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72 Current Cancer Drug Targets, 2009, 9, 72-80

The Role of Downstream Signaling Pathways of the Epidermal Growth


Factor Receptor for Artesunate’s Activity in Cancer Cells
V. Badireenath Konkimalla1, James A. McCubrey2 and Thomas Efferth*,1

1
German Cancer Research Center, Pharmaceutical Biology, Heidelberg, Germany; 2Department of Microbiology and
Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA
Abstract: Epidermal growth factor (EGF) and its receptor (EGFR) as well as the EGFR-coupled Ras>Raf>MEK>ERK
pathway are known to affect the survival of cancer cells upon chemotherapeutic treatment. In the present investigation, we
analyzed the role of EGFR signaling pathways for the activity of artesunate towards cancer cells. The microarray-based
mRNA expression of genes involved in EGFR signaling pathway was correlated with the 50% inhibition concentrations
(IC50) of 55 tumor cell lines for artesunate. The log10IC50 values were in a range of -6.609 to -4.0M. Candidate genes
identified by this approach were then experimentally validated by transfecting cell lines with corresponding cDNA vectors
and treating them with artesunate. Indeed, we observed that the Ras>Raf>MEK>ERK pathway is an important signaling
route for the response of tumor cells to artesunate. As exemplarily shown for artesunate, the application of such a com-
bined approach to identify signal transduction pathways involved in the response of tumor cells to cytotoxic compounds
might foster the development of novel molecular targeted therapies for cancer treatment.
Keywords: Artesunate, cancer, chemotherapy, EGFR, molecular pharmacology, natural products.

INTRODUCTION receptor (EGFR), can cause multiple drug resistance of anti-


cancer drugs [8]. The classical Ras>Raf>MEK>ERK path-
Cancer is the second leading cause of death in the west- way is involved in driving the proliferation and promoting
ern world, surpassed only by cardiovascular diseases. De- the survival of cancer cells [9]. Moreover, the role of this
spite considerable advancements in research and medical pathway to drug resistance has been unambiguously shown
care during the past decades, there is still a great need for during the past years [10]. The PI3K/PTEN/AKT signaling
improvement in the treatment options for patients afflicted route provides proliferative and anti-apoptotic signals and its
with cancer. Deciphering of various signaling transduction deregulation has often been linked with malignant transfor-
pathways gave a thorough insight into the processes of how mation and drug resistance too [11, 12].
cancer cells escape normal growth control. The concept of
The development of drug resistance and severe side ef-
molecular targeted chemotherapy has been developed based
fects of currently established anti-cancer drugs urges the
on the understanding of complex signaling networks. Here,
the objective is to specifically alter the function of those tar- need for search of novel drugs with optimized features. It is a
well–known fact that many medicinal plants do elicit diverse
get proteins that play a critical role in tumor growth and pro-
pharmacological activities and may, thus, serve as a valuable
gression upon binding to antibodies or small molecule in-
resource for procuring novel lead compounds. In our earlier
hibitors. Through this approach, we hope that new treatment
studies, we have systematically analyzed medicinal plants
adjuncts can be developed with higher specificity for tumor
used in traditional Chinese medicine [13-16]. Here, we found
cells and less side effects on normal tissues, a problem often
encountered with existing conventional chemotherapy. Most that the active principle of Artemisia annua L., artemisinin,
and its semi-synthetic derivative, artesunate, not only exert
cancer-related genetic alterations involve genes encoding
anti-malarial activity but also profound cytotoxicity against
signal transducers, particularly protein kinases [1]. Hence,
tumor cells. The inhibitory activity of artemisinin and its
the development of protein kinase inhibitors came into focus
derivatives towards cancer cells is in the nano- to micromo-
as novel cancer therapeutics. During recent years, prominent
lar range [17, 18]. Candidate genes that may contribute to the
new treatment options were implemented in clinical setting
resulting in a substantial improvement in survival time of sensitivity and resistance of tumor cells to artemisinins were
identified by pharmacogenomic and molecular pharmacol-
cancer patients [2-5]. Recently, ten protein kinase inhibitors
ogical approaches [19, 20]. Target validation was performed
have been approved for clinical use, and more than 100
using cell lines transfected with candidate genes or corre-
kinase-inhibiting agents are in different phases of clinical
sponding knockout cells. These genes are from classes with
trials in the United States [6].
different biological function; for example, regulation of pro-
Moreover, deregulated protein kinase activities and im- liferation (BUB3, cyclins, CDC25A), angiogenesis (vascular
balanced signaling routes foster the development of resis- endothelial growth factor and its receptor, matrix metallopro-
tance towards anti-tumor agents [7]. Growth factors and their teinase-9, angiostatin, thrombospondin-1) or apoptosis
receptors, i.e., epidermal growth factor (EGF) and the EGF- (BCL-2, BAX) [15]. Artesunate triggers apoptosis both
through p53-dependent and -independent pathways [21].
Anti-oxidant stress genes (thioredoxin, catalase, -glutamyl-
*Address correspondence to this author at the German Cancer Research cysteine synthetase, glutathione S-transferases) as well as
Center, Pharmaceutical Biology (C015), Im Neuenheimer Feld 280, 69120
Heidelberg, Germany; Tel: 0049-6221- 42 34 26; Fax: 0049-6221- 42 34 33;
EGFR confer resistance to artesunate [19, 22]. Cell lines
E-mail: t.efferth@dkfz.de over-expressing genes that confer resistance to established

1568-0096/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.


EGFR Signaling Pathways and Artesunate Current Cancer Drug Targets, 2009, Vol. 9, No. 1 73

anti-tumor drugs (MDR1, MRP1, BCRP, dihydrofolate re- ing, Elmira, NY, USA) with initially 104 cells/well that were
ductase, ribonucleotide reductase) were not cross-resistant to incubated for one day in the presence of the indicated sup-
artesunate, indicating that artesunate is not involved in plements. During the last 6h of incubation, DNA prolifera-
multidrug resistance [19, 20]. The anti-cancer activity of tion was measured by adding [3H]-thymidine (6.7 Ci/mmol;
artesunate has also been shown in human xenograft tumors NEN, Boston, MA, USA) as described [27].
in mice [23]. First encouraging observations in the clinical
treatment of patients suffering from uveal melanoma [24] Flow Cytometric Analysis
suggest further clinical trials with artesunate for cancer
treatment in the near future. The cells were recovered after trypsinization using
EDTA-free trypsin (Life Technologies, Inc.). The cells were
The observation that EGFR (or Erb-B1) represents a de- washed once with EDTA-free PBS and then incubated for 15
terminant of resistance of tumor cells towards artesunate [21, min with a mixture containing annexin-V and propidium
25] can be indicative that the EGFR-coupled Ras>Raf> iodide (Roche Diagnostics, Indianapolis, IN) in binding
MEK>ERK pathway might also be involved in resistance buffer (19mM HEPES (pH 7.4), 140mM NaCl, and 5mM
towards artesunate. The aims of the current investigation are, CaCl2). After the incubation period, the supernatants were
therefore, to establish the role of protein kinases for artesu- removed, and 500l of binding buffer was added to each
nate’s activity against cancer cells. Therefore, we correlated sample. The fluorescence was measured using a Becton
the microarray-based mRNA expression of genes involved in Dickinson FACScan flow cytometer (Becton Dickinson,
EGFR signaling with the 50% inhibition concentration (IC50) Franklin Lakes, NJ). Compensation on FL1 and FL2 chan-
for artesunate in 55 cell lines of the National Cancer Institute nels was performed as required to account for auto-
(NCI), USA. The candidate genes identified by this in silico fluorescence.
approach were experimentally validated. Transfected cell
lines over-expressing these candidate genes were treated
Statistical Analysis
with artesunate to analyze whether these cells reveal resis-
tance towards artesunate. By applying this combined meth- The mRNA expression values of the NCI cell lines of
odology, we observed that the Ras>Raf>MEK>ERK path- genes of interest were selected from the N.C.I. database
way is an important signaling route for the determination of (http://dtp.nci.nih.gov). The mRNA expression has been de-
resistance of tumor cells to artesunate. termined by microarray analysis [30, 31]. Fisher’s exact test
was used to calculate significance values as a relative meas-
MATERIALS AND METHODS ure for the linear dependency of two variables.

Cell Lines RESULTS


The maintenance of the IL-3/GM-CSF-dependent murine
myeloid cell line FDC-P1 has been described earlier [26]. We investigated the association of EGFR signaling-
Estradiol-dependent FD/v-ErbB:ER, FD/Raf-1:ER, FD/ related genes to the response of tumor cells to artesunate. For
MEK1:ER, FD/MEK1:ER+Akt1, FD/Bcr-Abl, and FD/ this reason, genes that belong to ERB-B/EGFR and MAPK
A-Raf:ER, transfectant cell lines were grown in the pres- pathways were selected, as shown in Fig. (1). We then used
ence of 1M ß-estradiol (Sigma, St Louis, MO, USA). The the IC50 values of the NCI cell line panel for artesunate [17]
transfection of FDC-P1 cells with plasmid cDNAs contain- and correlated them with the microarray-based mRNA ex-
ing recombinant retroviruses has been described [27]. pression values for these EGFR pathway-related genes. The
log10IC50 values were in a range of -6.609 to -4.0M. The
The panel of 55 human tumor cell lines of the Develop- genome-wide mRNA expression profiling of the NCI cell
mental Therapeutics Program of the NCI consisted of leu- lines has been reported [30, 31] and deposited at the NCI
kemia, melanoma, tumors of the central nervous system, database (http://dtp.nci.nih.gov). As shown in Table (1), the
colon, prostate, lung, kidney, ovary, and breast. Their origin mRNA expression of genes belonging to the Ras>Raf>
and processing have been previously described [28]. MEK>ERK pathway significantly correlated with the IC50
values for artesunate, indicating that this signaling route
Sulforhodamine B Assay might play a role for the response of tumor cells to artesu-
The determination of drug sensitivity in the NCI cell nate. Furthermore, the gene expression of the
lines by the sulforhodamine B assay has been reported [29]. PIK3R2>Akt1>BAD or >RPS6KA5 and CBLC pathways
The 50% inhibitory concentration (IC50) values for artesu- also significantly correlated with response of tumor cell lines
nate have been deposited in the DTP-NCI database to artesunate (Table 1). The mRNA expression of genes be-
(http://dtp.nci.nih.gov). longing to the CRK>ABL1 signaling route did not correlate
with artesunate’s activity in cancer cells (see Table (1)).
[3H]-Thymidine Incorporation Assay To test this hypothesis experimentally, we investigated
whether these signal transducers affect cellular sensitivity to
Cells were washed three times with phosphate buffered artesunate in cell lines transfected with corresponding cDNA
saline (PBS) before being set up for growth curves or prolif- constructs. As shown in Fig. (2), transfected cell lines selec-
eration assays. Growth curves were performed in 5mL of tively expressing RAF-1, MEK, or AKT-1 under appropriate
medium with the indicated supplements. Cell proliferation conditional stimulation were treated with or without artesu-
assays were performed in 96-well flat bottom plates (Corn-
74 Current Cancer Drug Targets, 2009, Vol. 9, No. 1 Konkimalla et al.

Fig. (1). Putative signaling routes identified by correlation analyses between microarray-based mRNA expression of 55 cell lines of the NCI,
USA, and the 50% inhibition concentrations (IC50) of these cell lines for artesunate. Black boxes indicate significant correlations (P<0.05,
Fisher exact test) between the corresponding genes and artesunate.

Table 1. Relationship Between mRNA Expression of Signal Transducers and Response to Artesunate in Tumor Cells Lines of the
NCI Drug Screening Panel. The Microarray-Based mRNA Expression of the Cell Lines is Deposited in the NCI Database
(http://dtp.nci.nih.gov)

Gene Response to Artesunate Fisher Exact Test Alternate Gene Symbols Description
Expres-
sion Sensitive Resistant
( -5.336 )* (> -5.336)*

EGFR low 18 10 P=0.039 ERBB. ERBB1. Epidermal growth factor receptor (eryth-
( 75.45) roblastic leukemia viral (v-erb-b) onco-
gene homolog. avian)
high 10 17
(> 75.45)
Pathway 1:
PIK3R2 low 18 9 P=0.014 p85-BETA. P85B Phosphoinositide-3-kinase. regulatory
(166.76) subunit 2 (p85)
high 9 18
(>166.76)
AKT1 low 17 10 P=0.050 AKT. MGC99656. PKB. V-akt murine thymoma viral oncogene
(479.04) PRKBA. RAC. RAC-ALPHA homolog 1
high 10 17
(>479.04)
EGFR Signaling Pathways and Artesunate Current Cancer Drug Targets, 2009, Vol. 9, No. 1 75

(Table 1). Contd.....

Gene Response to Artesunate Fisher Exact Test Alternate Gene Symbols Description
Expres-
sion Sensitive Resistant
( -5.336 )* (> -5.336)*

BAD low 18 9 P=0.014 BBC2. BCL2L8 BCL2-antagonist of cell death


(96.29)
high 9 18
(>96.29)
RPS6KA5 low 18 10 P=0.039 KS6A5. MGC1911. MSK1. ribosomal protein S6 kinase. 90kDa. poly-
(121.90) MSPK1. RLPK peptide 5
high 10 17
(>121.90)
Pathway 2:
SHC low 17 6 P=0.004 FLJ26504. p52SHC. p66. SHC (Src homology 2 domain containing)
(-0.041) p66SHC. SHC. SHCA transforming protein 1
high 11 21
(>-0.041)
SOS1 low (5.60) 18 10 P=0.039 GF1. GGF1. GINGF. HGF Son of sevenless homolog 1 (Drosophila)
high 10 17
(>5.60)
RAF1 low (-15) 14 5 P=0.014 c-Raf. CRAF. Raf-1 V-raf-1 murine leukemia viral oncogene
homolog 1
high (>-15) 14 22
MAP2K1 low 18 10 P=0.039 MAPKK1. MEK1. MKK1. Mitogen-activated protein kinase kinase 1
(0.1004) MP2K1. PRKMK1
high 10 17
(>0.1004)
MAPK1 low 20 10 P=0.010 ERK. ERK2. ERT1. MAPK2. Mitogen-activated protein kinase 1
(-0.017) MK01. p38. p40. p41. p41mapk.
P42MAPK. PRKM1. PRKM2
high 8 17
(>-0.017)
MYC low 10 18 P=0.020 c-Myc V-myc myelocytomatosis viral oncogene
(153.90) homolog
high 18 9
(>153.90)
Pathway 3:
CRK low (72) 17 11 P=0.11 CRKII. p38 v-crk sarcoma virus CT10 oncogene ho-
molog
high (>72) 11 16
ABL1 low 17 11 P=0.086 ABL. JTK7. c-ABL. p150. v-abl v-abl Abelson murine leukemia viral
(60.06) oncogene homolog 1
high 10 16
(>60.06)
Pathway 4:
CBLC low 18 10 P=0.039 C-CBL. CBL2. RNF55 Cas-Br-M (murine) ecotropic retroviral
(253.1) transforming sequence c
high 10 17
(>253.1)
*The median log10IC50 value for artesunate was used as a cut-off to separate tumor cell lines as being “sensitive“ or „resistant“.
The normalization has been performed either by pooling equal amounts of mRNA from HL-60, K562, NCI-H226, COLO205, SNB-19, LOX-IMVI, OVCAR-3, OVCAR-4, CAKI-1,
PC-3, MCF7 and Hs578T cell lines (Synteni microarrays) and analyzing by ScanAlyze program or using Affymetrix U95Av2 GeneChips. Data were normalized using the Affymetrix
Normalization 5.0 software.

nate and analyzed by the annexin-V/propidium iodide apop- apoptosis induced by 3 to 300pg/mL artesunate. Similarly,
tosis assay. The RAF-1 gene, which was under the control of activated MEK or AKT-1 caused reduced rates of apoptosis
the promoter for the estrogen receptor gene was condition- induced by artesunate (see Fig. (2)).
ally activated by -estradiol. This resulted in reduced rates of
76 Current Cancer Drug Targets, 2009, Vol. 9, No. 1 Konkimalla et al.

Fig. (2). Effects of activated protein kinases on the induction of apoptosis in response to artesunate. Annexin V/propidium iodide apoptosis
binding assays of FDC-P1, FD/v-ErbB:ER, FD/Raf-1:ER, FD/MEK1:ER, and FD/MEK1:ER+Akt1 cells measured in the presence of
1M -estradiol treated with or without 3.3 or 33pg/mL artesunate for 24h. The cells were harvested after trypsinization with EDTA-free
trypsin and incubated for 20min in a mixture containing annexin V-FITC and propidium iodide. After removing the staining solution, the
cells were re-suspended in the binding buffer, and the fluorescence was detected with a Becton Dickinson FACScan flow cytometer. The X
axis shows annexin V-FITC fluorescence and the Y axis shows the propidium iodide fluorescence. The percentages of cells in each quadrant
are presented. Lower left quadrant, viable cells; lower right quadrant, early apoptotic cells; upper left quadrant, necrotic cells; upper right
quadrant, late apoptotic and necrotic cells.
Since these data point to a role of signal transducers con- sponse of tumor cells to artesunate. Initially developed as
nected to the ERB-B/EGFR signaling network, we directly anti-malarial drug, artesunate turned out to reveal profound
tested the effect of artesunate on v-ERB-b:ER constructs. inhibitory activity against cancer cells [17, 18].
3
Using a [ H]-thymidine incorporation assay to measure pro-
We focused on four major pathways, which are linked to
liferative activity of cells, we found that the activation of v-
EGFR signalling. The key molecules of pathway 1, AKT1 is
ERB-B expression in FD cells by -estradiol-induced stimu-
3 well known from the literature to be involved in resistance to
lation led to a reduced inhibition of [ H]-thymidine incorpo-
standard chemotherapy [35]. Its role in artesunate resistance
ration by artesunate compared to cells without conditionally is new. BAD belongs to the Bcl-2 family of apoptosis regu-
activated v-ERB-B (see Fig. (3A)). Furthermore, conditional
lators, whose function for sensitivity and resistance to stan-
expression of RAF-1 also induced resistance to artesunate
dard chemotherapy and also to artesunate is well known [14,
(see Fig. (3B)), an observation that confirms the results ob-
19, 36]. The PIK3R2 and RPS6KA5 gene have not been as-
tained with the annexin-V/propidium iodide apoptosis assay.
signed to drug resistance yet. It is, however, plausible that
these genes also contribute to resistance to artesunate as parts
DISCUSSION of the entire signaling cascade.
Since protein kinases play a crucial role for many funda- The second pathway included SHC, RAF1, MAP2K1,
mental cellular processes such as proliferation, apoptosis, and MAPK1, all of which have been linked to therapy unre-
differentiation etc. [32, 33], it is noteworthy to speculate that sponsiveness [37-40]. The SOS1 gene found in the present
they are also involved in drug resistance. Indeed, the associa- investigation have not been described in the context of che-
tion of several kinases involved in signaling downstream of motherapy resistance yet. MYC, an important transcription
EGFR with resistance towards established anti-cancer drugs factor and oncogene, which regulates the cell cycle machin-
is well documented [7, 34]. ery also affect endocrine and cytotoxic cancer therapy [41,
42]. Our results enlarge MYC’s role for therapy response to
In the present investigation, we applied a combination of artesunate.
a biostatistical approach followed by experimental validation
to study the role of EGFR downstream pathways for the re- EGFR-related pathway 3 contained ABL1 and CRK
genes, both of which were not related to artesunate resistance
EGFR Signaling Pathways and Artesunate Current Cancer Drug Targets, 2009, Vol. 9, No. 1 77

Fig. (3). Effects of artesunate on DNA synthesis in oncogene-transformed and -estradiol-activated FD cells. [3H]-Thymidine incorporation
in cells over-expressing (A) v-ERB-B or (B) RAF-1. The cells were deprived of -estradiol for 24h. Three 100L aliquots per data point were
cultured at the indicated conditions and incubated in three wells of 96 well plates. Cells were incubated in medium supplemented with
(squares) or without 1M -estradiol (triangles) and artesunate at a concentration range of 0.1 to 100pg/mL.

in our investigation. ABL1 represents the non-activated duced rates of apoptosis in artesunate-treated transfected cell
wild-type form og the gene. While the bcr-abl fusion gene is lines, therefore, indicate that genes of the MAPK pathway
constitutively activated and mediates drug resistance by the confer resistance to artesunate.
Raf/MEK/ERK pathway signalling [7], the wild-type ABL1
The MAPK pathway represents an important signaling
gene does not. We also did not find a correlation between
route for receptors of the ERB family [8]. To prove whether
CRK and artesunate resistance. The v-CRK gene has been there is a direct connection between ERB genes and the
shown to protect against apoptosis [43]. Therefore, it could
MAPK pathway, we analyzed cell constructs carrying
be speculated that it should also play a role for drug resis-
cDNAs for v-ERB or RAF-1 for their responsiveness to arte-
tance. The lack of resistance induction in our study may in-
sunate. For this analysis, we used an [3H]-thymidine incorpo-
dicate that the influence of CRK in the entire regulatory sig-
ration assay as an independent method in addition to the an-
nalling network leading to cell death or survival after drug
nexin-V/propidium iodide apoptosis assay. [3H]-thymidine
exposure might not be as strong as other genes involved in incorporation is a measure for cell proliferation of tumor
our analysis.
cells, and tumor growth is the net result of cell proliferation
CBLC belongs to another EGFR-connected pathway, and apoptosis. Therefore, this assay is a supplement to fur-
which was significantly correlated to artesunate responsive- ther validate the results obtained by the in silico correlation
ness. There are no reports in the literature that CBLC plays a analyses and annexin-V/propidium iodide apoptosis assays.
role for chemosensitivity or resistance, indicating that this Addressing the question, whether expression of these en-
gene might be of minor importance. From the data presented
zymes leads to the activation of the enzymes of the signal
in the present investigation and the reports in the literature
pathway, we determined the protein levels of the key signal-
we conclude that the AKT1- and MAPK-pathways (path-
ing proteins and found that they did not increase upon activa-
ways 1 and 2) were the most relevant ones associated with
tion of the v-Erb-B:ER, Raf-1:ER, MEK1:ER and Akt:ER
resistance of cancer cells to artesunate.
constructs in the cells. However, importantly their activity
The fact that we identified mainly genes belonging to the did increase upon addition of the appropriate ligand (estro-
MAPK pathway speaks for some specificity of artesunate’s gen or tamoxifen) [46-75], which was linked to a prolifera-
mode of action towards tumor cells. The correlation of mi- tive response. This has been determined by western blot
croarray-based mRNA expression profiles with the IC50 val- analysis which examined the phosphorylation states and total
ues of cancer cell lines for artesunate represents a hunting protein levels of downstream substrates such as MEK1 and
strategy for the identification of candidate genes. It does, ERK1,2 [46-48] and correlated with increase in proliferation
however, not provide evidence for a causative link. For this (cell division and DNA synthesis).
reason, we treated cell lines transfected with the correspond-
Furthermore, we have established the effects of cytokines
ing candidate genes with artesunate. Indeed, we could verify
such as IL-3 and the conditional v-Erb-B:ER [46-48], Raf-
that the genes identified by our correlation analysis mediated
1ER (4-24), Akt:ER [54, 56, 58, 60], and MEK1:ER [69-75]
reduced rates of apoptosis by artesunate. We and other
constructs on the induction of downstream signaling cas-
authors have shown that artesunate-type drugs kill cancer cades in these cells (FDC-P1) [46-48, 50-53, 55, 56, 59-63,
cells by the induction of apoptosis [14, 16, 21, 44, 45]. Re-
70, 71, 73-75] as well as other hematopoietic (FL5.12, TF-1)
78 Current Cancer Drug Targets, 2009, Vol. 9, No. 1 Konkimalla et al.

[46, 49, 51, 53, 54, 57, 58, 71, 72], fibroblast (NIH-3T3) [46, [5] Sandler, A.; Gray, R.; Perry, M. C.; Brahmer, J.; Schiller, J. H.;
64-66], prostate (DU145) [67] and breast (MCF-7) [68, 69] Dowlati, A.; Lilenbaum, R.; Johnson, D. H. Paclitaxel-carboplatin
alone or with bevacizumab for non-small-cell lung cancer. N. Engl.
cells. In all cases, introduction of the conditional oncogene J. Med. 2006, 355, 2542-2550.
resulted in activation of the downstream signaling cascade. [6] Sebolt-Leopold, J. S.; Herrera, R. Targeting the mitogen-activated
Thus, the conditional oncogene system is an effective tool of protein kinase cascade to treat cancer. Nat. Rev. Cancer 2004, 4,
monitor the effects of a drug on the growth in response to 937-947.
activation by a particular oncogene, frequently mutated in [7] McCubrey, J. A.; Steelman, L. S.; Chappell, W. H.; Abrams, S. L.;
Wong, E. W.; Chang, F.; Lehmann, B.; Terrian, D. M.; Milella, M.;
human cancer. Tafuri, A.; Stivala, F.; Libra, M.; Basecke, J.; Evangelisti, C.; Mar-
In our system, we have selected for cells which are able telli, A. M.; Franklin, R. A. Roles of the Raf/MEK/ERK pathway
in cell growth, malignant transformation and drug resistance. Bio-
to efficiently recognize the different viral promoter systems
chim. Biophys. Acta 2007, 1773, 1263-1284.
(retroviral LTRs, SV40 and CMV encoded promot- [8] Roberts, P. J.; Der, C. J. Targeting the Raf-MEK-ERK mitogen-
ers/enhancers). We have determined in the v-Erb-B:ER, Raf- activated protein kinase cascade for the treatment of cancer. Onco-
1:ER, MEK1:ER and Akt:ER transfected cells, the chimeric gene 2007, 26, 3291-3310.
kinases are essentially inactive in the absence of ligand (e.g. [9] Steelman, L. S.; Pohnert, S. C.; Shelton, J. G.; Franklin, R. A.;
estrogen, tamoxifen). Upon addition of estrogen or ta- Bertrand, F. E.; McCubrey, J. A. JAK/STAT, Raf/MEK/ERK,
PI3K/Akt and BCR-ABL in cell cycle progression and leu-
moxifen, they become active. Therefore in our system we are kemogenesis. Leukemia 2004, 18, 189-218.
not examining the effects of different levels of mRNA ex- [10] McCubrey, J. A.; Steelman, L. S.; Franklin, R. A.; Abrams, S. L.;
pression, however, we are examining the effects of activation Chappell, W. H.; Wong, E. W.; Lehmann, B. D.; Terrian, D. M.;
of the chimeric proteins upon either addition or removal of Basecke, J.; Stivala, F.; Libra, M.; Evangelisti, C.; Martelli, A. M.
estrogen or tamoxifen. This represents a valid model to ex- Targeting the RAF/MEK/ERK, PI3K/AKT and p53 pathways in
hematopoietic drug resistance. Adv. Enzyme Regul. 2007, 47, 64-
amine the effects of drugs such as artesunate. 103.
We observed that both v-ERB- or RAF-1-transfected cells [11] Cuni, S.; Perez-Aciego, P.; Perez-Chacon, G.; Vargas, J. A.; San-
chez, A.; Martin-Saavedra, F. M.; Ballester, S.; Garcia-Marco, J.;
were more resistant to artesunate than control cells. This Jorda, J.; Durantez, A. A sustained activation of PI3K/NF-kappaB
indicates that v-Erb may confer artesunate resistance via the pathway is critical for the survival of chronic lymphocytic leuke-
MAPK pathway. The relevance of the ERB family for re- mia B cells. Leukemia 2004, 18, 1391-1400.
sponsiveness towards artesunate is further substantiated by [12] Kubota, Y.; Ohnishi, H.; Kitanaka, A.; Ishida, T.; Tanaka, T. Con-
previous results demonstrating that transfected U- stitutive activation of PI3K is involved in the spontaneous prolif-
eration of primary acute myeloid leukemia cells: direct evidence of
87.MGEGFR glioblastoma cells expressing a constitutively PI3K activation. Leukemia 2004, 18, 1438-1440.
activated deletion mutant human Erb-B1 construct is also [13] Efferth, T.; Fu, Y. J.; Zu, Y. G.; Schwarz, G.; Konkimalla, V. S.;
more resistant to artesunate than mock vector transfected U- Wink, M. Molecular target-guided tumor therapy with natural
87.MG control cells [25]. products derived from traditional Chinese medicine. Curr. Med.
Chem. 2007, 14, 2024-2032.
The identification of members of the EGFR family and [14] Efferth, T.; Giaisi, M.; Merling, A.; Krammer, P. H.; Li-Weber, M.
associated signaling pathways as determinants of artesunate Artesunate induces ROS-mediated apoptosis in doxorubicin-
resistance poses the question as to whether silencing of ERB- resistant T leukemia cells. PLoS ONE 2007, 2, e693.
related signaling routes renders tumor cells sensitive to arte- [15] Efferth, T.; Li, P. C.; Konkimalla, V. S.; Kaina, B. From traditional
Chinese medicine to rational cancer therapy. Trends Mol. Med.
sunate. We have previously shown by isobologram analyses 2007, 13, 353-361.
that treatment of U-87. MGEGFR cells treated with the [16] Efferth, T.; Rücker, G.; Falkenberg, M.; Manns, D.; Olbrich, A.;
EGFR tyrosine kinase inhibitor erlotinib (OSI-774) sensi- Fabry, U.; Osieka, R. Detection of apoptosis in KG-1a leukemic
tizes tumor cells to artesunate in a synergistic manner [25]. cells treated with investigational drugs. Arzneimittel-Forschung
1996, 46, 196-200.
[17] Efferth, T.; Dunstan, H.; Sauerbrey, A.; Miyachi, H.; Chitambar, C.
ACKNOWLEDGEMENT R. The anti-malarial artesunate is also active against cancer. Int. J.
Oncol. 2001, 18, 767-773.
This work was supported by a grant of the Dietmar [18] Kelter, G.; Steinbach, D.; Konkimalla, V. B.; Tahara, T.; Taketani,
Hopp-Stiftung to V.B.K. S.; Fiebig, H. H.; Efferth, T. Role of transferrin receptor and the
ABC transporters ABCB6 and ABCB7 for resistance and differen-
tiation of tumor cells towards artesunate. PLoS ONE 2007, 2, e798.
REFERENCES [19] Efferth, T.; Briehl, M. M.; Tome, M. E. Role of antioxidant genes
for the activity of artesunate against tumor cells. Int. J. Oncol.
[1] Hanahan, D.; Weinberg, R. A. The hallmarks of cancer. Cell 2000, 2003, 23, 1231-1235.
100, 57-70. [20] Efferth, T.; Davey, M.; Olbrich, A.; Rücker, G.; Gebhart, E.;
[2] Carpiuc, K. T.; Stephens, J. M.; Botteman, M. F.; Feng, W.; Hay, J. Davey, R. Activity of drugs from traditional Chinese medicine to-
W. A review of the clinical and economic outcomes of imatinib in ward sensitive and MDR1- or MRP1-overexpressing multidrug-
Philadelphia chromosome-positive acute lymphoblastic leukemia. resistant human CCRF-CEM leukemia cells. Blood Cells Mol. Dis.
Expert Opin. Pharmacotherapy 2007, 8, 2775-2787. 2002, 28, 160-168.
[3] Eiermann, W. Trastuzumab combined with chemotherapy for the [21] Efferth, T.; Sauerbrey, A.; Olbrich, A.; Gebhart, E.; Rauch, P.;
treatment of HER2-positive metastatic breast cancer: pivotal trial Weber, H. O.; Hengstler, J. G.; Halatsch, M. E.; Volm, M.; Tew, K.
data. Ann. Oncol. 2001, 12 , S57-S62. D.; Ross, D. D.; Funk, J. O. Molecular modes of action of artesu-
[4] Moore, M. J.; Goldstein, D.; Hamm, J.; Figer, A.; Hecht, J. R.; nate in tumor cell lines. Mol. Pharmacol. 2003, 64, 382-394.
Gallinger, S.; Au, H. J.; Murawa, P.; Walde, D.; Wolff, R. A.; [22] Efferth, T.; Oesch, F. Oxidative stress response of tumor cells:
Campos, D.; Lim, R.; Ding, K.; Clark, G.; Voskoglou-Nomikos, T.; microarray-based comparison between artemisinins and anthracy-
Ptasynski, M.; Parulekar, W. Erlotinib plus gemcitabine compared clines. Biochem. Pharmacol. 2004, 68, 3-10.
with gemcitabine alone in patients with advanced pancreatic can- [23] Dell'Eva, R.; Pfeffer, U.; Vene, R.; Anfosso, L.; Forlani, A.; Albini,
cer: a phase III trial of the National Cancer Institute of Canada A.; Efferth, T. Inhibition of angiogenesis in vivo and growth of Ka-
Clinical Trials Group. J. Clin. Oncol. 2007, 25, 1960-1966.
EGFR Signaling Pathways and Artesunate Current Cancer Drug Targets, 2009, Vol. 9, No. 1 79

posi's sarcoma xenograft tumors by the anti-malarial artesunate. cell chemoresistance. J. Natl. Cancer Inst. Monogr. 2001, 28, 30-
Biochem. Pharmacol. 2004, 68, 2359-2366. 37.
[24] Berger, T. G.; Dieckmann, D.; Efferth, T.; Schultz, E. S.; Funk, [41] Butt, A. J.; Caldon, C. E.; McNeil, C. M.; Swarbrick, A.; Musgrove
J.O.; Baur, A.; Schuler, G. Artesunate in the treatment of metastatic E. A.; Sutherland R. L. Cell cycle machinery: links with genesis
uveal melanoma--first experiences. Oncol. Rep. 2005, 14, 1599- and treatment of breast cancer. Adv. Exp. Med. Biol. 2008, 630,
1603. 189-205.
[25] Efferth, T.; Ramirez, T.; Gebhart, E.; Halatsch, M. E. Combination [42] Biliran, H.; Banerjee, S.; Thakur, A.; Sarkar, F. H.; Bollig, A.;
treatment of glioblastoma multiforme cell lines with the anti- Ahmed, F.; Wu, J.; Sun, Y.; Liao, J. D. c-Myc-induced chemosen-
malarial artesunate and the epidermal growth factor receptor tyro- sitization is mediated by suppression of cyclin D1 expression and
sine kinase inhibitor OSI-774. Biochem. Pharmacol. 2004, 67, nuclear factor-kappa B activity in pancreatic cancer cells. Clin.
1689-1700. Cancer Res. 2007, 13, 2811-2821.
[26] McCubrey, J.; Holland, G.; McKearn, J.; Risser, R. Abrogation of [43] Stam, J. C.; Geerts, W. J.; Versteeg, H. H.; Verkleij, A. J.; Hene-
factor-dependence in two IL-3-dependent cell lines can occur by gouwen, P. M. The v-Crk oncogene enhances cell survival and in-
two distinct mechanisms. Oncogene Res. 1989, 4, 97-109. duces activation of protein kinase B/Akt. J. Biol. Chem. 2001, 276,
[27] McCubrey, J. A.; Smith, S. R.; Algate, P. A.; DeVente, J. E.; 25176-25183.
White, M. K.; Steelman, L. S. Retroviral infection can abrogate the [44] Huan-huan, C.; Li-Li, Y.; Shang-Bin, L. Artesunate reduces
factor-dependency of hematopoietic cells by autocrine and non- chicken chorioallantoic membrane neovascularisation and exhibits
autocrine mechanisms depending on the presence of a functional antiangiogenic and apoptotic activity on human microvascular
viral oncogene. Oncogene 1993, 8, 2905-2915. dermal endothelial cell. Cancer Lett. 2004, 211, 163-173.
[28] Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwin- [45] Wu, G. D.; Zhou, H. J.; Wu, X. H. Apoptosis of human umbilical
ski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. vein endothelial cells induced by artesunte. Vasc. Pharmacol. 2004,
H.; Boyd, M. R. Feasibility of drug screening with panels of human 41, 205-212.
tumor cell lines using a microculture tetrazolium assay. Cancer [46] McCubrey, J. A.; Shelton, J. G.; Steelman, L. S.; Franklin, R. A.;
Res. 1988, 48, 589-601. Sreevalsan, T.; McMahon, M. Conditionally active v-ErbB:ER
[29] Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.; transforms NIH-3T3 cells and converts human and mouse cells to
Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, A.; Boyd, M. R. cytokine-independence. Oncogene 2004, 23, 7810-7820.
Comparison of in vitro anticancer-drug-screening data generated [47] Shelton, J. G.; Steelman, L. S.; Abrams, S. L.; White, E. R.; Akula,
with a tetrazolium assay versus a protein assay against a diverse S. M.; Bertrand, F. E.; Franklin, R. A.; McCubrey, J. A. Effects of
panel of human tumor cell lines. J. Natl. Cancer Inst. 1990, 82, endogenous epidermal growth factor receptor signaling on DNA
1113-1118. synthesis and ERK activation in a cytokine –dependent hema-
[30] Scherf, U.; Ross, D. T.; Waltham, M.; Smith, L. H.; Lee, J. K.; topoietic cell line. Cell Cycle 2005, 4, 818-821.
Tanabe, L.; Kohn, K. W.; Reinhold, W. C.; Myers, T. G.; Andrews, [48] Shelton, J. G.; Steelman, L. S.; Abrams, S. L.; White, E. R.; Akula,
D. T.; Scudiero, D. A.; Eisen, M. B.; Sausville, E. A.; Pommier, Y.; S. M.; Bertrand, F. E.; Franklin, R. A.; McCubrey, J. A. Condi-
Botstein, D.; Brown, P. O.; Weinstein, J. N. A gene expression da- tional EGFR promotes cell cycle progression and prevention of
tabase for the molecular pharmacology of cancer. Nat. Genet. 2000, apoptosis in the absence of autocrine cytokines. Cell Cycle 2005, 4,
24, 236-244. 822-830.
[31] Staunton, J. E.; Slonim, D. K.; Coller, H. A.; Tamayo, P.; Angelo, [49] McCubrey, J. A.; Steelman, L. S.; Hoyle, P. E.; Blalock, W. L.;
M. J.; Park, J.; Scherf, U.; Lee, J. K.; Reinhold, W. O.; Weinstein, Weinstein-Oppenheimer, C.; Franklin, R. A.; Cherwinski, H.;
J. N.; Mesirov, J. P.; Lander, E. S.; Golub, T. R. Chemosensitivity Bosch, E.; McMahon, M. Differential Abilities of Activated Raf
prediction by transcriptional profiling. Proc. Natl. Acad. Sci. USA Oncoproteins to Abrogate Cytokine-Dependency, Prevent Apopto-
2001, 98, 10787-10792. sis and Induce Autocrine Growth Factor Synthesis in Human He-
[32] Grant, S.; Qiao, L.; Dent, P. Roles of ERBB family receptor tyro- matopoietic Cells. Leukemia 1998, 12, 1903-1929.
sine kinases, and downstream signaling pathways, in the control of [50] Hoyle, P. E.; Moye, P. W.; Steelman, L. S.; Blalock, W. L.; Frank-
cell growth and survival. Front. Biosci. 2002, 7, d376-389. lin, R. A.; Pearce, M.; Cherwinski, H.; Bosch, E.; McMahon, M.;
[33] Shaul, Y. D.; Seger, R. The MEK/ERK cascade: from signaling McCubrey, J. A. Differential abilities of the Raf family of protein
specificity to diverse functions. Biochim. Biophys. Acta 2007, kinases to abrogate cytokine-dependency and prevent apoptosis in
1773, 1213-1226. murine hematopoietic cells by a MEK1-dependent mechanism.
[34] Navolanic, P. M.; Steelman, L. S.; McCubrey, J. A. EGFR family Leukemia 2000, 14, 642-656.
signaling and its association with breast cancer development and [51] McCubrey, J. A.; Steelman, L. S.; Moye, P. W.; Hoyle, P. E.;
resistance to chemotherapy. Int. J. Oncol. 2003, 22, 237-252. Weinstein-Oppenheimer, C. L.; Chang, F.; Pierce, M.; White, M.
[35] Martelli, A. M.; Tabellini, G.; Bortul, R.; Tazzari, P. L.; Cappellini, K.; Franklin, R.; Blalock, W. L. Effects of deregulated Raf and
A.; Billi, A. M.; Cocco, L. Involvement of the phosphoinositide 3- MEK1 expression on the cytokine-dependency of hematopoietic
kinase/Akt signaling pathway in the resistance to therapeutic treat- cells. Adv. Enzyme Regul. 2000, 40, 305-337.
ments of human leukemias. Histol. Histopathol. 2005, 20, 239-252. [52] Moye, P. W.; Blalock, W. L.; Hoyle, P. E.; Chang, F.; Franklin, R.
[36] Poeta, D. G.; Bruno, A.; Principe, M. I.; Venditti, A.; Maurillo, L.; A.; Weinstein-Oppenheimer, C.; Pearce, M.; Steelman, L.; McMa-
Buccisano, F.; Stasi, R.; Neri, B.; Luciano, F.; Siniscalchi, A.; de hon, M.; McCubrey, J. A. Synergy Between Raf and BCL2 in Ab-
Fabritiis, P.; Amadori, S. Deregulation of the mitochondrial apop- rogating the Cytokine-Dependency of Hematopoietic Cells. Leu-
totic machinery and development of molecular targeted drugs in kemia 2000, 14, 1060-1079.
acute myeloid leukemia. Curr. Cancer Drug Targets 2008, 8, 207- [53] Weinstein-Oppenheimer, C.; Steelman, L. S.; Algate, P. A.; Blal-
222. ock, W. L.; Burrows, C.; Hoyle, P. E.; Lee, J. T.; Moye, P. W.;
[37] Frackelton, A. R.; Lu, L.; Davol, P. A.; Bagdasaryan, R.; Hafer, L. Shelton, J. G.; Franklin, R. A.; McCubrey, J. A. Effects of Deregu-
J.; Sgroi, D. C. p66 Shc and tyrosine-phosphorylated Shc in pri- lated Raf Activation on Integrin, Cytokine-Receptor Expression
mary breast tumors identify patients likely to relapse despite ta- and the Induction of Apoptosis in Hematopoietic Cells. Leukemia
moxifen therapy. Breast Cancer Res. 2006, 8, R73. 2000, 14, 1921-1938.
[38] Swanton, C.; Downward, J. Unraveling the complexity of endo- [54] McCubrey, J. A.; Steelman, L. S.; Blalock, W. L.; Lee, J. T.; Moye,
crine resistance in breast cancer by functional genomics. Cancer P. W.; Chang, F.; Pearce, M.; Shelton, J. G.; White, M. K.; Frank-
Cell 2008, 13, 83-85. lin, R. A.; Pohnert, S. C. Synergistic Effects of PI3K/Akt on Abro-
[39] Woessmann, W.; Chen, X.; Borkhardt, A. Ras-mediated activation gation of Cytokine-Dependency Induced by Oncogenic Raf. Adv.
of ERK by cisplatin induces cell death independently of p53 in os- Enzyme Regul. 2001, 289-323.
teosarcoma and neuroblastoma cell lines. Cancer Chemother. [55] Chang, F.; McCubrey, J. A. p21Cip1 induced by Raf is associated
Pharmacol. 2002, 50, 397-404. with increased Cdk4 activity in hematopoietic cells. Oncogene
[40] Deng, X.; Kornblau, S. M.; Ruvolo, P. P.; May, W. S. Regulation 2001, 20, 4353-4364.
of Bcl2 phosphorylation and potential significance for leukemic [56] McCubrey, J. A.; Lee, J. T.; Steelman, L. S.; Blalock, W. L.; Moye,
P. W.; Chang, F.; Pearce, M.; Shelton, J. G.; White, M. K.; Frank-
80 Current Cancer Drug Targets, 2009, Vol. 9, No. 1 Konkimalla et al.

lin, R. A.; Pohnert, S. C. Interactions between the PI3K and Raf associated herpesvirus infected human B cells. Blood 2005, 105,
signaling pathways can result in the transformation of hema- 4516-4522.
topoietic cells. Cancer Detect. Prevent. 2001, 25, 375-393. [67] Lee, J. T.; Steelman, L. S.; McCubrey, J. A. Modulation of
[57] Chang, F.; Steelman, L. S.; McCubrey, J. A. Raf-Induced Cell Raf/MEK/ERK pathway in prostate cancer drug resistance. Int. J.
Cycle Progression in Human TF-1 Hematopoietic Cells. Cell Cycle Oncol. 2005, 26, 1637-1645.
2002, 1, 220-227. [68] Weinstein-Oppenheimer, C. R.; Henríquez-Roldán, C. F.; Davis, J.;
[58] Shelton, J. G.; Steelman, L. S.; Lee, J. T.; Knapp, S. L.; Blalock, Navolanic, P. M.; Saleh, O. A.; Steelman, L. S.; Franklin, R. A.;
W. L.; Moye, P. M.; Franklin, R. A.; Pohnert, S. C.; Mizra, A. M.; Robinson, P. J.; McMahon, M.; McCubrey, J. A. Role of the Raf
McMahon, M.; McCubrey, J. A. Effects of the Raf/MEK/ERK and signal transduction cascade in the in vitro resistance to the antican-
PI3K signal transduction pathways on the abrogation of cytokine cer drug doxorubicin. Clin. Cancer Res. 2001, 7, 2892-2907.
dependence and prevention of apoptosis in hematopoietic cells. [69] Davis, J. M.; Weinstein-Oppenheimer, C. R.; Steelman, L. S.;
Oncogene 2003, 24, 2478-2492. Navolanic, P. N.; Hu, W.; Konopleva, M.; Blagosklonny, M. V.;
[59] Shelton, J. G.; Chang, F.; Lee, J. T.; Franklin, R. A.; Steelman, L. McCubrey, J. A. Raf-1 and Bcl-2 Induce Distinct and Common
S.; McCubrey, J. A. B-Raf and insulin synergistically prevent Pathways Which Contribute to Breast Cancer Drug Resistance.
apoptosis and induce cell cycle progression in hematopoietic cells. Clin. Cancer Res. 2003, 9, 1161-1170.
Cell Cycle 2004, 3, 189-196. [70] Blalock, W. L.; Pearce, M.; Steelman, L. S.; Franklin, R. A.;
[60] Shelton, J. G.; Steelman, L. S.; White, E. R.; McCubrey, J. A. McCarthy, S. A.; Cherwinski, H.; McMahon, M.; McCubrey, J. A.
Synergy between PI3K/Akt and Raf/MEK/ERK Pathways in IGF- A conditionally-active form of MEK1 results in autocrine trans-
1R Mediated Cell Cycle Progression and Prevention of Apoptosis formation of human and mouse hematopoeitic cells. Oncogene
in Hematopoietic Cells. Cell Cycle 2004, 3, 372-379. 2000, 19, 526-536.
[61] Demidenko, Z. N.; Halicka, D.; Kunicki, J.; McCubrey, J. A.; [71] Blalock, W. L.; Moye, P. W.; Chang, F.; Pearce, M.; Steelman, L.
Darzynkiewicz, Z.; Blagosklonny, M. V. Selective killing of S.; McMahon, M.; McCubrey, J. A. Combined Effects of Aberrant
adriamycin-resistant (G2 checkpoint-deficient and MRP1- MEK1 Activity and BCL2 Overexpression on Relieving the Cyto-
expressing) cancer cells by docetaxel. Cancer Res. 2005, 65, 4401- kine-Dependency of Human and Murine Hematopoietic Cells. Leu-
4407. kemia 2000, 14, 1080-1096.
[62] Demidenko, Z. N.; An, W. G.; Lee, J. T.; Romanova, L. Y.; McCu- [72] Blalock, W. L.; Pearce, M.; Chang, F.; Lee, J.; Pohnert, S.; Burrows,
brey, J. A.; Blagosklonny, M. V. Kinase-Addiction and Bi-Phasic C.; Steelman, L. S.; Franklin, R. A.; McMahon, M.; McCubrey, J.
Sensitivity-Resistance of Bcr-Abl- and Raf-1-Expressing Cells to A. Effects of Inducible MEK1 Activation on the Cytokine-
Imatinib and Geldanamycin. Cancer Biol. Ther. 2005, 4, 484-490. Dependency of Lymphoid Cells. Leukemia 2001, 15, 794-807.
[63] Konopleva, M.; Shi, Y.; Steelman, L. S.; Shelton, J. G.; Munsell, [73] Blalock, W. L.; Steelman, L. S.; Shelton, J. G.; Moye, P. W.; Lee,
M.; Marini, F.; McQueen, T.; Contractor, R.; McCubrey, J. A.; An- J. T.; Franklin, R. A.; Mirza, A.; McMahon, M.; White, M. K.;
dreeff, M. Development of a conditional in vivo model to evaluate McCubrey, J. A. Requirement for the PI3K/Akt Pathway in MEK1-
the efficacy of Small Molecule Inhibitors for the Treatment of Raf Mediated Growth and Prevention of Apoptosis: Identification of an
Transformed Hematopoietic Cells. Cancer Res. 2005, 65, 9962- Achilles Heel in Leukemia. Leukemia 2003, 17, 1058-1067.
9970. [74] Shelton, J. G.; Moye, P. W.; Steelman, L. S.; Blalock, W. L.; Lee,
[64] Hamden, K. E.; Ford, P. W.; Whitman, A. G.; McCubrey, J. A.; J. T.; Franklin, R. A.; McMahon, M.; McCubrey, J. A. Differential
Akula, S. M. Raf induced Vascular Endothelial Growth Factor effects of kinase cascade inhibitors on neoplastic and cytokine-
Augments Kaposi’s Sarcoma-Associated Herpesvirus mediated cell proliferation. Leukemia 2003, 17, 1765-1782.
(KSHV/HHV-8) Infection. J. Virol. 2004, 78, 13381-13390. [75] Shelton, J. G.; Blalock, W. L.; White, E. R.; Steelman, L. S.;
[65] Akula, S. M.; Ford, P. W.; Whitman, A. G.; Hamden, K. H.; Shel- McCubrey, J. A. Ability of the Activated PI3K/Akt Oncoproteins
ton, J. G.; McCubrey J. A. Raf promotes human herpesvirus-8 to Synergize with MEK1 and Induce Cell Cycle Progression and
(HHV-8/KSHV) infection. Oncogene 2004, 23, 5227-5241. Abrogate the Cytokine-Dependence of Hematopoietic Cells. Cell
[66] Akula, S. M.; Ford, P. W.; Whitman, A. G.; Hamden, K. E.; Bryan, Cycle 2004, 3, 503-512.
B. A.; Cook, P. P.; McCubrey, J. A. B-Raf dependent expression of
vascular endothelial growth factor-A in Kaposi's sarcoma-

Received: August 11, 2008 Revised: November 03, 2008 Accepted: November 25, 2008

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