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  • br Materials and methods br


    Materials and methods
    Discussion CRC and PC occupy a good share in cancer-related deaths worldwide. CRC is a common gastrointestinal malignancy with a growing incidence whereas PC is one of the most dreadful malignancies with extremely poor survival rates. Though over the past decade there has been a remarkable improvement in the management and treatment of these cancers by understanding their underlying molecular mechanisms, the metastasis still remains a problem (Gryfe et al., 1997; Ryan et al., 2014). The battery of molecular biomarkers that would aid in early diagnosis and improve patient survival is required. SHH signalling pathway has essential roles in embryogenesis, maintenance of normal adult tissues and carcinogenesis (Yang et al., 2010). SHH protein, the main component of this pathway is expressed in the stomach, intestines and pancreas (van den Brink, 2007). Dysregulation of the SHH pathway via SHH overexpression has pathogenic effects in many solid malignancies like breast, gastric and pancreatic cancer. (Lee et al., 2007; Bailey et al., 2009; Noman et al., 2016). Apart from very few studies demonstrating that aberrant activation of SHH signalling is not common in CRC cell lines (Chatel et al., 2007), several studies established that active HH signalling is essential for tumour growth, recurrence, metastasis and survival of CRC dinaciclib (Varnat et al., 2009; Mazumdar et al., 2011). Therefore, we investigated SHH mRNA expression by real-time quantitative RT-PCR and protein expression by western blotting detection in primary CRC tissue specimens and their adjacent normal tissues. Results showed that the SHH mRNA and protein levels were increased by 39.34% (24/61) and 29.5% (18/61) respectively, compared with the levels in the adjacent normal tissue samples. All the samples with SHH overexpression at protein level also had overexpression of SHH at mRNA level suggesting SHH is upregulated at both transcriptional as well as translational level. Several studies have also reported upregulation of SHH in CRC. Monzo et al reported very high levels of SHH mRNA 84.21% (48/57) in CRC tissues (Monzo et al., 2006). Xu et al. have reported SHH overexpression in 59.6% (138/228) of CRC cases while Douard et al. reported SHH hedgehog mRNA overexpression in 86% (38/44) of the CRC cases (Douard et al., 2006; Xu et al., 2012). Our study was carried out on an ethnic Kashmiri population which may be the reason for the difference in our results with previously reported studies. SHH signalling cascade is known to promote cell proliferation and prevent apoptosis through binding of SHH to the transmembrane receptor protein Patched (PTCH1) which otherwise inhibits the activity of protein Smoothened (SMO), which in turn controls the downstream GLI transcriptional effectors responsible for transcription of genes causing cell proliferation and growth (Cohen et al., 2015; Jeong and McMahon, 2002). In our study, SHH protein overexpression was significantly associated with lymph node metastasis in CRC. Support for our study comes from a previous report where SHH protein over-expression correlated with lymph node metastasis in colon cancer (Xu et al., 2012). Importantly, we established the significant association of SHH overexpression with late-stage tumours indicating that SHH may be involved in cancer progression thereby functioning as a prognostic factor in CRC. According to a study done by Ghamdi et al. overexpression of SHH protein was not associated with any of clinicopathological parameters except low-grade CRC (Al Ghamdi et al., 2015) Douard et al. also did not observe a correlation between SHH protein over-expression and any of the clinicopathological variables of CRC (Douard et al., 2006). The reason for contradictory correlations may be due to the ethnicity of our population and considerably small sample size of our study. In PC, we also examined SHH mRNA and protein expression respectively. We observed that SHH mRNA was overexpressed in 81.81% (27/33) of PC cases and at the protein level, it was overexpressed in 72.7% (24/33) of PC cases compared to their adjacent normal tissues. These results support previous studies that SHH acts as an oncogene and plays a role in tumorigenesis or progression of pancreatic cancer. Activation of hedgehog signalling by SHH overexpression is known to enhance the proliferation and survival of pancreatic duct epithelial cells, the cells which give rise to PDAC (Morton and Lewis, 2007). A study by Thayer et al. reported SHH over-expression in 70% of PC's which is in consistency with our study (Thayer et al., 2003). Morton et al. reported SHH signalling to be crucial in multistage carcinogenesis of the pancreas (Morton et al., 2007). Ohuchida et al. proposed that SHH is a developmental marker in mucinous neoplasm of the pancreas (Ohuchida et al., 2006).