| Abstract |
Background: Colorectal cancer (CRC) is one of the commonest cancers and the third leading
cause of cancer death in both sexes. The disease progresses as a multi-step process
and is associated with genetic alterations and epigenetic changes. Throughout this mutation
process telomerase activation (hTERT) plays a catalytic role in the tumor progression cascade.
During the last decade multiple studies have linked the cycloxygenase-2 (COX-2) expression
with the pathogenesis of various types of cancers, included the CRC. Although the
actual mechanisms that control COX-2 expression have not been widely understood, it has
been shown that stroma cells of both, tumors and the adjacent normal tissues, contribute to
COX-2 over-expression resulting in PGE2 synthesis from arachidonic acid. Peptide hormone
somatostatin and its receptors (sstr) have a wide range of physiological functions and play a
role in the treatment of numerous human diseases, including CRC. Somatostatin and its synthetic
analogue, octreotide, are potentially active against CRC due to their anti-proliferative
and apoptosis-induced activity. Octreotide inhibits growth of colonic cancer cells. Insulin is a
peptide hormone produced by the beta pancreatic cells, and it has also been shown to initiate
mitogenic signals in certain cell types, acting as a trophic factor in tumor cells.
Methods – Aims: The aim of this study is to evaluate the in-vivo telomerase activity by [Real-
Time Polymerase Chain Reaction (RT-PCR)] in colon and rectal cancer tissues and their
corresponding adjacent normal mucosal tissues [10cm away from the tumor]. We compared
hTERT in tumors located in different colon locations [right colon, left colon and rectum] and
analyzed the association between hTERT with patients’ clinicopathological features [sex,
age, BMI, smoking habits, tumor differentiation (grade), Dukes & TNM stage] and survival.
We also studied MLH1 as an indicator of Microsatellite Instability (MIS), while p53 and other
molecular markers [Ki-67, Bcl-2] were also studied and correlated with telomerase activity.
Our aim also is to study the immunohistochemistry expression of COX-2 together with PGE2
synthesis on CRC and to correlate with a) clinicopathological parameters of the patients and
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b) telomerse activity (hTERT). Furthermore the in-vitro aim of study was to subsequently
study the effects of the somatostatin analogue octreotide with or without the trophic effects of
insulin on cellular proliferation of colon cancer cell lines (Caco-2 & HT-29) and octreotide action
in correlation with hTERT. We also investigated the effect of the protein of tyrosine
phosphatases (PTPs) and telomerase activity on cellular proliferation.
Results - Discussion: In our study we included 49 patients (21 women) with a median age
of 74 (range 49–87) years with biopsy-confirmed CRC. There was a significantly higher telomerase
activity in colon cancer tissue compared to normal tissue (p=0,006). Adenocarcinomas
of the colon cancers express significantly higher hTERT compared to rectal cancers
(p=0,012). The same was repeated in the adjacent normal tissues (p=0,035). Furthermore
colon cancers and their adjacent normal tissues had significantly more telomerase and were
more positive to MLH1 compared to rectal cancers. The expression of p53 was negatively
correlated to telomerase activity but was linked to better patient survival. As far as the immunohistochemistry
expression of COX-2 is concerned, that was positively expressed in 40
out of 49 cancer tissues (81.6%). The immunostaining was cytoplasmic, heterogeneous and
its intensity was ranged from mild to intense. COX-2 was also expressed in less extend in
tumor stroma. The same COX-2 expression was also observed in the adjacent normal tissue
stroma cells. The latter was also confirmed when PGE2 synthesis was found to be higher in
normal mucosa tissues compared to the adjacent cancer tissues (p=0,013). There was no
statistical significant relation of COX-2 expression with the above-mentioned patients’ clinicopathological
parameters. Interestingly, adjacent normal tissue of Dukes-C/D stage tumors
had significantly less PGE2 synthesis (p=0,036) and lower telomerase activity (hTERT)
(P=0,005), than adjacent normal tissue of Dukes A/B-stage tumors. These results might imply
that in early stages normal epithelium or its surrounding stroma cells might be more susceptible
to new telomerase or COX-2 respectively, driven tumor formation while in late stages
the extent of the systemic illness may decrease this potency or that there might be a less
powerful regeneration process of the normal colonic epithelium in late stages of colon cancer.
When tumors were grouped by a) right colon (cecum, ascending colon, transverse co49
lon), we observed higher hTERT activity and less PGE2 synthesis and b) by left colon (splenic
flexure, descending colon, sigmoid and rectal cancers), we observed more but not significant
PGE2 synthesis and significantly lower hTERT activity (p=0,012). The in-vitro experimental
protocol, determined elevated expression of sst1, sst2 and sst5 in both colon cancer
cell lines (Caco-2 & HT-29) by RT-PCR. Immunocytochemistry detected sst1, sst2A, sst2B,
sst3, and sst4 and sst5 protein expression in the membranes of both cell lines. Octreotide
treatment inhibited the proliferation of Caco-2 and HT-29 cells through the protein of tyrosine
phosphatases (PTPs). Insulin treatment exerted proliferative effects while octreotide reversed
its effect in both cell lines. Sodium orthovanadate (Na3VO4) suppressed the antiproliferative
effect of octreotide both in Caco-2 and HT-29 cells. After octreotide treatment,
colonic cancer cell-lines (Caco-2 & HT-29), that grow in cultured medium (10%FBS), exert
their action on hTERT, through different signaling pathways.
Conclusions - Applications: Our findings further support the concept of multiple molecular
entities within the spectrum of CRC. The differences that others and we have identified in
telomerase activity and COX-2 expression, in colon and rectal cancers may be related to the
diversity of carcinogenesis and disease progression mechanisms in these distinct locations
that can be attributed to their discrete embryology, anatomy, physiology and molecular biology.
We confirmed that colon and rectum behave as different entities (organs) with different
characteristics and properties. Furthermore it seems like COX-2 is implicated in the initial
steps of colorectal carcinogenesis progression, affecting initially the adjacent normal colon
surrounding stroma cells and then move to colon cancer epithelial cells. Treatment should
only be given for the prevention of CRC, especially for the left colon and rectum where COX-
2 was highly expressed. The difference in p53 expression between right – left colon and rectum
together with p53 prognostic role, will positively contribute to a better design of chemotherapy
treatment. It would be thus important to investigate in which way p53 expression
could benefit patient’s response on particular chemotherapeutic regimens. Our in-vitro data
indicate that the treatment of metabolic syndrome (insulin resistance), may suppress carcinogenesis
at least in certain locations on colon and rectum. The use of octreotide could pro50
vide a possible therapeutic approach for the treatment of patients with colon cancer at certain
locations in the colon and rectum.
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