Heterocyclic Cyclohexanone Curcumin Analogues Inhibit Growth of Ovarian Cancer Cells, in-vitro
Background: Ovarian cancer is the most lethal gynaecological cancer in women. Nearly a quarter of a million women are newly diagnosed globally, with up to 140,200 lives lost annually. The high mortality is attributed to vague symptomatology and lack of reliable markers in the early stages of cancer. There also exist challenges of resistance to currently available therapies such as carboplatin and paclitaxel owing to gross heterogeneity of the ovarian tumours. This study examined the efficacy of second generation heterocyclic cyclohexanone analogues of curcumin in ovarian cancer models. Methods: SKOV-3 and OVCAR-3 cells were used to model ovarian cancer. Various concentrations of the 14 analogues were used to treat the cells for 72 h, and IC50 curves were established using the SRB assay and Graphpad Prism analysis. Further studies were conducted using the two most potent compounds, namely RL112 and RL117. Flow cytometry was performed to determine their effect on cell cycle and apoptosis. The effect of treatment on the expression of key proteins was also determined using western blotting at 24, 48 and 72 h time points. The ability to inhibit migration was determined through a scratch assay. Results: RL112 and RL117 had the highest potency. In SKOV-3 cells, the respective IC50 were 0.52 and 0.97 µM, while in OVCAR-3 they were 0.64 and 0.99 µM. A time- and concentration- dependant, cell-specific effect was observed following the time course. Treatment with RL112 and RL117 caused S- and G2/M-phase increases of between 1 and 186% in SKOV-3 cells, and the effect lasted up to 72 h. No cell-cycle effect was detected in OVCAR-3 cells. In addition to the treatment-induced cell cycle effects, the morphological outlook of both cell types was indicative of apoptosis in both cell lines, and this was confirmed via flow cytometry showing time and concentration-dependent, statistically-significant increases in proportion of apoptotic cells. Western blot results showed reduced expression of the proteins AKT, phosphorylated AKT, NF-κB, ERK and phosphorylated ERK in RL112 treated SKOV-3 cells. Highest inhibition of AKT activity was at 72 h (48%), while that of NF-κB was at 24 h (72%). Inhibition of ERK activity was greatest at 24 h (68%). RL112 also inhibited migration of SKOV-3 cell in a concentration-dependent manner. Conclusions: The results have shown that the heterocyclic cyclohexanone analogues RL112 and RL117 are plausible lead compounds. Cytotoxicity to SKOV-3 and OVCAR-3 cells, both of which are resistant to cisplatin, was demonstrated. The mechanisms of action were shown to be potentially via multiple pathways, namely AKT, ERK and NF-κB, by reducing total and phosphorylated protein expression, which resulted in cell cycle arrest and apoptosis, as well as inhibiting the cells’ migratory capacity.
Advisor: Rosengren, Rhonda Joy
Degree Name: Master of Science
Degree Discipline: Pharmacology and Toxicology
Publisher: University of Otago
Keywords: Ovarian; cancer; Heterocyclic; Cyclohexanone; Analogues; Inhibit Growth; in-vitro; RL112; RL117; gynaecological cancer; second generation; SKOV-3; OVCAR-3; Cytotoxicity; AKT; ERK; NF-κB
Research Type: Thesis