Date of Award

11-1-2022

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Kulsoom Ghias

Second Advisor

Dr. Azhar Hussain

Third Advisor

Dr. Mati Ur Rehman

Department

Biological and Biomedical Sciences

Abstract

Background: Ranked as the third most common cancer worldwide and the fifth most common cause of cancer in Pakistan, colorectal cancer has a high incidence and mortality rate despite advancements in its treatment and management. Majority of the patients reporting with CRC present with late or end-stage disease that is refractory to treatment and has a poor overall prognosis. Multiple studies show that cancer stem cells (CSCs) play a significant role in tumor pathogenesis and chemo-resistance which results in tumor relapse and metastasis. ALDH1, a CSC like marker, has been shown to be up-regulated in various cancers including CRC. This up-regulation leads to development of a unique tumor micro-environment or CSC niche, that nurtures and protects these cells against chemotherapy. In addition, alterations in the levels of TMIGD1, a cell cycle checkpoint protein, also increases chemo-resistance leading to a poor survival in cancer.
Methods and Results: Using three established CRC cell lines that are representative of various molecular sub-types, the expression of ALDH1 and TMIGD1 was determined by RT-PCR and immune cytochemistry. MTT assay was done to assess cell viability and apoptosis was measured using annexin V/propidium iodide dual staining. Data showed maximum baseline expression of ALDH1 in HT-29 cell line followed by HCT 116 and HCT-15 cells by RT-PCR. Baseline TMIGD1 levels were comparable across all three cell lines, with HT-29 expressing the highest transcript levels followed by HCT-15 and HCT 116. ICC confirmed this data at the protein level. Treatment of all three CRC cell lines with ALDH1 specific inhibitor nifuroxazide and chemotherapeutic agent oxaliplatin showed a dose-dependent decrease in cell viability. At the transcript level, ALDH1 expression decreased with nifuroxazide, oxaliplatin or combination (nifuroxazide + oxaliplatin) treatment in all three cell lines. However, oxaliplatin treatment alone increased expression of ALDH1 in HT-29 cells while nifuroxazide and combination treatment down-regulated this expression. TMIGD1 levels were altered by treatment with the drugs either alone or in combination but did not inversely correlate with ALDH1 transcript levels, indicating a possible independent regulatory mechanism. Flow cytometry analysis showed that after being treated with nifuroxazide, oxaliplatin or a combination of both, the reduction in cell viability in the CRC cell lines was due to induction of apoptosis.
Conclusion: Colorectal cancers with high ALDH1 expression may benefit from the addition of ALDH1 specific inhibitors in combination with chemotherapy to induce a stronger treatment response and/or overcome resistance.

First Page

1

Last Page

70

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