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Fig. 1: Primary tumor growth after implantation

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 I have to ask you to be patient and to follow the mental processes and the experiments that I made. 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 caused

Tab. 1: Pulmonary metastasis formation in mice

a high rate of malformations in offspring, but no tumor induction. Once organogenesis was complete the frequency of tumor induction rose with a concomitant decrease in the rate of malformations (1, 2, 3, 4, 5). The question was why these different effects took place. The answer was immediately clear: during organogenesis all processes of differentiation took 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 regulators are perhaps present to prevent the indiscriminate multiplication of cells. These regulators are able to differentiate the mutated embryonic cells. Could these regulators control the multiplication of tumor cells? Were tumor cells similar to mutated embryonic cells? In order to answer these questions some experiments were carried out.

The experiments in animals

Different homogenates (embryos at day 9 of pregnancy, uteri at day 9 of pregnancy, non pregnant uteri and

normal liver) with 1 million cells of Lewis Lung Carcinoma were administrated to different groups of C57BL6/6 mice. The results of these experiments were published in "Cancer Letters" (6) and are reprinted here in fig.1 and in table 1. The homogenates of pregnant uteri dramatically inhibited the growth of primary tumor and the spontaneous pulmonary metastasis. The stop of tumor growth was not connected to the cytotoxicity (fig. 2). I was very surprised not to find any effect on tumor growth by embryonic homogenates. The experiments were repeated and they showed the same results. I thought then that the uterus could be a regulatory organ through the process of giving some “information” to the embryo. I made the

same experiments using embryos of Drosophyla which have all information to complete the differentiation and the development. These experiment, published in the “Biologische Medizine” (7), showed the results of tab. 2 e fig. 3. Tumor growth was significantly delayed in the group of treated mice in comparison with the control group; the survival time showed the same results. Even when the used embryos were philogenetically very different from mice, the homogenates were effective in delaying tumor growth. These experiments got stronger the hypothesis that tumor cells could respond to regulatory control induced by some substances which could be present in the course

of cell differentiation. In mammals this regulatory activity could be present in uterus, while in ovipari could be present in embryo.

An outline on embryonic differentiation

I will prove this hypotesis by further experiments. However in order to understand the function of this regulatory activity I first have to outline the embryonic differentiation

                 .

Shortly after fertilization, generally during gastrulation, the processes of differentiation begin. There are three postulates of cell differentiation:

1) every cell nucleus contains the complete genome established in the fertilized egg. In molecular terms the DNAs of all differentiated cells are identical

2) the unused genes in the differentiated cells are not destroyed or mutated and they retain the potential for being expressed

3) only a small percentage of the genome is expressed in each cell and a portion of the RNA synthesized is

specific for that cell type. Briefly, the differentiation that leads pluripotent cells to specialization, consists in a differential regulation of genes that restricts the expressed genome. The regulators are generally factors that cooperate in a network and this network promotes and controls differentiation of each kind of cell. All cells communicate with each other through this network. Cell differentiation is a very complex process that takes place at different levels:

A) differential gene transcription which regulates how the nuclear genes are transcripted into RNA

B) selective nuclear RNA processing which regulates how the transcripted RNAs get into cytoplasm to become messenger RNAs

C) selective messenger RNA translation which regulates how messenger RNAs in cytoplasm get translated into proteins.

D) differential modification of proteins, which regulates how proteins are allowed to function in the cells.

The network of differentiation is made up of many different substances that cooperate in several cascades. For example the transcription factors in Drosophila that contribute to making the anterior region of the embryo operate in a cascade. Briefly i.e.: the bicoid protein activates the hunchback gene; the new synthesized protein by hunchback gene activates Kruppel gene; the Kruppel protein activates odd-skipped gene and so on. Simultaneously many other cascades of regulators occur in the posterior region of the embryo (fig.4) that lead to the production of nanos proteins. These proteins stop the translation of the hunchback messenger RNA. These complex chemical messages that control the differential expression of the genes are responsible for the harmonious development of the embryo. In fact if the nanos gene is not present, the hunchback protein is made in all embryo and this protein inhibits the formation of abdomen in Drosophila. The hunchback gene is the most important gene in the formation of the anterior and the posterior regions of the embryo. The studies of the expression of the bicoid and the nanos genes clarify the data of literature, such as the formation of a second

abdomen after the destruction of the messenger bicoid RNA (with U.V. light or Rnasi). Transcription factors are very important in controlling the differential expression of genes, but in Eukariotes selective nuclear RNAs processes are more important. These selective processes clarify how the same gene can produce two different proteins in different cells or in the same cell at different times. Besides selective degradation or, otherwise, selective stabilization of messenger RNAs are responsible for further specifications of proteins. Today we have a dynamic vision about the regulation of genes expression. We think that the gene is not an independent and autonomous center of control of the synthesis of proteins. The gene is also controlled directly or indirectly by the synthesized proteins. Certainly the interactions between nucleus and cytoplasm and between cytoplasm and microenvironment are so wide that they constitute a marvelous example of complexity. Developing embryo is an excellent example of what the Santa Fe Institute had come to call “complex adaptive systems”. In fact embryo 1) is a network of many cells acting in parallel, 2) has many levels of organization that are constantly revising and rearranging, 3) has an implicit prediction encoded in its genes and 4) is always in transition and is characterized by perpetual nolvelty.

The experiments in “vitro”

We have seen that cell differentiation consist of a differential regulation of genes that restricts the expressed

genome. Are the substances present in the course of cell differentiation able to regulate also the genes that are important in controlling tumor growth, as I supposed on the basis of results of the previous experiments in animals? To answer this question I made some experiments “in vitro” that evaluated the regulation of some genes in cancer cells: p53; bcl-2; c-jun, and of some oncogenes: ras and myc. I am illustrating here only the results about the activation of p53.

Materials and Methods

Embryos of Zebrafish and trout at the stages of middle-blastula-gastrula, 5 somites, 20 somites were taken and

washed in distilled water and placed in a solution of pure glycerine and 30 % ethylic alcohol at the ratio 4 to 1. The embryos of Zebrafish were sonicated with 2 cycles of 10 seconds each and further treated with a turboemulsifier. The embryos of trout were processed with a turboemulsifier for 3 minutes and then were vacuum filtered through millipore 90 microns membranes and subsequently 10 microns membranes. 100 microliters of these solutions were incubated for 48 hours with the following cultures of stabilized tumoral cells: 1) glioblastoma, 2) melanoma, 3) hepatocarcinoma. The cells of glioblastoma and hepatocarcinoma were grown in Ham F12 containing antibiotics and 5% heat-inactivated fetal serum (FCS), while melanoma was cultured in the same medium containing 20% FCS. Other technical details of cell preparation for analysis have already been described (8).The evaluation of p53 protein was effected with analytical flow cytometry technique and with immunohistochemical method using the alkaline phosphatase detection system. With this last method the intensity of red immunostaining was evaluated in a four step scale (from 0 to +++) and precisely: 0 = non activation of p53 ; + = low activation of p53; ++ = medium activation of p53; +++ = high activation of p53. The different extracts were blind tested and labelled from X1 to X6. I took embryos of Zebrafish at the stages of middle-blastula-gastula, 5 somites, 20 somites for these reasons: Zebrafish is usually taken as study-model of cell differentiation; Zebrafish at the stage of middle-blastula-gastrula, 5 somites, 20 somites demonstrates some peaks of regulation (a lot of genes are repressed or activated in these stages). I thought that the embryos of trout would have silmilar behaviour.

Results

In fig.5 the activation of p53 in the treated glioblastoma cells with X1 extract is reported in comparison with the untreated cells. The treated cells showed with analytical flow cytometry technique a 23.5% higher activation than the untreated cells. In fig. 6 I report the activation of p53 in the treated cells with X3 extract in comparison with the untreated melanoma cells. The treated cells showed with analytical flow cytometry technique a 20% higher activation than the untreated cells. In fig. 7 we can see the untreated cells and in fig. 8 the treated cell with X3 extract of melanoma in which the immunohistochemical method was used. Before the treatment the high activated cells for p53 were 35%, the medium activated cells were 15%, the low activated cells were 5%, the non activated cells were 45%. After the treatment the high activated cells were 75%, the medium activated cells were 10%, the low activated cells were 5%, the non activated cells 10%. In fig. 9 we can see the untreated cells and in fig. 10 the treated cells with X3 extract of hepatocarcinoma in which the immunohistochemical method was used. Before the treatment the non activated cells were 100%; after the treatment the high activated cells were 25%, the medium activated cells 5%, the low activated cells 5%, the non activated cells 65%. Nevertheless the activation of p53 wasn't possible with all extracts, but only with some extracts. In fig. 11 I report the results of p53 activation in the treated glioblastoma cells with 5 different extracts. Only X5 extract increased significantly the level of p53. So it is possible conclude that 2 extracts (X1, X5) increased the level of p53 in the glioblastoma cells. The other 4 extracts were ineffective.

Discussion

These results prove that substances present in the embryo during cell differentiation are able to activate p53 in

different in vitro tumor cells. This activation takes place only after the treatment with specific embryonic extracts. Theoretically, I suggest the hypothesis that each type of tumor is controlled by a specific network of regulators present in the embryo during each step of cell differentiation. So it is possible that the more the tumor is indifferentiated, the sooner the regulators must be taken into the embryo of ovipari (in mammals these regulators could be taken into the uterus wall and into the placenta at the first stages of development). On the other hand we know many factors of differentiation present in the embryo (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) and in the decidua of the uterus (25). Some of these factors present in the embryo (26) and in the uterus (27) are able to control the differentiation and the multiplication of specific tumor cell lines. Generally, the factors that cause cell differentiation are represented by a network or by some family of substances that cooperate in several cascades. It is possible that only when the network of differentiation factors is complete,

does the control of

multiplication and differentiation of tumor cells take place. On the other hand this point of view is the same as that concerning the diet/cancer relationship. Today we know that the complex composition of many factors, no just a single factor of the diet, play an important role in the prevention of malignancy (28,29). The microenvinroment is very important in controlling the multiplication and the differentiation of normal and tumor cells. The microenvinroment of the embryo in ovipari (of the pregnant uterus in mammals ) appears to be the most effective in this kind of control. In fact this microenvinroment is quite capable of leading the totipotent stem cells to a complete differentiation. In the course of cell differentiation the administration of known carcinogens can't induce any tumors in the embryo, probably because the genome control system is always active. Recent studies (30, 31, 32, 33, 34) record that the function of p53 in the embryo is to prevent the malformations. Some authors (35, 36) have therefore defined p53 as “guardian of the baby” similar to the teratological supressor gene. Nevertheless, when embryonic stress is very severe and a large number of

mutations are present, p53 can't repair DNA and causes the apoptosis of all cells (abortion). These processes take place also in tumor cells when p53 is activated. In this sense tumor cells are similar to embryonic mutated cells. On the other hand tumor cells and embryonic cells have the same antigens (37). Today we know that

cancer is constituted by some different diseases in wich many changes are present: activation of proto-oncogenes or, more frequently, of oncogenes; repression or mutation of anti-oncogenes, production of growth-factors. Practically the program of cell differentiation is disactivated in tumor cells. Cancellation of this program involves a renewed expression of embryonic genes and it means that only the program of multiplication

is active. For this reason tumor cells can be considered as cells with “genes configurations” (see box 1) similar to those present in embryo during cell differentiation. Now, if we are to look at cancer therapy, we have to challenge the objective of previous cancer therapy, where its sole objective was the distruction of cancer cells. I suggest we focus on the normalisation and restoration of tumor cells. To achieve this object, we have to change the scientific paradigm by shifting the emphasis from reductionism to complexity. In fact in the disorder of cancer it is impossible to regulate the process with single molecules. We have to use specific networks of regulation. Only a therapy based on a redundancy of specific regulatory factors of cell differentiation can restore the program that is destroied in tumor cells, by passing over the mutations that give rise to malignancy. In this way it is possible to “create” some “cell actractors” which can change DNA and make compatible the code of tumor cells with the code of differentiated cells (see box 2). On the other hand the paradigm of complexity clarifies how biological networks in some conditions are auto-organizing, self repaering and able to create order.

Clinical Trial. Preliminary Results

Briefly, because the main subject of this report is to demonstrate that we need the paradigm of complexity to

understand cancer processes, I am illustrating the results of humans therapy with a redundancy of regulatory factors a very low doses (300-900 microgrammes/day

of total protein) obtained from embryo of Zebrafish. The therapy consists in the somministration sublingually of a mixture of proteins obtained from the stages of medio-blastula-gastrula, 5 somites, 20 somites. The study was made in different clinical centers (38). The treated cases were seriously ill patients, whom the oncologists stopped the traditional therapy of tested efficacy against cancer and patients with a lot of metastasis but still treated with chemotherapy and/or radiotherapy. Patients in the initial stages of their illness and well controlled by traditional therapy were not included in this trial. In 3 years we treated about 400 patients. Here we reported the results of therapy in 200 patients (we have been treated the other patients for a short time and no consideration is possible at the moment). The preliminary report can be sintetized in this way: 1) about 80% of cases showed an improvement of performance-status (we used the Eastern Cooperative Oncology Group system of evaluation) 2) in some cases it was possible to form the hypotesis that the survival time was increased (we described the survival curves with the Kaplan-Meier method

figg.12-13-14-15-16) 3) a small percentage of cases demonstrated a reduction of tumoral masses. This reduction was reported for breast cancer (4 cases), skin tumor (2 basal cell epitheliomas and 1 squamous cell carcinoma), lymphoma non-Hodgkin (2 cases), lymphoma of Hodgkin (1 case), cancer of colon (2 cases), of stomach (1 case), of laryngx (1 case), of lung (1 case), of kidney (1 case), of prostate (1 case), of bladder (1 case), of liver (1 case),

osteosarcoma (1 case). This clinical trial represents a open study, usually made before a case-control study. Without the control group we cannot draw definitive conclusions, except for the non toxicity of the proposed therapy. In fact no adverse effects were observed in all treated patients. This study indicates that it is very important in future to make a control-case study. This therapy at this moment can be considered as complementary to chemotherapy or radiotherapy and still aspecific. Besides the doses, that are intentionally very low to avoid adverse effects, can't be considered sufficient to control completely the clinical situation. We hope that it is possible in future to prepare more specific and effective products.

Open problems and suggestions of future researches

Fig. 16

The acquired experience indicates that the methods of preparation of embryonic extracts is very important. If the method is not well standardized, the extracts could have different effects. For example we practically didn't use the extracts of trout, because many substances were lost during the operations of preparation and purifications and the different extracts demonstrated different effects in vitro. The time of picking up the extracts in embryo is important too: we have to avoid the stages of multiplication that take place between the stages of differentiation. The future of researches could follow these suggestions:

1) production of more active extracts for specific tumors. Probably each kind of tumor requires a specific network of regulation. Our experience indicates that the most aggressive tumors require regulatory factors present at the beginning of embryonic differentiation. In these cases the most effective extracts are taken in middle-blastula-gastrula stage;

2) studies of the effects on tumor growth of specific extracts of decidua of pregnant uterus and of placenta. In experiments in animals these extracts were more effective than the extracts of ovipari embryos in controlling tumor growth;

3) identification of specific regulatory factors of the network for specific tumors. Even if the regulatory program of tumor is complex, it is possible that the single points of the network are more effective in regulating the tumor progression. These factors could be taken in high concentration.

4) studies of effects on tumor growth, when the level of complexity of embryonic or placental extracts increases. We can define with precision the different time of differentiation of each embryonic cell line and take the regulatory factors of this cell line at each different stage of differentiation. Then we can use a mixture that contain the complete differentiative network of this cell line (from the stage of stem cell to a completely differentiated cell);

5) production of high doses of factors that activate p53, by stressing embryo (i.e. with x radiations);

6) studies of effects on tumor growth by embryonic or placental extracts when known, specific growth factors are taken away.

Pier Mario Biava

Scuola di Specializzazione in Medicina del Lavoro

Università di Trieste

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