A New Approach to Metastatic CancerPrevention: Modified Citrus Pectin (MCP)

Alternative Medicine Review ◆ Volume 1, Number 1 ◆ 1996

Parris M. Kidd, PhD

Abstract
Citrus pectin (CP) is a commercially available, water-soluble fiber with provenhealth benefits. The branching polysaccharide structure of CP can be altered to produce a lower molecular weight, galactose-rich, modified citrus pectin (MCP) which hasunique properties. Specifically, MCP, but not CP, might help retard cancer metastasisby combining with an array of galactose-specific proteins on the cancer cell surfacecalled galectins (for galactose-specific lectins). As with many human cancer cell linesthat have been studied, the potentially metastatic B16-F1 (mouse melanoma) and MLL(rat prostate) cells carry galectins, cell surface proteins that bind to galactose on neighboring cancer cells andoligosaccharides on the host cell surface. MCP inhibits metastasis by the cells in the mouse and the rat, respectively. Unlike the much larger CPpolysaccharide, galactose-rich MCP may be small enough to access and bind tightlywith galectins on the cancer cell surface, saturating the galactose binding sites of thecancer cell lectins, and thereby inhibiting both aggregation of tumor cells and adhesionto normal cells. Thus deprived of adhesion, the cancer cells fail to metastasize. Undeniably, important gaps still exist in the current understanding of MCP’s clinical efficacyand its mode(s) of action. But MCP’s apparent safety and proven anti-metastatic action, and the lack of proven therapies against metastasis, together may justify its inclusion into comprehensive orthomolecular anticancer regimens.(Alt Med Rev 1996;1:04-10.)

Citrus Pectin (CP) and Modified Citrus Pectin (MCP)

Pectin is a water-soluble fiber that is present in the cell walls of all plant tissues, whereit functions as a cementing material to hold adjacent cells together. As a human dietary supplement, pectin prepared from citrus (CP) offers multiple health benefits.1-3 Results from preliminary animal studies indicated that the biological effects of pectin could be altered by modifyingits methyl group distribution patterns. More recent evidence indicates that a more extensivelymodified citrus pectin (Modified Citrus Pectin, MCP) may be capable of retarding cancer cellmetastasis. MCP may specifically combine with, and block, lectin molecules on the cell surface that mediate metastasis. These findings and their implications for human cancer management are the subject of this review

FIGURE 1.
Cell-to-cell interactions mediated by lectins. (A) Between cells of the same type; (B) between cells of different types

Chemically, pectin is the collectivename for a group of heterogeneous, highmolecular weight, branched polygalacturonic acid polysaccharides. Thegalacturonic acid makes up the bulk of a central chain, which is composed of partiallyesterified D-anhydrogalacturonic acid(AGA) units, linked through alpha (1—4)glycosidic bonds and interrupted in placesby (1—2) linked L-rhamnose sugars. Theside chains are composed mostly of the sugars D-galactose, L-arabinose, Dglucose, andD-xylose. Typically, some of the hydroxylgroups of the sugars are methoxylated, andthe most common pectin as isolated fromcitrus (citrus pectin, CP) contains up to 10%methyl ester groups.

Commercially available CP is extensively branched and averages 70-100 kDa.MCP was first produced by the relativelycrude technique of high-pH treatment todegrade the main galacturonic acid chainsfollowed by low-pH treatment to partiallydegrade the carbohydrates. These steps result in more or lessnonbranched,smaller carbohydrate chains averaging 10 kDa.4 Moreelegant techniques (e.g., U. S. Patent5,498,702) have since been developed thatmore effectively standardize the product.

Modified Citrus Pectin andExperimental Metastasis
MCP produces markedly different biological activity than does CP, since MCP contains relatively more sugar groups able to specifically block the surface carbohydrate binding proteins known as lectins. Galactose-binding lectins (galectins) are thought to give cancer cells much of their metastatic potential.Figure 1 illustrates various types of cell-tocell interactions mediated bylectins.

Metastasis, the spread of cancerthroughout the body, has been described eloquently5 as the “apotheosis of neoplastic transformation.” Whatever one’s philosophicalperspective on this pathophysiologic process,metastasis is undeniably one of the mostlifethreatening aspects of cancer. Atthis time,very few effective therapeutic tools againstmetastasis exist. Preliminary research findingssuggest MCP does have value for this application, especially since animalstudies suggestthat there may be no acute toxic effects in humans.Research aimed at elucidating themechanisms of metastasis has been ongoingfor more than a century, and this process (orfamily of processes) has been well studied invarious animal and “in vitro” experimentalmodels.

Metastasis proceeds in stages:
• A malignant tumor develops somewhere in the body;
• At some point in the tumor’s development, groups of cells break away(called detachment);
• These detached tumor cells invadethrough the surrounding solid tissue toreach body cavities, the lymphatics, orthe blood stream;
• Cells are transported by blood orlymph to distant tissues;
• Cells arrest on the distant tissues,invade, and proliferate.
The essence of metastasis is the transfer of cells from one location within the bodyto another. With respect to how groups ofmalignant cells become translocated, twoschools of thought exist, both of which appearto have validity to clinical metastasis.5,6 Oneis the “anatomical-mechanical” model, whichmaintains that translocation has to do morewith physical events that “trap” cells in certain locations. The other is the “seed-soil”model, which propounds that metastatic tumorcells take root in another tissue only as theyare allowed by favorable conditions for attachment and proliferation at that location. Thecurrent consensus position is that these suggested mechanisms are not mutually exclusive.Both are seen clinically, and in the very sametumor system some malignant cells can metastasize mainly by “anatomical-mechanical”and others by “seed-soil” mechanisms.6

The life-threatening nature of metastasis dictates that virtually all the studies on itsmechanisms must be done in animal modelsrather than in human subjects. However, animal models can at best offer only clues to understanding human disease. In the case of metastasis, the usual limitations of animal models are further complicated by the profoundchanges that tumor cell lines undergo whenmaintained in culture. For animal cancer models to have clinical relevance, they must firstbe carefully evaluated and refined. In thiscontext, one of the better animal models forthe study of metastasis is the mouse B16-F1melanoma, which is frequently used to studymetastasis, in part because it is highly metastatic in predictable patterns.

Following injection into the mouse tailvein, the B16-F1 melanoma cells consistentlymetastasize to the lung. Platt and Raz,7 tocompare the anti-metastatic potential of CP toMCP, used this system in two experiments withthe C57BL/6 mouse. The first utilized 53 micein 5 groups, and the second, 125 mice in 3groups. After 17 days, the mice were autopsied and the tumor colonies in the lungs werecounted. The results were clear-cut: CP failedto block metastasis, whereas MCP blockedmetastasis in the concentration ranges of 0.05-0.50% contained within the 0.2 cc used forinjection.

This study successfully demonstratedthe anti-metastatic potential of MCP. However, the one-time administration of cancercells into the circulation of the mouse is notlikely to be representative of the situation inthe human cancer patient. Such a patient islikely to have one or more primary tumors thatseed off a significant number of cells on manyoccasions over a prolonged period of time.Results from more experiments with MCP, thistime done with rats, do give further supportfor a clinical anti-metastatic action of MCP.

Raz and his collaborators8 have alsostudied metastasis in the Dunning rat prostatemodel. Dunning developed this metastasismodel from a spontaneously occurring prostate adenocarcinoma found in the maleCopenhagen inbred rat. Selection in culturefrom the primary tumor led to an aggressivelycancerous subline called MAT-LyLu (herecalled MLL). Injection of 1 million MLL cellsinto the male rat predictably leads to deathwithin 27 days. Metastasis begins at 10-12days after the inoculation. If the primary tumor is removed by limb amputation before thispoint, the animals do not die; if amputation isperformed after day 12 the animals die of lungand lymph node metastases by day 40.

Pienta, Raz, and their collaborators8used this MLL-Copenhagen rat model toevaluate the efficacy of orally administeredMCP against metastasis. On day 4 after injection with the MLL cells, groups were put ondrinking water containing 0.00%, 0.01%,0.10%, or 1.00% MCP. On day 14 the tumorbearing hind limbs were amputated under anesthesia, and the tumors weighed. At day 30the animals were sacrificed and the numbersof MLL secondary tumors counted.

Orally administered MCP did not affect primary tumor growth in the rats. However, MCP did inhibit metastases. In the 2control groups (0.00%, 0.01% MCP), 15 ofthe 16 rats had lung metastases (the 16th hadonly lymph node metastases). Of the groupsof rats receiving 0.10% and 1.0% of MCP intheir water, roughly half in each group had nometastases. In addition, the 1.0% MCP grouphad markedly fewer metastases in the lungs(P<.001) and lymph nodes (P<.01) when compared to controls.

The effective concentrations of MCPfell within the range of the earlier mouse experiment, in which MCP reduced the metastatic viability of the B16-F1 melanoma cellswhen mixed together with them prior to injection. Pienta et al8 note that “the concentrations of modified citrus pectin that inhibitmetastasis in vivo are similar to the concentrations of modified citrus pectin that inhibitadhesion and colony formation in vitro. Thismay reflect that concentrations in the bloodstream may need to be similar to those observed “in vitro.” To better critique these experimental findings in the context of clinicalmetastatic cancer, it is necessary to explore thecell-level mechanisms of the biological actionof MCP.

The Biology of “Fit”: Cell SurfaceLectins and Metastasis

Both the “anatomical-mechanical” and“seed-soil” hypotheses for cancer cell metastasis require that cancer cells first must adhereto a new tissue into which they have been transported, then they must stabilize and proliferate to establish new tumor foci.6 For adherence to occur, the cancer cells must have somecapacity to specifically recognize a particulartype of host tissue. Specific molecules arelocated on the cancer cells’ surfaces to ensurethese functions. Although the basic processesof cell-to-cell recognition appear to be thesame in normal and abnormal cells, the cancerous cells seem to have their own specificsurface patterns that favor specific recognitionof host tissues compatible with their survival.6,9-13

Cell-to-cell recognition occurs by wayof molecules that interact following the principles of “lock-and-key” correspondence, orsimply “fit.” This concept was first championed by Emil Fischer almost a century ago, toaccount for the specific interactions betweenenzymes and their substrates in solution. Later,it was extended by Ehrlich, then by Lillie, tothe interactions between cells in the solidphase.9 It is now known that a type of “fit”between cell surface recognition molecules underlies the preferential associations betweenmetastasizing cancer cells and their target organs.10-13

The modern concept of “fit” in cellto-cell adhesion involves two major components of the cell surface: a carbohydrate-richprotein (glycoprotein) and a carbohydrate-poorprotein (lectin).9 Details of the recognitionphenomenon still remain to be elucidated, butit appears to be dependent upon the characteristic surface patterning of thousands of glycoprotein and lectin molecules carried by eachcell. As one cell comes into contact with another, the information inherent in the patternsof the glycoprotein sugars can be “recognized”by lectins on the other cell. The multiple“keys” on the two cells can then fit into themultiple “locks” of both, and recognition occurs (see Figure 1). Structural and functionalchanges are then triggered in both cells. Thetwo cells have a number of options for furtherassociation: they can proceed into a morestable, closer association; they can move awayfrom each other; or, (as in the case of an immune cell coming in contact with a cancer cellor a bacterium) they can begin to conduct hostile actions toward each other. Interestingly,the “homing” of lymphocytes to lymph nodeendothelium is a process closely related to metastasis.14

Malignant cells capable of metastasistend to differ in their surface glycoprotein andlectin distribution patterns from normalcells.15,16 Two pro-metastatic lectins havebeen identified that have molecular weightsof about 14,500 and 34,000 kDa, respectively.Also, tumor cells with higher metastatic potential tend to carry more of these lectins ontheir surfaces,10-13,17 although there may beexceptions to this trend.18,19 These differencesappear to be the basis for the efficacy of MCPin blocking metastasis.11

There is a consistent correlation between the capacities of B16-F1 cells to aggregate and their potential to metastasize in vivo—the greater the aggregability, the greater thecapacity to metastasize.20 In their study, Plattand Raz7 found that CP increased the aggregation of the cells. This could help explainwhy data from the coinjection experiment indicated CP may have increased metastasis overcontrols. In contrast, MCP blocks cancer cellaggregation and reduces metastatic potential.

In these B16-F1 aggregation experiments, MCP almost completely blocked cellaggregation.7 This outcome is consistent withMCP’s almost complete inhibition of metastasis into the mouse lung.8 Furthermore, Pientaet al8 measured the capacity of MLL cells toadhere to rat aortic endothelial cells, an assaywhich simulates metastasis via blood circulation.21 MCP proved to be a potent inhibitorof MLL adhesion.
Pienta et al8 also cultured MLL cellsin a semi-solid medium of agarose to simulate the cells’ ability to proliferate and establish new metastatic foci. Here also MCPblocked malignant cell colony formation, animportant stage of the metastatic process, atconcentrations of 0.01-1.00%. The investigators searched the MLL cell surfaces for a particular lectin that targets galactose residues,known as gal-lectin or galectin, and confirmedits presence. Similarly, the B16-F1 melanomacells also were found to carry galectins on theirsurfaces.22 To further confirm the relevanceof these experimental findings to human prostate cancer, Pienta et al8 also looked forgalectin in human prostate cancer tissue biopsies specimens. They found galectin in copious quantities in this variety of human cancerspecimens.

Conclusion: MCP’s Promise andLimitations

Commercially available citrus pectin(CP) can be structurally altered using a seriesof pH changes to separate the large, branching, tree-like pectin molecule into smaller andmore linear pieces that average 10,000 kDa.This modified citrus pectin consists of smallpolysaccharides rich in galactose residues.The potentially metastatic B16-F1 melanoma,MLL, and numerous other neoplastic linescarry galectins -- cell surface proteins that seekout galactose. MCP blocks metastasis of thesecells in both mouse and rat models. Unlikethe much larger CP polysaccharide, the galactose-rich MCP may be small enough to access and bind tightly with galectins on the cancer cell surfaces, thus blocking their access tothe galactose of the host cell surfaces or theintercellular matrix. Deprived of a closeenough approach to these surfaces, the cancercells fail to adhere or proliferate and thus failto metastasize.

The significant reduction of experimental lung metastasis by the coinjection ofMCP with B16-F1 melanoma cell is notunique, since other substances are known toblock cancer cell metastasis in this system.Simple sugars like galactose and lactose canblock pro-metastatic lectins and interfere withmetastasis in vivo ,23,24 but these are unlikelyto be suitable for chronic oral intake due tothe likelihood of development of gastric intolerance and the rapid metabolism of simplecarbohydrates. However, in another animalmodel more representative of clinical metastasis, MCP did largely prevent metastasis whengiven orally with the drinking water, beginning 4 days after inoculation of the cancer. Asa soluble fiber, MCP poses minimal risk oftriggering intolerance. MCP still has not beenput to the major challenge that the clinicianmust constantly face, namely to prevent metastases. But MCP is deserving of further research to assess just this capacity, for severalreasons:
• Currently, few other promising antimetastatic interventions are available;
• MCP inhibits metastasis in 2 animalmodels;
• Although unproven against established metastases, MCP could providebenefit to the cancer patient solely byblocking further metastasis;
• The mechanism of action of MCP isrationally understood;
•MCP has been found to be effectivewith both oral and intravenous administration; and
• MCP is unlikely to be toxic, even athigh levels of intake.

Due to the heterogeneity of humancancers and the variability and complexity oftheir metastatic patterns it is unlikely that asingle agent will be discovered that preventsall metastasis. However, in a recent presentation to the American Association for CancerResearch, Naik, Pienta, Raz, and others25 reported that MCP blocked the adhesion to bloodvessel endothelia of 5 different human cancercell lines: human prostate adenocarcinoma PC-3, human breast carcinoma MCF-7 and T-47D,human melanoma A-375, and human laryngeal epidermoid carcinoma HEp-2.

Decades of intense study into themechanisms of metastasis have finally begunto spawn rational approaches to its clinicalmanagement. Whether MCP will ever attainthe status of a major breakthrough for themanagement of metastatic cancer awaits thenecessary clinical trials. Nonetheless, fromits record to date, MCP should be a prime candidate for further clinical investigation.

It would be counterproductive to denythat important gaps exist in the current understanding of MCP, its clinical efficacy and itsmode(s) of action. But MCP’s apparent safetyand proven anti-metastatic action, and the lackof proven therapies against metastasis, wouldjustify its inclusion into comprehensive orthomolecular anticancer regimens.

References
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摘要

柑橘果胶（CP）是市售的水溶性纤维，被证明有利于健康。 CP的分支多糖结构可以被改变来产生一种低分子量，富含半乳糖，有独特性质的改良柑橘果胶（MCP）。具体来说，MCP，但不是CP，通过结合癌细胞表面上叫做半乳糖凝集素（为特定的半乳糖凝集素）的癌细胞表面半乳糖特定蛋白质阵列，可以帮助延缓癌细胞转移。根据很多已研究的人类癌细胞株，潜在转移性瘤B16-F1，（小鼠黑色素瘤）和MLL（大鼠前列腺细胞）携带半乳糖凝集素，细胞表面的蛋白质结合附近癌细胞的半乳糖和宿主细胞表面的寡糖。 MCP通过分别在小鼠和大鼠的细胞抑制​​转移。不同于大得多的CP多糖，半乳糖丰富的MCP足够小来接触和紧紧结合癌细胞表面的半乳糖凝集素，饱和的半乳糖结合癌症细胞凝集素位点，从而抑制肿瘤细胞的聚集和对正常细胞的粘附。因此剥夺了附着力，癌细胞转移失败。无可否认，在目前尚无有效抗肿瘤转移的疗法的情况下，虽然MCP的作用机理尚未完全明确。但由于MCP所具有的安全性和可靠的抑制肿瘤转移的作用，将其作为一种全面正分子抗肿瘤转移疗法具有合理性。