Brief Communication Potent inhibitory effect of the cyclolignan picropodophyllin (PPP) on human adrenocortical carcinoma cells prolifera- tion
Mabrouka Doghman1, Magnus Axelson2, Enzo Lalli1
1Institut de Pharmacologie Moléculaire et Cellulaire CNRS UMR 6097 and Université de Nice - Sophia Antipolis, Val- bonne, France; 2Department of Clinical Chemistry, Karolinska University Hospital, Stockholm, Sweden.
Received December 14, 2010; accepted January 4, 2011; Epub January 5, 2011; Published February 15, 2011
Abstract: Adrenocortical carcinoma (ACC) is a very aggressive tumor with a poor prognosis. Available treatments for this type of cancer are far from being satisfactory. The IGF signalling pathway represents an important mechanism for ACT growth and constitutes a relevant therapeutic target. We investigated the effect of picropodophyllin (PPP), a member of the cyclolignan family and a new inhibitor of IGF-1R, on proliferation of human adrenocortical cell lines H295R and SW-13. PPP inhibits proliferation and induces an important accumulation in G2/M phase and apoptosis of H295R and SW-13 cells. Our data suggest that PPP may be a promising candidate for drug development for adrenocortical carcinoma.
Keywords: Adrenal cortex, cancer, IGF receptor, cell lines
Introduction
Adrenocortical carcinoma (ACC) is a rare and very aggressive tumor with an incidence of ap- proximatively 1-2 per million people per year [1, 2]. Its poor prognosis depends mainly upon a limited number of therapeutic tools. A complete surgical resection after an early diagnosis is the most valuable option for treatment. The use of the adrenolytic agent, mitotane (o,p-DDD), asso- ciated or not with DNA-damaging drugs, is the only medical therapy available up to date [3].
The molecular mechanisms of tumorigenesis of the adrenal cortex are to be found in a multistep process where genetic alterations and signaling pathways deregulation are combined. The insu- lin-like growth factor system is one of the best- investigated molecular pathways involved in adrenal growth. Several genetic alterations such as loss of imprinting or loss of heterozygosity of the 11p15 gene locus causing a strong IGF2 overexpression have been demonstrated in the majority of adult and childhood ACCs [4-7]. IGF2
binds two distinct receptors, type I (IGF-1R) and type II (IGF-IIR). Similar to the insulin receptor, IGF-1R is a receptor tyrosine kinase composed of two heterodimeric subunits that possesses an intrinsic tyrosine kinase activity, and acti- vates a variety of downstream effectors associ- ated with this receptor family. Since overexpres- sion of IGF-1R has been found in a substantial proportion of ACCs, it is likely that locally pro- duced IGF2 acts as an autocrine or paracrine growth factor in adrenocortical tumorigenesis [8 -10]. On the basis of the pivotal role of IGF-1R in IGF2 signaling, it becomes evident that this re- ceptor represents a promising target for adreno- cortical tumors therapy. It has been recently reported that suppression of IGF2 /IGF-1R sig- naling, through the use of the IGF-1R inhibitor NVP-AEW541 or by using blocking antibodies, inhibits ACC cell line proliferation in vitro and in vivo in a human ACC xenograft model [9, 10].
Picropodophyllin (PPP), a member of the cyclo- lignan family, has recently been described as an inhibitor of IGF-1R. PPP inhibits phosphorylation
PPP inhibits ACC cells proliferation
of IGF-1R without interfering with the highly ho- mologous insulin receptor or tyrosine kinases of other relevant growth factor receptors relevant for cancer cells [11]. PPP induces tumor regres- sion and inhibition of metastasis in several models of human cancer and its administration is well tolerated in vivo [12]. These data prompted us to investigate the effect of PPP on the growth of two established human ACC cell lines (H295R and SW-13).
Materials and Methods
Chemicals
PPP was synthetized in an ultrapure form as described [11]. NVP-AEW541 [13] was provided by Novartis. Stock solutions of both compounds were prepared in DMSO (50 mM and 10 mM, respectively).
Cell culture and proliferation assays
H295R cells were cultured in DMEM/F-12 sup- plemented with 2% NuSerum, 1% ITS Plus and antibiotics, as described [14]. SW-13 cells were cultured in DMEM/F12 supplemented with 10% FCS and antibiotics. To measure proliferation, cells were seeded in duplicate in 24-well plates at the density of 3x104 cells/well and cultured in complete medium in the presence of the indi- cated concentration of the different compounds or DMSO added to the culture medium. Cells were counted after 3 days of culture using the COUNTESS automate instrument (Invitrogen).
Immunoblots
H295R and SW-13 cells were treated with the indicated concentrations of different com- pounds or with DMSO vehicle. Protein extracts were prepared by harvesting cells in RIPA buffer [(50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP- 40, 0.5% sodium deoxycholate, 1 mM EDTA, 50mM NaF, Protein Inhibitor Cocktails 1 and 2 (Sigma)]. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblot was performed using a chemilumi- nescence system for protein detection (ECL Plus, GE Healthcare). Antibodies used were as follows: anti-IGF1Rß; anti-Akt (total) and phos- pho-Akt(Ser473); anti-p44/p42 mitogen-activated protein kinase and anti-phospho-p44/p42 mito- gen-activated protein kinase (all from Cell Sig- naling Technology); anti-phosphotyrosine PY20 (Sigma).
Flow cytometry
H295R and SW-13 cells were fixed in 70% etha- nol and then treated with RNAse A (50 µg/mL) for 30 min at 37℃. DNA was stained with propidium iodide (50 µg/mL) and cells were analyzed for cell-cycle distribution with a FAC- Scan instrument (Becton Dickinson).
Results
In this study, we used two established human ACC cell lines: the well-differentiated H295R cells, that retain the ability to synthetize steroid hormones, and SW-13 derived from a stage IV tumor that are not steroidogenic. Both cell lines represent suitable models to study the effects of IGF-1R inhibitors since they express high lev- els of IGF-1R. However, H295R cells, but not SW -13, produce high levels of IGF2, which acts in an autocrine manner to trigger their prolifera- tion [8, 9].
We first evaluated the effect of different doses of PPP on H295R and SW-13 cell proliferation and compared it with the NVP-AEW541 IGF-1R inhibitor. After 3 days of treatment, PPP inhib- ited cell growth of H295R (IC50 1.8 x 10-7M) and SW-13 (IC50 1.4 x 10-7M) cells in a dose- dependent manner, having a more potent effect than NVP-AEW541 on both cell lines (IC50 4.6 x 10-7M for H295R cells and IC50 1.6 x 10-6M for SW-13 cells; Figure 1A).
We next evaluated the effect of PPP on cell cy- cle distribution after a 24h treatment. Exposure of H295R cells to 1 mM PPP increased the frac- tion of cells in G2/M-phase (from 25% of DMSO control to 38%) and sub-G1 (hypodiploid apop- totic cells; from 10% of DMSO control to 19%) with a corresponding decrease of the fraction of cells in the G1-phase (from 47% of DMSO con- trol to 22%). The PPP-induced G2/M accumula- tion (from 22% of DMSO control to 74%) and apoptosis (from 2% of DMSO control to 8%) were also observed in SW-13 cells (Figure 1B).
Surprisingly, PPP did not significantly modulate phosphorylation of IGF-1RB in H295R cells (Figure 2A). The apoptotic effect of PPP has been shown to be associated with an important inhibition of PI3K/Akt pathway and a moderate effect on the ERK pathway in other cell types [11, 15]. After a 48h serum starvation, H295R and SW13 cells were treated with or without 10- 5M PPP for 2h and finally stimulated with 10%
PPP inhibits ACC cells proliferation
A
B
H295R
SW-13
120
% cell growth inhibition
PPP
120
PPP
100
A NVP-AEW541
% cell growth inhibition
100
& NVP-AEW541
80
80
60
60
40
40
20
20
0
0
D - 8
7
- 6
- 5
- 4
D - 8
- 7
- 6
- 5
- 4
Log drug concentration (mol/L)
Log drug concentration (mol/L)
125
Sub-G1
125
Sub-G1
% of total cells
100
G1
G1
S
% of total cell
100
S
75
G2/M
75
G2/M
50
50-
25
25.
0
0
DMSO
PPP
DMSO
PPP
serum. We observed that PPP exerts no signifi- cant effect on Akt and ERK1/2 phosphoryla- tions whereas NVP-AEW541 exerts a substantial inhibition of Akt and a moderate effect on ERK1/2 phosphorylations. These results sug- gest that PPP effect does not involve inhibition of the Akt and ERK1/2 effect in adrenocortical cell lines (Figure 2B).
Discussion
The factors responsible for the incidence of be- nign adrenocortical tumors and its malignant transformation are not well understood. In addi- tion to IGF2 overexpression, increased levels of the IGF-1R have been found in adrenocortical carcinomas, suggesting an important role for the IGF-system in adrenocortical carcinogenesis [9, 10]. These results indicated that IGF-1R may
represent an important target for cancer ther- apy. Recently some studies using IGF-1R inhibi- tors such as NVP-AEW541 or an anti-IGF-1R monoclonal antibody produced inhibition of ACC cell growth in vitro and in vivo [9, 10]. The re- sults of a phase I clinical study of an anti-IGF-1R monoclonal antibody in patients with advanced ACC have been reported [16].
In this report we have studied the effect of PPP, a member of the cyclolignan family described as a specific inhibitor of the IGF-1R, on ACC cells proliferation [11]. PPP has been shown to block the phosphorylation of the IGF-1R without af- fecting the homologous insulin receptor [11]. This represents an obvious advantage over some other IGF-1R inhibitors. Moreover, PPP has been shown to be well tolerated in vivo after oral administration [11, 12]. For these reasons,
A
PPP
P-IGF-1RB
IGF-1Rß
B
H295R
SW-13
10’
30’
60’
10’
30’
60’
PPP
NVP
P-Akt
Akt
P-p42/p44
p42/p44
PPP may represent a compound potentially in- teresting for drug development for ACC.
We have shown that PPP inhibits growth of two different human ACC cell lines (H295R and SW13) in vitro. Cell cycle analysis revealed that a 24h treatment with PPP drastically increased the fraction on G2/M and sub-G1 phases. These effects on cell cycle have been also observed in multiple myeloma cells after PPP treatment [17]. PPP exerts an important effect on prolif- eration at lower concentrations than NVP- AEW541. Nevertheless, we could not detect any effect of PPP on rapid phosphorylation of Akt and ERK1/2, whereas it has been largely asso- ciated with an inhibition of PI3K/Akt pathway in other cell lines [15]. These results suggest that in ACC cell lines, the inhibitory effect of PPP does not primarily involve these signaling path- ways. Recently, other reports revealed that PPP may act with mechanisms different from Akt/ Erk inhibition. PPP is a stereoisomere of podo-
phyllotoxin (PPT), an established inhibitor of microtubule assembly leading to mitotic arrest presenting a general toxicity [18, 19]. However, PPP used here is an ultrapure compound. It was shown that PPP does not bind to tubulin at con- centrations up to 50 uM while PPT does. The effect of PPP observed in our cellular model is observed at a concentration of 0.1 µM.
Further studies are necessary to further charac- terize the molecular mechanism of the inhibitory action of PPP on adrenocortical cell lines prolif- eration. This compound may represent an inter- esting therapeutic tool to be associated to newer drugs in ACT chemotherapy [14, 20-22], in order to develop more selective and specific treatments for clinical use.
Acknowledgements
We thank J. Cazareth for help with cell cycle analysis and Novartis for NVP-AEW541. Re-
PPP inhibits ACC cells proliferation
search in E.L. laboratory is funded by Institut National du Cancer, CNRS (LIA NEOGENEX) and FP7 ENS@T-CANCER.
Abbreviations: ACC, adrenocortical carcinoma; PPP, picropodophyllin; IGF-1R, type 1 IGF receptor.
Please address correspondence to: Dr. Mabrouka Doghman, Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 6097, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France, Tel: 33-(0) 493957751, Fax: 33-(0)493957708, E-mail: dogh- man@ipmc.cnrs.fr, or Dr. Enzo Lalli, Institut de Phar- macologie Moléculaire et Cellulaire, CNRS UMR 6097, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France, Tel: 33-(0)493957755, Fax: 33-(0)493957708, E-mail: ninino@ipmc.cnrs.fr
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