2007 NF Conference (June 10-12, Utah)

 

Signal Therapy of NF (Neurofibromatosis) by Natural PAK1 Blockers

 

Demestre, M^a., Messerli, S^b., Shahhossini, M^a., Kluwe, L^a., Mautner, V^a. and Maruta, H^a*.

^aUKE, Hamburg, Germany, and ^bMBL, Woods Hole, USA

 

Dysfunction of NF1 gene coding a RAS GAP is the major cause of type 1 NF, whereas type 2
NF is caused primarily by dysfunction of NF2 gene product called merlin that inhibits directly
PAK1, a Rac/ CDC42-dependent Ser/Thr kinase (see Fig. 1). More than a decade ago, we
demonstrated that both NF1 and NF2 gene products block the oncogenic RAS signalling,
reversing RAS trans-formation. Several years ago, NF1-deficient tumors were shown to
require PAK1 for their growth in vivo, and more recently we found that NF2-deficient tumors
also require the same kinase for their growth in vitro.

 

Among the anti-PAK1 drugs/ingredients that we have identified or developed for the treat-
ment of NF1 and NF2, a natural ring peptide FK228 appears to be the most potent both in
vitro and in vivo.  The IC50 for NF tumor cells is around 5 pM in vitro, and causes the
complete regression of NF1-deficient MPNST xenograft in mice (see Fig. 3). Its direct target
is an HDAC (histone deacetylase), and activates a specific set of tumor suppressor genes such
as p21 (a CDK inhibitor) and RAP1a (a RAS antagonist), which eventually block both up-stream
and down-stream of PAK1 (see Fig. 2). Unfortunately, however, FK228 is still in
clinical trials (phase 2) for cancers, but not for NF, and it would take several more years for
this powerful drug to enter NF trials, and eventually give any benefit to NF patients.

 

So we have recently started developing anti-PAK1 ingredients from natural products which
are inexpensively available in the market, hoping to give the immediate benefit to NF patients.
The first natural anti-PAK1 ingredient(s) was found in an ethanol extract of Chinese
(Sichuan) pepper which selectively blocks PAK1 activation, but not another kinase called
AKT. The IC50 for MPNST cells is around 10 mg/ml, and this extract suppresses the growth
ofNF1-deficient cancer xenograft in mice by 50%. More recently we found in the market a
much more potent natural anti-NF therapeutic called “Bio 30”. The IC50 for NF tumor cells is
2-8 mg/ml (depending on cell lines), and this product suppresses completely the growth in
mice of both MPNST xenograft over 100 days (see Fig. 4) and NF2-deficient Schwannoma
xenograft over 30 days (see Fig. 5). It also blocks the metastasis of MPNST (see Fig. 6).
Bio 30 is a water-miscible CAPE-rich extract of NZ (New Zealand) propolis (see Table 1).

 

CAPE (caffeic acid phenethyl ester) is the first identified anti-cancer ingredient in propolis, an
extract from bee hives, and is so far the most potent natural derivative of CA (caffeic acid)
that  down-regulates the GTPase Rac, a direct activator of PAK1 (see Fig. 2).
However, CAPE alone has never been in clinical trials, because the poor bioavailability (due to its water-insolubility).
Propolis contains a plenty of lipids which solubilize CAPE and other anti-cancer
polyphenols such as galangin, chrysin and apigenin (see Table 1).
Interestingly, CAPE appears to work synergistically with these other polyphenols in Bio 30 to suppress the growth
of these NF tumors both in vitro and in vivo. Bio 30 is a very safe healthcare food supplement
and costs only a dollar for daily treatment of both NF and formidable cancers such as pancreatic cancers. 

 

This work was supported in part by funds from DFG (Deutsche Forschungsgemeinschaft) to
HM and NF CURE Japan to VM and SM. Bio 30 was kindly provided by Manuka Health.

 

 

 

 

Fig. 1.  Both NF1 and NF2 gene products are tumor suppressors that block the oncogenic
RAS signalling, by attenuating either the GTPase RAS or the kinase PAK1, respectively.

 

 

 

Fig. 2.  FK228, the most potent HDAC inhibitor, blocks both down-stream and up-stream of
the kinase PAK1, while CAPE (caffeic acid phenethyl ester) down-regulates the GTPase Rac,
a direct activator of PAK1.  Thus, both FK228 and CAPE suppress the PAK1-dependent
anchorage-independent growth, metastasis and angiogenesis of more than 70% of human
cancers and NF tumors which are highly addicted to PAK1 for their growth.

 

 

 

Fig. 3.  FK228 (2.5 mg/kg, i.p., twice a week) causes the complete regression of human
MPNST xenograft in mice (Hirokawa, Y. et al :  Cancer Biol. & Therapy, 4, 379- 381, 2005).

 

 

 

 

 

 

Fig. 4.  The human NF1-deficient tumor (MPNST)-bearing nude mice were treated
with Bio 30 (100 mg/kg) ,i.p., twice a week. Once the control MPNST started to grow rapidly around
day 70 with a support of sufficient blood vessel formation around this poorly angiogenic
 solid tumor, the therapeutic effect of Bio 30 became apparent. 

 

 

 

Fig. 5. The human NF2-deficient Schwannoma-bearing nude mice were treated with Bio 30
(100 mg/kg) ,i.p., twice a week (since day 12). This treatment caused an almost
complete regression of the Schwannoma.

 

 

Fig. 6.  The MPNST-bearing mice were treated with Bio 30 (100 mg/kg) alone or CAPE 60
(Bio 30 100 mg/kg plus extra CAPE 5 mg/kg), i.p., twice a week. Bio 30 alone caused the
complete regression of two (out of six) original tumors, while CAPE 60 caused the complete
regression of four (out of six) original tumors.  
Also these treatments delayed or reduced  the metastasis of MPNST.

 

 

 

 

 

Fig. 7.  Propolis from bee hives as a 100 million years’ wisdom of bee society protecting their
larva from a variety of diseases. Propolis has been used since the ancient Egypt  
as a traditional medicine for the treatment of infection and inflammation as well as preparing
mummies.  In modern era, propolis was first identified as an anti-cancer remedy in a
late 1980s when Dezider Grunberger’s group at Columbia University found that CAPE is
the major anti-cancer ingredient in a propolis sample from Israel (Grunberger, D. et al: Experientia, 44, 230-232, 1988).    

                        

Table 1.  The content of CAPE and anti-cancer polyphenols in “Bio 30” Liquid,
an extract of New Zealand (NZ) propolis which is among the richest natural source of CAPE available
inexpensively in the market (Nicola Celli at Negri Sud, Italy, unpublished data).
CAPE works synergistically with other anti-cancer gradients in this extract to suppress the growth of these
NF (both NF1 and NF2) tumor cells. CAPE comes mainly from young buds of poplar trees.

 

Notes:

 

Both Brazilian “green” and “red” propolis samples contain no CAPE, but are rich in another
anti-cancer ingredient, artepillin C (ARC) and di-terpene, respectively. There is an indirect
evidence suggesting that, like the Chinese (Sichuan) pepper extract, ARC also blocks the
PAK1 cascade somehow without affecting the kinase AKT. ARC inhibits the growth of
MPNST cells in vitro (IC50 : around 25 mM), and blocks the angiogenesis with the same IC50
(Ahn, M.R. et al : Cancer Lett. 2007, in press). Thus, it would be worth testing a potential synergy
between Bio 30 and ARC-rich propolis (such as Brazilian ”green”) in suppressing the
growth of NF tumors.

 

 

Materials & Methods

 

Tumor Xenografts in Mice

 

6 weeks old female nu/nu mice (Charles River) received 5 million MPNST cells (S-462)
or NF2-deficient Schwannoma cells (HEI-193) of human origin in 0.2 ml of 50%
Matrigel per mouse subcutaneously, and when the average size of tumors reached
around 5 mm in a diameter, groups of 5-7 mice were treated with either Bio 30
( 100 mg/kg) alone or CAPE 60 (Bio 30 plus extra 5 mg/kg CAPE) i.p., twice a week,
while the control mice were treated with the vehicle (11% PEG and 26% DMSO in PBS).
  The size of each tumor (both the original and metastasized) was measured twice a week by a calliper.
None of these treatments caused any adverse effect on mice.

 

 

*Corresponding author:  Hiroshi Maruta

E-mail: maruta19420@hotmail.com  or  maruta19420@mac.com