Withanolides are Potent Novel Targeted Therapeutic Agents Against Adrenocortical Carcinomas
Chitra Subramanian . Huaping Zhang .
Robert Gallagher . Gary Hammer . Barbara Timmermann . Mark Cohen
Published online: 24 April 2014 @ Société Internationale de Chirurgie 2014
Abstract
Background Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with poor prognosis, as a majority of patients present with advanced disease. Current adjuvant strategies for metastatic patients include mitotane or other cytotoxic agents and carry a significant morbidity as well as a low (<10 %) 5-year survival. Withanolides, including withaferin A, are novel chemotherapeutic agents with potent targeted effects in medullary thyroid cancer and a number of solid malignancies with low toxicity in vivo. We hypothesize that novel naturally derived wit- hanolides will have potent targeted anti-cancer activity against ACCs.
Methods In vitro cell viability of ACC cell lines (Y1 and SW13) was measured using MTS cell proliferation assay. Cell cycle and apoptotic analysis studied using annexin V/propidium iodide staining on flow cytometry (FC) and targeted molecular mechanisms of withanolide cytotoxicity were assessed using standard Western blot analysis.
C. Subramanian Division of Endocrine Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, USA
H. Zhang · R. Gallagher · B. Timmermann Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, USA
G. Hammer
Division of Metabolism, Endocrinology and Diabetes, Department of Medicine, University of Michigan, Ann Arbor, MI, USA
M. Cohen Division of Endocrine Surgery, Department of Surgery, University of Michigan Hospital and Health Systems, Ann Arbor, MI, USA
e-mail: cohenmar@med.umich.edu
Results All the withanolides potently reduced ACC cell viability on MTS assay with 7- to 185-fold higher selec- tivity than normal fibroblasts. Cell cycle analysis demon- strated a shift in cell cycle arrest from G1/G0 to G2/M with induction of apoptosis at nanomolar concentrations of withanolides. Unlike current ACC therapeutics, withano- lides modulated expression of several key oncogenic pathway proteins in ACCs by Western blot, including Jagged 1, MAPK, and Akt/mTOR pathway proteins in a dose-dependent manner after 24 h drug treatment of SW13 cells.
Conclusion These results demonstrate the first evidence of the anticancer efficacy of withanolides in ACC cells and provide support for future translational evaluation of these compounds as novel therapeutic agents for ACC patients.
Introduction
Adrenocortical carcinoma (ACC) is a rare and heteroge- neous endocrine malignancy with an estimated worldwide incidence per million population of approximately 1-2 cases per year [1]. Because of difficulties in early detection and the lack of effective long-term treatment options, ACC is characterized by high recurrence rates, poor prognosis, and an overall 5-year survival rate of only 10-20 % [1]. Although long-term survival is possible with complete operative resection of early-stage disease, 70 % of ACC patients unfortunately will present with metastases at the time of diagnosis [2]. For metastatic disease, first-line standard systemic therapy involves the adrenolytic agent mitotane. However, mitotane, either as a monotherapy or in combination with other chemotherapeutic agents such as doxorubicin, etoposide, and cisplatin (known as the Italian regimen), does not yield complete responses or durable
long-term results. Current therapies fail either due to dis- ease progression or toxicities associated with these cyto- toxic drugs [3], leading to a critical need in this disease for discovery of novel therapies with lower toxicity profiles.
Results from genetic profiling of ACCs have implicated dysregulation of multiple signal transduction pathways, including insulin-like growth factor (IGF)-2, the ligand for notch 1 (jagged 1), and the Wnt/ß-catenin signaling path- ways during the development of ACC [4-6]. Current tar- geted drugs being evaluated in ACCs include agents targeting IGF-1R, phosphoinositide 3-kinase (PI3K), or mammalian target of rapamycin (mTOR) pathways, either alone or as a combination therapy [7]. In addition drugs, targeting the vascular endothelial growth factor receptor (VEGF-R), the Wnt/ß-catenin pathway, MDR/P-glyco- protein, peroxisome proliferator-activated receptor (PPAR)-y, and steroidogenic factor (SF)-1 are also being tested for their efficacy in ACC [8, 9]. While many of these agents are still in development or in early clinical trials, none have yet demonstrated a significant impact on the fatality of advanced ACC. Given the limitations of thera- peutics targeting a single pathway and our growing understanding of the multiple molecular signaling path- ways that drive ACCs, a novel approach to drug develop- ment in this disease would include agents with a large therapeutic index that simultaneously target multiple sig- naling pathways critical for ACC proliferation and aggressiveness.
Discovery of anti-cancer compounds from natural product sources is a greatly untapped resource for identi- fication of novel compounds with anti-cancer benefits and low toxicity [10]. Withanolides are naturally occurring C-28-steroidal lactones present in the Solanaceae plant species, including the genera Physalis and Withania (which includes the Ashwagandha plant) [11-13]. Our group has reported on the anti-cancer effects of several natural and semi-synthetic withanolides in breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, melanoma, and glioblastomas [13-21]. We have also defined the structure-activity relationships for these compounds, demonstrating that acetate derivatives of withanolides such as withalongolide A (WGA)-4,19,27-triacetate (WGA-TA) and withalongolide B (WGB)-4,19-diacetate (WGB-DA) have significantly higher cytotoxic activity than unmodi- fied withaferin A (WA) or WGA [22]. Furthermore, studies from our group and others have reported that withanolides, including WA, exert their anti-cancer properties through a combination of oxidative stress and heat shock chaperone protein modulation, leading to induction of G2/M arrest, reactive oxygen species-dependent apoptosis, and sup- pression of multiple oncogenic pathways such as nuclear factor (NF)-KB, notch, PI3K, protein kinase B (Akt)/ mTOR, and mitogen-activated protein kinase (MAPK)
among others [13-23]. Given that the multi-targeted anti- cancer mechanism of withanolides includes the major signaling pathways and proteins important for ACC growth and aggressiveness, we hypothesize that the natural wit- hanolides will have significant potency against ACCs and could represent a novel therapeutic strategy to develop translationally. The main objective of this study is to define the first application of withanolides in ACCs and to investigate the efficacy of WA, WGA, WGA-TA, and WGB-DA from the Physalis longifolia plant in adreno- cortical cancer cells.
Materials and methods
Cell lines and reagents
The ACC cell lines SW13 and Y1 were generously pro- vided by Dr. Gary D. Hammer (University of Michigan, Ann Arbor, MI, USA). The human ACC cell line SW13 was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf serum (FCS), 100 IU/mL of penicillin, and 100 µg/mL of streptomycin. Murine Y1 cell line was cultured in DMEM medium (Life Technologies, Grand Island, NY, USA) supplemented with 2.5 % FCS, 7.5 % horse serum, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin. The cell lines were incubated in a humidified atmosphere of 5 % CO2 in air at 37 °C. The cells were maintained as monolayer culture, and the experiments were performed on exponentially growing cultures. Withanolides involved in the study, namely WA, WGA, WGA-TA, and WGB-DA, were obtained from Dr. Barbara Timmermann’s laboratory (Lawrence, KS, USA), and their structures have been previously described [15]. Reagents used in flow cytometry (FC) analysis such as propidium iodide (PI) and ribonu- clease (RNase) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-fluorescein isothiocyanate (FITC) used for analysis of apoptosis was obtained from BD Biosciences (San Diego, CA, USA).
MTS cell proliferation assay to evaluate the in vitro potency of WA, WGA-TA, and WGB-DA on the ACC cell lines
For in vitro testing of drug efficacy, two ACC cell lines, SW13 and Y1, and the normal fibroblast cell line MRC5 were used, and the viability was determined by Cell Titer 96 AQueous Non-Radioactive Cell proliferation assay (Promega, Fitchburg, WI, USA). Approximately 2,000 cells per well were seeded in a 96-well micro titer plates in 100 µL of growth media and were allowed to attach overnight. On the second day, serial dilutions of the
withanolides (WA, WGA, WGA-TA, and WGB-DA) were added in replicates of three to the wells. The cells were then incubated for 72 h at 37 ℃ in a CO2 humidified chamber. The number of viable cells was determined by measuring A490 of the dissolved formazan reagent on a BioTek Synergy Neo plate reader (BioTek, Winooski, VT, USA) after the addition of 20 uL of the MTS reagent for 2 h as per the manufacturer’s protocol. All experiments were carried out at three independent time points and the viability of the cells was expressed as the ratio of the number of viable cells with withanolide treatment com- pared with control untreated cells. The half-maximal inhibitory concentrations (IC50) were calculated from the MTS assay curves using GraphPad Prism 5 software. The two tailed p values were calculated using Microsoft® Excel.
Flow cytometry for cell cycle analysis
The ACC cell line SW13, grown in 60 mm plates to 60-80 % confluence, was treated with varying concentrations of WA, WGA-TA, and WGB-DA for 24 h. The cells were then trypsinized and harvested by centrifugation. The pelleted cells were re-suspended in 0.43 mL of ice-cold 1X phos- phate buffered saline (PBS) followed by 1 mL of ice-cold ethanol to get a 70 % ethanol fixative solution. The cells were stored at -20 ℃ until analysis. Before analysis, the cells were collected by centrifugation, stained with PI solu- tion (40 µg/mL PI and 100 µg/mL RNaseA), and incubated at 37 ℃ for 30 min. The cell cycle analysis was carried out by capturing 10,000 events per sample using CyAn™M ADP Analyzer (Beckman Coulter, Inc., Brea, CA, USA) at the University of Michigan FC Core. Only viable cells without DNA fragmentation were analyzed to establish the true cell cycle in living cells using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Each experiment was repeated at three independent time points to confirm the results.
Analysis of apoptosis
For the analysis of cell death, SW13 cells grown in 60 mm plates were treated with varying concentrations of WA, WGA-TA, and WGB-DA for 24 h. The cells were tryp- sinized, washed, and re-suspended in annexin binding buffer [14]. Cells were stained using annexin V-FITC/PI staining according to the manufacturer’s protocol, and induction of apoptosis was measured using the CyAn™M ADP Analyzer. The apoptotic and necrotic cells were dif- ferentiated after treatment with drugs by phosphatidylser- ine staining with annexin on the outer leaflet of the cell membranes for apoptotic cells, and DNA staining by PI for necrotic and late apoptotic cells. Data from three inde- pendent experiments were analyzed to confirm the results.
Immunoblot analysis of cells treated with withanolides
ACC cell line SW13, grown to 70-80 % confluence, was treated with varying concentrations of withanolide deriva- tives WA, WGA-TA, and WGB-DA for 24 h. The cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 % (v/v) NP-40, 0.5 % (w/v) sodium deoxycholate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, and 0.1 % (w/v) sodium dodecyl sulfate (SDS) supplemented with protease inhibi- tor solution (Sigma-Aldrich, St. Louis, MO, USA). After lysis, the cells were centrifuged at 14,000 rpm for 20 min, the supernatant was collected, and the proteins were quantified using the BCA Protein Assay (Thermo Scien- tific, Rockford, IL, USA). Equal amounts of proteins were separated using SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto a Hybond nitrocellulose membrane (GE Healthcare Life Sciences, Piscataway, NJ, USA). The membranes were blocked using 5 % milk and probed overnight with appropriate dilutions of the primary antibodies for proteins as previously described [14]. The blots were then washed three times with PBS-T (PBS- Tween 20) and incubated with 1:5,000 dilutions of horse- radish peroxidase (HRP) conjugated secondary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). To ensure equal loading of proteins, actin was used as a control. The bands were visualized using Super signal Chemiluminescence Reagent (Thermo Scientific), and the images were captured on Kodak X-ray film (Midsci, St. Louis, MO, USA). Densitometric analysis was performed using Image J software (National Institutes of Health [NIH]).
Results
Withanolides have potent anti-proliferative effects against ACCs in vitro
The viabilities of two adrenocortical cell lines, SW13 and Y1, and the normal fibroblast cell line MRC5 were deter- mined after treatment with increasing concentrations of withanolides (WA, WGA, WGA-TA, and WGB-DA) ranging from 0.0195 to 10 uM for 72 h using MTS dye reduction assay (Fig. 1) and the IC50 values were deter- mined for each compound (Table 1). For all the withano- lides tested, the human SW13 cell lines were much more sensitive than the murine Y1 cell lines. Of the withanolides tested in the experiments, WGA-TA was the most potent compound in both the cell lines, with an IC50 value of 29 nM for SW13 and 117 nM for Y1 cells. Addition of acetyl groups to WGA to form WGA-TA and to WGB to
(a) 120
WA
100
WGA
WGA-TA
% Viability
80
WGB-DA
60
40
20
0
-8
-7
-6
-5
Log[Drug] M
(b)120
WA
100
WGA
% Viability
WGA-TA
80
WGB-DA
60
40
20
0
-8
-7
-6
-5
Log[Drug] M
| Withanolides | Yl IC50 (nM) | SW 13 IC50 (nM) | MRC-5 fibroblast IC50 (nM) | Fold selectivity for ACC against MRC5 | p value |
|---|---|---|---|---|---|
| WA | 369 ± 14 | 31 ± 4 | 5,750 ± 45 | 15.6-185.5 | <0.001 |
| WGA | 570 ± 11 | 119 ± 12 | 12,700 ± 140 | 22.3-106.7 | <0.001 |
| WGA-TA | 117 ± 0.9 | 29 ± 3 | 1,580 ± 22 | 13.5-54.5 | <0.01 |
| WGB-DA | 337 ± 16 | 62 ± 7 | 2,510 ± 141 | 7.4-40.5 | <0.01 |
The ACC cells were treated with varying concentrations of WA, WGA-TA and WGB-DA. Viability of the cells were determined by MTS assay and the two-tailed p values were calculated using Microsoft® Excel
ACC adrenocortical carcinoma, IC50 half-maximal inhibitory concentration, WA withaferin A, WGA-TA withalongolide A-4,19,27-triacetate, WGB-DA withalongolide B-4,19-diacetate
form WGB-DA changed the IC50 values from 570 nM for WGA to 117 nM for WGA-TA and 337 nM for WGB-DA for Y1 cells. The IC50 values for the human SW13 ACC cells changed from 119 nM for WGA to 29 nM for WGA- TA and 62 nM for WGB-DA. These results indicate that the addition of the acetate group significantly increases the potency of the parent compounds in both the ACC cell lines used in the present study. In addition, the fold selectivity of withanolides for ACC cell lines were much higher by a factor of 15.6-185.5 for WA, 22.3-106.7 for WGA, 13.5-54.5 for WGA-TA, and 7.4-40.5 for WGB- DA compared with normal MRC5 cell lines, indicating that the drugs do not affect the normal cells at optimal con- centrations. The human SW13 cell lines, as well as the withanolides WA, WGA-TA, and WGB-DA were used in all the subsequent mechanistic studies.
Withanolides shift the cell cycle towards G2/M arrest in ACC cells
The effect of withanolides on cell cycle progression in human SW13 ACC cells was analyzed by FC after treat- ment with the compounds for 24 h. The results are graphed
in Fig. 2a and b. They indicate that the control vehicle- treated cells have a normal cycling pattern of distribution, with 65.78 % of cells at G0/G1 phase, 11.99 % at S phase, and 22.23 % at G2/M phase, whereas withanolide treat- ment of SW13 cells led to a concentration-dependent accumulation of G2/M phase, with concomitant losses in the G0/G1 phase and little or no variation in the S phase. WA treatment after 24 h resulted in G2/M shift from 22.23 % for untreated cells to 47.7 % at 2 µM WA (p < 0.01). Although 24 h treatment of WGA-TA and WGB-DA resulted in decreases in the levels of G0/G1 at low concentrations of 1 µM, a shift in GO/G1 to G2/M was not seen for WGA-TA and WGB-DA. The apoptotic sub- G1 population, with a characteristic hypodiploid DNA content peak, increased at a concentration of 1 µM for WGA-TA compared with 2 and 4 uM for WGB-DA and WA.
Cell cycle progression was examined by Western blot analysis of the cell cycle regulatory protein cyclin B1 after treatment with withanolides for 24 h to confirm our FC analysis. As shown in Fig. 2c, SW13 cells treated with withanolides demonstrate a dose-dependent induction of cyclin B1 expression, with elevation starting at 2 uM for
(a) 100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0
0
0
20K
40K
60K
0
20K
40K
60K
0
20K
40K
60K
(b)
GUM
100
T
-
x
I
# GOGI
S
100
I
GUM
100
I
GZA
= S
% Gated Cell Populations
80
C
I
% Gated Cell Populations
SO
# GO GI
I
I
T
% Gated Cell Populations
SO
GOGI
I
E
60
I
60
I
2
60
I
x
T
T
40
x
I
40
I
I
40
x
x
x
I
x
20
20
I
I
20
I
0
0
Control
1.0 µM
2.0 uM
4.0 μ.Μ
0
Control
1.0 μ.Μ
2.0 uM
4.0 µM
Control
1.0 µM
2.0 μ.Μ
4.0 µM
WA
WGA-TA
WGB-DA
(c)
Withaferin A
WGA-TA
WGB-DA
0.25
1.00
2.00
4.00
0.25
1.00
2.00
4.00
0.25
1.00
2.00
4.00
0
4
Cyclin B1
1
ß-actin
WA and at 1 µM for WGA-TA and WGB-DA. These results suggest that the addition of acetate derivatives to the withalongolide backbone leads to much faster G2/M tran- sition than the parent compound.
Withanolides induce apoptosis, PARP cleavage, and caspase activation in SW13 cells
To explore the mechanism through which withanolides induce cell death, we performed annexin V-FITC/PI double staining on flow cytometry. The necrotic, early apoptotic, and late apoptotic cells were distinguished by Annexin V staining of phosphatidylserine on the outer leaflet of apop- totic cells. ACC cell line SW13 was treated with either dimethyl sulfoxide (DMSO) or 0.5-2 uM of each withano- lide for 24 h, and apoptosis was assessed as shown in Fig. 3a. Evaluation of the results indicated that, compared with baseline apoptotic levels, increasing concentrations of
cyclin B1 after treatment of SW13 cells with increasing concentra- tions of withanolides by Western blot analysis. Actin was used as a loading control. PI propidium iodide, WA withaferin A, WGA-TA withalongolide A-4,19,27-triacetate, WGB-DA withalongolide B-4,19-diacetate
withanolide treatment led to enhanced cell death in SW13 cells. The percentage of cells in early apoptosis (annexin V-FITC+/PI-) were 5.1 and 7.3 % for 1 and 2 uM WA, respectively; 5.38, 4.24, and 21.03 % for 0.5, 1, and 2 µM WGA-TA, respectively; and 4.88, 10.17, and 21.76 % for 0.5, 1, and 2 µM WGB-DA, respectively. Late apoptosis (annexin V-FITC-/PI-) percentages were 9.23 and 43.41 % for 1 and 2 µM WA, respectively; 23.58, 53.78, and 76.14 % for 0.5, 1, and 2 µM WGA-TA, respectively; and 20.75, 43.81, and 70.72 % for 0.5, 1, and 2 µM WGB-DA, respectively. The necrosis (annexin V-FITC-/PI+) per- centages were 1.7 and 9.85 % for 1 and 2 uM WA, respec- tively; 3.59, 6.66, and 3.96 % for 0.5, 1, and 2 µM WGA-TA, respectively; and 2.65, 4.07, and 1.97 % for 0.5, 1, and 2 µM WGB-DA, respectively, as shown in Fig. 3b. Based on these in vitro results for apoptosis, cell viability, and cell cycle modulation, ranking of withanolides in order of increasing potency would be WA < WGB-DA < WGA-TA.
(a)
Control
WA 1.0pM
WA 2.0 pM
(b)
SO
% Gated Cell Populations
70
Necrotic
1
Lse apoptotic
60
Early apoptotic
-
-
-
50
I
1
”
1
40
1
-
1
-
-
-
-
-
-
-
4
-
-
30
WGA-TA 0.5pM
WGA-TA 1.0UM
WGA-TA 2.0UM
20
T
I
4
Propidium iodide
10
I
-
-
-
0
E
Control
1.0
2.0
0.5
1.0
2.0
0.5
1.0
2.0
”
”
WA
WGA-TA
WGB-DA
P
1
-
1
e
-
NP
«
-
1
-
NP
Drugs (IM)
WGB-DA 0.5UM
WGB-DA 1.0UM
WGB-DA 2.0UM
(c)
Withaferin A
WGA-TA
WGB-DA
N
-
4
0.25
1.00
2.00
4.00
0.25
1.00
2.00
4.00
0.25
1.00
2.00
4.00
0
Caspase 3
-
-
#
”
-
-
12
1ª
MP
N
-
-
.
-
NHÀ
v
PARP
Cleaved PARP
Annexin V-FITC
B-actin
Because we observed induction of apoptosis after wit- hanolide treatment, we next examined treated cells for evidence of poly ADP ribose polymerase (PARP) cleavage and caspase-3 activation by Western blot analysis after 24 h treatment of SW13 cells with increasing concentra- tions of each withanolide. Even though cleavage of PARP, which is a substrate of caspase-3, was seen starting from 1 µM withanolide treatment of SW13 cells for all wit- hanolides, caspase-3 activation evidenced by decrease in the level of pro-caspase-3 was seen only with the acetate derivatives (WGA-TA and WGB-DA) starting from 2 uM concentrations of drugs after 24 h treatment (Fig. 3c). These in vitro data support the rationale that the anti-pro- liferative effect of withanolides in SW13 adrenocortical cancer cells is partially mediated through dose-dependent induction of apoptosis.
Assessment of withanolides’ effect on intracellular regulatory pathways in SW13 cells
Recent reports have shown that up-regulation of jagged 1, IGF2 and IGF1R play a critical role in the pathophysiology of ACC [6, 24, 25]. Moreover, WA has been shown to
c Novel withanolides induce cleavage of PARP and down-regulation of pro-caspase 3 in a concentration-dependent manner after 24 h of treatment as shown in the Western blot. Actin served as loading control. FITC fluorescein isothiocyanate, PARP poly ADP ribose polymerase, WA withaferin A, WGA-TA withalongolide A-4,19,27- triacetate, WGB-DA withalongolide B-4,19-diacetate
modulate proteins involved in notch and other signaling pathways [14, 26]. Therefore, in an effort to identify the mechanism thorough which withanolides suppress viability and growth and induce cell cycle arrest and apoptosis in ACC cells, we treated SW13 cells with varying concen- trations of withanolides and evaluated the effects of these withanolides on key regulatory pathway proteins by Wes- tern blot analysis. Comparison of modulations of key pro- teins in notch, Akt/mTOR and MAPK for WA, WGA-TA, and WGB-DA are shown in Fig. 4a and b. First, we assessed the expression of jagged 1, a ligand for notch after treating SW13 cells with the different withanolides for 24 h. Interestingly, at the lower concentrations of 0.25 and 1 µM WGA-TA and WGB-DA treatment, the levels of jagged 1 increased and then decreased to the basal level at higher concentrations above 2 uM. When the cells were treated with WA, the levels of jagged 1 increased modestly only at 4 µM drug treatment. Down-regulation of total Akt, phos- phorylated Akt, and the downstream mTOR effector pro- teins phosphor-p70/S6K (Thr389) and 4EBP1 was seen starting at 2 uM for the more potent WGA-TA and WGB- DA analogues, whereas only a slight change in the levels of expressions was seen for WA, even at the highest
(a)
Withaferin A
WGA-TA
WGB-DA
0.25
1.00
2.00
4.00
0.25
1.00
2.00
4.00
0.25
1.00
2.00
4.00
0
A
Jagged 1
p-AKT
A
T-AKT
A
p-p85-S6K
A
p-p70-S6K
AA
p85-S6K
p70-S6K
A
p-4EBP1
4
T-4EBP1
B-actin
-
Withaferin A
WGA-TA
WGB-DA
0.25
1.0
2.0
4.0
0.25
1.0
2.0
4.0
0.25
1.0
2.0
4.0
0
HSP90
1.0
0.9
0.7
0.4 1 0.3
0.4
0.40.3
.3
0.4
0. 0.5 0.
0.3
HSF1
1.0
0.81.2
1.0
0.9
1.1
0.8
S 0.6
.4 1
.8
1.1
1.1
0.8
HSP70
1.0 2.9 5.4 9.913.1 6.9 8.5 15 15 8.0 9.8 11 11
HSP32
1.0
S.3
15
22
24
16
20
20
20
12
28
28
28
HSP27
.. 0 0.9
0.9
1.1
O.S
1.
1.0
1.0
1.0
0.9
21.
0.6
B-actin
concentration of 4 uM that was used. In addition, Raf 1 protein expression was also down-regulated in a dose- dependent manner in all the withanolides tested, reaching maximal inhibition at 4 uM, while the expression levels of total- and phospho-ERK increased after 24 h. Taken toge- ther, these results indicated that the withanolides modulate several proliferative proteins involved in notch, MAPK, and the PI3 K/Akt/mTOR pathways that are implicated in ACC pathogenesis.
(b)
Withaferin A
WGA-TA
WGB-DA
0.25
1.00
2.00
4.00
0.25
1.00
2.00
4.00
0.25
1.00
0
2.00
4.00
Raf-1
A
p-ERK
M
T-ERK
-
-
-
4
ß-actin
estimated by Western blot using appropriate primary antibodies. The results indicate modulation of proteins with increasing concen- trations of withanolides. ACC adrenocortical carcinoma, Akt protein kinase B, MAPK mitogen-activated protein kinase, mTOR mammalian target of rapamycin, PI3 K phosphoinositide 3-kinase
Withanolide treatment modulates the heat shock stress response proteins
WA has been suggested to exert its anti-cancer properties through inhibition of heat shock protein (HSP)-90 [14]. In addition, WA is known to bind to the c-terminus of HSP90 and target its clients for degradation [27]. Furthermore, in glioma cells, using a reporter cell line assay, it has been suggested that WA inhibits tumor growth by activating the heat shock response [28]. Therefore, we investigated the efficacy of withanolides in modulating heat shock response proteins in ACCs. Consistent with the above reports, the current study also demonstrates down-regulation of HSP90 and heat shock factor (HSF)-1as well as induction of HSP70 and -32 after withanolide treatment in SW13 cells (Fig. 5). As seen from the figure, the HSP90 levels decreased starting at 1 µM of WA, whereas the more potent acetate derivatives, WGA-TA and WGB-DA, showed reduction in the levels of HSP90 starting from 250 nM. While the levels of HSP27 did not change, the expression levels of both HSP70 and HSP32 increased for all the withanolides in a dose-dependent manner starting at 250 nM.
Discussion
ACC is a rare and routinely fatal disease that is highly resistant to conventional chemotherapeutic agents. There remains a critical need, therefore, for development of novel therapeutic strategies and drugs that are able to simulta- neously target multiple oncogenic pathways vital to the pathogenesis of these cancers. Even though current multi- kinase inhibitors and combination strategies adding
tyrosine kinase inhibitors (TKIs) and mTOR inhibitors make mechanistic sense, they have yet to demonstrate a significant long-term impact on efficacy and survival in ACC patients. To bridge this critical gap in treatment, the discovery and development of novel multi-targeted drug compounds with low toxicity profiles remains a high pri- ority, especially if these compounds can selectively and simultaneously target the critical pathways for ACC growth and survival.
Our group and others have studied the anti-proliferative effect of these natural withanolides, especially WA, in multiple cancer models both in vitro and in small animal efficacy and toxicity studies. In various cancer models withanolides have been shown to target notch, MAPK, PI3K/Akt/mTOR, and other oncogenic pathways that are highly implicated in the pathogenesis of ACC. Therefore, in the current study, we evaluated the efficacy of WA, WGA, WGA-TA, and WGB-DA derived from the P. lon- gifolia plant on the ACC cell lines SW13 and Y1. The results from our experiments demonstrate for the first time the anti-proliferative properties of withanolides on ACC cells. In addition, the acetylated analogues WGA-TA and WGB-DA were more potent than their parent compounds (WGA and WA) and were able to induce a dose-dependent progression of cell cycle and apoptosis in SW13 cells.
Since withanolides produced potent levels of apoptosis at the concentrations tested in our studies, we investigated several key regulatory proteins that are involved in the development and pathophysiology of ACC. Our results indicate that the levels of Akt, p-Akt, and downstream effectors of the mTOR pathway (p70S6K and 4EBP1) decrease, whereas phospho-ERK levels increase after treatment with increasing concentrations of withanolides. Similar to our report, activation of ERK phosphorylation has been detected after treatment with PI3K/Akt/mTOR inhibitors in other cancer models due to activation of the Raf pathway [29-31]. Our observed results therefore indicate that natural and semi-synthetic withanolides modulate the P13K/Akt/mTOR pathway and the MAPK pathway.
In addition to hyper-activation of IGF2 and IGF1R, upregulation of the notch ligand jagged 1 in ACCs has been shown to enhance cell proliferation and tumor aggressive- ness through activation of notch signaling. Even though increased levels of jagged 1, IGF2, and Wnt/ß-catenin pathway signaling are implicated in ACC, the exact inter- play between the Wnt and notch pathways in this disease is not clearly known [6]. In ovarian cancer, it has been reported that jagged 1 is activated by both Wnt/B-catenin and notch 3 pathways [32]. In addition, the Wnt/ß-catenin pathway regulating the expression of jagged 1 has also been reported in colorectal and intestinal cancers [33, 34]. Hence, to investigate how jagged 1 and the Wnt/B-catenin pathway
are inter-related in ACC, we looked at the expression levels of jagged 1 after treatment with withanolides in SW13 cells that have low levels of ß-catenin [35]. Our results indicate an increase in the level of jagged 1 in SW13 cells after 24 h of treatment, especially in the potent WGA-TA and WGB- DA analogues. Based on these observations, we hypothe- size that the Wnt pathway is upstream of notch and is activated by withanolides at lower concentrations. The reason why the robustness of this effect is not seen with WA may be due to its weaker potency than the acetate deriva- tives as well as slight variances in structure-activity func- tions between these compounds. However, future investigations are needed to better define the mechanism of notch and ß-catenin pathway modulation by withanolides in ACC.
Many of the key regulatory proteins involved in the critical signaling pathways for ACC proliferation and sur- vival are either clients or targets of HSP90. We have pre- viously observed down-regulation of HSP90 expression in papillary and anaplastic thyroid cancers with WA treatment [20]. Furthermore, recent studies have reported an induc- tion of the heat shock response following WA exposure [14, 28]. In the current study, we observe down-regulation of HSP90 and HSF1 protein expression, as well as induc- tion of HSP32 and HSP70 after exposure to increasing concentrations of withanolides. Consistent with previous reports, these results indicate that withanolides exert many of their anti-cancer/anti-proliferative effects in ACCs through modulation of HSP90 chaperone function and oxidative stress response. Inhibition of HSP90 and knockdown of its many client proteins, including Akt, HSF-1, and Raf-1, demonstrate that withanolides may
Withanolides
AKT
Heat shock proteins
Wnt/ß-catenin
mTOR
Jagged-1
S6K, 4EBP-1V
Translation of protein, regulation of apoptosis and cell cycle
Springer
modulate ERK1/2 in ACC through upstream proteins in the MAPK pathway.
These results demonstrate the first application of natural product withanolides for the treatment of ACCs in vitro. Based on the in vitro results reported and the known mechanisms of action of the drug, we have proposed a schematic as a potential mechanism of action in ACCs (Fig. 6). Natural withanolides are potent anti-proliferative agents against ACCs, inducing cell cycle arrest and apop- tosis as well as modulating key proteins in the notch, MAPK, and PI3K/Akt/mTOR pathways along with down- regulation of HSP90 function and expression. Given their potency and selectivity in ACCs in vitro, further transla- tional evaluation of these natural compounds is warranted in vivo to better assess their potential for future clinical applications in patients with advanced ACC.
Acknowledgments The authors would like to thank the Department of Surgery at the University of Michigan and the Department of Medicinal Chemistry at the University of Kansas for providing resources to facilitate this research. This research was supported in part through funding from the Department of Surgery at the Uni- versity of Michigan, as well as from research grant support from the National Center for Research Resources: NIH COBRE P20RR015563 (PI: B.N.T.), and a Cancer Center Support Grant Award (PI: M.S.C.) from the University of Michigan Comprehensive Cancer Center Support Grant (NCI : P30CA046592).
References
1. Michalkiewicz E, Sandrini R, Figueiredo B et al (2004) Clinical and outcome characteristics of children with adrenocortical tumors: a report from the international pediatric adrenocortical tumor registry. J Clin Oncol 22:838-845
2. Schteingart DE, Doherty GM, Gauger PG et al (2005) Manage- ment of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 12:667-680
3. Berruti A, Terzolo M, Sperone P et al (2005) Etoposide, doxo- rubicin and cisplatin plus mitotane in the treatment of advanced adrenocortical carcinoma: a large prospective phase II trial. Endocr Relat Cancer 12:657-666
4. Berthon A, Sahut-Barnola I, Lambert-Langlais S et al (2010) Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development. Hum Mol Genet 19:1561-1576
5. Heaton JH, Wood MA, Kim AC et al (2012) Progression to adrenocortical tumorigenesis in mice and humans through insu- lin-like growth factor 2 and beta-catenin. Am J Pathol 181:1017-1033
6. Simon DP, Giordano TJ, Hammer GD (2012) Upregulated JAG1 enhances cell proliferation in adrenocortical carcinoma. Clin Cancer Res 18:2452-2464
7. Demeure MJ, Bussey KJ, Kirschner LS (2011) Targeted therapies for adrenocortical carcinoma: iGF and beyond. Horm Cancer 2(6):385-392
8. Stigliano A, Cerquetti L, Sampaoli C et al (2012) Current and emerging therapeutic options in adrenocortical cancer treatment. J Oncol 2012:408131
9. Giordano TJ (2010) Adrenocortical tumors: an integrated clinical, pathologic, and molecular approach at the University of Michi- gan. Arch Pathol Lab Med 134:1440-1443
10. Sarkar FH, Li Y (2009) Harnessing the fruits of nature for the development of multi-targeted cancer therapeutics. Cancer Treat Rev 35:597-607
11. Gunasekera SP, Kinghorn AD, Cordell GA, Farnsworth NR (1981) Plant anticancer agents. XIX Constituents of Aquilaria malaccensis. J Nat Prod 44(5):569-572
12. Sahai M, Kirson I (1984) Withaphysalin D, a new withaphysalin from Physalis minima Linn. var. indica. J Nat Prod 47(3): 527-529
13. Zhang H, Samadi AK, Rao KV, Cohen MS, Timmermann BN (2011) Cytotoxic oleanane-type saponins from Albizia inundata. J Nat Prod 74(3):477-482
14. Grogan PT, Sleder KD, Samadi AK, Zhang H, Timmermann BN, Cohen MS (2013) Cytotoxicity of withaferin A in glioblastomas involves induction of an oxidative stress-mediated heat shock response while altering Akt/mTOR and MAPK signaling path- ways. Invest New Drugs 31(3):545-557
15. Samadi AK, Bazzill J, Zhang X, Gallagher R, Zhang H, Golla- pudi R, Timmermann BN, Cohen MS (2012) Novel withanolides target medullary thyroid cancer through inhibition of both RET phosphorylation and the mammalian target of rapamycin path- way. Surgery 152(6):1238-1247
16. Samadi AK, Mukerji R, Shah A, Timmermann BN, Cohen MS (2010) A novel RET inhibitor with potent efficacy against med- ullary thyroid cancer in vivo. Surgery 148(6):1228-1236
17. Samadi AK, Tong X, Mukerji R, Zhang H, Timmermann BN, Cohen MS (2010) Withaferin A, a cytotoxic steroid from Vassobia breviflora, induces apoptosis in human head and neck squamous cell carcinoma. J Nat Prod 73(9):1476-1481
18. Zhang X, Mukerji R, Samadi AK et al (2011) Down-regulation of estrogen receptor-alpha and rearranged during transfection tyro- sine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells. BMC Complement Altern Med 11:84
19. Samadi AK, Cohen SM, Mukerji R et al (2012) Natural wit- hanolide withaferin A induces apoptosis in uveal melanoma cells by suppression of Akt and c-MET activation. Tumour Biol 33:1179-1189
20. Samadi A, Loo P, Mukerji R et al (2009) A novel HSP90 mod- ulator with selective activity against thyroid cancers in vitro. Surgery 146:1196-1207
21. Zhang H, Samadi AK, Gallagher RJ et al (2011) Cytotoxic wit- hanolide constituents of Physalis longifolia. J Nat Prod 74:2532-2544
22. Zhang H, Samadi AK, Cohen MS, Timmermann BN (2012) Antiproliferative withanolides from the Solanaceae: a structure- activity study. Pure Appl Chem 84:1353-1367
23. Mayola E, Gallerne C, Esposti DD et al (2011) Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2. Apoptosis 16:1014-1027
24. Almeida MQ, Fragoso MC, Lotfi CF et al (2008) Expression of insulin-like growth factor-II and its receptor in pediatric and adult adrenocortical tumors. J Clin Endocrinol Metabolism 93: 3524-3531
25. Barlaskar FM, Spalding AC, Heaton JH et al (2009) Preclinical targeting of the type I insulin-like growth factor receptor in adrenocortical carcinoma. J Clin Endocrinol Metab 94:204-212
26. Koduru S, Kumar R, Srinivasan S, Evers MB, Damodaran C (2010) Notch-1 inhibition by Withaferin-A: a therapeutic target against colon carcinogenesis. Mol Cancer Ther 9(1):202-210
27. Yu Y, Hamza A, Zhang T et al (2010) Withaferin A targets heat shock protein 90 in pancreatic cancer cells. Biochem Pharmacol 79:542-551
28. Santagata S, Xu YM, Wijeratne EM et al (2012) Using the heat- shock response to discover anticancer compounds that target protein homeostasis. ACS Chem Biol 7:340-349
29. Gotink KJ, Broxterman HJ, Labots M et al (2011) Lysosomal sequestration of sunitinib: a novel mechanism of drug resistance. Clin Cancer Res 17:7337-7346
30. Manara MC, Nicoletti G, Zambelli D et al (2010) NVP-BEZ235 as a new therapeutic option for sarcomas. Clin Cancer Res 16:530-540
31. Roccaro AM, Sacco A, Husu EN et al (2010) Dual targeting of the PI3 K/Akt/mTOR pathway as an antitumor strategy in Wal- denstrom macroglobulinemia. Blood 115:559-569
32. Chen X, Stoeck A, Lee SJ et al (2010) Jagged1 expression reg- ulated by Notch3 and Wnt/beta-catenin signaling pathways in ovarian cancer. Oncotarget 1:210-218
33. Rodilla V, Villanueva A, Obrador-Hevia A et al (2009) Jagged 1 is the pathological link between Wnt and notch pathways in colorectal cancer. Proc Natl Acad Sci USA 106:6315-6320
34. Guilmeau S, Flandez M, Mariadason JM et al (2010) Heteroge- neity of Jagged1 expression in human and mouse intestinal tumors: implications for targeting Notch signaling. Oncogene 29:992-1002
35. Durand J, Lampron A, Mazzuco TL et al (2011) Characterization of differential gene expression in adrenocortical tumors harboring beta-catenin (CTNNB1) mutations. J Clin Endocrinol Metab 96:E1206-E1211