13.
41. Wang T, Niu G, Kortylewski M, et al. Regulation of the innate and
adaptive immune responses by Stat-3 signaling in tumor cells. Nat Med
2004;10:48 – 54.
57. Potapova O, Haghighi A, Bost F, et al. The Jun kinase/stressactivated protein kinase pathway functions to regulate DNA repair and
inhibition of the pathway sensitizes tumor cells to cisplatin. J Biol Chem
1997;272:14041 – 4.
42. Turkson J, Bowman T, Adnane J, et al. Requirement for Ras/Rac1mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional
activity induced by the Src oncoprotein. Mol Cell Biol 1999;19:7519 – 28.
58. Song H, Sondak VK, Barber DL, Reid TJ, Lin J. Modulation of Janus
kinase 2 by cisplatin in cancer cells. Int J Oncol 2004;24:1017 – 26.
Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Inhibition of constitutive signal transducer and activator of
transcription 3 activation by novel platinum complexes with
potent antitumor activity
James Turkson, Shumin Zhang, Jay Palmer, et al.
Mol Cancer Ther 2004;3:1533-1542.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://mct.aacrjournals.org/content/3/12/1533
This article cites 57 articles, 23 of which you can access for free at:
http://mct.aacrjournals.org/content/3/12/1533.full#ref-list-1
This article has been cited by 25 HighWire-hosted articles. Access the articles at:
http://mct.aacrjournals.org/content/3/12/1533.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.