Anticancer Effects of Fucoxanthin through Cell Cycle Arrest, Apoptosis Induction, and Angiogenesis Inhibition in Triple-Negative Breast Cancer Cells.

The absence of progesterone receptors, estrogen receptors, and human epidermal growth factor receptor-2 restricts the therapy choices for treating triple-negative breast cancer (TNBC). Moreover, conventional medication is not highly effective in treating TNBC, and developing effective therapeutic agents from natural bioactive compounds is a viable option. In this study, the anticancer effects of the natural compound fucoxanthin were investigated in two genetically different models of TNBC cells: MDA-MB-231 and MDA-MB-468 cells. Fucoxanthin had a significant anticancer effect in both cell lines at a concentration range of 1.56-300 µM. The compound decreased cell viability in both cell lines with higher potency in MDA-MB-468 cells. Meanwhile, proliferation assays showed similar antiproliferative effects in both cell lines after 48 h and 72 h treatment periods. Flow cytometry and Annexin V-FITC apoptosis assay revealed the ability of fucoxanthin to induce apoptosis in MDA-MB-231 only. Cell cycle arrest analysis showed that the compound also induced cell cycle arrest at the G1 phase in both cell lines, accompanied by more cell cycle arrest in MDA-MB-231 cells at S-phase and a higher cell cycle arrest in the MDA-MB-468 cells at G2-phase. Wound healing and migration assay showed that in both cell lines, fucoxanthin prevented migration, but was more effective in MDA-MB-231 cells in a shorter time. In both angiogenic cytokine array and RT-PCR studies, fucoxanthin (6.25 µM) downregulated VEGF-A and -C expression in TNF-α-stimulated (50 ng/mL) MDA-MB-231, but not in MDA-MB-468 cells on the transcription and protein levels. In conclusion, this study shows that fucoxanthin was more effective in MDA-MB-231 TNBC cells, where it can target VEGF-A and VEGF-C, inhibit cell proliferation and cell migration, and induce cell cycle arrest and apoptosis-the most crucial cellular processes involved in breast cancer development and progression.

Involvement of AKT/PI3K Pathway in Sanguinarine's Induced Apoptosis and Cell Cycle Arrest in Triple-negative Breast Cancer Cells.

BACKGROUND/AIM: Chemotherapy resistance in triple-negative breast cancer (TNBC) cells is well documented. Therefore, it is necessary to develop safer and more effective therapeutic agents to enhance the outcomes of chemotherapeutic agents. The natural alkaloid sanguinarine (SANG) has demonstrated therapeutic synergy when coupled with chemotherapeutic agents. SANG can also induce cell cycle arrest and trigger apoptosis in various cancer cells. MATERIALS AND METHODS: In this study, we investigated the molecular mechanism underlying SANG activity in MDA-MB-231 and MDA-MB-468 cells as two genetically different models of TNBC. We employed various assays including Alamar Blue to measure the effect of SANG on cell viability and proliferation rate, flow cytometry analysis to study the potential of the compound to induce apoptosis and cell cycle arrest, quantitative qRT PCR apoptosis array to measure the expression of different genes mediating apoptosis, and the western system was used to analyze the impact of the compound on AKT protein expression. RESULTS: SANG lowered cell viability and disrupted cell cycle progression in both cell lines. Furthermore, S-phase cell cycle arrest-mediated apoptosis was found to be the primary contributor to cell growth inhibition in MDA-MB-231 cells. SANG-treated TNBC cells showed significantly up-regulated mRNA expression of 18 genes associated with apoptosis, including eight TNF receptor superfamily (TNFRSF), three members of the BCL2 family, and two members of the caspase (CASP) family in MDA-MB-468 cells. In MDA-MB-231 cells, two members of the TNF superfamily and four members of the BCL2 family were affected. The western study data showed the inhibition of AKT protein expression in both cell lines concurrent with up-regulated BCL2L11 gene. Our results point to the AKT/PI3K signaling pathway as one of the key mechanisms behind SANG-induced cell cycle arrest and death. CONCLUSION: SANG shows anticancer properties and apoptosis-related gene expression changes in the two TNBC cell lines and suggests AKT/PI3K pathway implication in apoptosis induction and cell cycle arrest. Thus, we propose SANG's potential as a solitary or supplementary treatment agent against TNBC.

The Anticancer Effects of Marine Carotenoid Fucoxanthin through Phosphatidylinositol 3-Kinase (PI3K)-AKT Signaling on Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks specific targets such as estrogen, progesterone, and HER2 receptors. TNBC affects one in eight women in the United States, making up 15-20% of breast cancer cases. Patients with TNBC can develop resistance to chemotherapy over time, leading to treatment failure. Therefore, finding other options like natural products is necessary for treatment. The advantages of using natural products sourced from plants as anticancer agents are that they are less toxic, more affordable, and have fewer side effects. These products can modulate several cellular processes of the tumor microenvironment, such as proliferation, migration, angiogenesis, cell cycle arrest, and apoptosis. The phosphatidyl inositol 3-kinase (PI3K)-AKT signaling pathway is an important pathway that contributes to the survival and growth of the tumor microenvironment and is associated with these cellular processes. This current study examined the anticancer effects of fucoxanthin, a marine carotenoid isolated from brown seaweed, in the MDA-MB-231 and MDA-MB-468 TNBC cell lines. The methods used in this study include a cytotoxic assay, PI3K-AKT signaling pathway PCR arrays, and Wes analysis. Fucoxanthin (6.25 µM) + TNF-α (50 ng/mL) and TNF-α (50 ng/mL) showed no significant effect on cell viability compared to the control in both MDA-MB-231 and MDA-MB-468 cells after a 24 h treatment period. PI3K-AKT signaling pathway PCR array studies showed that in TNF-α-stimulated (50 ng/mL) MDA-MB-231 and MDA-MB-468 cells, fucoxanthin (6.25 µM) modulated the mRNA expression of 12 genes, including FOXO1, RASA1, HRAS, MAPK3, PDK2, IRS1, EIF4EBP1, EIF4B, PTK2, TIRAP, RHOA, and ELK1. Additionally, fucoxanthin significantly downregulated the protein expression of IRS1, EIF4B, and ELK1 in MDA-MB-231 cells, and no change in the protein expression of EIF4B and ELK1 was shown in MDA-MB-468 cells. Fucoxanthin upregulated the protein expression of RHOA in both cell lines. The modulation of the expression of genes and proteins of the PI3K-AKT signaling pathway may elucidate fucoxanthin's effects in cell cycle progression, apoptotic processes, migration, and proliferation, which shows that PI3K-AKT may be the possible molecular mechanism for fucoxanthin's effects. In conclusion, the results obtained in this study elucidate fucoxanthin's molecular mechanisms and indicate that fucoxanthin may be considered a promising candidate for breast cancer-targeted therapy.

Anticancer Effects of Thymoquinone through the Antioxidant Activity, Upregulation of Nrf2, and Downregulation of PD-L1 in Triple-Negative Breast Cancer Cells.

The variety of therapies available for treating and preventing triple-negative breast cancer (TNBC) is constrained by the absence of progesterone receptors, estrogen receptors, and human epidermal growth factor receptor 2. Nrf2 (nuclear factor-erythroid 2-related factor), and PD-L1 (program cell death ligand 1), a downstream signaling target, have a strong correlation to oxidative stress and inflammation, major factors in the development and progression of TNBC. In this study, the genetically distinct MDA-MB-231 and MDA-MB-468 TNBC cells were treated with the natural component thymoquinone (TQ). The results show that TQ exhibits considerable antioxidant activity and decreases the generation of H2O2, at the same time increasing catalase (CAT) activity, superoxide dismutase (SOD) enzyme, and glutathione (GSH). Additionally, the results show that TQ treatment increased the levels of the different genes involved in the oxidative stress-antioxidant defense system PRNP, NQO1, and GCLM in both cell lines with significant large-fold change in MDA-MB-468 cells (+157.65 vs. +1.7, +48.87 vs. +2.63 and +4.78 vs. +2.17), respectively. Nrf2 mRNA and protein expression were also significantly increased in TQ-treated TNBC cells despite being higher in MDA-MB-468 cells (6.67 vs. 4.06). Meanwhile, TQ administration increased mRNA levels while decreasing PD-L1 protein expression in both cell lines. In conclusion, TQ modifies the expression of multiple oxidative-stress-antioxidant system genes, ROS, antioxidant enzymes, Nrf2, and PD-L1 protein, pointing to the therapeutic potential and chemopreventive utilization of TQ in TNBC.

Sanguinarine Inhibition of TNF-α-Induced CCL2, IKBKE/NF-κB/ERK1/2 Signaling Pathway, and Cell Migration in Human Triple-Negative Breast Cancer Cells.

Angiogenesis is a process that drives breast cancer (BC) progression and metastasis, which is linked to the altered inflammatory process, particularly in triple-negative breast cancer (TNBC). In targeting inflammatory angiogenesis, natural compounds are a promising option for managing BC. Thus, this study was designed to determine the natural alkaloid sanguinarine (SANG) potential for its antiangiogenic and antimetastatic properties in triple-negative breast cancer (TNBC) cells. The cytotoxic effect of SANG was examined in MDA-MB-231 and MDA-MB-468 cell models at a low molecular level. In this study, SANG remarkably inhibited the inflammatory mediator chemokine CCL2 in MDA-MB-231 and MDA-MB-468 cells. Furthermore, qRT-PCR confirmed with Western analysis studies showed that mRNA CCL2 repression was concurrent with reducing its main regulator IKBKE and NF-κB signaling pathway proteins in both TNBC cell lines. The total ERK1/2 protein was inhibited in the more responsive MDA-MB-231 cells. SANG exhibited a higher potential to inhibit cell migration in MDA-MB-231 cells compared to MDA-MB-468 cells. Data obtained in this study suggest a unique antiangiogenic and antimetastatic effect of SANG in the MDA-MB-231 cell model. These effects are related to the compound's ability to inhibit the angiogenic CCL2 and impact the ERK1/2 pathway. Therefore, SANG use may be recommended as a component of the therapeutic strategy for TNBC.

Thymoquinone Anticancer Effects Through the Upregulation of NRF2 and the Downregulation of PD-L1 in MDA-MB-231 Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a metastatic nature. TNBC lacks the expression of the progesterone receptor, estrogen receptor, and human epidermal growth factor receptor 2. The absence of these three receptors limits the therapy options. Meanwhile, conventional medication is not very effective in the treatment and prevention of TNBC. Developing innovative therapeutic agents from natural bioactive compounds is a viable option. In the current study, the natural compound thymoquinone (TQ) was used in MDA-MB-231 TNBC cells. A dose response to TQ (0-50 µM) was obtained following a 24-hour exposure. An Annexin V-FITC apoptosis detection was used to study the apoptotic effect of TQ.  With increasing TQ concentrations from 0-20 µM, flow cytometer examination revealed an increase in apoptotic cells. Nearly 80% of the cells studied were in the apoptotic phase at 20 µM. (early and late apoptosis). Meanwhile, at 30 µM, there was a significant drop in apoptotic cells, which may be interpreted as an increase in necrotic cells. Molecular-targeted therapy is a new approach in treating cancer. The effect of TQ on the expression of Nrf2(Nuclear factor erythroid 2- related factor 2) and PD-L1 (Programmed death-ligand 1) was investigated using a specific primary antibody against these proteins. A Western blot analysis confirmed TQ's ability to change the expression of both proteins under investigation. According to normalized data, TQ had the ability to elicit more than 2-fold increase in Nrf2 expression in IFN-γ stimulated MDA-MB-231 cells. In contrast, co-treated cells (IFN-γ + TQ) showed a 65 percent reduction in PD-L1 expression. In conclusion, TQ was suggested as a promising anticancer option for treating  TNBC.

Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells.

Breast cancer (BC) is the most common cancer in women worldwide, and it is one of the leading causes of cancer death in women. triple-negative breast Cancer (TNBC), a subtype of BC, is typically associated with the highest pathogenic grade and incidence in premenopausal and young African American (AA) women. Chemotherapy, the most common treatment for TNBC today, can lead to acquired resistance and ineffective treatment. Therefore, novel therapeutic approaches are needed to combat medication resistance and ineffectiveness in TNBC patients. Thymoquinone (TQ) is shown to have a cytotoxic effect on human cancer cells in vitro. However, TQ's mode of action and precise mechanism in TNBC disease in vitro have not been adequately investigated. Therefore, TQ's effects on the genetically different MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were assessed. The data obtained show that TQ displayed cytotoxic effects on MDA-MB-468 and MDA-MB-231 cells in a time- and concentration-dependent manner after 24 h, with IC50 values of 25.37 µM and 27.39 µM, respectively. Moreover, MDA-MB-231 and MDA-MB-468 cells in a scratched wound-healing assay displayed poor wound closure, inhibiting invasion and migration via cell cycle blocking after 24 h. TQ arrested the cell cycle phase in MDA-MB-231 and MDA-MB-468 cells. The three cell cycle stages in MDA-MB-468 cells were significantly affected at 15 and 20 µM for G0/G1 and S phases, as well as all TQ concentrations for G2/M phases. In MDA-MB-468 cells, there was a significant decrease in G0/G1 phases with a substantial increase in the S phase and G2/M phases. In contrast, MDA-MB-231 showed a significant effect only during the two cell cycle stages (S and G2/M), at concentrations of 15 and 20 µM for S phases and all TQ values for G2/M phases. The TQ effect on the apoptotic gene profiles indicated that TQ upregulated 15 apoptotic genes in MDA-MB-231 TNBC cells, including caspases, GADD45A, TP53, DFFA, DIABLO, BNIP3, TRAF2/3, and TNFRSF10A. In MDA-MB-468 cells, 16 apoptotic genes were upregulated, including TNFRSF10A, TNF, TNFRSF11B, FADD TNFRSF10B, CASP2, and TRAF2, all of which are important for the apoptotic pathway andsuppress the expression of one anti-apoptotic gene, BIRC5, in MDA-MB-231 cells. Compared to MDA-MB-231 cells, elevated levels of TNF and their receptor proteins may contribute to their increased sensitivity to TQ-induced apoptosis. It was concluded from this study that TQ targets the MDA-MB-231 and MDA-MB-468 cells differently. Additionally, due to the aggressive nature of TNBC and the lack of specific therapies in chemoresistant TNBC, our findings related to the identified apoptotic gene profile may point to TQ as a potential agent for TNBC therapy.

A Rare Presentation of a Non-Asian Female With Metastatic Non-small-cell Lung Cancer Harboring EGFR L747P Mutation With Clinical Response to Multi-targeted Epigenetic and EGFR Inhibition.

BACKGROUND: Activating mutations of the epidermal growth factor receptor (EGFR) gene have been utilized to predict the effectiveness of EGFR tyrosine kinase inhibitor (TKI) therapy. The most common EGFR mutations are exon 19 deletion and exon 21-point mutation, which are sensitive to EGFR TKI. However, rare/complex EGFR mutations still exist, data of which are scarce and controversial. Hence, their role in response to standard therapy remains uncertain. CASE REPORT: We present the case of a patient diagnosed with stage IV lung adenocarcinoma for whom standard chemotherapies, including platinum agents, had failed. The patient was found to have an EGFR exon 19 (L747P) mutation, as evident in her liquid biopsy. This alteration has not been described before in the literature on non-Asian females. Data from the current case study highlight the aggressive nature of this type of EGFR mutation as indicated by the complete resistance to erlotinib. Using standard first-generation EGFR inhibitors in treating this point mutation was considered inadequate. However, this patient showed a substantial response when treated with erlotinib combined with epigenetic therapies, consisting of DNA methyltransferase and histone deacetylase inhibitors. For more than 8 years, the patient has been responding to combination therapy with a normal quality of life. CONCLUSION: This case represents a possible novel approach to reducing resistance in patients harboring this rare EGFR mutation which may translate to better outcomes.

Polyphenols Modulating Effects of PD-L1/PD-1 Checkpoint and EMT-Mediated PD-L1 Overexpression in Breast Cancer.

Investigating dietary polyphenolic compounds as antitumor agents are rising due to the growing evidence of the close association between immunity and cancer. Cancer cells elude immune surveillance for enhancing their progression and metastasis utilizing various mechanisms. These mechanisms include the upregulation of programmed death-ligand 1 (PD-L1) expression and Epithelial-to-Mesenchymal Transition (EMT) cell phenotype activation. In addition to its role in stimulating normal embryonic development, EMT has been identified as a critical driver in various aspects of cancer pathology, including carcinogenesis, metastasis, and drug resistance. Furthermore, EMT conversion to another phenotype, Mesenchymal-to-Epithelial Transition (MET), is crucial in developing cancer metastasis. A central mechanism in the upregulation of PD-L1 expression in various cancer types is EMT signaling activation. In breast cancer (BC) cells, the upregulated level of PD-L1 has become a critical target in cancer therapy. Various signal transduction pathways are involved in EMT-mediated PD-L1 checkpoint overexpression. Three main groups are considered potential targets in EMT development; the effectors (E-cadherin and Vimentin), the regulators (Zeb, Twist, and Snail), and the inducers that include members of the transforming growth factor-beta (TGF-ß). Meanwhile, the correlation between consuming flavonoid-rich food and the lower risk of cancers has been demonstrated. In BC, polyphenols were found to downregulate PD-L1 expression. This review highlights the effects of polyphenols on the EMT process by inhibiting mesenchymal proteins and upregulating the epithelial phenotype. This multifunctional mechanism could hold promises in the prevention and treating breast cancer.

The Anticancer Effects of Flavonoids through miRNAs Modulations in Triple-Negative Breast Cancer.

Triple- negative breast cancer (TNBC) incidence rate has regularly risen over the last decades and is expected to increase in the future. Finding novel treatment options with minimum or no toxicity is of great importance in treating or preventing TNBC. Flavonoids are new attractive molecules that might fulfill this promising therapeutic option. Flavonoids have shown many biological activities, including antioxidant, anti-inflammatory, and anticancer effects. In addition to their anticancer effects by arresting the cell cycle, inducing apoptosis, and suppressing cancer cell proliferation, flavonoids can modulate non-coding microRNAs (miRNAs) function. Several preclinical and epidemiological studies indicate the possible therapeutic potential of these compounds. Flavonoids display a unique ability to change miRNAs' levels via different mechanisms, either by suppressing oncogenic miRNAs or activating oncosuppressor miRNAs or affecting transcriptional, epigenetic miRNA processing in TNBC. Flavonoids are not only involved in the regulation of miRNA-mediated cancer initiation, growth, proliferation, differentiation, invasion, metastasis, and epithelial-to-mesenchymal transition (EMT), but also control miRNAs-mediated biological processes that significantly impact TNBC, such as cell cycle, immune system, mitochondrial dysregulation, modulating signaling pathways, inflammation, and angiogenesis. In this review, we highlighted the role of miRNAs in TNBC cancer progression and the effect of flavonoids on miRNA regulation, emphasizing their anticipated role in the prevention and treatment of TNBC.

Gene Expression Alterations Associated with Oleuropein-Induced Antiproliferative Effects and S-Phase Cell Cycle Arrest in Triple-Negative Breast Cancer Cells.

It is known that the Mediterranean diet is effective in reducing the risk of several chronic diseases, including cancer. A critical component of the Mediterranean diet is olive oil, and the relationship between olive oil consumption and the reduced risk of cancer has been established. Oleuropein (OL) is the most prominent polyphenol component of olive fruits and leaves. This compound has been shown to have potent properties in various types of cancers, including breast cancer. In the present study, the molecular mechanism of OL was examined in two racially different triple-negative breast cancer (TNBC) cell lines-African American (AA, MDA-MB-468) and Caucasian American (CA, MDA-MB-231). The data obtained showed that OL effectively inhibits cell growth in both cell lines, concomitant with S-phase cell cycle arrest-mediated apoptosis. The results also showed that OL-treated MDA-MB-468 cells were two-fold more sensitive to OL antiproliferative effect than MDA-MB-231 cells were. At lower concentrations, OL modified the expression of many apoptosis-involved genes. OL was more effective in MDA-MB-468, compared to MDA-MB-231 cells, in terms of the number and the fold-change of the altered genes. In MDA-MB-468 cells, OL induced a noticeable transcription activation in fourteen genes, including two members of the caspase family: caspase 1 (CASP1) and caspase 14 (CASP14); two members of the TNF receptor superfamily: Fas-associated via death domain (FADD) and TNF receptor superfamily 21 (TNFRSF21); six other proapoptotic genes: growth arrest and DNA damage-inducible 45 alpha (GADD45A), cytochrome c somatic (CYCS), BCL-2 interacting protein 2 (BNIP2), BCL-2 interacting protein 3 (BNIP3), BH3 interacting domain death agonist (BID), and B-cell lymphoma/leukemia 10 (BCL10); and the CASP8 and FADD-like apoptosis regulator (CFLAR) gene. Moreover, in MDA-MB-468 cells, OL induced a significant upregulation in two antiapoptotic genes: bifunctional apoptosis regulator (BFAR) and B-Raf proto-oncogene (BRAF) and a baculoviral inhibitor of apoptosis (IAP) repeat-containing 3 (BIRC3). On the contrary, in MDA-MB-231 cells, OL showed mixed impacts on gene expression. OL significantly upregulated the mRNA expression of four genes: BIRC3, receptor-interacting serine/threonine kinase 2 (RIPK2), TNF receptor superfamily 10A (TNFRSF10A), and caspase 4 (CASP4). Additionally, another four genes were repressed, including caspase 6 (CASP6), pyrin domain (PYD), and caspase recruitment domain (CARD)-containing (PAYCARD), baculoviral IAP repeat-containing 5 (BIRC5), and the most downregulated TNF receptor superfamily member 11B (TNFRSF11B, 16.34-fold). In conclusion, the data obtained indicate that the two cell lines were markedly different in the anticancer effect and mechanisms of oleuropein's ability to alter apoptosis-related gene expressions. The results obtained from this study should also guide the potential utilization of oleuropein as an adjunct therapy for TNBC to increase chemotherapy effectiveness and prevent cancer progression.

Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells.

Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including, anti-inflammatory, immunomodulatory, and neuroprotective, as well as having antioxidant and anticancer activities. This study evaluated the effects and mechanisms of RA in two racially different triple-negative breast cancer (TNBC) cell lines. Results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. Remarkably, RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Further, in MDA-MB-231 cells, RA significantly upregulated the mRNA expression of three genes: harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, the compound induced a significant transcription activation in three genes, including TNF, growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in MDA-MB-468 cells.

Molecular mechanism of gossypol mediating CCL2 and IL­8 attenuation in triple­negative breast cancer cells.

Chronic inflammation associated with cancer is characterized by the production of different types of chemokines and cytokines. In cancer, numerous signaling pathways upregulate the expression levels of several cytokines and evolve cells to the neoplastic state. Therefore, targeting these signaling pathways through the inhibition of distinctive gene expression is a primary target for cancer therapy. The present study investigated the anticancer effects of the natural polyphenol gossypol (GOSS) in triple­negative breast cancer (TNBC) cells, the most aggressive breast cancer type with poor prognosis. GOSS effects were examined in two TNBC cell lines: MDA­MB­231 (MM­231) and MDA­MB­468 (MM­468), representing Caucasian Americans (CA) and African Americans (AA), respectively. The obtained IC50s revealed no significant difference between the two cell lines' response to the compound. However, the use of microarray assays for cytokine determination indicated the ability of GOSS to attenuate the expression levels of cancer­related cytokines in the two cell lines. Although GOSS did not alter CCL2 expression in MM­468 cells, it was able to cause 30% inhibition in TNF­α­stimulated MM­231 cells. Additionally, IL­8 was not altered by GOSS treatment in MM­231 cells, while its expression was inhibited by 60% in TNF­α­activated MM­468 cells. ELISA assays supported the microarray data and indicated that CCL2 expression was inhibited by 40% in MM­231 cells, and IL­8 expression was inhibited by 50% in MM­468 cells. Furthermore, in MM­231 cells, GOSS inhibited CCL2 release via the repression of IKBKE, CCL2 and MAPK1 gene expression. Additionally, in MM­468 cells, the compound downregulated the release of IL­8 through repressing IL­8, MAPK1, MAPK3, CCDC88A, STAT3 and PIK3CD gene expression. In conclusion, the data obtained in the present study indicate that the polyphenol compound GOSS may provide a valuable tool in TNBC therapy.

The Antiproliferative Effects of Flavonoid MAO Inhibitors on Prostate Cancer Cells.

Prostate cancer (PCa) patients commonly experience clinical depression. Recent reports indicated that monoamine oxidase-A (MAO-A) levels elevate in PCa, and antidepressant MAO-Is show anti-PCa properties. In this work, we aimed to find potential drugs for PCa patients suffering from depression by establishing novel anti-PCa reversible monoamine oxidase-A inhibitors (MAO-AIs/RIMA); with an endeavor to understand their mechanism of action. In this investigation, twenty synthesized flavonoid derivatives, defined as KKR compounds were screened for their inhibitory potentials against human MAO-A and MAO-B isozymes. Meanwhile, the cytotoxic and antiproliferative effects were determined in three human PCa cell lines. MAO-A-kinetics, molecular docking, SAR, cell morphology, and cell migration were investigated for the most potent compounds. The screened KKRs inhibited MAO-A more potently than MAO-B, and non-toxically inhibited LNCaP cell proliferation more than the DU145 and PC3 cell lines, respectively. The results showed that the three top MAO-AI KKRs compounds (KKR11, KKR20, and KKR7 (IC50s 0.02-16 µM) overlapped with the top six antiproliferative KKRs against LNCaP (IC50s ~9.4 µM). While KKR21 (MAO-AI) and KKR2A (MAO-I) were ineffective against the PCa cells. Furthermore, KKR21 and KKR11 inhibited MAO-A competitively (Kis ≤ 7.4 nM). Molecular docking of the two compounds predicted shared hydrophobic and distinctive hydrophilic interactions-between the KKR molecule and MAO-A amino acid residues-to be responsible for their reversibility. The combined results and SAR observations indicated that the presence of specific active groups-such as chlorine and hydroxyl groups-are essential in certain MAO-AIs with anti-PCa effects. Additionally, MAO-A inhibition was found to be associated more with anti-PCa property than MAO-B. Distinctively, KKR11 [(E)-3-(3,4-dichlorophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one] exhibited anti-metastatic effects on the DU145 cell line. The chlorine substitution groups might play vital roles in the KKR11 multiple actions. The obtained results indicated that the flavonoid derivative KKR11 could present a novel candidate for PCa patients with depression, through safe non-selective potent inhibition of MAOs.

Effects of gossypol on apoptosis­related gene expression in racially distinct triple­negative breast cancer cells.

Apoptosis is a gene­directed mechanism that regulates cell proliferation and maintains homeostasis. Moreover, an aberrant apoptotic process can lead to several pathological conditions, such as tumorigenesis and cancer metastasis. In the present study, the apoptotic effect of the natural polyphenol compound gossypol GOSS) was investigated in triple­negative breast cancer TNBC) cells. The effect of GOSS was evaluated in two cell lines representative of a Caucasian­American and African­American origin, MDA­MB­231 MM­231) and MDA­MB­468 MM­468), respectively. A similar response to both cytotoxicity and proliferation was observed in the two cell lines. However, MM­468 cells were 2­fold more sensitive to the apoptotic effect of the compound, which was accompanied by a longer delay in colony formation. Furthermore, GOSS was found to alter the mRNA expression of many apoptosis­related genes. The compound significantly upregulated growth arrest and DNA damage­inducible 45 alpha protein (GADD45A), tumor necrosis factor receptor superfamily 9 (TNFRSF9) and BCL2 interacting protein 3 BNIP3) in MM­231 cells. Similarly, GADD45A and BNIP3 were upregulated in MM­468 cells. A significant finding in this study is the profound 159­fold increase in TNF gene expression that was observed in MM­468 cells. Moreover, the apoptosis­suppressor gene baculoviral IAP repeat containing 5 BIRC5) was significantly repressed (by more than 90%) in both cell lines, as well as death­associated protein kinase 1 (DAPK1) in MM­231 cells and tumor protein 73 (TP73) in MM­468 cells. In conclusion, the data obtained in this study provide a molecular understanding of the GOSS­induced apoptosis effect and suggest the importance of this polyphenol compound targeted towards TNBC treatment, particularly in African­American women.

The inhibitory effects of plumbagin on the NF-қB pathway and CCL2 release in racially different triple-negative breast cancer cells.

Breast cancer (BC) is the second leading cause of death among women in the US, and its subtype triple-negative BC (TNBC) is the most aggressive BC with poor prognosis. In the current study, we investigated the anticancer effects of the natural product plumbagin (PL) on racially different TNBC cells. The PL effects were examined in two TNBC cell lines: MDA-MB-231 (MM-231) and MDA-MB-468 (MM-468), representing Caucasian Americans and African Americans, respectively. The results obtained indicate that PL inhibited cell viability and cell proliferation and induced apoptosis in both cell lines. Notably, MM-468 cells were 5-fold more sensitive to PL than MM-231 cells were. Testing PL and Taxol® showed the superiority of PL over Taxol® as an antiproliferative agent in MM-468 cells. PL treatment resulted in an approximately 20-fold increase in caspase-3 activity with 3 µM PL in MM-468 cells compared with an approximately 3-fold activity increase in MM-231 cells with 8 µM PL. Moreover, the results indicate a higher sensitivity to PL in MM-468 cells than in MM-231 cells. The results also show that PL downregulated CCL2 cytokine expression in MM-468 cells by 30% compared to a 90% downregulation in MM-231 cells. The ELISA results confirmed the array data (35% vs. 75% downregulation in MM-468 and MM-231 cells, respectively). Moreover, PL significantly downregulated IL-6 and GM-CSF in the MM-231 cells. Indeed, PL repressed many NF-қB-regulated genes involved in the regulation of apoptosis, proliferation, invasion, and metastasis. The compound significantly downregulated the same genes (BIRC3, CCL2, TLR2, and TNF) in both types of cells. However, PL impacted five more genes in MM-231 cells, including BCL2A1, ICAM1, IKBKE, IL1ß, and LTA. In conclusion, the data obtained in this study indicate that the quinone compound PL could be a novel cancer treatment for TNBC in African American women.

The attenuating effects of plumbagin on pro-inflammatory cytokine expression in LPS-activated BV-2 microglial cells.

Activated microglial cells produce the pro-inflammatory mediators such as nitric oxide (NO) and cytokines. The excessive release of these mediators can lead to neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Inhibition of the release of these pro-inflammatory molecules may prevent or halt the progression of these diseases. Plumbagin (PL), a naphthoquinone compound in the roots of the traditional medicinal plant Plumbago zeylanica L., showed anti-inflammatory effects on macrophages. However, PL effects on activated microglia remain unknown. In the present study, PL has been examined for its anti-inflammatory effect on LPS - activated microglial BV-2 cells. In this study, NO and iNOS expression were investigated in BV-2 microglial cells in the presence of PL or the selective iNOS inhibitor L-N6-(1-iminoethyl) lysine (L-NIL). The results obtained indicate that PL was >30-fold potent than L-NIL in inhibiting NO production with an IC50 of 0.39µM. Our immunofluorescence study confirmed the ability of PL to significantly inhibit iNOS expression in the activated microglia. Furthermore, the extracellular microglial pro-inflammatory cytokine expression in the presence of 2µM of PL was detected, quantified, and validated using cytokine antibody protein arrays and quantitative ELISA. The results obtained showed that PL significantly downregulated the expression of many cytokines including IL-1α, G-CSF, IL-12 p40/p70, MCP-5, MCP-1, and IL-6. In conclusion, PL potency in attenuating multiple pro-inflammatory agents indicates its potential to be used for neurodegenerative diseases.

Natural Products Screening for the Identification of Selective Monoamine Oxidase-B Inhibitors.

AIMS: Monoamine oxidase-B inhibitors (MAO-BIs) are used for the initial therapy of Parkinson's disease. Also, MAO-BIs have shown to be effective neuroprotective agents in several neurodegenerative diseases. However, some concerns exist regarding the long-term use of these compounds. Meanwhile, natural compounds showed potential MAO-B selective inhibitions. To date, few selective natural MAO-BIs have been identified. Therefore, the current study is designed to identify plants with potent and specific MAO-B inhibition. STUDY DESIGN: In this work, we utilized high throughput screening to evaluate the different plants ethanolic extract for their effectiveness to inhibit recombinant human (h)MAO-A and hMAO-B and to determine the relative selectivity of the top MAO-BI. METHODOLOGY: Recombinant human isozymes were verified by Western blotting, and the 155 plants were screened. A continuous fluorometric screening assay was performed followed by two separate hMAO-A and hMAO-B microtiter screenings and IC50 determinations for the top extracts. RESULTS: In the screened plants, 9% of the extracts showed more than 1.5-fold relative inhibition of hMAO-B (RIB) and another 9% showed more than 1.5-fold relative inhibition of hMAO-A. The top extracts with the most potent RIBs were Psoralea corylifolia seeds, Phellodendron amurense bark, Glycyrrhiza uralensis roots, and Ferula assafoetida roots, with the highest RIB of 5.9-fold. Furthermore, extensive maceration of the promising extracts led to increase inhibitory effects with a preserved RIB as confirmed with luminescence assay. The top four extracts hMAO-BIs were equally potent (IC50= 1.3 to 3.8 µg/mL) with highly significant relative selectivities to inhibit hMAO-B (4.1- to 13.4-fold). CONCLUSION: The obtained results indicate that Psoralea corylifolia seeds, Ferula assafoetida, Glycyrrhiza uralensis roots, and Phellodendron amurense ethanolic extracts have selective inhibitions for human MAO-B. Investigating these plant extracts as natural resources for novel selective MAO-BIs may lead to the development of molecules that can be used in the therapeutic management of neurodegenerative diseases including Parkinson's disease.

Evaluation of the Isoflavone Genistein as Reversible Human Monoamine Oxidase-A and -B Inhibitor.

Monoamine oxidases inhibitors (MAOIs) are effective therapeutic drugs for managing Parkinson's disease (PD) and depression. However, their irreversibility may lead to rare but serious side effects. As finding safer and reversible MAOIs is our target, we characterized the recombinant human (h) MAO-A and MAO-B inhibition potentials of two common natural isoflavones, genistein (GST) and daidzein (DZ) using luminescence assay. The results obtained showed that DZ exhibits partial to no inhibition of the isozymes examined while GST inhibited hMAO-B (IC50 of 6.81 µM), and its hMAO-A inhibition was more potent than the standard deprenyl. Furthermore, the reversibility, mode of inhibition kinetics, and tyramine oxidation of GST were examined. GST was a time-independent reversible and competitive hMAO-A and hMAO-B inhibitor with a lower K i of hMAO-B (1.45 µM) than hMAO-A (4.31 µM). GST also inhibited hMAO-B tyramine oxidation and hydrogen peroxide production more than hMAO-A. Docking studies conducted indicated that the GST reversibility and hMAO-B selectivity of inhibition may relate to C5-OH effects on its orientation and its interactions with the threonine 201 residue of the active site. It was concluded from this study that the natural product GST has competitive and reversible MAOs inhibitions and may be recommended for further investigations as a useful therapeutic agent for Parkinson's disease.

Evaluation of the Inhibitory Effects of Bavachinin and Bavachin on Human Monoamine Oxidases A and B.

Monoamine oxidase B inhibitors (MAO-BIs) are used in the early management of Parkinson's disease (PD). Long-term suspected side effects of MAO-B classical inhibitors established the need for safer alternative therapeutic agents. In our study, the flavanone bavachinin (BNN) and its analog bavachin (BVN) found in the seeds of Psoralea corylifolia L. ethanolic extract (PCSEE) were investigated for their human MAO-A and MAO-B (hMAO-A and hMAO-B) inhibition. Both PCSEE and BNN effectively reduced hMAO-B activity more than hMAO-A while BVN had activating effects. BNN showed selective hMAO-B inhibition (IC50 ~ 8.82 µM) more than hMAO-A (IC502009;~ 189.28 µM). BNN in the crude extract was determined by HPLC, also validated by TLC showing a yield of 0.21% PCSEE dry weight. BNN competitively inhibited hMAO-A and hMAO-B, with a lower hMAO-B K i than hMAO-A K i by 10.33-fold, and reduced hMAO-B K m /V max efficiency ratio to be comparable to the standard selegiline. Molecular docking examination of BNN and BVN predicted an indirect role of BNN C7-methoxy group for its higher affinity, selectivity, and reversibility as an MAO-BI. These findings suggest that BNN, which is known to be a potent PPAR-γ agonist, is a selective and competitive hMAO-B inhibitor and could be used in the management of PD.