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• COLORECTAL CANCER •
Honokiol: A potent chemotherapy candidate for human colorectal
Fei Chen, Tao Wang, Yi-Feng Wu, Ying Gu, Xiao-Li Xu, Shu Zheng, Xun Hu
Fei Chen, Tao Wang, Ying Gu, Xiao-Li Xu, Shu Zheng, Xun Hu,
Cancer Institute, Second Affiliated Hospital of Zhejiang University,
Hangzhou 310009, Zhejiang Province, China
Yi-Feng Wu, Life Science College, Zhejiang University, Hangzhou
310027, Zhejiang Province, China
Supported by Cheung Kong Scholars Programme of National Ministry
of Education, China, and Li Ka Shing Foundation, Hong Kong
Co-first-authors: Fei Chen and Tao Wang
Correspondence to: Professor Xun Hu, Cancer Institute, Second
Affiliated Hospital of Zhejiang University, Hangzhou 310009, Zhejiang
province, China. firstname.lastname@example.org
Telephone: +86-571-87783868 Fax: +86-571-87214404
Received: 2004-02-14 Accepted: 2004-02-24
AIM: To investigate the anticancer activity of Honokiol on RKO,
a human colorectal carcinoma cell line in vitro and in vivo,
and to evaluate its possible use in clinic.
METHODS: In vitro anticancer activity of honokiol was
demonstrated by its induction of apoptosis in tumor cells.
We analyzed cell proliferation with MTT assay, cell cycle with
flow cytosmeter, DNA fragment with electrophoresis on
agarose gels. To test the mechanism of honokiol-induced
apoptosis, Western blotting was used to investigate the factors
involved in this process. The pharmacokinetics study of
honokiol was tested by high phase liquid chromatography.
In in vivo study, Balb/c nude mice were incubated with
RKO cells. Honokiol was injected intraperitoneally every
other day into tumor bearing Balb/c nude mice.
RESULTS: Our results showed that honokiol induced apoptosis
of RKO cells in a time- and dose-dependent manner. At
5-10 ug/mL for 48 h, honokiol induced apoptosis through
activating Caspase cascades. Pharmacokinetics study
demonstrated that, honokiol could be absorbed quickly by
intraperitoneal injection, and maintained in plasma for more
than 10 h. In nude mice bearing RKO-incubated tumor, honokiol
displayed anticancer activity by inhibiting tumor growth and
prolonging the lifespan of tumor bearing mice.
CONCLUSION: With its few toxicity to normal cells and
potent anticancer activity in vitro and in vivo, honokiol might
be a potential chemotherapy candidate in treating human
Chen F, Wang T, Wu YF, Gu Y, Xu XL, Zheng S, Hu X. Honokiol:
A potent chemotherapy candidate for human colorectal
carcinoma. World J Gastroenterol 2004; 10(23): 3459-3463
In traditional Chinese medicine, Houpu (Magnolia officinalis)
has long been one of the important herbs. It is widely used by
Chinese people in treating thrombotic stroke, typhoid fever,
anxiety and nervous disturbance when used in combination
with other herbs. With its major active constituent extracted
from the bark of Houpu, honokiol has been found having a
variety of pharmacological effects, such as anti-inflammatory,
antithrombotic, anti-arrhythmic, antioxidative and
anxiolytic effects. Recently, honokiol has been reported to
exhibit a potent cytotoxicity by inducing cell apoptosis in rat
and human leukemia cells[7,8], human fibrosarcoma cells,
human squamous lung cancer CH27 cells and human SVR
angiosarcoma cells, yet there has been no report on honokiol
in the treatment of human colorectal carcinoma.
Previous studies have shown that honokiol can induce
apoptosis with characteristic morphological changes and DNA
fragments, involvement of Caspase family and Bcl-2 family.
It could reduce tumor volume of SVR angiosarcoma in nude
mice. However, it is still unclear whether honokiol can be
used as a monomer in clinic. In this study, we chose colorectal
carcinoma cells to investigate its possible application in clinical
MATERIALS AND METHODS
Cell line and reagents
Human colorectal cell line RKO was provided by the Cancer
Institute of Zhejiang University. Cells were maintained in RPMI-
1640 medium (Gibco BRL) supplemented with 100 mL/L heat-
inactivated fetal bovine serum (Si-Ji-Qing Biotechnology Co,
Hangzhou, China), 100 U/mL penicillin and 100 µg/mL
streptomycin at 37 in a 50 mL/L CO2 atmosphere. Antibodies
used in this study including Caspases-3, -9 and pan-actin were
purchased from NeoMarkers, Fremont, CA, USA. Honokiol was
obtained from the National Institute for Pharmaceutical and
Biological Products, Beijing, China. The drug was dissolved in
dimethyl sulfoxide (DMSO) at the stock concentration of
10 g/L. It was further diluted in culture medium at the final
DMSO concentration <1%. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) was purchased from
Sigma Chemical Corporation, USA. Six weeks old female Balb/
c mice and female BALB/c nude mice (weighing 20±2 g each)
were provided by the Experimental Animal Center of Zhejiang
Cell proliferation assay
Cells (1×104 in 100 µL) were seeded on 96-well plates in triplicate.
Following a 24-h- culture at 37
with fresh medium at various concentrations of honokiol in a
final volume of 200 µL. Cells were incubated at 37
Then 50 µL of MTT (2 mg/mL in PBS) was added to each well,
incubated for an additional 4 h, the plate was centrifuged at
1 000 r/min for 10 min, then the medium was removed. MTT
formazan precipitate was dissolved in 100 µL of DMSO, shaken
mechanically for 10 min and then read immediately at 570 nm in
a plate reader (Opsys MR, Denex Technology, USA).
, the medium was replaced
for 68 h[12,13].
Morphological changes and detection of DNA fragmentation
RKO cells were exposed to a variety of concentrations of
honokiol for 24 h, then examined under reverse microscope
(Olympus) and imaged with a digital camera. To detect DNA
fragments, the cells were collected and lysed with lysis buffer
containing 50 mmol/L Tris-HCL (pH 7.5), 20 mmol/L EDTA, and
10 g/L NP-40. Then 10 g/L SDS and RNase (5 µg/mL) were
added to the supernatants, and incubated at 56
followed by incubation with proteinase K (2.5 µg/mL) at 37
for 2 h. After the DNA was precipitated by addition of both
ammonium acetate (3.3 mol/L) and ethanol (99.5 %), it was
dissolved in a loading buffer. DNA fragmentation was detected
by electrophoresis on 15 g/L agarose gels and was visualized
with ethidium bromide staining.
for 2 h,
Cell cycle analysis by FCM
Honokiol-treated RKO cells and vehicles were fixed with
700 mL/L alcohol for 15 min at 4
propidium dodide (PI, Sigma, USA). The red fluorescence of
DNA-bound PI in individual cells was measured at 488 nm with
a FACSCalibur (Becton Dickinson, USA) and the results were
analyzed using ModFit 3.0 software. Ten thousand events were
analyzed for each sample.
, then stained with 1.0 µg/mL
Western blot assay
RKO cells (5×106) were lysed by 4 g/L trypsin containing 0.2 g/L
EDTA, then collected after washed twice with phosphate-
buffered saline (PBS, pH 7.4). Total protein extracts from the
cells were prepared using cell lysis buffer [150 mmol/L NaCl,
0.5 mol/L Tris-HCL (pH 7.2), 0.25 mol/L EDTA (pH 8.0), 10 g/L
Triton X-100, 50 mL/L glycerol, 12.5 g/L SDS]. The extract (30 µg)
was electrophoresed on 120 g/L SDS-PAGE gel and electroblotted
onto polyvinydene difluoride membrane (PVDF, Millipore Corp.,
Bedford, MA) for 2 h in a buffer containing 25 mmol/L Tris-HCL
(pH 8.3), 192 mmol/L glycine and 200 mL/L methanol. The blots
were blocked with 50 g/L nonfat milk in TBST washing buffer
for 2 h at room temperature and then incubated at 4
with anti-caspase-3 and -9 antibodies (NeoMarkers), all of which
were diluted 1:400 in TBST. After washed at room temperature
with washing buffer, they were labeled with peroxidase-
conjugated secondary antibodies.
Cell toxicity on primary cells
Primary human fibroblast cells were derived from fresh skin.
Human monocytes were isolated from umbilical blood by Ficoll-
Hyaque separation method while seeded in 96-well microplates
at the concentration of 5 µg/mL phytohemagglutinin (PHA).
HUVECs isolated from fresh human umbilical cords were
inoculated into 96-well microplates at 5 000 cells/well. Following
treatment with the concentrations of 5, 10, 20, 40 µg/mL of honokiol
for 24 h, cell viability was estimated by trypan blue exclusion.
Three wells were measured at each time point/concentration.
Six wells were measured for each concentration of test compound.
All toxicity experiments were at least repeated three times.
For intraperitoneal (ip) pharmacokinetics study, honokiol was
mixed with PEG400/dextrose by 7:3 in volume at a concentration
of 20 g/L. Thirty Balb/c mice received honokiol by i.p. at a dose of
250 mg/kg. Blood samples were collected as described. The
plasma concentrations were tested by the total fluorescence
intensity at 290 nm with high phase liquid chromatograph (HPLC,
HEWLETT PACKARD). Chromatography was carried out
using a Hypersil C18 column (5 mm×100 mm×2.1 mm) with a flow
rate of 0.2 mL/min. Pharmacokinetic parameters were estimated
by Modkine programs (Biosoft, UK).
In vivo efficacy evaluation
Effect of honokiol on ascites formation in Balb/c nude mice
Five Balb/c nude mice in each group were transplanted with
1×107 RKO cells by ip. Honokiol was dissolved in PEG400/
Tween 20 (9:1 by volume). Honokiol-treated group was
intragastrically administered 2 mg of honokiol per mice on d 0,
2, 4, 6, 8 and 10 after inoculation of RKO cells. While the control
group was given the same volume of PEG400/Tween20. Animals
were regularly monitored for the appearance of peritoneal bulge
and body weight.
Effect of honokiol on solid tumor growth in Balb/c nude mice
RKO cells (5×106) were injected subcutaneously at the axilla of
Balb/c nude mice. When tumors became visible about one week
after implant, the animals were randomized into four groups:
Adriamycin-treated, honokiol-treated, vehicle and control. All
mice received ip injection on days 8-11, 14-17, 21-24 and 28-31.
Each mouse of honokiol-treated group received 80 mg/(kg/d)
of honokiol suspended in PEG400/dextrose (7:3 by volume)
intraperitoneally, while vehicle given equivalent solvent of
PEG400/dextrose. Adriamycin dissolved in saline was injected
ip at a dose of 2 mg/kg. Mice of control group were given the
same volume of saline. Tumor growth was monitored with
calipers every other day, and tumor volume was calculated
using the modified ellipsoid formula: A/6×A×B2, where A is the
longer axis and B is the axis perpendicular to A (Figure 1).
Figure 1 Chemical structure of honokiol (C18H18O2, MW = 266.33).
Values were given as mean±SD. Statistical comparisons were
made by Student’s t-test, and P<0.05 was taken as significant.
Inhibition of RKO cell proliferation
Cells treated with honokiol resulted in a dose- and time- dependent
cytotoxicity in RKO cells. As shown in Figure 2A, honokiol-
mediated cytotoxicity occurred at the concentration of 5 µg/mL
and above. A significant decrease in cell number was seen at
10 µg/mL. The concentration leading to a 50% decrease in cell
number (IC50) was about 12.47 µg/mL. Moreover, treatment of
RKO cells with 5 µg/mL or 10 µg/mL of honokiol resulted in a
significant growth inhibition at various time points (Figure 2B).
Morphological changes and DNA fragmentation detection in
According to MTT results, we chose 5, 10, 15 µg/mL of honokiol
to detect molecular changes. Under an inverted phase contrast
microscope, honokiol-treated cells exhibited morphological
features of apoptosis (Data not shown): rounded and granulated
morphology, some vacuoles coming from cytoplasm, cell shrinkage
and eventually detached from culture plates. In honokiol-
treated cells, a degradation of chromosomal DNA into small
internucleosomal fragments was evidenced by the formation
of 180-200 bp DNA ladders on agarose gels (Figure 3), hallmark
of cells undergoing apoptosis. No DNA ladders were detected in
the samples isolated from control cultures. These results indicated
that honokiol induced an apoptotic cell death in RKO cells.
3460 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol December 1, 2004 Volume 10 Number 23
Figure 2 Concentration- and time-dependent inhibition of RKO
cells exposed to honokiol shown by MTT assay. (A) RKO cells
were plated in quadruplicate in 96-well plates and treated with
increasing concentrations of honokiol for 68 h. (B) RKO cells
treated with 5, 10 and 15 µg/ mL of honokiol were tested at
different time points.
Figure 3 Differences in vehicle or honokiol induced apoptotic
DNA laddering of RKO cells. lane 1: control; lane 2: 5 µg/ mL;
lane 3: 10 µg/ mL; lane 4: 15 µg/ mL.
Effect of honokiol on cell cycle analysis of RKO cells
RKO cells were exposed to increasing concentrations of
honokiol (5-15 µg/mL) for 48 h, and the growth of cells was
analyzed with flow cytometry. In the absence of honokiol, the
cell populations were at G1, S, and G2/M phases (Figure 4),
accompanied with increased concentrations of honokiol by a
concomitant increase of the G1 phase (Table 1). From Figure 4,
the peak areas of subdiploid were enlarged with increased
concentrations of honokiol. This observation led to a suggestion
of G1 arrest. DNA fragmentation was seen when the cells were
exposed to honokiol at 10 µg/mL and above (14.10% and 20.31%,
Table 1 Effect of honokiol on cell cycle distribution and
apoptosis of RKO cells
Cell cycle distribution (%)
Groups Apoptosis (%)
G2/ M G0/ G1 S
5 µg/ mL
10 µg/ mL
15 µg/ mL
aP<0.05, bP<0.01 vs corresponding control group. Cell cycle
distribution was determined after 48 h of treatment in each
group. The tabulated percentages were an average calculated
on the results of three separate experiments. The results were
represented by mean±SD (n = 3).
Caspase -3 and -9 expression by Western blot
Since Caspases are the main factors in the apoptotic pathway,
we investigated whether Caspases were involved in inducing
apoptosis of RKO cells treated with honokiol. Cells induced for
48 h were analyzed for protein expression by Western blot. The
results showed that Caspase-3 and -9 were up-regulated in a
dose-dependent manner (Figure 5).
Effects of honokiol on primary cultured cells
As shown in Figure 6, honokiol had little cytocidal effect
on primary human fibroblast cells and human lymphocytes
even up to 40 µg/mL. HUVEC cells after honokiol treatment
resulted in a sharply dose-dependent cytotoxicity. These
Chen F et al. Honokiol for treatment of human colorectal carcinoma 3461
Cell viability (% control)
0 5 10 15 20 25 30 35 40
12 24 36 48 72
1 2 3 4
Figure 4 Apoptosis of RKO cells detected by FCM. (A) control; (B) 5 µg/ mL; (C) 10 µg/ mL; (D) 15 µg/ mL.
0 20 40 60 80 100
0 20 40 60 80 100
0 20 40 60 80 100
0 20 40 60 80 100
results demonstrated that human fibroblast cells and
lymphocytes were more resistant to the honokiol-mediated
cytotoxicity than HUVECs.
Figure 5 Western blot analysis for the expression of Caspase-
3 and -9 in human colorectal carcinoma cell line RKO cells. Lane
1: vehicle; lane 2: 5 µg/ mL; lane 3: 10 µg/ mL.
Figure 6 Cytocidal effect of honokiol on the growth of primary
cultured human umbilical vein endothelial cells, primary hu-
man fibroblast cells and human lymphocytes.
The pharmacokinetics of honokiol was evaluated after
intraperitoneal injection of 250 mg/kg to BALB/c mice. The
maximum plasma concentration of honokiol was observed at
27.179±6.252 min after administration (Figure 7). The plasma
disappearance curve could best be described by a first-order
absorption one-compartment model, with an absorption
half-life of 10.121±2.761 min, and an elimination half-life of
5.218±0.461 h (Figure 7).
Figure 7 Honokiol concentration in plasma of BALB/ c mice.
Inhibition of tumor growth in nude mice implanted with RKO
We studied the effect of honokiol on the growth of RKO tumor.
There was significant inhibition of tumor growth by honokiol
(Table 2). All the control mice developed peritoneal bulge and
died by d 12. However, no peritoneal bulge was observed in
80% of the honokiol-treated animals and the mice survived for
up to 30 d. These observations indicated the anti-tumor activity
of honokiol in vivo (Table 2).
Table 2 Effect of honokiol on ascites growth of tumor in Babl/
c nude mice
Group Ascites MST Dead Living Survival percentage
(d) (D) (L) (L/ (D+L)×100%)
Control 5 10.7
Honokiol 1 34.3
MST: Mean survival time. RKO cells were transplanted
intraperitoneally. Honokiol was administered 2 mg per mice
on d 0, 2, 4, 6, 8, and 10 after tumor transplantation. The mice
were monitored for peritoneal bulge and survived for up to 30
d. Control mice died by d 12 of RKO inoculation.
Inhibition of solid tumor growth in nude mice bearing RKO cells
From Figure 8, animals in control and vehicle groups showed a
progressive increase in tumor volume, with a growth rate of
1627.6% and 1408.2% respectively on d 28. While in treated
groups, tumor growth rate was increased to 968.9% in adriamycin
group and 709.9% in honokiol-treated group. There was a
significant difference between honokiol-treated group and its
control (treated with PEG400/dextrose) (P<0.05). Similar results
were found between adriamycin-treated group and its control
(P<0.05). These data further confirmed that honokiol had an
effective anticancer activity in vivo.
Figure 8 Effect of honokiol on the growth of xenografted RKO
in Balb/ c nude mice. The Y-axis represents tumor volume, X-
axis represents time (day) after RKO cell inoculation.
Prolongation of life-span in nude mice bearing RKO solid tumor
The lifespan of mice in honokiol-treated group (80 mg/kg) was
monitored and compared to the vehicle and adriamycin-treated
group (Table 3). The mean survival time was 50.9 d in honokiol-
treated group, with a significant prolongation compared to vehicle
group (29.7 d, P<0.05). The survival rate in honokiol-treated group
was 176.7%, much higher than that in vehicle group (P<0.01).
There was no significant difference between control and vehicle
groups and between adriamycin-treated and honokiol-treated
groups. The results demonstrated that honokiol had a similar
effect to adriamycin in prolongation of lifespan of tumor-bearing
Table 3 Effect of honokiol on MST and T/ C% in Balb/ c nude
mice bearing RKO-incubated tumor
Group Number of mice MST (d) T/ c (%)
MST, mean survival time. The survival rate (T/ C%) was cal-
culated according to the following equation: T/ C (%) = [average
survival period in the test group/ average survival period in
the control group]×100. aP<0.05; bP<0.01 vs vehicle or control.
3462 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol December 1, 2004 Volume 10 Number 23
1 2 3
d 7 d 14 d 21 d 28 d 35
Tumor volume (cm3)
5 10 20 40 80
Cell viability (% control)
0 5 10 15 20 25
Many anticancer drugs kill tumor cells by inducing apoptosis.
Previous studies suggest that growth inhibition by honokiol
resulted from the induction of apoptosis in several cell lines[7-11].
A further study reported that in human squamous lung cancer
cells, honokiol induced apoptosis by down-regulating Bcl-XL
and sequentially activating Caspase cascade. Our results
confirmed this honokiol-mediated apoptotic progression in
cultured RKO cells.
A variety of compounds with potent anticancer activity in
vitro could not be used in clinic, one probable reason was due
to their strong toxicities to normal cells[17,18]. Therefore, we first
investigated honokiol’s effects on the toxicity of primary cultured
human cells. Our data demonstrated that the IC50s were much
higher in human fibroblasts and lymphocytes than in RKO
cells, with the exception that HUVECs were more sensitive to
honokiol. The safe doses for fibroblasts and lymphocytes could
be up to 40 µg/mL (Figure 6), much greater than that for RKO
and other tumor cells (data not shown). The phenomenon that
primary cultured endothelial cells were more sensitive to
honokiol might be related to the finding that honokiol had
antiangiogenesis activity by inhibiting VEGF and its receptor 2
in vitro. In chemotherapy, antiangiogenesis is another
important target besides apoptosis induction in tumor cells.
Based on the fewer toxicity to normal fibroblasts and
lymphocytes, we thus proposed that honokiol could be a safe and
potent candidate of chemotherapy for colorectal cancer in vivo.
Another major obstacle was that many potent agents were
poorly absorbed and quickly cleared in vivo, such as curcumin.
Curcumin was difficult to absorb in the gastrointestinal tract
and, even when systemically administered, it was rapidly cleared
by hepatic metabolism.
We then established two in vivo models with Babl/c nude
mice bearing RKO cells, the ascitic tumor model and the solid
tumor model. In RKO ascitic tumor model, honokiol exhibited a
strong efficiency in prolonging the lifespan of ascitic tumor
bearing mice and was highly effective on inhibiting intraperitoneal
ascites in Balb/c nude mice. In our another model, honokiol
also exhibited a potent efficiency in inhibiting solid tumor
growth. In RKO incubated tumor bearing mice, honokiol at
80 mg/kg significantly inhibited the tumor growth and
prolonged the lifespan compared to the vehicle (P<0.05). The
antitumor efficiency of honokiol was similar to that of the
commonly used chemotherapy drug, adriamycin.
The results of the present study are encouraging because
honokiol has shown significant inhibition of tumor growth
in vitro and in vivo, as well as prolongation of lifespan in
tumor-bearing mice in vivo. Together with its safety to human,
honokiol might be a promising chemotherapy candidate in
treating colorectal carcinoma or other cancers in clinic.
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Chen F et al. Honokiol for treatment of human colorectal carcinoma 3463