Studies on the role of neutrophil gelatinase-associated lipocalin (NGAL) in oral squamous cell carcinoma”, is a presentation of my original research work carried out at the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, India, under the supervision of Dr. I would also like to thank the current and former heads of the Department of Biosciences and Bioengineering, IIT Guwahati. Prof. I would like to acknowledge the support received from other teaching and non-teaching staff of the Department of Biosciences and Bioengineering, IIT Guwahati.
1 Chapter
Introduction
- Multistep process of development of oral cancer
- Preneoplastic lesions
- Individual cancers of oral cavity 1. Lip cancer
- Floor of the mouth cancer
- Buccal mucosa cancer
- Tongue cancer
- Hard palate cancer
- Salivary gland tumours
- TNM (Tumour lymph node metastasis) staging
- Etiology of oral cancer
- Tobacco
- Alcohol
- Virus infection
- Molecular alterations in oral cancer
- Therapies available
- Radiation therapy [external beam radiotherapy (EBRT)]: Radiation therapy is the most common in oral and oropharyngeal cancers and sometimes it is given after
- Chemotherapy
- Problems associated with therapies
- Chemoresistance: The multidrug resistance proteins ABCB1 (also known as MDR1 or P-gp), ABCC1 (also known as MRP1), ABCG2 (also known as BCRP
- Tumour recurrence: Local and regional recurrence is one of the reasons for poor prognosis of oral cancer. The reported reasons for tumour recurrence are tumour
- Neutrophil Gelatinase Associated Lipocalin (NGAL)
- Structure of NGAL
- Function of NGAL
- NGAL in Malignancy
- Expression of NGAL in different cancers
- Role of NGAL in cancer cell survival and proliferation
- Role of NGAL in EMT, invasion and metastases
- Role of NGAL in chemoresistance
- Importance of the study
- The main objectives of this study are
However, clear histological features of OMSF are not reported in the literature (Isaac et al., 2008). Coles et al., 1999 elucidated the three-dimensional structure of NGAL using NMR. Furthermore, NGAL overexpression was independent of LCN2 promoter methylation in bladder cancer (Dokun et al., 2008).
2 Chapter
Introduction
- Materials and Methods 1. Tissue microarray
- Tissue microarray details
- Immunohistochemistry (IHC)
- Scoring
- Statistical analysis
- Results and Discussion
- Expression of NGAL is downregulated in oral cancer
- Expression of NGAL is downregulated in different tumours of oral cavity
- Expression of NGAL is inversely correlated with degree of differentiation of oral cancer
- Expression of NGAL is downregulated in different stages of oral cancer
- Expression of NGAL is downregulated in different grades of oral cancer
- Expression of NGAL is downregulated in different process of the development of oral cancer
- Expression of NGAL is downregulated in different pathological types of oral cancer
- Expression of NGAL with respect to gender and age of oral cancer patients
- Conclusion
T2N1M0 2 III D9a 58 M Larynx Squamous cell carcinoma of the piriformis sinus T3N0M0 2 III D10a 71 M Larynx Squamous cell carcinoma of the larynx. T2N1M0 - III F5a 51 M Larynx Squamous cell carcinoma of the larynx (rare) T2N1M0 2 III F6a 58 M Larynx Squamous cell carcinoma of the piriformis sinus. Therefore, we analyzed the expression of NGAL in different stages of oral cancer development.
3 Chapter
Introduction
- Materials and methods 1. Materials
- Cell culture
- Cell proliferation
- Western blot analysis
- Statistical analysis
- Results and Discussion
- Nicotine and the expression of NGAL in OSCC cells
- Tobacco-specific nitrosamines and the expression of NGAL
- Conclusion
However, the role of tobacco in downregulating NGAL expression has not been investigated to date. Therefore, the main objective of this chapter is to investigate the role of tobacco on NGAL expression in oral cancer cells. Western blot was performed to analyze the expression of NGAL in tobacco-treated cells.
This is the first report showing the effect of tobacco components on NGAL expression in any type of cancer. Our results showed that nicotine upregulated the expression of NGAL in KB-CHR-85 cells in a dose-dependent manner. Therefore, we determined the effect of these tobacco-specific nitrosamines on NGAL expression in oral cancer cells.
We then treated SAS and KB-CHR-85 cells with the above-mentioned concentrations of NNK and NNN and analyzed the expression of NGAL. Therefore, we determined the effect of 4-NQO on NGAL expression in oral cancer cells. This is the first report showing the effect of tobacco components on NGAL expression.
Similarly, in KB-CHR-85 cells nicotine, NNK and 4-NQO decreased NGAL expression suggesting that NGAL is involved in oral cancer development.
4 Chapter
Introduction
- Materials and Methods
- Cell culture
- shNGAL stable knockdown
- Cell proliferation assay
- Cell cycle analysis
- Cell survival assay
- Cell invasion and migration assay
- Western blot analysis
- RNA isolation and Reverse transcriptase PCR
- Propidium Iodide Flow Cytometry (PI/FACS) analysis
- Statistical analysis
- Results and Discussion
- Confirmation of knockdown of NGAL
- Silencing of NGAL increases proliferation and survival of oral cancer cells
- Silencing of NGAL increases invasion and migration of oral cancer cells In the second chapter we found that the expression of NGAL is significantly
- Silencing of NGAL activates mTOR signaling and suppresses autophagy
- Silencing of NGAL selectively induces resistance against cisplatin
- Curcumin restored the expression of NGAL in oral cancer cells
- Conclusion
However, the role of NGAL in various processes of oral cancer development is poorly understood. The migratory potential of NGAL clot cells was determined by the in vitro wound healing assay. Therefore, we determined the effect of NGAL silencing on oral cancer cell proliferation.
Therefore, we hypothesized that downregulation of NGAL may lead to oral cancer invasion and metastasis. In vitro wound healing assay showed that the migratory potential of solid NGAL cells was higher. In contrast, in our study we found that down-regulation of NGAL increased invasion and migration of SAS cells.
In the previous chapter, we observed that treatment of oral cancer cells with tobacco components upregulated the expression of NGAL (Figure 3.1-3.4). Curcumin is believed to be one such agent that can regulate NGAL expression. Therefore, we examined the effect of curcumin on NGAL expression and oral cancer cell proliferation in vitro.
Interestingly, in elucidating the mechanism involved, we found that curcumin can upregulate the expression of NGAL in SAS cells.
5 Chapter
Discussion and Conclusion
- Limitations of the study
- Future prospective
However, in other cancers such as pancreatic and oral cancer, downregulation of NGAL protects against disease progression (Monisha et al., 2014). In a recent study, Lin et al., 2016 reported that NGAL expression was decreased in OSCC patients with lymph node metastasis compared to non-metastatic patients. Similarly, in colon cancer, NGAL silencing decreased cell-matrix attachment, motility, and cell invasion (Hu et al., 2009).
In contrast, in our study we observed that knockdown of NGAL in oral cancer cells increased cell proliferation, survival, invasion and migration. Similar results were observed in colon cancer, where upregulation of NGAL suppresses invasion and liver metastasis (Lee et al., 2006). The downstream target of mTOR, S6, also serves as a potential diagnostic biomarker for oral cancer (Chaisuparat et al., 2013).
The expression of caspase-3 was found to be downregulated or completely absent in dysplastic epithelium and the majority of OSCC (Veeravarmal et al., 2016). The expression of NGAL in oral cancer tissues was studied using TMA which did not contain tissues from Indian population. Establishment of the expression of NGAL in oral cancer tissues of Indian population of different stages, grades, types, etc.
Deciphering the expression pattern of NGAL in large cohorts would establish the prognostic significance of NGAL in oral cancer.
BIBLIOGRAPHY
Ribosomal protein S6 phosphorylation is associated with epithelial dysplasia and oral squamous cell carcinoma. Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma. Expression of Bcl-2 family proteins and association with clinicopathological characteristics of oral squamous cell carcinoma.
Neutrophil gelatinase-associated lipocalin and its receptor: independent prognostic factors of esophageal squamous cell carcinoma. Phosphorylated mTOR expression is associated with poor prognosis for patients with esophageal squamous cell carcinoma. Upregulation of neutrophil gelatinase-associated lipocalin in oral squamous cell carcinoma: relation to cell differentiation.
The phosphorylated mammalian target of rapamycin is associated with adverse outcome in oral squamous cell carcinoma. Stromal expression of neutrophil gelatinase-associated lipocalin correlates with poor differentiation and unfavorable prognosis in oral squamous cell carcinoma. Downregulation of neutrophil gelatinase-associated lipocalin in head and neck squamous cell carcinoma correlated with tumorigenesis, not metastasis.
Upregulation of neutrophil gelatinase-associated lipocalin in esophageal squamous cell carcinoma: significant correlation with cellular differentiation and tumor invasion.
List of Abbreviations
FAK : Focal adhesion kinase FGF-2 : Fibroblast growth factor FOS : FBJ murine osteosarcoma FOXP3 : Forkhead box P3. LRP1B : LDL receptor-related protein 1b MDMX : mouse double minute X MDR1 : multidrug resistance protein 1 MEC : mucoepidermoid carcinoma MEK : mitogen-activated protein kinase. NF-κB : Light chain enhancer of nuclear factor kappa of activated B cells NGAL : Neutrophil gelatinase-associated lipocalin.
NP-40 : 4-nonylphenyl poly (ethylene glycol) Nrf2 : Nuclear factor (erythroid-derived 2) 2 NSCLC : Non-small cell lung carcinoma.
LIST OF TABLES
PRESENTATION AND PUBLICATION LIST
Padmavathi G, Bordoloi D, Banik K, Monisha J, Singh AK and Kunnumakkara AB, "Mechanism of Chemoresistance in Bone Cancer and Different Chemosensitization Approaches", In Cancer Cell Chemoresistance and Chemosensitization. Banik K, Sailo BL, Thakur KK, Jaiswal A, Monisha J, Bordoloi D, and Kunnumakkara AB, "Potential of different sensitizers to overcome chemoresistance in cervical cancer," In Cancer Cell Chemoresistance and Chemosensitization. Bordoloi D, Banik K, Khwairakpam AD, Sharma A, Sailo BL, Monisha J, and Kunnumakkara AB, “Different approaches to overcome chemoresistance in esophageal cancer,” in Cancer Cell Chemoresistance and Chemosensitization.
Harsha C, Bordoloi D, Prakash J, Manteghi N, Padmavathi G, Monisha J and Kunnumakkara AB, 'Different chemosensitization methods in gastric cancer, in Cancer cell chemoresistance and chemosensitization'. Khwairakpam AD, Monisha J, Banik K, Harsha C, Sharma A, Bordoloi D and Kunnumakkara AB, 'Chemoresistance in Brain Cancer and Different Chemosensitization Approaches', In Cancer Cell Chemoresistance and Chemosensitization. Singh AK, Roy NK, Anand A, Banik K, Monisha J, Bordoloi D and Kunnumakkara AB, 'Different approaches to inhibit chemoresistance in hepatocellular carcinoma', in Cancer Cell Chemoresistance and Chemosensitization'.
మోనిషా జె, శర్మ ఎ, బానిక్ కె, పద్మావతి జి, బోర్డోలోయ్ డి, మరియు కున్నుమక్కర AB, "కెమోథెరపీ-రెసిస్టెంట్ మెలనోమా సెల్స్ యొక్క హైపర్సెన్సిటివిటీ ఆఫ్ కెమోథెరపీటిక్ ఏజెంట్స్", కెమోరెసిస్టెన్స్ మరియు కెమోసెన్సిటైజేషన్ ఆఫ్ క్యాన్సర్ సెల్స్లో. పద్మావతి జి, మోనిషా జె, బానిక్ కె, ఠాకూర్ కెకె, చౌదరి హెచ్, బోర్డోలోయ్ డి, మరియు కున్నుమక్కర AB, "ప్రోస్టేట్ క్యాన్సర్లో కెమోరెసిస్టెన్స్ని అధిగమించడానికి డిఫరెంట్ కెమోసెన్సిటైజేషన్ అప్రోచ్స్", కెమోరెసిస్టెన్స్ మరియు కెమోసెన్సిటైజేషన్ ఆఫ్ క్యాన్సర్ సెల్స్లో. కెమోరెసిస్టెన్స్ మరియు కెమోసెన్సిటైజేషన్లో రాయ్ ఎన్కె, శర్మ ఎ, సింగ్ ఎకె, బోర్డోలోయ్ డి, సైలో బిఎల్, మోనిషా జె మరియు కున్నుమక్కర AB, బ్లాడర్ క్యాన్సర్.
Padmavathi G, Monisha J, Harsha C and Kunnumakkara AB, "FGF and FGFR protein translocations and their effect in cancer", In Fusion Genes and Cancer.
Cell Science &
Molecular Biology
Neutrophil Gelatinase-Associated Lipocalin (NGAL): A Promising Biomarker for Cancer
Therapeutics
Review Article
Negligible expression of NGAL is observed in normal pancreas, endometrial glands and peripheral blood leukocytes. Overexpression of NGAL was first identified in primary kidney cells of mice infected with tumor virus SV40 [10]. Provatopoulou et al reported the presence of NGAL and MMP9 in the serum of breast cancer patients and concluded that it can be used in non-invasive monitoring of breast cancer progression, supporting its role as a breast cancer biomarker.
High expression of NGAL was observed in both colorectal carcinoma cell lines and xenograft mouse models of this disease [26,27]. Upregulation of NGAL led to activation of differentiation pathway and invasive progression of esophageal squamous cell carcinoma [ 32 ]. Expression of NGAL was found to be high in mildly invasive (SCCKN, HSC-2 and OSC-20), weak in mildly invasive (HSC-3, OSC-19 and SCC-25) and negative in highly invasive (HOC-313) and TSU) oral squamous cell carcinoma cell lines.
Furthermore, the expression of NGAL was found to be inversely correlated with E-cadherin and MMP-9 [49]. In hepatocellular carcinoma, overexpression of NGAL and/or NGALR resulted in poor prognosis and poor survival. High expression of NGAL was observed in advanced gastric cancer patients, resulting in poor prognosis [57].
Du et al investigated the mechanism of expression of NGAL in gastric cancer before and after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction.
NGAL is Downregulated in Oral Squamous Cell Carcinoma and Leads to Increased Survival,
Proliferation, Migration and Chemoresistance
However, the role of NGAL in oral cancer has not been well established so far. To understand the role of NGAL in oral cancer, we first determined the expression of NGAL in oral cancer tissues. Furthermore, the expression of NGAL was inversely associated with the degree of differentiation of tumors.
Similarly, the expression of NGAL was significantly downregulated with an increase in grade of oral cancer compared to normal tissues (Figure1D,E). Similarly, the expression of NGAL was significantly downregulated with an increase in grade of oral cancer compared to normal tissues (Figure 1D,E). Expression of NGAL (neutrophil gelatinase-associated lipocalin) in oral cancer. A) Representative images of expression of NGAL in oral cancer (left panel).
To study the role of NGAL in oral cancer cell proliferation and survival, we silenced the expression of NGAL (Figure 3A). To study the role of NGAL in oral cancer cell proliferation and survival, we silenced the expression of NGAL (Figure 3A). Western blot analysis showing the expression of NGAL in SAS cells after knockdown (right panel).
We studied the expression of NGAL in oral cancer tissues and found that NGAL was decreased in primary and metastatic tumor tissues.
