A Review On Molecular Biology Of Genes And Diseases

IGF Pathway in Cancer Treatment

Discuss about the Molecular Biology for Genes & Diseases.

The insulin-like growth factor family consists of three peptide hormones that include insulin, IGF-1 and IGH-2. They have 50% of their amino acid sequences in common (Le Roith 1997). They play a critical role in growth, differentiation and metabolism in almost every cell of the human body. Due to its diverse and far reaching roles it has been associated with several diseases and disorders and is a potential target for therapeutic intervention (Forbes 2016). IGF signaling is associated with decreased cell death and increased cell proliferation. Activation of IGF-1R leads to the activation of two pathways, PI3K/AKT and Ras/MAPK. PI3K/AKT pathway ultimately leads to decreased apoptosis, increased glucose metabolism, increased protein synthesis. On the other hand Ras/MAPK pathway leads to increased cellular proliferation by activating different transcription factors, such as ELK1.

Cancer cell proliferation, migration and invasion have been related to the activation of IGF-1R in many studies previously (Sachdev and Yee 2007). Cancers in lung, breast, colon, prostate are showed to be associated with increased level of plasma IGF (Pollak, Schernhammer and Hankinson 2004). Other members of IGF pathway are also proved to be involved with malignancies. This provides a way to treat cancers via modulating IGF pathway and various ligands involved with it. Drug resistance by tumors is one of the critical conditions in new age cancer treatment. As IGF pathway is involved in cell proliferation it shows a new era of treating these kinds of situations. Many studies have shown that modulating IGF pathway has promising effect in these cases (Denduluri et al. 2015). Ovarian cancer is the fifth most frequent cause of cancer death in women. In nearly half of ovarian and breast cancer patients, RAB25 mRNAs were found to be selectivey overexpressed at stage 3 and 4 serous epithelial ovarian cancers in comparison to other genes located in the amplified region. Decreased expression of two proteins BAK and BAX were found to be associated with an inhibition of apoptosis in such cells. Simultaneously activation of PI3K/AKT pathway provided potential mechanisms to employ the effects of RAB25 on tumor aggressiveness (Lee et al. 2015). Involvement of PI3k/AKT pathway in the RAB25 related tumor aggressiveness leads to the way to treatment by modulating this pathway.

Recently various evidences show that IGF-1 promotes atherosclerotic plaque stability and consequently reduces atherosclerosis in animal models. IGF-1 induces reduction of oxidative stress, cell apoptosis, proinflammatory signaling, and endothelial dysfunction, that helps prevent the disease. Aging has been widely linked with increased vascular oxidative stress and certain vascular diseases (Barzilai et al. 2012). It has been postulated IGF-1 may play a crucial role on vascular aging processes. There are various evidences that IGF-1 exerts pleiotropic effects on the vasculature resulting in reduced vascular oxidative stress, apoptosis, and inflammatory signaling pathway (Higashi et al. 2012). By modulating IGF signaling disease related to stress or premature cell death or decreased cell death can be prevented in future.

IGF Pathway in Cellular Stress-Related Diseases

Further studies on signaling pathway of IGF show its potential role in regulation of autoimmune diseases. Though this area of treatment requires a lot more research and practical approaches, the role of IGF signaling is competent enough. Studies show that IGF-I can potentially protect animal models from developing insulin-deficient diabetes mellitus (Gao et al. 2011). On the contrary, patients with Graves’ disease have shown an overexpression of the IGF-1R in many cell types and antibodies against the receptor were prevalent in such patients.  The frequency of IGF-IR positive B and T cells were found to be relatively high in patients with the disease. Hence, as IGF-1 and IGF-1R is found to be linked with the mentioned autoimmune disease modulation of the pathway might be a potential therapeutic target for managing the diseases and alleviating the health consequences (Smith 2010).

The modulation of IGF pathway opens new ways to treat various diseases like different types of cancer, cellular stress-related diseases and immunological diseases also. It depends totally on massive research and practically showing the results on animal models to prove the modulation of IGF pathway actually showing some positive results.

Comparative Genomic Hybridization: Comparative Genomic hybridization is a comparative study of two genomes by in situ hybridization technique that allows detection of differences in DNA sequences between the two genomes. Two genomes, one study and one reference genomes are differentially labeled after being denatured into single stranded DNA (Pinkel and Albertson 2005). These two genomes are then hybridized to normal metaphase spreads or to microarray. Ratio of the fluorescence intensity are then measured And plotted on a graphical manner. The graph can reveal locations of copy number changes in along the target chromosome or DNA sequences. It has been extensively used to identify genetic differences between normal cells and certain diseased cells mostly in cancer cell biology. It efficiently detects any gain or loss of genetic material in human genetic disorders by comparing the same with a reference genome, often a relevant normal cell genome.

Primarily the effect of stable expression of Rab25, a hallmark of cancer has been assayed in all the parts of Figure 2. Other hallmarks that were also assayed were Bak and Bax nuclear proteins. Bak and Bax are two nuclear-encoded proteins that are capable of moving through mitochondrial membrane and induce cell death by apoptosis. Phosphorylation of AKT was also assayed during stable expression of Rab25 and in presence of RNAi. AKT or protein kinase B (PKB) is a protein kinase that plays key roles several cellular functions including apoptosis.

IGF Pathway in Immunological Diseases

Viral-mediated transduction is the most potent technique to produce stable cell lines with a overexpressing protein. Vectors are utilized for this purpose. Vectors are autonomously replicating DNA molecules that can be used to transfer DNA fragments to cells of interest. For transfecting mammalian cells, vectors derived from mammalian viruses are used that include Simian viruses (SV40), polyomavirus, herpesvirus and papovirus (Khan 2013). The Rab25 gene can be inserted into any one of the vector mentioned and the same gene can be transferred to an appropriate mammalian cell line by viral transfection. Mammalian cell lines that are extensively used for this purpose are HeLa, HEK293T, U2OS so on and so forth. Compared to transient transfection, stable transfected cell lines are produced to analyze impact of altered gene expression over a long time period. Generally, antibiotic resistance or fluorescent reporter cell markers are incorporated into plasmid or vector DNA to create selection criteria. Clonal Selection can be done by appropriate selection controls. The selected colony is used to produce monoclonal cell line development and consequently producing a stable cell line generation. The expression and stability is checked on a regular basis. The construct expression is then validated by qRT-PCR and/or Western blot techniques.

Assay (e) was performed to evaluate the levels of Bak and Bax proteins in the four different ovarian cancer cell lines namely A2780 and hey and immortalized ovarian cancer cell lines T29 and T80 where there is overexpression of the protein Rab25. Bak protein levels are found to be lower in Rab25 expressing A2780, HEY and T29 cells as compared to pcDNA cells. In T80 cell lines Bak was overexpressed showing that Bak levels are regulated by other factors in certain cells. Bax levels were low in A2780 and Hey cells but not in T29 and T80 cells. In assay (f) Bak and Bax levels were measured in A2780 cells were Rab25 expression is downregulated by RNAi. The downregulation of Rab25 nullified Rab25 mediated inhibition of Bak and Bax and hence the two protein levels were found to be high in the cell lines.

Bak and Bax are two nuclear proteins that can travel to mitochondrial outer membrane upon activation of relevant signaling pathway and is essential for mitochondrial permiabilisation during apoptosis (Dewson and Kluck 2009). These two proteins play a significant role in intrinsic apoptotic pathway in mammalian cells and as a result they have being extensively studied for therapeutic purposes to block apoptosis.

Comparative Genomic Hybridization

Motility of cells where Rab25 is overexpressed or knocked down can be assayed by standard apoptotic assay methods. Cleavage of the genomic DNA into discrete fragments and consequently membrane disintegration are key features that occur during apoptosis and cell-mediated cytotoxicity. DNA cleavage being hallmark of apoptosis is often measured to assay apoptotic cell death. The DNA fragments can be assayed by ELISA quantification of histone-complexed DNA fragments. Furthermore, caspases are a class of protein that are expressed and involved in the early stages of apoptosis. Activation of the caspase proteins can be assay to determine the stage of apoptosis in a cell (Suzuki, Nakabayashi and Takahashi 2001). This can be done by in vitro enzyme assay or by detection of cleavage of an in vivo caspase substrate. Furthermore to assess the motility transwell migration assay can be performed to assess cell movement and migration. In Transwell migration assay there are two chambers; the cell suspension placed on the upper chamber and chemoattractants are placed in the lower chamber. Cells that are motile will migrate through the membrane and get attached to the lower side of the upper chamber; non-migratory cells will remain on the upper chamber. The migratory cells can then be labeled and quantified to assess the motility of the cell lines with overexpressed and knocked out Rab25 protein.

References

Barzilai, N., Huffman, D.M., Muzumdar, R.H. and Bartke, A., 2012. The critical role of metabolic pathways in aging. Diabetes, 61(6), pp.1315-1322.

Denduluri, S.K., Idowu, O., Wang, Z., Liao, Z., Yan, Z., Mohammed, M.K., Ye, J., Wei, Q., Wang, J., Zhao, L. and Luu, H.H., 2015. Insulin-like growth factor (IGF) signaling in tumorigenesis and the development of cancer drug resistance. Genes & Diseases, 2(1), pp.13-25.

Dewson, G. and Kluck, R.M., 2009. Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis. Journal of cell science, 122(16), pp.2801-2808.

Forbes, B.E., 2016. Two years in IGF research. Growth Hormone & IGF Research.

Gao, J., Chesebrough, J.W., Cartlidge, S.A., Ricketts, S.A., Incognito, L., Veldman-Jones, M., Blakey, D.C., Tabrizi, M., Jallal, B., Trail, P.A. and Coats, S., 2011. Dual IGF-I/II–neutralizing antibody MEDI-573 potently inhibits IGF signaling and tumor growth. Cancer research, 71(3), pp.1029-1040.

Higashi, Y., Sukhanov, S., Anwar, A., Shai, S.Y. and Delafontaine, P., 2012. Aging, atherosclerosis, and IGF-1. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, p.gls102.

Khan, K.H., 2013. Gene expression in mammalian cells and its applications. Adv Pharm Bull, 3(2), pp.257-63.

Lee, J.S., Kang, J.H., Boo, H.J., Hwang, S.J., Hong, S., Lee, S.C., Park, Y.J., Chung, T.M., Youn, H., Lee, S.M. and Kim, B.J., 2015. STAT3-mediated IGF-2 secretion in the tumour microenvironment elicits innate resistance to anti-IGF-1R antibody. Nature communications, 6.

Pinkel, D. and Albertson, D.G., 2005. Comparative genomic hybridization. Annu. Rev. Genomics Hum. Genet., 6, pp.331-354.

Pollak, M.N., Schernhammer, E.S. and Hankinson, S.E., 2004. Insulin-like growth factors and neoplasia. Nature Reviews Cancer, 4(7), pp.505-518.

Sachdev, D. and Yee, D., 2007. Disrupting insulin-like growth factor signaling as a potential cancer therapy. Molecular cancer therapeutics, 6(1), pp.1-12.

Smith, T.J., 2010. Insulin-like growth factor-I regulation of immune function: a potential therapeutic target in autoimmune diseases?. Pharmacological reviews, 62(2), pp.199-236.

Suzuki, Y., Nakabayashi, Y. and Takahashi, R., 2001. Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death. Proceedings of the National Academy of Sciences, 98(15), pp.8662-8667.