Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Nutrition and Food Science

First Advisor

Smiti V. Gupta

Abstract

Lung cancer is one of the leading causes of death among cancers, with non-small cell lung cancer (NSCLC) accounting for 80-85% of all lung cancers and a five-year survival rate of 5 % at stage IIIB. Delta-tocotrienol (δT) including other tocotrienol isomers has been shown to exhibit anti-tumor activity via inhibition of different signaling pathways in tumors including NSCLC. Previously we reported that δT reduced cell invasion via inhibition of the Notch-1 and NF-κb pathway.

Matrix metallopeptidase 9 (MMP9) dependent cell migration and invasion are key processes in cancer metastasis. Hence, its suppression is a promising strategy for cancer therapeutics. The objective of specific aim 01 was to investigate the possibility of MMP9 inhibition as the underlying mechanism behind the anti-metastatic effects of δT on NSCLC cell lines A549 and H1299. Effects of δT on cell migration, invasion, cell adhesion and aggregation capability were investigated. MMP-9 activity was determined using gel zymography. The various proteins, genes, and miR involved in the Notch-1 and uPA signal transduction pathways have been studied for anti-metastatic activity by RT-PCR and western blot. Our findings showed that δT reduced cell migration, invasion, and adhesion in a dose and time-dependent manner. δT significantly inhibited MMP-9 activity in gel zymography. Further, δT inhibited Notch-1 mediated NF-κb and urokinase plasminogen activator (uPA) pathways which lead to the down-regulated expression of MMP-9 and increased miR 451 expressions. Our data suggests that δT attenuates tumor aggressiveness, invasion, and metastasis by down-regulation of the MMP-9 gene via Notch1 and uPA pathways

Further, the primary energy source of NSCLC is glutamine, and this cancer exhibits a high rate of glutamine dependency during its growth and development. Glutamine and essential amino acids (EAA) in NSCLC are reported to upregulate mTOR, a bioenergetics sensor which regulates cell growth, cell survival, and protein synthesis. SLC1A5/SLC7A5 transporters that allow glutamine and EAA to enter the proliferating tumors and send a regulatory signal to mTOR are novel concepts in cancer cell growth and development. Therefore, inhibiting glutamine uptake via blocking or downregulating glutamine transporters would be an excellent therapeutic target for NSCLC treatment. The Specific Aim:2 of this study, was to verify the metabolic dysregulation of glutamine and its derivatives in NSCLC using cellular 1H-NMR metabolomics approach while investigating the effect of δT on NSCLC growth and development, glutamine transporters, and mTOR pathway. Endometabolome of NSCLC was analyzed followed by cellular metabolomics analysis using SIMCA+ multivariate analyzing software. The results in metabolomics analysis showed significant inhibition in the uptake of glutamine, its derivatives; glutamate and glutathione, and some EAA in both cell lines with δT treatment providing their potential use as robust surrogate biomarkers for δT intervention in NSCLC. To further validation, NMR spectrums were quantified using Chenomx NMR Suite and metabolites were further analyzed using metaboanalyst 3.0 software. The results in metaboanalyst 3.0 indicated that δT directly impacted on the metabolism of glutamine and its derivatives where further validating results at previous analysis. Therefore, expression of glutamine transporters and mTOR pathway proteins were explored, and dose-dependent inhibition of glutamine transporters (SLC7A5 and SLC1A5) and mTOR pathway proteins (P-mTOR, mTOR, S6K, c-MYC and Bcl-XL) was evident in western blot analysis. Our findings suggest that δT works by inhibiting glutamine uptake into proliferating cells through glutamine transporter inhibition thus resulting in inhibition of cell proliferation and induction of apoptosis via downregulation of the mTOR pathway.

As the specific aim, one and two reported that δT shows anti-cancer properties by targeting different anti-cancer mechanisms. However, the δT are presently not available in quantities required for animal or clinical studies. Therefore, the objective of specific aim 03 is to investigate the interactions and effects of commercially available tocotrienols mixture directly isolated from palm oil (a mixture of isomers) in adenocarcinoma (A549) and squamous cell carcinoma lung cancer (H520) cell lines. A dose-dependent decrease in all growth was observed in both cancer cell lines with the addition of tocotrienols by MTS and colonogenic assay. Furthermore, a significant reduction in cell migration and tumor invasiveness was seen in both cell lines. Additionally, a significant induction of apoptosis was observed in Annexin V stain in flow cytometry analysis. Since tocotrienols showed effects against proliferation, apoptosis, migration, and invasiveness, RT-PCR and western blot analysis were used to explore molecular mechanisms behind above regulations by testing the expression of Notch-1 and its downstream stream genes. A dose-dependent decrease in expression was observed in Notch-1, Hes-1, Survivin, MMP-9, VEGF, and Bcl-XL proteins. Also, we found a mechanism linking the NF-kB pathway and Notch-1 down-regulation from NF- Кb colorimetric assay. Thus, our data suggests that commercially available tocotrienols inhibit cell growth, migration, and tumor cell invasiveness via down-regulation of Notch 1 and NF -КB while inducing apoptosis. Hence, these commercially available tocotrienol rich capsule could be an effective therapeutic for lung cancer prevention as same as pure δT.

These Anticancer effects and mechanisms of δT warrant further investigation of δT as a potential natural therapeutic approach to prevent NSCLC.

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