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Can Plants’ Ability for DNA Repair and Stress Defense be Used against Patients’ Circulating Tumor Cells?

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Introduction: It was suggested that specific plants may reduce cancer's resistance to chemotherapy. Resistance inhibits apoptosis, as well as other fundamental anti-cancer protective mechanisms. Soy bean has been found to reduce cellular stress and repair DNA damage caused by drought or parasites, and can transfer this defense mechanism to other plant species as well. The aim of this study is therefore to conduct a systematic comparison of the effect of soy bean formulation (FSWW08) on gene expression in in vitro human breast cancer cell line, and in in vivo in blood circulating tumor cells (CTC), after oral consumption of FSWW08 by patients suffering from breast-, ovarian-, and prostate cancer. Method: In vitro gene expressions studies were conducted with the human breast cancer cell line BT-474 that was exposed to doxorubicin or FSWW08, either alone or in combination. Ovarian-, prostate-, and breast cancer patients received FSWW08 for 30 days. CTC were extracted from their blood according to an established protocol. Gene expression evaluations were conducted before and after treatment. Results: In vitro, the multi-drug resistance (MDR) protein was reduced by FSWW08, but was increased by doxorubicin. The combination of FSWW08 and doxorubicin, however, showed a protective effect against the increase of MDR in physiologic concentrations, increased, however, also in high experimental concentrations of both agents. The expression of several cancer-related protective genes, such as tumor suppressor factors p21, p38 and p53, was improved by FSWW08 in vitro and in vivo, which helped cell differentiation and new tissue formation. Additionally, the BAX/Bcl2 ratio was improved, in vitro, as well as gene expression of estrogen receptor beta, NF-κB, MAP kinase, c-JUN, and matrix metalloproteinase 9, together with an increase of VEGF expression in vivo in CTC.
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Journal of Pharmacy and Nutrition Sciences, 2015, 5, 000-000 1
ISSN: 2223-3806 / E-ISSN: 1927-5951/15 © 2015 Lifescience G lobal
Can Plants’ Ability for DNA Repair and Stress Defense be Used
against Patients’ Circulating Tumor Cells?
C.D. Volko and U.D. Rohr*
Endobal Medical Research Company, 1103 E. Tropicana Ave. #3006, Las Vegas, Nevada 89119, USA
Abstract: Introduction: It w as suggested that specific plants may reduce canc er's r esistance to c hemotherapy .
Resistance inhib its apo ptosis, as well as other fundamental an ti-cancer protective mechanisms. Soy bean has been
found to reduce cellular stress and re pair DNA dama ge caused by drought or parasit es, and can tr ansfer t his def ense
mechanism to other plant species as well. The aim of this study is therefore to conduct a systematic comparison of the
effect of s oy bean formulation (FSWW08) on gene expression i n in vitro human breast cancer cell line, and in in vivo in
blood circulating t umor ce lls (CTC), after oral consumptio n of FSWW08 by patients suffering from breast-, ovar ian-, and
prostate cancer.
Method: In vitro gene expressions studies were conducted wit h the human breast cancer c ell line BT-474 that was
exposed to doxorubicin or FSWW08, either alone or in combination. Ovarian-, prostate-, and breast cance r patients
received FSWW08 for 30 days. CTC were extracted from their blood according to an established protocol. Gene
expression evaluations were conducted before and after treatment.
Results: In vitro, the multi-drug resistance (MDR) protein was reduced by FSWW08, but was increased by doxorubicin.
The combination of F SWW08 and doxorubicin, however, showed a protective effect against the increase of MDR in
physiologic conce ntrations, increased, however, also in high experimental co ncentrat ions of bo th agents. The expr essio n
of several cancer-related protective genes, such as tumor suppressor factors p21, p38 and p 53, was improved by
FSWW08 in vitro and in vivo, which helped cell differentiation and new tissue formation. Additionally, the BAX/Bcl2 ratio
was improv ed, in vit ro, as well as gene expression of estrogen recept or beta, NF-B, MAP kinase, c-JUN, and matrix
metalloproteinase 9, together with an increase of VEGF expression in vivo in CTC.
Conclusion: It was demonstrated that FSWW08 improved the gene functions related to DNA re pair and s tress in hu man
blood CTC and resistance marker, in vitro, when applied in combination with doxorubicin. As such, larger in vitro and in
vivo clinical stu dies that investigate single botanical compounds from other pl ants, ar e warranted.
Keywords: Tp53, Tp21, Bax/Bcl2, MAP kinase, VEGF, CTC, circulating tumor cell, fermented soy, MDR protein,
estrogen receptor beta, NF-B, RT-PCR technique, human breast cancer cell line BT-474, ovarian cancer, breast
cancer, prostate cancer.
INTRODUCTION
During World War II, the US government developed
protective measures against cancer [1]. This served as
the model for a long series of similar agents (alkylating
agents) killing rapidly growing cancer cells by
damaging their DNA - the beginning of chemotherapy
[1].
According to experts in the field two problems have
to be solved to improve chemotherapy: Resistance and
side effects [2-6]. Several new approaches are now
being studied, including new drugs, new combinations
of drugs, new delivery techniques, novel approaches
that target drugs more specifically at the cancer cells,
first, drugs to reduce side effects and second, agents
that overcome multi-drug resistance [2-6]. Several
reviewers suggested that plant based formulations may
inhibit resistance against cancer therapy, including
Isoflavones, curcuma, vitamin A, and some extracted
ingredients from Chinese herbs [4].
*Address correspondenc e to this author at the 1101 E. Tropicana Ave. #3006 ,
Las Vegas, Nevada 89119, USA; Tel: 1 202 321 6621;
E-mail: uwerohr@gmail.com
A new concept suggests that cancer develops from
a small group of cells similar to stem cells, which
means that they are not fully differentiated, show
impaired hormone receptor expression (Figure 1) and
are both the origin of cancer (Figure 2) and responsible
for metastasis through migration (Figure 3) [6]. The
cancer stem cell theory has profound implications for
cancer chemotherapy because it explains why it is
effective in more differentiated cells but ineffective in
cancer stem cells [6]. The spread of cancer is
facilitated via migration of the blood (circulating tumor
cells or CTC) or the lymph system to other organs
(Figure 3). It was suggested that plant based natural
agents may be particularly effective managing CTC,
which was shown in vitro [7]. Several chemotherapeutic
agents, which do stop fast growing tumors, have the
opposite effect on cancer stem cells, causing them to
divide too rapidly [8], as well as increasing stress
related oxygen species (ROS), causing resistance to
the therapy, whereas some plant ingredients are able
to reduce ROS and reduce stress resistance to cancer
therapy [9].
Scientists explain human cancers which sustained
proliferation, escape from apoptosis, and genomic
2 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
instability by mutations of the TP53, ATM (ataxia-
telangiectasia mutated (ATM) gene, and MDM2
(Mouse double minute 2 homolog) genes [10]. It is
defined that DNA replication stress generates genomic
instability and causes suppressed apoptosis [10-21].
Figure 1: Hormone receptor distribution in stem cells, cancer
stem cells, and normal fully differentiated Schwann cells,
taken from [28].
Recently, researchers have suggested that dietary
compounds such as curcumin, sulforaphana, soy
Isoflavones, epigallocatechin-3-gallate, resveratrol,
lycopene, piperine and vitamin D3 can directly or
indirectly affect cancer stem cell self-renewal pathways
[9], as well as reduce unfavorable cellular stress
caused by chemotherapy and making chemotherapy
more effective [23, 24]. Plants have evolved to live in
environments where they are often exposed to different
stress factors in combination and are subject to high
levels of DNA damage resulting from the plant’s
obligatory dependence on environmental stresses like
solar UV radiation, attacks of yeasts to digest plants,
drought, chilling injury, and other air and soil pollutants
including heavy metals and metabolic by-products from
endogenous processes [24]. Therefore, to survive
under frequent and extreme environmental stress
conditions, plant cells have evolved with highly efcient
and wide-ranging mechanisms for the detection and
repair of DNA damage to eliminate the chances of
permanent genetic alterations and to maintain genome
stability for faithful transfer of genetic information over
generations [26-28].
Classically plant based anticancer medicine is toxic,
often inhibits mitosis, follows the first generation
anticancer drugs made from mustard gas and has
severe side effects. The here outlined mechanism of
DNA repair differs completely from this strategy: it is a
non-toxic mechanism, since plants under
environmental stress attack are already severely
immunologically compromised and need no further
reduction of defense mechanisms [25] The DNA repair
under stress is a mechanism to strengthen impaired
plants. Yeasts and plants share genes for cell repair
and DNA protection with mammals like MAPkinase and
heat shock protein (Figure 4) [25]. MAPkinase
cascades are crucial in eukaryotes for transducing the
perception of environmental stimuli into internal
signaling pathways [30]. Plants have a particularly
Figure 2: Schematic depiction of invasive breast cancer. Primary breast cancer consists of two types of cancer cells. Cancer
stem cells have only low estrogen receptor expression, whereas primary cancer consists of cancer cells expressing 62%
estrogen receptor. Chemo-, radio-, or adjuvant therapy cannot reduce cancer stem cells, because they have virtually no
estrogen receptor expression. Therefore, cancer recurrence is difficult to predict. (See ref [28]).
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 3
large number of MAPkinase components, allowing for
the control over a wide range of stress response
pathways [31]. Interestingly, in Arabidopsis, a unique
plant-specic transcription factor Suppressor of
Gamma Response 1 (SOG1) acts as the main
regulator in DNA damage repair and performs
analogous functions to mammalian tp53, which is
involved in a majority of the plant’s response to DNA
damage, such as transcriptional response, activation of
cell cycle checkpoint and programmed death of stem
cells [32]. It is a tremendously important finding that in
plants a factor similar to tumor suppressor factor tp53
exists, which is a hallmark gene factor for cell
differentiation in mammals, since in more than 50% of
all tumors tp53 is missing [33]. Transcription analyses
in Arabidopsis plants and many others have revealed
that a fairly high proportion of the genes are associated
with DNA damage repair [34, 35].
Transcription factors are controlled by plant
hormones and small proteins (Figure 4): The
expression of soybean miRNAs was investigated in
response to water decit and infection with soybean
rust fungus, where all miRNAs were differentially
regulated by each stress, usually in an opposite
direction [36]. It has been shown that soybeans are
able to affect stress response in other species too, like
tomato plants as well as tobacco plants [37]. There are
numerous studies showing that soy can alter the
genome in human cancer cells [38]. This paper
therefore systematically investigates the effect of
fermented soy bean formulation on cancer cells in vitro,
Figure 3: Tumor spread is facilitated via cancer stem cells, which have very low estrogen receptor expression and might not
respond to chemo-, radio-, or adjuvant therapy.
Figure 4: Key events in the signal transduction pathway
activated in response to combined biotic and abiotic stresses
(taken from ref. [17]).
4 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
as was shown by others also, in vivo in CTC extracted
from blood from cancer patients, which has not been
shown before. We detected and reported earlier that
the effectiveness of chemotherapy was increased by
fermented soy formulation, side effects were reduced,
and survival increased [39, 40]. This study investigates
in vitro whether a combination of the classic cytotoxic
compound doxorubicin may be combined with
fermented soy bean and counterbalance some of the
side effects of classical chemotherapy on the genetic
level. For the first time ever stress DNA repair genes
are investigated in blood circulating tumor cells (CTC)
in vivo altered by a processed plant.
MATERIALS AND METHODS
a. IRB
The study protocol was reviewed by a local
institutional review board (IRB) to protect the rights of
patients, safety and well-being of humans involved in a
clinical trial by reviewing all aspects of the trial and
approving its startup and was published previously [24,
39, 40]. All patients had to sign an Informed Consent
Form after the physician explained the study and he
read the informed consent form where the study goal
was outlined in a written form, in which the study was
outlined and participation was voluntarily and it was
explained individually to all patients that they could
leave the study at any time even without explanation.
No patient received a financial compensation for
participating in the study, besides received the
medication for free. The study fulfilled the Convention
of Helsinki in its current form. The data were stored at
the Hospital and the data were processed anonymous
to protect the identity of the patient.
b. In Vitro Gene Expression Investigation
All in vitro procedures determining the gene
expression determination have been described before
[24, 39, 40, 44], however a more thorough investigation
and discussion about in vi tro and in vivo comparison
and DNA repair is missing. Investigations of the human
breast cancer cell line BT-474 were conducted, which
were purchased from the German National Resource
Centre for Biological Material (DSMZ, Leibniz Institute
DSMZ-German Collection of Microorganisms and Cell
Cultures, Inhoffenstraße 7B, 38124 Braunschweig,
GERMANY, EU). It is a human breast tumor-cell line
and was established from a solid invasive ductal
carcinoma of the breast (DSMZ No.: 64.
WWW.DSMZ.de).
Doxorubicin hydrochloride was solved as 10 mg in 5
ml volume aqueous solution and purchased from
HEXAL AG, 83607 Holzkirchen, Germany, EU.
FSWW08 (Haelan 951, Organic NON-GMO Soy), a
fermented Soy Bean Beverage made in China, is
distributed by Haelan Products INC., 18568 142nd Ave.
N.E. Bldg.F. Woodinville, WA 98072 U.S.A.,
www.haelan951.com. FSWW08 was given as a
donation. FSWW08 contains 250 mg soy Isoflavones
per 235ml.
Culture Medium
A RPMI Medium with FBS was employed according
to the recommended condition of DSMZ Cell Culture
Data. For every test 1x105 BT-474 tumor cells were
used. Growth was induced on a 24 Well-plate
(CELLSTAR) purchased by Greiner bio-one. After
adding the drug substances, the cells were incubated
over a period of 72 h at a temperature of 37° Celsius in
an incubator.
Processing of Harvested Cells
Cells were processed according to recommended
procedures by Quiagen with a QIAmp RNA Blood Mini
Kit to obtain total-RNA. This was obtained by
cryopreservation and Freeze-Drying Protocols of the
cell pellet with Quiagen RLT-buffer. Further isolation of
RNA was performed by QIA shredder column and QIA
spin column. The RNA containing elute was evaluated
by reverse transcription. The reaction was conducted
with the help of random hexamers and M-MLV
transcriptase, so that RNA was transformed to cDNA.
Gene-Expression Determination
Quantitative Real-Time RT-PCR was employed to
measure mRNA gene expression. Gene expression
measurements were conducted with Sequence
Detector 7700 by Applied Biosystems (ABI) Life
Technologies, 3175 Staley Rd, Grand Island, NY
14072, USA. A 5´-Nuclease Assay was employed,
which was developed by ABI. The method is based on
measurement of quantitative determination of cDNA as
an equivalent amount of mRNA. For quantitative
purposes every gene expression determination was
done with a control cell line, which contained mRNA as
a standard. The dilution of the control cell line was
done in ratio of 1:10.
The measurement was done in a relation as a
fraction to an amount of cDNA 2x106. The value
obtained in the Real-Time RT-PCR is the cell
equivalent (CEQ). All measurements were done in
triplicate.
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 5
The measured gene expression determination was
related to GAPDH and normalized to allow comparison
among each other. As an additional method all
obtained values were normalized to 1 as an untreated
tumor, so that a relative gene expression is expressed
in all figures.
Dosing of Test Substances
Doxorubicin is used in cancer mono therapy usually
in a dose of 50-80mg/m body surface. The average
body surface of an adult is usually 2 m. The injection
solution is composed of 2μg Doxorubicin/μl. The
amount of blood of an adult person is 5-7 Liter on the
average. The dose of FSWW08 is one bottle containing
236 ml per day, half a dose 118 ml twice a day.
Substances were tested alone and in combination and
compared with an untreated cell line. Gene expression
of parameters (BCL2, BAX, TopoII, MDR1, ERBB2 and
others, Table 2) in different concentrations were
measured. The questions to answer are:
a) What are the effects of Doxorubicin monotherapy
on the expression of genes related to the
mamma carcinoma cell line BT-474?
b) What are the effects of the combination of
Doxorubicin and FSWW08 on the gene
expression in the cell line BT-474?
c) Is it possible to obtain similar results when
combining Doxorubicin with FSWW08 as in
using Doxorubicin alone on BT-474?
d) In vivo gene expression of tumor cells circulating
in blood extracted from patients
Patients' Characteristics
A pilot study was conducted with 5 ovarian cancer
patients, 5 prostate cancer patients, and 7 breast
cancer patients (pT1 to pT4, pN0 to PN2, Mo).
Blood Sampling
Two samples of 5 ml ethylenediamine tetraacetic
acid blood were collected for isolation of CTCs before
the application of therapeutic substances with an S-
Monovette® (Sarstedt AG & Co, Nürmbrecht,
Germany) and were stored at 4°C until further
examination. The samples were processed
immediately or not later than 4 hours after blood
withdrawal. An additional serum sample was collected
to determine serum tumor markers.
Extracting Cancer Tumor Cells from Blood
The methods are described in [40].
Method of Determination of Gene Expression
The method is described above as in vitro and was
used with the same laboratory protocols and
equipment. The lower detection limit of this assay is
based on spiking experiments two cells per 5 ml [41].
Examination of blood samples of healthy donors (n =
20) yielded a specificity of 95%, which corroborates
values in the literature [10]. We detected in all ovarian
(100%), all prostate cancer (100%) as well as in 5 from
7 women suffering from breast cancer CTC (71%),
which is higher than reported in the literature [41].
c. Gene Expression in CTC in Cancer Patients
Extracted from Blood
We relate gene expression results to two clinical
trials, where we investigated a) in a pilot trial over a 4
year period survival [40] and well-being of prostate-,
breast- and ovarian cancer, and b) in a multicenter
placebo controlled trial in 268 patients [39]. We
compared FSWW08 with a casein solution. Besides
cachexia, white blood counts and other blood
parameters were determined. Patient characteristics
can be found in the literature [39, 40].
Statistical Evaluation
A t-test compares the means of two groups. For
example, compare whether relative gene expression
differs between a control and treated group. The t test
compares one variable between two groups. The t-
tests (and related nonparametric tests) compare
exactly two groups. Software was used of the 2014
version of GraphPad Software, Inc., 7825 Fay Avenue,
Suite 230, La Jolla, CA 92037 USA,
RESULTS
The effects of Doxorubicin and FSWW08 on gene
expression on GAPDH of BT-474 cells were
investigated separately and compared to untreated BT-
474 cells (Table 1) (Figure 5). Both Doxorubicin and
FSWW08 resulted in reduction of the cell morphology
and gene expression of BT-474 (Table 1, Figure 5).
Doxorubicin: The expression of GAPDH was
decreased by 50% with low DOX concentration and by
more than 90% at higher DOX concentrations. This
implies a significant reduction of the number of
surviving tumor cells (Table 1, Figure 5). FSWW08:
FSWW08 resulted in a 5-fold decrease of GAPDH
gene expression compared to untreated tumor cells
and caused up to a 10-fold decrease. This means that
FSWW08 had a significant effect on the number of
surviving tumor cells. Both Doxorubicin and FSWW08
have to be considered cytotoxic (Table 1, Figure 5).
6 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
Table 1: Summary of in vitro and in vivo gene expression changes by FSWW08. In vitro investigation were conducted
with normalized numbers of cells from cell line BT-474, purchased from the German National Resource
Centre for Biological Material (DSMZ, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell
Cultures, Inhoffenstraße 7B, 38124 Braunschweig, G
GENE Function Cell Line
BT-474
CTC from
breast
tumor
CTC from
Prostate
Tumor
CTC form
ovarian
tumor
GADPH GAPDH is a house-keeping gene. Gen eexpression is equal
in tumor cells and in healthy cells. It is a gene for
normalisation and quantification because it is expressed only
in living c ells. A reduction of GADPH means that cells have
died [42]
-80%
(± 4)
-18%
(± 3)
-22%
(± 4)
-19%
(±4)
BAX BAX is a pro-apoptotic mitochondrial membrane protein. It
inhibits Bcl2 an d accelerates the programmed cell d eath
(apoptosis). Re duced expressi on of BAX in re lation to Bcl2
correlates with non-response to 5-flu orouracil, epirubicin and
cyclophosphamide [47]
Up to 4 fold No change 8 to 60 fold
increase
No
change
Bcl2 Bcl2 is coding for an anti-apoptotic mitochondrial membrane
protein. Bc l 2 is overexpressed in many tumors and
consequently causes resistance to apoptosis-inducing drugs
(e.g. intercalat ing agents, alkylating agents, plati num
compounds.
No change at
low
concentration
No change No change No
change
ERBB2 Erbb2 (also HER2/neu) is a tyrosine kinase involved in cell
proliferation and differentiation. Tumors overexpressing Erb b2
can be treated with Herceptin (an Erbb2-anti body).
No change
In low
concentration
No change No change No
change
MDR1 Multidrug r esistance 1 (MDR 1) is a glycoprotein cap able to
transport anticancer drugs out of the cells. Tumor cells
overexpressing MDR1 are resistant to multi ple dru g types like
vinca-alkaloides, anthracyclines, taxanes or mitomycin C.
-44 % - 25%
(±6%)
- 31%
(±6%)
-33 %
(±5%)
Tp21 The cyclin- dependent kinase inhi bitor p21 (als o known as
p21WAF1/Cip1) promotes cell cycle arrest in response to many
stimuli. It is well positioned to function as both a sensor and
an effector of multiple anti-pr oliferative si gnals.
8 fold
increase
6 fold
increase
22 fold
increase
6 fold
increase
tp53 tp53 is a tumor suppressor gene and regulates the cell cycle.
Mutations in p53 are the most frequent genetic alterations in
human mali gnancies. Genetic alterations in p53 (mutati ons,
LOH, expression) are therefore used as molecular tumor
markers.
2,4 fold
increase
18 fold
increase
16 fold
increase
150 fold
[57]
increase
c-jun c-Jun is an immunity mark er and is involved in ma ny allerg ic
and inflammatory reactions
-55%
(±16%)
-54%
(±22%)
-62%
(±36%)
-22%
(±12%)
Estrogen
receptor beta
Estrogen receptor beta is related to lower prostate c ancer
however its meaning in breast and in ovarian cancer is
debated
Incr. 80%
(±41%)
80 fold
increase
82 fold
increase
72 fold
increase
Estrogen
receptor alpha
The classical estrogen receptor relat ed to breast c ancer
survival, against tamoxife n protects.
No change No change No change No
change
Topo IIa Topoisomerase II alpha is catalyzing controlled cu ts and
reconnection of DNA-double strands during DNA- replication.
Inhibitors of Topoisomerase II (anthrazyclines, mitoxantron,
etoposid) are provoking faulty action of Top o II, leaving
behind DNA-damages. If Topo II is under expressed in tu mor
cells, lesser DNA-damages occur and the therapy may fail.
On the ot her han d, over expression of Topo II sensitizes the
cells to Topo II inhibitors.
-34% (±6%)
ND ND ND
VEGF Vascular endothe lial gr owth factor (VEGF) is up-regulated in
many tumors and is inducing angiogenesis by paracrine
stimulati on of endothelial cells. High expression lev els of
VEGF in tumor cells are associated with poor response to
hormone therapy with t amoxif en and c ombination
chemotherapy with FAC or CMF. New anticancer agents like
bevacizumab (Avastin) are targeted s pecifically against
VEGF.
Not
determined
± 0 % 37 fold
increase
7,6 fold
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 7
Figure 5: Effects of Doxorubicin and fermented soy (FSWW08) on the GADPH expression of cell line BT-474. GADPH gene
expression of untreated cells expressed as 1.
Figure 6: The role of c-myc, Bax and Bcl2 in apoptosis. c-myc: a regulator gene that codes for a transcription factor, a mutated
version of which is found in many cancers. NF-kB: a protein complex that controls the transcription of DNA, important in stress,
immunity and cancers.
Change in the gene expression of anti-apoptotic
BCL2 (Figure 6): Doxorubicin: The effect of
Doxorubicin on the expression of BCL2 was very
strong. With increasing concentration of DOX the BCL2
expression values dwindled down to less than 10% of
what was found in the untreated BT-474 tumor cells
8 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
(only reported). FSWW08: FSWW08 had at low
concentrations no effect on regulation of the anti-
apoptotic BCL2 expression values (Table 1).
BAX gene expression (Table 1, Figure 7).
Doxorubicin had a low effect on the pro-apoptotic BAX
expression (not shown here, only reported). Application
of FSWW08 on the cell line BT-474 led to an increase
of pro-apoptotic (Figure 6) BAX expression, and the
increase of BAX expression was even higher: almost
the 4-fold number was achieved compared to untreated
tumor cells.
TOPO IIa gene expression with increasing
concentration of agents: Doxorubicin led to a 1.8-fold
increase of TOPO IIa gene expression, gene
expression diminished again at high concentrations
(Table 1). TOPO IIa gene expression displayed only
the 0.6-fold value of untreated BT-474 cells. FSWW08
does not cause increase of TOPO IIa. On the contrary,
it causes an up to 0.3-fold decrease (Table 1).
MDR1 (multidrug resistance) gene expression
(Table 1, Figure 8), a marker of tumor resistance. An
increase of Doxorubicin led to significant increase of
the expression of the MDR1 gene, up to 17-fold
increase in the gene expression of MDR1. The
expression in the low concentrations of FSWW08 alone
decreased by 40%, comparable to untreated tumor
cells.
Figure 7: The effect of Doxorubicin (D) and FSWW08 on BAX gene expression in breast tumor cell line BT474. BAX gene
expression of untreated cells expressed as 1.
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 9
Figure 8: The effect of doxorubicin and fermented soy on
MDR1 gene expression (multi-drug resistance) in breast
tumor cell line BT474. MDR1 is a cell membrane protein that
pumps substances out of cells. MDR1 gene expression of
untreated cells expressed as 1. Upper figure doxorubicin is
added. Lower Figure doxorubicin is added and FSWW08 in
increasing concentrations, denoted at the x-axis.
Combination of Doxorubicin and FSWW08
With the low concentration of 10 DOX, an
increasing concentration of FSWW08 also caused an
up regulation of the expression of MDR1. With 10
DOX/60 FSWW08 it was possible to achieve an
expression corresponding to a 60 FSWW08 mono
therapy. A high concentration of Doxorubicin 100 DOX
combined with 10 HAL had an effect comparable to
Doxorubicin mono treatment. Increasing the amount of
FSWW08 (10, 30, 60 FSWW08) caused a decrease in
the expression of MDR1. 60 FSWW08 had the effect of
a 3-4-fold increase while the available amount of 100
DOX would have caused a 17-fold increase in mono
therapy. Apparently the effect of one of the two agents
on the MDR1 gene prevailed. If Doxorubicin and
FSW008 were given in average concentrations, no
increase in MDR1 gene expression was achieved. This
means that at low physiologic concentrations
fermented soy bean (FSWW08) eliminates drug
resistance caused by doxorubicin.
ERBB2 Gene Expression
Doxorubicin alone caused a continuously
decreasing ERBB2 expression (not shown here, only
reported). While at low concentrations for DOX is still
more than 0.7 times of untreated BT-474, higher DOX
concentrations already down regulates this receptor
tyrosine kinase by 10 times (Table 1).
Estrogen Receptors Alpha and Beta Gene
Expression
Estrogen receptor alpha is the classical Estrogen
receptor alpha to describe breast cancer risk. As can
be seen in Table 1, FSWW08 did not change Estrogen
receptor alpha gene expression, but did statistically
significantly increase Estrogen receptor beta gene
expression (Figure 9).
Figure 9: Circulating Tumor Cells (CTCs) of prostate cancer
patients were analyzed for expression of ER-alpha (Estrogen
receptor-alpha) and ER-beta, n = 6 mean and SEM. Asterisk
denotes differences reach statistically significant different on
a 5% probability level.
Gene expression studies with from blood extracted
CTC from cancer patients (Table 1). Table 1 compares
several gene expression investigations obtained by oral
consumption of fermented soy bean FSWW08 after 3
month consumptions with those seen in vitro cell
culture. Estrogen receptor alpha and beta (Figures
9,10): In agreement with in v itro experiments, Estrogen
receptor alpha was not increased in CTC of prostate,
ovarian, and breast cancer, however Estrogen receptor
beta (Table 1).
BAX / Bcl2 Gene Expression
The BAX / Bcl2 ratio is statistically significantly
raised in CTC of prostate cancer patients, however not
in ovarian and breast cancer in vivo (Table 1, Figures
10 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
11-13), suggesting that FSWW08 increases a pro-
apoptotic state of CTC in prostate cancer. BAX was
increased in vitro in breast cancer cells, suggesting a
pro-apoptotic effect as well (Table 1).
Figure 10: Circulating Tumor Cells (CTCs) of ovarian cancer
patients were analyzed for expression of ER-alpha (Estrogen
Receptor-alpha) and ER-beta n=5 (mean and SEM). Asterisk
denotes differences reach statistically significant different on
a 5% probability level.
Figure 11: CTCs (circulating tumor cells) in prostate cancer
patients showed an increased gene expression ratio of
apoptosis-triggering genes Bax/Bcl2 ratio after FSWW08
(fermented soy) therapy.
GAPDH gene expression: All three different CTC
from breast, prostate, and ovarian cancer patients are
decreased (Table 1), although somewhat smaller than
in vitro.
Tp53 Gene Expression
tp53 protein function is considered a gate keeper
and hall mark for cell repair and controls many
important markers in cancer, as well as cell cycle arrest
(Figure 14) and apoptosis. Mutation of tp53 and loss of
gene expression have been detected in many cancers.
It was found that gene expression of tp53 in CTC was
increased in vitro a well as in vivo (Table 1, Figure 15).
The increase in CTC was rather pronounced,
suggesting that in the in vivo case, either additional
mechanisms occur in CTC or fermented soy may
cause additional effects. As is depicted in Figure 15,
tp53 gene expression is sustained over several years.
Tp21 Gene Expression
tp21 is also a key protein in cell repair controlling
apoptosis and cell cycle arrest. It may act together with
Figure 12: CTCs of mama cancer patients showed no
change of gene expression ratio of apoptosis-triggering
genes Bax/Bcl2 after FSWW08 consumption. (Mean and
Standard deviation). Average are not statically significantly
different on a 10% level of significance.
Figure 13: CTCs of breast cancer patients showed no
change of gene expression ratio of apoptosis-triggering
genes bax/bcl2 after 100 days of FSWW08 (fermented soy)
therapy. (Mean and Standard error of the mean; (n = 5).
Mean values are statistically not significant on a 5% level of
significance.
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 11
p53 and is important in controlling cancer (Figure 14).
Tp21 is increased rather strongly in vitro as well as in
vivo in CTC. Clearly, the effect of FSWW08 on CTC is
more pronounced than in vitro in all tumors (Table 1,
Figures 16-18).
MAPK
The expression of the MAPK (Figure 19), was
positively altered by FSWW08 in breast cancer cells.
Additionally reduced inflammation in cancer was
detected with conventional staining technique,
Figure 14: Schematic representation of the relationship between tumor suppressor factors p21 and p53 and their involvement in
apoptosis and cell cycle progression.
Figure 15: Relative gene expression changes of in-blood circulating cancer cells ovarian cancer patients compared to baseline,
compared to expression after 1.5 months, after 3 months and after 4 years (A) prostate cancer patients, (B) breast cancer
patients (C) ovarian cancer patients. Numbers denote individual patients. Taken from ref [11]. All average levels after
consumption reach statistically of significance on the 5 % level of significance.
12 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
corroborating the gene expression results (Figure 20),
where a histological tissue sample of a breast cancer
patient was excised and stained. The conventional
staining technique supports the genetic findings that
immunity is favorably increased in the tumor.
c-Jun Gene Expression
The intracellular release of cytokines in asthma or
allergic reactions is linked to increased c-Jun kinase,
p30 MAP-kinase and NFB in local immune cells [12].
The first-generation antihistamines have similar
immune modulating responses to FSWW08 and
dexamethasone shares similar ability to alter the gene
expression of p38, MAPkinase, c-Jun and NFB in
myeloma cells, prostate, breast and ovarian cancers
[40]. The reduction of c-Jun in CTC extracted from
Breast tumors supports the findings that immunity is
increased (Figure 19).
It is believed that the decline of TNF alpha in the
blood of patients (Figure 21) is caused by improved
barrier function, so that larger molecules cannot
permeate the cellular barrier (Figure 22). The reduction
in cachexia (Figure 23) by fermented soy bean may
very well be related to improved barrier function,
documented by the improvements of matrix
metalloproteinase 9 gene expression [24].
Figure 16: Individual relative gene expression changes of tumor suppressor factors p21 in blood CTC (circulating tumor cells),
of prostate cancer patients.
Figure 17: The gene expression of tumor suppressor factors p21 in blood CTC of mamma cancer patients (n=7).
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 13
Figure 18: The individual relative gene expression of tumor suppressor factors p21 in blood CTC extracted form ovarian cancer
patients (n=5).
Figure 19: Gene expression changes by fermented soy (FSWW08) on blood circulating tumor cancer from breast cancer cells.
The cells were extracted from pooled blood.
VEGF Gene Expression
As is depicted in Figure 24, a statistically significant
increase of VEGF gene expression was detected in
CTC extracted from prostate and ovarian cancer
patients.
Clinical Effects
In cancer patients, appetite and immune status are
significantly weakened. A fermented soy formulation
FSWW08 (group A, consumed FSWW08 orally) and a
placebo formulation (group B) was tested in a clinical
14 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
Figure 20: Histology of a breast cancer sample of a 38-year-old woman. Upper row was taken before consumption and lower
row was taken 14 days later. ‘‘H&E’’ denotes hematoxylin and eosin. Hematoxylin and eosin stain is frequently used for routine
tissue preparation. Eosin is an acid aniline dye. It binds to and stains basic structures (or negatively charged structures), such as
cationic amino groups on proteins. It stains them pink. It can be interpreted as DNA activity and the loss of color as a reduced
DNA activity. F8 as a vascular marker is discussed by Folkman [79]. KI67 is a common marker of anti-angiogenesis. Taken from
ref. [38].
Figure 21: TNF-a blood levels before, during, and after daily consumption of fermented soy. Taken from ref [28].
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 15
Figure 22: A conceptual model of cytokines in cancer. Cancer and immune cells are sources of cytokines, which support the
growth of cancer and lead to psych behavioral symptoms (fatigue, depression, and cognitive impairment), drug toxicity, drug
resistance, anorexia and cachexia, pain, and cancer recurrence and progression. Genetic background, cancer treatment, and
psychological distress can corroborate the production of cytokines. In cancer survivors, hyperactive immune cells might be the
major source of cytokines in psych behavioral symptoms. Taken from ref: [28].
Figure 23: Influence of daily consumption of a fermented soy formulation on appetite loss (cachexia) in cancer patients under
chemotherapy compared to a group receiving placebo solution containing casein. This was a double-blind study Ref [27].
study, conducted in six cancer hospitals where cancer
patients underwent radio or chemotherapy (patients
undergoing radiation therapy n = 78, patients
undergoing chemotherapy n = 184, total 262) [39]
(Figure 23). Group A experienced statistically
significant increases in lymphocyte transformation
rates, whereas group B did not (Figure 25).
Formulations A inhibited or lessened statistically
significant decreases in white blood counts, whereas
the placebo group experienced substantial decreases.
Hemoglobin and platelet decreases were inhibited in
group A, although not statistically significantly [39].
Patients in group A received no blood transfusions,
whereas many patients from the placebo group
received blood transfusions undergoing chemotherapy
(Figure 24). Appetite loss was reduced in group A from
57.9% to 13.3%. In the placebo group, an increase in
appetite loss was detected under chemo- and radiation
16 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
therapy from 41.8% to 70.9 % (Figure 26). This study
was conducted in China, but similar results were
obtained in the USA at Harvard Medical School [44].
FSWW08 caused in a small pilot study a higher
survival of ovarian cancer patients compared to
standard therapy, although not statistically significant
[40]. All prostate cancer patients treated with FSWW08
are still alive [40]. The survival of breast cancer
patients increased and the survival rate under
consumption of FSWW08 can be related to breast
cancer patients with high gene expression of tumor
suppressor factor p53, which also show a higher
survival rate than compared to those without it.
DISCUSSION
The World Health Organization (WHO) reported that
cancer causes 63% of deaths worldwide and is the
second cause of death in western countries [45].
Cancer is a complex genetic disease with alterations in
multiple cellular signaling pathways [46]. Two traits are
shared in most cancers: genome instability and
mutation tumor-promoting inflammation in the
surrounding tissue so that the tumor can grow into
surrounding tissue [47]. Chemotherapy against the
tumor is mostly conducted with agents inhibiting mitosis
or by other cell toxic mechanisms, for example with
doxorubicin [48]. Only recently it was found that plants
Figure 24: VEGF gene expression in circulating tumor cells extraxted from patients.
Figure 25: Mean and standard deviation of leukocytes before (grey-filled squares) and after (black-filled squares) chemotherapy
with FSWW08, or FSWW08 with yam root extract or a casein placebo solution. Dashed line limits lower range of normal range.
**Denotes a statistically significant difference on the p < 0.01 error level of significance. Taken from ref [27].
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 17
regulate cell growth similar to animals and humans
[78]. Animals developed later in the evolutionary
development other gene regulating compounds like the
steroidal hormones, where estradiol was the first
steroidal hormone (and was later diversified into the
hormone cascade [79]).
Plants protect themselves against the growth of a
parasite, what is mechanistically similar to a growth of
a tumor into a surrounding tissue (Figure 26), by DNA
repair mechanism, what is defined as environmental
biotic stress [29]. It is of interest that a tumor may be
mechanistically defined as an unwanted intruder that
imposes stress on its tissue environment to facilitate its
growth (Table 2, Figure 26). Only recently plant
scientists have revealed that many agricultural plants
protect themselves against environmental drought
stress and injuring yeasts or parasite by similar repair
DNA mechanisms, which exist in humans too, like
MAPkinase (Table 2, Figures 4, 19). Hormones and
small RNA actively alter the genomes within a plant
and they have even the capability to conduct this in
other species too, because it was shown that soybean
can pass protective genome mechanism to other plant
species too, raising the question whether this
mechanism can protect against cancer too [37].
The clinical importance of stress repair mechanism
and the difference to chemotherapy may be illustrated
by the fact that the fermented soy formulation
discussed here in this study caused also a strong and
long lasting reduction of mental stress in soldiers
suffering from PTSD, the posttraumatic stress disorder
[49]: Severe mental stress is a characteristic of PTSD
and cellular and mental stress are related [50]. The
same fermented soy bean formulation has been shown
clinically in humans to stabilize Amyotrophic Lateral
Sclerosis, ALS, what is by definition a progressive cell
disease [82]: Although no complete normalization was
attained, an inhibition of the progressive state was
Figure 26: Schematic comparison of similar growth pattern of a cancer into tissue and a parasite into a leaf.
Table 2: Comparison of cellular gene markers and hormones related to cancer compared to gene described in the
plant as a cell repair gene and determined in plant biology. All genes were determined in soy with the
exception of VEGF like genes and protein. This was found in almost all species with larger tissue besides
yeasts. Foe explanation see text and literature quotation and Figure 4
Plants
Biotic Stress Defense
Human
Cancer Markers
SUPPRESSOR OF GAMMA RESPONSE 1 (SOG 1) Tp53, Tp21 Human Cancer
NO production co ntrol via glutathione peroxidase, phenylalanine-ammonia lyase (PAL), and
PR-1, genes (ref [81])
IKK/NFB-like signaling pathway
BAX BAX
VEGF/PDGF (platelet-derived growth factor) group of the cystine-knot superfamily. (Ref [80]) VEGF
MAPKinas e [30] MAPkinas e
Matrix metalloprotein ases ( MMP (ref [29]) MMP
c-JUN c-JUN
18 Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 Volko and Rohr
seen over several years and for the first time ever. As
was outlined above this plant cell repair mechanism
through abiotic and biotic stress response is non-toxic
and no side effects were reported in cancer multicenter
trial studies and in the ALS study conducted with the
same fermented soy bean formulation, tested here in
vitro gene expression studies.
Clinical improvements in cancer patients, relating to
mental and cellular stress, have been reported
previously by other authors too, investigating the soy
ingredient isoflavone genistein, which resulted in
increased patient survival time, better quality of life,
and reduced adverse effects related to cancer
chemotherapy delivering a dose of 1000 to 1200 mg
Isoflavones a day [51]. We previously reported also
that fermented soy bean (FSWW08), which reduced
stress and improved well-being in soldiers, increased
quality of life in cancer patients as well, reducing
cachexia (Figure 22), reduced side effects of cancer
treatment like cachexia (Figure 23) and leukocytopenia
(Figure 25) and increased the survival time of cancer
patients [40].
A direct comparison of gene expression changes by
FSWW08 formulation was conducted by comparing the
effect on cancer tumor cells (CTC) extracted from
blood of cancer patients to in vitro cancer cell line
treated with cytotoxic doxorubicin (Table 1). Gene
expressions were conducted with the same protocol,
method, and the same laboratory. On the other side
CTC of breast cancer patients and the in vitro breast
cancer cell line are not from the same donor
population. Also, CTC subpopulations might exist with
different properties, because cancer cells having the
ability to form micro metastases and metastases are
likely emerge from CTC, a high medical need exists to
develop new methods to characterize the genotype and
phenotype of CTC [11]. Although we have not
investigated whether subpopulations in our CTC exist,
the alterations by fermented soy bean formulations
were pronounced.
Comparisons of gene expression cancer marker by
fermented soy bean in CTC with the breast cancer cell
line BT shows that many anticancer effects like
increase of BAX, increase of tp53, c-Jun, tp21,
silencing of MAPkinase are increased in a similar
manner in vitro cell lines and in CTC (Table 1). This
investigation corroborates other in v itro investigators,
who also saw an improvement of tp53 gene expression
in vitro [51], as well as MAPkinase [52], NF-B [38],
matrix metalloproteinase [53-55], investigating soy
ingredients.
This here presented investigation is unique since for
the first time ever gene expression in circulating tumor
cells extracted from patient blood are represented and
are additionally related to new findings in the area of
genetic plant science (Table 2), where plants show a
mechanism for cell repair under stress not using any
toxic mechanism.
A very important finding is the simultaneous
increase of tp53 and tp21 gene expression in CTC,
which corroborates the strong apoptotic effect of
fermented soy bean formulation, because tp53 reduces
cell cycling, increases cell repair and diminishes
cellular stress (Figure 14) [56, 58]. Modification and
loss of tp53 has been described as a hall mark for
cancer and missing in about 50% of all cancer patients
[51]. Clearly, there are differences between in vitro and
in vivo: in vivo CTC cells have to be considered to be
less differentiated, so that the increase of tp21 and
tp53 gene expression may have been more
pronounced in vivo (Table 1). Tp53 is related to
increased apoptosis of white blood cell formation too
[56], which we saw in our clinical experiments (Figure
24). Only recently it was found that plants do express
very similar repair genes to tp53 to overcome
environmental stress [29].
The additional improvement in barrier function
documented by increase of matrix metalloproteinase
gene by soy by us and others [39, 55] in combination
with strong increase in immunity (Figure 20) may very
well explain the reduction in cancer side effects through
inhibiting cytokine release from the tumor into the blood
(Figure 23) but also modify the cancer stem cells
(CSC) in the tumor, which are believed to drive cancer
growth and metastasis. Improving barrier function of
plants may be an important mechanism by plants to
protect themselves against parasites, injuries, and cuts
and they may not have lost its ability when applied in
humans.
A special fermented soy bean FSWW08 did
increase VEGF gene expression in CTC of prostate
and in ovarian cancer patients (Table 1, Figure 24).
Precursor of VEGF have not been identified in yeasts,
but in almost all species, where larger tissue is formed
(Table 2) [80].
The concept and limitation of “anti-VEGF” therapy in
anticancer treatment is currently strongly debated
among researchers, as it failed in breast cancer and
there may be even an acquired resistance to anti-
VEGF therapy in glioblastoma, which may even
increase the invasiveness of Glioblastoma [59]. VEGF
Can Plants’ Ability for DNA Repair and Stress Defense Journal of Pharmacy and Nutrition Sciences, 2015, Vol. 5, No. 3 19
is a marker for cellular stress in humans: If oxygen
consumption is low, then VEGF is increased in
pregnancy to promote blood vessel growth and oxygen
supply of the fetus [61-64]. VEGF helps to reduce
cellular stress. As can be seen in Figure 20 where
reduced angiogenesis marker and DNA activity have
been identified in breast cancer tissue after
consumption of the fermented soy bean formulation
FSWW08. The recued angiogenesis and improved
barrier functions preventing the diffusion of cytokines
into the general blood stream (Figure 21), which are
normally responsible for depression, inflammation,
fatigue and most of all for cachexia, the appetite loss
(Figure 22), were also improved (Figure 22).
Both substances, Doxorubicin and fermented soy
bean, had a cytotoxic effect in the human breast cancer
cell line BT-474 (Table 1). Both substances increased
at high doses the expression of MDR1, Doxorubicin
even more. At low physiologic doses, however,
fermented soy bean reduced multiple drug resistance
gene MDR1 even in combination with doxorubicin.
The fact that FSWW08 led to a strong increase in
the expression of the pro-apoptotic gene BAX [64, 65]
(Figure 7) in cancer, in vitro, while the traditional
chemotherapeutic agent Doxorubicin left the
expression of this gene unaltered shows that it might at
least be beneficial to combine FSWW08 with
Doxorubicin for a more effective treatment of cancer. It
is not known why FSWW08 did increase BAX in CTC
extracted from blood of prostate cancer (Figure 11), but
left those extracted from ovarian and breast cancer
unchanged (Figures 12, 13). Programmed cell death to
reduce unnecessary or unwanted cells during
development or disease is similar in plants and animals
[66, 67]. Common pathways in plants and animals
leading to cell death has not been proven [68, 69]. In
plants, the triggering of cell death in response to an
invading pathogen results in the formation of a zone of
dead cells in the vicinity of the infection site [70]. This
killing of host cells is frequently associated with
resistance to further pathogen multiplication and
spread [71]. The tobacco mosaic virus is a model plant
parasite and causes host cells death around a
pathogenic intruder to prevent from further migrating
into the cell. Bcl-2/BAX heterodimers gene controls
death and survival of cells in yeasts and in plants [72,
73] and organizes the protection of a plant against an
intruder [74, 75].
NF-B (nuclear factor kappa B) family transcription
factors are master regulators of immune and
inflammatory processes in response to both injury and
infection [76]. NF-B exists not in plant cells. However
direct precursor cascades were found in plants
regulating NO production and is therefore an important
factor in plant physiology [81]. It was shown by many
investigators that many plants can reduce NF-B in
human tissue and cells, as was shown by fermented
soy beans also [77].
No clinically relevant health claims can be made
from these in vitro and in vivo studies incorporating
FSWW08, since larger statistically relevant studies
have to be conducted. It may be, however, suggested
that more studies have to be conducted combining
plant based medicine with DNA repair with those
conducting DNA cytotoxic mechanism. The here tested
formulation is not palatable for patients used to
Western food. Also it needs to be investigated, which of
the ingredients besides Isoflavones may also have
caused the gene modifications described in this study.
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... For example, when fish conquered land for the first time, the only oxygen source was salt water in the ocean and not the surrounding air [17]. Evolution added a steroidal hormone called aldosterone to increase potassium to keep water in [1,2].) the lungs of the fish who had moved on to dry land, thus they recovered oxygen from the water and not from the surrounding air [18]. Stress edema in the lung and legs, in today's severe human immune diseases, in cancer, severe mental diseases and swollen legs in PMS in the female hormone cycle have to be considered unnecessary, as we can retrieve enough oxygen from the surrounding air causing hypertension in humans [18,19]. ...
... Programed cell death to reduce unnecessary or unwanted cells during development or disease is similar in plants and animals [49]. In plants, the triggering of cell death in response to an invading pathogen results in the formation of a zone of dead cells in the vicinity of the infection site [1]. This killing of host cells is frequently associated with resistance to further pathogen multiplication and spread [50]. ...
... The immune modulation during pregnancy makes it necessary to reduce the cellular immunity in the first trimester of the mother, allowing the trophoblast to grow into the uterus [1]. The growth of the blastocyst into the uterus is similar to a bacterial infection and has to be considered as a stressful event for the mother [58]. ...
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