Pseurotin A Validation as a Metastatic Castration-Resistant Prostate Cancer Recurrence-Suppressing Lead via PCSK9-LDLR Axis Modulation.

Metastatic castration-resistant prostate cancer (mCRPC) cells can de novo biosynthesize their own cholesterol and overexpress proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 proved to contribute to mCRPC cell motility since PCSK9 knockdown (KD) in mCRPC CWR-R1ca cells led to notable reductions in cell migration and colony formation. Human tissue microarray results proved a higher immunohistoscore in patients ≥ 65 years old, and PCSK9 proved to be expressed higher at an early Gleason score of ≤7. The fermentation product pseurotin A (PS) suppressed PCSK9 expression, protein-protein interactions with LDLR, and breast and prostate cancer recurrences. PS suppressed migration and colony formation of the CWR-R1ca cells. The progression and metastasis of the CWR-R1ca-Luc cells subcutaneously (sc) xenografted into male nude mice fed a high-fat diet (HFD, 11% fat content) showed nearly 2-fold tumor volume, metastasis, serum cholesterol, low-density lipoprotein cholesterol (LDL-C), prostate-specific antigen (PSA), and PCSK9 levels versus mice fed a regular chow diet. Daily oral PS 10 mg/kg treatments prevented the locoregional and distant tumor recurrence of CWR-R1ca-Luc engrafted into nude mice after primary tumor surgical excision. PS-treated mice showed a significant reduction in serum cholesterol, LDL-C, PCSK9, and PSA levels. These results comprehensively validate PS as an mCRPC recurrence-suppressive lead by modulating the PCSK9-LDLR axis.

Towards Developing Novel Prostate Cancer Recurrence Suppressors: Acute Toxicity of Pseurotin A, an Orally Active PCSK9 Axis-Targeting Small-Molecule in Swiss Albino Mice.

The proprotein convertase subtilisin kexin type 9 (PCSK9) emerged as a molecular target of great interest for the management of cardiovascular disorders due to its ability to reduce low density lipoprotein (LDL) cholesterol by binding and targeting at LDLR for lysosomal degradation in cells. Preliminary studies revealed that pseurotin A (PsA), a spiro-heterocyclic γ-lactam alkaloid from several marine and terrestrial Aspergillus and Penicillium species, has the ability to dually suppress the PCSK9 expression and protein-protein interaction (PPI) with LDLR, resulting in an anti-hypercholesterolemic effect and modulating the oncogenic role of PCSK9 axis in breast and prostate cancers progression and recurrence. Thus, a preliminary assessment of the PsA acute toxicity represents the steppingstone to develop PsA as a novel orally active PCSK9 axis modulating cancer recurrence inhibitor. PsA studies for in vitro toxicity on RWPE-1 and CCD 841 CoN human non-tumorigenic prostate and colon cells, respectively, indicated a cellular death shown at a 10-fold level of its reported anticancer activity. Moreover, a Western blot analysis revealed a significant downregulation of the pro-survival marker Bcl-2, along with the upregulation of the proapoptotic Bax and caspases 3/7, suggesting PsA-mediated induction of cell apoptosis at very high concentrations. The Up-and-Down methodology determined the PsA LD50 value of >550 mg/kg in male and female Swiss albino mice. Animals were orally administered single doses of PsA at 10, 250, and 500 mg/kg by oral gavage versus vehicle control. Mice were observed daily for 14 days with special care over the first 24 h after dosing to monitor any abnormalities in their behavioral, neuromuscular, and autonomic responses. After 14 days, the mice were euthanized, and their body and organ weights were recorded and collected. Mice plasma samples were subjected to comprehensive hematological and biochemical analyses. Collected mouse organs were histopathologically examined. No morbidity was detected following the PsA oral dosing. The 500 mg/kg female dosing group showed a 45% decrease in the body weight after 14 days but displayed no other signs of toxicity. The 250 mg/kg female dosing group had significantly increased serum levels of liver transaminases AST and ALT versus vehicle control. Moreover, a modest upregulation of apoptotic markers was observed in liver tissues of both animal sexes at 500 mg/kg dose level. However, a histopathological examination revealed no damage to the liver, kidneys, heart, brain, or lungs. While these findings suggest a possible sex-related toxicity at higher doses, the lack of histopathological injury implies that single oral doses of PsA, up to 50-fold the therapeutic dose, do not cause acute organ toxicity in mice though further studies are warranted.

S-(-)-Oleocanthal Ex Vivo Modulatory Effects on Gut Microbiota.

Compelling evidence points to the critical role of bioactive extra-virgin olive oil (EVOO) phenolics and gut microbiota (GM) interplay, but reliable models for studying the consequences thereof remain to be developed. Herein, we report an optimized ex vivo fecal anaerobic fermentation model to study the modulation of GM by the most bioactive EVOO phenolic S-(-)-oleocanthal (OC), and impacts therefrom, focusing on OC biotransformation in the gut. This model will also be applicable for characterization of GM interactions with other EVOO phenolics, and moreover, for a broadly diverse range of bioactive natural products. The fecal fermentation media and time, and mouse type and gender, were the major factors varied and optimized to provide better understanding of GM-OC interplay. A novel resin entrapment technique (solid-phase extraction) served to selectively entrap OC metabolites, degradation products, and any remaining fraction of OC while excluding interfering complex fecal medium constituents. The effects of OC on GM compositions were investigated via shallow shotgun DNA sequencing. Robust metabolome analyses identified GM bacterial species selectively altered (population numbers/fraction) by OC. Finally, the topmost OC-affected gut bacterial species of the studied mice were compared with those known to be extant in humans and distributions of these bacteria at different human body sites. OC intake caused significant quantitative and qualitative changes to mice GM, which was also comparable with human GM. Results clearly highlight the potential positive health outcomes of OC as a prospective nutraceutical.

Small extracellular vesicle-associated miR-6068 promotes aggressive phenotypes of prostate cancer through miR-6068/HIC2/SIRT1 axis.

Early diagnosis and treatment of patients with aggressive prostate cancer (PCa) remains a clinically unmet need. We aimed to determine the levels of small extracellular vesicle (sEV)-associated microRNAs (miRs); miR-4737, miR-6068, and miR-6076 in a large panel of PCa cells and delineate the biological significance of miR-6068 in promoting PCa cells. sEVs were isolated from the conditioned medium of PCa cells, followed by RNA extraction and quantitative Real-Time PCR analysis. Functional assays were performed, and the protein expression of hypermethylated in cancer 2 (HIC2), as a potential miR-6068 target gene, was evaluated in PCa tissues by immunohistochemistry. sEV-associated miR-6068, miR-4737, and miR-6076 levels displayed large and significant differences compared to normal cells. miR-6068 was explicitly upregulated in sEV of PC-3 and CWR-R1ca cells (P<0.010). Suppression of miR-6068 in CWR-R1ca cells decreased cell proliferation, colony formation, and cell migration. In contrast, upregulation of miR-6068 in RC77T/E cells decreased HIC2 levels and increased cell aggressive phenotypes. The overexpression of HIC2 in PCa tissues was primarily observed in the cytoplasm compared to benign prostatic hyperplasia (BPH) and normal tissues (P<0.0001). This study confirms the differential packaging of miR-4737, miR-6068, and miR-6076 in sEVs of PCa cells. MiR-6068 promotes PCa cells to acquire aggressive phenotypes by inhibiting the HIC2/Sirtuin 1 (SIRT1) axis.

Oleocanthal Attenuates Metastatic Castration-Resistant Prostate Cancer Progression and Recurrence by Targeting SMYD2.

Metastatic castration-resistant prostate cancer (mCRPC) is the most aggressive prostate cancer (PC) phenotype. Cellular lysine methylation is driven by protein lysine methyltransferases (PKMTs), such as those in the SET- and MYND-containing protein (SMYD) family, including SMYD2 methylate, and several histone and non-histone proteins. SMYD2 is dysregulated in metastatic PC patients with high Gleason score and shorter survival. The Mediterranean, extra-virgin-olive-oil-rich diet ingredient S-(-)-oleocanthal (OC) inhibited SMYD2 in biochemical assays and suppressed viability, migration, invasion, and colony formation of PC-3, CWR-R1ca, PC-3M, and DU-145 PC cell lines with IC50 range from high nM to low µM. OC's in vitro antiproliferative effect was comparable to standard anti-PC chemotherapies or hormone therapies. A daily, oral 10 mg/kg dose of OC for 11 days effectively suppressed the progression of the mCRPC CWR-R1ca cells engrafted into male nude mice. Daily, oral OC treatment for 30 days suppressed tumor locoregional and distant recurrences after the primary tumors' surgical excision. Collected OC-treated animal tumors showed marked SMYD2 reduction. OC-treated mice showed significant serum PSA reduction. For the first time, this study showed SMYD2 as novel molecular target in mCRPC, and OC emerged as a specific SMYD2 lead inhibitor. OC prevailed over previously reported SMYD2 inhibitors, with validated in vivo potency and high safety profile, and, therefore, is proposed as a novel nutraceutical for mCRPC progression and recurrence control.

The Tobacco ß-Cembrenediol: A Prostate Cancer Recurrence Suppressor Lead and Prospective Scaffold via Modulation of Indoleamine 2,3-Dioxygenase and Tryptophan Dioxygenase.

Prostate cancer (PC) is the second leading cause of death in men in the US. PC has a high recurrence rate, and limited therapeutic options are available to prevent disease recurrence. The tryptophan-degrading enzymes 2,3-indoleamine dioxygenase (IDO1) and tryptophan dioxygenase (TDO2) are upregulated in invasive PC. (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (ß-CBT) and its C-4 epimer α-CBT are the precursors to key flavor ingredients in tobacco leaves. Nearly 40-60% of ß- and α-CBT are purposely degraded during commercial tobacco fermentation. Earlier, ß-CBT inhibited invasion, reversed calcitonin-stimulated transepithelial resistance decrease, and induced tighter intercellular barriers in PC-3M cells. This study demonstrates the in vitro ß-CBT anti-migratory (wound-healing assay) and anti-clonogenicity (colony-formation assay) activities against five diverse human PC cell lines, including the androgen-independent PC-3, PC-3M, and DU-145, the castration-recurrent CWR-R1ca, and the androgen-dependent CWR-22rv1. Meanwhile, ß-CBT potently suppressed in vivo locoregional and distant recurrences after the primary tumor surgical excision of PC-3M-Luc cell tumor engrafted in male nude mice. ß-CBT treatments suppressed organ and bone metastasis and lacked any major toxicity over the 60-day study course. ß-CBT treatments significantly suppressed IDO1, TDO2, and their final metabolite kynurenine levels in PC-3M cells. ß-CBT treatments significantly suppressed the tumor recurrence marker PSA and kynurenine levels in treated animals' plasma. ß-CBT emerges as a promising PC recurrence suppressive lead.

PCSK9 Axis-Targeting Pseurotin A as a Novel Prostate Cancer Recurrence Suppressor Lead.

Prostate cancer (PC) is the most common malignancy and the second leading cause of cancer death in men. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in the cholesterol metabolism by regulating the LDL receptor (LDLR) degradation. The PCSK9 axis is proved to be a potential novel therapeutic target in multiple cancer types. Pseurotin A (PS) is a small-molecule natural-product inhibitor of PCSK9 expression and PCSK9-LDLR protein-protein interaction (PPI). The in vitro results of this study show that PS treatments caused dose-dependent suppression of migration, colony formation, and PCSK9 expression in the PC cell lines PC-3 and 22Rv1. PS suppressed the in vivo progression of PC-3 cells orthotopically xenografted in nude mice and prevented locoregional and distant tumor recurrences after primary tumor surgical excision. Western blot analysis showed decreased PCSK9 expression in collected primary and recurred PC-3 tumors in PS-treated mice. PS treatments also reduced the hemoglobin content in collected treated tumors and the Matrigel-plug angiogenesis mouse model. PS treatments prevented metastasis to distant organs compared to vehicle-treated control mice. A reduction in mice plasma cholesterol levels was observed. Microarray analysis of collected treated primary PC-3 tumors showed a distinct gene signature that confirmed the targeting of PCSK9 and cholesterol metabolism. Thus, the PCSK9 axis is proposed as a novel PC pathogenesis molecular target, and PS is defined as a novel effective PCSK9-targeting lead potentially useful for the control of the castration-resistant PC recurrence and metastasis.

(-)-Oleocanthal Nutraceuticals for Alzheimer's Disease Amyloid Pathology: Novel Oral Formulations, Therapeutic, and Molecular Insights in 5xFAD Transgenic Mice Model.

Alzheimer's disease (AD) is a complex progressive neurodegenerative disorder affecting humans mainly through the deposition of Aß-amyloid (Aß) fibrils and accumulation of neurofibrillary tangles in the brain. Currently available AD treatments only exhibit symptomatic relief but do not generally intervene with the amyloid and tau pathologies. The extra-virgin olive oil (EVOO) monophenolic secoiridoid S-(-)-oleocanthal (OC) showed anti-inflammatory activity through COX system inhibition with potency comparable to the standard non-steroidal anti-inflammatory drug (NSAID) like ibuprofen. OC also showed positive in vitro, in vivo, and clinical therapeutic effects against cardiovascular diseases, many malignancies, and AD. Due to its pungent, astringent, and irritant taste, OC should be formulated in acceptable dosage form before its oral use as a potential nutraceutical. The objective of this study is to develop new OC oral formulations, assess whether they maintained OC activity on the attenuation of ß-amyloid pathology in a 5xFAD mouse model upon 4-month oral dosing use. Exploration of potential OC formulations underlying molecular mechanism is also within this study scope. OC powder formulation (OC-PF) and OC-solid dispersion formulation with erythritol (OC-SD) were prepared and characterized using FT-IR spectroscopy, powder X-ray diffraction, and scanning electron microscopy (ScEM) analyses. Both formulations showed an improved OC dissolution profile. OC-PF and OC-SD improved memory deficits of 5xFAD mice in behavioral studies. OC-PF and OC-SD exhibited significant attenuation of the accumulation of Aß plaques and tau phosphorylation in the brain of 5xFAD female mice. Both formulations markedly suppressed C3AR1 (complement component 3a receptor 1) activity by targeting the downstream marker STAT3. Collectively, these results demonstrate the potential for the application of OC-PF as a prospective nutraceutical or dietary supplement to control the progression of amyloid pathogenesis associated with AD.

Comparative Gene Signature of (-)-Oleocanthal Formulation Treatments in Heterogeneous Triple Negative Breast Tumor Models: Oncological Therapeutic Target Insights.

Triple negative breast cancer (TNBC) heterogeneity and limited therapeutic options confer its phenotypic aggressiveness. The discovery of anti-TNBC natural products with valid molecular target(s) and defined pharmacodynamic profile would facilitate their therapeutic nutraceutical use by TNBC patients. The extra-virgin olive oil (EVOO) is a key Mediterranean diet ingredient. S-(-)-Oleocanthal (OC) leads the bioactive anti-tumor EVOO phenolic ingredients. A previous study reported the solid dispersion formulated OC with (+)-xylitol (OC-X) suppressed the in vivo progression and recurrence of the TNBC MDA-MB-231 cells. This study investigates the ability of OC-X formulation to suppress the in vivo heterogeneous BC initiation and progression utilizing advanced preclinical transgenic MMTV-PyVT and TNBC PDX mouse models. Furthermore, the clustering of the gene expression profiles in MMTV-PyVT and PDX mouse tumors treated with OC-X acquired by a Clariom S microarray analysis identified the distinctly affected genes. Several affected novel signature genes identified in response to OC-X treatments and proved overlapped in both mouse and human tumor models, shedding some lights toward understanding the OC anticancer molecular mechanism and assisting in predicting prospective clinical outcomes. This study provides molecular and preclinical evidences of OC-X potential as a nutraceutical suppressing heterogeneous TNBC model and offers preliminary gene-level therapeutic mechanistic insights.

Spontaneous In Vitro and In Vivo Interaction of (-)-Oleocanthal with Glycine in Biological Fluids: Novel Pharmacokinetic Markers.

Since the first discovery of its ibuprofen-like anti-inflammatory activity in 2005, the olive phenolic (-)-oleocanthal gained great scientific interest and popularity due to its reported health benefits. (-)-Oleocanthal is a monophenolic secoiridoid exclusively occurring in extra-virgin olive oil (EVOO). While several groups have investigated oleocanthal pharmacokinetics (PK) and disposition, none was able to detect oleocanthal in biological fluids or identify its PK profile that is essential for translational research studies. Besides, oleocanthal could not be detected following its addition to any fluid containing amino acids or proteins such as plasma or culture media, which could be attributed to its unique structure with two highly reactive aldehyde groups. Here, we demonstrate that oleocanthal spontaneously reacts with amino acids, with high preferential reactivity to glycine compared to other amino acids or proteins, affording two products: an unusual glycine derivative with a tetrahydropyridinium skeleton that is named oleoglycine, and our collective data supported the plausible formation of tyrosol acetate as the second product. Extensive studies were performed to validate and confirm oleocanthal reactivity, which were followed by PK disposition studies in mice, as well as cell culture transport studies to determine the ability of the formed derivatives to cross physiological barriers such as the blood-brain barrier. To the best of our knowledge, we are showing for the first time that (-)-oleocanthal is biochemically transformed to novel products in amino acids/glycine-containing fluids, which were successfully monitored in vitro and in vivo, creating a completely new perspective to understand the well-documented bioactivities of oleocanthal in humans.

Correction: Triple-negative breast cancer suppressive activities, antioxidants and pharmacophore model of new acylated rhamnopyranoses from Premna odorata.

[This corrects the article DOI: 10.1039/D0RA01697G.].

HER2 Kinase-Targeted Breast Cancer Therapy: Design, Synthesis, and In Vitro and In Vivo Evaluation of Novel Lapatinib Congeners as Selective and Potent HER2 Inhibitors with Favorable Metabolic Stability.

HER2 kinase as a well-established target for breast cancer (BC) therapy is associated with aggressive clinical outcomes; thus, herein we present structural optimization for HER2-selective targeting. HER2 profiling of the developed derivatives demonstrated potent and selective inhibitions (IC50: 5.4-12 nM) compared to lapatinib (IC50: 95.5 nM). Favorably, 17d exhibited minimum off-target kinase activation. NCI-5-dose screening revealed broad-spectrum activities (GI50: 1.43-2.09 µM) and 17d had a remarkable selectivity toward BC. Our compounds revealed significant selective and potent antiproliferative activities (∼20-fold) against HER2+ (AU565, BT474) compared to HER2(-) cells. At 0.1 IC50, 15i, 17d, and 25b inhibited pERK1/2 and pAkt by immunoblotting. Furthermore, 17d demonstrated potent in vivo tumor regression against the BT474 xenograft model. Notably, a metastasis case was observed in the vehicle but not in the test mice groups. CD-1 mice metabolic stability assay revealed high stability and low intrinsic clearance of 17d (T1/2 > 145 min and CLint(mic) < 9.6 mL/min/kg).

Olive Oil Lignan (+)-Acetoxypinoresinol Peripheral Motor and Neuronal Protection against the Tremorgenic Mycotoxin Penitrem A Toxicity via STAT1 Pathway.

Penitrem A, PA, is an indole diterpene alkaloid produced by several fungal species. PA acts as a selective Ca2+-dependent K-channels (Maxi-K, BK) antagonist in brain, causing motor system dysfunctions including tremors and seizures. However, its molecular mechanism at the peripheral nervous system (PNS) is still ambiguous. The Mediterranean diet key ingredient extra-virgin olive oil (EVOO) provides a variety of minor bioactive phenolics. (+)-Pinoresinol (PN) and (+)-1-acetoxypinoresinol (AC) are naturally occurring lignans in EVOO with diverse biological activities. AC exclusively occurs in EVOO, unlike PN, which occurs in several plants. Results suggest that PA neurotoxicity molecular mechanism is mediated, in part, through distortion of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. PA selectively activated the STAT1 pathway, independently of the interferon-γ (IFN-γ) pathway, in vitro in Schwann cells and in vivo in Swiss albino mice sciatic nerves. Preliminary in vitro screening of an EVOO phenolic compounds library for the ability to reverse PA toxicity on Schwann cells revealed PN and AC as potential hits. In a Swiss albino mouse model, AC significantly minimized the fatality after intraperitoneal administration of PA fatal doses and normalized most biochemical factors by modulating the STAT1 expression. The olive lignan AC is a novel lead that can prevent the neurotoxicity of food-contaminating tremorgenic indole alkaloid mycotoxins.

Pseurotin A as a novel suppressor of hormone dependent breast cancer progression and recurrence by inhibiting PCSK9 secretion and interaction with LDL receptor.

Hypercholesterolemia has been documented to drive hormone-dependent breast cancer (BC) progression and resistance to hormonal therapy. Proprotein convertase subtilisin/kexin type-9 (PCSK9) regulates cholesterol metabolism through binding to LDL receptor (LDLR) and targeting the receptor for lysosomal degradation. Inhibition of PCSK9 is an established strategy to treat hypercholesterolemia. Pseurotin A (PS) is a unique spiro-heterocyclic γ-lactam alkaloid isolated from the fungus Aspergillus fumigatus. Preliminary studies indicated that PS lowered PCSK9 secretion in cultured HepG2 hepatocellular carcinoma cells, with an IC50 value of 1.20 µM. Docking studies suggested the ability of PS to bind at the PCSK9 narrow interface pocket that accommodates LDLR. Surface plasmon resonance (SPR) showed PS ability to inhibit the PCSK9-LDLR interaction at a concentration range of 10-150 µM. PS showed in vitro dose-dependent reduction of PCSK9, along with increased LDLR levels in hormone-dependent BT-474 and T47D breast cancer (BC) cell lines. In vivo, daily oral 10 mg/kg PS suppressed the progression of the hormone-dependent BT-474 BC cells in orthotopic nude mouse xenograft model. Immunohistochemistry (IHC) investigation of BT-474 breast tumor tissue proved the PS ability to reduce PCSK9 expression. PS also effectively suppressed BT-474 BC cells locoregional recurrence after primary tumor surgical excision. Western blot analysis showed decreased PCSK9 expression in liver tissues of PS-treated mice compared to vehicle-treated control group. PS treatment significantly reduced PCSK9 expression and normalized LDLR levels in collected primary and recurrent breast tumors at the study end. PS-treated mice showed reduced plasma cholesterol and 17ß-estradiol levels. Inhibition of tumor recurrence was associated with significant reductions in plasma level of the human BC recurrence marker CA 15-3 in treated mice at the study end. Histopathological examination of various PS-treated mice organs indicated lack of metastatic tumor cells and any pathological changes. The results of this study provide the first evidence for the suppression of the hormone-dependent breast tumor progression and recurrence by targeting the PCSK9-LDLR axis. PS is a novel first-in-class PCSK9-targeting lead appropriate for the use to control hormone-dependent BC progression and recurrence.

Safety Evaluations of Single Dose of the Olive Secoiridoid S-(-)-Oleocanthal in Swiss Albino Mice.

Epidemiological and clinical studies compellingly showed the ability of Mediterranean diet rich in extra-virgin olive oil (EVOO) to reduce multiple diseases such as cancer, cardiovascular diseases, and aging cognitive functions decline. The S-(-)-Oleocanthal (OC) is a minor phenolic secoiridoid exclusively found in extra-virgin olive oil (EVOO). OC recently gained notable research attention due to its excellent in vitro and in vivo biological effects against multiple cancers, inflammations, and Alzheimer's disease. However, OC safety has not been comprehensively studied yet. This study reports for the first time the detailed safety of oral single OC dose in Swiss albino mice, applying the OECD 420 procedure. Male and female Swiss albino mice (n = 10) were orally treated with a single OC dose of either 10, 250, or 500 mg/kg bodyweight or equivalent volumes of distilled water. Mice fed a regular diet, and carefully observed for 14 days. Further, mice were then sacrificed, blood samples, and organs were collected and subjected to hematological, biochemical, and histological examinations. OC 10 mg/kg oral dose appears to be without adverse effects. Further, 250 mg/kg OC, p.o., is suggested as a possible upper dose for preclinical studies in the future.

Triple-negative breast cancer suppressive activities, antioxidants and pharmacophore model of new acylated rhamnopyranoses from Premna odorata.

Phytochemical investigation of Premna odorata Blanco "Lamiaceae" young stems afforded four new acylated rhamnopyranoses 1-4, along with fourteen known compounds 5-19. The structures of the new compounds were confirmed using extensive 1D, 2D NMR, and HRESIMS analysis. The isolated compounds were tested for their cell proliferation and migration inhibition activities against the invasive human triple-negative breast cancer cells MDA-MB-231 and MCF-7, and the normal human breast cell line MCF-10A. In addition, free radical scavenging activities using 2,2'-diphenyl-1-picrylhydrazyl (DPPH) were studied. Compound 1 was the most active as an antiproliferative agent, showing a high to moderate antiproliferative effect with an IC50 value of 4.95 and 17.7 µM against MCF-7 and MDA-MB-231, respectively. The antiproliferative activities of compounds 1-5 against the normal breast cell line MCF-10A were moderate to low with IC50 values of 13.91 to 27.70 µM. On the other hand, compounds 1 and 10 suppressed MDA-MB-231 cell migration in the wound-healing assay at 10 µM concentration. Meanwhile, compounds 1-5 exhibited the highest value of DPPH radical scavenging activities with an IC50 value range of 17.5-20.43 ± 0.5 µg mL-1. The pharmacophore model generated using Molecular Operating Environment (MOE) for compounds 1-5 showed three hydrogen bond acceptors (HBAs), one hydrogen bond donor (HBD), one aromatic ring (Aro), and one hydrophobic (Hyd.) group. The central HBA feature lies at a distance of 4.36 Å and 6.38 Å from the remaining two HBA features. Also, the HBD feature maintains a distance of 2.74 Å from the aromatic feature. Acylated rhamnopyranoses can be considered good scaffolds for developing new anti-breast cancer and antioxidant compounds.

Design, synthesis, and validation of novel nitrogen-based chalcone analogs against triple negative breast cancer.

Great strides have been made in triple negative breast cancer (TNBC) treatment, which represents 20% of total predicted annual US breast cancer (BC) cases. Despite the development of several therapeutics, TNBC patients have poor overall survival rate, compared to other BC patients, justifying the urgent need to discover new entities for use to control TNBC. Chalcones are important natural products with diverse bioactivities including anticancer effects. This study aimed to design, synthesize and validate novel chalcone leads as potential therapies for TNBC. Fourteen novel chalcone analogs were designed and synthesized comprising alicyclic amines (pyrrolidine, morpholine and piperidine) or nitrogen mustard (Bis-(2-chloroethyl) amine) substituents. Among them, compound 14((E)-3-(4-(Bis(2-chloroethyl) amino) phenyl)-1-(3-methoxyphenyl) prop-2-en-1-one) was identified as the most effective against TNBC and other BC phenotypes, with anti-proliferative IC50 values ranging between 3.94 and 9.22 µM against the TNBC cell lines MDA-MB-231 and MDA-MB-468, as well as against the estrogen positive MCF-7 cell line. Chalcone 14 effectively suppressed the colony formation capacity of MDA-MB-231, MDA-MB-468, and MCF-7 cell lines at 5 and 10 µM treatment concentrations. Furthermore, compound 14 has significantly inhibited cell invasion and migration of MDA-MB-231 and MCF-7 BC cell lines. Additionally, compound 14 had significantly promoted apoptosis by upregulating BAX and downregulating Bcl-2 proteins. Compound 14 induced significant cell cycle arrest of TNBC cells at the G2/M phase. It also induced a reversal of Epithelial Mesenchymal Transition (EMT) by upregulating the epithelial markers E-cadherin and Pan-cadherin and downregulating FAK. Furthermore, it had dramatically diminished new vessel formation (vasculogenesis) in chick chorioallantoic membrane (CAM) model by 60.20 ± 8.47%. Chalcone 14 inhibited 46.41 ± 0.71% of the TNBC MAD-MB-231 cells growth in a nude mouse orthotopic xenograft model in comparison with vehicle control treated animals. Collectively, this study results propose chalcone 14 as a promising lead molecule for the control of TNBC as well as other breast cancer phenotypes.

Optimization of Taste-Masked (-)-Oleocanthal Effervescent Formulation with Potent Breast Cancer Progression and Recurrence Suppressive Activities.

S-(-)-Oleocanthal (OC), a naturally occurring phenolic secoiridoid exclusively found in extra-virgin olive oil (EVOO), is a potential nutraceutical therapeutic for inflammation, neurodegenerative diseases, and many malignancies, especially breast cancer (BC). The oral delivery of OC is challenging because of its irritative, bitter, and pungent taste and exceptional chemistry, including two reactive aldehydes, phenolic, and ester groups. OC irritation did not correlate with CO2-induced irritation, and hence, OC was not exerting generalized acid-sensing irritation. The objective of this study was to develop an effervescent formulation of OC with an effective CO2-induced masked taste maintaining the efficacy against the estrogen receptor (ER) and HER2 positive BC. Several ratios of acid and carbonate sources were screened, and five effervescent formulations EF1-EF5 were selected and prepared based on their pH and effervescence time. OC formulations were characterized using differential scanning calorimetry, FT-IR spectroscopy, and scanning electron microscopy analyses. OC formulations exhibited acceptable flowability and effervescence time. Based on physical characteristics and improved OC release, formulation EF-2 was selected for subsequent studies. EF-2 showed effective OC taste masking, as suggested by electronic artificial tongue and mouse preference tests. EF-2 suppressed more than 70% of the hormone and HER2-positive BT-474 BC cell growth in a nude mouse xenograft model. Furthermore, EF-2 demonstrated significant inhibition of BT-474 tumor cell locoregional recurrence after primary tumor surgical excision. EF-2-treated mouse sera had significantly reduced CA 15-3 levels, the human BC recurrence marker, compared to the placebo control group at the end of the study. These results highlight the potential of the OC formulation EF-2 as a prospective nutraceutical for the control and prevention of ER+/HER+ BC progression and locoregional recurrence.

Novel olive oil phenolic (-)-oleocanthal (+)-xylitol-based solid dispersion formulations with potent oral anti-breast cancer activities.

Epidemiological studies have compellingly documented the ability of the Mediterranean diet rich in extra-virgin olive oil to reduce the incidence of certain malignancies, and cardiovascular diseases, and slow the Alzheimer's disease progression. S-(-)-Oleocanthal (OC) was identified as the most bioactive olive oil phenolic with documented anti-inflammatory, anticancer, and anti-Alzheimer's activities. OC consumption causes irritating sensation at the oropharynx via activation of TRPA1. Accordingly, a taste-masked formulation of OC is needed for its future use as a nutraceutical while maintaining its bioactivity and unique chemistry. Therefore, the goal of this study was to prepare a taste-masked OC solid formulation with improved dissolution and pharmacodynamic profiles, by using (+)-xylitol as an inert carrier. Xylitol was hypothesized to serve as an ideal vehicle for the preparation of OC solid dispersions due to its low melting point and sweetness. The optimized OC-(+)-xylitol solid dispersion was physically and chemically characterized and showed effective taste masking and enhanced dissolution properties. Furthermore, OC-(+)-xylitol solid dispersion maintained potent in vivo anti-breast cancer activity. It effectively suppressed the human triple negative breast cancer development, growth, and recurrence after primary tumor surgical excision in nude mice orthotopic xenograft models. Collectively, these results suggest the OC-(+)-xylitol solid dispersion formulation as a potential nutraceutical for effective control and prevention of human triple negative breast cancer.

(-)-Oleocanthal Prevents Breast Cancer Locoregional Recurrence After Primary Tumor Surgical Excision and Neoadjuvant Targeted Therapy in Orthotopic Nude Mouse Models.

Breast cancer (BC) recurrence represents a challenge for survivors who have had their primary tumors surgically excised, and/or have completed radiation, neoadjuvant, or adjuvant therapeutic regimens. Current BC treatments mostly lack the ability to reduce the risk of disease recurrence. About 70% of BC patients will subsequently suffer disease relapse, manifesting as local, regional, or distant tumor recurrence, which clearly underscores the urgent need to discover novel recurrence inhibitors. (-)-Oleocanthal (OC) is a natural phenolic, found so far exclusively in extra-virgin olive oil (EVOO). OC exerts documented bioactivities against diverse cancer types, inflammation, and neurodegenerative diseases. Herein we report the novel activity of daily oral treatment with OC (10 mg/kg) in preventing BC locoregional recurrence in a nude mouse xenograft model generated by orthotopic inoculation with BT-474 cells as a luminal type B model. We further report inhibition of tumor recurrence by OC after completion of a lapatinib neoadjuvant regimen. However, in a recurrence model of triple-negative breast cancer (TNBC), OC treatment (10 mg/kg) did not effectively prevent tumor recurrence, but rather, was seen to significantly reduce the growth of recurrent tumors as compared to vehicle control-treated animals. Inhibition of tumor recurrence was associated with significant serum level reductions of the human BC recurrence marker CA 15-3 at the study end in animals treated with OC. OC treatment upregulated the expression of the epithelial marker E-cadherin and downregulated the levels of the mesenchymal marker vimentin in recurrent tumors vs. untreated control animals. OC treatment also reduced the activation of MET and HER2 receptors, as indicated by reduced phosphorylation levels of these proteins in recurrent tumors vs. controls. Collectively, the results of our studies provide the first evidence for suppression of BC tumor recurrence by oral OC treatment in an animal model for such recurrence, and furthermore, highlight favorable prospects for this natural product to emerge as a first-in-class BC recurrence inhibitor.