Synthesis of 7-O-methylpunctatin, a novel homoisoflavonoid, and its molecular mechanisms in attenuating basal and COX-2-induced malignant phenotype of breast cancer cells

Abstract

Background: Breast cancer is the leading cause of female morbidity and mortality worldwide. A substantial amount of evidence suggests a major role for inflammation in the initiation and progression of various malignancies including breast cancer. Among the different inflammatory markers, cyclooxygenase-2 (COX-2) is the most investigated and is overexpressed in tumor breast tissue yet undetectable in normal tissue. In fact, phytochemical polyphenols have been reported to decrease the proliferation of cancer cells, block pro-inflammatory cytokines as well as hinder transcription factors that mediate cancer progression.

Methods/Aims: Breast adenocarcinoma cell lines MCF-7 and MDA-MB-231 were employed to study the effects of the synthesized homoisoflavonoid (7-O-methylpunctatin). H1NMR and C13NMR were done to confirm the structure of our synthesized 7-O-methylpunctatin (7MP). The effects of 7MP were studied on COX-2 overexpressing and basal-breast cancer cells. First, the proliferative (MTT) and migratory (wound-healing assay) capacities of breast cancer cells treated with 7MP were assessed compared to control cells. Following that, prostaglandin E2 (PGE2) levels were measured using Enzyme Linked Immunosorbent Assay (ELISA) in COX-2 overexpressing cells. Western blotting was used to evaluate 7MP’s selectivity for COX-2 or COX-1. In addition, cell cycle analysis (flow cytometry) and apoptosis markers (caspase-glo assay) were examined on treated cells to evaluate the effect of 7MP on cell cycle and apoptotic machinery. Finally, the phosphorylation level of ERK1/2 (In-cell ELISA) and NF-κB activity (NF-κB luciferase reporter assay) were measured.

Results: The H1NMR and C13NMR spectra confirmed that our synthesized 7MP has the desired structure as that extracted from Bellevalia eigii. 7MP significantly reduced the basal and COX-2-induced proliferation of breast cancer cells. It also reduced the basal migratory capacity of MDA-MB-231 and MCF-7 cells. We also showed that 7MP selectively targets COX-2 but not COX-1 and decreases the amount of released PGE2 from parent MDA-MB-231 cells and COX-2 overexpressing cells. The cell cycle analysis demonstrated that treatment with 20 µM 7MP caused G0/G1 arrest of breast cancer cells and induced apoptosis by increasing the activity of caspases 3/7. Finally, 7MP inhibited basal and COX-2 induced ERK1/2 phosphorylation and NF-κB activity in MDA-MB-231 cells.

Conclusion: Our results show that COX-2 potentiates the tumorigenic capacity of breast cancer cells. We also demonstrate that the homoisoflavonoid synthesized 7MP attenuates both basal and inflammation-potentiated malignant phenotype of breast cancer cells.