Evaluation of the antitumour activity of novel flavonoids on pre-clinical models of breast and ovarian cancer
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Date
08/07/2017Author
Martínez Pérez, Carlos
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Abstract
New drugs are needed for better cancer management. Clinical trials are currently
underway to assess the use of flavonoids (natural polyphenols) as anticancer agents. Among
them, myricetin has been shown to induce cell cycle arrest and apoptosis in pre-clinical
cancer models. We hypothesised that myricetin-derived novel flavonoids designed to
enhance this natural potential and improve on the drug-likeness limitations of myricetin
might have increased potential for their application in the management of breast and ovarian
cancer.
The effect of a library of novel flavonoids was screened on 3 panels of breast and ovarian
cancer cell lines, representing different molecular subtypes and phenotypes, to assess their
potency. The second-generation bi-methoxylated analogue AO-1530-OMe (Oncamex) was
identified as the most effective candidate in the library, with sub-micromolar concentrations
exerting a strong antiproliferative effect across almost all models studied. Results suggested
that changes in the hydroxylation profile, the addition of methoxylations and a decyl alkyl
chain were some of the structure-activity relationships contributing to this improved efficacy.
Plate assays showed 8 h treatment with Oncamex reduced cell viability and induced
cytotoxicity and apoptosis, concomitant with caspase activation and PARP cleavage. Pre-incubation
with an antioxidant partially blocked these effects, suggesting the possible
involvement of ROS modulation in the mechanism of action of Oncamex. Fluorescence
microscopy reported the quick and stable delivery of Oncamex to the mitochondria.
Fluorescent probes showed that Oncamex can induce mitochondrial superoxide production
at concentrations associated with its antiproliferative effects. Study of the electrochemical
properties of Oncamex by cyclic voltammetry supported this.
Differential gene expression analysis following a microarray experiment showed
Oncamex induces changes in the expression of genes controlling cell cycle and apoptosis.
Together with previous results, the findings from this analysis led to the postulation of a
model for the mechanism of action of Oncamex: due to its enhanced reactivity and
mitochondrial targeting, Oncamex can generate mitochondrial superoxide, leading to
mitochondrial dysfunction, membrane permeabilisation and the activation of the JNK
pathway and the transcription factor FOXO3, which together contribute to the induction of
intrinsic apoptosis and the inhibition of proliferation.
Further proliferation assays on cell culture models also reported enhanced effect of
Oncamex when administered in combination with paclitaxel and TRAIL. These improved
responses were observed in breast and ovarian cancer models, including cells lines
characterised by their treatment-resistant phenotype. Cotreatment with Oncamex also
improved the effect of tamoxifen on anti-oestrogen resistant LCC9 breast cancer cells.
Results from preliminary in vivo studies in mice implanted with the MDA-MB-231 breast
cancer xenograft were consistent with an antiproliferative effect of Oncamex (25mg/kg/day)
in vivo, as treatment inhibited tumour growth and reduced the expression of the marker of
proliferation Ki-67 without signs of systemic toxicity. Tissues from this experiment also
allowed for preliminary in vivo validation of the proposed mechanism of action of Oncamex
by immunohistochemistry. The in vivo cytostatic effect of Oncamex was confirmed in a
second in vivo experiment, which also investigated the effect of Oncamex at higher doses or
in combination with paclitaxel.
In conclusion, the novel flavonoid Oncamex has shown a promising antiproliferative
effect in pre-clinical models of breast and ovarian cancer, including models of treatment-resistant
cancers. Preliminary in vivo studies have demonstrated a partial recapitulation of
the effect of Oncamex. A mechanistic model has been proposed by which Oncamex induces
intrinsic apoptosis through its redox reactivity and mitochondrial targeting. These results
support the potential of this prototypic candidate, although possible work in the structure
and formulation of this candidate and further study and validation of its mechanism of action
is needed for its continued development as an anticancer agent.
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