Carcinogenesis vol.32 no.4 pp.568–575, 2011
Advance Access publication February 8, 2011
2#-Hydroxyflavanone inhibits proliferation, tumor vascularization and promotes
normal differentiation in VHL-mutant renal cell carcinoma
Lokesh Dalasanur Nagaprashanthay, Rit Vatsyayany,
Jyotsana Singhal, Poorna Lelsani, Laszlo Prokai, Sanjay
Awasthi and Sharad S.Singhal?
Department of Molecular Biology and Immunology, University of North
Texas Health Science Center, Fort Worth, TX 76107, USA
?To whom correspondence should be addressed. Tel: þ1 817 735 2236;
Fax: þ1 817 735 2118;
Renal cell carcinoma (RCC) is one of the top tencancers prevalent
in USA. Loss-of-function mutations in the von Hippel–Lindau
(VHL) gene constitute an established risk factor contributing to
75% of total reported cases of RCC. Loss-of-VHL leads to a highly
vascularized phenotype of renal tumors. Intake of citrus fruits has
been proven to reduce the risk of RCC in multicenter interna-
tional studies. Hence, we studied the effect of 2#-hydroxyflavanone
(2HF), an active anticancer compound from oranges, in RCC.
Our in vitro investigations revealed that 2HF suppresses VHL-
mutant RCC to a significantly greater extent than VHL-wild-type
RCC by inhibiting epidermal growth factor receptor signaling,
which is increased due to VHL mutations in RCC. Our results
also revealed for the first time, that 2HF inhibits glutathione S-
transferase pi activity. 2HF reduced cyclin B1 and CDK4 levels
and induced G2/M phase arrest in VHL-mutant RCC. Impor-
tantly, 2HF inhibited the angiogenesis in VHL-mutant RCC by
decreasing vascular endothelial growth factor expression. Our in
vivo studies in mice xenografts confirmed our in vitro results as
evident by decreased levels of proliferation marker, Ki67 and
angiogenic marker, CD31, in 2HF-treated mice xenografts of
VHL-mutant RCC. 2HF also increased the expression of E-cad-
herin in VHL-mutant RCC, which would be of significance in
restoring normal epithelial phenotype. Collectively, our in vitro
and in vivo results revealed the potent antiproliferative, anti-
angiogenic and prodifferentiation properties of 2HF in VHL-
mutant RCC, sparing normal cells, which could have significant
implications not only in the specific management of VHL-mutant
RCC but also towards other VHL syndromes.
Renal cell carcinoma (RCC) is a frequently lethal cancer that affects
patients who carry inherited or somatic mutations in the von Hippel–
Lindau (VHL) gene, which contributes to 75% of total RCCs (1–3).
RCC arises from epithelial cells of the proximal renal nephron and is
characterized by its many different cytological and histological var-
iants (3). Tumor vascularity is of specific significance in RCC because
of constitutively active hypoxic signaling in majority of renal tumors
as a consequence of VHL mutations. According to National Cancer
Institute, 1 in 67 men and women harbor the lifetime risk for RCC.
Current chemotherapeutic choices for the advanced kidney cancer are
limited, with a low chance of temporary remission, small improve-
ment in average survival and substantial toxicity (4). The association
of lifestyle habits like tobacco smoking with RCC along with the
increased risk for RCC in VHL-mutant populations makes the chemo-
prevention of RCC an important public health necessity (3–5). In this
regard, validation of the potential VHL-mutant RCC-specific antican-
cer compounds attains contemporary significance in renal oncology.
Flavonoids are a large group of polyphenolic compounds present in
foods and beverages of plant origin, which have antioxidant, anti-
inflammatory, antimutagenic and antiproliferative properties (6–8).
2#-Hydroxyflavanone (2HF) is a flavanone belonging to the larger
family of flavonoids. The multicenter international RCC studies have
established that the intake of citrus fruits is associated with decreased
risk of RCC (9). 2HF is known for its antimetastatic effects in lung
cancer (10). In the present report, we show that 2HF, an active anti-
cancer compound in oranges and citrus fruits, predominantly inhibits
the growth of VHL-mutant RCC, a major subtype of RCC. Our inves-
tigations addressed the impact of 2HF on oncogenic processes of
importance in loss-of-VHL induced renal carcinogenesis like regula-
tion of tumor proliferation and specifically angiogenesis in addition to
investigating the impact on differentiation of 2HF-treated VHL-
mutant RCC tumors in vivo. Our collective in vitro and in vivo inves-
tigations elucidated the anticancer potential and novel mechanisms of
action of 2HF in VHL-mutant RCC.
Materials and methods
3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and
2HF were obtained from Sigma (St Louis, MO). AKRIC, VHL, CD31, Ki67,
cyclin B1, CDK4, Akt, epidermal growth factor receptor (EGFR), PI3K and E-
cadherin antibodies were purchased from Santa Cruz Biotechnology (Colum-
bus, OH) and Cell Signaling Technologies (Danvers, MA). ELISA kit for
vascular endothelial growth factor (VEGF) expression was procured from
R&D Systems. Source of glutathione S transferase p (GSTp) antibody was
the same as described previously (11). Matrigel was procured from BD Bio-
sciences (San Jose, CA). Terminal deoxynucleotidyl-transferase deoxyuridine
triphosphate nick-end labeling (TUNEL) fluorescence and avidin/biotin com-
plex (ABC) detection kits were purchased from Promega (Madison, WI) and
Vector (Burlingame, CA), respectively.
Cell lines and cultures
Human RCCs (Caki-2) was purchased from American Type Culture Collection
(Manassas, VA), and Caki-1, A-498 and 786-O cells were kindly authenticated
and provided by Dr William G.Kaelin, Dana–Farber Cancer Institute, Harvard
Medical School, Boston, MA. Human kidney normal (mesangial) cells were
a generous gift from Dr Rong Ma, University of North Texas Health Science
Center (Fort Worth, TX). All cells were cultured at 37?C in a humidified
atmosphere of 5% CO2in RPMI-1640 medium supplemented with 10% fetal
bovine serum and 1% P/S solution. All cells were tested for Mycoplasma once
every 3 months.
Proteomic analysis, database searching and comparison of protein expression
RCC cells were lysed in buffer containing 20 mM Tris–HCl, 50 mM NaCl and
6 M urea, 10 mM NaPP, 1 mM NaF and 1 mM Na3VO4. The lysate (200 lg
protein) was subjected to reduction and alkylation of cysteines using 2.5 mM
dithiothreitol and 7 mM idoacetamide followed by trypsin digestion and solid
phase extraction using a C18cartridge (Supelco, Bellefonte, PA). The digested
peptides were analyzed using reverse-phase liquid chromatography-tandem
mass spectrometry analysis using a hybrid Linear ion trap (LTQ)–Fourier
transform ion cyclotron resonance (FTICR, 7T) mass spectrometer (LTQFT;
Thermo, San Jose, CA), which is equipped with nanospray ionization source
and operated by XCalibur (version 2.2) data acquisition software as described
previously(12).A 120min gradientprovideby nano-LC2D (Eksigent,Dublin,
CA) was carried out to 40% acetonitrile at 250 nl/min. An electrospray ioni-
zation spray voltage of 2.0 kV and a capillary temperature of 250?C were
maintained during the run. We employed a data-dependent mode of acquisition
in which accurate mass/charge (m/z) survey scan was done in FTICR cell
followed by a parallel MS/MS linear ion trap analysis. FTICR full-scan mass
Abbreviations: AKR1C1, aldoketo reductase family 1, member C1; EGFR,
epidermal growth factor receptor; GSTp, glutathione S-transferase pi; 2HF, 2#-
hydroxyflavanone; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazo-
lium bromide; RCC, renal cell carcinoma; TUNEL, terminal deoxynucleotid-
yl-transferase deoxyuridine triphosphate nick-end labeling; VEGF, vascular
endothelial growth factor; VHL, von Hippel–Lindau.
yThese authors contributed equally to this work.
? The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org
spectra were acquired at 100 000 mass resolving power (at m/z 400) from m/z
350 to 1500 using the automatic gain control mode of ion trapping. Collision-
induced dissociation in the linear ion trap was performed using a 3.0 Tn iso-
lation width and 35% normalized collision energy with helium as the collision
gas. MS/MS spectra were searched against a human protein database by the
analysis was guided first by normalized spectral counts from the Scaffold
program (Proteome Software, Portland, OR) with previously validated method
(12). Extracted ion chromatograms (areas under the corresponding chromato-
graphic peaks) of isoform-specific doubly or triply charged tryptic peptides
from the full-scan high-resolution mass spectra were then used as quantitative
measures of respective protein expression levels selected for evaluation in this
Drug sensitivity (MTT) assay
Cell density measurements were performed using a hemocytometer to count
reproductive cells resistant to staining with trypan blue. Approximately 20
000 cells were plated into each well of 96-well flat-bottomed microtiter plates.
After 12 h incubation at 37?C, medium containing 2HF (ranging 0–200 lM)
were added to the cells. After 72 h incubation, 20 ll of 5 mg/ml MTT were
introduced to each well and incubated for 2 h. The plates were centrifuged and
medium was decanted. Cells were subsequently dissolved in 100 ll dimethyl
sulfoxide with gentle shaking for 2 h at room temperature, followed by mea-
surement of optical density at 570 nm (13–15). Eight replicate wells were used
at each point in each of three separate measurements.
Colony formation assay
Cell survival was evaluated using a standard colony-forming assay. In total, 1 ?
105cells/ml were incubated with 2HF (50 lM) for 24 h, and aliquots of 50 or
100 ll were added to 60 mm size petri dishes containing 4 ml culture medium.
After 10 days, adherent colonies were fixed, stained with 0.5% methylene blue
for 30 min and colonies were counted using the Innotech Alpha Imager HP (16).
Effect of 2HFon apoptosis by TUNEL assay
In total, 1 ? 105cells were grown on the coverslips for ?12 h followed by
treatment with 2HF (50 lM) for 24 h. Apoptosis was determined by the
labeling of DNA fragments with TUNEL assay using Promega apoptosis
detection system according to the protocol described previously (15).
Flow cytometry analysis
The effect of 2HF on cell cycle distribution was determined by fluorescence
activated cell sorting analysis. In total, 2 ? 105cells were treated with 2HF
(ranging from 25 to 50 lM) for 18 h at 37?C. After treatment, floating and
adherent cells were collected, washed with phosphate-buffered saline and fixed
counted and same numbers of cells were resuspended in 500 ll phosphate-
buffered saline in flow cytometry tubes. Cells were then incubated with 2.5 ll
of RNase (stock 20 mg/ml)at37?C for 30minafter whichthey were treated with
10 ll of propidium iodide (stock 1 mg/ml) solution and then incubated at room
temperature for 30 min in the dark. The stained cells were analyzed using the
Beckman Coulter Cytomics FC500, Flow Cytometry Analyzer. Results were
processed using CXP2.2 analysis software from Beckman Coulter.
In vitro migration assay
Cell migration was determined using a scratch assay (17). In total, 2 ? 104
Caki-2 and 786-O cells were seeded in six-well plates to reach 100% conflu-
ence within 24 h and then treated with 10 lM mitomycin C for 2 h. Sub-
sequently, a similarly sized scratch was made with a 200 ll pipette tip
across the center of each well and immediately imaged at baseline and then
at 24 h under an Olympus Provis AX70 microscope. The rate of cell migration
was determined by comparing the sizes of scratch area using Image J software.
In vitro angiogenesis assay (tube formation assay)
Tube formationassaywasperformed asfollows:96-wellplateswerecoatedwith
100 ll of Matrigel (10 mg/ml) and incubated at 37?C for 30 min to promote
gelling. Fourteen thousand cells were resuspended in medium (serum concen-
tration 10%) and added to each well. Tube formation in the presence of 50 lM
2HF wascompared. The number and lengthoftubesformedwere countedunder
an Olympus Provis AX70 microscope for analysis between both the groups.
Assessment of angiogenesis, proliferation and apoptosis
Renal tumors (control as well as 2HF treated) were harvested from mice-
bearing tumors for 60 days. Tumor samples fixed in buffered formalin for 12
h were processed conventionally for paraffin-embedded tumor sections (5 lm
thick). Hematoxylin and eosin staining was performed on paraffin-embedded
tumor sections. Histopathologic analyses with anti-E-cadherin, anti-CD31 and
anti-Ki67 IgG were also performed, using Universal ABC detection kit (Vec-
tor). The sections were examined under Olympus Provis AX70 microscope
connected to a Nikon camera.
In vivo xenograft studies
Hsd: athymic nudenu/numicewere obtained fromHarlan(Indianapolis, IN) and
were acclimated for a week before beginning the experiment. All animal experi-
ments were carried out in accordance with a protocol approved by the Institu-
tional Animal Care and Use Committee (IACUC). Twenty-eight 11-weeks-old
mice were divided into four groups of seven animals (treated with vehicle only
i.e. corn oil and 2HF at the doses of 0.0025, 0.005 and 0.01% wt/wt). All 28
animals were injected with 2 ? 106786-O (VHL mutant) cells in 100 ll of
phosphate-buffered saline, subcutaneously into one flank of each mouse. At
the same time, animals were randomized into control and treatment groups.
Treatment was started 10 days after the 786-O cells implantation to see palpable
tumor growth. Treatment consisted of 2HF at the doses of 0.0025, 0.005 and
0.01% (wt/wt), equivalent to 25, 50 and 100 mg/kg body wt. respectively, in 200
ll cornoil byoralgavage alternate day.Control groupswere treatedwithcornoil
only. In parallel, we also performed Caki-2 (VHL-wild-type) RCC xenografts
studies. Animals were examined daily for signs of tumor growth. Tumors were
measured in two dimensions using calipers and body weights were recorded.
Each mouse in every group was monitored on alternate days for signs of distress
andareasofswellingor redness.Photographsofanimalswere takenatday1,day
10, day 20, day 40 and day 60 after subcutaneous injection are shown for all
groups. Photographs of tumors were also taken at day 60.
All data were evaluated with a two-tailed unpairedStudent’s t-test or compared
by one-way analysis of variance and are expressed as the mean ± standard
deviation. A P-value of ,0.05 was regarded as statistically significant.
2HF inhibits proliferation and stimulates apoptosis in VHL-mutant
The MTT assay following the treatment of 2HF in RCC cell lines
revealed the potent inhibition of survival of VHL-mutant RCC in
the presence of 2HF [IC50at 72h: VHL-mutant RCC (786-O and
A498): 28 ± 4 lM, VHL-wild-type RCC (Caki-1 and Caki-2): 90 ±
6 lM] (Figure 1A). In accordance with MTTassay, 2HF inhibited the
clonogenic survival of VHL-mutant RCC (?70% inhibition) in colony
formation assay to significantly greater extent when compared with
VHL-wild-type RCC cells (?20% inhibition) (Figure 1B). Following
our initial investigations in four RCC cell lines, we investigated the
detailed mechanisms of action of 2HF in Caki-2 (VHL wild-type) and
786-O (VHL mutant) cells. Our initial cytotoxicity studies revealed
that 2HF inhibits the growth of VHL-mutant RCC to a greater extent
when compared with its inhibitory effect on VHL-wild-type RCC.
Hence, we focused on investigating the preceding cellular events that
determine the eventual cytotoxicity of 2HF in RCC. The cytotoxicity
of 2HF treatment was also determined at 24 h by the MTTassay (IC50
at 24 h: 786-O 5 72 ± 6 lM, caki2 5 148 ± 11 lM). We used 50 lM
of 2HF for 24 h treatment for both the cell lines as cell death should be
minimal formechanistic and imaging studies focused on early cellular
events that contribute to eventual cytotoxicity at 72 h. The 50 lM of
2HF treatment for 24 h effectively induced apoptosis in VHL-mutant
RCC to a greater extent, sparing normal mesangial cells, when com-
pared with VHL-wild-type RCC as determined by enhanced DNA
fragmentation in TUNEL apoptotic assay (Figure 1C). The enhanced
cytotoxicity of 2HF in VHL-mutant RCC along with the absence of
any cytotoxicity towards normal mesangial cells in MTT, clonogenic
survival and TUNEL apoptotic assays revealed that 2HF is a potential
flavonoid that could have significant therapeutic relevance in specif-
ically targeting VHL-mutant RCC.
2HF inhibits activation of EGFR, PI3K and Akt signaling in VHL-
Loss-of-VHL leads to upregulation of EGFR signaling in renal can-
cers (18). Activation of EGFR is involved in the growth and progres-
sion of many types of solid tumors, including RCC by upregulating
PI3K and Akt signaling (19). Hence, we investigated the effect of 2HF
2HF inhibits VHL-mutant RCC
on EGFR signaling in VHL-mutant RCC. Western blot analysis re-
vealed that 2HF significantly inhibits pEGFR (Y1068), PI3K (Y458/199)
and pAkt (S473) in VHL-mutant RCC (Figure 1D). The 2HF treatment
also increased poly ADP-ribose polymerase-cleavage in VHL-mutant
RCC (786-O) to a significantly greater extent when compared with
VHL-wild-type (Caki-2) RCC.
Detection of differential expression of AKR1C1 and GSTp in RCC
In order to understand the differences in the VHL-wild-type and VHL-
mutant RCC, we performed proteomic analysis of whole cell proteome
using a hybrid linear ion trap–Fourier transform ion cyclotron reso-
nance tandem mass spectrometer (LTQFT; Thermo) operated with
nano-electrospray ionization and coupled to an Eksigent nano-LC
system (12). MS/MS spectra were searched against a human protein
database by the Mascot software (Matrix Science) and label-free
quantification was guided first by spectral counts from the Scaffold
software (Proteome Software, Version 2) with our previously vali-
dated method (12). Caki-2 (VHL wild-type) and 786-O (VHL mutant)
cells, revealed differential expression of aldoketo reductase family 1,
member C1 (AKR1C1; selectively detected in Caki-2 RCC) and
GSTp (selectively detected in 786-O RCC). The MS/MS spectra of
isoforms-specific representative peptides for these proteins are shown
in the top with corresponding peptide sequence below (Figure 2A). The
of doubly and triply charged tryptic peptides detected for AKR1C1 and
GSTp, respectively, are represented in the bar diagrams. This observed
differential expression of AKR1C1 and GSTp was also revalidated by
western blot analysis using specific antibodies (Figure 2B).
2HF inhibits GSTp activity, angiogenesis and migration of VHL-
The enhanced growth inhibitory effect of 2HF, a well characterized
AKR1C family inhibitor, in VHL-mutant RCC which does not express
AKR1C1 was an interesting finding (20). We investigated the effect of
2HFon the enzymatic activity of GSTp towards GSH and 1-chloro 2,4-
dinitro benzene (1, chloro 2, 4-dinitro benzene), a model substrate
routinely used for GST activity (11). 2HF inhibited the total GST ac-
tivity to a significant extent in the VHL-mutant RCC (Figure 3A).
Human recombinant purified GSTp was used as a standard in enzyme
which mediates xenobiotic resistance by detoxifying administered che-
established marker of many aggressive cancers like lung and prostate
cancers (21,22). GSTp-mediated detoxification of toxic end products of
Fig. 1. Enhanced anticancer effects of 2HF in VHL-mutant RCC sparing normal cells. Drug sensitivity assays were performed by MTTassay using 2HF at 72 h
posttreatment to determine IC50. Values are presented as mean ± standard deviation from two separate determinations with eight replicates each (n 5 16) (panel
A). Colony-forming assay was performed and the colonies were counted using Innotech Alpha Imager HP as detailed in Materials and Methods.?P , 0.001
compared with control (panel B). For TUNEL apoptosis assay, cells were grown on coverslips and treated with 50 lM 2HF for 24 h. TUNEL assay was performed
using Promega fluorescence detection kit and examined using Zeiss LSM 510 META laser scanning fluorescence microscope with filters 520 and .620 nm.
Photographs taken at identical exposure at ?400 magnification are presented. Apoptotic cells showed green fluorescence (panel C). Effect of 2HF on poly ADP-
ribose polymerase cleavage, EGFR, PI3K and Akt activation: VHL-wild-type (Caki-2) and VHL-mutant (786-O) control and 50 lM 2HF-treated cells were lysed
and analyzed by western blot forpoly ADP-ribose polymerase cleavage, pEGFR (Y1068), pAkt (S473) and PI3K (Y458/199) by using specific antibodies. Membranes
were stripped and reprobed for glyceraldehyde 3-phosphate dehydrogenase as a loading control (panel D).
L.D.Nagaprashantha et al.
lipid peroxidation like 4-Hydroxy-2-nonenal (4-HNE) leads to buffer-
ing of tumor-toxic oxidative stress and favors tumor survival and pro-
liferation in hypoxic environment (23). GSTpalso has posttranslational
regulatory role in S-glutathionylation of various cell proteins, which is
implicated in regulating cell adhesion and proliferation (24). In this
regard, the ability of 2HF to inhibit GSTp and total GST activity in
VHL-mutant RCC, which has high levels of expression of GSTp rep-
resents an important anticancer effect of 2HF given its cytotoxic poten-
tial in VHL-mutant RCC. Further detailed studies would reveal the role
of GSTp and oxidative stress pathways in mediating the anticancer
effects of 2HF in RCC. As there are no chemopreventive strategies
reported for the VHL-mutant RCC, which is a highly prevalent malig-
nancy in USA and given the ability of 2HF to effectively inhibit the
survival of VHL-mutant RCC as revealed by our initial studies, we
specifically focused on studying the impact of 2HF in regulating the
proliferative potential, angiogenic response and differentiation of VHL-
mutant RCC both in vitro and in vivo.
VHL-null/mutant renal tumors are characterized by an angiogenic
phenotype due toconstitutiveHIF2a upregulation as aconsequence of
loss-of-VHL function (25). Hence, the investigation of the regulation
of tumor angiogenesis is important in the characterization of effective
anticancer compounds and further drug development. We studied the
effect of 2HF on angiogenic signaling in vitro by examining VEGF
expression (26). 2HF treatment caused significant reduction in the
levels of VEGF expression in VHL-mutant RCC when compared with
VHL-wild-type RCC (Figure 3B). 2HF treatment lead to specific and
significant decrease in angiogenesis as determined by change in both
the number and size of cellular tubes formed in invitro tube formation
assay in VHL-mutant RCC (Figure 3C). Following invitro angiogenic
assay, we studied the effect of 2HF on the migratory potential of RCC
in vitro. 2HF treatment also caused significant inhibition of cell mi-
gration in wound-healing assay in VHL-mutant RCC (Figure 3D).
2HF inhibits cell cycle progression in VHL-mutant RCC
The mechanism of cytotoxicity of 2HF was further assessed by de-
termining apoptosis through cell cycle fluorescence activated cell
sorting analysis. The 50 lM of 2HF treatment for 18 h caused G2/
M phase arrest, which was predominant in VHL-mutant RCC (?61%
cells accumulated in G2phase, P , 0.01) (Figure 4A). Please note
that the use of even higher concentration of 2HF (50 lM) in Caki-2
RCC was not effective in inhibiting cell cycle when compared with
cell cycle results obtained with 25 lM of 2HF in 786-O RCC. We
further analyzed the morphology of RCC cells after 2HF treatment.
The VHL-mutant and VHL-wild-type RCC were treated with 50 lM
of 2HF for 24 h and the cell morphology was observed by live cell
imaging in Zeiss phase contrast microscope. The 2HF-treated VHL-
mutant RCC cells were less adherent and more rounded compared
with the controls and VHL-wild-type RCC. The initial morphological
observation of control and 2HF-treated VHL-mutant RCC cells in-
dicated impaired cell division in 2HF-treated cells. 2HF-treated
VHL-mutant RCC cells had more cells that were unable to complete
cytokinesis compared with the control cells (Figure 4B). These re-
sults confirmed G2/M phase arrest and potential inhibition of the
completion of cytokinesis in 2HF-treated VHL-mutant RCC. The
2HF treatment reduced the levels of cyclin B1 and CDK4 in VHL
mutant but not in VHL-wild-type RCC (Figure 4C). Some of the
natural anticancer compounds like silibinin are known to cause G2/
M phase arrest by inhibiting cyclin B1 (27). CDK4, commonly as-
sociated with G1transition, has been also investigated for its role in
G2/M transition and it has been shown that overexpression of dom-
inant-negative CDK4 leads to arrest of G2phase progression (28).
Some of the anticancer compounds like apigenin and thiomersal also
cause inhibition of CDK4 along with cyclin B1 while causing G2/M
phase arrest (29,30). Collectively, our in vitro results strongly vali-
dated the specific antiproliferative, anti-angiogenic and antimeta-
static effects of 2HF in VHL-mutant RCC which lead to further
investigation of 2HF in vivo mice xenografts.
2HF induces potent tumor regression in vivo mice xenografts
VHL-mutant 786-O RCC cells bearing animals with established sub-
cutaneously implanted tumors (?20 mm2) were treated with 0.0025,
0.005 and 0.01% (wt/wt) (equivalent to25, 50 and 100 mg/kg body wt,
Fig. 2. Differential expression of AKR1C1 and GSTp in RCC as detected by LC-MS and MS/MS. Caki-2 and 786-O cells were subjected to proteomic analyses
as described in Methods section. MS spectra were searched against a human protein database by Mascot software (Matrix Science) and label-free quantification:
upper panel shows the MS/MS spectrum of one of the sequenced, isoforms-specific tryptic peptides for the respective proteins with the sequence coverage displayed
below. The bar diagrams indicating the quantitative levels of AKR1C1 and GSTp, respectively, were based on integration of extracted ion chromatograms, n 5 4
in each group, for the triply and doubly charged isoform-specific tryptic peptides; n.d. denotes that the peptide was not detected in the samples (panel A). Differential
protein expression was confirmed by performing western blot using 50 lg of cell lysates and antibodies against AKR1C, GSTp and VHL. for glyceraldehyde 3-
phosphate dehydrogenase was used as internal loading control. The experiment was repeated three times and similar results were obtained (panel B).
2HF inhibits VHL-mutant RCC
respectively) of 2HF in corn oil by oral gavage on alternate days. In
the present studies, doses of 2HF were well tolerated by the mice and
did not result in any weight loss compared with age-matched controls
(Figure 5A). Photographs of animals were taken at day 1, 10, 20, 40
and 60 after subcutaneous injection. Tumors grew more slowly in
VHL-mutant RCC mice xenografts administered with 2HF than in
respective untreated control mice. At day 60, tumor cross-sectional
area and tumor weight of mice bearing VHL-mutant RCC was signif-
icantly lower in 0.01% (wt/wt) dose-treated group as compared with
thevehicle only (corn oil) treated group (19.8 ± 3 versus 122 ± 7 mm2
and 0.07 ± 0.01 g versus 2.14 ± 0.24 g, respectively; P , 0.001). More
importantly, in vivo studies showed that administration of 2HF at
0.01% (wt/wt), to nude mice-bearing VHL-mutant RCC completely
arrested tumor progression, whereas uncontrolled growth was ob-
served in the animals treated with vehicle only (Figure 5B and C).
The 2HF-treated animals with VHL-mutant RCC were still alive at
139 days. In comparison, all animals treated with vehicle only were
censored by day 71 ± 3. These results indicated that dietary 2HF
administration inhibits VHL-mutant RCC growth and prolongs sur-
vival without causing side effects. To rule out the possibility that the
observed invivo effects of 2HF were specific to VHL-mutant RCC, we
also evaluated the antineoplastic effects of 2HF on the VHL-wild-type
(Caki-2) RCC. We observedtumorgrowth arrest due to 2HF treatment
in VHL-wild-type RCC but to a lesser extent compared with VHL-
mutant RCC (at day 60, tumor cross-sectional area and tumor weight,
2HF treated versus control; 98 ± 12 versus 115 ± 7 mm2and 1.84 ±
0.12 g versus 2.25 ± 0.18 g, respectively; non-significance) (Figure 5).
Also, even 100 mg/kg body wt of 2HF caused only ?18% reduction in
the tumor growth of VHL-wild-type Caki-2 RCC, whereas only 25
mg/kg body wt of 2HF caused 41% tumor regression in VHL-mutant
786-O RCC (P , 0.001).
In our in vitro studies, 2HF effectively inhibited the angiogenic
process and clonogenic potential besides causing apoptosis in VHL-
mutant RCC. In order to assess the degree of impact of 2HFinvivo on
these processes of specific importance in VHL-mutant RCC progres-
sion and metastasis, we performed histopathological examination of
the resected tumor xenografts.
2HF inhibits the expression of proliferative and angiogenic markers
while promoting normal epithelial differentiation in VHL-mutant
The histopathological examination of paraffin-embedded tumor xe-
nograft sections as observed by initial hematoxylin and eosin stain-
ing revealed that 2HF treatment reduces the number of tumor blood
vessels and restores the normal morphology specifically in VHL-
mutant RCC when compared with controls (Figure 6). Following
this observation, we probed the tumor sections for specific markers
of proliferation, angiogenesis and differentiation. 2HF treatment de-
creased the levels of proliferation marker, Ki 67 and angiogenesis
marker, CD31, in VHL-mutant RCC, which further supported the
observed in vitro antiproliferative and anti-angiogenic effects of
2HF. Another important finding was that the 2HF treatment predom-
inantly increased the expression of E-cadherin in VHL-mutant RCC
Fig. 3. 2HF inhibits GSTpactivity and angiogenesis. GSTactivity towards GSH and 1-chloro 2,4-dinitro benzene and its inhibition by 2HF was performed in 28
000g crude supernatant prepared from Caki-2 and 786-O cells. Recombinant purified GSTpwas used as a control (panel A and inset). The inhibitory effect of 2HF
on GSTwas studied at a fixedconcentration of GSH and GSH and 1-chloro 2,4-dinitro benzene (1 mM each) and varying concentrations ofinhibitor. The enzymes
were preincubated with the inhibitor for 5 min at 37?C prior to the addition of the substrates (panel A). VEGF expression in control and 2HF-treated cells by
enzyme-linked immunosorbent assay kit (R&D System) (panel B). Effect of 2HF (50 lM) on tube formation of Caki-2 and 786-O cells on matrigel was assessed
(panel C) and wound-healing assay shows that the 2HF inhibits 786-O cell migration as detailed in Materials and Methods (panel D).
L.D.Nagaprashantha et al.
xenografts. E-cadherin is considered a suppressor of invasion and
growth of many epithelial cancers because of its role in the inhibi-
tion of epithelial–mesenchymal transition (EMT) and promoting
normal epithelial phenotype (31–34). E-cadherin is frequently
downregulated during cancer progression and correlates with poor
prognosis (35). Loss of E-cadherin is associated with incidence and
progression of many epithelial tumors (35–37). In this regard, over-
expression of E-cadherin consequent to 2HF treatment represents
a highly significant and novel mechanism of action of 2HF in VHL-
mutant RCC (Figure 6).
RCC is one of the frequently incident cancers in USA with an in-
creasing current trend of incidence. Intake of citrus fruits has been
shown to reduce RCC risk in clinical trials (9). In this regard, we
investigated the anticancer effects and the respective mechanisms of
action of 2HF, a natural compound found in citrus fruits and oranges,
in RCC. Our studies demonstrate that 2HF exhibits potent antiproli-
ferative and pro-apoptotic effects to a significantly greater extent in
VHL-mutant RCC when compared with VHL-wild-type RCC. The
Fig. 5. Effect of oral administration of 2HF on tumor regression of RCC in nude mice: For 786-O RCC, mice were divided into four groups treated with corn oil
(i.e. vehicle) and 2HF 0.0025, 0.005 and 0.01% (wt/wt) (equivalent to 25, 50 and 100 mg/kg body wt, respectively). For Caki-2 RCC, mice were divided into two
groups treated with corn oil, and 2HF 0.01% (wt/wt) (equivalent to 100 mg/kg body wt). Experimental details are given in the Methods section. Animals were
examined daily for signs of tumor growth and body weights were recorded (panels A). Photographs of animals were taken at day 1, day 10, day 20, day 40 and day
60 after subcutaneous injection are shown for all groups (data not shown). Weights and photographs of tumors were also taken at day 60 (panels B). Tumors were
measured in two dimensions using calipers (panels C).
Fig. 4. Effect of 2HF on cell cycle progression in RCC. Inhibitory effect of 2HF on cell cycle distribution was determined by fluorescence activated cell sorting
analysis. Experimental details are given in the Methods section. The stained cells were analyzed using the Beckman Coulter Cytomics FC500, Flow Cytometry
Analyzer. The experiment was repeated three times and similar results were obtained (panel A). The cell morphology was observed and imaged at ?40
magnification in phase contrast microscope (Axio observer A1; Carl Zeiss microimaging, Thornwood, NY). Arrows in the panel point towards cells arrested in or
completingcytokinesis (panel B). VHL-wild-type (Caki-2) and VHL-mutant(786-O) control and 50 lM 2HF-treatedcells were processed for western blot analysis
for cyclin B1 and CDK4 expression by using specific antibodies. Membranes were stripped and reprobed for GAPDH as a loading control (panel C).
2HF inhibits VHL-mutant RCC
antiproliferative effects of 2HF were mediated by the inhibition of
EGFR, PI3K and pAkt signaling in VHL-mutant RCC. The growth
inhibitory effects of 2HF also included the inhibition of cell cycle
progression, which was mediated by reduction in the levels of cyclin
B1 and CDK4 in VHL-mutant RCC.
2HF was shown in our studies, for the first time, to be a novel GSTp
inhibitor. 2HF also effectively inhibited both recombinant GSTp
and .50% of total GST activity in VHL-mutant RCC at concentra-
tions toxic to tumors but well tolerated by normal cells. Posttransla-
tional S-glutathionylation of proteins which is an emerging topic of
investigation in apoptosis and enhanced oxidative signaling due to
constitutively active HIF2a in VHL-null background signify the role
of GSTp function in regulating differential cytotoxicity of 2HF in
RCC. Enhanced expression of GSTp can lead to increased detoxifi-
cation of products of lipid peroxidation and administered chemother-
apy drugs by conjugation with glutathione (GSH) to form glutathione
adducts (GS-E) which are eventually transported out of the cells by an
active, ATP dependent process mediated by the membrane trans-
porter, RLIP76 or Ral-binding protein 1 (RalBP1) (14–16). GSTpalso
catalyzes the S-glutathionylation of active site nucleophilic cysteines
in many phosphatases conferring additional negative charge to the
active site (24). S-glutathionylation induced redistribution of charge
at active site of proteins has been known to influence both substrate
accessibility and catalytic efficiency of target proteins with pro-
nounced effects on the tumor-signaling pathways (38). In this regard,
further knockin and knockout follow-up studies of GSTp, VHL and
HIF2a could characterize the impact of differential regulation of ox-
idative stress pathways in regulating the anticancer effects of 2HF.
One of the significant observation was that 2HF caused effective
inhibition of VEGF expression in VHL-mutant RCC sparing normal
mesangial cells. Angiogenesis is essential for the growth of rapidly
proliferating tumors whose centers are usually hypoxic and specifi-
cally in renal tumors with VHL mutations, which have high HIF2a
levels. HIF2a initiates a compensatory angiogenic response to match
the rapid rate of tumor growth by stimulating VEGF expression (39).
Normally, VHL binds to HIF2a and targets it for degradation. Loss or
inactivation of VHL leads to loss of substrate recognition required for
binding to HIF2a which effectively leads to enhanced levels and
activity of HIF2a and consequent tumor angiogenesis (40,41). In this
regard, 2HF represents a safe and novel anti-angiogenic natural com-
pound without normal tissue cytotoxicity with specific significance in
the management of highly vascular VHL-mutant RCC.
Our in vivo studies further provided corroborative evidence to the
anticancer effects and mechanisms of action of 2HF of particular rele-
vance to VHL-mutant RCC. It is possible that potential pharmacokinetic
differences in drug uptake and metabolism between cell types which in
turn influence the duration of drug action at intracellular targets could be
contributing to some of the terminal events due to 2HF treatment be-
tween the VHL-wild-type and VHL-mutant genotypes of RCC which
need further absorption, distribution, metabolism and excretion (ADME)
studies. However, from a clinical point of view, the ability of 2HF to
effectively target VHL-mutant RCC, which contributes to most common
form of RCCs is of potential significance in the chemoprevention of
RCC. Our mice xenograft studies also confirmed that the orally admin-
istered 2HF is effective in vivo to exert its anticancer effects in VHL-
mutant RCC as observed similarly in the in vitro studies. The dose of
2HF required to cause effective tumor regression (?90% reduction in
tumor growth, Figure 5) in VHL-mutant RCC was 0.01% (wt/wt), which
is well in comparable range to other flavonoids being tested in clinical
trials (27). Also, 2HF caused inhibition of Akt signaling and increased
poly ADP-ribose polymerase-cleavage in mice xenografts of VHL-mu-
tant RCC, which revealed that the orally administered 2HF can effec-
tively induce an in vivo antimitogenic and pro-apoptotic response (data
not shown). 2HF also decreased the expression of proliferative marker,
Ki 67 and angiogenic marker, CD31, in the VHL-mutant RCC. One of
the significant findings was that 2HF treatment increased the levels of E-
cadherin specifically in VHL-mutant tumors in vivo. Loss of E-cadherin
is associated with incidence and progression of many epithelial tumors
(42). Given the renal tubular epithelial origin of RCC, the increased
expression of E-cadherin after 2HF treatment assumes mechanistic sig-
nificance in the chemoprevention of VHL-mutant RCC.
VHL syndrome is an autosomal dominant condition caused by mu-
tation or deletion of the VHL gene and characterized by highly vas-
cular neoplasms including RCC, cafe ´-au-lait spots, angiomatosis,
hemangioblastomas and pheochromocytomas. VHL protein is an
Fig. 6. Histopathologic analyses of the markers of proliferation, angiogenesis and differentiation in tumor sections after 2HF treatment. Control and 2HF-treated
RCC-bearing nude mice tumor sections were used for histopathologic analyses. Presented are hematoxylin and eosin stained sections, immuno-histochemistry
analyses for Ki-67 expression (marker of cellular proliferation), CD31 (angiogenesis marker) and E-cadherin (tumor suppressor) from tumors in mice of control
and 2HF-treated groups. Statistical significance of differencewas determined by two-tailed Student’s t-test. P , 0.001, 786-O 2HF-treated compared with control.
However, these differences were not significant in Caki-2. Immunoreactivity is evident as a dark brown stain, whereas non-reactive areas display only the
background color. Sections were counterstained with hematoxylin (blue). Photomicrographs at ?40 magnification were acquired using Olympus Provis AX70
microscope. Percent staining was determined by measuring positive immunoreactivity per unit area. Arrows represent the area for positive staining for an antigen.
The intensity of antigen staining was quantified by digital image analysis. Bars represent mean ± standard error (n 5 5);?P , 0.001 compared with control.
L.D.Nagaprashantha et al.
ubiquitin E3 ligase that targets HIF to proteasomal degradation and
thus prevents constitutive activation of hypoxic and angiogenic
signaling (43). Our findings providing strong evidence for the pro-
apoptotic, anti-angiogenic and pro-differentiation effects of 2HF in
VHL-mutant RCC could also have additional potential implications
towards other VHL-related tumor syndromes in general. Taken to-
gether, in the light of pathogenetic mechanisms of loss-of-VHL driven
renal carcinogenesis, the anticancer properties of 2HF like inhibition
of survival, proliferation and tumor vascularization without causing
any overt toxicity towards normal tissues provide sound scientific
rationale for the role of 2HF in the chemoprevention of VHL-mutant
This work was supported by the National Institutes of Health grant
(CA 77495 to S.A. & S.S.S.) and ISIORR018999-01A1; the Welch
Foundation endowment (BK-0031 to L.P.); the Cancer Research
Foundation of North Texas; Institute for Cancer Research & the Joe
& Jessie Crump Fund for Medical Education to S.S.S.
The authors thank Dr Xiangle Sun, core facility at the University of North
Texas Health Science Center, Fort Worth, TX, for helping with flow cytometry
and laser capture microdissection (supported by NIH Grant ISIORR018999-
01A1). We also thank Dr Sumihiro Suzuki,Department of Biostatistics, School
of PublicHealth, University of North Texas HealthScience Center, Fort Worth,
TX, for his assistance in the statistical analyses of the data.
Conflict of Interest Statement: None declared.
1.Linehan,W.M. et al. (2004) Genetic basis of cancer of the kidney: disease-
specific approaches to therapy. Clin. Cancer Res., 10, 6282S–6289S.
2.Atkins,M.B. et al. (2007) Innovations and challenges in renal cell carci-
noma: summary statement from the Second Cambridge Conference. Clin.
Cancer Res., 13, 667s–670s.
3.Kuroda,N. et al. (2003) Review of renal oncocytoma with focus on clinical
and pathobiological aspects. Histol. Histopathol., 18, 935–942.
4.Hunt,J.D. et al. (2005) Renal cell carcinoma in relation to cigarette smok-
ing: meta-analysis of 24 studies. Int. J. Cancer., 114, 101–108.
5.Van Dijk,B.A. et al. (2006) Cigarette smoking, von Hippel-Lindau gene
mutations and sporadic renal cell carcinoma. Br. J. Cancer., 95, 374–377.
6.Choquenet,B. et al. (2009) Flavonoids and polyphenols, molecular families
with sunscreen potential: determining effectiveness with an in vitro
method. Nat. Prod. Commun., 4, 227–230.
7.Woodman,O.L. et al. (2004) Vascular and anti-oxidant actions of flavonols
and flavones. Clin. Exp. Pharmacol. Physiol., 31, 786–790.
8.Benavente-Garcia,O. et al. (2008) Update on uses and properties of
citrus flavonoids: new findings in anticancer, cardiovascular, and anti-
inflammatory activity. J. Agric. Food Chem., 56, 6185–6205.
9.Wolk,A.et al. (1996) International renal cell cancer study. VII. Role of diet.
Int. J. Cancer., 65, 67–73.
10.Hsiao,Y.C. et al. (2007) Flavanone and 2’-OH flavanone inhibit metastasis
of lung cancer cells via down-regulation of proteinases activities and
MAPK pathway. Chem. Biol. Interact., 167, 193–206.
11.Singhal,S.S. et al. (1992) Glutathione S-transferases of human lung: char-
acterization and evaluation of the protective role of the alpha-class iso-
zymes against lipid peroxidation. Arch. Biochem. Biophys., 299, 232–241.
12.Prokai,L. et al. (2009) Rapid label-free identification of estrogen-induced
differential protein expression in vivo from mouse brain and uterine tissue.
J. Proteome Res., 8, 3862–3871.
13.Singhal,S.S. et al. (2010) Rlip76 transports sunitinib and sorafenib and
mediates drug resistance in kidney cancer. Int. J. Cancer., 126, 1327–1338.
14.Singhal,S.S. et al. (2009) RLIP76: a target for kidney cancer therapy.
Cancer Res., 69, 4244–4251.
15.Singhal,S.S. et al. (2008) Hsf-1 and POB1 induce drug sensitivity and
apoptosis by inhibiting Ralbp1. J. Biol. Chem., 283, 19714–19729.
16.Singhal,J. et al. (2008) RLIP76 in defense of radiation poisoning. Int.
J. Radiat. Oncol. Biol. Phys., 72, 553–561.
17.Rosenberg Zand,R.S.et al. (2002) Flavonoidscan block PSAproductionby
breast and prostate cancer cell lines. Clin. Chim. Acta., 317, 17–26.
18.Lee,S.J. et al. (2008) Von Hippel-Lindau tumor suppressor gene loss in
renal cell carcinoma promotes oncogenic epidermal growth factor receptor
signaling via Akt-1 and MEK-1. Eur. Urol., 54, 845–853.
19.Merseburger,A.S. et al. (2008) Activation of PI3K is associated with re-
duced survival in renal cell carcinoma. Urol. Int., 80, 372–377.
20.Skarydova,L. et al. (2009) AKR1C3 as a potential target for the inhibitory
effect of dietary flavonoids. Chem. Biol. Interact., 178, 138–144.
21.Ritchie,K.J. et al. (2007) Glutathione transferase pi plays a critical role in
the development of lung carcinogenesis following exposure to tobacco-
related carcinogens and urethane. Cancer Res., 67, 9248–9257.
22.Kollermann,J. et al. (2006) Impact of hormonal therapy on the detection of
promoter hypermethylation of the detoxifying glutathione-S-transferase P1
gene (GSTP1) in prostate cancer. BMC Urol., 6, 15.
23.Awasthi,Y.C. et al. (2008) Self-regulatory role of 4-hydroxynonenal in
signaling for stress-induced programmed cell death. Free Radic. Biol.
Med., 45, 111–118.
24.Barrett,W.C. et al. (1999) Regulation of PTP1B via glutathionylation of the
active site cysteine 215. Biochemistry., 38, 6699–6705.
25.Ohh,M. et al. (2000) Ubiquitination of hypoxia-inducible factor requires
direct binding to the beta-domain of the von Hippel-Lindau protein. Nat.
Cell Biol., 2, 423–427.
26.Leung,D.W. et al. (1989) Vascular endothelial growth factor is a secreted
angiogenic mitogen. Science/, 246, 1306–1309.
27.Tyagi,A.K. et al. (2002) Silibinin strongly synergizes human prostate car-
cinoma DU145 cells to doxorubicin-induced growth Inhibition, G2-M ar-
rest, and apoptosis. Clin. Cancer Res., 8, 3512–3519.
28.Gabrielli,B.G. et al. (1999) A cyclin D-Cdk4 activity required for G2 phase
cell cycle progression is inhibited in ultraviolet radiation-induced G2 phase
delay. J. Biol. Chem., 274, 13961–13969.
29.Yin,F. et al. (2001) Apigenin inhibits growth and induces G2/M arrest by
modulating cyclin-CDK regulators and ERK MAP kinase activation in
breast carcinoma cells. Anticancer Res., 21, 413–420.
30.Woo,K.J. et al. (2006) Thimerosal induces apoptosisand G2/Mphase arrest
in human leukemia cells. Mol. Carcinog., 45, 657–666.
31.Scholzen,T. et al. (2000) The Ki-67 protein: from the known and the un-
known. J. Cell. Physiol., 182, 311–322.
32.Folkman,J. (1971) Tumor angiogenesis: therapeutic implications. N. Engl.
J. Med., 285, 1182–1186.
33.Wheelock,M.J. et al. (2003) Cadherins as modulators of cellular pheno-
type. Annu. Rev. Cell Dev. Biol., 19, 207–235.
34.Yang,J. et al. (2008) Epithelial-mesenchymal transition: at the crossroads
of development and tumor metastasis. Dev. Cell., 14, 818–829.
routine immunohistochemistry panel in breast invasive ductal carcinoma.
Cancer Biomark., 5, 1–8.
36.Cheng,J.C. et al. (2010) Hydrogen peroxide mediates EGF-induced down-
regulation of E-cadherin expression via p38 MAPK and snail in human
ovarian cancer cells. Mol. Endocrinol., 24, 1569–1580.
of ovarian cancer cells by decreasing the expression of E-cadherin, beta-
catenin, and caveolin-1. Anat. Rec. (Hoboken), 293, 1134–1139.
38.Xie,Y. et al. (2009) S-glutathionylation impairs signal transducer and acti-
vator of transcription 3 activation and signaling. Endocrinology., 150,
39.Nilsson,M.B. et al. (2010) Multiple receptor tyrosine kinases regulate
HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma:
implications for antiangiogenic mechanisms of multikinase inhibitors.
Oncogene., 29, 2938–2949.
40.Del Rey,M.J. et al. (2009) Human inflammatory synovial fibroblasts induce
enhanced myeloid cell recruitment and angiogenesis through a hypoxia-in-
ducible transcription factor 1alpha/vascular endothelial growth factor-medi-
ated pathway in immunodeficient mice. Arthritis Rheum., 10, 2926–2934.
41.Kumar,A. et al. (2009) Antiangiogenic and antiproliferative effects of
substituted-1,3,4-oxadiazole derivatives is mediated by down regulation
of VEGF and inhibition of translocation of HIF-1alpha in Ehrlich ascites
tumor cells. Cancer Chemother. Pharmacol., 6, 1221–1233.
42.Almeida,P.R.et al. (2010) E-cadherin immunoexpression patterns inthe char-
acterisation of gastric carcinoma histotypes. J. Clin. Pathol., 63, 635–639.
43.Kaelin,W.G.Jr. (2008) The von Hippel-Lindau tumour suppressor protein:
O2 sensing and cancer. Nat. Rev. Cancer., 8, 865–873.
Received December 1, 2010; revised January 12, 2011;
accepted January 22, 2011
2HF inhibits VHL-mutant RCC