Differentiation Therapy by Extracts of Embryo Cells: A Possibility for Cancer Treatment

Various chemical and biochemical differentiation compounds have been shown to revert to normal state several kinds of cancer cells. As embryonic and cancer cells present similarities of behavior and phenotype and as regulatory factors in young embryos have been found, a treatment of cancer cells by embryo cell extracts could be possible. These factors were prepared from rat and mouse embryonic cell nuclei and partially purified by high performance liquid chromatography. They inhibited up to 90% DNA synthesis of LFCl2A, an established cell line from a hepatocarcinoma and of Raji, a B cell line at the stage blast 1 from a Burkitt lymphoma. They prolonged the survival time of rats injected with LFCl2A and increased the percentage of survivors at one month. In the same way, the inhibitory and apoptotic effects of Zebra fish proteins on human colon cancer cells and hepatocellular carcinoma have been reported by Italian researchers. As these factors were not cytotoxic and were active at low concentration, they could be used repeatedly on the remaining cancer cells, after surgery, radiotherapy or chemotherapy.


Introduction
Surgery, chemotherapy, and radiotherapy, (at total 88.5% of the studies) [1], are the main methods used for the treatment of cancer, owing to a mean of 64% of the cancer patients survive at least 5 years after their diagnosis [2], with a huge variation in survival between cancer types. Their aim is to remove or to kill as many cancer cells as possible, while preserving the normal tissues, their flaw is to leave inevitably some cancer cells or to

Regulatory Factors in Embryonic Cells
The embryo contains differentiation factors that regulate cellular gene expression and enable the normal development of the different organs.
This ability of regulation can be applied to certain types of cancer cells: a blastocyst regulates one injected teratocarcinoma cell consistently, which contributes thereafter to the normal tissues of the adult [22,23]. However, the results are less decisive when several cells are injected together or when B16 melanoma cells are used [24].
Similarly, when injected into 10 day old mouse embryos, myeloid leukemia cells loose their malignant character, and the apparently healthy adult mice have granulocytes containing a marker derived from the cancer cells [25].
This regulating capacity of mouse embryos decreases with age: towards myeloid leukemic cells, it disappears after the11 th day [25].
Thus, it seems that differentiation factors, probably proteins, are present in the young embryo and are able to revert certain types of cancer cells into normal, non pathogenic, differentiated ones. They are in general absent or ineffective in adult animals, since ascite tumours or teratocarcinoma develop in adult mice. In the latter case, some cells differentiate (teratoma), while the other ones divide rapidly (carcinoma), likely by depletion of regulatory factors from the embryo following the destruction of its structure and by lack of differentiation factors from the adult receiver.
The factors of differentiation working by binding on DNA must be mainly localized in the nuclei of the cells of the embryo [26]. Moreover, the separation of the nuclei has the advantage of eliminating the cytoplasmic proteins.

Extracts of Rat and Mouse Embryo Cell Nuclei
This hypothesis has been tested with rat and mouse embryo nuclei [27,28], prepared by a modification of the method of Widnell and Tata [29] and the activity of their extracts have been tested on LFCl 2 A, an established cell line derived from a hepato carcinoma induced in the Commentry rat by 4 dimethylamino-azobenzene (Villejuif, France) and on Raji, a B cell line at the stage blast 1 originating from a Burkitt lymphoma which expresses EBV early viral antigens. [30,31].
To evaluate the biological effect of the different fractions studied, the variation of the DNA synthesis, measured by the incorporation of 3

Some particular points must be emphasized
The corresponding oncogenes of different species (human, cat, rat, chicken, insects) present a great similarity [32]. One can conceive that the transcription factors have been conserved too during evolution and that it would be possible to regulate cancer cells of one species with differentiation factors from another one. Actually, we have observed that extracts of rat embryo cell nuclei were active on rat hepatoma and on human lymphoma cell line.

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Except perhaps in the case of organ regeneration [33], normal differentiated cells do not produce (or not enough) regulatory factors able to revert cancer cells: injected ascite tumours, as spontaneous tumours, can generally develop in adults.
Conversely, are the extracts of embryo cell nuclei active towards the normal differentiated and stem or germ cells of the organism? This point ought to be the subject of further study and could eventually be solved by chromatographic separation.

Biological Activities of Zebrafish Embryo Extracts
The same approach has been followed by Italian researchers, who have shown to inhibit proliferation and induce apoptosis in colon tumours [35].
Out of 38 patients with advanced hepatocellular carcinoma, treated with these stem cell differentiation stage factors from Zebrafish embryos, 5 of them presented a sustained complete response, and no side effects occurred [36].
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 [37,38].

Conclusion
These data show that extracts from mouse and rat or Zebrafish embryo cells contain factors that inhibit the proliferation of cancer cells, prolong the survival time of rats injected with them, and enhance the apoptosis rate. It seems that they can be obtained from different species and purified by the general chromatographic methods. They are not cytotoxic since the number of cells incubated with them does not decrease, and not immunogenic, as they are in very low concentration. Consequently, this treatment could be repeated several times, in order to revert the remaining cancer cells, in the cases of treatments by surgery, radio or chemotherapies.