THYMOQUINONE INDUCES APOPTOSIS AND DNA DAMAGE IN 5-FLUOROURACIL-RESISTANT COLORECTAL CANCER STEM /PROGENITOR CELLS AND SENSITIZES THEM TO RADIATION

Abstract

Background: Colorectal cancer (CRC) is one of the most prevalent cancers among men and women. It is a heterogenous disease that arises from a combination of genetic and epigenetic alterations. Despite the advances in treatment options, CRC is associated with high mortality and recurrence rates. High recurrence rates have been recently attributed to a population of cancer stem cells (CSCs) characterized by self-renewal capacity and resistance to chemo and radiation therapy. Isolation of colorectal CSCs remains to be a challenge due to the lack of universally characterized markers. Therefore, it is essential to adopt new approaches for the treatment of this disease by identifying and effectively targeting colorectal CSCs. The black seed extract Thymoquinone (TQ) has shown promising antitumor properties on numerous cancer systems both in vitro and in vivo; however, its effect on colorectal CSCs is poorly established. In addition, studies on the effect of TQ as a radiosensitizer against CSCs are limited.

Objective: The overall aim of this thesis was to investigate the effect of TQ on targeting colorectal cancer stem/progenitor cells either alone or in combination with radiation. Our first aim was to demonstrate the inhibitory effect of TQ on 5FU-sensitive and resistant human colorectal cancer HCT116 cells cultured in 2D. Considering that three-dimensional (3D) sphere cultures are rapidly emerging as an in vitro model to study CSC properties, our second aim was to employ 3D sphere formation assay to isolate and enrich for colorectal CSCs from both cell lines and study the effect/mechanism of action of TQ on proliferation and self-renewal capacity of colonospheres. The third aim was to establish the effect of TQ on sensitizing 2D and 3D cultured cells to radiation. Our last aim was to characterize the in vivo tumor initiation capacity of colonospheres and determine cellular/molecular effects of TQ on resistant colonospheres in vivo.

Methods: We first assessed the response of TQ on 2D cultures, and then tested its efficacy in a 3D sphere-formation assay against an enriched population of colorectal cancer stem/progenitor cells in which single cell suspensions were plated in a 3D environment using Matrigel as an extracellular matrix. Immunofluorescent analysis, immunohistochemistry and western blot were used to determine TQ’s mechanism of action. The effect of TQ and radiation combination in 2D and 3D cultures was established using immunofluorescence staining for γ-H2AX and xenotransplantation mouse model of colorectal cancer along with molecular assays were used to characterize TQ’s effect in vivo. Statistical analysis was performed using Graphpad prism 7.

Results: Our results showed that TQ significantly decreased self-renewal potential of CSC populations enriched from 5FU-sensitive and resistant HCT116 cells at 10-fold lower concentrations when compared to 2D monolayers. TQ decreased the expression levels of colorectal stem cell markers CD44 and EpCAM and proliferation marker Ki67 in colonospheres derived from both cell lines and reduced cellular migration and invasion. Further investigation revealed that TQ treatment led to increased TUNEL positivity and a dramatic increase in the amount of the DNA damage marker γ-H2AX particularly in 5FU-resistant colonospheres, suggesting that the diminished sphere forming ability in TQ-treated colonospheres is due to induction of DNA damage and apoptotic cell death. Interestingly, combination of TQ and radiation induced a dramatic increase in γ-H2AX 24 hours after radiation, suggesting that TQ sensitizes 5FU-sensitive and resistant cells to radiation. TQ and radiation treatment also significantly reduced the number of 5FU-sensitive and resistant colonospheres when compared to radiation alone. The intraperitoneal injection of TQ in mice inhibited subcutaneous tumor growth of spheres derived from 5FU-sensitive and 5FU-resistant HCT116 cells. Also, TQ treatment induced apoptosis and inhibited NF-κB and MEK signaling in these tumors.

Conclusion: This study underscores the importance of 3D sphere culture assay for studying colorectal CSC properties. Moreover, this study demonstrates TQ’s potential as an effective treatment strategy against colorectal cancer stem/progenitor cells either alone or in combination with radiation.