Cancer Therapy Vol 1,
31-37, 2003
Vascular
endothelial growth factor modulates cisplatin sensitivity in human ovarian
carcinoma cells
Research Article
Guodong
Hu, Sean Ryan, Yunfeng Zhu, Eddie Reed, Xiping Li, Gangduo Wang, and Qingdi Q.
Li*
The Mary Babb Randolph Cancer Center and
Department of Microbiology, Immunology and Cell Biology, West Virginia
University School of Medicine and Robert C. Byrd Health Sciences Center,
Morgantown, WV 26506, USA
__________________________________________________________________________________________________
*Corresponding Author:
Qingdi Q. Li, M.D., Ph.D., 1831 Mary Babb Randolph Cancer Center, West Virginia
University, Health Sciences Center, P.O. Box 9300, Morgantown, WV 26506-9300,
USA; Tel: 304-293-6870; Fax: 304-293-4667; e-mail: qli@hsc.wvu.edu
Key words:
Angiogenesis, VEGF, ovarian cancer, Caov3 cells, cisplatin resistance.
Abbreviations: VEGF, vascular endothelial growth
factor; VPF, vascular permeability factor; GSH, glutathione; cisplatin (CDDP),
cis-diamminedichloroplatinum (II); bFGF, basic fibroblast growth factor; HGF,
hepatocyte growth factor; PGF, placenta growth factor; PDEGF, platelet-derived
endothelial growth factor; PBS, phosphate-buffered saline; DMSO, dimethyl
sulfoxide; MTT, 3-(4,5-dimethylthuazole-2-yl)-2,5 diphenyl tetrazolium bromide;
RT-PCR, reverse transcriptase polymerase chain reaction; FasL, Fas ligand; NER,
nucleotide excision repair; JNK, c-Jun N-terminal kinase; ERK, extracellular
signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK,
mitogen-activated protein kinase/ERK kinase; AP-1, activator protein 1.
Received: 11
February 2003; Accepted: 19 February 2003; electronically published: April 2003
Summary
Cisplatin is
among the most effective and widely used chemotherapeutic agents employed for
treatment of human cancers, and a major limitation of cisplatin chemotherapy is
serious drug resistance. Vascular endothelial growth factor (VEGF), a potent
angiogenic factor, plays an important role in cell growth and survival of
endothelial cells and tumor cells. However, the role of VEGF in cisplatin
resistance in human cancers is unclear. Therefore, the present study sought to
examine the effect of VEGF on cisplatin-induced cytotoxicity in human ovarian
cancer CaOV3 cells. We show in this report that VEGF mediated cytoprotection
against cisplatin-caused cell killing and significantly increased cell survival
in CaOV3 cells exposed to cisplatin. VEGF was found to reduce cisplatin
cytotoxicity and decrease cisplatin sensitivity in these cells, which are
dependent upon the concentrations of cisplatin. The effect of VEGF was also
sequence-dependent. Concurrent treatment of VEGF and cisplatin markedly
increased cell viability as compared to cells exposed to cisplatin alone. By
contrast, only a little effect of VEGF was observed when cells were treated
with VEGF after or prior to cisplatin. These findings suggest that VEGF may
contribute to the chemoresistance to cisplatin in patients with ovarian cancer
and other tumors, and hence highlight that potential therapeutic strategies of
anti-angiogenesis which specifically inhibit VEGF activity may reverse drug
resistance to cisplatin.
I.
Introduction
Human ovarian cancer is the fifth leading cause of
cancer death among women in the United States and the most common cause of
death in women in whom gynecologic cancer develops. The mainstay of therapy for
advanced stage ovarian cancer is cisplatin-based systemic chemotherapy (Young
et al, 1993; Reed, 1993; Reed, 1996; Reed et al, 1996; Reed, 1998). However,
long-term disease-free survival following appropriate aggressive initial
treatment ranges from 10 to 20% (Young et al, 1993; Omura et al, 1991). The
disappointing survival statistics stem from the fact that while most patients
have a response to initial therapy, the majority of these responses are
transient. Most patients will have cisplatin-resistant disease. The precise
mechanism of cisplatin resistance in human cancers is, however, still not fully
understood although substantial efforts have been made to solve this enigma.
Multiple mechanisms have been implicated in the development of cisplatin
resistance including reduced accumulation of the drug, elevated levels of
glutathione (GSH), enhanced expression of metallothionein, increased DNA
repair, enhanced tolerance of cisplatin damage, increased levels of
Bcl-2-related anti-apoptosis genes, and alterations in signal transduction
pathways involved in apoptosis (Reed et al, 1996;
Gosland et al, 1996; Dabholkar and Reed, 1996; Kerbel, 1997; Reed, 1998; Reed,
1998).
Angiogenesis is the process of new blood vessel
growth and is necessary for growth of solid malignant tumors (Folkman, 1991).
Angiogenesis not only allows a tumor to increase in size, but also increases the
probability of metastasis (Folkman, 1993). Vessel growth is controlled by a balance of
endogenous inhibitors and stimulators (Folkman, 1991). A number of growth factors and cytokines have been
identified as potential positive inducers of angiogenesis, including vascular
endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF),
hepatocyte growth factor (HGF), placenta growth factor (PGF), and
platelet-derived endothelial growth factor (PDEGF) (Kerbel, 2000; Slodkowska et
al, 2000; Liekens et al, 2001). Recently, an increasing number of studies both in
vitro and in mice demonstrated that
angiogenic growth factors augment tumor cell survival and confer drug
resistance by inhibiting apoptosis (Borsellino et al, 1995; Volm et al, 1999;
Grothey et al, 1999; Coleman et al, 2000). For instance, evidence showed that
HGF reduces sensitivity to chemotherapeutic agents and stimulates cell invasion
and migration (Meng et al, 2000). Other investigations indicated that elevated
levels of intracellular bFGF correlate with resistance to fludarabine in
chronic lymphocytic leukemia (Menzel et al, 1996). Furthermore, overexpression
of bFGF is associated with resistance to cisplatin in a human bladder cancer
cell line (Miyake et al, 1998). Moreover, the addition of exogenous bFGF to
endothelial cells inhibits apoptosis induced by DNA damage from ionizing
radiation (Fuks et al, 1994). However, the role of VEGF and other angiogenic
factors in the development of cisplatin drug resistance is unknown at the
present time. The goal of the current study was to evaluate the effect of VEGF
on cisplatin antitumor activity in human ovarian cancer cells. We show in this
paper that VEGF decreases drug sensitivity and increases cell survival in human
CaOV3 ovarian tumor cells exposed to cisplatin.
II.
Materials and methods
A.
Cell line and cell culture conditions
The human ovarian carcinoma cell line CaOV3 (HTB-75;
American Type Culture Collection, Manassas, VA) that has been described
previously was used in all of the experiments. Cells were cultured in
monolayers using a RPMI 1640 medium supplemented with 10% (v/v) fetal calf
serum, 2 mM L-glutamine, 0.2 units/ml human insulin, 50 units/ml penicillin,
and 50 mg/ml
streptomycin (Life Technologies, Inc, Gaithersburg, MD). Cells were grown in
logarithmic growth at 37 ûC in a humidified atmosphere consisting of 5% CO2 and
95% air. Cells were routinely tested for mycoplasma infection using a
commercial assay system (MytoTect; Life Technologies); new cultures were
established monthly from frozen stocks. All media and reagents contained
<0.1 ng/ml endotoxin as determined by Limulus polyphemus amebocyte lysate
assay (Whittaker Bioproducts, Walkersville, MD). Cell viability was determined
in triplicate by trypan blue dye exclusion. Before starting the experiments,
cells were grown to 70-90% confluence after subculturing. Cisplatin
(Sigma-Aldrich Co., St. Louis, MO) was initially dissolved in
phosphate-buffered saline without Ca2+ or Mg2+ at 1.0
mg/ml (3.33 mM
cisplatin), and dilutions from this solution were made in medium to obtain the
desired drug treatment concentrations. VEGF (Human Recombinant VEGF165) was
purchased from Oncogene Research Products (Cambridge, MA). VEGF was initially
dissolved in phosphate-buffered saline (PBS) at 5 mg/ml, and dilutions from this solution were made in
medium to obtain the desired cytokine treatment concentrations.
CaOV3 cells were assayed for
sensitivity to cisplatin by measurement of the inhibition of growth following
24 to 48-h exposure to cisplatin ranging from 20 to 40 mM. Cells were seeded at an initial cell density of 2 X
104 cells/ml. Cells were starved for 48 h with the medium containing
0.2% fetal bovine serum. Cells were then treated with VEGF or cisplatin alone,
or the combination of VEGF and cisplatin in different sequences. After
continuous contact with cisplatin for 24-48 h, medium was removed, and cell
viabilities were determined using the MTT cell viability assay. Cells treated
similarly in the absence of VEGF and/or cisplatin served as controls.
B. Cell toxicity
assay
The effect of VEGF and/or
cisplatin on antitumor activity in human CaOV3 ovarian carcinoma cells was
determined by the MTT survival assay, or using a commercial MTT assay kit
(CellTiter 96‰ Aqueous One Solution
Cell Proliferation Assay; Promega Corporation, Madison, WI) according to the
manufacturerÕs instructions. The MTT survival assay was performed as described
previously (Yu et al, 2000). The MTT assay is a commonly used
method in evaluation of cell survival, based on the ability of viable cells to
convert MTT, a soluble tetrazolium salt [3-(4,5-dimethylthuazole-2-yl)-2,5
diphenyl tetrazolium bromide], into an insoluble formazan precipitate, which is
quantitated by spectrophotometry following solubilization in dimethyl sulfoxide
(DMSO). Briefly, CaOV3 cells untreated and treated with VEGF or cisplatin
alone, or the combination of VEGF and cisplatin in 96-well tissue culture
dishes were incubated with MTT (2 mg/ml) for 4 h. The cells were then solubilized in 125 ml of DMSO and absorbance readings
were taken using a 96-well Opsys MRŠ Microplate Reader (ThermoLabsystems; Chantilly, VA). The amount of MTT
dye reduction was calculated based on the difference between absorbance at 570
nm and at 630 nm. Cell viability in treated cells was expressed as the amount
of dye reduction relative to that of untreated control cells. The wells which
contained only medium and 10 ml of MTT were used as blanks for the plate reader. Three sets of
experiments were performed in 8-12 wells for each treatment.
III. Results
A major goal of the ongoing project is to understand
whether angiogenic growth factors that induce angiogenesis might reverse the
drug resistance to cisplatin in human ovarian cancer and better understand the
underlying mechanisms in the process. In the present investigation, we first
determined whether the angiogenic factor VEGF could influence the cisplatin
anticancer activity in human ovarian carcinoma cells. VEGF was found to
significantly reduce cell susceptibility to cell killing caused by cisplatin
and augment cell survival in the CaOV3 human ovarian tumor cell line. As shown
in Fig. 1, concurrent treatment of
both VEGF and cisplatin for 24 h dramatically decreased cisplatin-induced cell
killing in these cells from 80% cells to 32-47% cells. This amounts to
approximately a 2.5-fold reduction in the amount of cell killing as compared to
the control in which cells were treated with cisplatin alone. A greater effect
of VEGF was observed in cells exposed to VEGF plus cisplatin for 24 h, and then
fresh medium containing only VEGF was replenished for an additional 24 h. In
contrast, only a little effect of VEGF in this regard was seen when VEGF was
given after or prior to cisplatin.
We also examined the cytoprotective effect of VEGF
in cells exposed to 20 mM cisplatin for a longer time (48 h) and in cells
exposed to a higher concentration of cisplatin at 40 mM. In
each case, the effect of VEGF was virtually identical to the effect seen in
cells exposed to 20 mM cisplatin for 24 h. Fig. 2 shows that there was marked cell kill in the
cisplatin-treated group, in which approximately 87% of the cells were killed in
a 48-h incubation time. By contrast, VEGF treatment significantly diminished
the cell kill in this model system, yielding a 4.7 to 5.8-fold higher level of
cell viability than in cells exposed to cisplatin alone. The same was true when
CaOV3 cells were exposed to 40 mM of cisplatin.
Figure 1. Effect of VEGF on cytotoxicity by CDDP (20
mM for 24 h) in
human ovarian carcinoma cells as assessed by the MTT survival assay.
2 X 104 cells per well from CaOV3 cells were evenly
distributed in 96-well plates, and were starved for 48 h in culture medium
containing 0.2% fetal bovine serum. Cells were then treated as the following:
Control, treated with medium only; VEGF alone, treated with 50 ng/ml VEGF only;
CDDP alone, exposed to CDDP at 20 mM for 24 h, and fresh medium was then replenished;
CDDP-VEGF, exposed to CDDP for 24 h, and then fresh medium containing VEGF was
replenished; CDDP+VEGF, exposed to CDDP and VEGF for 24 h, and then fresh
medium was replenished; CDDP+VEGF-VEGF, exposed to CDDP and VEGF for 24 h, and
then fresh medium containing VEGF was replenished; VEGF-CDDP, treated with VEGF
for 24 h, changed to fresh medium containing CDDP for another 24 h, and then
fresh medium was replenished; VEGF-CDDP-VEGF, treated with VEGF for 24 h,
changed to fresh medium containing CDDP for an additional 24 h, and then fresh
medium containing VEGF was replenished. All the cells were harvested 48 h from
the time when 20 mM CDDP was added to the culture. Cell viability was measured by the MTT
assay and is expressed as a percentage of untreated control. CDDP, cisplatin.
Our data in Fig. 3 shows that VEGF, at the concentration of 50 ng/ml,
both decreased cisplatin-induced cytotoxicity and increased cell survival in
these cells. Table I is the
comparison of the effect of VEGF on cell viability between different
concentrations of cisplatin, or different exposure time to the drug in human
CaOV3 ovarian cancer cells.
As seen in the table, there is no significant
difference in the effect of VEGF on cell toxicity between the cells exposed to
cisplatin for 24 h or for 48 h. However, the cell viability following cisplatin
and VEGF treatment was much lower in cells exposed to 40 mM
cisplatin than in cells exposed to 20 mM cisplatin, indicating
that the higher the concentration of cisplatin, the lower the protective effect
of VEGF. Together, these results suggest that VEGF has strong cytoprotective
activity against cisplatin-caused cell death and promotes cell survival in
cisplatin-treated human CaOV3 ovarian cancer cells.
Figure 2. Effect of VEGF on cytotoxicity by CDDP (20
mM for 48 h) in
human ovarian cancer cells as determined by the MTT cell viability assay.
2 X
104 cells per well from CaOV3 cells were evenly plated in 96-well
plates, and were starved for 48 h in culture medium containing 0.2% fetal
bovine serum. Cells were then treated as the following: Control, treated with
medium only; VEGF alone, treated with 50 ng/ml VEGF only; CDDP alone, exposed
to CDDP at 20 mM for 48 h, and fresh medium was then replenished;
CDDP-VEGF, exposed to CDDP for 48 h, and then fresh medium containing VEGF was
replenished; CDDP+VEGF, exposed to CDDP and VEGF for 48 h, and then fresh
medium was replenished; CDDP+VEGF-VEGF, exposed to CDDP and VEGF for 48 h, and
then fresh medium containing VEGF was replenished; VEGF-CDDP, treated with VEGF
for 24 h, changed to fresh medium containing CDDP for 48 h, and then fresh
medium was replenished; VEGF-CDDP-VEGF, treated with VEGF for 24 h, changed to
fresh medium containing CDDP for 48 h, and then fresh medium containing VEGF
was replenished. All the cells were harvested 72 h from the time when 20 mM CDDP was added to the culture. Cell viability was
measured by the MTT assay and is expressed as a percentage of untreated
control. CDDP, cisplatin.
Figure 3. Effect of VEGF on cell toxicity by CDDP
(40 mM
for 24 h) in human ovarian tumor cells as measured by the MTT survival assay.
2 X
104 cells per well from CaOV3 cells were evenly distributed in
96-well plates, and were starved for 48 h in culture medium containing 0.2%
fetal bovine serum. Cells were then treated as the following: Control, treated
with medium only; VEGF alone, treated with 50 ng/ml VEGF only; CDDP alone,
exposed to CDDP at 40 mM for 24 h, and fresh medium was then replenished;
CDDP-VEGF, exposed to CDDP for 24 h, and then fresh medium containing VEGF was
replenished; CDDP+VEGF, exposed to CDDP and VEGF for 24 h, and then fresh
medium was replenished; CDDP+VEGF-VEGF, exposed to CDDP and VEGF for 24 h, and
then fresh medium containing VEGF was replenished; VEGF-CDDP, treated with VEGF
for 24 h, changed to fresh medium containing CDDP for an additional 24 h, and
then fresh medium was replenished; VEGF-CDDP-VEGF, treated with VEGF for 24 h,
changed to fresh medium containing CDDP for another 24 h, and then fresh medium
containing VEGF was replenished. All the cells were harvested 48 h from the
time when 40 mM
CDDP was added to the culture. Cell viability was determined by the MTT assay
and is expressed as a percentage of untreated control. CDDP, cisplatin
IV. Discussion
VEGF, also known as vascular permeability factor
(VPF), is a cytokine/growth factor, and has been known to be a potent,
endothelial-cell-specific angiogenic mitogen. VEGF is secreted from tumor cells
and other cells via its specific binding to its tyrosine kinase receptors
(VEGFR1/Flt-1 and VEGFR2/Flk-1/KDR). Binding of VEGF to its receptors leads to
intracellular propagation of a mitogenic signal through activation of the PI3
kinase-Akt and the ras-raf-MAP kinase pathways. VEGF and its receptors are
expressed in angiogenic tissues during development, wound healing, and other
situations such as neoplasm (Boocock et al, 1995) when
angiogenesis occurs. Evidence has been accumulated that VEGF and its receptor
mRNAs or proteins have been identified by reverse transcriptase polymerase
chain reaction (RT-PCR), in situ
hybridization, or immunohistochemistry in a number of tumors, including ovarian
cancer (Boocock et al, 1995). The spatial and temporal patterns of expression
of VEGF and its receptors as well as the results of targeted mutagenesis
support that they are required for both normal and pathological angiogenesis
during development. Similarly, the role of VEGF in tumor angiogenesis has been
clearly demonstrated using tumor models in rodents. Moreover, recent studies
also found that VEGF plays a role in the regulation of apoptosis induction and
cell survival. Thus, VEGF contributes to the development and progression of
malignant tumors.
However, the role of VEGF and its receptor tyrosine
kinases in the formation and development of drug resistance in human cancers
remains unknown. In the present study, we demonstrated for the first time that
VEGF plays an important role in the modulation of cisplatin antitumor activity
in human ovarian carcinoma cells.
The addition of exogenous VEGF to the growth medium of
CaOV3 cells markedly enhanced the dose-dependent survival of cells exposed to
increasing concentrations of cisplatin, and therefore directly reduced the
sensitivity of CaOV3 cells to this chemotherapy drug. The cytoprotective effect
of VEGF against cisplatin toxicity is sequence-dependent, with maximal effect
seen in cells exposed to VEGF and cisplatin simultaneously, suggesting that
VEGF may exert its action through reducing cisplatin-caused cell damage. A
higher survival rate was observed in cells treated with VEGF plus cisplatin for
24 h, followed by VEGF only for an additional 24 h, as compared to the cells
incubated with medium only after VEGF and cisplatin were removed from the
cultures. This appears to suggest that continuous exposure of the cells to VEGF
after cisplatin damage may prevent the cells from further damage or apoptosis
caused by cisplatin, or it may enhance cell repair of cisplatin-induced DNA
damage leading to a higher rate of cell viability. Exogenous VEGF did not
produce any cytotoxic effects in the absence of cisplatin, and it had the
expected stimulatory effect on cell growth (Figs. 1 and 2).
Similar effect of VEGF was also observed in TOV-21G human ovarian cancer cell
line, indicating that augmented cell survival and decreased cisplatin
sensitivity appear to be a common effect of VEGF in different ovarian cancer
cells.
The mechanism underlying the
effect of VEGF in ovarian tumor cells is, however, unclear at this point. Given
its broad spectrum of activities, VEGF may exert its effect in mediating the
development of drug resistance through several ways. First of all, VEGF may be
involved in cisplatin drug resistance via anti-apoptotic activity. Recently,
experimental and clinical studies showed that VEGF was related not only to
angiogenic activity, but also to the inhibition of apoptotic activity
(Slodkowska et al, 2000). For example, the effects of VEGF on delaying
apoptosis and prolonging the survival of tumor cell may be indirect, via the
inhibition of specific genes that promote apoptosis, such as down-regulating
Fas and Fas ligand (FasL) proteins, or decreasing levels of cytochrome c in the
cytoplasm (Volm et al, 1996; Coleman et al, 2000). Alternatively, VEGF may
block cisplatin-induced apoptosis through reducing cisplatin-caused DNA damage.
Cisplatin-induced apoptosis has been closely tied to its ability to cause DNA
damage (Eastman, 1990).
In addition, VEGF-mediated
protection of tumor cells against cisplatin may result not only from activation
of an anti-apoptosis pathway, but also from an increase in repair of DNA
damage. In other words, VEGF may modulate cisplatin sensitivity indirectly
through the regulation of DNA repair activity. Although we do not have direct
evidence at this point that VEGF mediate this effect by enhancing DNA repair,
we showed in a separate study that SU5416, a selective inhibitor of VEGF
receptors, counteracted the effect of VEGF by augmenting cisplatin cytotoxicity
and increasing cisplatin sensitivity in human ovarian tumor cells. We further
found that the effect of SU5416 on the increase in cell death or reduction of
cell survival of cisplatin-treated cells is due in part to the reduction in
repair efficiency of cisplatin-caused DNA damages. It is broadly accepted that
the antitumor activity of cisplatin results from the formation of cisplatin-DNA
adducts that strongly interfere with the processing of genomic information
(Rosenberg, 1979; Reed et al, 1993; Dabholkar and Reed, 1996). Cisplatin-DNA
damage is repaired predominantly by the nucleotide excision repair (NER)
machinery. Enhanced DNA repair capacity contributes to the formation of drug
resistance to cisplatin in a wide variety of tumor cells.
Our and other previous studies
revealed that cisplatin may increase NER repair gene expression and DNA repair
activity through a JNK-AP1 pathway leading to cell survival (Potapova et al,
1997; Li et al, 1998; Li et al, 1998; Li et al, 1999). On the other hand, a great deal of studies have
supported the general view that activation of the ERK pathway delivers a
survival signal. Consistent with such a prosurvival function for ERK, studies
have shown that an inhibition of ERK signaling leads to increased sensitivity
of ovarian cancer cell lines to cisplatin (Hayakawa et al, 1999; Persons et al,
1999). Therefore, it is possible that JNK and ERK may act collaboratively or
synergistically to enhance survival of cisplatin-treated cells, as inhibition
of either pathway accentuated cisplatin toxicity (Hayakawa et al, 1999). Based
on these observations, we propose that VEGF may stimulate a ras-raf-MEK-ERK
or PI3K-Akt cascade activity that enhances cisplatin-induced activation of
JNK-AP1. Such a mechanism might serve to integrate the actions of receptor
protein tyrosine kinases and non-receptor protein tyrosine kinases, which may
underlie the mechanism of VEGF and cisplatin mediated DNA repair and cell
survival in human ovarian cancer and other carcinomas. Studies are in progress
to explore whether VEGF mediates cytoprotection against cisplatin-induced
apoptosis in human cancer cells by upregulating apoptosis-rescue signals,
assess the effect of VEGF on DNA repair activity, and elucidate the role of
PI3K-Akt, ERK or JNK in the signal transduction pathways through which VEGF
modulates DNA repair activity or apoptotic activity in human carcinoma cells.
In conclusion, we provided in vitro evidence for the first time that VEGF
mediated cytoprotection against cisplatin-caused cell killing and significantly
increased cell survival in human ovarian cancer cells exposed to cisplatin.
Taken together with previous studies, our results strengthen the case that VEGF
contributes to the carcinogenesis and chemoresistance of the chemotherapeutic
agent cisplatin. Strategies targeting VEGF signaling pathway or the activity of
VEGF, or down-regulating its expression could be employed to reduce drug
resistance, increase tumor cell apoptosis, and enhance the chemotherapeutic
effectiveness of cisplatin.
Acknowledgements
This project was
supported by grants from the National Institutes of Health, Bethesda, Maryland
(No. 1P20RR016440-010003) and West Virginia University Research Development
Grant (to Q.Q.L.).
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Front row from left: Qingdi
Q. Li and Sean Ryan
Rear row from left: Hang Hu, Guodong Hu, Xiping Li and Gangduo Wang
