Breast cancer is the second most common cancer in women in the United States, accounting for approximately 30% of newly diagnosed female cancers every year. In 2023, it is estimated that around 297,790 invasive breast cancers will be diagnosed as new cases with nearly 43,700 women deaths. The average lifetime risk of a woman in the United States accruing a breast cancer diagnosis is approximately 13%, meaning that there is a 1 in 8 chance of developing breast cancer. Classification of breast cancers is distinguished based on the presence of three receptors: HER2, estrogen, and progesterone. Absence of these receptors is categorized as triple negative breast cancer and accounts for about 15% of all breast cancers, thus is the most aggressive and difficult to treat. In this study, research involving two flavonoids, CT1 and CT3 show cytotoxic effects against cell lines MCF7 (ER+, PR+, HER2-) and SKBr3 (ER-, PR-, HER2+), that represent the most common breast cancers. CT1 and CT3 were extracted from the leaves of Chromolaena tacotana using a Soxhlet extractor, followed by isolation and purification by chromatography. The cells were seeded and then treated with CT1 or CT3 at concentrations of 5, 10, 20, 40 and 80 mM for cytotoxicity assays, and 40mM for analysis of mechanism of action via immunoblotting and TUNEL. These two flavonoids differ on the presence of a double bond between positions 2 and 3. At the concentrations tested, CT1 has cytotoxic activity against MCF7 but no significant effect on SKBr3, while CT3 has cytotoxic activity against SKBr3 but not on MCF7. CT1 and CT3 target the activated forms of ERK, c-JUN and SP6; however, the effect of CT1 appears to be significantly stronger than CT3 and does not involve the survival pathway. CT1 and CT3 inhibit cell viability in MCF7 and SKBr3 breast cancer cells by activating an extrinsic apoptotic pathway. Additional studies using a triple negative breast cancer cell line has shown that this activation is independent of the presence of estrogen and progesterone receptors or the upregulation of HER2.

TRPM3 (melastatin-related transient receptor potential 3) is a calcium-permeable nonselective cation channel that is expressed in various tissues, including insulin-secreting β-cells and a subset of sensory neurons from trigeminal and dorsal root ganglia (DRG). TRPM3 can be activated by the neurosteroid pregnenolone sulphate (PregS) or heat. TRPM3α2 mice display an impaired sensation of noxious heat and inflammatory thermal hyperalgesia. A calcium-based screening of a compound library identified four natural compounds as TRPM3 blockers. Three of the natural compounds belong to the citrus fruit flavanones (hesperetin, eriodictyol and naringenin), the forth compound is a deoxybenzoin that can be synthesized from an isoflavone of the root of Ononis spinosa (ononetin). The IC50 for the substances ranged from upper nanomolar to lower micromolar concentrations. Electrophysiological whole-cell measurements as well as calcium measurements confirmed the potency of the compounds to block TRPM3 in DRG neurones. To further improve the potency and the selectivity of TRPM3 block and to identify the pharmacophore within the flavanone structure, we conducted a hit optimisation procedure by re-screening a focussed library. The library composed of several flavanones with different substitutions on relevant chemical positions and of representatives from different flavonoid subgroups. Within this secondary screen, we identified isosakuranetin and liquiritigenin as active blockers of PregS-induced Ca2+ entry through TRPM3. Isosakuranetin, a flavanone that can be found in blood oranges and grapefruits, displayed an IC50 of 50 nM, and is the most potent inhibitor of TRPM3 identified so far. The novel compounds exhibited a marked specificity for TRPM3 compared with other thermosensitive TRP channels, and blocked PregS-induced [Ca2+]i signals and ionic currents in freshly isolated DRG neurones. Furthermore, isosakuranetin and hesperetin reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Since the physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

TRPM3 (melastatin-related transient receptor potential 3) is a calcium-permeable nonselective cation channel that is expressed in various tissues, including insulin-secreting β-cells and a subset of sensory neurons from trigeminal and dorsal root ganglia (DRG). TRPM3 can be activated by the neurosteroid pregnenolone sulphate (PregS) or heat. TRPM3α2 mice display an impaired sensation of noxious heat and inflammatory thermal hyperalgesia. A calcium-based screening of a compound library identified four natural compounds as TRPM3 blockers. Three of the natural compounds belong to the citrus fruit flavanones (hesperetin, eriodictyol and naringenin), the forth compound is a deoxybenzoin that can be synthesized from an isoflavone of the root of Ononis spinosa (ononetin). The IC50 for the substances ranged from upper nanomolar to lower micromolar concentrations. Electrophysiological whole-cell measurements as well as calcium measurements confirmed the potency of the compounds to block TRPM3 in DRG neurones. To further improve the potency and the selectivity of TRPM3 block and to identify the pharmacophore within the flavanone structure, we conducted a hit optimisation procedure by re-screening a focussed library. The library composed of several flavanones with different substitutions on relevant chemical positions and of representatives from different flavonoid subgroups. Within this secondary screen, we identified isosakuranetin and liquiritigenin as active blockers of PregS-induced Ca2+ entry through TRPM3. Isosakuranetin, a flavanone that can be found in blood oranges and grapefruits, displayed an IC50 of 50 nM, and is the most potent inhibitor of TRPM3 identified so far. The novel compounds exhibited a marked specificity for TRPM3 compared with other thermosensitive TRP channels, and blocked PregS-induced [Ca2+]i signals and ionic currents in freshly isolated DRG neurones. Furthermore, isosakuranetin and hesperetin reduced the sensitivity of mice to noxious heat and PregS-induced chemical pain. Since the physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

Citrus and other fruits produce secondary metabolites that are synthesized, regulated, and modified in part by a class of enzymes called glycosyltransferases. This class of enzymes is of substantial interest to this lab due to their unique structural and functional properties. Glycosides of flavonoids produced by glycosyltransferases have emerged in recent years as a critical part of plant metabolism, thus impacting every aspect of their growth, cultivation, production, and utilization. One such glycosyltransferase, found in Duncan Grapefruits (Citrus paradisi), was previously identified, recombinantly expressed, and shown through biochemical characterization to exclusively glycosylate the flavonol class of flavonoids. The structural basis that accounts for a glycosyltransferase's selectivity has been determined by protein crystallization in other labs, yet no structural basis currently exists for the specificity exhibited by this flavonol-specific glycosyltransferase. Currently, the WT enzyme and two mutants were expressed in E. coli, where they underwent site-directed mutagenesis to insert thrombin cleavage tags for removal of protein purification vectors, with the goal of transforming into yeast for adequate protein production. Subsequent purification and crystallization screens will allow for formation and acquisition of glycosyltransferase crystals, whose x-ray diffraction patterns will be decoded, thus revealing the enzyme's complete structure. We hypothesize that obtaining a crystal structure for this enzyme will illuminate the structural basis of its specificity. Additionally, we hypothesize that a thrombin- cleavage gene vector inserted for removal of purification tags will have no impact on enzyme activity or specificity.

<p>In this study two medicinal plant species, namely Galenia africana and Elytropapus rhinocerotis, the former belonging to the family Aizoceae and the latter belonging to the family Asteraceae, have been investigated and different compounds isolated and characterized. Both species are important plants used in traditional medicine in Africa and particularly in South Africa. Flavanoids are secondary metabolites found in plants. They have a protective function against UV radiation and have a defence against invading illnesses due to their important antioxidant activity. Much of the food we eat and some beverages we drink contain flavonoids. The aim of this study was to investigate the electrochemistry of flavanoids isolated from these species.</p>

<p>In this study two medicinal plant species, namely Galenia africana and Elytropapus rhinocerotis, the former belonging to the family Aizoceae and the latter belonging to the family Asteraceae, have been investigated and different compounds isolated and characterized. Both species are important plants used in traditional medicine in Africa and particularly in South Africa. Flavanoids are secondary metabolites found in plants. They have a protective function against UV radiation and have a defence against invading illnesses due to their important antioxidant activity. Much of the food we eat and some beverages we drink contain flavonoids. The aim of this study was to investigate the electrochemistry of flavanoids isolated from these species.</p>

Magister Scientiae - MSc / In this study two medicinal plant species, namely Galenia africana and Elytropapus rhinocerotis, the former belonging to the family Aizoceae and the latter belonging to the family Asteraceae, have been investigated and different compounds isolated and characterized. Both species are important plants used in traditional medicine in Africa and particularly in South Africa. Flavanoids are secondary metabolites found in plants. They have a protective function against UV radiation and have a defence against invading illnesses due to their important antioxidant activity. Much of the food we eat and some beverages we drink contain flavonoids. The aim of this study was to investigate the electrochemistry of flavanoids isolated from these species. / South Africa