| Abstract |
Colon cancer is one of the leading causes of cancer-associated death among
men and women worldwide. It has been shown that genomic instability, as well as
environmental factors can be correlated to the risk of colon cancer however, the
pathogenesis of the malignancy still remains unknown. Many colorectal cancers are
thought to arise from adenomatous polyps in the colon.
The complex interactions among the cancer cells, their extracellular matrix
(ECM) and the surrounding normal cells are thought to play a pivotal role in tumor
development and progression. The composition of the ECM affects various cellular
functions, such as proliferation, adhesion, migration and differentiation.
Proteoglycans (PGs), major constituents of the ECM, through their core proteins or
via their glycosaminoglycan (GAG) chains are able to interact with collagens, growth
factors, growth factor receptors and adhesion molecules and through these
interactions may participate in the regulation of many cellular events.
The molecular characteristics of colon cancer cells and their stroma, as well as
their association with the development and the progression of the malignancy have
not been adequately studied. The structural analysis and the role of GAGs/PGs that
are produced by colon cancer cells and the surrounding cells may contribute to the
better understanding of these glycocomplexes, which participate in the regulation of
numerous cellular events, as well as in the cell malignant transformation. The
understanding of the interactions among cancer cells and their microenvironment may
contribute to colon cancer treatment.
In this study, the synthesis and the distribution of GAGs among the cell
membrane and the culture medium by two human colon cancer cells, HT29 and
SW1116 were examined. The results demonstrated that both estrogen receptorpositive
(ER+) cancer cell lines produced hyaluronan (HA), both extracellular and
membrane-associated galactosaminoglycans (GalAGs) and heparan sulfate (HS), with
the HT29 cells producing all GAG fractions at significantly higher rates.
Abstract
VIII
The effects of genistein on the synthesis of these secreted or cell-associated
macromolecules, as well as on the growth of HT29 and SW1116 cell lines were also
studied. It is well known that the soy isoflavone genistein can affect cell metabolism
by specifically inhibiting protein tyrosine kinase (PTK) and/or interacting with the
estrogen receptors (ERs). The observed dose-dependent inhibitory effect of genistein
on the synthesis of both secreted and cell-associated GAG/PG by the SW1116 cells,
as well as on their growth, was suggestive of a PTK mechanism. On the other hand,
the synthesis of GAGs/PGs by HT29 cells in the presence of genistein was dependent
on their type and localization which implies the active participation of the ERs, which
was further supported by the observed growth stimulation at low concentrations of
genistein.
Quantitative and qualitative changes in GAGs/PGs production, and
consequently in the ECM composition, have been suggested to have a role in the
development of some types of cancer. Therefore, the effects of endogenous and
exogenously added GAGs on the proliferation of HT29, SW1116 and HCT116 human
colon cancer cell lines were examined. For this purpose exogenously added GAGs
(chondroitin sulfate-CS, dermatan sulfate-DS, hyaluronan-HA and heparin), an
inhibitor of endogenous GAG sulfation and specific glycosidases to cleave cellassociated
glycosaminoglycans were utilized. The obtained results showed that colon
cancer cell growth was exclusively stimulated by exogenously added heparin and
insensitive to endogenous GAGs/PGs production, in a sulfation pattern-related
manner. Numerous mechanisms of heparins’ action have been postulated including
the regulation of growth factors / growth factor receptors, mitogen activated protein
kinases (MAPKs) activities or the modulation of cell cycle progression. Specifically,
HSPGs/heparin have been demonstrated to increase the affinity of FGF-2 to its
respective, FGFR1-4, receptors. The active role of FGF-pathways has been suggested
in the early stages of colorectal tumorigenesis. Moreover, it has been shown that HS
chains stimulate the proliferation of colon cancer cells via a mechanism involving
increased expression of tyrosine kinase receptors of EGF. Aim of this study was to
examine the possible involvement of the FGF-2 and EGF signaling on the heparininduced
effect on HT29, SW1116 and HCT116 colon cancer cell lines. Treatment of
Abstract
IX
the tested cell lines with the FGF-2 neutralizing antibody or an EGFR tyrosine kinase
inhibitor showed that the stimulatory effect of heparin on the cells’ growth was not
FGF-2 or EGFR dependent.
In view of the fact that MAPKs signaling involvement on colon cancer cell
growth is well established, three major MAPK pathways, JNK, ERK1/2 and p38,
were examined for their participation on the mitogenic effect of heparin. The results
showed that heparin-induced stimulation of colon cancer cell growth was correlated to
increased phosphorylation of p38 MAP kinase. Treatment with a highly specific p38
kinase inhibitor significantly inhibited colon cancer cell heparin-induced growth,
while administration of JNK or MEK1/2 kinase inhibitors had no significant effect.
Furthermore, heparin through a p38-dependent mechanism modulated the expression
of p21WAF1/cip1, p53 and cyclin D1 cell cycle regulators in a manner supportive of
colon cancer cell proliferation. Heparin treatment also promoted cell cycle
progression in cells with increased S-phase entry via a p38-dependent mechanism. On
the other hand, treatment with heparin did not appear to affect HT29, SW1116 and
HCT116 cell apoptosis rates. This study introduces a new role of heparin in the
regulation of colon cancer cell proliferation through p38 MAP kinase signaling.
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