3.3. Results and discussion
3.3.5. Effect of BaP on the expression of TIPEs in human lung epithelial cells
and progression of lung cancer. However, further in depth studies are requisite to unveil the underlined molecular mechanism of action.
Chapter 3
80 Figure 3.5. Effect of BaP on the expression of TIPE, TIPE2 and TIPE3 in lung epithelial cells A. Structure of BaP, B. MTT assay showing non-cytotoxic concentrations of BaP in L132 lung epithelial cells, C. RT-PCR analysis of the expression of TIPE, TIPE2 and TIPE3 after treating L132 cells with BaP for 24 h, D. Graphical representation of the expression of TIPE, TIPE2 and TIPE3 in BaP treated L132 cells as measured by densitometry scanning. GAPDH was used as internal control. Data are represented as Mean±SE, * denotes p<0.05 compared to control.
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2015a). Besides, this constituent of tobacco was also reported to cause upregulation of mesenchymal markers such as N-cadherin and vimentin and reduce the expression of E-cadherin, an epithelial marker (Chen et al., 2017). In addition, BaP is reported to cause alteration in p53 signaling cascade in human lung cancer cells as well cancer stem Cells (Bak et al., 2018). Additionally, NF-κB is also reported to have strong involvement in the BaP-promoted p53 expression (Pei et al.,1999). Therefore, examining the effect of BaP on the expression of TIPEs may give insight towards understanding the role of TIPEs in BaP induced lung carcinogenesis. Therefore, initially we determined the non-toxic concentrations of BaP which were found to be 0.05, 0.1, 0.25 and 0.5 μg/ml. Hence, those concentrations were used to treat L132 human lung epithelial cells for 24h and then expression of TIPE, TIPE2 and TIPE3 were analyzed with the help of RT-PCR. The mRNA level of TIPE was found to be upregulated in a dose dependent manner. In the case of TIPE2 and TIPE3, slight upregulation was observed at 0.05 μg/ml; however beyond that concentration, downregulation of both the genes were observed (Figure 3.5). As mentioned earlier, BaP activates MAPK signaling pathway through AhR. Deregulated MAPK pathway in turn gives rise to alterations in diverse cellular processes like differentiation, proliferation, apoptosis etc. (Vázquez-Gómez et al., 2018). Therefore, MAPK can be presumed as a downstream target of TIPE in BaP mediated lung carcinogenesis.
Further, BaP is reported to exert varied effect on the basis of the growth kinetics of the target cell population, tissue type and also genetic variations (Hamouchene et al., 2011).
Therefore, it can be considered to be responsible in part for TIPE2 and TIPE3 to get upregulated at lower concentration i.e. 0.05 µg/ml followed by their downregulation beyond that concentration when checked upto 0.5 µg/ml of BaP.
Chapter 3
82 3.4. Conclusion
This is the first report which showed the correlation between tobacco and its constituents and the regulation of TIPE, TIPE2 and TIPE3 in human lung cancer. These results are in accordance with our previous findings from IHC data. Treatment of L132 human lung epithelial cells with the water extract of tobacco called tuibur and other important constituents of tobacco such as NNK, NNN, nicotine and BaP resulted in upregulation of TIPE2 and TIPE3 notably. Additionally, tuibur, NNK, NNN and nicotine treated L132 cells showed upregulation of TIPE as well. This supports our IHC results, where upregulation of TIPE, TIPE2 and TIPE3 was observed in lung cancer tissues compared to normal lung tissues. Therefore, it can be presumed that deregulation of TIPE proteins may serve as one of the key molecular events in the development and progression of lung cancer as tobacco, which is responsible for 90%
of all lung cancer cases is strongly involved in the modulation of the expression of these proteins. Nevertheless, our results provide only a preliminary indication of the involvement of TIPE, TIPE2 and TIPE3 in tobacco induced lung carcinogenesis.
Therefore, mechanistic studies are certainly warranted to decipher the upstream regulators as well as downstream targets of TIPEs in tobacco induced lung carcinogenesis.
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Chapter 4
Role of TIPE family of
proteins in the development and progression of lung
cancer
Chapter 4
83 4.1. Introduction
In the preceding chapters, we have shown that TIPE family of proteins were differentially expressed in lung cancer tissues. TIPE, TIPE2 and TIPE3 were found to be significantly upregulated and TIPE1 showed marked downregulation in lung cancer tissues compared to the normal lung tissues. Further, treatment with tuibur and other tobacco related components were found to upregulate the expression of TIPE, TIPE2 and TIPE3 markedly. Noteworthy, treatment with NNK, the potent lung carcinogen which has been used customarily for inducing lung carcinogenesis in different animal models resulted in significant upregulation of TIPE, TIPE2 and TIPE3 in human lung epithelial cells. Altogether, these studies provide us a clear idea that TIPE, TIPE2 and TIPE3 are strongly involved in the positive regulation of lung carcinogenesis, in particular tobacco induced lung cancer. On the other hand, TIPE1 is found to be involved in the negative regulation of lung tumorigenesis. However, to elucidate the exact role of this family of proteins and their downstream targets, it is imperative to silence or disrupt them. Notably, siRNA or shRNA mediated silencing of these proteins was found to influence cell growth, proliferation, invasion and metastasis of lung cancer cells as evinced by a few studies carried out thus far which are discussed in the first chapter. However, no study reported the comparative analysis upon individual knockout of all the four proteins. Therefore, in this chapter, we have done knockout of TIPE, TIPE1, TIPE2 and TIPE3 individually in NCIH460 human lung cancer cells using CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9- method of gene editing and determined the effect of individual gene knockout on different hallmarks of cancer as well as a comparative analysis of them in lung cancer cells. Further, we determined their downstream targets which are involved in the
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pathogenesis of lung cancer. In addition, we found their involvement in tobacco mediated lung carcinogenesis and the underlined mechanism of action.