The main objective of this proposal was to investigate the effect of vitamin B6 on colon carcinogenesis in vivo. Two in vivo studies were conducted to determine the role of vitamin B6 as a biological modifier of colon carcinogenesis. It is hypothesized that vitamin B6 may serve as an antioxidant in vivo and will modulate colon carcinogenesis. In the first study, a 2X3 factorial experimental design was used to determine if three different levels of vitamin B6, classified as low, normal and high in conjunction with two different levels of protein intake, classified as normal or high, will affect post-initiation stages of colon carcinogenesis, in Sprague-Dawley rats. Male Sprague-Dawley male were injected with azoxymethane for two weeks (15mg/kg/week) and then one week later they were allocated to different dietary treatment groups. After eight weeks, the effects of dietary treatment on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of preneoplastic lesions, aberrant crypt foci (ACF), were evaluated. The lowest level of vitamin B6 intake with a high protein diet reduced the growth and development of ACF. Vitamin B6 had no significant effect on the oxidative stress markers. The level of protein was an important variable in modulating the levels of 3-nitrotyrosine and 8-OH-DG which were lower in high protein groups than normal protein counterparts. The objective of the second study was to investigate if a supraphysiological (5 fold higher than normal level) dosage of vitamin B6 could have an antioxidant effect in a metabolically compromised state like obesity and thereby lower the risk of colon cancer. Female Zucker obese (Zk-OB) rats received normal (Zk-OBN, 7 mg/kg) or high (Zk-OBH, 35 mg/kg) vitamin B6 (Pyridoxine-HCl) diets two weeks prior to, during and six weeks following injection with colon carcinogen AOM. The effects of supplemental vitamin B6 on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of ACF were carried out. High intake of vitamin B6 significantly lowered liver weights and plasma cholesterol compared to the normal intake (p≤0.05). Zk-OBH rats had significantly reduced number of ACF compared to Zk-OBN (p≤0.05). Hepatic GSH increased in the ZK-OBH group with a concomitant decrease in GPx activity. The findings demonstrate that in Zucker Obese rats, a high B6 intake augmented the antioxidant potential and decreased sensitivity to colon carcinogenesis. These findings suggest that high vitamin B6 plays an important therapeutic role in the compromised state of obesity.

Obesity increases the risk of colon cancer as well as the expression of many cancer markers, ostensibly due to the interaction between insulin resistance and adipocyte production of hormones, mitogens and cytokines which collaborate to enhance proliferation signaling and impair the DNA damage response. Cdc7 and PCNA are both proteins involved in the DNA damage response as well as DNA replication. Both have also been shown to be upregulated in human tumours. To assess Cdc7 and PCNA roles during the DNA damage response in obese and lean animals, we administered azoxymethane (AOM), a colon-specific carcinogen, to obese and lean rats. Cdc7 and PCNA levels in colonic mucosal protein extracts from obese Zucker rats were compared with those from their lean counterparts. Significant differences were seen between lean and obese animals 3 hours post-AOM (lean Cdc7 levels > obese Cdc7 levels) and 24 hours post-AOM (lean PCNA levels > obese PCNA levels). This result suggests an impaired checkpoint response in obese animals relative to lean animals and supports a previously reported early role for Cdc7 in the checkpoint signaling cascade relative to a later role of PCNA in DNA damage repair. At the time tumours appeared (32 weeks post-AOM), colonic mucosal Cdc7 levels of obese rats exceeded that of their lean counterparts, suggesting that the obese metabolic environment causes upregulation of Cdc7 in obese rat epithelia. Cdc7 and PCNA levels were then compared between tumours and mucosa in obese and Sprague Dawley rats. Tumour Cdc7 levels were upregulated relative to mucosal levels in more samples than tumour PCNA levels, suggesting Cdc7 may be a more sensitive tumour marker. No significant differences in Cdc7 levels were seen between obese tumours and mucosa, likely due to elevation of obese mucosal Cdc7 levels. However, Sprague Dawley (non-obese) rats showed significantly higher Cdc7 and PCNA levels in tumours than mucosa, consistent with previous studies in human tissues. These results suggest that Cdc7 may be a more sensitive tumour marker than PCNA, but that its utility as a biomarker of colon cancer is dependent on the metabolic state (leanness) of the individual.

Colon cancer and obesity are two significant and related pathological states with multiple etiological factors. In this dissertation, it was hypothesized that tumor growth is accelerated in the altered state of obesity due to their resistance towards tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity. Physiologically elevated TNF-alpha in an obese state induces increased nuclear transcription factor-kB (NF-kB) activity, known to transcribe genes crucial to cell survival. Insulin resistance, oxidative stress, and a pro-inflammatory environment are few of the biological consequences of TNF-alpha and NF-kB pathway activation, and further contribute to disease progression. <br /><br /> Three major studies were conducted to investigate phenotypical changes in obesity associated tumors. Firstly, characteristics of the TNF-alpha resistant phenotype were preliminarily assessed by evaluating the effects of exogenous TNF-alpha treatment to HT-29 cells. Elevated levels of NF-kB in response to exogenous TNF-alpha gave an indication that this pathway is critical for cell survival. Furthermore, upregulation of TNF-alpha receptor 2 (TNFR2) suggested another strategy by which the cells were utilizing exogenous TNF-alpha for a survival advantage. Inhibition of NF-kB via St. John?s Wort treatment demonstrated that HT-29 cells may be sensitized towards TNF-alpha mediated cytotoxicity. <br /><br /> Zucker obese (Zk-Ob), Zucker lean (Zk-Ln), and Sprague Dawley (SD) animal models were used to assess tumor phenotype <em>in vivo</em>. Remarkable physiological differences between genotypes were observed. Zk-Ob rats had significantly higher body and organ weights as well as plasma TNF- alpha, insulin, leptin, and oxidative markers than Zk-Ln and SD animals. Tumor incidence and multiplicity were also notably higher in Zk-Ob rats. Protein analyses demonstrated increased levels of TNF-alpha, TNFR2, NF-kB, IkB kinase beta (IKKbeta), insulin receptor (IR), insulin like growth factor-I-receptor (IGF-IR), and mitogen activated protein kinase (MAPK) in Zk-Ob tumors than Zk-Ln counterparts. In all groups, tumors generally had higher protein expression than surrounding, normal appearing colonic mucosa. It is well known that these molecules are involved in signaling pathways that influence and co-operate with each other in rendering growth autonomy to tumor tissue. <br /><br /> A higher number of lesions in the distal than proximal colon in Zk-Ob rats was observed, supporting the emerging concept that genotype/physiological state of the host affects development and distribution of tumors. Thus, a third study was conducted to explore differences between distal and proximal tumor phenotype. Results demonstrated that expression of TNFR2, NF-kB, IR, IGF-IR, and MAPK p44 were significantly higher in distal than proximal tumors. This observation suggested that development of tumors in different regions of the colon varied under the same physiological conditions. Moreover, phenotype of distal tumors appeared to be upregulating survival pathways in comparison to proximal lesions, possibly explaining the higher tumor incidence in the distal colon. <br /><br /> Research documented in this thesis supported the hypothesis that the physiological status of the host intricately affects tumor phenotype. In particular, the TNF-alpha resistant phenotype was most prominent in Zk-Ob tumors, and appeared to be associated with upregulation of multiple signaling pathways cooperating towards tumorigenesis.

The main objective of this proposal was to investigate the effect of vitamin B6 on colon carcinogenesis in vivo. Two in vivo studies were conducted to determine the role of vitamin B6 as a biological modifier of colon carcinogenesis. It is hypothesized that vitamin B6 may serve as an antioxidant in vivo and will modulate colon carcinogenesis. In the first study, a 2X3 factorial experimental design was used to determine if three different levels of vitamin B6, classified as low, normal and high in conjunction with two different levels of protein intake, classified as normal or high, will affect post-initiation stages of colon carcinogenesis, in Sprague-Dawley rats. Male Sprague-Dawley male were injected with azoxymethane for two weeks (15mg/kg/week) and then one week later they were allocated to different dietary treatment groups. After eight weeks, the effects of dietary treatment on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of preneoplastic lesions, aberrant crypt foci (ACF), were evaluated. The lowest level of vitamin B6 intake with a high protein diet reduced the growth and development of ACF. Vitamin B6 had no significant effect on the oxidative stress markers. The level of protein was an important variable in modulating the levels of 3-nitrotyrosine and 8-OH-DG which were lower in high protein groups than normal protein counterparts. The objective of the second study was to investigate if a supraphysiological (5 fold higher than normal level) dosage of vitamin B6 could have an antioxidant effect in a metabolically compromised state like obesity and thereby lower the risk of colon cancer. Female Zucker obese (Zk-OB) rats received normal (Zk-OBN, 7 mg/kg) or high (Zk-OBH, 35 mg/kg) vitamin B6 (Pyridoxine-HCl) diets two weeks prior to, during and six weeks following injection with colon carcinogen AOM. The effects of supplemental vitamin B6 on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of ACF were carried out. High intake of vitamin B6 significantly lowered liver weights and plasma cholesterol compared to the normal intake (p≤0.05). Zk-OBH rats had significantly reduced number of ACF compared to Zk-OBN (p≤0.05). Hepatic GSH increased in the ZK-OBH group with a concomitant decrease in GPx activity. The findings demonstrate that in Zucker Obese rats, a high B6 intake augmented the antioxidant potential and decreased sensitivity to colon carcinogenesis. These findings suggest that high vitamin B6 plays an important therapeutic role in the compromised state of obesity.

Obesity increases the risk of colon cancer as well as the expression of many cancer markers, ostensibly due to the interaction between insulin resistance and adipocyte production of hormones, mitogens and cytokines which collaborate to enhance proliferation signaling and impair the DNA damage response. Cdc7 and PCNA are both proteins involved in the DNA damage response as well as DNA replication. Both have also been shown to be upregulated in human tumours. To assess Cdc7 and PCNA roles during the DNA damage response in obese and lean animals, we administered azoxymethane (AOM), a colon-specific carcinogen, to obese and lean rats. Cdc7 and PCNA levels in colonic mucosal protein extracts from obese Zucker rats were compared with those from their lean counterparts. Significant differences were seen between lean and obese animals 3 hours post-AOM (lean Cdc7 levels > obese Cdc7 levels) and 24 hours post-AOM (lean PCNA levels > obese PCNA levels). This result suggests an impaired checkpoint response in obese animals relative to lean animals and supports a previously reported early role for Cdc7 in the checkpoint signaling cascade relative to a later role of PCNA in DNA damage repair. At the time tumours appeared (32 weeks post-AOM), colonic mucosal Cdc7 levels of obese rats exceeded that of their lean counterparts, suggesting that the obese metabolic environment causes upregulation of Cdc7 in obese rat epithelia. Cdc7 and PCNA levels were then compared between tumours and mucosa in obese and Sprague Dawley rats. Tumour Cdc7 levels were upregulated relative to mucosal levels in more samples than tumour PCNA levels, suggesting Cdc7 may be a more sensitive tumour marker. No significant differences in Cdc7 levels were seen between obese tumours and mucosa, likely due to elevation of obese mucosal Cdc7 levels. However, Sprague Dawley (non-obese) rats showed significantly higher Cdc7 and PCNA levels in tumours than mucosa, consistent with previous studies in human tissues. These results suggest that Cdc7 may be a more sensitive tumour marker than PCNA, but that its utility as a biomarker of colon cancer is dependent on the metabolic state (leanness) of the individual.

Introduction: Colon cancer is the third leading cause of cancer death among women in Canada. Although regular screening beginning at age 50 years can significantly decrease risk of colon cancer mortality, many eligible Canadian women have never obtained screening. Cancer screening rates among immigrant women to Canada are even lower than for native-born women. Disparities in the use of preventive cancer services by immigrants have been linked to limited acculturation and speaking a language other than English. Poor prose comprehension may frustrate access and use of preventive cancer information by older ESL immigrant women to Canada. In order to develop useful and actionable cancer prevention information, it is necessary to understand the barriers ESL immigrant women face in obtaining and using health and cancer information. Therefore, the primary objective of this research was to assess the relationship of acculturation on the prose comprehension of older ESL immigrant women to Canada. Methods: Interviews were conducted with 78 older adult Spanish-speaking immigrant women (aged 45 to 73 years) residing in Kitchener-Waterloo, Ontario. Acculturation was inferred by length of Canadian residency and measured using the Bidimensional Acculturation Scale (BAS). Several measures were used to assess comprehension including the shortened Test of Functional Health Literacy in Adults (S-TOFHLA), the Rapid Estimate of Literacy in Medicine (REALM), the Newest Vital Sign (NVS), and a modified Cloze procedure. The modified Cloze procedure was constructed using a one-page colon cancer information sheet created for the public by the Canadian Cancer Society. Four multiple linear regression analyses were used to test the relationship between the independent variables of acculturation (BAS) and length of Canadian residency, age, Spanish language education, employment, and media variables (television and internet use) on each measure of prose comprehension (i.e., dependent variables of scores on S-TOFHLA, NVS, REALM and Cloze). Logistic regression was used to test whether acculturation and comprehension predicted screening intentions. Semi-structured interviews were conducted to identify the women’s cancer information needs and preferences as well as the barriers they experienced in obtaining and understanding English language cancer information. Results: Three significant models predicting comprehension of colon cancer and health information were identified. The independent variables BAS, Spanish language education and television viewing explained 23% of the variation in Cloze comprehension scores (F=6.76; df=3; p < 0.01; R2= 0.23). Approximately 42% of the variation in S-TOFHLA scores among older ESL immigrant women was explained by the independent variables BAS, age, television viewing and internet use (F=12.13; df=5; p < 0.01; R2=0.42). Using REALM as the dependent measure of comprehension, BAS and television viewing explained 17% of the variation in scores (F=7.54; df=2; p<0.01; R2=0.17). BAS was the only significant predictor of the dependent comprehension measure NVS (F=5.36; df=1; p=0.02; R2=0.07). Logistic regression models predicting colon cancer screening intentions were not significant. Qualitative data analyses revealed that women’s colon cancer information needs, preferences and perceived barriers accessing English language information did not vary according to BAS score or duration of residency in Canada. All women requested actionable information that was community and culturally specific. Additional factors related to older ESL immigrant women’s comprehension of cancer information were identified. These included self-efficacy, social networks and mode of information delivery. These additional modes of information delivery included receiving health information orally as compared to written information. Conclusion: Acculturation (as measured by BAS) significantly predicted prose comprehension by older ESL Spanish speaking immigrant women across four separate measurement tools (Cloze, S-TOFHLA, REALM, NVS). Yet, the proportion of the variance in comprehension scores explained by acculturation and other demographic variables was low to modest, ranging from 7-42%. In addition to acculturation, self-efficacy and social networks may also be associated with prose comprehension. Low self-efficacy among older ESL immigrant women may be a barrier to information seeking and perceived comprehension. However, strong social networks may provide women with the confidence and resources necessary to access health information and services. These results highlight the need for the additional research regarding the influence of self-efficacy on ESL immigrants’ ability to find and use health and cancer information. Recommendations: This research has important implications for public health educators. Health educators are encouraged to develop cancer and health information for ESL speakers in Canada that is community, culturally and linguistically specific and which provides actionable information. This is especially salient given the changing demographic and cultural profile of Canadians. Also, among older ESL immigrants who struggle with language barriers, receiving health information orally may be preferred.

This dissertation examines the effects of postharvest treatment and processing on biologically active compounds of orange juice, and ??Rio Red?? grapefruit and their ability to prevent chemically induced colon cancer in rat model. The first study evaluated the differences in flavonoid content of commercial ??made from concentrate?? (MFC) orange juices and ??not from concentrate?? (NFC) orange and grapefruit juices. Total flavonoid content of MFC orange juices (53 mg/100 mL; n = 12) was significantly (P &#8804; 0.05) higher than NFC orange juices (36.5 mg/100 mL; n = 14). The second study investigated the ionizing radiation and storage effects on bioactive compounds and quality of ??Rio Red?? grapefruit. Results showed that storage and irradiation significantly (P &#8804; 0.05) affected the bioactive compounds in grapefruit, however, the effect of storage was prominent. The third study examined the influence of irradiation and freeze drying on bioactive compounds of grapefruit. Irradiation of grapefruit prior to freeze drying resulted in enhanced (P &#8804; 0.05) flavonoid content (naringin and narirutin). Freeze drying markedly reduced (P &#8804; 0.05) lycopene content. Freeze drying and irradiation reduced (P &#8804; 0.05) volatile compounds (d-limonene and myrcene), with the exception of ethanol. In the fourth study suppression of colon cancer development in Sprague Dawley rats by natural and irradiated grapefruits and their functional compounds, naringin and limonin, were evaluated.The total number of aberrant crypts (AC; P = 0.02), number of high multiplicity AC foci (ACF; P = 0.01), and proliferative index (P = 0.02) were lower and apoptosis (P = 0.02) was higher in azoxymethane (AOM) injected rats on experimental diets. However, only natural grapefruit and limonin only suppressed AOM induced expansion (P = 0.008) of proliferative zone and also enhanced apoptosis more effectively than other experimental diets indicating that natural grapefruit and limonin may serve as better chemopreventive agents compared to IGFPP and naringin. The present study indicates that postharvest quarantine doses of irradiation slightly alter composition of bioactive compounds and in turn marginally reduce the chemopreventive ability of grapefruit against the promotion stage of colon cancer. These results warrant the necessity of testing the impact of post harvest treatments on fruits and vegetables chemopreventive ability.

<p>Metastasis is a feared aspect of cancer and little is known about the underlying mechanisms. It is proposed that metastasis is caused by cell fusion between tumour and immune active phagocyte cells, for example macrophages. Such hybrid cells could then develop immortality and chemo tactic mobility. In two different systems it was examined whether it is possible to detect variation in cancer cells that would explain an initial fusion between tumour cells and leukocyte cells. Both systems included use of STR markers. Human colon carcinoma cells, which originally had been grown in nude mice, were investigated with mouse specific primers. These showed no trace of mouse DNA, which they most probably would have if cell fusion had occurred. Human breast cancer cells grown in nude mice, that had received injection of stem cell from male blood, showed no presence of Y-chromosomes. Blood, which was analyzed from one of the mice, showed a weak presence of something else than just mouse DNA. The result was however vague and hard to evaluate, and tries to reproduce the positive outcome failed. No evidence, which indicated that cell fusion occurred, was possible to demonstrate. On the other hand, there are previous studies that show how metastases can express macrophage specific properties, which gives all reason for further investigations.</p>

Inflammation and aberrant microRNAs expressions promote colon cancer growth and progression. However, the molecular mechanisms that link these pathways remain to be determined. In this dissertation, the causal relationship between inflammation and aberrant microRNAs expressions were explored. Elevated expression of prostaglandin E₂ (PGE₂) receptor EP4 has been seen in human colon cancer. However, the mechanism by which EP4 receptor protein is deregulated is not known. Experiments in this dissertation demonstrate, for the first time, that the EP4 receptor is negatively regulated by miR-101.In previous work, we show that S100P is induced by stimulation of the PGE₂/EP4 receptor signaling pathway. S100P is a ligand for Receptor for Advance Glycation End-products (RAGE). However, little is known about the downstream targets of S100P/RAGE signaling. Here, we demonstrated that S100P/RAGE receptor signaling induces expression of miR-155 via the transcription factor AP-1. In addition, we investigated the genes that are downstream of S100P/RAGE/miR-155 pathway. Our microarrays and bioinformatics analyses identified two novel miR-155 targets, WNK1 and ZNF493 that are down-regulated upon activation of the S100P/RAGE/miR-155 pathway. Lastly, we investigated whether inhibition of S100P/RAGE signaling pathway would be beneficial as a cancer therapy using methyl-2-acetamidoacrylate (M2AA). M2AA treatments decreased colon cancer cells viability and also suppressed colon tumor growth and metastasis in vitro and also in the CAM assay in vivo. Taken together, our results suggest that modulation of S100P/RAGE signaling by M2AA offers therapeutic potential as anti-metastatic agents. In summary, this dissertation provides new insights on the molecular events that link inflammation pathways and microRNAs to colon cancer as well as show that therapeutic strategies targeting these pathways could be effective in treatment of neoplasia.

Με σκοπό να διερευνήσουμε τους μηχανισμούς επαγωγής απόπτωσης από την TRAIL και την επιλεκτικότητα την οποία δείχνει απέναντι στα καρκινικά κύτταρα, χρησιμοποιήσαμε κυτταρικές σειρές, προερχόμενες από το παχύ έντερο ανθρώπου, οι οποίες αντιπροσωπεύουν τα διάφορα στάδια της καρκινογένεσης, από το πρώιμο αδένωμα στο καρκίνωμα. Παρατηρήθηκε μια ανθεκτικότητα των πρώιμων αδενωμάτων απέναντι στην TRAIL σε αντίθεση με τα κύτταρα που προέρχονται από καρκίνους προχωρημένου σταδίου, ενώ σημαντικό ρόλο φαίνεται να παίζει, εκτός από το στάδιο καρκινογένεσης και η ύπαρξη ορισμένων ογκογονιδίων στον καθορισμό της ευαισθησίας των κυττάρων απέναντι στην TRAIL. Συγκεκριμένα, οι διάφορες ισομορφές του ογκογονιδίου RAS παίζουν ιδιαίτερο ρόλο τόσο όσο αφορά την ανταπόκριση του κύτταρου στην επαγωγή με TRAIL, όσο και στη ρύθμιση των μορίων που εμπλέκονται άμεσα στο μονοπάτι σηματοδότησης του TRAIL. Τα ογκογονίδια RAS παίζουν σημαντικό ρόλο στην καρκινική εξαλλαγή ενεργοποιώντας μια σειρά από μονοπάτια σηματοδότησης που οδηγούν στην ανεξέλεγκτη κυτταρική διαίρεση και στην προστασία από τα αποπτωτικά σήματα. Εντούτοις, η TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand) έχει τη δυνατότητα να προκαλεί απόπτωση κυρίως στα καρκινικά κύτταρα, ενεργοποιώντας τους υποδοχείς DR4 και DR5 (Death Receptors). Σε αυτή τη μελέτη δείξαμε ότι σε αδενοκαρκινώματα του παχέος εντέρου ανθρώπου η έκφραση των υποδοχέων της TRAIL ρυθμίζεται από την ενεργότητα της κινάσης MEK (MAPK/ERK Kinase). Η ευαισθησία στην TRAIL των κυτταρικών σειρών που χρησιμοποιήσαμε φάνηκε να συσχετίζεται με το βαθμό κακοήθους εξαλλαγής. Συγκεκριμένα, οι κυτταρικές σειρές AAC1 και RGC2, που προέρχονται απο πρώιμα αδενώματα και η κυτταρική σειρά Caco2, που προέρχεται απο μέσο αδένωμα παρουσίασαν μεγάλη ανθεκτικότητα στην TRAIL. Αντίθετα, οι κυτταρικές σειρές DLD-1 και HT-29, που προέρχονται απο αδενοκαρκινώματα προχωρημένου στάδιου ήταν ευαίσθητες στην TRAIL. Επιλέχθηκε η κυτταρική σειρά Caco2 η οποία είναι ανθεκτική στην TRAIL και οι δύο κυτταρικές σειρές που προέρχονται από προχωρημένα αδενοκαρκινώματα για να εξεταστεί η έκφραση σημαντικών μορίων για την αποπτωτική σηματοδότηση από την TRAIL. Φάνηκε μια αντιστοιχία της έκφρασης των υποδοχέων DR4 και DR5 με την ευαισθησία των κυττάρων στην TRAIL, ενώ τα επίπεδα έκφρασης των FADD, κασπάση 8, κασπάση 3 και FLIP δεν παρουσίασαν παρόμοια αντιστοιχία. Επιπλέον, παρατηρήθηκε παρατεταμένη ενεργοποίηση των κινασών MEK, ERK, JNK1/2 και p38 μετά από επαγωγή με TRAIL, σε αντίθεση με την αντίστοιχη κινητική ενεργοποίησης των ίδιων κινασών μετά από επαγωγή με ορό. Αντίστοιχη κινητική με αυτή των ΜΕΚ και ERK παρουσίασε η έκφραση του γονιδίου c- FOS μετά από επαγωγή με TRAIL ή ορό. Τα κύτταρα Caco2, DLD-1 και HT- 29 επωάστηκαν με χημικούς αναστολείς των κινασών ΜΕΚ για 16 h και ελέγχθηκε η έκφραση των DR4 και DR5. Τα επίπεδα των DR4 και DR5 μειώθηκαν και στις τρεις κυτταρικές σειρές ως συνέπεια της αναστολής της ΜΕΚ, ενώ η αναστολή των ΜΕΚ δεν επιρρέασε τα επίπεδα των FADD, FLIP, κασπάση 8, κασπάση 3 και τον υποδοχέα FAS που ανήκει στην ίδια οικογένεια με τους DR4 και DR5. Επιπλέον, ελέγχθηκε με ανάλυση FACS η έκφραση των υποδοχέων DR4, DR5 και FAS in vivo σε ζωντανά κύτταρα που προεπωάστηκαν με τον αναστολέα της ΜΕΚ και στους αντίστοιχους μάρτυρες και τα αποτελέσματα ήταν αντίστοιχα με αυτά των in vitro πειραμάτων. Οι παραπάνω παρατηρήσεις αποτελούν ισχυρές ενδείξεις για το συσχετισμό της ενεργοποίησης της οδού MEK/ERK/FOS με με την έκφραση των υποδοχέων DR4 και DR5 και με την ευαισθησία των κυττάρων στην TRAIL, καθώς κύτταρα HT-29 που προεπωάστηκαν με τον αναστολέα των ΜΕΚ παρουσίσαν μειωμένη ευαισθησία στην TRAIL. Για τον προσδιορισμό των διακριτών επιδράσεων των ογκογονιδίων RAS στον καρκίνο του παχέος εντέρου, διαμολύνθηκαν κύτταρα που προέρχονται από μέσο αδένωμα του παχέος εντέρου (Caco2) με τα ογκογονίδια Ki- και Ha-RAS. Μετά από εξέταση πολλών διαφορετικών κλώνων επιλέχθηκαν για λεπτομερέστερη ανάλυση κλώνοι οι οποίοι δεν υπερεκφράζουν την RAS σε πρωτεϊνικό επίπεδο πάνω από 3 φορές σε σχέση με την ενδογενή RAS των μητρικών κυττάρων. Επιπλέον, ελέγχθηκε αν οι κλώνοι που υπερεκφράζουν τις ογκογόνες RAS παρουσιάζουν αυξημένη σηματοδότηση προς μονοπάτια κυτταρικής σηματοδότησης που είναι γνωστό οτι ενεργοποιούνται από τις RAS. Τόσο οι κλώνοι Ki-RAS, όσο και οι Ha-RAS, έδειξαν να ενεργοποιούν τα μονοπάτια σηματοδότησης RAF/MEK/ERK και PI3K/AKT με τους Ha-RAS να προκαλούν ισχυρότερη ενεργοποίηση. Σχεδιάστηκαν πειράματα για τον καθορισμό του βαθμού της in vitro και in vivo κακοήθους εξαλλαγής. Τα πειράματα σε μαλακό άγαρ δείχνουν την αποτελεσματικότητα των καρκινικών κυττάρων να αναπτύσσονται δημιουργώντας αποικίες χωρίς να προσκολλώνται σε επιφάνεια. Οι δυο ισομορφές της RAS αύξησαν σημαντικά την ιδιότητα των Caco2 να σχηματίζουν αποικίες σε μαλακό άγαρ, ενώ η Ha-RAS παρουσίασε τη μεγαλύτερη ικανότητα. Για τον προσδιορισμό του βαθμού κακοήθους εξαλλαγής των κλώνων in vivo έγινε υποδόριος εμβολιασμός με περίπου 106 κύτταρα από τον κάθε κλώνο σε ποντίκια SCID. Τόσο η Κi-RASV12 όσο και η Ha-RASV12 αύξησαν την ικανότητα των Caco2 να σχηματίζουν όγκους σε ποντίκια SCID με την Ha-RAS να προκαλεί τον σχηματισμό περισσότερων και μεγαλύτερων όγκων. Για την περαιτέρω κατανόηση των μηχανισμών εξαλλαγής των κυττάρων από τα ογκογονίδια RAS αξιολογήθηκαν αποτελέσματα από ανάλυση γονιδιακών μικροσυστοιχιών. Μετά από ανάλυση του γονιδιακού προφίλ των CACO-RAS κλώνων και τα δυο ογκογονίδια βρέθηκαν να επάγουν την έκφραση γονιδίων που εμπλέκονται στην αγγειογένεση και στην προώθηση του καρκίνου, όπως τα γονίδια που κωδικοποιούν τους υποδοχείς VEGF και TGFβ. Σημειώνεται ότι μεταστατικοί δείκτες, όπως η Vimentin, που είναι και δείκτης της μετάβασης από επιθηλιακό σε μεσεγχυματικό φαινότυπο, βρέθηκαν να υπερεκφράζονται μόνο στους κλώνους Ha-RASV12. Ο in vitro και in vivo χαρακτηρισμός εξαλλαγμένων κυττάρων από τα ογκογονίδια RAS, καθώς και η ανάλυση μικροσυστοιχειών γονιδίων έδειξε ότι στο κυτταρικό μοντέλο που χρησιμοποιήθηκε το ογκογονίδιο Ha-RAS έχει αυξημένες εξαλλακτικές ιδιότητες. Τα ογκογονίδια RAS αύξησαν το βαθμό κακοήθους εξαλλαγής των κυττάρων Caco2 και ελέγχθηκε αν αυτό συνοδεύεται από αυξημένη ευαισθησία των κλώνων CACO2-RAS στην TRAIL. Μετρήθηκε η βιωσημότητα των κλώνων CACO2-RAS μετά από επαγώγη με TRAIL και φάνηκε ότι και τα δύο ογκογονίδια αύξησαν την ανταποκρισιμότητα των κυττάρων Caco2 στην TRAIL. Τα Caco2 που εξαλλάχθηκαν με την Ki- RASV12 ήταν λιγότερο ευαίσθητα στην TRAIL από ότι αυτά με τη Ha- RASV12 σε όλους τους κλώνους που ελέγχθηκαν. Ο έλεγχος της έκφρασης σημαντικών παραγόντων για την απόπτωση από την TRAIL έδειξε ότι οι πρωτεΐνες που παρουσίασαν τις μεγαλύτερες διαφοροποιήσεις στους κλώνους που υπερεκφράζουν τις RAS, σε σχέση με τα κύτταρα μάρτυρα, ήταν οι υποδοχείς DR4 και DR5. Παρόλο που οι κλώνοι Ha-RAS παρουσίασαν μεγαλύτερη ευαισθησία στην TRAIL, τα επίπεδα έκφρασης των DR4 και DR5 ήταν παρόμοια μεταξύ των κλώνων Ki-RAS και Ha-RAS. Για να ελεγχθεί κατά πόσο το μονοπάτι σηματοδότησης MEK/ERK παίζει ρόλο στην υπερέκφραση των υποδοχέων από τα ογκογονίδια RAS χρησιμοποιήθηκαν χημικοί αναστολείς της ΜΕΚ και ελέγθηκε η επίδρασή τους στα επίπεδα των DR4 και DR5. H χημική αναστολή της ΜΕΚ προκάλεσε μείωση των επιπέδων των φωσφορυλιωμένων ERK1/2 και των επιπέδων έκφρασης των DR4 και DR5 τόσο στους CACO-Κ15, όσο και στους CACO-Η2. Τέλος, ελέγχθηκε κατά πόσο η μείωση των DR4 και DR5 από την χημική αναστολή των ΜΕΚ μπορέι να έχει επίδραση στη δράση της TRAIL απέναντι στους κλώνους CACO2-RAS και βρέθηκε ότι προεπώση των Ha-RASV12 κλώνων για 16 h με χημικό αναστολέα των ΜΕΚ μέιωσε σημαντικά την ευαισθησία τους στην TRAIL. / In order to study the mechanism by which TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing Ligand) induces apoptosis almost exclusively to cancer cells we used human colon cancer cell lines that represent different stages of carcinogenesis, from early adenoma to carcinoma. It was observed that early adenoma cells were resistant to TRAIL-induced apoptosis. On the contrary, cells that were derived from carcinomas were sensitive; while a correlation of the sensitivity of the cells to TRAIL-induced apoptosis to the presence of certain activated oncogenes was observed. In particular, the various isoforms of RAS oncogenes appear to play an important role in the responsiveness of the cancer cells to TRAIL as well as in regulating specific components which are essential for TRAIL signaling. RAS oncogenes play an important role in oncogenic transformation by activating various signaling pathways that favor tumor growth also by controlling cell division and resistance to apoptotic stimuli. TRAIL, however, has the unique ability to cause apoptosis preferentially to cancer cells by activating DR4 and DR5 receptors. In this study we show that in human colorectal adenocarcinomas cells the expression of DR4 and DR5 is partially regulated by the activity of MEK (MAPK/ERK Kinase). The sensitivity of the cell lines to TRAIL seemed to be correlated with the level of oncogenic transformation of the cells. In particular, the AAC1 and RGC2 cell lines that were derived from early adenomas and the Caco2 cell line that was derived from an intermediate adenoma were very resistant to TRAIL induced apoptosis. On the contrtary, the DLD-1 and HT-29 cell lines, which came from advanced stage carcinomas were sensitive to TRAIL. The Caco2 cell line, which is resistant to TRAIL and the cell lines that derived from advanced stage adenocarcinomas were chosen for expression analysis of important molecules in TRAIL-induced apoptosis. There was a correlation of DR4 and DR5 expression with the sensitivity of the cell lines to TRAIL, while the expression levels of FADD, Caspase 3, Caspase 8 and FLIP did not seem to correlate with TRAIL sensitivity in these cell lines. In addition, it was observed a prolonged activation of MEK, ERK1/2, JNK1/2 and p38 after induction with TRAIL, which did not follow the kinetics of serum-induced activation of ERK1/2. The activation of MEK and ERK1/2 was correlated to the kinetics of c-FOS proto-oncogene expression induced by TRAIL or serum. The expression levels of DR4 and DR5 were analysed after incubation for 16 h of the Caco2, DLD-1 and HT-29 cell lines with MEK inhibitors. There was a decrease of DR4 and DR5 expression levels in response to MEK inhibition, while the expression levels of FADD, Caspase 3, Caspase 8, FLIP and FAS receptor were not affected. Moreover, FACS analysis of living cells showed that MEK inhibition reduces the levels of DR4 and DR5 but not of other receptors of the same family, in vivo. It appears that the activation of the MEK/ERK/FOS axis plays a role in the positive feedback loop of TRAIL its receptors DR4 and DR5. To determine the distinct effects of different RAS oncogenes in cancer cells colon intermediate adenoma cells (Caco2) were chosen for transfection with the Ki- and Ha-RAS oncogenes. Clones that did not express more than 3 times the endogenous levels of RAS proteins were selected for further analysis. In addition it was examined whether the CACO2-RAS clones are able to activate the RAF/MEK/ERK and PI3K/AKT pathways, which are known effectors of RAS proteins. Both RAS isoforms activated these two pathways with the Ha-RASV12 clones presenting better potential in activating both RAF/MEK/ERK and PI3K/AKT pathways. In order to characterize the in vitro and in vivo oncogenic potential the ability of the CACO2-RAS clones to grow in soft agar and to grow tumors in nude mice was examined. The in vitro and in vivo characterization of the Caco2 RASV12-transformed cells showed that the Ha-RAS oncogene has a higher in vitro and in vivo transforming ability relative to the Ki-RAS oncogene in these cells. Results from cDNA microarray analysis were evaluated in order to further understand the mechanisms by which the RAS oncogenes cause oncogenic transformation. Gene expression profile analysis of the CACO2-RAS clones showed that both oncogenes induced expression of genes involved in angiogenesis and tumor promotion such as VEGF and TGFβ. Notably, metastatic markers, such as Vimentin, which is also an epithelial to mesenchymal transition marker, were overexpressed only in the Caco2 cells transformed with the Ha-RAS oncogene. The RAS oncogenes increased the oncogenic potential of the Caco2 cells and it was examined if the increased oncogenic potential correlated with increased sensitivity to TRAIL. Moreover, the examination of the expression levels of molecules important for TRAIL singnaling showed that Ki-RAS as well as Ha-RAS oncogenes can induce DR4 and DR5 expression with similar efficiency. However, in spite of similar induction of DR4 and DR5 by the two RAS oncogenes, the Ki-RAS-transformed cells were less susceptible to TRAIL induced apoptosis. Finally, in order to see whether the increased MEK/ERK activation observed in the CACO-RAS clones played a role in increasing DR4 and DR5 levels the CACO-RAS clones were incubated for 16h in the presence of MEK inhibitors. MEK inhibition resulted in decreased DR4 and DR5 expression levels, as well as decreased sensitivity of the clones to TRAIL induced apoptosis.