NATUR-LEAF ®

Sterols and Sterolins - a
Natural Health Supplement

APOSTOLIC SCHOOL OF NATURAL MEDICINE
Peoples University of the Americas
Ponce, Puerto Rico

XIX. CARIBBEAN CONVENTION OF SCIENCES
VOLUME 18, 16 /1999

EXTRACELLULAR MICRO ENVIRONMENT

PETER R. ROTHSCHILD

Copyright © 1999 by Peter R. Rothschild. All rights reserved.
No part of this publication may be used or reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system without written permission from Peter R. Rothschild.

ABSTRACT

Extracellular Micro Environment

Author: Peter R. Rothschild; Source: XIX. Caribbean Convention of Sciences Volume 18, 16/1999

Discussion: Sprout-derived sterols, when associated to sterolins, yield manifold, revolutionary combinations of antibody-regulating enzymes, that energize the "Fc" fractions of antibodies in general, teaming up with sprout-grown biochemical factors, which, in turn stimulate the "Fab" fraction of the same antibodies, thus completing a natural immune-enhancement cycle that also prevents antibodies from initiating auto-aggressive phases, that create autoimmune diseases. Such sterol/sitosterolin combinations are available in capsule form that are easily administered and are totally void of toxicity and side effects.

THE INFLUENCE OF THE EXTRACELLULAR MATRIX ON THE TISSULAR MICRO-ENVIRONMENT

--Peter R Rothschild

When I was a young student, I never ceased to wonder why don't the cells of our ears turn into knee cells? How do they know to what morphology and functions they have to conform to when they develop? The answers I harvested invariably gravitated around genetic codes and other such sundries. Alas, none of these explanations did render intelligible the mechanisms that are responsible for these biological feats.

Yet, the biotechnological pursuits in which I was involved since the very beginning of my professional life consistently required coherent answers to these questions. Eventually it became obvious to me that, for reasons unknown, both cytologists and pathologists steadily dismissed the complex microenvironment that surrounds all cells in any given tissue. Ever since the field of histo-pathology evolved, all that was examined were clusters or individual cells, carefully removed from the dense fibrous grid of proteins and sugars that usually enfold them.

However, this only became obvious about 20 years ago, and the extracellular matrix was not recognized until recently. Nonetheless, it became gradually evident merely a short while ago. For, researchers considered these integral tissue parts as mere filler and padding materials

Eventually it was discovered that protein components - called lamiriin and fibronectin, for instance - bind to specific molecules known as integrins on the cell surface By way of these over 20 integrins the matrix emits specific signals to the cells, that regulate with high precision which genes are to go active, and at length determine whether the cell will differentiate - specialize, that is - mitose, migrate, or, in some instances even carry out apoptosis (virtual, biochemically committed suicide).

Recent research revealed that the extracellular matrix unquestionably possesses the ability to command cells to activate and de-activate both tissue and function-specific genes. Thus, cells from totally different organs and tissues surveyed by conventional microscopic methods are almost indistinguishable when they are removed from their specific extracellular matrix - their normal habitat - and placed on a substrate or vitreous surface under a microscope. Not until pertinent molecules are attached to their surfaces do cells begin to differentiate. In other words, cells acquire their organ - and tissue-specificity under the explicit influence of the appropriate molecules of the matrix.

These findings eventually crystallized into the prevalent new concept that the congruity of the microenvironment outside the cells endows these with the tissue's particular specialization. Quite reasonably, nearly all investigations focused on the matrix's influence on normal cells and tissues. Nonetheless, more recently widening research efforts are being dedicated to explore these mechanisms in the development of cancer.

The hitherto achieved results have produced some stunning surprises. For instance Mina J. Bissell and her team, working at Lawrence Berkeley National Laboratory in California, has demonstrated not long ago that antibodies blocking the functions of certain integrins, appear to transform malignant breast cells into normal mammary cells. Needless to say, that such findings suggest entirely new anti-cancer strategies.

The majority of human cancer forms originate in the epithelium. That is, in the layers of cells, which constitute the external stratum of the skin and also line both, internal and external surfaces of most organs, populating a laminin-rich basal layer, called the base-membrane, which separates them from the rest of the organ to which they belong.

Researchers have known since quite some time that malignant epithelial cells are frequently able to perforate the base-membrane. Once they achieved to get through, they are enabled to metastasize and expand to further areas of the body. The proteolytic enzymes that allow malignant cells to break down the matrix are called proteases. Now, while proteases - used in appropriate combinations, doses, and forms of administration - are unquestionably useful in a variety of cancer therapies, recent findings suggest that they can also be vital in early tumor development.

Bissell's team, collaborating with the research group headed by Zena Werb at the University of California in San Francisco, administered mice a copy of the gene for stromolysin-1, which is a matrix-degrading proteolytic enzyme. Breast cells usually secrete this protein during involution; that is, during the metamorphosis that takes place in the extracellular matrix after pregnancy, when the breast reduces its size and ceases producing milk.

The teams designed the added gene to become active much earlier, during the middle, or the end of the pregnancy. Quite unexpectedly an important percentage of the test animals developed breast tumors. The surprise stemmed from the fact that stromolysin-1 is not considered an oncogene - carcinogen - yet, the animals developed tumors.

The exact mechanism through which stromolysin-1 induces cancer, is not quite clear yet. Nevertheless, the very fact that tumors can develop merely by damaging the extracellular matrix insinuates that a normally functioning matrix does indeed incorporate tumor-suppressing capabilities.

Additional data that supports this postulate was obtained from a test developed again by Bissell's group and a team led by Ole W. Petersen from the Panum Institute in Copenhagen. At the time the researchers were struggling with precisely the practical, yet complex problem, which I have lust mentioned, and which cell biologists were now facing with increasing despair: How does one tell a breast cancer cell apart from a normal mammary cell?

The answer should apparently be easy. Pathologists perform this task with relative ease, as a daily routine. They achieve this by removing a fragment of tissue from the breast - performing a biopsy - and looking for telltale differences in the cell shape. Yet, if the researchers employ enzymes to break down the extracellular matrix and place the freed live cancer cells along normal cells on top of a substrate, they will find it almost impossible to tell them apart.

The thusly-handled cells will look identical and will grow at the same rate. No assay has been designed yet to distinguish the healthy and malignant cells placed within the same culture. To overcome this handicap, researchers founded their investigations on what was known about the mechanisms by which the extracellular matrix determines how specific cells differentiate. For instance, capillary epithelial cells enfold to form a blood vessel, but only if they grow under the direct influence of pertinent matrix molecules. Bissell & Cols., therefore, attempted to build a "virtual breast" by formulating a solution that contained laminin and other basal membrane molecules. Then they mixed mammary cells into the compound and allowed it to gel. It was quite surprising that within this gelatinous mass both, normal and cancerous mammary cells behave in an entirely different fashion. The healthy cells immediately begin synthesizing the components of the basal membrane, creating splendid, magnificently organized structures, that appear totally identical to normal breast cells, which even began secreting milk proteins.

The cells also ceased mitosing - dividing - which was very remarkable because healthy human epithelial cells placed on a substrate do not stop growing.

Whereas, malignant breast cells, when placed amidst such a gel environment, continued dividing and forming enormous masses with little or no structural organization at all. In other words, the tumor cells appear to generate a massive extracellular matrix, but without any functional organization.

Such discoveries inevitably lead us to wonder why cancer cells do not form normal tissue structures. Investigations carried out by the Author & Cols., working at the Apostolic School of Natural Medicine of the Peoples University of the Americas in Puerto Rico, made it obvious that any reasonable research program required means to create cancer cells in vitro, rather than use specimens obtained from live cancer tissues. Thus, experiments were designed in which the researchers grew healthy mammary cells without being aided by any appropriate chemical or biochemical growth factors that are ordinarily available in the auxostatic cell culture medium. As expected, gradually, these cells developed mutations.

The ensuing genetic anomalies ranged from reiterations of entire chromosomes, to specific, recognized carcinogenic gene mutations. The criteria determining when a cell turns malignant, was based on periodical testing their ability of inducing tumors when injected into mice.

It was very interesting to observe that the distribution of a variety of integrins changed eloquently when the cells turned malignant. For, in healthy cells integrins appear in separate, well-defined sectors. Whereas, in cancer cells integrins materialize at random on their surfaces. Furthermore, integrins known as beta-1, were much more abundant than beta-4.

By and large, this was not quite surprising, because the team has already observed that healthy and cancerous cells generate different kinds and quantities of integrins. What we initially did not know was the significance of these differences.

By using three-dimensional cell cultures, Bissell & Cols. discovered that when they blocked the beta-1 integrins with appropriate antibodies, malignant breast cells reorganized within a few days into cells that seemingly were structurally normal. Particularly important was the observation that the cells stopped growing.

Now this was astonishing, because the cells necessarily still carried genetic mutations that incited their malignant behavior. Nevertheless, it was undeniable that the antibodies effectively reduced the beta-1 intern signals within the cells, and that it was this decrease that brought about the stunning cessation of malignant growth. Bissell and her team proved beyond any doubt that both the ratio and composition of integrins prevailing on the cells' surface governs the entire spectrum of tissue morphology and growth.

Since then, we and all those researchers who followed her example, were even able to switch the cells back and forth between normal and malignant-looking tissues, by treating them with antibodies, alternately cleansing them from the same. Eventually, we treated mammary cancer cells with beta-1 integrin-blocking antibodies and injected these into a small group of mice. Only 6 from 17 such animals developed tumors; whereas, 16 of 17 mice, who were parenterally given untreated cancer cells, developed tumors.

A research group in Japan recently used the same integrin-blocking antibodies to grapple with cancer in a most effective way. They injected such antibodies into mice with already well-established tumors. The treatment not only unquestionably arrested the cancerous growth, but also shrank it considerably. All researchers who follow tip Bissell's lead agree that also other integrins seem to be vital for tumor growth. While beta-4 integrins evidently do not inhibit or correct malignant cells, healthy cells treated with the same, lose their organized structure. This discovery suggests that by eliminating the aptitude for recognizing the beta-4 integrins in the extracellular matrix may be an important method for inhibiting cancer development.

The copious variety of existing integrins signifies that the extracellular matrix's influence on cells is not only extensive, but also quite complex. As a matter of fact, we were able to gradually determine that integrin synthesis may be as important for tumor-genesis as the widely studied cell surface proteins that react specifically to human chemical growth factors.

Alan F. Horwitz working at the University of Illinois at Urbana-Champaign, reports that integrin-inhibiting compounds used during tumor formation of blood vessels are already being tested on humans. His contention is that without a steady blood supply, tumors cannot grow to dangerous sizes.

Bissell is convinced that her team's recent experiments with integrin-blocking antibodies offer an impressive argument that cancer research must concentrate not only on the tumor cells' altered links to their microenvironment, but also on the internal shifts that stimulate their proliferation.

Our experiments show that there is little doubt that the structure of the extracellular matrix exerts a paramount influence of the expression of genes in the cell. On the other hand, the very notion that that cancer is reversible, was sheer heresy but five years ago.

The immediate goal of our endeavors focused on finding a non-toxic, a biological, if possible vegetal compound that would act as an all-natural integrin inhibitor. First of all, it had to be determined which of all possible non-toxic substances could be assimilated by human metabolic mechanisms without losing its effective ability to constructively act within the extracellular environment.

Research targeting metabolic mechanisms in charge of regulating endogenous cholesterol synthesis, carried out in Finland beginning over ten years ago ended up targeting sterols and sitosterols yielded by tall pine oils. Sterols represent fatty substances that, in one form or another are produced by all living organisms. In mammals - to which also humans belong - the most frequently occurring form is cholesterol. The main building blocks of all cell walls - membranes - of the 30-odd trillion cells of a human body are built with this lipid construction material. In a far smaller volume, the same cholesterol also serves as precursors for both sex and adrenal cortex hormones (cortisone). Sterols synthesized by plants are called phytosterols, which, though in lesser percentages, represent important factors in plant development, particularly at their genetic level. Sitosterols are fatty substances synthesized exclusively by plants. Notwithstanding, when sitosterols are paired with sterolins - which constitute their specific glucosides - they are able to exert paramount chemical and biochemical influences over the mammalian metabolism and manifold immune mechanisms.

Harvey Schipper from the University of Manitoba at Winnipeg, as well as other researchers, have questioned whether physicians do have to kill every single cancer cell in the body, or wouldn't it be easier to find treatments that acquire control over malignant cell growth. Bissell's work certainly provides plentiful proof that such a procedure is indeed possible to accomplish.

Regrettably, all cancer tissues appear to have the irrepressible ability to eventually outmaneuver their host. Bert Vogelstein from the Johns Hopkins Medical Institutions in Baltimore explains that some cells are capable of developing genetic mutations that enable them to oppose or evade even the most powerful growth-restraints imposed by the extracellular matrix.

The paramount importance of the extracellular matrix research consists in that it emulates the microenvironment in which the tumor evolves, and corroborates the evidence that the matrix can indeed activate or deactivate the genes in the tumor cells. However, though investigators certainly identified scores of genes inducing cancerous mutations, only seldom were they able to explain satisfactorily which are the ultimate mechanisms capable of affecting such genes. Indeed, no clue is revealed when the cells are placed under the microscope or into an artificial auxostatic environment. The only hint the hitherto accomplished research offers is that the extracellular matrix can and indeed does act upon the chromosomal expression.

Curiously, all the research performed in conjunction with the extracellular matrix, is solely dedicated to tumor genesis. We know of no means other than sterols and sitosterolin compounds that would address the potentials of the matrix concerning other pathologies that are regarded resistant to all conventional forms of therapies. Nonetheless, if we accept the unquestionable influence of the matrix over cancer cells, it would appear not less rational that the same mechanisms should also affect all tissues in the body.

After scrupulously analyzing the possible scope of such rationale, we decided to focus our attention on tissues involved in chronic degenerative diseases, in particular on Rheumatoid Arthritis, Lupus Erythematosus, Multiple Sclerosis and Chronic Fatigue Syndrome.

Our first goal was to identify the prevalent mechanism that brings about the unquestionable intracellular influence of the extracellular matrix. Needless to say, such influence necessarily involves information transfer. Thus, it was completely unquestionable that, whichever the mechanism that allows such interference may be, must definitely be based on complex data processing and traffic. After examining several possible avenues of reasoning, our team came to the conclusion that only a process analogous to micro-chimerism could bring about phenomena reported from the afore-mentioned research.

Now, micro-chimerism enacts the much-debated theory that advocates another truly heretic proposition. The postulate upholds that, under given circumstances, exogenous immune cells are capable of virtually discretional alterations of their surface idiosyncrasy, to an extent at which they effectively emulate the host's cells, and operate alongside - though not necessarily cooperate with - his tissues.

The dynamics of the process seem to entail the synthesis of odd chromosomal proteins that reside in centrosomes, kinetochores and nucleic membranes - structures involved in the dispersal of chromosomes in the mitosing cells - that appear to imply the potentials for differentiation due to variable electron spin of the peripheral protein atoms. Imperfect spin combinations produce chromosomal mis-segregation models, which constitute the ultimate etiologic factors of the pathologies in question.

Similar ideas prompted Huntington Potter, a geneticist at the Harvard Medical School, to suggest that some, perhaps many cases of Alzheimer Disease result from abnormal accumulations of certain chromosome proteins - presenilin-1 and 2 - in the affected cells. Potter's studies with antibodies seem to indicate that the abnormal proteins reside in the endoplasmic reticulum (ER), which is the site where the cell synthesizes proteins, and in the Golgi complex, where proteins are frequently modified.

Yet, such studies are often misleading, because they are always carried out on cells isolated from their extracellular matrix. For instance, centrosomes and kinetochores play a crucial role in distributing identical sets of chromosomes within the mitosing cell. The two centrosomes organize microtubules - filaments - along which the chromosomes, led by intracellular light signals, trek to opposite sides of the dividing cell. The kinetochores represent the sites at which the chromosomes attach to the microtubules. Bioavailable sterols and sterolins appear to both stimulate and regulate the adequate synthesis of kinetochores.

In non-mitosing cells, sterols and sterolins probably adhere to the inner surface of the nucleic membrane, and cinch the chromosomes through the kinetochores. When normal cells initiate their mitosis, these presumably assist the process by liberating the chromosomes. Now, while this seems to furnish strong evidence that the process yields specific proteins that participate in chromosome segregation, it is not less obvious that the undesirable mutant versions of the same occur through pathogen influences exerted by the abnormally altered extracellular matrix. Moreover, in case of the non-dividing cells, such chromosome mutations may induce overall cell destruction by attempting to induce the non-mitosing cell to divide.

Peter Davies of the Albert Einstein College of Medicine in New York, published specific data last year hinting that brain cells in Alzheimer's patients may precisely attempt to make them mitose, destroying them thereby.

Our team stipulates that all these parameters are yet quite unexplored and subject to endless challenges. Nonetheless, I find it odd that as to this date no research team has recognized the undeniable potentials of these sterols and sterolins, which clearly indicate that chronic degenerative diseases could possibly be helped quite effectively by correcting and reorganizing the pertinent extracellular matrixes, based on the hitherto obtained above narrated knowledge, and using the appropriate tools developed by contemporary advanced biotechnology.

Currently ongoing clinical studies with orally administered sterols and sterolins address precisely these encouraging issues.