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[Cancer Biology & Therapy 5:3, 305-309, March 2006]; ©2006 Landes Bioscience
Sichuan Pepper Extracts Block the PAK1/Cyclin D1 Pathway
and the Growth of NF1-Deficient Cancer Xenograft in Mice
Research Paper
ABSTRACT
There is increasing evidence that more than 70% of cancers including pancreatic,
breast and prostate cancers as well as neurofibromatosis (NF) are highly addicted to
abnormal activation of the Ser/Thr kinase PAK1 for their growth. So far FK228 is the most
potent among the HDAC (histone deacetylase) inhibitors that block the activation of both
PAK1 and another kinase AKT, downstream of PI-3 kinase. However, FK228 is still in clinical
trials (phase 2) for a variety of cancers (but not for NF as yet), and not available for most
cancer/NF patients. Thus, we have been exploring an alternative which is already in the
market, and therefore immediately useful for the treatment of those desperate cancer/NF
patients. Here we provide the first evidence that extracts of Chinese/ Japanese peppercorns
(Zanthoxyli Fructus) from the plant Zanthoxylum piperitum called “Hua Jiao”/“Sansho”,
block selectively the key kinase PAK1, leading to the downregulation of cyclin D1. Unlike
FK228, these extracts do not inhibit AKT activation at the concentrations that block either
cancer growth or PAK1 activation. The Chinese pepper extract selectively inhibits the
growth of NF1-deficient malignant peripheral nerve sheath tumor (MPNST) cells, without
affecting the growth of normal fibroblasts, and suppresses the growth of NF1-deficient
human breast cancer (MDA-MB-231) xenograft in mice. Our data suggest that these
peppercorn extracts would be potentially useful for the treatment of PAK1-dependent NF
such as MPNST, in addition to a variety of PAK1-dependent cancers including breast cancers.
2University Hospital Eppendorf; Hamburg University; Hamburg, Germany
3Chemical Genetics Laboratory; RIKEN; Wako, Saitama, Japan
4Kitasato Institute; Minato-ku, Tokyo, Japan
*Correspondence to: Hiroshi Maruta; Ludwig Institute for Cancer Research;
Parkville/Melbourne, Australia 3050; Tel.: 613.9341.3155; Fax: 613.9341.3104;
Email: [email protected] or [email protected]
.D
Received 11/25/05; Accepted 12/12/05
INTRODUCTION
CE
Published previously as a Cancer Biology & Therapy E-publication:
http://www.landesbioscience.com/journals/cbt/abstract.php?id=2404
Neurofibromatosis (NF), types 1 and 2 (NF1 and NF2), are disorders predisposing
patients to tumors in their nerves, from which more than 1.5 million people on this planet
are suffering.1,2 So far there is no effective therapeutic available for the treatment of NF. It
is caused by loss-of -function mutations of either NF1 or NF2 tumor suppressor genes.1,2
NF1 gene product has been long known a RAS GAP (attenuator), and NF2 gene product
called Merlin has recently been identified an inhibitor of PAK1, the key Ser/Thr kinase
downstream of RAS.3 Thus, inactivation of either NF1 or NF2 causes abnormal activation
of PAK1, and indeed the malignant growth of both NF1 and NF2 tumors was proven to
require PAK1 as RAS transformation.3-5
Subsequently, at least two types of anti-PAK1 drugs have been found to suppress effectively neurofibroma xenografts in mice: (1) the most potent histone deacetylase (HDAC)
inhibitor FK2285,6 and (2) a unique combination of two Tyr-kinase inhibitors, PP1 and
GL-2003.7-10 However, none of these drugs has been available to NF patients as yet:
FK228 is still at phase 2 of clinical trials only for cancers (excluding NF), while the
PP1/GL-2003 combination has not entered even the phase 1 of clinical trials. In an
attempt to find an immediate benefit for NF patients, we have started exploring alternative
therapeutics available in the market, which would be potentially useful for the treatment
of NF patients. There are several HDAC inhibitors/downregulators in the market, including
the anti-epileptic drugs valproate (VPA), carbamazepine (CBZ), and curcumin (a yellow
spicy ingredient of Indian curry),11-13 which eventually block PAK1 activation. However,
their IC50 for HDAC is rather high (above 1 µM), and these anti-epileptic drugs cause a
few side effects such as rash, sleepiness, dizziness, and loss of short memory. Thus, a more
potent and safer (with less side effect) compound that blocks PAK1 activation would be
desirable for the effective signal therapy of NF.
Natural red/purple dyes (anthocyanins/anthocyanidins or other polyphenols) from a
variety of vegetables such as bilberry, grapes, and purple corn have been recently reported
to have an anti-cancer activity in cell culture or animal models.14,15 Some of anthocyanidins
(aglycons) have been found to block EGF receptor/ErbB1-dependent pathways.16
IEN
KEY WORDS
ON
1Ludwig Institute for Cancer Research; Parkville/Melbourne, Australia
OT
D
IST
RIB
UT
E
.
Yumiko Hirokawa1
Thao Nheu1
Kirsten Grimm2
Victor Mautner2
Satoko Maeda3
Minoru Yoshida3
Kanki Komiyama4
Hiroshi Maruta1,*
Pepper, PAK1, Cyclin D1, NF1, MPNST, Cancer
BIO
SC
ACKNOWLEDGEMENTS
©
20
06
LA
ND
ES
We are grateful to Drs. Hong Ji and Ryuya
Horiuchi for their generous supply of Chinese and
Japanese peppers, respectively, during an early stage
of this study, Dr. Lan Kluwe for her technical
supervision and critical reading of this manuscript,
and Prof. Tony Burgess for his continued support.
This work was supported in part by the Denise
Terrill Classic Award from Texas NF Foundation (to
HM) and by Deutsche Krebshilfe 70-2794 (to VM).
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Cancer Biology & Therapy
305
Anti-PAK1 Pepper Extracts Block the Cancer Growth In Vivo
However, due to the high IC50 of these compounds (above 25 µM),
it is unlikely that they are practical for the treatment of either cancers
or NF. Resveratrol (RSVL) or its metabolite (piceatannol),
di-hydroxyl derivative RSVL-2 or methoxy derivatives such as MR3
and MR4 block the Tyr-kinase Syk, upstream of PI-3 kinase, leading
to the inactivation of both PAK1 and another Ser/Thr kinase
AKT.17-19 Again the IC50 of the natural polyphenol RSVL for both
normal and cancer cell growth is far above 1 µM, although the synthetic
derivatives MR3 and MR4 are much more potent (IC50 around
100 nM), and their toxicity is more selective towards cancer cells,
with little effect on normal cell growth.18,19
The major spicy water-insoluble ingredient called “Sanshool” of
Chinese/Japanese peppers (Zanthoxyli Fructus) has been used for
more than two thousand years in East Asia as seasonings or traditional
medicines for stimulating digestion or removing intestinal
worms/parasites. In this brief report, we provide the first evidence
that the water/ethanol-soluble red/brownish extracts of these peppers
selectively block the PAK1/ cyclin D1 pathway, and suppress the
growth of MPNST cells in vitro and NF1-deficient (multidrugresistant) cancer xenograft in mice.
MATERIALS AND METHODS
Preparation of extracts from Chinese/Japanese peppercorns (Zanthoxyli
Fructus). The optimal conditions for the extraction of anti-PAK1 activity
from Chinese peppercorns called “Hua Jiao” in Chinese (or “Kasho” in
Japanese) are: 100 g of Zanthoxyli Fructus from Sichuan Province, China,
purchased at an oriental grocery shop in Melbourne (or Hamburg) were
soaked in 400 ml of 70% ethanol in water (v/v). The extraction was performed
at 25˚C by constantly rotating a 500 ml plastic container for several hours.
The peppercorns were removed by filtering through a very fine stainless steel
mesh for tea extraction, and the filtrate was then centrifuged at 2000 rpm
for 5 min to remove the remaining insoluble powders. This clear extract
(around 250 ml) is called “Kasui” (meaning a water-soluble extract of
“Kasho” in Japanese) as the anti-PAK1 ingredient(s) is soluble in a warm
(around 45˚C) water as well. This extract contains 28 mg of dry materials
per ml (measured after drying by a speed-vac at 50˚C). The optimal conditions for the extraction of anti-PAK1 activity from Japanese pepper called
“Sansho” in Japanese are: 5 g of the peppercorns (grinded powder) from
Wakayama Prefecture, Japan, purchased from Tsumura Juntendo in Tokyo
were suspended in 50 ml of 1% HCl in methanol (v/v), and the extraction
was performed at 45˚C in a water bath by occasional shaking for one hour.
The mixture was then centrifuged at 2000 rpm for 5 min to obtain the clear
supernatant (extract). This extract is called “Sansui” (meaning a water-soluble
extract of “Sansho” in Japanese), again as the anti-PAK1 ingredient(s) is
soluble in a warm (around 45˚C) water as well. This extract (around 40 ml)
contains around 25 mg of dry material per ml. The extract was then
lyophilized to remove both HCl and methanol, and reextracted with 10 ml
of 70% ethanol in water (v/v) at room temperature. The latter extract
contains 52 mg of dry materials per ml. Both extracts were stored for a week
in a refrigerator, and then kept frozen at minus 20˚C.
Cells lines. The human NF1-deficient breast cancer cell line MDAMB-231 was obtained from the ATCC. Another NF1-deficient cell line
S-462 was derived from a human malignant peripheral nerve sheath tumor
(MPNST) of an NF1 patient, and is closely related to the NF1-deficient
MPNST cell line S-805 described previously.5 In these MPNST cell lines
both NF1 alleles are inactivated.20 The normal fibroblast cell line
(FB1144,1) was derived from a healthy (non-NF) person who was operated
for maxillofacial surgery.
Analysis of anti-mitotic activity of pepper extracts in vitro. 103 cells of
MDA-MB-231 were seeded in a 96-well plate and incubated in 200 µl of
culture medium RPMI-1640 containing 10% fetal calf serum (FCS) in the
presence of a variety of concentrations of Chinese/ Japanese pepper extracts
306
Figure 1. Chinese pepper extract inhibits the growth of NF1-deficinet cancer
cells. MDA-MB-231 cells were cultured for eight days in the presence of the
indicated final concentrations of the extract as described under Materials
and Methods. The cell number in the control (no extract added), 9 × 104
cells/well, was taken as 100%.
for eight days, and cells were trypsinized and counted by a hemocytometer
periodically. Each point (in both time and concentration) was analyzed in
triplicates, and the average was calculated. The standard deviation was less
than 5%. 2 x 103 cells of either NF1-deficient MPNST cell line S-462 or the
normal fibroblasts were seeded in a 96-well plate and cultured in the DMED
(Gibco, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) and
2 mM Gln in the presence of a variety of concentrations of Chinese pepper
extract for three days. Their growth was measured using the Cell proliferation
kit II XTT (Roche, Basel, Switzerland), according to the manufacturer’s
instruction. Briefly, tetrazolium salt (XTT) was added to the culture medium,
where the viable cells reduce XTT through mitochondrial dehydrogenase to
a purple formazan product, which is measured spectrophotometrically at
450 nm.21 The OD450 obtained for the nontreated cells was taken as 100%
and all other values were normalized accordingly. Each point was repeated in
four wells, and the average was calculated.
Kinase assay and immunoblot of PAK1, AKT and cyclin D1 in cancer
cells. The MDA-MB-231 cells were treated with 70% ethanol extracts of
Chinese or Japanese pepper (the final concentrations, 20 and 100 µg/ml,
respectively) for 48 hours, and then cell lysates containing 1 mg of proteins
(measured by Bradford assay) were subjected to PAK1 kinase assay and
immuno-blotting with antibodies against PAK1, AKT, phospho AKT (Ser
473) and cyclin D1 as described previously.6,22
Xenograft experiments. BALB/c nu/nu mice (female, seven week old)
were purchased from Animal Resources Center (Western Australia).
MDA-MB-231 (5 x 106 cells per mouse) in 0.1 ml of a culture medium containing 50% Matrigel (BD Biosciences, Bedford, MA) were injected subcutaneously into two groups of eight nude mice. Twelve days later each group
was treated intra-peritoneally with the 70% ethanol extract of Chinese pepper
(110 mg/kg) or vehicle alone (0.4 ml of 5% DMSO in PBS) as the control,
twice a week for 2–3 weeks. Under these conditions no mice suffered from
any detectable adverse effect from these extracts. It should also be noted that,
in accordance with Australia’s strict regulation of animal experimentation, all
mice carrying solid tumors larger than 1 cm in diameter were immediately
terminated for the animal welfare. Palpable tumor diameters were measured
twice a week. Tumor volumes were calculated as:
(π/48) x [length (mm) + width (mm)].3
Statistical analysis. Data are expressed as means ± SD (standard deviation),
unless otherwise stated. Statistic analysis was performed mainly by means of
Student’s t-Test. Differences were considered significant at p < 0.05.
Cancer Biology & Therapy
2006; Vol. 5 Issue 3
Anti-PAK1 Pepper Extracts Block the Cancer Growth In Vivo
Figure 2. Cancer-specific anti-mitotic action of the Chinese pepper extract.
MPNST and normal fibroblasts were treated with the indicated final concentrations of the Chinese pepper extract for 72 hrs. The OD450 of the control
(no extract added) of MPNST and normal fibroblasts, 2.1 and 1.7,
respectively, were taken as 100%. Only the growth of MPNST (but not the
normal cells) was strongly inhibited by the extract 10-20 µg/ml) in a similar
manner to MDA-MB-231 (see Fig. 1).
RESULTS AND DISCUSSION
Zanthoxyli fructus extracts selectively inhibit the growth of
human NF1-deficient cancer cells. The Chinese pepper extract
strongly inhibits the growth of several human cancer cell lines
including the NF1-deficient breast cancer cell line MDA-MB-231
where normal RAS is abnormally activated, due to no expression of
NF1 gene encoding a RAS GAP , and the IC50 is around 10 µg/ml
(see Fig. 1).
To test further the selectivity of the Chinese pepper extract’s
effects, its anti-mitotic activity was compared between NF1-deficient
malignant peripheral nerve sheath tumor (MPNST) cells and normal
fibroblasts of human origin. As shown in Figure 2, the pepper extract
at 20 µg/ml has little effect on the growth of normal fibroblasts,
while it inhibits the growth of MPNST by around 90%. The results
clearly suggest that its effect is highly specific on the NF1-deficient
cancer cells in which both RAS and PAK1 are abnormally activated.5,10
At the concentrations higher than 320 µg/ml, the extract causes
the cell death (apoptosis), suggesting that one of its targets might be
the PI-3 kinase pathway, which is essential for cell survival.
Chinese/Japanese pepper extracts selectively block PAK1 activation and cyclin D1 upregulation in cancer cells. In order to
understand the molecular mechanism underlying the anti-mitotic
activity of these extracts, we examined their effects on both kinases
PAK1 and AKT which act downstream of PI-3 kinase. As shown in
(Fig. 3), both Chinese and Japanese pepper extracts around the IC50
for cell growth (0.02 mg/ml and 0.1 mg/ml, respectively) selectively
block the activation of PAK1, without affecting either its protein
level, AKT protein levels or activation of AKT (phospho AKT levels).
PAK1 is essential for RAS-induced upregulation of cyclin D1.22 As
predicted, both extracts strongly block cyclin D1 upregulation (see
Fig. 3), which is essential for the activation of cyclin-dependent
kinases (CDKs), leading to the G1 to S transition of cell cycle for the
initiation of DNA replication (see Fig. 4).
Chinese pepper extract suppresses the growth of NF1-deficient
cancer xenograft in mice. The human cancel cell line MDA-MB-231
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Figure 3. Chinese/Japanese pepper extracts block PAK1 activation and
cyclin D1 upregulation. The lysates from MDA-MB-231 cells, pretreated with
Chinese or Japanese pepper extracts (at 20 and 100 µg/ml, respectively)
for 48 hours, were subjected to the in vitro PAK1 kinase assay, and
immuno-blotting with antibodies against the indicated proteins as described
under Materials and Methods.
has mainly three unique properties. It does not express NF1, a RAS
GAP, and therefore its tumor can serve as a model of the type 1 neurofibromatosis in vivo. Furthermore, although it was derived from a
breast cancer, its growth is independent of estrogen, and it is resistant
to a variety of anti-cancer drugs including FK228.6 We have examined whether the Chinese pepper extract has any therapeutic effect
on this NF1-deficient experimental NF tumor in vivo. This extract
(110 mg/kg, i.p., twice a week) suppressed almost completely the
growth of this NF1-deficient tumor xenograft in mice at least during
the first two weeks treatment (see Fig. 5), without any detectable
adverse effect on the treated animals. However, thereafter the tumor
started to grow slowly even with this herbal “Kasui” extract treatment.
The result suggests at least two possibilities: (1) a “Kasui”-resistant
mutant population of this cell line began taking over the whole
tumor population, or (2) this extract upregulates a multi-drug resistant
(MDR) protein in the cells that would pump the anti-PAK1 ingredient
out. A similar result was obtained independently, by another group
with the warm water-soluble extract of Japanese pepper, testing on
tumor xenografts derived from MDA-MB-231 and a few other cancer
cell lines, but at apparently higher doses.23
These results suggest that the Chinese/Japanese pepper extracts
would be among the first effective NF therapeutics available in the
market. However, to handle the emerging drug-resistance problem,
we are currently examining whether another herbal product called
diindolylmethane (DIM), the major anti-cancer metabolite of indole
3 carbinol (I3C) derived from broccoli and other cruciferous vegetables, which blocks RAS-induced oncogenic/angiogenic signaling
pathway involving both AKT and PAK1 by upregulating the PIP3
phosphatase PTEN,24-27 or any other PAK1 blockers such as
GL-2003, can overcome such a “Kasui”-resistance of this tumor.
Interestingly, when used together GL-2003 and another Tyr-kinase
inhibitor called PP1, both of which eventually block RAS-induced
PAK1 activation, appear to overcome the resistance to each drug of
tumors derived from both this cell line and human pancreatic cancer
cell line.10 Also both purification and chemical identification of the
water/ethanol-soluble responsible (anti-PAK1) ingredient in the
Cancer Biology & Therapy
307
Anti-PAK1 Pepper Extracts Block the Cancer Growth In Vivo
Figure 4. The oncogenic PAK1 signal pathway that Chinese pepper extract
blocks. The insert (right, bottom) is a color picture of the matured Chinese
pepper fruits just before the harvest. The red extract blocks the activation of
PAK1 in the cancer cells, leading the downregulation of cyclin D1.
red-brown Chinese/Japanese pepper extracts are under way, in an
attempt to separate first the anti-PAK1 ingredient(s) from the rest in
this extract. So far no water-soluble natural compound has ever been
identified that selectively blocks PAK1 activation, Thus, it is likely
that the anti-mitotic component in these extracts would be a new
compound(s).
Regarding an orthotopic animal model of NF1 and NF2, very
recently Breakefield’s group is reportedly successful in demonstrating
the first effective therapeutic effect of herpes simplex virus (HSV) on
Schwannomas developed in NF2 transgenic mouse.28 Thus, we are
planning to collaborate with this group to test the therapeutic effect
of Chinese peppercorn extract, DIM, or their combination on this
NF2 mouse model. Although an NF1 transgenic mouse model was
also developed several years ago,29 so far nobody has published any
successful therapeutic effect on this mouse model, which is reportedly
good for leukemia, but not suitable for solid tumors such as neurofibromas and Schwannomas.
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