Geraniin selectively promotes cytostasis and apoptosis in human colorectal cancer cells by inducing catastrophic chromosomal instability

Abstract

Homeostasis of chromosomal instability (CIN) facilitates the origin and evolution of abnormal karyotypes that are critical for the survival and proliferation of cancer cells, but excessive CIN can result in cellular toxicity. Geraniin is a multifunctional ellagitannin found in some species of Geranuim and Phyllanthus. We employed the cytokinesis-block micronucleus cytome assay to evaluate the CIN, nuclear division index (NDI) and apoptosis induced by geraniin in human colorectal adenocarcinoma cells (Colo205 and Colo320) and human colon mucosal epithelial cells (NCM460). Cells were exposed to 25, 50 or 100 μg/ml geraniin for 24, 48 or 72 h. 0.05 μg/ml mitomycin C was used as a positive control and media as a negative control. The results showed that, compared to negative controls, geraniin significantly reduced NDI (P < 0.01) and increased CIN (P < 0.01) and apoptosis (P < 0.05) in Colo205 and Colo320 cells in a dose- and time-dependent manner. Conversely, geraniin significantly increased NDI (P < 0.05) and decreased CIN (P < 0.001) and apoptosis (P < 0.01) in NCM460 cells. Moreover, CIN was positively associated with apoptosis (r = 0.437, P < 0.001) and negatively associated with NDI (r = -0.744, P < 0.001) in these cells. Together, our results highlight that the induction of catastrophic CIN may underlie the antitumor potential of geraniin. Our data also suggest that geraniin can decrease the risk of oncogenic transformation via decreasing CIN in normal cells.

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271
Mutagenesis, 2018, 33, 271–281
doi:10.1093/mutage/gey016
Original Manuscript
Original Manuscript
Geraniin selectively promotes cytostasis and
apoptosis in human colorectal cancer cells by
inducing catastrophic chromosomal instability
XihanGuo1, HanWang1, JuanNi1, ZiqingLiang1, XiayuWu1, JinglunXue2
and XuWang1*
1School of Life Sciences, The Engineering Research Center of Sustainable Development and Utilization of Biomass
Energy, Yunnan Normal University, Kunming, Yunnan 650500, China and 2State Key Laboratory of Genetic Engineering,
Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China
*To whom correspondence should be addressed. Tel:+86 137 08892237; Fax: +86 0871 65941366; Email: wangxu@fudan.edu.cn
Received 14 April 2018; Revised 02 July 2018; Accepted 13 July 2018.
Abstract
Homeostasis of chromosomal instability (CIN) facilitates the origin and evolution of abnormal
karyotypes that are critical for the survival and proliferation of cancer cells, but excessive CIN
can result in cellular toxicity. Geraniin is a multifunctional ellagitannin found in some species
of Geranuim and Phyllanthus. We employed the cytokinesis-block micronucleus cytome assay
to evaluate the CIN, nuclear division index (NDI) and apoptosis induced by geraniin in human
colorectal adenocarcinoma cells (Colo205 and Colo320) and human colon mucosal epithelial
cells (NCM460). Cells were exposed to 25, 50 or 100μg/ml geraniin for 24, 48 or 72h. 0.05μg/ml
mitomycin C was used as a positive control and media as a negative control. The results showed
that, compared to negative controls, geraniin significantly reduced NDI (P< 0.01) and increased
CIN (P<0.01) and apoptosis (P<0.05) in Colo205 and Colo320 cells in a dose- and time-dependent
manner. Conversely, geraniin significantly increased NDI (P<0.05) and decreased CIN (P<0.001)
and apoptosis (P<0.01) in NCM460 cells. Moreover, CIN was positively associated with apoptosis
(r=0.437, P<0.001) and negatively associated with NDI (r=-0.744, P<0.001) in these cells. Together,
our results highlight that the induction of catastrophic CIN may underlie the antitumor potential
of geraniin. Our data also suggest that geraniin can decrease the risk of oncogenic transformation
via decreasing CIN in normal cells.
Keywords: Geraniin, cytokinesis-block micronucleus cytome assay, chromosomal instability, colorectal cancer, apoptosis
Introduction
Chromosome is the largest unit of organisation of the eukaryotic
genome. Its stability ensures the preservation of genome integrity
and promotes faithful genome propagation. Although numerous
processes are tightly coordinated to ensure chromosomal stability
in normal cells, cells are constantly under attack by chromosome
damaging agents of both exogenous and endogenous origin and
these assaults can lead to changes in the number and structure of
chromosomes—referred to as chromosomal instability (CIN). It has
been rmly established that CIN contributes to a variety of complex
developmental disorders such as cancer (1).
Cancer is one of the major public health problems worldwide
and the leading cause of death in economically developed countries
(2). CIN and cancer are intertwined in a complex web: CIN is a
hallmark of most solid tumours and is highly associated with cancer
stage transition. It has been suggested that CIN may contribute to
the initiation and progression of cancer by facilitating the appear-
ance of cancer-causing karyotypes (3). These abnormal karyotypes
Advance Access publication 06 August, 2018

will activate and/or enhance multiple pathways that are integral to
the survival and proliferation of the cancer cells, such as inamma-
tion, immune system evasion or apoptosis resistance (3). However,
this intrinsic feature that distinguishes cancer cells from their normal
counterparts carries the promise to selectively target them, because
it confers a state of increased basal CIN stress, making cancer cells
vulnerable to further augment CIN generation (4–6). However, the
traditional genotoxic agents used for cancer chemotherapy not only
induce CIN to tumour cells but also to normal cells. The induced
CIN in normal cells can promote pro-oncogenic mutations in cancer-
chemotherapy survivors, leading to an increased risk of secondary
cancers (7). Therefore, identication of novel agents that target can-
cer cells by selectively elevating CIN beyond the therapeutic thresh-
old, while leaving normal cells unaffected provides a new window of
opportunity to improve the outcomes of cancer treatment (1).
Polyphenols constitute one of the most numerous and ubiquitous
groups of plant metabolites and provide a rich source for new drug
development, particularly drugs in the area of cancer (8). Tannins
are one subgroup of polyphenols that can be further divided into
three major groups: hydrolysable tannins, non-hydrolysable/con-
densed tannins and phlorotannins. Ellagitannins, which belong to
the hydrolysable tannin group, are polyesters of a sugar moiety and
various numbers of hexahydroxydiphenoyl. Geraniin (Figure 1A)
is a typical ellagitannin because it is made up of a glucopyranose
esteried to three common acyl moieties, namely a galloyl, a hexahy-
droxydiphenoyl and a dehydrohexahydrodiphenoyl group (9). The
natural occurrence of geraniin has been veried in ethnopharmaco-
logical important plants, such as a broad species of genus Geranium
(e.g. Geranium sibiricum and G. thunbergii) and Phyllanthus (e.g.
Phyllanthus emblica and P.amarus), highlighting its role as an active
ingredient in traditional medicines (10). Alarge number of in vitro
and in vivo animal experiments have reported that geraniin exhibits
highly antioxidative, antihypertensive, antihyperglycemic, antiviral
and antibacterial, antimutagenic and radioprotective, hepatoprotec-
tive, anti-inammatory, immunomodulatory and antitumor activi-
ties (9,10). Although various biological and pharmacological effects
of geraniin have been revealed, the possible role of geraniin on mod-
ulation of CIN in human cancer cells and their normal counterparts
has not been elucidated.
In the present study, we intend to determine whether geraniin has
potential to selectively elevate CIN in cancer cells. Two human colo-
rectal cancer (CRC) cell lines (Colo205 and Colo320) and a noncan-
cerous colon cell line (NCM460) were treated by geraniin and their
CIN levels were measured via the cytokinesis-block micronucleus
cytome (CBMN-Cyt) assay. In CBMN-Cyt assay, the endpoints of
micronucleus (MN), nucleoplasmic bridge (NPB) and nuclear bud
(NB) provide a reliable measure of numerical CIN and structural
CIN at a post-mitotic interphase (11). Moreover, the proliferation
Figure1. Influence of geraniin treatment on the proliferation of Colo320, Colo205 and NCM460 cells. (A) Structure of geraniin. The chemical formula of geraniin
is C41H28O27 with a molecular mass of 952.64g/mol. (B-D) Colo320, Colo205 and NCM460 cells with a seeding density of 1× 105/ml were incubated without or
with geraniin (1.6–100μg/ml) for 24 (B), 48 (C) and 72 h (D), then cells were harvested and the total cell numbers were determined by counting live cells on a
hemacytometer with the inclusion of trypan blue. Comparison was made by using one-way ANOVA (P=0.004) with Tukey’s post hoc test. Values are presented
as mean ± S.E.M. (n=3). *P<0.05, **P<0.01 and ***P<0.001 compared with corresponding control.
272 X.Guo etal.

(nuclear division index, NDI) and cell death (apoptosis and necrosis)
were also determined by CBMN-Cyt assay.
Materials and methods
Chemicals
Geraniin (purity: 99%) was obtained from Amresco (Cleveland,
OH). Astock solution of geraniin was prepared by dissolving the
powder in RPMI 1640 medium at 1mg/ml. The solution was ltered
through a 0.22-μm pore size hydrophilic polyethersulfone mem-
brane (Merck Millipore, MA) and stored at –80°C. The stock solu-
tion was thawed at 4°C and diluted to the desired concentration in
medium immediately before use.
Cell lines and cell culture
Two CRC cell lines, Colo320 and Colo205, were obtained from
Cell Bank of Kunming Institute of Zoology, the Chinese Academy
of Sciences (Kunming, China). Colo320 was derived from a 55-year-
old Caucasian female with Duke’s type C (lymph-node positive)
CRC. Colo205 was derived from a 70-year-old Caucasian male with
Duke’s type D (distantly metastasis) CRC. NCM460, a noncancerous
human colon mucosal epithelial cell line that derived from a 68-year-
old Hispanic male, was obtained from INCELL (San Antonio, TX,
USA). Colo320, Colo205 and NCM460 are rapid growing cells,
with a double time <24h.
Colo205, Colo320 and NCM460 cells were maintained as a
monolayer in 25cm2 asks (Corning, NY) in RPMI 1640 medium
(Gibco) supplemented with 10% fetal bovine serum (Gibco), 1%
penicillin [5000 IU/ml]/ streptomycin [5 mg/ml] solution (Gibco),
1% L-glutamine (2mM) (Sigma), and kept at 37°C in a 5% CO2
environment. To avoid changes of cell characteristics produced by
prolonged culture, only cells from specic passages were used for
all studies (passage 30–35 for Colo320, passage 15–20 for Colo205,
and passage 10–15 for NCM460).
Cell viabilitytests
Cell viability was tested by trypan blue exclusion. NCM460, Colo320
and Colo205 were seeded into 24-well plates (Corning, NY, USA)
at a density of 1 × 105 cells/ml and exposed to different geraniin
doses (0, 1.6, 3.1, 6.2, 12.5, 25, 50, 100μg/ml) in triplicates. After
every 24 h of incubation, one replicate was used for cell viability
testing. NCM460, Colo320 and Colo205 cells were detached from
plates with 0.25% trypsin (Gibco, NY) and cells were suspended
with medium after trypsin discarded. Cell suspension (5μl) were
stained with 5μl trypan blue (Boster, Wuhan, China) and counted in
a hemocytometer. This procedure was repeated three times.
Cytokinesis-block micronucleus cytome
(CBMN-Cyt)assay
CBMN-Cyt assay is a well-established system that allows CIN,
cytotoxic and cytostatic events to be captured within one assay(11).
CBMN-Cyt assay was performed as described previously(12,13).
Briey, NCM460, Colo320 and Colo205 cells were seeded into
24-well plates at a density of 1×105 cells/ml and allowed to attach
overnight. After this period, cells were cultured in RPMI1640 medium
containing 0, 25, 50, 100μg/ml geraniin for 24, 48 or 72h, respec-
tively. Mitomycin C (MMC; Sigma, MO), a direct-acting clastogen
and a commonly used chemotherapeutic agent(14), was used as the
positive control at a nal concentration of 0.05μg/ml. The medium
was discarded after treatment, and cells were washed twice with
Hanks’ balanced salt solution (HBSS). Geraniin-free fresh medium
with cytochalasin-B (Cyto-B; 4.5μg/ml; Sigma, MO) was added to
each culture to block cytokinesis. Cyto-B was rinsed with HBSS after
a further 24h, cells were detached from plates with 0.25% trypsin
and resuspended in 200μl fresh medium. Cells were centrifuged onto
glass slides using a cyto-centrifuge for 5min at 800 rpm (100× g).
The nal cell density per slide was kept between 0.5×105 and 1×105
cells. After drying briey in air, slides were xed in fresh 3:1 metha-
nol and glacial acetic acid for 10min and stained with 10% Giemsa
(San’ersi Reagent CO., LTD, Shanghai, China), which could stain
cytoplasm and nucleus differentially. The slides were washed twice in
ddH2O, then allowed to air dry and coverslip.
Stained slides were encoded to ensure a blind microscopic
analysis, and such a code was removed until the whole micro-
scopic analysis was nished. All biomarkers of CBMN-Cyt assay
were scored under 1000× magnication with optical microscope
(Olympus, Tokyo, Japan) using previously described criteria(15).
CIN are scored in cytochalasin B-induced binucleated cells
(BNCs) containing MN, NPB and NB. Cytotoxicity is measured
via necrotic and apoptotic cell ratios and the cytostasis of viable
cells is measured with NDI (Figure2). Briey, 1000 BNCs were
scored per group for the frequency of BNCs with MN, NPB and
NB. Five hundred cells were scored for the percentage of necrosis
and apoptosis. NDI was calculated as NDI=(M1+2 M2+3 M
≥3)/N, where M1, M2, and M ≥ 3 represent the number of cells
with 1, 2 or ≥ 3 nuclei and N is the total number of viable cells
scored. Percent cytostasis and proliferation were calculated by the
following formula (16): % cytostasis = 100 – 100 (NDI T – 1)/
(NDI C – 1); % proliferation=100– 100 (NDI C – 1)/ (NDI T – 1).
NDI T and NDI C represent the NDI in geraniin-treated and con-
trol cultures, respectively.
Statistical analysis
The Kolmogorov–Smirnov (KS) test was used to test the normality
of all data sets. The differences of observed values among the control
and geraniin-treated groups were analyzed using One-way analysis
of variance (ANOVA). First, Levene’s test was performed to examine
the homogeneity of variances among the control and geraniin-treated
groups. Post-hoc tests [A Tukey’s test was used when the equality of
variances assumption holds (P > 0.05), and the Dunnett T3 test was
used otherwise (P<0.05)] once a signicant effect was detected. The
correlation between the biomarkers investigated was evaluated by
Pearson correlation analysis. Data in all gures and tables represent
the means ± the standard error of the mean (S.E.M.) of three independ-
ent experiments. Only differences having a P-value (two-tailed) lower
than 0.05 were considered as being signicant. All statistical analyses
were performed using SPSS 17.0 for windows (SPSS, Chicago, IL) and
graphed by GraphPad PRISM 5.0 (GraphPad, San Diego, CA).
Results
The choice of appropriate concentrations of geraniin
To obtain the appropriate concentrations of geraniin for this study,
we obtained the concentration–response curves of geraniin to
NCM460, Colo320 and Colo205 cells. As shown in Figure 1B–D,
treatment of Colo205 cells with geraniin resulted in a dose- and time-
dependent reduction of cell number (P < 0.05). Geraniin showed
a moderate toxicity with a median inhibitory concentration (IC50)
48.20, 41.50 and 34.78 μg/ml after 24, 48 and 72 h treatment,
respectively. Geraniin at 1.6–12.5μg/ml had no obvious cytotoxicity
to Colo320 cells, beyond which (25–100μg/ml), geraniin exhibited
Geraniin selectively promotes cytostasis and apoptosis in human colorectal cancercells 273

a dose- and time-dependent cytotoxicity to Colo320 cells (P<0.05;
Figure1B–D). Compared to Colo205, Colo320 cells were found to
be slightly less susceptible to geraniin, with an IC50 50.33μg/ml after
72h treatment.
On the other hand, the response of NCM460 to geraniin fol-
lowed a bell shaped dose–response curve at all time intervals. As
shown in Figure1B–D, NCM460 cells treated with low concentra-
tions of geraniin (1.6–12.5 μg/ml) had higher viability compared
with that from untreated control or that treated with high geraniin
concentrations (25–100μg/ml) (P<0.05). Within the dose and time
range evaluated, geraniin had essentially no inhibitory impact on the
viability of NCM460 cells, indicating that geraniin could selectively
killCRC.
Based on these results, we chose geraniin at concentrations of
25, 50 and 100μg/ml (26.25, 52.5 and 105 μM, respectively) for
the following experiments. Under this concentration range, geraniin
had a high cytotoxicity to CRC while no cytotoxicity to noncan-
cerous colon cells, thus providing a suitable condition to test our
hypothesis.
Oppsite roles of getaniin in regulating CIN between
CRC and noncancerouscells
The effects of geraniin on CIN of NCM460, Colo320 and Colo205
were investigated by the well-estimated CBMN-Cyt assay(11).
Photomicrographs of the different endpoints scored in CBMN-Cyt
assay are shown in Figure2A–D. The results showed that geraniin
signicantly increased the frequency of MN and NPB in Colo320
cells in a dose- and time-dependent manner (P<0.01; Table1). The
potential of geraniin in increasing MN frequency maximised after
72 h treatment, with a 4.42-fold increase at the highest scorable
dose (50μg/ml; P < 0.001); while the potential in increasing NPB
frequency maximised after 48h treatment, with a 7.11-fold increase
at 100μg/ml (P< 0.001). However, geraniin was found to have no
obvious impact on the NB frequency (Table1).
As shown in Table2, a similar dose- and time-dependent increase
of MN and NPB by geraniin was also observed in Colo205 cells. The
potential of geraniin in increasing MN and NPB frequencies maximised
after 48h treatment, with a 4.58- and 5.73-fold increase at 100μg/ml,
respectively (P< 0.001). Moreover, geraniin showed potential to sig-
nicantly increase NB frequency in Colo205 cells, with a 4.10-, 4.10-
and 4.08-fold increase at 100μg/ml after 24, 48 and 72h treatment,
respectively (P<0.05, P<0.001 and P<0.01, respectively).
When compared with the control group, a dose- and time-
dependent decrease in MN, NPB and NB frequencies was observed
in NCM460 cells after geraniin treatment (Table3). The potential
of geraniin in decreasing MN, NPB and NB maximised after 24h
treatment, with a 76.59, 53.71 and 70.22% decrease at 100μg/ml,
respectively (P < 0.001). Taken together, these data demonstrated
that geraniin could specically increase CIN in CRC cells while
decreasing it in noncancerous coloncells.
Selective cytostatic effect of geraniin onCRC
The NDI in CBMN-Cyt assay provides a measurement of the
proliferative status after geraniin treatment (11). The results in
Figure3A and 3B showed that geraniin treatment could decrease
the NDI (increase % cytostasis) in Colo320 and Colo205 cells in
a dose- and time-dependent manner (P<0.01), when compared to
corresponding untreated controls. Geraniin exhibited the most pre-
dominant potential (P<0.001) in increasing cytostasis in Colo205
and Colo320 after 48 and 72 h, respectively. At these intervals,
geraniin at 100μg/ml caused a nearly 90% of cytostasis in both
CRC celllines.
The NDI of NCM460 was found to be signicantly increased
after geraniin treatment, in a way depended on the treated dose and
time (P< 0.01; Figure3C). After 72h treatment, geraniin showed
the most predominant capacity to promote NCM460 proliferation,
with a 72.96% promotion at 100μg/ml (P<0.001). Taken together,
these data demonstrated that geraniin could selectively induce cyto-
stasis in CRC cells while promoting proliferation in noncancerous
colon cells.
Selective pro-apoptotic effect of geraniin onCRC
The effects of geraniin on cell death (apoptosis and necrosis) of
Colo320, Colo205 and NCM460 were investigated by CBMN-Cyt
Figure2. Photomicrographs of various endpoints scored in cytokinesis-block
micronucleus cytome assay. (A) Anormal binucleated cell (BNC) induced
by cytochalasin B.Examples of BNCs containing a micronucleus (MN, B), a
nucleoplasmic bridge (NPB, C) or a nuclear bud (NB, D) are shown (indicated
by arrows). Amononucleated cell (E) and a BNC (F) at early stage of apoptosis
with chromatin condensation and intact cytoplasmic and nuclear boundaries
as well as cells exhibiting nuclear fragmentation into smaller bodies within
an intact cytoplasmic membrane. Amononucleated cell (G) and a BNC (H)
at late stage of necrosis with cytoplasm loss, swelled nuclear and damaged
nuclear membrane with only a partially intact nuclear structure and often
with nuclear material leaking from the nuclear boundary. Giemsa-stained
DNA is in red, and the cytoplasm is in blue. Bar, 10μm.
274 X.Guo etal.

assay (Figure 2E–H). The results showed that geraniin could sig-
nicantly induce apoptotic cell death in CRC cells. As shown in
Figure4A–C, the percentages of morphologically apoptotic cells in
Colo320 and Colo205 were signicantly increased as geraniin con-
centration elevated (P<0.05). The apoptotic response to geraniin
was stronger in Colo320 than that in Colo205. After 72h treatment,
geraniin at 100 μg/ml induced an over 11-fold increase of apop-
tosis in Colo320 (P< 0.001), whereas only a 3.44-fold increase in
Colo205 (P<0.001). In contrast, apoptosis frequency in NCM460
cells was found to be signicantly decreased after geraiin treatment,
in a dose- and time-dependent manner (P<0.01). When compared
to the corresponding controls, geraniin at 100μg/ml caused a 27.76,
48.53 and 63.83% decrease in apoptosis of NCM460 cells after
24, 48 and 72h treatment, respectively (P< 0.001). In contrast to
apoptosis, geraniin was found to signicantly decrease the necrosis
frequency in Colo320 after 24h treatment (P <0.001; Figure 4D)
and in Colo205 cells after 48h treatment (P<0.001; Figure4E), as
well as in NCM460 cells after 24h treatment (P<0.001; Figure4F).
Associations of apoptosis and NDI withCIN
To determine whether the alteration of CIN induced by geraniin
was associated with the changes in apoptosis and cytostasis, we
analyzed the relationships between CIN and apoptosis, CIN and
NDI using data from Colo320, Colo205 and NCM460 cells. Of
note, we used the relative changes of these biomarkers for the cor-
relation analysis due to the different baselines of CIN, apoptosis and
NDI among these cell lines. As expected, there was a statistically
signicant (P<0.001) positive association (Pearson correlation coef-
cient=0.437) between CIN and apoptosis (Figure5A). In addition,
a statistically signicant (P<0.001) negative association (Pearson
correlation coefcient= -0.744) was found between CIN and NDI
(Figure5B). Together, these results suggested that the induction of
apoptosis and cytostasis in CRC cells by geraniin might be attributed
to the increased CIN inthem.
Discussion
In this study, we show, for the rst time, that geraniin has potential
to selectively promote catastrophic CIN in CRC cells followed by
cytostasis and apoptosis. Intriguingly, although geraniin is highly
genotoxic to CRC cells, it reduces CIN in noncancerous colon cells
at the same concentrations.
One predominant hallmark that distinguishes cancer cells from
their noncancerous counterparts is CIN. The acquisition of CIN
Table1. The effects of geraniin on the frequency of binucleated cells (BNCs) displaying micronuclei (MN), nucleoplasmic bridges (NPB) or
nuclear buds (NB) per 1000 BNCs in Colo320 cells (n=3 per treatment).
Biomarkers Treatment
interval
Dose of geraniin (μg/ml) MMC
(0.05μg/ml)
0 25 50 100
MN 24 h 24.49±1.17 41.66±2.08* 47.20±4.95** 48.36±1.65** 56.14±1.24***
48 h 17.64±2.66 17.66±1.04 23.02±1.18 45.06±4.12*** 65.73±1.27***
72 h 9.48±1.99 35.89±3.24** 41.96±1.64*** NA 74.93±2.07***
NPB 24 h 23.82±2.28 54.44±3.07** 73.06±5.52*** 85.02±4.41*** 43.94±2.13*
48 h 16.69±3.40 65.75±5.03** 89.59±4.23*** 118.67±13.27*** 54.46±2.32*
72 h 36.64±1.06 51.04±7.12 73.47±3.94** NA 64.53±2.52**
NB 24 h 1.93±0.57 0.98±0.57 1.93±0.55 2.92±0.09 2.35±0.45
48 h 0.32±0.32 0.98±0.00 0.97±0.00 0.97±0.01 3.26±0.27
72 h 0 0.97±0.56 1.63±0.33 NA 3.86±0.34***
Data represented the mean frequency ± the standard error of the mean (S.E.M.) per 1000 BNCs from three independent experiments. Signicant differences
between geraniin-treated groups and controls at each treatment interval are indicated by *P<0.05, **P<0.01 and ***P<0.001. MMC (mitomycin C) was used
as a positive control. NA, not analyzed.
Table2. The effects of geraniin on the frequency of binucleated cells (BNCs) displaying micronuclei (MN), nucleoplasmic bridges (NPB) or
nuclear buds (NB) in Colo205 cells (n=3 per treatment).
Biomarkers Treatment
interval
Dose of geraniin (μg/ml) MMC
(0.05μg/ml)
0 25 50 100
MN 24 h 8.91±1.66 31.12±3.10*** 37.56±1.40*** 43.37±2.74*** 71.60±1.66***
48 h 16.58±2.05 51.41±2.88*** 61.05±4.00*** 75.70±3.35*** 87.29±2.82***
72 h 19.30±2.24 32.04±2.09* 40.35±3.03*** 60.19±5.92*** 105.39±3.32***
NPB 24 h 2.56±0.66 10.04±1.13** 10.90±1.63** 9.46±0.86** 5.35±0.46*
48 h 6.49±0.29 28.00±0.48*** 26.08±2.04*** 37.21±3.42*** 15.69±1.49**
72 h 11.16±1.59 24.03±0.58** 31.95±2.26*** 35.02±2.09*** 26.40±2.30**
NB 24 h 1.91±0.53 6.80±1.67 5.45±1.69 7.83±1.13* 4.84±0.64*
48 h 7.77±1.99 21.54±4.07* 25.41±0.84** 31.89±2.85*** 16.67±0.89*
72 h 6.54±0.98 19.23±2.56* 21.27±0.81** 26.69±3.63** 25.13±2.09**
Data represented the mean frequency ± the standard error of the mean (S.E.M.) per 1000 BNCs from three independent experiments. Signicant differences
between geraniin-treated groups and controls at each treatment interval are indicated by *P<0.05, **P<0.01 and ***P<0.001. MMC (mitomycin C) was used
as a positive control.
Geraniin selectively promotes cytostasis and apoptosis in human colorectal cancercells 275

is an important mechanism for tumour evolution and associates
with a poor prognosis(1). However, it has been suggested that there
is an optimal level of CIN in tumours required for tumour adap-
tation and progression, beyond which CIN becomes unfavourable
because it destroys the genome and, therefore compromises clonal
expansion (17). To date, only limited studies provided evidence
that specic cellular stress resulting from massive CIN induced by
genetic methods can indeed be utilised as a strategy to kill tumour
cells (4–6), as well as a relationship between high levels of CIN and
survival outcome in cancers (18). In fact, we have previously noted
that P.emblica uses this strategy to selectively kill Colo320 cells (16).
In this study, we conrmed this notion by highlighting that geraniin
kills CRC cells through inducing catastrophic CIN in them, indicat-
ing a new mechanism underlying the anticancer action of geraniin.
The exact mechanisms underlying geraniin-induced catastrophic
CIN in Colo320 and Colo205 cells remain to be determined. One
potential explanation is that geraniin inhibited Heat shock protein
90 (HSP90) in these cells (19). HSP90 is a molecular chaperone
participates in maturating, stabilizing and activating numerous cli-
ent proteins involved in cell signaling and survival (20). In particu-
lar, HSP90 involves in the spindle assembly checkpoint (SAC) that
governs the delity of chromosome transmission (21). Moreover,
HSP90 interacts with several other pathways that could affect chro-
mosome transmission delity, including the kinetochore assembly
(22). Geraniin is able to inhibit the in vitro HSP90 ATPase activity in
a dose-dependent manner, with an inhibitory efciency higher than
that measured for 17-AAG, a well-known HSP90 inhibitor currently
undergoing phase II/III clinical trials. In yeast, HSP90 inhibition by
radicicol and Macbecin II is the most potent inducer of CIN among
the various types of stresses tested, including oxidative stress, osmotic
stress, endoplasmic reticulum stress, replicative stress, translational
stress, membrane integrity defect and spindle assembly defect (21).
Previously, 17-AAG was found to induce CIN in HeLa and HCT116
cells in vitro (23) and in a mouse sarcomatosis model (24).
In addition to SAC, checkpoint that govern sister chromatids
segregation is decatenation checkpoint. DNA topoisomerase II
(TOP2) plays an important role in activation of this checkpoint
(25). Geraniin has found to been a potent inhibitor of TOP2, which
completely inhibit TOP2 activity at a concentration of 500nM in
vitro (26). When TOP2 function is blocked, cells fail to separate sis-
ter chromatids due to a failure to resolve sister chromatid cohesion
(27). In addition, simultaneously perturbing HSP90 and TOP2 can
synergistically induce catastrophic CIN in CRC cells (28). In light of
these ndings, we predict that geraniin-induced CIN in Colo320 and
Colo205 cells may be a combined effect of interfering the function of
HSP90 and TOP2. If so, we speculate that geraniin-induced MN usu-
ally derived from whole chromosomes. Further study aims to deter-
mine whether the geraniin-induced MN harbor whole chromosomes
or chromatin fragments will provide insights into our speculation.
Our data highlighted that CIN was positively associated with
cytostasis and apoptosis. These data looks like paradoxical consider-
ing that division is needed to ‘express’ CIN biomarkers in CBMN Cyt
assay. Apotential explanation for our results is that HSP90 inhibition
induces downregulation of critical HSP90 protein clients and results in
mitotic arrest (29). Upon prolonged mitotic arrest, cancer cells exhibit
multiple fates: Some cells slipped through mitosis without any signs of
division, while others clearly died in mitosis. Some of those that slipped
through mitosis died in interphase (30). In addition, a small propor-
tion of cells divided and incorrectly separated their chromosomes,
resulting CIN in daughter cells that display a high frequency to die in
interphase (30). In light of this view, we speculated that CRC carry-
ing catastrophic CIN induced by geraniin will lose their proliferation
capacity and/or be cleared by apoptosis. In line with this, 17-AAG has
been shown to induce cytostasis and apoptosis in human CRC (29).
Although this nding was only obtained from CRC, we expect that the
observed phenomena is generally applicable to different cancer cells.
CIN is frequently seen in many human tumours and these cells express
more HSP90 and TOP2 to overcome stresses generated from CIN
(27,31). Therefore, induction of catastrophic CIN, possible through
inhibition of HSP90 and TOP2, may be a mechanism underlying gera-
niin-induced apoptosis seen in human glioma (32), breast cancer (33),
ovarian cancer (34), lung cancer cells (35) and melanoma cells (36).
As mentioned, since many client proteins of HSP90 are involved in
cell cycle regulation, some Hsp90 inhibitors may perturb physiologi-
cal functions in normal cells. Recently, HSP90 inhibitor CH5164840
is found to induce MN in human TK-6 lymphocytes via an aneugenic
mechanism (37). In this study, however, we do not nd any genotoxic
effect of geraniin to NCM460 cells. The selective induction of CIN
in CRC distinguishes geraniin from other HSP90 inhibitors, and
could be partially explained by geraniin may possess a much higher
binding afnity for HSP90 from cancer cells than does HSP90 from
normal cells, as occurs in 17-AAG (38). Moreover, an interesting nd
is that geraniin-reduced CIN in NCM460, although the underlying
mechanisms remain to be further explored. We previously showed
Table3. The effects of geraniin on the frequency of binucleated cells (BNCs) displaying micronuclei (MN), nucleoplasmic bridges (NPB) or
nuclear buds (NB) per 1000 BNC in NCM460 cells (n=3 per treatment).
Biomarkers Treatment
interval
Dose of geraniin (μg/ml) MMC
(0.05μg/ml)
0 25 50 100
MN 24 h 51.31±2.37 22.59±1.96*** 16.93±0.98*** 12.01±2.58*** 192.56±12.33***
48 h 48.10±2.49 13.41±1.88*** 13.45±0.97*** 25.15±2.42 *** 242.67±12.24***
72 h 70.74±4.61 65.32±4.30 48.84±2.12* 33.07±3.62*** 310.84±30.96***
NPB 24 h 7.69±0.46 5.47±0.58 5.54±0.68 3.56±0.65** 22.66±2.86***
48 h 6.42±2.04 2.23±0.63 2.22±0.87 7.27±3.80 34.50±4.87***
72 h 28.11±2.28 10.79±1.99** 9.69±2.13** 12.31±1.99** 49.41±4.63***
NB 24 h 25.05±5.01 14.22±1.79 12.74±2.68 7.46±1.98* 52.39±3.88***
48 h 19.02±1.18 10.21±2.75* 10.05±2.73* 18.04±3.95 54.92±3.72***
72 h 24.77±3.05 19.22±2.37 17.46±2.15 13.45±1.66* 58.74±2.68**
Data represented the mean frequency ± the standard error of the mean (S.E.M.) per 1000 BNCs from three independent experiments. Signicant differences
between geraniin-treated groups and controls at each treatment interval are indicated by *P<0.05, **P<0.01 and ***P<0.001. MMC (mitomycin C) was used
as a positive control.
276 X.Guo etal.

that P.emblica, a functional food and folk medicine contains gera-
niin, decrease the CIN via activating the SAC of NCM460 cells (39).
Therefore, we speculate that geraniin might activate the SAC in
NCM460 cells. It will be interesting to explore whether geraniin has
the potential to recapitulate the phenomenon observed in P.emblica.
Anyhow, this nding suggests geraniin is also a promising candidate
for cancer chemoprevention since pharmacological reinforcement of
surveillance mechanisms against CIN in noncancerous cells offers a
promising strategy for reducing oncogenic transformation (40). In
this context, increased proliferation and decreased apoptosis, two
hallmarkers of cancer, seen in geraniin-treated NCM460 cells do not
mean geraniin has potential to induce oncogenic transformation of
noncancerou colon cells, but indicate that it will help to maintain
colonic epithelial barrier function, thereby preventing the mucosal
invasion of intraluminal microorganisms.
A striking unexpected nding was that geraniin suppressed
necrosis in CRC cells. Necrosis is a type of programmed cell death
that has a prominent role in multiple physiological and pathologi-
cal settings (41). Remarkably, necrosis involves in cell survive when
tumour become oxygen- and nutrient-deprived. For example, necro-
sis is accompanied by mitochondrial dysfunction with enhanced
generation of reactive oxygen species (42), which exerts a key role
in several hallmarks of cancer such as self-sufciency in prolifera-
tion signals (43). Moreover, the necrotic centre of a tumour releases
angiogenesis-promoting factors that diffuse out of the tumour, reach
the sprout tips, guide their chemotaxis and branching behaviour,
Figure3. Influence of geraniin treatment on the nuclear division index (NDI) of Colo320, Colo205 and NCM460 cells. Colo320 (A), Colo205 (B) and NCM460 cells
(C) were incubated with the indicated concentrations of geraniin for 24, 48 and 72h, followed by 24h of recovery time incubated with cytochalasin B.Then cells
were fixed and the NDI (black bars) and % cytostasis (the numbers above black bars in Aand B) or % proliferation (the numbers above black bars in C) were
determined. Values are presented as mean ± S.E.M. (n =3). *P<0.05, **P<0.01 and ***P<0.001 compared with the corresponding control.
Geraniin selectively promotes cytostasis and apoptosis in human colorectal cancercells 277

and ultimately determine the vascular topology (44). These insights
strongly support the notion that, by impairing the proliferation and
metastasis of cancer cells, the inhibition of necrotic cell death may be
a more promising target for effective antitumour therapy, although
the inducers of necrosis might be theoretically useful for the treat-
ment of apoptosis-resistant tumours (45).
The in vivo effects of geraniin are largely dependent on its
pharmacokinetics and bioavailability. Existing evidence on the
absorption and metabolism of geraniin upon oral ingestion is fairly
limited. Generally, intact geraniin is rarely found in the circulation
after oral dosing because of its large molar mass, which does not
facilitate simple diffusion very effectively (9). Rather, geraniin is
hydrolysed by the intestinal bacteria to various metabolites (gallic
acid, pyrogallol, ellagic acid and urolithins) which are subsequently
absorbed by the small intestine and the colon and persists in the
body for a relatively long duration (46). These metabolites exhibit
Figure4. Influence of geraniin treatment on the apoptosis and necrosis frequencies of Colo320, Colo205 and NCM460 cells. Colo205, Colo320 and NCM460
cells were incubated with the indicated concentrations of geraniin for 24, 48 or 72h followed by 24h of recovery time for cell death assay. Then cells were fixed
and the apoptosis frequency (A-C) and necrosis frequency (D-F) were determined. All measurements were set relative to the values measured in corresponding
untreated control cells, which were arbitrarily set at 1.Values are presented as mean ± S.E.M. (n=3). *P<0.05, **P<0.01 and ***P<0.001 compared with the
corresponding control.
278 X.Guo etal.

more potent antioxidant activity compared to geraniin, and may be
the key players (particularly urolithins) that account for the bioac-
tivities and nal health benets of geraniin (47).
Our present ndings have two major potential implications. First,
we previously found the extract of P.emblica fruit can elevate CIN in
Colo320 cells (16) while decrease CIN in NCM460 cells (15,16). The
faithful recapitulation of these observations by geraniin, therefore, pro-
vides compelling evidence that geraniin is one major bioactive compo-
nent of P.emblica. Further studies, therefore, can be conducted to test
whether geraniin is also responsible for many other benecial effects
of this functional food and folk medicine on human health. Second,
our present results will provide some clues to investigate the anticancer
Figure 5. Relationships between chromosomal instability (CIN) and apoptosis (A), CIN and nuclear division index (NDI; B). Pearson correlation analysis
performed using data from NCM460, Colo320 and Colo205 cells. Due to the different baselines of CIN, apoptosis and NDI among these cell lines, relative changes
of CIN, apoptosis and NDI were used for analysis. The solid line is the weighted regression. r, Pearson’s correlation; n, the total quantified pairs.
Figure6. Model summarizing our data. Homeostasis of chromosomal instability (CIN) can facilitate the evolution of advantageous karyotypes that are integral
to the survival and proliferation of cancer cells. Geraniin treatment disrupts this homeostasis by elevating the frequency of catastrophic CIN, which results
in cellular toxicity and apoptosis in cancer cells. CIN, on the other hand, can also contribute to the tumorigenicity of normal cells. Geraniin reduces CIN in
normal cells, and therefore reduces their risk of oncogenic transformation. Although the underlying molecular mechanisms remain to be fully understood, we
hypothesise that the inhibition of HSP90 and TOP2 and the activation of spindle assembly checkpoint (SAC) may account for the opposite roles of geraniin in
regulating CIN between cancer and normal cells.
Geraniin selectively promotes cytostasis and apoptosis in human colorectal cancercells 279

mechanisms of other natural occurring HSP90 and/or TOP2 inhibitors,
such as epigallocatechin gallate, curcumin and resveratrol.
There are, however, two limitations in this study. First, there is only
limited number of CIN endpoints were investigated. Some other CIN
events that cannot be detected by CBMN-Cyt assay, such as multinu-
cleation/polyploidy, centrosome aberrations (amplication, fragmen-
tation and declustering) and mitotic multipolarity, need to be further
characterised. Second, we focused this study on detailed analysis of the
impacts of geraniin on CIN using in vitro cell models. Whether these
effects are applicable to in vivo models need to be determined.
In summary, geraniin shows potential to selectively kill CRC by
further augmenting CIN stress, while it is nontoxic to noncancerous
colon cells but may also act on pathways that prevent against CIN
(Figure6). Hence, our ndings not only uncover a basis for the anti-
cancer effect of geraniin, but also suggest the potential application of
geraniin for cancer chemoprevention.
Funding
This work was supported by by the National Natural Science
Foundation of China (#31260268 and #31560307).
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