Stem cells maturation arrest
and differentiation therapy.

S. Sell

All tissues of the body arise from proliferation and differentiation of stem cells. The concept that adult tissues contain embryonic remnants that generally lie dormant, but may be activated to become cancer was first formulated as the “embryonal rest” theory of cancer in the mid 1800’s.
This hypothesis was revisited in the 1960’s when Roy Stevens demonstrated that germinal cells from the adult testes could give rise to teratocarcinomas and Barry Pierce showed that development of teratocarcinomas resulted from a failure of the embryonal cancer stem cells to differentiate, i.e. “maturation arrest”. However, up to 99% of a teratocarcinoma may be composed of terminally differentiated mature tissue cells. If cancer results from maturation arrest of proliferating cancer stem cells, then removal of this arrest should permit the cancer cells to resume differentiation. The maturation arrest of embryonal cancer stem cells can be overcome and the cancer stem cells induced to differentiate by transplantation of the embryonal cancer stem cells into normal blastocysts or by treatment with retinoids.
This led to the concept of “differentiation therapy” of cancer.
An illustrative example of maturation arrest and differentiation therapy is the gene translocations in hematopoietic stem cells that lead to specific alterations in gene expression manifested as maturation arrest at a specific stage of white cell differentiation (leukemias) that can be reversed by specific treatments that remove the block to differentiation. The origin of other cancers from tissue stem cells is now being documented. For example, “promotion” of an epidermal cancer may be accomplished months or even years after the initial exposure to carcinogen (“initiation”).
Since all skin cells except the skin stem cell are replaced every 2 weeks, this implies that the original carcinogenic event occurs in a long-lived epithelial stem cell population. Even organs with very slow normal cell renewal such as the liver, contain tissue stem cells. Although the normal cell turnover in the liver is very slow (once every 1-2 years) as compared to the GI tract (once a week), the liver can respond to injury by rapid proliferation of mature hepatocytes, a finding that led to the belief that the liver did not contain stem cells like other organs. However, there is rapid proliferation of putative liver stem cells in response to injury when proliferation of mature hepatocytes is inhibited, and the cellular events during chemical hepatocarcinogenesis using different protocols supports the conclusion that cancers may arise from liver progenitor cells at various stages of differentiation in the hepatocyte lineage. Recent results also reinforce the conclusion that lung cancer arises from stem cells in the terminal bronchioles. Results will be presented which indicate that stem cells from the bone marrow may give rise to the precursor cells of liver cancer and to gastrointestinal cancer.
Understanding the signals that control normal development and differentiation of various tissue lineage cells may eventually lead us to ways to treat many cancers by differentiation therapy. In breast cancers, where the cancer stem cells may be identified by the side population cells which exclude Hoescht 33342 dye the number of proliferating cancer stem cells varies from less than 1 % to 100%. If the signals that allow these cells to remain stem cells can be blocked, it should be possible to force the cells to differentiate.

S. Sell
Wadsworth Center and
Ordway Research Institute,
Albany,
NY, USA

 

The use of stem cells differentiation stage
factors in controlling tumor growth-a new model of cancer.

P. M. Biava

Our previous studies suggest that tumor development in embryo is reduced or suppressed when processes of cell differentiation are in progress. In fact, these studies demonstrated that tumor growth can be delayed or even suppressed by factors present in the embryos of ovipara and in the pregnant uteri of mammals.
In vivo subcutaneous inoculation of Lewis lung carcinoma cells into C57BL/6J mice falied to originate primary masses or metastases when tumor cells were mixed with uterine mucosa extracts from 9-days pregnant mice, whereas mixing of tumor cells with ovipara embryos picked at the blastodermal stage significantly reduced the masses of primary tumor.
These in vivo findings were confirmed by in vitro results obtained after the challenge of several human tumor cell lines of different origin with Zebrafish embryonic factors, especially with those taken during the stage in which the totipotent stem cells are differentiating into the pluripotent stem cells (50% epiboly stage).
The cells, which were treaded with the Zebrafish substances taken during precise stages of embryonic differentiation, showed a significant decrease in the growth rate, with average growth inhibition values ranging from 25% to 80%, Further studies were carried out in order to address which cell regulation pathways are involved in this mechanism of tumor growth inhibition.
It was demonstrated that key-role cell cycle regulator molecules, such as p53 and pRb are involved through transcriptional and post-translational events.
Changes in the expression levels of p53 after treatment of cells with the embryonic differentiation factors, as well as changes in the phosphorylation pattern of pRb , were observed the latter leading to a modification of the hyperphosphorylated/hypo-phosphorylated pRb forms ratio within the whole population of treated cells.
While studying the effects of pregnant uterine mucosa extracts on tumor cells. the interest was tocused on a low-molecular weight fraction (5 kDa) having antiproliferative properties which was isolated from pregnant pig uterine mucosa and which was named “life-protecting factor ( LPF ) Prelimmary in vitro studies showed that the fraction was as effective in delaying tumor cell qrowth through apoptotic events as the whole uterine mucosa extract.
On the basis of these in vitro and in vivo studies a new model of cancer is here proposed In this model cancerogenesis is conceived as a process of deterministic chaos. It is a branching process, that lead the cell to a rampant genetic instability: the final attractor is a new stable “gene configuration” similar to that present in the embryos during the stages of multiplication between two stages of differentiation.
In fact cancer cells have
1) activated proto-oncogenes or oncogenes,
2) produce embryonic growth-factor,
3) are insensitive to anti-growth signal,
4) have several surface antigens known as oncofetal antigens, maintained during phylogeny.
Cancer cells and embryonic cells s’hare some molecular pathways and their key-role effectors: e..g.the APC/be-ta catenin /TCF/ Wnt pathway and the Hedgehog /Smoothened/ Patched pathway.
In embryonic development these pathways lead cells to successful differentiation, in tumorigenesis their mutated counterparts lead cells to constant multiplication.
The use of embryonic substances taken during precise stages of cell differentiation especially during the stage in which the totipotent stem cells are differentiating into the pluripotent stem cells are able to stop or delay tumor growth, by-passing the mutation that give rise to malignancy.

P. M. Biava
Foundation for Research into
the Biological Therapies of Cancer
Sesto San Giovanni Hospital
Sesto S.Giovanni
Milan, Italy.

Apoptosis, phenotype differentiation and growth inhibition of colon cancer cells (Caco2) induced by Zebrafish embryo proteins.

M. Bizzarri

Previous studies showed that a set of regulatory proteins, extracted from fish embryo during differentiating processes, shared some cytostatic characteristics on several cancer cell lines.
An experimental study was carried out in order to ascertain the inhibitory and pro-apoptotic properties of this protein network. Whole cell-count, sulphoronamide b-test (SRB test), apoptotic-parameters (caspases 3 and 8, FLIP, pRb, p53, Myc, E2F), were obtained from colon cancer cells line (Caco2) treated with increasing concentration of the embryonic extracts (E1 3 µg/ml and E2 0.3 µg/ml) in association or not with 5-Fluorouracil (5FU) in the suboptimal therapeutic range (0.1 mg/ml, FU1).
Cancer cell proliferation was significantly inhibited in a dose-dependent manner by embryonic extracts and an additive enhancing effects was evidenced in association with 5FU.
At 48 h the inhibition rate increases from 68% (FU1 alone) to 75% and 82% respectively in E2+FU1 and E1+FU1 treated specimens; the strongest growth-inhibitory effect was recorded in the Fu+E1 arm (82% at 48 h and 92% at 96 h). This pattern is in fact strictly related to the pro-apoptotic characteristics shared by embryo extracts in association with 5FU. Caco2 cells treated with E1/2 showed a marked increase in the G2/M fraction. Embryo proteins shared a significant apoptotic effect (+128% and +147%, for E1 and E2 respectively).
In both Fu+E1 and Fu+E2 treated tumour cells, apoptotic index increases significantly from 41 to 46 respectively, evidencing an effective additive apoptotic effect between Fu and Embryo proteins. Both Fu and E1-E2 treated Caco2 cells presented an increase in caspases-8 activity, meanwhile a selective enhancing effect of caspases-3 activity was recorded only in embryo-treated cells.
A significant increase in pRb phosphorilated forms that leads to an increase in E2F-1 was induced by embryo proteins, jointly to a paradoxical c-Myc overexpression.
As reported for other colon cancer cell lines these data support the evidence of a specific apoptotic enhancing effect shared by embryo extracts. Finally, embryo proteins enhance E-cadherin synthesis - with a concomitant decrease in ß-catenin expression - so exerting a clear differentiating effect on colon tumour cells.
These preliminary results are encouraging and suggest that embryo differentiating factors could improve chemotherapy efficacy, even if exerting autonomous and complex interferences on cancer cells (growth inhibition, apoptotic-induction and differentiating effects) from that recorded with fluorouracil.

M. Bizzarri
Professor at “La Sapienza” University, Rome. Responsible of NMR unit of “Centro Interuniversitario Grandi Apparecchiature Biomediche nelle Neuroscienze (CIGABIN). Member of the Scientific Council of the International Society of Magnetic Resonance (ISMAR)and of the Scientific Committee of the European Experimental NMR Conference (EENC).

A. Cucina1 , M. Bizzarri2, L. Frati2, P. Coluccia1, F. D’Anselmo1,
B. Borriello2, F. Conti3, A. Miccheli3 ,A. Gulino2, P. M. Biava4
1 Dept. of Surgery “Pietro Valdoni”.
2 Dept. of Experimental Medicine and Pathology.
3 Dept. of Chemistry. 4 Foundation for research into the
biological therapies of cancer Sesto S. G. Hospital - Sesto S. Giovanni
Milan Italy

Efficacy of stem cells differentiation stage
factors in intermediate-advanced hepatocellular carcinoma.

T. Livraghi

Background and aim: There is no standard treatment for patients with advanced hepatocellular carcinoma. We developed a product containing substances taken from the stage in which embryonic stem cells are differentiating into adult stem cells that inhibit carcinogenesis in the laboratory. The aim of this open randomized study was to assess its efficacy in patients with hepatocellular carcinoma not suitable for resection, transplantation, ablation therapy or arterial chemoembolization.
Methods: A total of 179 consecutive patients was enrolled. We randomly assigned the patients to receive either regulators of stem cells differentiation or only conservative treatment. Primary end points were tumor response and survival. Secondary end points were performance status and patient tolerance.
Results: 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 with regulators of stem cells differentiation. The results obtained in 154 additional patients confirmed previous results. Analyzing 151 assessable patients, 4 complete responses, 26 regressions, 24 stable disease, and 97 progressions of disease were obtained. Evaluation of survival showed a significant difference between the group of patients with objective response and stable disease versus the group with progression. Of the 23 treated patients with a performance status of 1, 19 changed to 0. Conclusions: The study indicated that treatment with regulators of stem cells differentiation of the patients with intermediate-advanced hepatocellular carcinoma was efficacious, in terms of complete response or regression in 19.8% and in terms of stable disease in 16%, with an improvement in survival for responders versus non responders and in performance status for most patients.

T. Livraghi1, F. Meloni1, S. Lazzaroni2, A. Frosi3,
M. Bizzarri4, L. Frati4, P. M. Biava5
1 Department of Radiology, Civil Hospital of Vimercate,
Vimercate, Milan;
2 Department of Internal Medicine, San Biagio Hospital, Clusone, Bergamo;
3 Hepatology-Gastroenterology Unit, Internal Medicine Department,
Sesto S. Giovanni Civil Hospital, Milan;
4 Department of Experimental Medicine, La Sapienza University, Rome;
5 Foundation for Research into the Biological Therapies of Cancer,
Sesto S. Giovanni Civil Hospital,
Milan, Italy