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R E S E A R C H A R T I C L E Open Access
Kalanchoe tubiflora extract inhibits cell
proliferation by affecting the mitotic apparatus
Yi-Jen Hsieh
1,5
, Ming-Yeh Yang
2
, Yann-Lii Leu
4
, Chinpiao Chen
5
, Chin-Fung Wan
6,7
, Meng-Ya Chang
3,8
and
Chih-Jui Chang
2*
Abstract
Background: Kalanchoe tubiflora (KT) is a succulent plant native to Madagascar, and is commonly used as a
medicinal agent in Southern Brazil. The underlying mechanisms of tumor suppression are largely unexplored.
Methods: Cell viability and wound-healing were analyzed by MTT assay and scratch assay respectively. Cell cycle
profiles were analyzed by FACS. Mitotic defects were analyzed by indirect immunofluoresence images.
Results: An n-Butanol-soluble fraction of KT (KT-NB) was able to inhibit cell proliferation. After a 48 h treatment
with 6.75 μg/ml of KT, the cell viability was less than 50% of controls, and was further reduced to less than 10% at
higher concentrations. KT-NB also induced an accumulation of cells in the G2/M phase of the cell cycle as well as
an increased level of cells in the subG1 phase. Instead of disrupting the microtubule network of interphase cells,
KT-NB reduced cell viability by inducing multipolar spindles and defects in chromosome alignment. KT-NB inhibits
cell proliferation and reduces cell viability by two mechanisms that are exclusively involved with cell division: first
by inducing multipolarity; second by disrupting chromosome alignment during metaphase.
Conclusion: KT-NB reduced cell viability by exclusively affecting formation of the proper structure of the mitotic
apparatus. This is the main idea of the new generation of anti-mitotic agents. All together, KT-NB has sufficient
potential to warrant further investigation as a potential new anticancer agent candidate.
Keywords: Kalanchoe tubiflora, Multipolar spindle, Anti-proliferation
Background
The mitotic spindle is a complex molecular scaffold that
mediates proper sister-chromatid segregation during mi-
tosis and is essential in maintaining genomic integrity of
daughter cells. In animal cells, thus far two major path-
ways have been identified in mitotic spindle assembly.
First, the mitotic centrosomes function as microtubule-
organizing centers (MTOCs) [1], directing spindle
assembly from spindle poles through the regulation of
nucleating microtubules [2]. The second pathway is
dependent on a RanGTP gradient mediated by Aurora A,
which regulates spindle assembly [3,4]. The chromosomal
passenger complex (CPC) generates spindle assembly in
the absence of a RanGTP gradient [5,6]. Disruption of
these pathways and disruption of the mitotic spindle will
activate the spindle assembly checkpoint, causing mitotic
arrest and cell death [4].
Compoundsthattargetmicrotubuleassemblyareoften
very promising as anti-cancer medication. Vinca alkaloids
and taxanes, for example, are administered against both
solid tumors and haematological malignancies [7-9]. Many
of these compounds were originally isolated from a variety
of marine organisms or botanicals [10-12]. Microtubule-
targeting drugs are classified into two groups, microtubule-
destabilizing agents and microtubule stabilizing agents
[13,14]. Both microtubule -destabilizing and -stabilizing
agents inhibit cell proliferation by binding to tubulin, alter-
ing microtubule dynamics, and disrupting the mitotic spin-
dle. Cancer cells are generally more susceptible to tubulin
binding agents compared to normal cells [15]. Since micro-
tubules are present in cells that are in interphase or under-
going mitosis, these microtubule targeting agents not only
disrupt mitotic spindle formation but they can also disrupt
the microtubule network. The microtubule network
* Correspondence: cjchang@mail.tcu.edu.tw
2
Department of Molecular Biology and Human Genetics, Tzu Chi University,
No. 701, Zhongyang Rd., Sec. 3, Hualien 97004, Taiwan
Full list of author information is available at the end of the article
© 2012 Hsieh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Hsieh et al. BMC Complementary and Alternative Medicine 2012, 12:149
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mediates important cellular processes during interphase,
which include cellular migration and intracellular transport.
Disruption of the microtubule network can cause a diverse
range cytotoxic effects [16].
Recently, a new generation of anti-mitotic agents that
target kinesins and mitotic kinases, such as Eg5, Aurora
kinase and polo-like kinase, were developed as putative
anti-cancer medications [17,18]. Eg5 is a kinesin motor
that localizes to the centrosomes and MT asters during
prophase [19] and is required for centrosome separation
as well as bipolar spindle assembly [20,21]. The aurora
kinase family is crucial for the progression of cells from
mitosis to cytokinesis [22-25]. Aurora A localizes to
spindle poles and functions in centrosome maturation,
separation and spindle bipolarity [26,27]. Aurora B is the
enzymatically active member of the CPC, which localizes
along the chromosome arms and at the centromeres
during prophase. It is concentrated at the inner centro-
mere region from prometaphase to metaphase and
transfers to the spindle midzone, cell cortex and mid-
body during late mitosis and cytokinesis [25,28]. The
CPC is essential both for the assembly and stability of
the bipolar mitotic spindle [29]. Inhibition of Aurora B
and other components of CPC causes mitotic catastro-
phe [29-32], indicating that CPC may be involved in
both mitotic spindle assembly pathways.
Kalanchoe is a genus of the Family Crassulaceae. Vari-
ous species of Kalanchoe are often referenced in folk-
lore, and commonly used in traditional medicine
worldwide for the treatment of fever, abscesses, bruises,
contused wounds, coughs, skin diseases, infections,
hypertension, rheumatism and inflammation [33-36].
Kalanchoe species are also used by the Kerala tribes for
treating cancer symptoms [37]. A variety of bufadieno-
lide compounds were isolated from various Kalanchoe
species, which show strong anti-tumor promoting activ-
ity [38-43]. Kalanchoe tubiflora (KT), due to its wide
variety of potential biological activities, was selected for
this study. KT is one of the most common medicinal
plants used for wound healing in Southern Brazil. The
traditional uses of KT in wound healing coincide with
results from systematic biological assays [44]. Here we
show that an n-BuOH-soluble fraction of KT has anti-
proliferative activity, which is due to the induction of
multi-polar spindles and chromosomal misalignment of
mitotic cells. These abnormal mitotic events lead to mi-
totic catastrophe, a desirable effect of a cancer thera-
peutic drug.
Methods
Regents
All reagents were purchased from Sigma unless other-
wise stated. The primary antibodies were used as fol-
lowed: anti-alpha tubulin (mouse mAb B512, 1:2000;
Sigma, Taiwan); anti-Aurora A polyclonal antibody (1:
500, Cell signaling, Taiwan); anti-phospho-Histone 3
polyclonal antibody (1: 500, Upstate, Taiwan).
Preparation of extracts from Kalanchoe tubiflora
Fresh Kalanchoe tubiflora (KT) was chopped and boiled
three times with 95% EtOH under reflux and filtered.
The filtered broth was concentrated under reduced pres-
sure. The crude extract was resuspended in H
2
O and
partitioned successively with CHCl
3
and n-BuOH to give
a CHCl
3
-soluble fraction (KT-C), a n-BuOH-soluble
fraction (KT-NB), and a H
2
O-soluble fraction (KT-W).
53.79 g of dry (KT-NB) extract were obtained from
6638.76 g of raw KT plant tissue. The procedure of KT-
NB extraction is represented in figure 1. Stock solution
was prepared in DMSO and filtered through 0.22 μm
membrane. Cell culture medium was used to further di-
lute the extracts to a desired concentration for all cellu-
lar assays.
Cell lines and culture
Human lung carcinoma (A-549), human bladder papil-
lary transitional cell carcinoma (BFTC905), human
breast carcinoma (MCF-7), human larynx Epidermoid
carcinoma (HEp-2), human uterus sarcoma (MED-SA)
and HeLa cells were purchased from the Food Industry
Research and Development Institute (Hsinchu, Taiwan).
Cells were grown, according to supplier instructions, in
media supplemented with 10% fetal bovine serum (FBS).
Cells were maintained at 37°C in a humidified atmos-
phere of 5% CO2.
Figure 1 Flow chart of Kalanchoe tubiflora extract synthesis.
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Cell viability assay
The percentage of growth inhibition was determined by
the MTT cell proliferation/viability assay. A total of 2000
cells/well were seeded onto a 96-well plate for 16 h.
Cells were treated with various concentrations of KT-NB
(25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.125 μg/ml, and
1.5625 μg/ml) and incubated for an additional 3 days at
37°C. The MTT assay was performed in triplicate. Subse-
quently, the test solutions were removed and replaced
with culture medium containing 500 μg/ml of MTT
(Thiazolyl blue formazan, Sigma, Taiwan) for an add-
itional 4-6 h. The supernatant was aspirated, and 200 μl
of DMSO was added to the wells to dissolve any pre-
cipitate present. The optical density (OD) values were
measured in an ELISA reader (SUNRISE, TECAN,
Switzerland) at a wavelength of 570 nm. The mean and
standard deviation of each group were calculated. The
OD reading of every group was first subtracted by a
blank (background) control. Relative survival rate = [OD
(treatment groups)/OD (negative control)] x 100.
Wound-healing assay
Cell mobility was assessed using a wound-healing assay.
A-549 cells were seeded in six-well plates for 24 h. Con-
fluent monolayer cells were scratched by a 200 μl pipette
tip and then removed by washing the cells with serum-
free medium to clear cell debris and suspension cells.
Migration of cells into the wound was observed at differ-
ent time points. Cells that migrated into the wounded
area or cells with extended protrusion from the border
of the wound were visualized and photographed under
an inverted microscope.
FACS
Cells were treated with KT-NB or DMSO and harvested
at 24, 48, 72 h. Floating and adherent cells were col-
lected and fixed in cold 70% ethanol at 4°C overnight.
After washing, cells were treated with RNase and stained
with Propidium Iodide (PI) for 1 h in the dark at room
temperature. Flow cytometric analysis was performed by
using a FACScalibur flow cytometer (Becton Dickinson).
Cell cycle distribution was analyzed using Cell Quest
software (Becton Dickinson). Each experiment was con-
ducted three times.
Immunofluorescence and microscopy
For immunostaining, cells were fixed in 4% paraformalde-
hyde in PBS buffer for 10 min at room temperature. The
cells were permeabilized in 0.1% Triton X-100 in PBS for
5 min and then rinsed in PBS. Cells were blocked for
30 min at room temperature in PBS + 10% FBS. Antibody
incubations were performed in PBS for 1 h, followed by
four 10-min washes in PBS at room temperature. DNA
was stained with 0.1 mg/ml DAPI for 5 min at room
temperature and rinsed with PBS. Slides were mounted in
Vectashield mounting medium (Vectra) and sealed using
nail varnish.
Images were performed using a Zeiss Axiovert 200 M
microscope controlled by MetaMorph Software. Image
stacks were deconvolved and quick-projected. The
images were processed using Adobe Photoshop.
Results
KT-NB decreases cell viability
We treated HeLa cells with different doses of KT-NB to
analyze its effect of cell proliferation. Cells were exposed
to three different concentrations of KT-NB and DMSO,
which was used as a control. At each time point, the cell
number was determined using a haemocytometer. Com-
pared to control cells, 1.35 μg/ml of KT-NB only slightly
affected cell growth; however, cell growth was inhibited
at concentrations of 6.75 μg/ml and 13.5 μg/ml after a
48 h treatment (Figure 2A). We also used the MTT
assays to assess the anti-proliferative effect of KT-NB on
Hep2, MES-SA, BFTC905, MCF7, HeLa and A549 cells
(Figure 2B). These cells were treated with KT-NB
Figure 2 KT-NB inhibits cell proliferation and reduces cell
viability. (A) HeLa cells were treated with different concentration of
KT-NB (1.35 μg/ml, 6.75 μg/ml, 13.5 μg/ml). DMSO was used as
control. Cell numbers were determined using a haemocytometer at
different time points. (B) Human lung carcinoma (A-549), human
bladder papillary transitional cell carcinoma (BFTC905), human breast
carcinoma (MCF-7), human larynx Epidermoid carcinoma (HEp-2),
human uterus sarcoma (MED-SA) and HeLa cells were treated with
1.5625 μg/ml, 3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml of
KT-NB for 72 h and harvested for MTT assay. Results were based on
three independent experiments.
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(1.5625 μg/ml, 3.125 μg/ml, 6.25 μg/ml, 12.5 μg/ml, and
25 μg/ml) for 72 h and harvested for analysis. KT-NB
reduced the viability of the different cancer cells in a
dose dependant manner. The IC50 of KT-NB in A549
cells was less than 1.5625 μg/ml. KT-NB could also
inhibit the growth of other cancer cells at slightly
higher concentrations (6.25 μg/ml). Compared to cancer
cells, the toxicity of KT-NB was less in normal cells
(Additional file 1: Figure S1). It is worthy of note that
there was ambiguity whether the reduced cell growth rate
resulted from cell death or non-proliferating. Overall,
these results showed that KT-NB could effectively inhibit
the growth of cancer cells at concentrations less than
6.25 μg/ml.
KT-NB inhibits cell migration
A wound healing assay was performed to assess the
effect of KT-NB on A549 cell migration. In response to
a scratch wound, cells treated with KT-NB (3 μg/ml,
12.5 μg/ml, and 50 μg/ml) exhibited a concentration and
time dependent decrease in cell migration. Cell migra-
tion was slightly inhibited at lower KT-NB concentra-
tions (3 μg/ml), while at the concentration of 50 μg/ml,
KT-NB almost completely blocked the migration of cells
into the scratch wound (Figure 3). Note that this
inhibition effect might due to either inhibition of cell
migration or induction of cell death after KT-NB
treatment.
KT-NB induces cell cycle arrest
FACS analysis was performed to examine the effect of
KT-NB on the cell cycle. After 24 h treatment of KT-
NB, a progressive accumulation of cells were found in
the G2/M phase of the cell cycle, which was accompan-
ied with a decrease of cells in the G1 phase (Figure 4A).
The mitotic index in control and KT-NB treated cells
was examined by both anti-phospho-Histone H3 stain-
ing and DAPI staining under a fluorescence microscope.
The mitotic index increased from 8% in control cells to
20% in cells treated with 6.75 μg/ml of KT-NB at 24 h
(Figure 4B). We also observed that cells exposed to KT
exhibited higher levels of subG1 with DNA content less
than 2 N. The level of cells in the subG1 phase increased
to 35% after 72 h treatment (Figure 4C).
KT-NB induces multipolar spindles
Since KT-NB treatment induces cell cycle arrest during
mitosis, we analyzed whether KT-NB could cause mi-
totic spindle defects and regulate important mitotic
checkpoints. To analyze the structure of mitotic
Figure 3 KT-NB inhibits cell migration in a wound healing assay. A-549 cells were seeded in six-well plates and allowed to adhere for 24 h.
Cells were treated with 3 μg/ml, 12.5 μg/ml, 50 μg/ml of KT-NB. Migration of cells into the wound was observed at 24, 48, 72 h after monolayer
cells were scratched by a 200 μl pipette tip. Results are based on three independent experiments. Lines indicated the border of the wounds.
Imagines were processed using Photoshop software. The contrast of the images was adjusted to that the border could be identified without
ambiguity. The border lines were determined by eyes.
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spindles, we used HeLa cells to performed immunofluor-
escent staining. The mitotic phases of cells were care-
fully analyzed by DAPI and microtubule staining.
Majority of the control cells showed perfect bipolar
structure during metaphase (Figure 5A). In contrast,
cells treated with 6.75 μg/ml of KT-NB for 24 h exhib-
ited multipolar spindles (Figures 5B, 6A and 6B). More
than half of the mitotic cells had more than two spindle
poles (Figure 5C). To examine whether KT-NB treat-
ment can alter important components involved in the
mitotic spindle poles, the localization of Aurora A was
analyzed. Cells treated with KT-NB showed normal Aur-
ora A localization to spindle poles (Figure 6A).
Aurora B is required for stability of the bipolar mitotic
spindle [32] and functions through the phosphorylation
of histone H3 on Ser10 and Ser28 in prophase. Our
results indicated that KT-NB treated cells also did not
exhibit altered levels of Histone H3 phosphorylation
(Figure 6B). Immunofluorescence analysis of α-tubulin
and γ-tubulin revealed that the microtubule organization
and centrosome number were normal in cells treated
with KT-NB in interphase (data not shown). Taken to-
gether, these results suggested that KT-NB treatment
induced multipolar spindles but did not interfere with
the recruitment of essential components necessary for
functional spindle poles, nor did it disrupt the micro-
tubule network in interphase cells.
KT-NB induces chromosome misalignment in metaphase
KT-NB treatment induced a large amount of mitotic cells
with multipolar spindles, but 45% of the mitotic cells still
showed normal bipolar spindles. Unexpectedly, KT-NB-
treated cells with normal bipolar spindle assembly experi-
enced problems in the chromosomal congression to the
metaphase plate (Figures 7B and 7C). To specify this
phenotype, we only analyzed metaphase cells. In cells trea-
ted with KT-NB for 24 h, 37% of the metaphase cells with
bipolar spindles contained misaligned chromosomes
(Figure 7D). In contrast, control cells in metaphase exhib-
ited correct chromosomal alignment at the metaphase
plate (Figure 7A). It is worthy to note that high percentage
of mitotic cells with mitotic apparatus defects was
observed if all phases of mitotic cells were analyzed. By
grouping cells that exhibited misaligned chromosomes
with bipolar spindles or that exhibited multipolar spindles
(i.e. abnormal mitotic cells), the level of defective mitotic
Figure 4 KT-NB arrests cells in the M phase and increases sub-G1 cell population. (A) FACS analysis of DNA content after KT-NB treatment.
HeLa cells were treated with 6.75 μg/ml of KT-NB or DMSO as control. Cells were harvested for analysis after 24, 48 and 72-h treatment. Tables
showed the percentage of relative cell numbers of different cell cycle phases. (B) Cells were treated with 6.75 μg/ml KT-NB for 24 h. Quantitation
of the mitotic index in control and KT-NB treated cells (n> 500, three independent experiments). (C) Percentage of relative cell numbers of
sub-G1 cells. Cells were calculated based on the results shown in (A).
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Figure 5 KT-NB induces multipolar spindles. HeLa cells were treated with DMSO as a control (A) or 6.75 μg/ml of KT-NB (B) for 24 h. After
incubation, cells were fixed and processed for immunofluorescence analysis. DAPI was used for DNA staining, while anti-tubulin shows the
mitotic spindle. Merged images: (DAPI is blue, tubulin is red). (C) The percentage of mitotic cells with multipolar spindles was quantified in
control and KT-NB treated cells (n > 100, three independent experiments). Scale bar: 5 μm.
Figure 6 KT-NB does not affect Aurora A localization and histone H3 phosphorylation on serine
10
.HeLa cells were treated with 6.75 μg/ml
of KT-NB for 24 h and harvested for immunofluorescence analysis. DAPI was used for DNA staining, while anti-tubulin shows the mitotic spindle.
Cells were immunostained with. anti-Aurora A (A) or Anti-phospho-histone H3 (Ser10) (B). Merged images DAPI is blue, anti-tubulin is red, Aurora
A(A) or histone H3 (B) is green. Scale bar: 5 μm.
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cells after KT-NB treatment was, at 90%, much higher
than control cells (Figure 7E).
Discussion
Medicinal plants are an important source for the potential
development of effective anticancer agents [45]. In fact,
more than half of the today’s anticancer drugs were origin-
ally synthesized from natural products and their derivatives.
In the present study, we found that extracts from Kalan-
choe tubiflora (KT), exhibited significant anti-proliferative
effects against a variety of human cancer cell lines.
Majority of the effective anti-cancer or antibiotic con-
centrations of plant extracts are greater than 100 μg/ml
[45,46]; however, in our study, cell proliferation was
inhibited by 48 h treatment with 6.75 μg/ml KT-NB, and
the viability was less than 50% after a 72 h exposure.
KT-NB does affect normal diploid human cells that are
dividing but much less efficiently than most of the can-
cerous cell lines we tested. Therefore, KT-NB may be a
highly effective candidate as a future anti-cancer drug.
Drugs based on natural products that bind to tubulin or
microtubules remain an important component in chemo-
therapy. Anti-mitotic agents inhibit cell proliferation by
Figure 7 KT-NB induces chromosomal misalignment in cells with bipolar spindles. Cells were treated with DMSO as control (A) or 6.75 μg/
ml of KT-NB (B,C) for 24 h. After incubation, cells were fixed and processed for immunofluorescence analysis. (A) The control cell in metaphase
showed bipolar spindle with chromosome alignment to the metaphase plate. (B) The KT-NB treated cell showed bipolar spindle containing extra
centrosome (arrowhead). (C) The KT-NB treated cells exhibited bipolar spindle with the normal two centrosomes. Arrows points to the
misalignment chromosomes. DAPI was used for DNA staining, while anti-tubulin shows the mitotic spindle. Merged images: (DAPI is blue, tubulin
is red). Scale bar: 5 μm. (D) Only the metaphase cells were analyzed. The percentage of metaphase cells with misaligned chromosome(s) in the
bipolar spindle were quantified in control and KT-NB treated cells (n > 50, three independent experiments). (E) All phases of mitotic cells were
analyzed. The percentage of mitotic cells with multipolar spindles or misaligned chromosome(s) was quantified in control and KT-NB treated cells
(n > 100, three independent experiments). Bi-mis: cells with bipolar spindle and misaligned chromosomes. Multi: cells with multipolar spindles.
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suppressing microtubule dynamics [13]; however, based
on immunofluorescence staining images, we found that
KT-NB did not disrupt the microtubule organization in
interphase or spindle formation during mitosis, indicating
that it is not a microtubule destabilizing agent. [47]. Our
results also showed that KT-NB, which did not lead to the
formation of parallel microtubule alignment or packed
bundles of microtubules, is not a microtubule stabilizing
agent either [48]. Rather than acting on microtubule dy-
namics, we found that KT-NB induces multipolar spindles
and perturbs accurate mitosis.
Centrosomes increase both in size and in microtubule-
capacity in late G2 phase of the cell cycle. Aurora A, by
recruiting pericentriolar material (PCM) and interacting
with the Ran-TPX2 pathway, is required for the matur-
ation of centrosomes and mitotic-spindle assembly re-
spectively [3,49]. It was noted that depletion of TPX2
from Xenopus egg extracts results in the formation of
less compact spindles [50]. In vertebrate cells, depletion
of TPX2 using RNAi also caused the formation of multi-
polar spindles [51]; however, inhibition of Aurora A
activity results in monopolar spindles [24]. Our immu-
nostaining results showed that localization of Aurora A
was not disrupted by KT-NB. Since we did not observe a
high level monopolar spindles, an effect common with
Aurora A inhibitors, it is also unlikely that KT-NB inhi-
bits Aurora A activity.
Some of the KT-NB-treated cells showed normal bipo-
lar spindles with two centrosomes in mitosis. It was
noted that a few cells with more than two centrosomes
could assemble almost bipolar spindles (Figure 7B). With
KT-NB treatment, it did not matter whether spindles
were formed by the normal two centrosomes or by extra
centrosomes, as more than half of the cells with bipolar
spindles failed to exhibit properly aligned chromosomes
at the metaphase plate. Although, chromosomal mis-
alignment can result from the suppression of micro-
tubule dynamics by a stabilizing poison, such as taxol
[48], as discussed previously, this is an unlikely mechan-
ism for KT-NB.
The failure of non-bipolar attachment corrections dur-
ing the stochastic attachment process in prometaphase,
is another possibility for chromosomal misalignment.
Aurora B, the enzymatic member of CPC, destabilizes
the non-bipolar microtubule-kinetochore attachments
by phosphorylating several microtubule capture factors
on the kinetochore [52-54]. To examine if the misa-
ligned chromosome in KT-NB treatment was due to the
inhibition of Aurora B kinase, we analyzed the histone
H3 phosphorylation levels. In prophase, the CPC loca-
lizes to the chromosome arms where it phosphorylates
histone H3 on Ser10 and Ser28 [25]. Phosphorylation of
histone H3 was not affected in KT-NB treated cells
(Figure 6B).
Plk is a central regulator of the cell cycle, playing cru-
cial roles in mitotic events, including activation of
CDC25c phosphatase, regulation of microtubule nucle-
ation, centrosome maturation and kinetochore assembly
[55,56]. To ensure the accuracy of these processes, plk1
activity is subjected to complex regulation. It is activated
at mitotic entry by Aurora A kinase and its adapator
BORA, which phosphorylate plk1 in its T-loop [57,58].
Plk1 is also activated by Aurora B kinase at centromeres,
and that is ccrucial for polo function in regulating
chromosome dynamics in prometaphase [59]. In our
study, monopolar spindles were not observed in KT-NB
treated cells. Two other possible targets of KT-NB are
CENP-E and Mps1. CENP-E is a plus end-directed
motor protein that has a pivotal role in mitosis. It stabi-
lizes the interaction between microtubules and kineto-
chores of the mitotic spindle [60] and regulates the
mitotic checkpoints by modulating the function of
BuBR1 [61]. Complete inhibition of CENP-E leads to de-
fective mitosis with unaligned chromosomes and apop-
tosis [62]. Mps1 was originally discovered in a yeast
genetic screen for mutants producing monopolar spindle
[63]. It is a conserved multi-functional kinase that plays
roles in the SAC and chromosome bi-orientation [64].
Some small-molecular inhibitors of Mps1 were devel-
oped in 2010 [65]. Chemical inhibition of Mps1 leads to
chromosome misalignment and increases the frequency
of multipolar mitoses [66].
David Pellman’s group used live cell images to study
the fate of cells with extra centrosomes in 2009 [67].
According to their study, most of progeny of multipolar
cells died or arrested regardless of whether the cells
were mono- or poly-nucleated. It will be interesting to
use live cell images in the future to reveal the fate of
KT-NB treated cells.
Targeting of mitotic cells is one of the bases of therapy
for patients with multiple types of solid tumors. Some
antimitotic agents, taxanes or vinca alkaloids, affect both
dividing and nondividing cells. An essential characteris-
tic of the ideal new generation of antimitotic agents is to
target dividing cells but not non-dividing cells. As most
cancer cells have faster dividing rates, these drugs target
cancer cells preferentially. Potentially then, perturbing
the mitotic apparatus by KT-NB may be used to more
efficiently target rapidly proliferating cancer cells.
Conclusions
Here we report that KT-NB is a promising anti-cancer
agent candidate. Further in vivo research will be needed
to determine the effectiveness of KT-NB as an actual
anti-cancer compound. Unlike microtubule binding
agents, KT-NB inhibits cell proliferation and reduces cell
viability by two mechanisms: first, by inducing multipo-
larity; second, by disrupting chromosome alignment
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during metaphase. Both mechanisms disrupt mitotic
progression and inhibiting cell proliferation. The under-
lying mechanism of KT-NB is complicated and unclear.
Further research involving the identification and isola-
tion of pure compounds that parallel the phenotypes
observed in KT-NB treated cells will be necessary to de-
termine the cellular targets of KT-NB.
Additional file
Additional file 1: Figure S1. The toxicity of KT-NB was less in normal
cells. (A) Normal female embryonic lung cells (WI-38) and lung cancer
cells (A549) were treated with different concentrations of KT-NB (1.35
μg/ml, 6.75 μg/ml, 13.5 μg/ml and 25 μg/ml). DMSO was used as a
control. Cells were harvested for MTT assay at different time points. The
absorbance of the control group was defined as 100%. Results were
based on three independent experiments. (B) Cell morphology of WI-38
was unaffected after KT-NB treatment for 72 h.
Competing interests
The authors declare that there are no conflicts of interest.
Authors’contributions
YJH carried out extraction, wound healing assay, data analysis. MYY carried
out immunostaining, MTT assay. YLL collected plant and identified Kalanchoe
tubiflora. CPC and CFW participated in extraction design. MYC carried out
microscopy. CJC conceived of the study, and participated in its design and
coordination and helped to draft the manuscript. All authors read and
approved the final manuscript.
Acknowledgments
We like to thank Mar Carmena and Ji-Hshiung Chen for their critical reading
of the manuscript. We also thank Tang K. Tang for providing image systems.
Work in CJC’s laboratory was funded by the National Science Council,
Taiwan.
Author details
1
Department of Laboratory Medicine and Biotechnology, Tzu Chi University,
Hualien 97004, Taiwan.
2
Department of Molecular Biology and Human
Genetics, Tzu Chi University, No. 701, Zhongyang Rd., Sec. 3, Hualien 97004,
Taiwan.
3
Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan.
4
Graduate Institute of Natural Products, Chang Gung University, Taoyuan,
Taiwan.
5
Department of Chemistry, National Dong-Hwa University, Hualien,
Taiwan.
6
School of Applied Chemistry, Chung Shan Medical University,
No.110,Sec.1,Jianguo N.Road, Taichung City 40201, Taiwan.
7
Institute of
NanoEngineering and MicroSystems, National Tsing Hua University, No. 101,
Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
8
Department of Medical
Research, Buddhist Tzu-Chi General Hospital, Hualien, Taiwan.
Received: 26 March 2012 Accepted: 31 August 2012
Published: 10 September 2012
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doi:10.1186/1472-6882-12-149
Cite this article as: Hsieh et al.:Kalanchoe tubiflora extract inhibits cell
proliferation by affecting the mitotic apparatus. BMC Complementary and
Alternative Medicine 2012 12:149.
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