No abstract

Deoxyribonucleic acid (DNA) is the self-replicating hereditary material that provides a blueprint which, in collaboration with environmental influences, produces a structural and functional phenotype. As DNA coordinates and directs differentiation, growth, survival, and reproduction, it is responsible for life and the continuation of our species. Genome integrity requires the maintenance of DNA stability for the correct preservation of genetic information. This is facilitated by accurate DNA replication and precise DNA repair. DNA damage may arise from a wide range of both endogenous and exogenous sources but may be repaired through highly specific mechanisms. The most common mechanisms include mismatch, base excision, nucleotide excision, and double-strand DNA (dsDNA) break repair. Concurrent with regulation of the cell cycle, these mechanisms are precisely executed to ensure full restoration of damaged DNA. Failure or inaccuracy in DNA repair contributes to genome instability and loss of genetic information which may lead to mutations resulting in disease or loss of life. A detailed understanding of the mechanisms of DNA damage and its repair provides insight into disease pathogeneses and may facilitate diagnosis and the development of targeted therapies.

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds’ effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

Following exposure of cells to gamma-radiation, a cascade of intracellular consequences may be observed in a semitemporal manner. This includes deoxyribonucleic acid (DNA) damage and reactive oxygen species (ROS) accumulation initially, with consequent signaling for DNA repair and facilitative regulation of the cell cycle. Failure to rectify the damage or ROS levels leads to induction of senescence or apoptosis. 2-Ethyl-3-O-sulfamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), a 2-methoxyestradiole analog designed in silico for superior pharmacokinetics, was investigated for its potential to enhance apoptotic signaling and decrease the long-term survival of cells exposed to radiation. Sequential early intracellular effects within radiation-treated MCF-7 breast- and A549 lung cancer cells pre-exposed to low-dose ESE-15-ol were investigated using various flow cytometric protocols, spectrophotometry, and microscopy. Long-term cellular survival and proliferation was examined using clonogenic studies, which demonstrated a significant decrease in the presensitized cells. Combination-treated cells exhibited increased superoxide formation, and decreased Bcl-2 expression and -phosphorylation. Induction of apoptosis and elevation of the sub-G1 phase was evident in the pre-exposed MCF-7 cells, although only minimally in the A549 cells at 48-h. These results indicate that low-dose ESE-15-ol may increase tumor response to radiation. Future studies will investigate the effect of ESE-15-ol pre-exposure on radiation-induced DNA damage and repair mechanisms.

The recurrence and/or lack of response of certain tumors to radio- and chemotherapy has been attributed to a small subpopulation of cells termed cancer stem cells (CSCs). CSCs have been identified in many tumors (including solid and hematological tumors). CSCs are characterized by their capacity for self-renewal, their ability to introduce heterogeneity within a tumor mass and its metastases, genomic instability, and their insensitivity to both radiation and chemotherapy. The latter highlights the clinical importance of studying this subpopulation since their resistance to traditional treatments may lead to metastatic disease and/or tumor relapse. Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignancy worldwide with the highest incidence occurring in East Asia and eastern and southern Africa. Several cellular subpopulations believed to have CSC properties have been isolated from HNSCCs, but at present, identification and characterization of CSCs remains an experimental challenge with no established or standardized protocols in place to confirm their identity. In this review we discuss current approaches to the study of CSCs with a focus on HNSCCs, particularly in the context of what this might mean from a therapeutic perspective.