Elucidation of the WNT & AKT/Phosphoinositide-3-Kinase Pathways in Colorectal Carcinoma

Colorectal cancer (CRC) is the third most common cancer III Malaysia and is currently the commonest cancer in males. Genetics, experimental and epidemiological data suggest that CRC develops from complex interaction between inherited susceptibility and environmental factors. Accumulating evidence suggests that the Wnt and PI3K (phosphoinositide-3-kinase)/Akt signalling pathways playa causative role in tumorigenesis of colorectal cancer. By employing immunohistochemical method, the expressIOn and correlation of several key regulators or related biomolecules of the Wnt and PI3K1Akt signalling pathways in 47 archival formalin fixed, paraffin embedded tissues of surgically resected colorectal cancer (CRC) specimens performed at Kuala Lumpur Hospital (KLH) between 1999 and 2000, were studied. Laser captured microdissection technique, polymerase chain reaction and direct sequencing were used to investigate mutations in exon 3 of the p-catenin gene. Mutations in the mutation cluster region (MCR) of adenomatous polyposis coli (APC) gene were also investigated. The expressions of Wnt-l, WISP-l and FRAT-l mRNA were determined by reverse-transcription and real-time polymerase chain reaction method. The results showed that: The expressions ofWnt-l, FRAT-I, APC, nuclear p-catenin, cytoplasmic p-catenin, membrane p-catenin, membrane E-cadherin, cytoplasmic E-cadherin, WISP-I, cyclin-Dl, p-Aktl (Ser473), p-Akt1l2/3 (Thr308), p-BAD (Ser136), p-GSK 3p(Ser9) and survivin were found in 55.3%, 36.2%, 51.1% 44.6%, 95.7%,30.6%,46.8%,95.7%,31.9%, 10.6%,34%,44.7%,57.4% 44.7% and 59.6% of CRC tissues, respectively and 17.5%, 5% 100%, 0%, 75%, 100%, 100%, 50%, 12.5%, 0%, 5%, 12.5%, 22.5%, 22.5% and 32.5% of apparently normal adjacent tissues, respectively. The sum of scores for all biomolecules except APC, membrane p-catenin and membrane E-cadherin staining was significantly higher in CRC tissues in comparison to apparently normal adjacent tissues (p < 0.05). The sum of score for APC, membrane p-catenin and membrane E-cadherin staining was significantly lower in CRC tissues in comparison to apparently normal adjacent tissues (p < 0.05). The expression of Wnt and PI3K1Akt signalling pathway-related biomolecules was interrelated. The results ofnucleotide sequencing showed that no mutations at exon-3 of p-catenin were found. However, point mutations in the mutation cluster region of the APC gene leading to the formation of truncated APC protein, were found in four out eleven CRC tissues examined. A 1.43 to 21.26-foid and 1.11 to 109.14-fold increase in the level of expression of Wnt-l and FRAT-1 mRNA was found in eight out of eleven CRC tissues relative to apparently normal adjacent tissues. On the other hand, a 1.94 to 46.69-fold increase in the level of WISP-I mRNA was found in all the CRC tissues. This study has provided important information for researchers and clinicians in terms of clinical evidence of the involvement of the Wnt signalling pathway and PI3K1Akt signalling pathway in colorectal tumorigenesis. In addition, the present study also provided crucial information on the elucidation of the relationship between the biomolecules of these signalling pathways towards understanding their roles in colorectal tumourigenesis and the identification of potential targets for advance therapeutic intervention ofCRe. Based on our current results, we propose that Wnt-I, FRAT-1 and WISP-I could be served as potent therapeutic target for the treatment of CRe. On the basis of our present study, we conclude that the Wnt and PI3K1Akt signalling pathways are involved in tumourigenesis of CRC in Malaysia. These pathways are interrelated although they might also act independently in promoting tumour growth and inhibition of apoptosis. This study has also provided useful information for the search or design of better antitumour interventions

Characterisation of Plant Derived Damnacanthal and Nordamnacantbal Induced Cytotoxicity on Human HT29 Colon Adenocarcinoma Cell Line

Nordamnacanthal and damnacanthal are two anthraquinones isolated from the roots of Morinda elliptica. They were found to exhibit cytotoxic activity against HT29 human colon adenocarcinoma cells. The cytotoxic concentrations of damnacanthal and nordamnacanthal that inhibited 50% growth (IC₅₀) of HT29 were 17 µg/ml and 7 µg/ml respectively. For the comparative purposes, the ICsos of several cytotoxic drugs against HT29 were also determined. The inhibition effect of nordamnacanthal was found to be comparable to etoposide (IC₅₀ = 7 µg/ml). cisplatin (IC₅₀ = 5 µg/ml) and doxorubicin (IC₅₀ = 6 µg/ml). The compound was found to be less active than methotrexate (MTX) (IC₅₀ < 0.05 µg/ml) and leunase (IC₅₀ = 2 µg/ml). On the other hand, the cytotoxic effect of damnacanthal was less active as compared to all cytotoxic drugs. However both compounds were found to be less toxic against non-cancerous fibroblast 3T3 ceJJs with the ICsos of 30 /lglml (damnacanthal) and 21 /lglm] (nordamnacanthal) respectively. Furthermore, damnacanthal and nordamnacanthal were found to induce apoptosis on HT29 cells at their ICso concentration as demonstrated by conventional agarose gel electrophoresis and also morphological alterations. DNA laddering was obtained after 12 hours of treatment by both compounds in a dose-independent but time-dependent fashion. Both compounds also caused cell death with apoptotic features such as cell shrinkage, membrane blebbing, nuclear fragmentation, and the presence of apoptotic bodies. In addition, caspase-3 was found to be activated during the execution of apoptosis induced by these compounds. This caspase activation was inhibited by a peptide based general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-V AD-FMK). In conclusion, this study demonstrates the potential antitumor activites of damnacanthal and nordamnacanthal

Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer

Positive correlation between overexpression of phospho-BAD with phosphorylated Akt at serine 473 but not threonine 308 in colorectal carcinoma

The enhancement of cell proliferation and promotion of cell survival via the inhibition of apoptosis is thought to be the key to the initiation and progression of cancers. The phosphatidylinositol-3 kinase (PI3K)/Akt is an important survival signal pathway that has been shown to be crucial in the regulation of balance between pro-apoptotic and survival (anti-apoptotic) signal. In this study, the expression of phosphorylated Akt at Thr308 and Ser473, BCL-2-antagonist of cell death (BAD) at Ser136 and glycogen synthase kinase-3β (GSK-3β) at Ser9 in 47 paraffin-embedded human colorectal carcinoma (CRC) tissues were determined by immunohistochemical staining in order to dissect the alterations in the signal transduction pathways in CRC. Our results showed that there was a significant increase in the expression of these biomolecules in CRC tissues compared to the apparently normal adjacent tissues. The frequency of increased expression in tumor colonic mucosa were as follows: p-Akt1/2/3 (Thr308)=16/47 (34%); p-Akt1 (Ser473)=21/47 (44.7%); phospho-BAD (p-BAD) Ser136=27/47 (57.4%) and phospho-GSK-3β (p-GSK-3β)=21/47 (44.7%). Analysis of the total p-Akt1 (Ser473), p-Akt1/2/3 (Thr308), p-GSK-3β (Ser9) and p-BAD (Ser136) score found that there was a statistically significant relationship with each other. A statistically significant positive linear relationship was found between total p-Akt (Ser473) score and total p-GSK-3β (Ser9) score as well as with total p-BAD (Ser136) score. On the other hand, total p-Akt1/2/3 (Thr308) scores had a statistically significant positive linear relationship with p-GSK-3β (Ser9) only. The Akt targets, p-GSK-3β (Ser9) and p-BAD (Ser136) were positively correlated to each other. There was no significant correlation between clinico-pathological data with total p-Akt1 (Ser473), p-Akt1/2/3 (Thr308), p-GSK-3β (Ser9) and p-BAD (Ser136) score except for age. The total scores of p-GSK-3β were found to be higher in patients in the age group of greater than 60. This is the first report of p-Akt1/2/3 (Thr308) and p-BAD (Ser136) expression in primary colorectal tumor tissue. Our data further supports the role of PI3K/Akt signaling pathways in the pathogenesis of CRC and contributes to the identification of target molecules in the signal transduction pathway for cancer therapy

Elucidation of regulatory interaction networks underlying human prostate adenocarcinoma

10.5246/jcps.2015.01.002Journal of Chinese Pharmaceutical Sciences24112-2

Nrf2 expression is regulated by epigenetic mechanisms in prostate cancer of TRAMP mice.

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer

Inhibitory Effect of a γ-Tocopherol-Rich Mixture of Tocopherols on the Formation and Growth of LNCaP Prostate Tumors in Immunodeficient Mice

In the present study, we determined the effects of a γ-tocopherol-rich mixture of tocopherols (γ-TmT) on the growth and apoptosis of cultured human prostate cancer LNCaP cells. We also determined the effects of dietary γ-TmT on the formation and growth of LNCaP tumors in immunodeficient mice. In the in vitro study, we found that the activity of γ-TmT was stronger than α-tocopherol for inhibiting the growth and stimulating apoptosis in LNCaP cells. In the animal study, treatment of severe combined immunodeficient (SCID) mice with dietary γ-TmT inhibited the formation and growth of LNCaP xenograft tumors in a dose-dependent manner. Mechanistic studies showed that g-TmT administration inhibited proliferation as reflected by decreased mitosis and stimulated apoptosis as reflected by increased caspase-3 (active form) expression in LNCaP tumors. In addition, dietary administration of g-TmT increased the levels of a-, γ- and δ- tocopherol in plasma, and increased levels of γ- and δ- tocopherol were also observed in the prostate and in tumors. The present study demonstrated that g-TmT had strong anticancer activity both in vitro and in vivo. Additional studies are needed to determine the potential preventive effect of g-TmT for prostate cancer in humans