RADIOSENSITIZING EFFECT OF THYMOQUINONE IN 2D AND 3D CELL CULTURE MODELS OF HUMAN COLORECTAL CANCER

Abstract

Background: Colorectal cancer (CRC) is the third most common cancer affecting both men and women worldwide. Despite the great advances made in detecting and treating CRC cancer, it remains a major burden in many countries. Standard treatments for CRC include surgical resection, chemotherapy, radiotherapy, and ablative therapies for metastases. These treatments have increased the overall survival of patients; however, in many cases, tumor relapse occurs due to resistance of cancer stem cells (CSCs) to conventional therapies, including radiotherapy. Some natural compounds have been shown to induce radiosensitization of CSCs. Thymoquinone (TQ), the black seed extract, has shown numerous anti-cancer activities in many cancers, including CRC. In addition, a radiosensitizing effect of TQ has been documented in breast cancer, melanoma, and head and neck squamous cell carcinoma (HNSCC), yet no studies have investigated its radiosensitizing effects on CRC cells and stem cells.

Objective: The overall aim of this thesis was to investigate the radiosensitizing potential of TQ on CRC cells and stem/progenitor cells and in patient-derived organoids. Our first aim was to study the inhibitory effects of TQ and radiation on a panel of human colorectal cancer cells cultured in 2D. Our second aim was to isolate and enrich for CRC stem cells from HCT116 and HT29 cell lines using 3D sphere formation assay and study the effect/mechanism of action of TQ and radiation on self-renewal capacity of colonospheres. The third aim was to employ fresh CRC tissue specimens from treatment-naïve patients to establish 3D patient-derived organoids and study the response of the established organoids to TQ alone, radiation alone, and combinations of TQ and radiation.

Methods: We first assessed the radiosensitizing effects of TQ in 2D cultures of CRC cells (HCT116, HCT116 p53 null, HT29, and DLD1) with different mutations and sensitivity to TQ and radiation. We then tested the efficacy of the combination treatment in CRC stem/progenitor cells enriched in 3D sphere formation assay, in which single cell suspensions were plated using Matrigel as an extracellular matrix. Immunofluorescent analysis and western blot were used to determine the mechanism of radiosensitization by TQ in these 2D and 3D cultures. The effect of TQ and radiation combinations in patient-derived organoids was established using organoid forming assay and immunofluorescence staining for cancer stem cell markers CD44 and CK19. Statistical analysis was performed using Graphpad prism 6.

Results: Our results showed that TQ sensitized CRC cells to radiation and reduced cell proliferation, viability, clonogenic survival, and migration ability and was non-toxic to non-tumorigenic intestinal cells. TQ sensitizing effects were associated with G2/M arrest, DNA damage, oxidative stress, as well as changes in key signaling molecules involved in radioresistance, including p-mTOR, MEK, β-catenin, and NF-κB. Moreover, combination treatment upregulated p53 and p21 expression. Combining a low dose of TQ (3 µM) with ionizing radiation (IR) (2 Gy) resulted in complete eradication of CSC populations enriched from HCT116 and HT29 cells at generation 5 and this was associated with inhibition of survival, stemness, and DNA repair through targeting molecules involved in these processes, including CD44, CK8, CK19, CD133, β catenin, and NF-κB while upregulating γH2AX, p53, and p21. These doses also led to ~1.4- to ~3.4-fold decrease in organoid forming ability of patient-derived organoids and resulted in more than 1.3-fold decrease in stem cell markers CD44 and CK19 expression in organoids that were resistant to radiation. Our findings show that combining TQ and radiation could be a promising therapeutic strategy for eradicating CRC cells and stem/progenitor cells.

Conclusion: This study underscores the importance of 3D sphere and organoid culture assays for studying colorectal cancer stem/progenitor cell characteristics. Moreover, this study demonstrates TQ’s potential as a radiosensitizing molecule against colorectal cancer cells and stem cells, in addition to patient-derived organoids that are resistant to radiation alone.