Research Question Essays Examples
Critical appraisal of “Co-localization of β-catenin with Notch intracellular domain in colon cancer: a possible role of Notch1 signaling in activation of CyclinD1-mediated cell proliferation”
The increasing prevalence of colorectal cancer among Asians has been attributed to the westernization of their diet. The title of the article explicitly mentions the population, parameter and the expected outcome of the study. According to the authors, the protein β-catenin has been previously observed to localize in the sub-cellular regions followed by translocation to the nucleus. The authors believe that this step promotes the immortalization of the cells, thereby establishing the relevance of this study to current research. Currently, the studies on colorectal cancer have been focused on the Wnt and Notch 1 signaling pathways but have not considered the interaction of Notch intracellular domain (NICD) with β-catenin. Therefore, this study adds a new insight into the existing research (Gopalakrishnan et al. 281). According to past literature, Wnt signaling pathway is involved in tissue differentiation and development of various organs in the body. Dysregulation of this pathway has been thought to be associated with tumorigenesis. The protein under study, β-catenin, forms a vital component of the cell membrane and cytoskeleton by combining with E-cadherin. This adhesion promotes cell-cell adhesion in normal tissue. This establishes the fact that β-catenin has major role in controlling cell architecture. During Wnt signaling, β-catenin translocates to the nucleus and activates several cell proliferation genes. During the absence of Wnt signaling, β-catenin is phosphorylated upon complex formation with adenomatous polyposis coli (APC), glycogen synthase kinase (GSK) 3β and axin and degraded by the ubiquitin pathway. Wnt/β-catenin have been reported to have interaction with other pathways such as Notch pathway (Gopalakrishnan et al. 281). The rationale for this study comes from the evidence of direct Notch pathway and β-catenin interaction in Drosophila and evidence of synergistic effect of β-catenin and Notch pathway in the maintenance of intestinal stem cells. By extension of this fact, a dysregulated β-catenin and Notch pathway could interact in tumorigenesis as well (Gopalakrishnan et al. 282).
The hypothesis proposed by the authors in this paper is that the protein β-catenin interacts with NICD to activate the genes CyclinD1 and Hes1, which have been implicated in tumorigenesis. Therefore, the authors would have had to prove that a) Notch and β-catenin interact, b) this interaction activates Hes1 and CyclinD1, and c) inhibition of β-catenin and NICD is significant for treating colorectal cancer.
This research question would require an analytical observation research design that reports the occurrence of a phenomenon by comparing disease samples to control and determine the frequency of occurrence of the phenomenon. For this case-control study, the authors obtained 22 normal, 35 adenoma (benign tumor) and 63 adenocarcinoma (malignant tumor) colorectal biopsy samples from the same hospital. It is essential to draw control samples from the same population to eliminate bias. The tissue samples suggest that the authors wanted to study the disease progression from benign to malignant, which in case of cancer would be marked by immortalization of the benign tissue mass. To maintain the cancerous nature of the adenocarcinoma tissues, patients who underwent any form of therapy were excluded. The authors have mentioned that the Institutional Ethical Committee of their institution approved the study (Gopalakrishnan et al. 282).
Testing the hypothesis
According to the protocol of Immunohistochemical and immunofluorescence analysis, the samples were observed under a light microscope after treatment to elucidate the presence of β-catenin and NICD (Gopalakrishnan et al. 282). The researchers used human colon tumor cell line HT29 to test their hypothesis. To prove that β-catenin localization was tumor specific, the cytoplasmic concentration of the protein was compared with the adenoma and adenocarcinoma samples. The result showed that the β-catenin showed weak cytoplasmic staining in normal tissue and intense cytoplasmic as well as nuclear staining in cancerous samples. To prove that NICD was expressed mainly in colorectal tissues, immunohistochemical studies were performed that showed results similar to β-catenin. To prove the interaction between β-catenin and NICD, the authors had to prove that they co-localized in the nucleus and cytoplasm at the same time. For this step, coimmunofluorescence was performed with fluorescence antibodies specific for β-catenin (green) and NICD (red). Interaction of the two antibodies was observed as yellow fluorescence, thereby proving the interaction (Gopalakrishnan et al. 285). To analyze the level of protein expression, a total RNA study was performed. The RNA was isolated from all the three samples under study and the cell lines. A PCR was performed using primers for CyclinD1 mRNA and Hes 1 mRNA. This mRNA was compared with the mRNA levels after an inhibitor was used to arrest the Notch pathway. To prove that Notch signaling was essential for cell proliferation, the cell growth was inhibited using a cell growth inhibitor, N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The results showed decreased level of CyclinD1 and Hes 1 transcripts as well as decreased expression of cell replication proteins. DAPT arrests Notch signaling and the further cascade of events, indicating that Notch pathway, β-catenin, CyclinD1 and Hes 1 are related (Gopalakrishnan et al. 286). During DAPT treatment, the authors also observed an increase in the levels of the protective gene MUC2, which has been associated with promoting cell structure integrity (Gopalakrishnan et al. 281). This step mimics the actions that would be observed if a hypothetical drug would inhibit the Notch pathway.
Statistical analyses of the results
Significance of this study
Past literatures suggest that elucidating the Wnt and Notch pathways could help discover novel targets for colorectal cancer therapy. Current researches in the field of colorectal therapy focuses on inhibiting the Wnt and Notch pathways in the cancer stem cells. In order to develop a successful drug candidate, it is essential to know all the relevant targets and steps within these two pathways. This study is significant because previous studies have concentrated only on targeting Wnt/ β-catenin signaling for drug therapy because there was no knowledge on the Notch/ β-catenin signaling. The authors have, therefore, added another possible candidate pathway as a colorectal cancer drug development target. Accordingly, knowing that the protein β-catenin interacts with NICD could be another piece of the colorectal cancer therapy puzzle. The authors have successfully established that in presence of a Notch pathway inhibitor such as DAPT, there is a statistically significance decrease in the transcription of Hes1 (7.1 fold at 25μM concentration) and CyclinD1 (1.6 fold at 25μM concentration), which have been established as genes required for the progression of colorectal cancer (Gopalakrishnan et al. 281). The inhibitor also allows positive increase in the transcription of MUC2 gene (7.5 fold at 25μM concentration), which has been established to be a protector of the intestinal epithelium in past literatures. Therefore, if the inhibitor were to be replaced by an actual colorectal cancer drug that targeted the β-catenin or the NICD, we could expect similar results and that is what the authors have tried to prove via this paper (Gopalakrishnan et al. 290).
Cons of the study
The study, being an observational in nature, could have had confounding biases, data coarseness due to measurement errors, etc. These errors are not determinable through examination of the authors’ literature. The authors have not mentioned possible biases in the sample selection and collection. The construction of the paper, however, does have some issues. The introduction of new ideas in the discussion section warrants an explanation. The authors could have discussed the new ideas under literature review section or as a continuation of the introductory paragraphs. The ultimate purpose of this study-to find a drug therapy target-has not been mentioned at all.
Gopalakrishnan, Natarajan, et al. "Colocalization of β-catenin with Notch intracellular domain in colon cancer: a possible role of Notch1 signaling in activation of CyclinD1-mediated cell proliferation." Molecular and cellular biochemistry 396.1-2 (2014): 281-293.