THE STORY OF THE RESEARCH FOR A NEW TREATMENT OF CANCER, DEGENERATIVE DISEASES AND IN REGENERATION OF TISSUES.

The paradigm of complexity and some aspects of theory of information, of linguistics and of semiology are fundamental in understanding the process of cancerogenesis and in determining the correct therapeutic approach to tumoral diseases. To go into these problems in more depth way I have to ask you to be patient and to follow the mental processes and the experiments that I made.

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At the beginning of 1982….

At the beginning of 1982 I studied the relationship between the agents that cause cancer, mutations and malformations and focused my attention on some of the data of literature, that showed how carcinogens administrated during pregnancy had had different effects. The administration of carcinogens before or during organogenesis causes in fact a high rate of malformations in offspring, but no tumor induction. Once organogenesis is complete the frequency of tumor induction rises with a concomitant decrease in the rate of malformations.

MD. Pier Mario Biava

The question was why these different effects take place ?

The answer was immediately clear:

During organogenesis all processes of cell differentiation take place and they could stop the action of factors which cause cancer. Some malformations of tissues and organs are possible but these tissues and organs are made up of differentiated cells. During organogenesis some substances with regulatory properties are perhaps present to prevent the indiscriminate multiplication of cells. These regulators are able to differentiate the mutated stem cells. Could these regulators control the multiplication of tumor cells? Were tumor cells similar to mutated stem cells? In order to answer to these questions some experiments were carried out.

Here we summarize now several researches conducted over the past 35 years in vitro as well as in vivo and finally clinical studies on cases of hepatocellular carcinoma at intermediate-advanced stage having administered factors extracted during stem cells differentiating processes.

Lastly, we report recent experiments that showed that stem cells differentiating factors (SCDFs) are able to prevent neurodegenerative processes in mouse hippocampus cell line and to significantly ameliorate psoriasis.

EXPERIMENTAL RESEARCHES AND CLINICAL TRIALS

The treatment of oncologic and degenerative diseases:

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The first Experiments were published on Cancer Letter in 1988

Abstract

Based on the hypothesis that the development of cancer is actively inhibited during embryonic life, the effects on tumor growth of homogenates of different tissues (embryos, uteri at ninth day of pregnancy, non-pregnant uteri and normal liver) were investigated in syngeneic C57BL/6 female mice. Primary tumor growth and spontaneous pulmonary metastasis formation were completely suppressed in the group of mice treated with pregnant uteri homogenates. Embryos, non-pregnant uteri and normal liver homogenates were ineffective.

Click here to see: Effects of treatment with embryonic and uterine tissue homogenates on Lewis lung carcinoma development.

http://www.cancerletters.info/article/0304-3835%2888%2990287-X/abstract

The most important scientific articles in which were published the results of the researches here recorded are:

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Activation of anti-oncogene p53 produced by embryonic extracts in vitro tumor cells.

ACTIVATION OF ANTI-ONCOGENE P53 PRODUCED BY EMBRYONIC EXTRACT

  • Results demonstrate that substances present in the embryo during cell differentiation are able to activate p53

Download: Tumor Marker Oncology, volume 12 Winter 1997

The following are the main images related to the different studies carried out on embryo differentiation factors:

The state of the art of research includes both in vitro works on molecular dynamics involving stem differentiation factors and in vivo work: on mice and on men. It remains necessary to implement in-vivo research, especially clinical.

Specifically, studies have been conducted on the following aspects:

  • Slower growth of tumor cell lines
  • Block of the cell cycle, activatinig trascriptional way the oncosupressor gene p53
  • regulating in post-traslational way the Retinoblastoma protein (pRb)
  • Animal studies
  • Protein Analysis of Zebrafish Embryo Extract
  • Clinical study of 200 patients to evaluate possible side effects
  • Randomized clinical study in 179 patients with intermediate or advanced hepatocarcinoma

Slowed down and/or cell cycle block action

It has been shown that in the embryonic microenvironment there are factors that regulate the expression of the p53 co-repressor, activating it, and post-translationally pRb. In fact, with the differentiation of stem cell differentiating factors of different tumor lines, a block of cell cycle was observed in G1-S phase.

Activation of anti-oncogene p53

Through cytofluorometry and immunohistochemistry, a significant increase in the concentration of p53 protein has been demonstrated in specific lines of cellular tumor lines such as glioblastoma multiforme, melanoma and hepatocarcinoma treated with stem differentiation factors. This increase is a consequence of the transcriptional regulation of the p53 oncosoppress gene.

Download the publication: ACTIVATION OF ANTI-ONCOGENE P53 PRODUCED BY EMBRYONIC EXTRACT

Before:

Before

After:

After

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CELL PROLIFERATION CURVES OF DIFFERENT HUMAN TUMOR LINES AFTER IN VITRO TREATMENT WITH ZEBRAFISH EMBRYONIC EXTRCTS

Abstract:

Five tumor cell lines of different origin (glioblastoma, melanoma, kidney adenocarcinoma, breast carcinoma and lymphoblastic leukemia) were treated in vitro with the extracts from zebrafish embryos collected at four different developmental stages. All cell lines responded with a significant slowing of the proliferation when treated with the extracts taken during the stages of cell differentiation, while no slowing effect was observed when they were treated with the extract taken from a merely multiplicative stage. These results suggest that a complex network of molecular factors during embryo differentiation may help abnormally proliferating cells to normalize their cycle, and that the administration of embryonic cell differentiation factors may be a useful tool in cancer therapy. On the other hand, it is known that the stem cells can be differentiated into different types of cells in relationship to different kinds of embryonic microenvironment. Since this network of cell differentiation factors may normalize the altered expression of genes, we suggest it as a sort of physiological gene therapy.

ABSTRACT:
Five tumor cell lines of different origin (glioblastoma, melanoma, kidney adenocarcinoma, breast carcinoma and lymphoblastic leukemia) were treated in vitro with the extracts from zebrafish embryos collected at four different developmental stages. All cell lines responded with a significant slowing of the proliferation when treated with the extracts taken during the stages of cell differentiation, while no slowing effect was observed when they were treated with the extract taken from a merely multiplicative stage. These results suggest that a complex network of molecular factors during embryo differentiation may help abnormally proliferating cells to normalize their cycle, and that the administration of embryonic cell differentiation factors may be a useful tool in cancer therapy. On the other hand, it is known that the stem cells can be differentiated into different types of cells in relationship to different kinds of embryonic microenvironment. Since this network of cell differentiation factors may normalize the altered expression of genes, we suggest it as a sort of physiological gene therapy.

Click here to open the link to the abtract:

http://zfin.org/cgi-bin/webdriver?MIval=aa-pubview2.apg&OID=ZDB-PUB-021017-40

These blocking mechanisms of the cell cycle have been observed in several tumor cell lines.
Specifically, tumor cell lines were investigated of:

  • kidney adenocarcinoma
  • Lymphoblastic Leukemia
  • Breast cancer
  • Melanoma
  • Glioblastoma

kidney-adenocarcinoma:

Lymphoblastic-Leukemia:

Breast-cancer:

Melanoma:

Glioblastoma:

Still in 2001 : Xenopus laevis embryos share antigens with zebrafish embryos and with human malignant neoplasms.

Abstract:

Previous experiments have demonstrated that antigens present in Xenopus laevis embryos are shared with human malignant neoplasms. In fact it is known that antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to different antigens present in human tumors. The aim of the the present study was to evaluate whether these antisera react also to different antigens contained in zebrafish embryos at different stages of cell differentiation. This was done with the agar-gel immunodiffusion method performed according to an already described protocol. The results that we obtained show that both antisera raised in against pellet and supernatant fractions of Xenopus laevis embryos react to antigens of zebrafish at different stages of cell differentiation.

Previous experiments have demonstrated that antigens present in Xenopus laevis embryos are shared with human malignant neoplasms. In fact it is known that antisera raised in rabbit against pellet and supernatant fraction of Xenopus laevis embryos react to different antigens present in human tumors. The aim of the present study was to evaluate whether these antisera react also to different antigens contained in zebrafish embryos at different stages of cell differentiation. This was done with the agar-gel immunodiffusion method performed according to an already described protocol.
The results that we obtained show that both antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to antigens of zebrafish at different stages of cell differentiation.
It has already been described that antibodies produced by immunizing rabbit to Xenopus laevis embryo fractions react with a variety of human malignant neoplasms. In fact antisera raised in rabbit against pellet and supernatant fractions of Xenopus laevis embryos react to different antigens contained in 50 of 55 different tumors. These reactions were demonstrated histologically.1
The antigens in the supernatant fraction are probably different from those in the pellet fraction. Antisera raised against the pellet fraction react with carbohydrate antigens.2 The nature of the antigens in the supernatant is not known yet. These previous results suggested that Xenopus laevis ambryos may contain an “oncodevelopmental” carbohydrate re-expressed in human tumors and that Xenopus may be an useful source for tumor associated antigens of human malignant tumors.
It may be that the antigens of Xenopus laevis embryos are preserved during phylogeny and that they are shared with other different embryos. The aim of the present study is to evaluate if the antisera raised in rabbit to the pellet and supernatant fractions of Xenopus laevis embryos react with antigens of zebrafish embryos at different stages of cell differentiation.

Figure 1
Zebrafish: middle – blastula – gastrula
Antitsera R 750-I (supernatant) A R 755-I (pellet) B

Figure 2
Zebrafish: 5 somites
Antisera R 750-I (supernatant) A R 755-I (pellet) B

Figure3
Zebrafish: 20 somites
Antisera R 750-I (supernatant) A R 755-I (pellet) B

Materials and Methods
The embryos of zebrafish at the stages of middle-blastula-gastrula, 5 somites, 20 somites were washed in distilled water and placed in a solution of pure glycerol and 30% of ethanol at a 4:1 ratio. The embryos were sonicated with 2 cycles of 10 seconds each and further treated with a turboemulsifier. 35 microliters of these solutions were used in an agar-gel immunodiffusion test. The methods of preparation of antisera raised in rabbit to pellet and supernatant fractions of Xenopus laevis embryos have already been described.1,2 A solution of 1 gram of agar-gel diluted in 40 ml. of distilled water and in 10 ml. of TBE (Tris Boric acid EDTA) was put in a plastic support. The lyophilized antisera were resuspended in 500 microliters of distilled water and 35 microliters of this solution were distributed in different wells of agar gel. 35 microliters of extracts of zebrafih at different stages of cell differentiation were put in different wells, in front of the antisera at a distance of 1 cm. The agar-gel with antisera and embryonic extracts were incubated for 24 hours at 22 degrees centigrade. The agar gel was stained with Coomassie Blue for 15 minutes, then washed with distilled water and finally destained in a solution composed by 10% acetic, 45% ethanol, 45,% distilled water.

Results
The results that we obtained show that both antisera raised in rabbit to pellet and supernatant fraction of Xenopus laevis embryos react to antigens of zebrafish at the three stage of cell differentiation. In fact the Fig. 1, Fig.2 and Fig.3 show that the antigens of zebrafish embryos at the stages of 50% of epiboly, 5 somites, 20 somites react with antisera R750-1 raised in rabbit to supernatant (part A of the figure) and with antisera R755-1 raised in rabbit to pellet (part B).

Conclusions
These results suggest that Xenopus laevis embryos share antigens with zebrafish embryos. These antigens are conserved during phylogeny. They are expressed in the zebrafish embryo since the beginning of cell differentiation and are present until last organogenesis. These antigens are more expressed at the end than at the beginning of organogenesis, as the results illustrated in Fig. 3 show in comparison with the results of the Fig. 1 and 2. In any case, these antigens shared with different species of embryos are re-expressed in different human tumors as already described. Tumor cells re-express several others “oncodevelopmental” antigens in addition to those described from us. For this reason, tumor cells can be considered as mutated embryonic cells, in which “gene configurations” are similar to those present in embryo during the steps of multiplication comprised between two stages of cell differentiation. This hypothesis has been already put forward in previous reports.3,4,5,6,7,8 On the other hand, tumor cell genome is normally affected by a dramatically high number of altered genes, most of which playing an important role in normal embryo development. In fact, during tumorigenesis some embryonic genes are activated or mutated, leading the cell to an uncontrolled multiplication program.
Many substances present in embryo during organogenesis are able to reduce tumor growth in vitro or in vivo, because they regulate some important genes that control cell differentiation and multiplication.5,7 In fact, our previous works demonstrated that substances present in embryo during cell differentiation are able to reduce tumor growth in vivo4 and to activate the tumor suppressor p53 in different tumor cell lines in vitro.5 Other works led to the same hypothesis: in fact the transplantation of teratocarcinoma cells into the mouse blastocyst origins normal chimeric mice, since the teratocarcinoma cells are led to differentiate in various kinds of tissues9 Otherwise, the transplantation of embryonic stem cells in mice origins teratocarcinomas.10 Our results suggest that Xenopus laevis and zebrafish embryos may be an useful source for tumor associated antigens of human malignant tumors.

P. M. Biava,
Ospedale Civile SSG Milano,
Italia

A. Monguzzi,
Ospedale Civile SSG Milano,
Italia

D. Bonsignorio,
Ospedale Civile SSG Milano,
Italia

A. Frosi,
Ospedale Civile SSG Milano,
Italia

S. Sell,
Albany Medical College,
Albany, NY,USA;

J. V. Klavins,
Albert Einstein College Of Medicine,
New York, NY, US;

1. Klavins J. V., Sell S., Fuchs A.
J.Tumor Marker Oncol. 11/2: 36 (1996).
2. Zhang S., Sell S., Livingston P.O., Klavins J. V.
J. Tumor Marker Oncol. 12(2):52(1997).
3. Biava P. M., Fiorito A., Negro C.,
Mariani M. Cancer Lett. 41: 265-270 (1988).
4. Biava P. M., Carluccio A.
Biol. Medizin. 5:247-249(1995).
5. Biava P.M., Carluccio A.
J. Tumor Marker One. 12,4: 9-15 (1997).
6. Biava P. M.
Complessita e cancro. Leadership Medica – Anno XV. 1- (1999).
7. Biava P. M. Bonsignorio D.
J. Tumor Marker Onc. 17-47-54 (2002)
8. Biava P. M., et al
J. Tumor Marker Onc. 17-59-64 (2002). Stewart T. A., Mintz B.
9. Proc. Natl. Acad. Sci
USA, 78 :6314-6318.(1981)
10. Reubinoff B. E. et al.
Nat. Biotechnol. 18(4) : 399-404 (2000).

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Cancer and cell differentiation: a model to explain malignancy:

Cell Proliferation is affected by factors that are found in embryos and in pregnant uteri. This factors are organized in a network wohse complexity should be unscattered to retain its full efficacy. This is particulary true for embryos whose complex of molecular factors represents a closed microenviroment that can normalize the behavior of abnormously growing cell populations via a regulatory process involving key-role proteins of cell cycle homeostasis.

  • To read more, please download: Cancer and cell differentiation: a model to explain malignancy.

Still in 2002: Mother-embryo cross-talk: the anti-cancer substances produced by mother and embryo during cell differentiation. A review of experimental data: J. Tumor Marker Oncology

Introduction
During pregnancy, a close cross-talk between mother and developing embryo is formed, made of a complex network of circulating molecular factors. This cross-talk is necessary for the prevention of pregnancy-threatening events, including the establishment of abnormally proliferating cell clones which may damage the integrity of this cross-talk.

Animal Studies:

The effects of stem differentiation factors on inhibition of tumor growth were in vivo tested on females of singular C57BL / 6 mice from the weight of 18-20 grams to which a subcutaneous Lewis primary carcinoma injection was performed. Therefore, both the size of the primary tumor, and the survival time of the mice, have been evaluated. In terms of development of the primary tumor, an extremely significant difference (P <0.001) was observed between treated and control mice (Figure 1) and thus also with regard to the survival ratio, always in favor of the treated mouse.

Biava, P.M.; Bonsignorio, D.; Hoxha, M.; Impagliazzo, M.; Frosi,
A.; Larese, F.; Negro, C. (2002) Mother-embryo cross-talk: the anti-cancer substances produced by mother and embryo during cell differentiation. A review of experimental data. J. Tumor Marker Oncol., 2002, 17, 55-58

Still in 2002: Post-traslational modification of the retinoblastoma protein (pRb) induced by in vitro administration of Zebrafish embryonic extracts on human kidney adenocarcinoma cell line

Tumor cells share several key-role features with embryonic cells. Tumor development is reduced or even suppressed in embryos during early differentiation processes suggest that factors present in the developing embryo may effect tumor growth. Experiments carried out in our lab showed that treatment of several tumor cell lines with embryonic and/or pregnant uteri homogenates inhibits tumor growth both in vitro and in vivo.

Still in 2002: Embryonic differentiation factors with anticancer properties:preliminary clinical results in the therapy for advanced tumors

It is possible to activate the p53 onco-suppressor after treatment with embryonic extracts on different tumor cells. The evidence that embryonic factors of cell differentiation can be used as a physiological gene therapy of cancer constituted the objective basis to prepare a therapy to test in human cancer. As a result, different products containing specific embryonic differentiation factors were prepared.

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Treatment with stem cell differentiation stage factors of intermediate-advanced hepatocellular carcinoma: an open randomized clinical trial

Abstract

There is no standard treatment for patients with advanced hepatocellular carcinoma (HCC). We developed a product containing stem cells differentiation stage factors (SCDSF) that inhibits tumor growth in vivo and in vitro. The aim of this open randomized study was to assess its efficacy in patients with HCC not suitable for resection, transplantation, ablation therapy, or arterial chemoembolization. A total of 179 consecutive patients were enrolled. We randomly assigned the patients to receive either SCDSF or only conservative treatment. Primary end points were tumor response and survival. Secondary end points were performance status and patient tolerance. Randomization was stopped at the second interim analysis (6 months) of the first 32 patients recruited when the inspection detected a significant difference in favor of treatment (p = 0.037). The responses to the therapy obtained in 154 additional patients confirmed previous results. Evaluation of survival showed a significant difference between the group of patients who responded to treatment versus the group with progression of disease (p < 0.001). Of the 23 treated patients with a performance status (PS) of 1, 19 changed to 0. The study indicated the efficacy of SCDSF treatment of the patients with intermediate-advanced HCC.

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Zebrafish embryo proteins induce apoptosis in human colon cancer cells

Abstract

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G(2)/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.

Download from :

Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2).

Abstract

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G(2)/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.

 

SYNERGISTIC EFFECT BETWEEN CHEMOTHERAPY AND DIFFERENTIATION FACTORS

IMAGES OF TUMOR CELLS CaCo2

In order to verify the synergistic effect between chemotherapy and differentiation factors, cancer cell lines of colon were treated with:

  • 5-FLUORURACIL
  • DIFFERENTIATION FACTORS
  • 5-FLUOROURACIL + DIFFERENTIATION FACTORS

As can be seen from the table emerges a powerful synergistic activity in the association of Differentiation factors with Fluorouracil.

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Reprogramming of normal and cancer stem cells. Curr. Phar. Biotechnol

Abstract:

Over the last decade there has been an exponential rise in our understanding of the biochemical mechanisms controlling cell proliferation and differentiation. While the four transcription factors Oct4, Sox2, Klf4 and cMyc have shown to be sufficient to induce pluripotency in fibroblasts, there has in addition been much research into the mechanisms and pathways of cell differentiation and the specific properties of stem cells, namely their plasticity and capacity for trans-differentiation.

Download from: http://www.ingentaconnect.com/content/ben/cpb/2011/00000012/00000002/art00001

Abstract:

Over the last decade there has been an exponential rise in our understanding of the biochemical mechanisms controlling cell proliferation and differentiation. While the four transcription factors Oct4, Sox2, Klf4 and cMyc have shown to be sufficient to induce pluripotency in fibroblasts, there has in addition been much research into the mechanisms and pathways of cell differentiation and the specific properties of stem cells, namely their plasticity and capacity for trans-differentiation. These studies have allowed progress at very fundamental level, with the prospect of further progress – until recent years quite unimaginable – in the field of reparative, regenerative and transplant medicine. In fact, from the present time, the genetic engineering production of regulatory factors identified through such research, has allowed the production of new tissues and a new category of cell therapy products, in which the main biological action is carried out by cells or tissues, albeit in the presence of organic or inorganic matrices or coatings. Examples of this type of product are anti-tumor vaccines, in vitro cultivated skin, products made of structural and cellular elements for the reconstruction of bones, cartilages, teeth, etc. From the best, most analytical and detailed characterization of stem cells, then, it has become clear that some tumor cell behaviors – that have a crucial role in determining their malignity – can be attributed to the presence of cells with characteristics similar to those of stem cells. The field of cancer research is consequently also witnessing a surge in studies designed to identify the metabolic pathways common to tumor and stem cells. This will in turn cast light on which micro-environment factors can direct these pathways towards differentiation and induce cancerous cells to behave less aggressively. From this point of view, over recent years there has been a lively return to studies that were very significant in the 70’s and 80’s, on the role of the embryonic micro-environment in conditioning tumor cell behavior towards normal phenotypes. This research is now underway, and will in all probability lead to important results over the next few years. Against this background, this special issue on “Reprogramming of normal and cancer stem cells” focuses on research in terms of conditioning the fate of normal and tumor stem cells with a view to new prospects for therapies. The issue therefore begins with articles covering the possibility of reprogramming normal stem cells, including through use of biomaterials, and goes on to consider what characteristics of tumor stem cells can allow them to be identified and studied. This is followed by a series of further articles illustrating the role of the micro-environment in conditioning the fate of a tumor cell. A number of metabolic pathways characterizing and common to both stem and tumor cells are examined, in order to gain a better understanding of the possibilities of conditioning the fate of both cell types; in addition, the role played by infectious and inflammatory diseases in the genesis of tumor diseases is also considered. Today, in fact, we know that inflammatory processes can support rather than hinder tumor growth, and also that pro-inflammatory cytokines can promote tumor proliferation, inhibiting the cell pathways that are able to block the neoplastic growth. The special issue goes on with a series of articles taking a close look at the specific role played by the micro-environment in conditioning the destiny of the tumor stem cells present in some tumors, for example breast and retinoblastoma tumor, and the role played by the use of normal stem cells in treating disorders such as hematological diseases. One article also considers the risks run by some reprogramming techniques: for example, creating embryo cells via parthenogenesis can give rise to tumors. The issue continues with various articles illustrating in close detail the role of the embryonic micro-environment in conditioning the destiny of tumor cells. In this context, one review takes a look at general aspects, while others consider aspects that can help clarify the mechanisms underlying the capacity of factors of this type of microenvironment to reprogram a tumor cell. One mathematical model sets out from a description of the state of cell differentiation, making use of existing data from studies of tumor growth slowing, linked to the use of such factors, with the goal of shedding light on aspects such as fitness, dosage, and administration time for the differentiation factors on improvement in tumor inhibitory response. Other articles illustrate a number of clinical cases of full regression of hepatocellular carcinomas in intermediate-advanced stages observed following administration of stem cell differentiation factors, and describe the molecular mechanisms that might explain these inhibitory responses on the tumor growth. It should be noted that the randomized and controlled clinical studies launched to date using stem cell differentiation factors are limited to patients with intermediate-advanced stage of hepatocellular carcinomas where other therapies were no longer possible. These factors are at present used only for hepatocellular carcinomas, since it has been demonstrated that substances capable of slowing one tumor’s growth may be inefficacious for another type. Finally, it is important to note that research into the possibility of reprogramming normal and tumor stem cells requires a complex approach to the issue. In fact, the problem requires the study of networks of substances and genes involved in the reprogramming phase, demanding skills in a variety of different areas of research, not simply of medical/biological, but also mathematical/ computational and modeling, in view of the complexity and non-linearity of the processes being studied. A paradigm shift is underway, and the future will witness our engagement in increasing numbers of scientific studies requiring cross-disciplinary skills.

The new paradigm and the new ideas were well understood many years ago by Professor John Klavins, who has been for a long time President of the International Academy of Tumor Marker Oncology. Professor Klavins has always sustained my studies on reprogramming cancer cells, though the possibility of controlling tumor growth by using reprogramming factors was not considered realistic at the time I began studying it.

I wish to dedicate this work to my friend John Klavins.

ACKNOWLEDGEMENTS

Many thanks to Roberta Zorovini and Silvia Cefalo from Smile Tech srl, via Valdirivo 19 Trieste, Italy ([email protected]), for their accurate and thorough work as organizing and editing secretariat.

Document Type: Research Article

DOI: http://dx.doi.org/10.2174/138920111794295873

Publication date: 1 febbraio 2011

Still in 2011 : Cancer cell reprogramming: stem cell differentiation stage factors and an agent based model to optimize cancer treatment. Curr. Phar. Biotechnol.

Abstract

The recent tumor research has lead scientists to recognize the central role played by cancer stem cells in sustaining malignancy and chemo-resistance. A model of cancer presented by one of us describes the mechanisms that give rise to the different kinds of cancer stem-like cells and the role of these cells in cancer diseases. The model implies a shift in the conceptualization of the disease from reductionism to complexity theory. By exploiting the link between the agent-based simulation technique and the theory of complexity, the medical view is here translated into a corresponding computational model.

From: https://www.ncbi.nlm.nih.gov/pubmed/21044002

Abstract

The recent tumor research has lead scientists to recognize the central role played by cancer stem cells in sustaining malignancy and chemo-resistance. A model of cancer presented by one of us describes the mechanisms that give rise to the different kinds of cancer stem-like cells and the role of these cells in cancer diseases. The model implies a shift in the conceptualization of the disease from reductionism to complexity theory. By exploiting the link between the agent-based simulation technique and the theory of complexity, the medical view is here translated into a corresponding computational model. Two main categories of agents characterize the model, 1) cancer stem-like cells and 2) stem cell differentiation stage factors. Cancer cells agents are then distinguished based on the differentiation stage associated with the malignancy. Differentiation factors interact with cancer cells and then, with varying degrees of fitness, induce differentiation or cause apoptosis. The model inputs are then fitted to experimental data and numerical simulations carried out. By performing virtual experiments on the model’s choice variables a decision-maker (physician) can obtains insights on the progression of the disease and on the effects of a choice of administration frequency and or dose. The model also paves the way to future research, whose perspectives are discussed.

Download: Cancer Cell Reprogramming

Still in 2011 : Embryonic morphogenetic field induces phenotypic reversion in cancer cells. Curr. Phar. Biotechnol.

Abstract

Cancer cells introduced into developing embryos can be committed to a complete reversion of their malignant phenotype. It is unlikely that such effects could be ascribed to only few molecular components interacting according to a simple linear-dynamics model, and they claim against the somatic mutation theory of cancer. Some 50 years ago, Needham and Waddington speculated that cancer represents an escape from morphogenetic field like those which guide embryonic development. Indeed, disruption of the morphogenetic field of a tissue can promote the onset as well as the progression of cancer. On the other hand, placing tumor cells into a “normal” morphogenetic field – like that of an embryonic tissue – one can reverse malignant phenotype, “reprogramming” tumor into normal cells.

From: https://www.ncbi.nlm.nih.gov/pubmed/21044001

Abstract

Cancer cells introduced into developing embryos can be committed to a complete reversion of their malignant phenotype. It is unlikely that such effects could be ascribed to only few molecular components interacting according to a simple linear-dynamics model, and they claim against the somatic mutation theory of cancer. Some 50 years ago, Needham and Waddington speculated that cancer represents an escape from morphogenetic field like those which guide embryonic development. Indeed, disruption of the morphogenetic field of a tissue can promote the onset as well as the progression of cancer. On the other hand, placing tumor cells into a “normal” morphogenetic field – like that of an embryonic tissue – one can reverse malignant phenotype, “reprogramming” tumor into normal cells. According to the theoretical framework provided by the thermodynamics of dissipative systems, morphogenetic fields could be considered as distinct attractors, to which cell behaviors are converging. Cancer-attractors are likely positioned somewhat close to embryonic-attractors. Indeed, tumors share several morphological and ultra-structural features with embryonic cells. The recovering of an “embryonic-like” cell shape might enable the gene regulatory network to reactivate embryonic programs, and consequently to express antigenic and biochemical embryonic characters. This condition confers to cancer an unusual sensitivity to embryonic regulatory cues. Thus, it is not surprising that cancer cells exposed to specific embryonic morphogenetic fields undergoes significant modifications, eventually leading to a complete phenotypic reversion.

Still in 2011 :

Zebrafish stem cell differentiation stage factors suppress Bcl-xL release and enhance 5-Fu-mediated apoptosis in colon cancer cells.

Abstract

Stem cell differentiation stage factors (SCDSF), taken from Zebrafish embryos during the stage in which totipotent stem cells are differentiating into pluripotent stem cells, have been shown to inhibit proliferation and induce apoptosis in colon tumors. In order to ascertain if these embryonic factors could synergistically/additively interact with 5-Fluorouracil (5-Fu), whole cell-count, flow-cytometry analysis and apoptotic parameters were recorded in human colon cancer cells (Caco2) treated with Zebrafish stem cell differentiation stage factors (SCDSF 3 µg/ml) in association or not with 5-Fu in the sub-pharmacological therapeutic range (0.01 mg/ml). Cell proliferation was significantly reduced by SCDSF, meanwhile SCDSF+5-Fu leads to an almost complete growth-inhibition. SCDSF produces a significant apoptotic effect, meanwhile the association with 5-FU leads to an enhanced additive apoptotic rate at both 24 and 72 hrs.

From: https://www.ncbi.nlm.nih.gov/pubmed/21043999

Abstract

Stem cell differentiation stage factors (SCDSF), taken from Zebrafish embryos during the stage in which totipotent stem cells are differentiating into pluripotent stem cells, have been shown to inhibit proliferation and induce apoptosis in colon tumors. In order to ascertain if these embryonic factors could synergistically/additively interact with 5-Fluorouracil (5-Fu), whole cell-count, flow-cytometry analysis and apoptotic parameters were recorded in human colon cancer cells (Caco2) treated with Zebrafish stem cell differentiation stage factors (SCDSF 3 µg/ml) in association or not with 5-Fu in the sub-pharmacological therapeutic range (0.01 mg/ml). Cell proliferation was significantly reduced by SCDSF, meanwhile SCDSF+5-Fu leads to an almost complete growth-inhibition. SCDSF produces a significant apoptotic effect, meanwhile the association with 5-FU leads to an enhanced additive apoptotic rate at both 24 and 72 hrs. SCDSF alone and in association with 5-Fu trigger both the extrinsic and the intrinsic apoptotic pathways, activating caspase-8, -3 and -7. SCDSF and 5-Fu alone exerted opposite effects on Bax and Bcl-xL proteins, meanwhile SCDSF+5-Fu induced an almost complete suppression of Bcl-xL release and a dramatic increase in the Bax/Bcl-xL ratio. These data suggest that zebrafish embryo factors could improve chemotherapy efficacy by reducing anti-apoptotic proteins involved in drug-resistance processes.

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The zebrafish embryo derivative affects cell viability of epidermal cells: a possible role in the treatment of psoriasis.

Abstract

In patients affected by psoriasis, use of a topical formula containing a derivative of zebrafish embryos was associated with reduced skin inflammation and dermal turnover, as well as a generally better outcome. In an attempt to understand the molecular mechanisms lying beyond these findings, we investigated the anti-proliferative effects of the zebrafish embryos derivative by addressing the mitochondrial function (MTT assay) and cell nuclei distribution (Hoestch staining). In cell cultures stimulated with fetal calf serum (FCS) or epidermal growth factor (EGF), the zebrafish derivative significantly inhibited cell proliferation induced by either approach, although the effect was stronger in cells stimulated with FCS. These results suggest that the zebrafish embryos derivative may dampen increased cell proliferation; this observation may be relevant to cutaneous pathologies related to altered proliferative mechanisms, including psoriasis.

From: https://www.ncbi.nlm.nih.gov/pubmed/24005140

Abstract

In patients affected by psoriasis, use of a topical formula containing a derivative of zebrafish embryos was associated with reduced skin inflammation and dermal turnover, as well as a generally better outcome. In an attempt to understand the molecular mechanisms lying beyond these findings, we investigated the anti-proliferative effects of the zebrafish embryos derivative by addressing the mitochondrial function (MTT assay) and cell nuclei distribution (Hoestch staining). In cell cultures stimulated with fetal calf serum (FCS) or epidermal growth factor (EGF), the zebrafish derivative significantly inhibited cell proliferation induced by either approach, although the effect was stronger in cells stimulated with FCS. These results suggest that the zebrafish embryos derivative may dampen increased cell proliferation; this observation may be relevant to cutaneous pathologies related to altered proliferative mechanisms, including psoriasis.

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Still in 2013:

Rediscovering Meaning

Abstract

The present crisis of the Western countries involves not just the world of politics, finance, and the economy, but also the ecosystem balance, the world of the values on which the model of social organization and economic development is based. The crisis is therefore systemic, epoch-making, and overcoming it will require radical changes, above all in the way we think, in our scale of values, and therefore our culture. On the other hand, for decades the world—in particular the West—has been experiencing a cultural sea-change that is challenging many of the pillars of society that have stood for centuries. The young people of today and their use of technological social networks to develop non-profit initiatives for social change may create a more positive future while maintaining our human values.

From: World Futures

The Journal of New Paradigm Research

https://www.tandfonline.com/doi/full/10.1080/02604027.2012.762196

Abstract

The present crisis of the Western countries involves not just the world of politics, finance, and the economy, but also the ecosystem balance, the world of the values on which the model of social organization and economic development is based. The crisis is therefore systemic, epoch-making, and overcoming it will require radical changes, above all in the way we think, in our scale of values, and therefore our culture. On the other hand, for decades the world—in particular the West—has been experiencing a cultural sea-change that is challenging many of the pillars of society that have stood for centuries. The young people of today and their use of technological social networks to develop non-profit initiatives for social change may create a more positive future while maintaining our human values.

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A systemic approach to cancer treatment: tumor cell reprogramming focused on endocrine-related cancers.

Abstract

The term “cancer cell reprogramming” is used to define any kind of intervention aimed at transforming cancer cells into terminally differentiated cells. Using this approach, new technologies have been applied with different methods for a more systemic approach to cancer treatment. This review reports on advances of these technologies, including our personal contributions, mainly carried out on endocrine-related cancers. Some of the interventions, aimed at reverting cancer cells into a normal phenotype, are based on the evidence that tumor development is suppressed by the embryonic microenvironment. On the basis of this rationale, experiments have been conducted using stem cell differentiation stage factors (SCDSFs) taken at different stages of development of Zebrafish embryos, oocyte extracts, or naïve human umbilical cord matrix derived stem cells (UMDSCs).

From: https://www.ncbi.nlm.nih.gov/pubmed/24304275

Abstract

The term “cancer cell reprogramming” is used to define any kind of intervention aimed at transforming cancer cells into terminally differentiated cells. Using this approach, new technologies have been applied with different methods for a more systemic approach to cancer treatment. This review reports on advances of these technologies, including our personal contributions, mainly carried out on endocrine-related cancers. Some of the interventions, aimed at reverting cancer cells into a normal phenotype, are based on the evidence that tumor development is suppressed by the embryonic microenvironment. On the basis of this rationale, experiments have been conducted using stem cell differentiation stage factors (SCDSFs) taken at different stages of development of Zebrafish embryos, oocyte extracts, or naïve human umbilical cord matrix derived stem cells (UMDSCs). SCDSFs induce significant growth inhibition on different tumor cell lines in vitro, likely because of increases in cell cycle regulatory molecules, such as p53 and pRb. Treatment with these factors activates apoptosis and differentiation related to caspase-3. This is achieved via p73 apoptotic-dependent pathway activation with a concurrent normalization of the E-cadherin and beta-catenin ratio. Extracts from prophase amphibian oocytes could reprogram relevant epigenetic alterations in MCF-7 and HCC1954 breast cancer cell lines, while un-engineered (naïve) human UMDSCs attenuated growth of MDA-231 human breast carcinoma cells. A product prepared for human treatments, containing SCDSFs at very low doses, yielded favorable results in breast cancer and in intermediate-advanced hepatocellular carcinoma. Other reprogramming interventions used in the models of breast, prostate and ovarian cancer cell lines are described. Finally, current and future perspectives of this novel technology are discussed and a new hallmark of cancer is suggested: the loss of differentiation of cancer cells.

Still in 2014:

Changing the endocrine dependence of breast cancer: data and hypotheses.

Abstract

Among the most common human cancers, often only breast and prostate cancers have advantage of hormone dependence. For a long time, this advantage permitted breast cancer to be efficaciously managed in the adjuvant and metastatic settings with low side effects by endocrine therapy. Unfortunately, soon or afterward hormone dependence is lost in most patients. In breast cancer, de novo or acquired hormone resistance is an hot issue and the focus of endless debate. Although a lack of oestrogen receptors (ERs) is considered to be the main reason for de novo hormone resistance, many studies have been conducted and many different mechanisms have been hypothesised to account for acquired hormone resistance.

From: https://www.ncbi.nlm.nih.gov/pubmed/24304277

Abstract

Among the most common human cancers, often only breast and prostate cancers have advantage of hormone dependence. For a long time, this advantage permitted breast cancer to be efficaciously managed in the adjuvant and metastatic settings with low side effects by endocrine therapy. Unfortunately, soon or afterward hormone dependence is lost in most patients. In breast cancer, de novo or acquired hormone resistance is an hot issue and the focus of endless debate. Although a lack of oestrogen receptors (ERs) is considered to be the main reason for de novo hormone resistance, many studies have been conducted and many different mechanisms have been hypothesised to account for acquired hormone resistance. Thus far, hormone resistance appears to be occasionally delayed or avoided in “in vivo” experiments. However, this finding did not have a significant benefit in current clinical practice. The principal aim of this review article is to sum up and update the issue of changing the endocrine dependence of breast cancer. Recent molecular insights extensively elucidating and shedding new light on this very controversial issue are considered. Moreover, based on our recent reports, a new mechanistic interpretation of and a therapeutic approach for overcome hormone resistance are proposed.

Still in 2014:

The role of neuroendocrine cells in prostate cancer: a comprehensive review of current literature and subsequent rationale to broaden and integrate current treatment modalities.

Abstract

Neuroendocrine prostate carcinoma (NE-PCa) is a heterogeneous disease. Due to a high prevalence of NE (neuroendocrine) differentiation in patients who receive prolonged androgen deprivation treatment, the real incidence of NE-PCa remains unknown. Similarly, the biological steps from prostate carcinoma (PCa) toward NE differentiation are far less than definitive and, consequently, there is a lack of evidence to support any of the treatments as the “gold standard”.

From: https://www.ncbi.nlm.nih.gov/pubmed/24304274

Abstract

Neuroendocrine prostate carcinoma (NE-PCa) is a heterogeneous disease. Due to a high prevalence of NE (neuroendocrine) differentiation in patients who receive prolonged androgen deprivation treatment, the real incidence of NE-PCa remains unknown. Similarly, the biological steps from prostate carcinoma (PCa) toward NE differentiation are far less than definitive and, consequently, there is a lack of evidence to support any of the treatments as the “gold standard”.

MATERIALS AND METHODS:

A systematic literature search was conducted using the PubMed, Scopus, and Embase databases to identify original articles and review articles regarding NE-PCa . Keywords were “prostate cancer” and “neuroendocrine”. Articles published between 1995 and 2013, were reviewed and selected with the consensus of all of the authors.

RESULTS:

Fifty-one articles were selected by the authors for the purpose of this review. The principle findings were reported into some subsections: Epidemiology, Biological steps of NE differentiation (with some principle articles on animal and in vitro, since there is very little in the literature on human studies); for the treatment options, we had to expand the search on PubMed to a larger timeframe and selection since very little was specifically found in the first criteria: surgery, radiotherapy, ablative techniques, immunomodulation and epigenetic therapy were then reviewed. A multidisciplinary approach, advocated by many authors, although promising, has failed to demonstrate increased survival rates. Limitations of this review include the lack of a clear definition of NE-PCa and consequently, the lack of strong evidence provided by a large series with long-term follow-up.

CONCLUSIONS:

Supported from this extensive review, we propose it is worthwhile to investigate a new multimodal therapeutic approach to address advanced NE-PCa starting from a debulking (with radical intent) of the disease plus epigenetic therapy with stem cell differentiation stage factors (SCDSFs). In addition immunotherapy can be used to treat the cancer presenting phenotype in association with chemomodulation plus ablative therapies, in case of advanced or recurrent diseases. SCDSFs may be utilized to regulate cancer stem cells and possible new phenotypes could also be associated with ablative therapies. Hormonal deprivation, radiotherapy, chemotherapy, ex vivo vaccines and targeted therapies could also be used and reserved in case of failure.

Still in 2014:

Human Stem Cell Exposure to Developmental Stage Zebrafish Extracts: a Novel Strategy for Tuning Stemness and Senescence Patterning

Abstract

BACKGROUND: Zebrafish exhibits extraordinary ability for tissue regeneration. Despite growing investigations dissecting the molecular underpinning of such regenerative potential, little is known about the possibility to use the chemical invent