Eugenol potentiates cisplatin anti-cancer activity through inhibition of ALDH-positive breast cancer stem cells and the NF-κB signaling pathway.

Triple-negative breast tumors are very aggressive and contain relatively high proportion of cancer stem cells, and are resistant to chemotherapeutic drugs including cisplatin. To overcome these limitations, we combined eugenol, a natural polyphenolic molecule, with cisplatin to normalize cisplatin mediated toxicity and potential drug resistance. Interestingly, the combination treatment provided significantly greater cytotoxic and pro-apoptotic effects as compared to treatment with eugenol or cisplatin alone on several triple-negative breast cancer cells both in vitro and in vivo. Furthermore, adding eugenol to cisplatin potentiated the inhibition of breast cancer stem cells by inhibiting ALDH enzyme activity and ALDH-positive tumor initiating cells. We provide also clear evidence that eugenol potentiates cisplatin inhibition of the NF-κB signaling pathway. Indeed, the binding of NF-κB to its cognate binding sites present in the promoters of IL-6 and IL-8 was dramatically reduced, which led to potent down-regulation of the IL-6 and IL-8 cytokines upon combination treatment relative to the single agents. Similar effects were observed on proliferation, inhibition of epithelial-to-mesenchymal transition and stemness markers in tumor xenografts. These results provide strong preclinical justification for combining cisplatin with eugenol as therapeutic approach for triple-negative breast cancers through targeting the resistant ALDH-positive cells and inhibiting the NF-κB pathway.

Eugenol triggers apoptosis in breast cancer cells through E2F1/survivin down-regulation.

BACKGROUND: Breast cancer is a major health problem that threatens the lives of millions of women worldwide each year. Most of the chemotherapeutic agents that are currently used to treat this complex disease are highly toxic with long-term side effects. Therefore, novel generation of anti-cancer drugs with higher efficiency and specificity are urgently needed. METHODS: Breast cancer cell lines were treated with eugenol and cytotoxicity was measured using the WST-1 reagent, while propidium iodide/annexinV associated with flow cytometry was utilized in order to determine the induced cell death pathway. The effect of eugenol on apoptotic and pro-carcinogenic proteins, both in vitro and in tumor xenografts was assessed by immunoblotting. While RT-PCR was used to determine eugenol effect on the E2F1 and survivin mRNA levels. In addition, we tested the effect of eugenol on cell proliferation using the real-time cell electronic sensing system. RESULTS: Eugenol at low dose (2 µM) has specific toxicity against different breast cancer cells. This killing effect was mediated mainly through inducing the internal apoptotic pathway and strong down-regulation of E2F1 and its downstream antiapoptosis target survivin, independently of the status of p53 and ERα. Eugenol inhibited also several other breast cancer related oncogenes, such as NF-κB and cyclin D1. Moreover, eugenol up-regulated the versatile cyclin-dependent kinase inhibitor p21WAF1 protein, and inhibited the proliferation of breast cancer cells in a p53-independent manner. Importantly, these anti-proliferative and pro-apoptotic effects were also observed in vivo in xenografted human breast tumors. CONCLUSION: Eugenol exhibits anti-breast cancer properties both in vitro and in vivo, indicating that it could be used to consolidate the adjuvant treatment of breast cancer through targeting the E2F1/survivin pathway, especially for the less responsive triple-negative subtype of the disease.

PAC, a novel curcumin analogue, has anti-breast cancer properties with higher efficiency on ER-negative cells.

We have investigated here the anti-breast cancer properties of two novel curcumin analogues, EAC and PAC. Apoptosis was assessed by the annexin V/propidium iodide (PI) assay on different breast cancer and normal cells. Immunoblotting analysis determined the effects of these agents on different apoptotic and oncogenic proteins. Furthermore, flow cytometry and Elispot were utilised to investigate the effects on the cell cycle and the production of cytokines, respectively. Breast cancer tumour xenografts were developed in nude mice. Finally, (18)F-radiolabeled PAC and curcumin were produced to study their bioavailability and tissue biodistribution in mice. PAC is five times more efficient than curcumin and EAC in inducing apoptosis, mainly via the internal mitochondrial route. This effect was 10-fold higher against ER-negative as compared to ER-positive cells, and ectopic expression of ERα rendered ER-negative breast cancer cells more resistant to PAC. In addition, PAC delayed the cell cycle at G2/M phase with a stronger effect on ER-negative cells. Moreover, PAC exhibited strong capacity as an immuno-inducer through reducing the secretion of the two major Th2 cytokines IL-4 and IL-10. Importantly, PAC significantly reduced tumour size, and triggered apoptosis in vivo. Furthermore, PAC inhibited survivin, NF-kB and its downstream effectors cyclin D1 and Bcl-2, and strongly up-regulated p21(WAF1) both in vitro and in tumours. Besides, PAC exhibited higher stability in blood and greater biodistribution and bioavailability than curcumin in mice. These results indicate that PAC could constitute a powerful, yet not toxic, new chemotherapeutic agent against ER-negative breast tumours.

The RAD9-dependent gene trans-activation is required for excision repair of active genes but not for repair of non-transcribed DNA.

The Saccharomyces cerevisiae RAD9 and RAD24 are two cell cycle checkpoint genes required for UV-dependent up-regulation of a battery of genes involved in different metabolic pathways. RAD9 is also implicated in nucleotide excision repair (NER); however, its precise role is still unclear. For the present study, we made use of the high-resolution primer extension technique to show that the RAD9-deleted cells are deficient in the repair of both strands of the URA3 gene. Interestingly, this defect was suppressed by over-expressing the RAD24 gene, suggesting that the role of RAD9 in NER is indirect probably through the UV-dependent trans-activation of some NER factors. Accordingly, we present evidence that the inhibition of UV-related de novo protein synthesis by cycloheximide has no effect on the rad9Delta mutant while it suppresses the correcting effect of RAD24 over-expression. Importantly, we have also shown that RAD9 has no role in repair of transcriptionally inactive DNA sequences (URA3 promoter and transcriptionally silent GAL10 gene). Furthermore, de novo protein synthesis was not required for NER in the absence of transcription-coupled NER. This implies that RAD9-dependent gene up-regulation is required for NER only when this process is coupled to transcription.

Homologous recombination is involved in transcription-coupled repair of UV damage in Saccharomyces cerevisiae.

To efficiently protect the integrity of genetic information, transcription is connected to nucleotide excision repair (NER), which allows preferential repair of the transcribed DNA strands (TS). As yet, the molecular basis of this connection remains elusive in eukaryotic cells. Here we show that, in haploids, the RAD26 gene is essential for the preferential repair of the TS during G1. However, in G2/M phase there is an additional RAD51-dependent process that enhances repair of TS. Importantly, the simultaneous deletion of both RAD26 and RAD51 led to complete abolishment of strand-specific repair during G2/M, indicating that these genes act through two independent but complementary subpathways. In diploids, however, RAD51 is involved in repair of the TS even in G1 phase, which unveils the implication of homologous recombination in the preferential repair of the TS. Importantly, the abolishment of NER, by abrogation of RAD1 or RAD14, completely stopped repair of UV damage even during G2/M phase. These results show the existence of functional cross-talk between transcription, homologous recombination and NER.

UV-induced de novo protein synthesis enhances nucleotide excision repair efficiency in a transcription-dependent manner in S. cerevisiae.

DNA damage results in the up-regulation of several genes involved in different cellular physiological processes, such as the nucleotide excision repair (NER) mechanism that copes with a broad range of DNA alterations, including the carcinogenic ultraviolet (UV) light-induced pyrimidine dimers (PDs). There are two NER sub-pathways: transcription coupled repair (TCR) that is specific for the transcribed strands (TS) of active genes and global genomic repair (GGR) that repairs non-transcribed DNA sequences (NTD) and the non-transcribed strands (NTS) of expressed genes. To elucidate the role of UV-dependent de novo protein synthesis in nucleotide excision repair in the budding yeast, we investigated the effect of the protein synthesis inhibitor, cycloheximide, on the removal of PDs. Log phase as well as G(1)-synchronized cells were treated with the drug shortly before UV irradiation and immediately thereafter, and the repair of damaged DNA was assessed with the high resolution primer extension technique. The results show that in both cellular conditions, the inhibition of UV-dependent de novo protein synthesis by cycloheximide impairs the excision repair of the transcriptionally active GAL10 and URA3 genes, with a greater effect on the non-transcribed strands. This indicates that UV-mediated de novo protein synthesis is required for efficient nucleotide excision repair, but not for the preferential repair of the TSs. On the other hand, cycloheximide did not affect the repair of either strand of the repressed GAL10 gene or the non-transcribed promoter region of the URA3 gene, showing that UV-induced de novo protein synthesis is not required for PD removal from transcriptionally inactive DNA sequences. Together, these data show that despite the fact that NTD and NTSs are normally repaired by the GGR sub-pathway, their requirement for UV-dependent de novo protein synthesis is different, which may suggest a difference in the processing of UV lesions in these non-transcribed sequences of the genome.