Gene Expression Program for fibrogenesis induced pericellular fibrosis in rat liver and suppressive observation of metastasis in human carcinomas

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Gene Expression Program for fibrogenesis

induced pericellular fibrosis in rat liver and

suppressive observation of metastasis in human carcinomas


Hide
yasu Hirano, Takeshi Hirano, Kazunori Harada, Tetsuo Hamada,

Naoki Kunugita, Masahiro Nakano, Keiichi Ar
ashidani

University of Occupational & Environmental Health, School of Medicine

1
-
1 Iseigaoka Yahatanishi Kitakyushu FO 807
-
8555 Japan


Abstract

Vitamin C(VC), Vitamin B
1
(VB
1
), Proline(Pro), Glyci
ne(Gly),
Cysteine
(Cys) and Alanine(Ala)
increased the
gene ex
pression of profilin, collagen and elastin during
fibrogenesis in alcoholic rats. Fuzzy Chaos Algorithm
made it possible to regulate histology by fuzzy ON/OFF
repeats of regulator molecules. Fibrosis by VC, VB
1
,
Ca2+ was reinforced by protein expression p
rogramming
by Pro, Gly and Cys. Protein expression programming was
performed to increase the efficiency of
translation

by
reducing translational idling on ribosome for supplement
of aminoacyl
_
tRNAs
,
by targeting specific protein in here
collagens. Gene exp
ression program for fibrogenesis
applied to envelope the metastasized cancer cells ran
effectively in human. A scirrhus stomach patient carrying
signet ring cells have been suppressed for 6 years. Used
SF (steamed Small Fishes) and elastin enrich Ala. Ala
increased pericellular fibrosis in alcoholic rat liver
equivalents our program image of envelopment
metastasized cancer cells.


1. Introduction


In 1996, we succeeded in inducing alcoholic fibrosi
s in the livers of rats provided with high doses of alc
ohol
(50_70%) in their drinking water (1). In that ex
periment, the animals received an alternating autoclave
d (atCE2) and non_autoclaved standard diet (CE2). T
he alternating feeding pattern was based on the results

of a survey of the eating and drinking habit
s of alco
holic patients. The atCE2 diet was used as a specific

pathogen_free (SPF) diet and the CE2 diet was us
ed as a non_SPF diet. Autoclaving the diet degrades

VB
1
, VC, retinol (VA), and VB
12
.


To investigate the mechanism(s) of experimental
fibroge
nesis

inductio
n (
1
)
,


we


performed

a database



Figure 1. Fuzzy Chaos Algori
th
m illustrated by weekly al
ternating diet regimens in which vitamin C (VC) was used t
o produce ON/OFF gene expression cycles. The serum le
vels of VC reflected oral VC intake
: VC in the diets affecte
d these levels and subsequent cycles became fuzzy. Repea
ting the alternating feeding regimens at weekly intervals re
sulted in gene expression cycles that mimicked chaos. Th
e one_day time course in serum VC levels after the admini
s
tration of VC 1 g/person was analyzed at 10 and 30 min, a
nd at 1, 3, 6, 12, and 24 hr after administration. The bod
y weight of the 5 subjects was 53 ± 7.7 (SD) kg. Prior ma
ximum informed consent was obtained from all participant
s. Time course analysis
of orally administered VC showe
d maximum serum VC levels at 3 hr; they returned to the o
rdinal level after 24 hr.


analysis and found that ascorbic acid (VC) induces collage
n synthesis

(
2_4
)
(the ON switch for collagen genes) and V
B1 produces ATP energy

(
5
)

(the ON switch for cell activat
ion). Furthermore, decreased VA reduces the expression
of RARE_carrying genes such as the fibronectin gene who
se product plays a role in cell migration

(
6,7
)
, that is, reduc
ed VA induces the fixation of tissues (organ) to
stop tissue
(organ) gelation (the OFF switch for cell migration) by pre
venting the migration of functional cells from the tissue (or
gan). VB12 induces mitosis of megaloblastic bone marro
w_, stromal osteoprogenitor_ and osteoblastic cells

(
8,9
)
, s
uggesting

that decreased VB12 might switch OFF the mito
sis of structural cells such as hepatocytes in our liver fibros
is.

The results of our database analysis led to our Fu
zzy Set Chaos
Algorithm

(FSCA) which posits that histolo
gy can be regulated by way of vitamin
s, minerals, and dail
y nutrients whose concentration (Fuzzy Set) in the blood re
flects eating patterns and the velocity of absorption of thes
e substances from digested food. We also predicted that p
rolonged observation of animals fed experimental diets wo
uld allow us to plot the levels of these substances on a rada
r graph, and that the resulting pattern might be recognized
as a Fuzzy Chaos pattern.. The pattern

used in human

sh
own in Fig. 1 suggested that ON/OFF gene expression reg
ulation could be achieve
d. The inside region indicates fu
zzy

OFF, and the outside region indicates fuzzy ON
in vivo
.


In the current study we applied our FSCA to the s
uppression of metastasized cancer cells. If we were succe
ssful in enveloping metastasized cancer cells with fib
roblas
ts by commanding them to surround these cancer cells, pro
duce collagens, and bundle collagen fibers with post_activ
ated elastin using calcium for fixation, we might be able to
induce necrosis or death of metastasized malignant tumor c
ells. We assume
d that the induction of necrosis or cell de
ath in metastasized cancer cells is regulated by the high or
low dependence on nutrients of metastasized cells and surr
ounding fibroblasts.

P
rofilin cDNA was obtained by Fuzzy Set Cloning
(FSC
)(
10, 11
)
; activated

profilin gene expression was
detected by the defuzzification process in CCl4_induced
liver cirrhosis in rats.

(
12
)

In the same manner,
extracellular matrix (ECM) gene expression levels were
analyzed during granulation of flexor tendons

(
13
)
. The
fibronec
tin and collagen genes were expressed after time
lag
s (
10_15
)
. We speculated that for the artificial
regulation of histological reconstruction, we could use our
fuzzy set ON/OFF switching to program gene expression
for each gene set.


1.1


Gene expression mole
cules and information
obtained by searching the Medline database


To regulate gene expression, we wanted to
identify non_toxic, long_term usable, non_antigenic
molecules. We found in the Medline database (Fig. 2) the
following vitamins as gene expression
inducer molecules:
VA activates fibronectin

(
16
)
, laminin B1

(
17
)
, and the
bone sialoprotein

(
18
)

gene by binding to the retinoic acid
responsive element (RARE). VC activates collagen

(
2_4
)

and profilin (
data showed in reference 10: dsi [10]
).
One of bi
nding factors of VC is the core binding factor 1
(Cbfa1).

(
19
)

VD activates ECM metalloprote
in
ase gene
expression by binding VD responsive elements

(
20
).

VB1
functions in ATP energy

production in the tricarboxylic
acid (TCA) cycle, acting as the co_enzyme

of oxidative
decarboxylation and

_ketoglutarate dehydrogenase which
catalyzes transketolase reactions.



Figure 2. Image flow chart for gene and protein expression

programming for weekly switching to reconstruct histolog
ical events. Commands are translated into gene o
r protei
n expression molecules after database analysis and the hist
ological reconstruction is planned by positioning the molec
ules on a time schedule to form a one_week cycle after con
sidering the post_expressed character of the protein.


1.2
Experimentally an
alyzed molecules for fibrogenesis
induced in alcoholic rats


We further analyzed the progression to fibrogenesis in
alcoholic rats. When groups of 3 rats received the CE2 di
et, no fibrosis was induced in the course of several cycles o
f alternating weekly
diets with VC (1 g/50 kgbw/d) or VB1

(25 mg/50 kgbw/d) or both VC and VB1 supplementation
. Similar observations were made with groups of rats that

received dietary supplements of VC, VB1 and CaCl2 (0.6
3 g/50 kgbw/d). These results using the CE2 diet for b
asic
feeding indicated that CE2 was too high in nutrients to ach
ieve our goal.

In the next set of experiments, we used the vitamin_deplete
d atCE2 diet as the basic diet. When either VC alone or b
oth VC and VB1 were added, no fibrosis induction occurre
d.
However, VC, VB1, and CaCl2 supplementation induc
ed fibrosis (stained blue) in areas of steatosis (faintly peric
ellular fibrosis was also noted (
dsi [10]
). In rats receiving

VC, VB1, and CaCl2 supplementation, there was slightly
less fibrosis than in rats

receiving the alternating diets repo
rted earlier

(
1
)
.

We created a target protein expression system that induc
ed fibrous histologic changes. We chose type I, III, and I
V collagens as the target translational induction proteins

(
1
2_15
)
. The specificity
of the amino acid content of these
3 collagens, deduced from the PIR databases, was Proline (
Pro) : Glycine (Gly) : Cysteine (Cys) = 1 : 1 : 0.2 (molar ra
tios). We decided to use L_proline (0.52 g/50 kgbw/d), gl
ycine (0.4 g/50 kgbw/d), and L_cysteine (0.2

g/50 kgbw/d)

to increase collagen translation on polysomes in the cytop
lasm, and succeeded in inducing clear liver fibrosis in alco
holic rats (
dsi [10]

The pathologic appearance was almost t
he same as that seen when the rats received alternating CE
2 and a
tCE2 diets

(
1
)
.



In our alcoholic rats drinking 70% ethanol (v/v),

we found that the profilin (
d s i [ 1
0]
) and type III collagen (Fig. 3A) mRNA levels were incr
eased after intravenous injection of VC (1 g/50 kg). The
animals were injected on the last day o
f atCE2 feeding (on
e week after completion of a VC_supplemented diet regim
en). The effect of injected VC on ECM gene expression (fi
bronectin, Type IV collagen,

_actin) was negligible (
dsi [
10]
) within 24 hr.

We hypothesize that following a chain reaction, the
affected organ is in a state of acute distress and that an
increase in profilin gene expression, efflux of profilin from
injured cells, extracellular profi
lin_induced signal
transduction of fibroblasts, expression of growth factor
(b_FGF)

(
21
)
, and excreted growth factor(s) stimulate
fibroblasts.



1.3
Utilization of biological sensors to recognize
metastasized cancer cells

To enable the fibroblasts to recognize

migrating tumor
cells, we used receptor ligand recognition systems that are
known as biological sensors. Tumor cells express many
cytokines and chemokines, e.g. IL_1, IL_6, IL_8, MIP1,
TNF

, and TGF


(
22_26
)
. As fibroblasts have many
kinds of receptors for these cytokines and chemokines, we
posited that it would

be easy for them to recognize
metastasized cancer cells (Fig. 5)

(
27_30
)
.

1.4
Receiving commands from blood vessels around
metastasiz
ed cancer cells

Bradykinin

(
31
)
, vascular permeability factor (VEGF/VPF)

(
32
)

and other permeability proteins that are enriched
around tumor cells, increase the efflux of acute_phase
molecules including small molecular weight substances
such as VC, VB1, VB
2, VB6, VB12, and Ca
2+

(Fig. 5).
Thus, in gene expression programming projects, it is easy
for surrounding fibroblasts to receive the flow of
commands from the blood vessels.

1.5
The rainbow strategy


Table I. Gene expression programming us
ing our rainbow
strategy.

(
33
)

VC, calcium (Ca), and small fishes (SF) en
riched with skin [type I], bone [type I, III], blood vessels [t
ype III], and lens capsule of the eye [type IV] were used as

the source for collagen fiber production (ON switch colla
g
en genes and collagen production). The major collagen
s contained are shown in brackets. SF were eaten on the
day calcium was provided (5g SF is equivalent to
26.5

mg
calcium). Ca was used for the activation of elastin, a calci
um_binding protein. VB1, VB
2, VB6, and nicotinamide w
ere for ATP energy production and VB12 was for the prolif
eration of fibroblasts. The death of enveloped metastasiz
ed cancer cells is programmed to be forced by the withhold
ing of nutrients. Pantothenic acid (PA) was used to prev
e
nt intestinal paralysis induced by the chronic administratio
n of VB1. Doses are in mg/50 kgbw/d.

' [
ENVMETCAN.BAS
]_ Envelope Metasta
sized

Cancer
Cells_

*Start


For Week=1 to 24

' _ _ _ _ Monday _ _ _ _


Activate Ca
2+

signal transduction system_
s
et prot
eins

(
Ca
2+
ST_SP)


If Found Profilin, Cytokines and Chemokines Then
React Fibroblasts To These Molecules

' _ _ _ _ Tuesday _ To prevent from intestinal paralysis
caused by VB1 _ _ _


Activ
ate

Peristalsis

' _ _ _ _ Wednesday _ _ _ _ Synthesize collagens _

_ _ _


Switch ON
Profilin gene set (Prof_gs)


Switch ON Co
llagen gene set (Col_gs) and Collagen
protein set (Col_SP)


Activ
ate

TCA_cycle

' _ _ _ _ from Thursday to Sunday _ _ _ Synthesize
collagens and fix them _


For day=Thu to Sun


Fully Switc
h ON Prof_gs


Fully Switch ON
Col_gs and Col_SP


Fully Activ
ate

TCA_cycle


Proceed Mitosis


Activ
ate

Elasti
n (
Ca
2+
B_SP),
Ca
2+
ST_SP

and Proceed
Fixation


Next day


Next Week

*End



The technique we used to induced alcoholic fibrosis in rats
wa
s applied to envelope metastasized cancer cells with
fibroblasts. If we could devise a way of enveloping
metastasized cancer cells with fibroblasts, then we could
possibly induce their death by denying them the nutrients
required for their proliferation (
dsi [10]
). The commands
for fibroblasts to envelope the target cancer cells were
relayed via blood in blood vessels. To be applicable in
humans, more precise and speedy expression programming
would have to be deployed. Therefore, we used our
alcoholic r
at model to develop an advanced program of
ON/OFF switching of the cycle in the course of a week.
In humans, the medication changed daily, therefore, we
called this the
r
ainbow
strategy

because the packages
containing the tablets were of 7 colors (red, or
ange, yellow,
green, blue, indigo, and violet); one color for each day of
the week (Table I).

1.6
Actual programming and command (molecules)
roles extracted from databases for enveloping
metastasized cancer cells by the GEP therapy

The program to envelop metas
tasized cancer cells by
inducing fibroblasts to migrate around metastasized cancer
cells involved the administration of VC (800 to 1000
mg/patient) and calcium
in calcium lactate

(1
30

m
g

equivalent to Ca
2+
) for 4 days, followed by 2 days without
VC tablets

(Table I). The withholding of VC served to
initiate VD, VA, and VE relative increases that
incompletely activate ECM_metalloprote
in
ase, and to
incompletely inhibit collagen synthesis. Thus, only the
dietary VD, VA, and VE were able to convey plasticity t
o
the envelope wall. We posit that the plasticity of the
envelope wall prevents the
sudden
escape of metastasized
cells through an occasional opening in the
collagen_fortified wall envelope of a tumor mass.
Simultaneously, VA begins to activate RARE_carry
ing
genes called the

FN_
gs
. Fibronectin directs post_mitotic
fibroblasts to the weak part of the wall. This is essential
because fibronectin binds DNA, actin, and integrins that
are introduced into the exudate from broken cells.


Collagen gene activation

was programmed on
Wednesdays and at the same time, the calcium_binding
domain of the elastin protein
set (
Ca
2+
B_SP)
was activated
by the calcium ion that plays a role in collagen fiber
bundling. ECM fixes the fibroblasts tightly. Collagen,
elastin, and c
alcium_enriched foods such as partially dried,
steamed small fishes

(SF: Pro:Gly:Cys:Ala = 14:17:4.8:21
mg/g vs sirloin : Pro:Gly:Cys:Ala = 7.4:8.1:2.2:11 mg/g)

were made available in the diet to provide similar
advantageous conditions for fibroblasts as d
oes the
collagen gene switched on by VC. Furthermore, by
administering VB1, VB2, VB6, and VB12, our program
commands surrounding fibroblasts to enter mitosis

under
introduced fibrogeneisis
.


By Monday of the second week, the envelope
inflexibility is reduc
ed and post_mitotic fibroblasts are
induced to migrate to un_enveloped regions or delicate
zones. This induces fibroblasts to envelop metastasized
cancer cells smoothly and tightly but with some flexibility.
The side effect of VB1 administration, paralysi
s of
intestinal contractions

(constipation)
, was prevented by
administering pantothenic acid.

1.7
Judging the effect of gene expression programming
on stopping the growth of metastasized cancer cells

The envelopment of metastasized cancer cells
was found in

he
ad CT_scan (dsi [10]).
In this patient, we first applied o
ur GEP_therapy for Env_Meta_Can (enveloping metastasi
zed cancer cells); the effect of the envelopment was clear i
n the brain. After tumor resection, the memory disturban
ce was cleared, however, sev
eral enveloped masses of met
astasized tumors (3_4 cm in diameter) were found in the ab
domen after one month. At this point, no small metastasi
s was found in the lung on x_ray films and on CT scans.
This indicated that new metastasis was suppressed but we

could not suppressed the growth of an already metastasized

cancer mass within blood vessels. GEP_therapy was then
stopped, and the patient received one month of radiation th
erapy followed by 2 months of chemotherapy, a 2nd course

of GEP_therapy, and immun
otherapy consisting of injecti
on with young adult lymphocytes. This patient, a 51_year
old woman died (
dsi [10]
).


Our 1st surviving patient, an 8
9
_year old man (
dsi [10]
)
suffered from pathologically diagnosed scirrhous stomach
cancer

with signet ring cel
ls
. Death within one year from
his malignant cancer was predicted by his attending
surgeon who, based on his clinical experience, expected
the development of metastasized tumors.


Another patient, an
90
_year old woman was
diagnosed with lung cancer (small
_cell carcinoma); she
had a dry cough and elevated tumor marker IAP and NSE.
Her body temperature, tumor marker SCC (squamous cell
carcinoma) and CRP (C_reactive protein: marker for
infectious disease) levels were normal.



2. Conclusions

We were able to
cure efficiently early and/or operable
cases with metastasis to fewer than
several

lymph nodes as
determined by macroscopic inspection. Metastasis to fewer
than 3 lymph nodes (not pathological) seems to be the
determinant of whether a patient can be cured
or not at
present. If we could induce the speedy envelopment of
cancer cells by fibroblasts, we would be able to save
patients with much more advanced cancers.


VA, VD, and VE_enriched foods have to be
reduced to minimum levels because we noted that the d
aily
intake of pumpkin or cheese reduced the programming
effect. An advantageous situation for fibroblasts was
created when VA, VD, and VE were decreased to below
the usual level, because the function of VD, VE, and VA is
activation of ECM metalloprote
in
a
se, inhibition of
collagen synthesis, and induction of cell migration,
respectively.


Chemotherapy and radiation treatment may
affect the commands for gene expression programming.
In our program, the possible effects of radiation and/or
chemotherapy were
not considered. In the future, it is
necessary to evaluate gene expression programming in the
presence of one or more of these therapies.


In non_operable cases, we were able to improve
the patients'
quality of
lives

(QOL)
. However, with time
their tumor

masses increased and organ damage led to their
death. Our enveloping technique could stop the
cough_related bleeding from lung metastasis.


The envelopment by fibroblast may not be able
to save the lives of patients suffering from leukemia or
benign tumo
rs, and it may not be useful in non_operable
cases or in patients with late_phase malignant tumors with
metastasized foci receiving nutrients via the blood vessels.
However, the treatment method described here seemed to
improve the quality of the remainin
g life of these patients.


Based on the experience documented here, we
suggest that metastasis of malignant tumor cells could be
suppressed by using this expression programming after
surgical removal of the original tumor mass and of
suspicious lymph nodes

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