Effects In Triple-negative Breast Cancer Cells Research Articles

Inhibition of Cancer Stem-like Cells by Curcumin and Other Polyphenol Derivatives in MDA-MB-231 TNBC Cells.

Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancers and is highly aggressive. Despite an initial positive response to chemotherapy, most patients experience rapid disease progression leading to relapse and metastasis. This is attributed to the presence of breast cancer stem cells (BCSCs) within the tumor, which are characterized by self-renewal, pluripotency, and resistance mechanisms. Targeting BCSCs has become critical as conventional therapies fail to eradicate them due to a lack of specific targets. Curcumin, a polyphenol derived from turmeric (Curcuma longa), exhibits anticancer effects against breast cancer cells and BCSCs. The use of curcumin derivatives has been suggested as an approach to overcome the bioavailability and solubility problems of curcumin in humans, thereby increasing its anticancer effects. The aim of this study was to evaluate the cellular and molecular effects of six synthetic compounds derived from the natural polyphenol epigallocatechin gallate (EGCG) (TL1, TL2) and curcumin derivatives (TL3, TL4, TL5, and TL6) on a TNBC mesenchymal stem-like cell line. The activity of the compounds against BCSCs was also determined by a mammosphere inhibition assay and studying different BCSC markers by Western blotting. Finally, a drug combination assay was performed with the most promising compounds to evaluate their potential synergistic effects with the chemotherapeutic agents doxorubicin, cisplatin, and paclitaxel. The results showed that compounds exhibited specific cytotoxicity against the TNBC cell line and BCSCs. Interestingly, the combination of the curcumin derivative TL3 with doxorubicin and cisplatin displayed a synergistic effect in TNBC cells.

Exploring the regulatory role of FBXL19-AS1 in triple-negative breast cancer through the miR-378a-3p/OTUB2 axis.

The regulatory potential of long noncoding RNA (lncRNA) FBXL19-AS1 has been highlighted in various cancers, but its effect on triple-negative breast cancer (TNBC) remains unclear. Here, we aimed to elucidate the role of FBXL19-AS1 in TNBC and its underlying mechanism. RT-qPCR was employed to detect the expressions of FBXL19-AS1 and miR-378a-3p in tissues and cells. Immunohistochemical staining and western blot were utilized to detect the expression levels of proteins. Cell activities were detected using flow cytometry, CCK-8, and transwell assay. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were deployed to investigate interactions of different molecules. Protein-protein interaction (PPI) network, gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG) pathways were used to analyze the downstream pathway. In vivo xenograft model was conducted to detect the effect of FBXL19-AS1 on tumor growth. FBXL19-AS1 was overexpressed in TNBC tissues and cell lines compared with counterparts. FBXL19-AS1 knockdown suppressed TNBC cell activities, whereas its overexpression exhibited the opposite effect. Mechanistically, FBXL19-AS1 was found to interact with miR-378a-3p. Further analysis revealed that miR-378a-3p exerted tumor-suppressive effects in TNBC cells. Additionally, miR-378a-3p targeted and downregulated the expression of ubiquitin aldehyde binding 2 (OTUB2), a deubiquitinase associated with TNBC progression. In vivo experiments substantiated the inhibitory effects of FBXL19-AS1 knockdown on TNBC tumorigenesis, and a miR-378a-3p inhibitor partially rescued these effects. The downstream pathway of the miR-378a-3p/OTUB2 axis was explored, revealing connections with proteins involved in modifying other proteins, removing ubiquitin molecules, and influencing signaling pathways, including the Hippo signaling pathway. Western blot analysis confirmed changes in YAP and TAZ expression levels, indicating a potential regulatory network. In summary, FBXL19-AS1 promotes exacerbation in TNBC by suppressing miR-378a-3p, leading to increased OTUB2 expression. The downstream mechanism may be related to the Hippo signaling pathway. These findings propose potential therapeutic targets for TNBC treatment.

Withaferin A alters the expression of microRNAs 146a-5p and 34a-5p and associated hub genes in MDA-MB-231 cells.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer. Due to the absence of obvious therapeutic targets, microRNAs (miRNAs) provide possible hope to treat TNBC. Withaferin A (WA), a steroidal lactone, possesses potential anticancer activity with lesser side effects. The present study identifies hub genes (CDKN3, TRAF6, CCND1, JAK1, MET, AXIN2, JAG1, VEGFA, BRCA1, E2F3, WNT1, CDK6, KRAS, MYB, MYCN, TGFβR2, NOTCH1, SIRT1, MYCN, NOTCH2, WNT3A) from the list of predicted targets of the differentially expressed miRNAs (DEMs) in WA-treated MDA-MB-231 cells using in silico protein-protein interaction network analysis. CCND1, CDK6, and TRAF6 hub genes were predicted as targets of miR-34a-5p and miR-146a-5p, respectively. The study found the lower expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells, and further, it was observed that WA treatment effectively restored the lost expression of miR-34a-5p and miR-146a-5p in MDA-MB-231 cells. An anti-correlation expression pattern was found among the miR-34a-5p and miR-146a-5p and the respective target hub genes in WA-treated TNBC cells. In conclusion, WA might exert anti-cancer effect in TNBC cells by inducing miR-34a-5p and miR-146a-5p expressions and decreasing CCND1, CDK6, and TARF6 target hub genes in TNBC cells.

Toxoflavin analog D43 exerts antiproliferative effects on breast cancer by inducing ROS-mediated apoptosis and DNA damage

Triple-negative breast cancer (TNBC) is regarded as the deadliest subtype of breast cancer because of its high heterogeneity, aggressiveness, and limited treatment options. Toxoflavin has been reported to possess antitumor activity. In this study, a series of toxoflavin analogs were synthesized, among which D43 displayed a significant dose-dependent inhibitory effect on the proliferation of TNBC cells (MDA-MB-231 and HCC1806). Additionally, D43 inhibited DNA synthesis in TNBC cells, leading to cell cycle arrest at the G2/M phase. Furthermore, D43 consistently promoted intracellular ROS generation, induced DNA damage, and resulted in apoptosis in TNBC cells. These effects could be reversed by N-acetylcysteine. Moreover, D43 significantly inhibited the growth of breast cancer patient-derived organoids and xenografts with a favorable biosafety profile. In conclusion, D43 is a potent anticancer agent that elicits significant antiproliferation, oxidative stress, apoptosis, and DNA damage effects in TNBC cells, and D43 holds promise as a potential candidate for the treatment of TNBC.

Combination Curcuma longa and Phyllanthus niruri Extract Potentiate Antiproliferative in Triple Negative Breast Cancer MDAMB-231 Cells.

Triple negative breast cancer cells (TNBC) are a small part of cancer-inducing cells in breast cancer, which are characterized by high metastatic and self-renewal. Self-renewal has the ability to renew itself and loses control of proliferation. Curcuma longa extract (CL) and Phyllanthus niruri extract (PN) known to have anti-proliferative effects on cancer cells. However, the effects of combination CL and PN on TNBC proliferation still unclear. This study aimed to evaluate the antiproliferative effects of the combination CL and PN on TNBC MDAMB-231 and attempted to elucidate the underlying molecular mechanisms. The dried rhizomes of Curcuma longa and the herbs of Phyllanthus niruri were macerated with ethanol for 72 h.The antiproliferative and synergistic effects of combination CL and PN were investigated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Combination index values were calculated using CompuSyn (ComboSyn, Inc, Paramus, NJ). The cell cycle and apoptosis assay were determined by propidium iodide (PI) and PI-AnnexinV assay under flow cytometer, respectively. The intracellular ROS levels were evaluated using 2',7'-Dichlorodihydrofluorescein diacetate (DCFDA) assay. The mRNA expressions of proliferation-related genes in the cells were determined using bioinformatic assay. The CL and PN single treatment caused a potent and dose-dependent decrease in the percentage of viable cells with IC50 value of 13 μg/mL and 45 μg/mL for 24 h, respectively. The combination index values of the different combinations ranged from 0.08 - 0.90, indicating slightly strong to very strong synergistic effects. The combination of CL and PN also remarkably induced the S- and G2/M-phases cell cycle arrest that leading to apoptosis induction. Furthermore, the combination of CL and PN treatment induced the intracellular reactive oxygen species (ROS) levels. Mechanistically, the AKT1, EP300, STAT3 and EGFR signaling as potential targets of combination CL and PN in antiproliferation and antimetastatic of TNBC. The combination of CL and PN exerted promising antiproliferative effects in TNBC. Therefore, CL and PN may be considered a potential source for the development of potent anticancer drugs for breast cancer treatment.

Finding New Molecular Targets of Two Copper(II)-Hydrazone Complexes on Triple-Negative Breast Cancer Cells Using Mass-Spectrometry-Based Quantitative Proteomics.

Breast cancer is the most common cancer in women, with a high incidence estimated to reach 2.3 million by 2030. Triple-Negative Breast Cancer (TNBC) is the greatest invasive class of breast cancer with a poor prognosis, due to the side-effects exerted by the chemotherapy used and the low effectivity of novel treatments. In this sense, copper compounds have shown to be potentially effective as antitumor agents, attracting increasing interest as alternatives to the usually employed platinum-derived drugs. Therefore, the aim of this work is to identify differentially expressed proteins in MDA-MB-231 cells exposed to two copper(II)-hydrazone complexes using label-free quantitative proteomics and functional bioinformatics strategies to identify the molecular mechanisms through which these copper complexes exert their antitumoral effect in TNBC cells. Both copper complexes increased proteins involved in endoplasmic reticulum stress and unfolded protein response, as well as the downregulation of proteins related to DNA replication and repair. One of the most relevant anticancer mechanisms of action found for CuHL1 and CuHL2 was the down-regulation of gain-of-function-mutant p53. Moreover, we found a novel and interesting effect for a copper metallodrug, which was the down-regulation of proteins related to lipid synthesis and metabolism that could lead to a beneficial decrease in lipid levels.

Vitamin K2 enhances the tumor suppressive effects of 1,25(OH)2D3 in triple negative breast cancer cells

K vitamins are well known as essential cofactors for hepatic γ-carboxylation of coagulation factors, but their potential role in chronic diseases including cancer is understudied. K2, the most abundant form of vitamin K in tissues, exerts anti-cancer effects via diverse mechanisms which are not completely understood. Our studies were prompted by previous work demonstrating that the K2 precursor menadione synergized with 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to inhibit growth of MCF7 luminal breast cancer cells. Here we assessed whether K2 modified the anti-cancer effects of 1,25(OH)2D3 in triple negative breast cancer (TNBC) cell models. We examined the independent and combined effects of these vitamins on morphology, cell viability, mammosphere formation, cell cycle, apoptosis and protein expression in three TNBC cell models (MDA-MB-453, SUM159PT, Hs578T). We found that all three TNBC cell lines expressed low levels of the vitamin D receptor (VDR) and were modestly growth inhibited by 1,25(OH)2D3 in association with cell cycle arrest in G0/G1. Induction of differentiated morphology by 1,25(OH)2D3 was observed in two of the cell lines (MDA-MB-453, Hs578T). Treatment with K2 alone reduced viability of MDA-MB-453 and SUM159PT cells but not Hs578T cells. Co-treatment with 1,25(OH)2D3 and K2 significantly reduced viable cell number relative to either treatment alone in Hs578T and SUM159PT cells. The combination treatment induced G0/G1 arrest in MDA-MB-453 cells, Hs578T and SUM159PT cells. Combination treatment altered mammosphere size and morphology in a cell specific manner. Of particular interest, treatment with K2 increased VDR expression in SUM159PT cells suggesting that the synergistic effects in these cells may be secondary to increased sensitivity to 1,25(OH)2D3. The phenotypic effects of K2 in TNBC cells did not correlate with γ-carboxylation suggesting non-canonical actions. In summary, 1,25(OH)2D3 and K2 exert tumor suppressive effects in TNBC cells, inducing cell cycle arrest leading to differentiation and/or apoptosis depending on the specific cell line. Further mechanistic studies to clarify common and unique targets of these two fat soluble vitamins in TNBC are warranted.

Abstract 2539: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis

Abstract TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting that there are unknown modulatory mechanisms responsible for its lack of TRAIL activity in vivo. We hypothesized that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu, modulating the therapeutic efficacy. To investigate the hypothesis, we performed RNAseq analysis of MDA-MB231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. TRAIL treatment of the TNBC significantly induced expression of a number of cytokines, such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL11, IL6, which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by Death Receptor 5 and Caspase-8 protein, but not Caspase-8 enzymatic activity. Gene Set Enrichment Analysis of the RNAseq indicated that NFKB pathway-mediated TNF-alpha signaling was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL. However, siRNA knockdown experiments indicated that the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs’ (1, 2, 3, 8) and IL6 significantly increased neutrophil chemotaxis in a NFKB2-dependent manner. Moreover, neutrophils pre-incubated with supernatants from TRAIL-treated TNBC significantly inhibited its cytotoxic effect in TNBCs in a NFKB2-dependent pathway. Further RNAseq and RT-PCR of neutrophils incubated with by either TRAIL or supernatant of MDA-MB231 cells treated with TRAIL revealed significant enrichment of inflammatory pathway-related genes as well as increased expression of immune modulating cytokines. These results suggested that TRAIL exerts a pro-inflammatory role towards immune cells in tumor microenvironment. CODEX (CO-Detection by indEXing) analysis of in vivo TNBC xenografts from mice treated with the TRAIL agonist MEDI3039 confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Using TNBC organoids and humanized mice models, the changes in tumor immune environment caused by TRAIL are currently under investigation. Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production that affects neutrophil functions, leading to modulation of the immune response in TNBC. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa A. Ridnour, David A. Wink, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2539.

Combination of androgen receptor inhibitor enzalutamide with the CDK4/6 inhibitor ribociclib in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) that currently lacks specific therapy options. Thus, chemotherapy continues to be the primary treatment, and developing novel targets is a top clinical focus. The androgen receptor (AR) has emerged as a therapeutic target in a subtype of TNBC, with substantial clinical benefits shown in various clinical studies. Numerous studies have shown that cancer is associated with changes in components of the cell cycle machinery. Although cell cycle cyclin-dependent kinase (CDK) 4/6 inhibitors are successful in the treatment of ER-positive BC, they are not helpful in the treatment of patients with TNBC. We investigated the possibility of combining CDK4/6 inhibitor(ribociclib) with AR inhibitor(enzalutamide) in the AR-positive TNBC cell line. Ribociclib showed an inhibitory effect in TNBC cells. Additionally, we found that enzalutamide reduced cell migration/invasion, clonogenic capacity, cell cycle progression, and cell growth in AR-positive cells. Enzalutamide therapy could increase the cytostatic impact of ribociclib in AR+ TNBC cells. Furthermore, dual inhibition of AR and CDK4/6 demonstrated synergy in an AR+ TNBC model compared to each treatment alone.

Circular RNA dehydrodolichyl diphosphate synthase facilitated triple-negative breast cancer progression via miR-362-3p/DDX5 axis.

Triple-negative breast cancer (TNBC) is a common hypotype of breast cancer. Circular RNAs (circRNAs) are burgeoning serve as vital controllers in numerous tumors. Nevertheless, the expression and regulatory mode of circRNAs in TNBC are still indistinct. This paper aimed to reveal the function and molecular mechanism of circular RNA dehydrodolichyl diphosphate synthase (circDHDDS) in TNBC. The contents of circDHDDS, DHDDS mRNA, microRNA-362-3p (miR-362-3p) and DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 (DDX5) were indicated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were executed to assess cell proliferation. The flow cytometry assay was utilized to detect cell apoptosis. The transwell assay and tube formation assay were applied to measure cell migration, invasion and angiogenesis. The targeted relationships of miR-362-3p and circDHDDS or DDX5 were forecasted and detected by dual-luciferase reporter assay. The in vivo test was implemented to confirm the effect of circDHDDS. The contents of circDHDDS and DDX5 were increased, and miR-362-3p level was decreased in TNBC. CircDHDDS deficiency reserved cell proliferation, migration, invasion and angiogenesis, while facilitated cell apoptosis in TNBC cells. Furthermore, miR-362-3p was validated to exert a tumor repressive effect in TNBC cells by suppressing DDX5. Moreover, DDX5 could regulate the development of TNBC. The experimental data exposed that levels of miR-362-3p presented noteworthy negative correlation with circDHDDS and DDX5, while circDHDDS and DDX5 exhibited significant positive correlation. In mechanism, circDHDDS bound to miR-362-3p to modulate DDX5 expression. In addition, circDHDDS knock-down also attenuated tumor growth. CircDHDDS expedited TNBC by swelling DDX5 via adapting miR-362-3p.

ZNF703 promotes triple-negative breast cancer cells through cell-cycle signaling and associated with poor prognosis

BackgroundThe oncogenic drivers of triple-negative breast cancer (TNBC), which is characterized by worst prognosis compared with other subtypes, are poorly understood. Although next-generation sequencing technology has facilitated identifying potential targets, few of the findings have been translated into daily clinical practice. The present study is aimed to explore ZNF703 (Zinc finger 703) function and its underlying mechanism in TNBC.MethodsZNF703 expressions in tissue microarray were retrospectively examined by immunohistochemistry. The cell proliferation by SRB assay and colony formation assay, as well as cell cycle distribution by flow cytometry were assessed. The protein levels associated with possible underlying molecular mechanisms were evaluated by western blotting. Kaplan-Meier analysis was used to plot survival analysis.ResultsOur data suggest that ZNF703 expressed in 34.2% of triple-negative human breast tumors by immunohistochemistry. In vitro, ZNF703 knockdown had potent inhibitory effects on TNBC cell proliferation and cell cycle, with cyclin D1, CDK4, CDK6, and E2F1 downregulated, while Rb1 upregulated. Moreover, Kaplan-Meier analysis showed that high mRNA expression of ZNF703 was correlated to worse overall survival (HR for high expression was 3.04; 95% CI, 1.22 to 7.57, P = 0.017).ConclusionsTaken together, the results identified that targeting ZNF703 contributed to the anti-proliferative effects in TNBC cells, due to induced G1-phase arrest. This study is the first to identify ZNF703 as a potentially important protein that is involved in TNBC progression.

Anticancer and chemosensitization effects of cannabidiol in 2D and 3D cultures of TNBC: involvement of GADD45α, integrin-α5, -β5, -β1, and autophagy.

To date, promising therapy for triple negative breast cancer (TNBC) remains a serious concern clinically because of poor prognosis, resistance, and recurrence. Herein, anti-cancer potential of synthetic cannabidiol (CBD; Purisys, GA; GMP grade) was explored either alone or as a chemosensitizer followed by post-treatment with doxorubicin (DOX) in TNBC (i.e., MDA-MB-231 and MDA-MB-468) cells. In comparison to 2D cultures, CBD showed greater IC50 values in 3D (LDP2 hydrogel based) cultures of MDA-MB-231 (6.26-fold higher) and MDA-MB-468 (10.22-fold higher) cells. Next-generation RNA sequencing revealed GADD45A, GADD45G, FASN, LOX, and integrin (i.e., -α5, -β5) genes to be novelly altered by CBD in MDA-MB-231 cells. CIM-16 plate-based migration assay and western blotting disclosed that CBD induces anti-migratory effects in TNBC cells by decreasing fibronectin, vimentin, and integrins-α5, -β5, and -β1. Western blotting, RT-qPCR, and immunocytochemistry revealed that CBD inhibited autophagy (decreased Beclin1, and ATG-5, -7, and -16) of TNBC cells. CBD pre-treatment increased DOX sensitivity in TNBC cells. CBD pre-treatment accompanied by DOX treatment decreased LOX and integrin-α5, and increased caspase 9 protein respectively in MDA-MB-468 cells.

Combinative treatment of Curdione and docetaxel triggers reactive oxygen species (ROS)-mediated intrinsic apoptosis of triple-negative breast cancer cells

ABSTRACT Traditional Chinese medicine Curcuma zedoary has been used for treating various diseases and cancers. However, the therapeutic effect of Curdione, one of its major components in triple negative breast cancer (TNBC) is still obscure. This study is aimed to explore whether combination of Curdione and docetaxel (DTX) could strengthen the DTX-induced pro-apoptotic effects in TNBC cells and identify its involved signaling pathways. In this study, combination of Curdione and DTX intensified the inhibited MDA-MB-468 cell proliferation and increased cell apoptosis caused by DTX treatment alone. Moreover, the combinative treatment of Curdione and DTX synergistically potentiated DTX-induced cell apoptosis by triggering reactive oxygen species (ROS) generation. Co-treatment with NAC (ROS inhibitor) could mostly block the effects induced by combination of Curdione and DTX. SB203580 (p38 inhibitor) or SC-79 (Akt activator) could partly reverse the effects induced by co-treatment, indicating that mitogen-actived protein kinases (MAPKs) and the phosphatidylinositol 3-kinases (PI3K) /Akt signaling pathway were involved in the co-treatment induced ROS-mediated cell apoptosis. To sum up, combination of Curdione and DTX enhanced the chemotherapeutic efficacy on MDA-MB-468 cells by triggering ROS-mediated cell apoptosis via MAPKs and PI3K/Akt signaling pathways. Curdione combined with DTX might have potentials application as the therapeutic strategy for TNBC. Abbreviations TNBC: triple negative breast cancer; ROS: reactive oxygen species; NAC: N-acetyl-L-cysteine; DTX: docetaxel; MAPKs: mitogen-actived protein kinases; PI3K/Akt: phosphatidylinositol 3-kinases (PI3K) /Akt; NF-Κb: the nuclear factor κB (NF-κB)

Combination inhibition of triple-negative breast cancer cell growth with CD36 siRNA-loaded DNA nanoprism and genistein

Currently, a single treatment is less effective for triple-negative breast cancer (TNBC) therapy. Additionally, there are some limitations to the use of siRNA alone as a new method to treat breast cancer, such as its effective delivery into cells. In this study, we proposed a strategy that combines a siRNA-loaded DNA nanostructure and genistein for TNBC therapy. Both CD36 siRNA-loaded self-assembled DNA nanoprisms (NP-siCD36) and genistein knocked down CD36, resulting in enhanced anticancer efficacy through phosphorylation of the p38 MAPK pathway. In vitro studies showed that combination therapy could effectively enhance cell apoptosis and reduce cell proliferation, achieving an antitumor effect in TNBC cells. The current study suggests that NP-siCD36 combined with genistein might be a promising strategy for breast cancer and treatment.

Birinapant Enhances Gemcitabine's Antitumor Efficacy in Triple-Negative Breast Cancer by Inducing Intrinsic Pathway-Dependent Apoptosis.

Triple-negative breast cancer (TNBC) is the most aggressive subgroup of breast cancer, and patients with TNBC have few therapeutic options. Apoptosis resistance is a hallmark of human cancer, and apoptosis regulators have been targeted for drug development for cancer treatment. One class of apoptosis regulators is the inhibitors of apoptosis proteins (IAPs). Dysregulated IAP expression has been reported in many cancers, including breast cancer, and has been shown to be responsible for resistance to chemotherapy. Therefore, IAPs have become attractive molecular targets for cancer treatment. Here, we first investigated the antitumor efficacy of birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspases (SMACs), in TNBC. We found that birinapant as a single agent has differential antiproliferation effects in TNBC cells. We next assessed whether birinapant has a synergistic effect with commonly used anticancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), erlotinib (EGFR inhibitor), and gemcitabine, in TNBC. Among these tested drugs, gemcitabine showed a strong synergistic effect with birinapant. Birinapant significantly enhanced the antitumor activity of gemcitabine in TNBC both in vitro and in xenograft mouse models through activation of the intrinsic apoptosis pathway via degradation of cIAP2 and XIAP, leading to apoptotic cell death. Our findings demonstrate the therapeutic potential of birinapant to enhance the antitumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins.

Effects of Wild Yam Root (Dioscorea villosa) Extract on the Gene Expression Profile of Triple-negative Breast Cancer Cells

Wild yam extract [Dioscorea villosa, (WYE)] is consistently lethal at low IC50s across diverse cancer-lines in vitro. Unlike traditional anti-cancer botanicals, WYE contains detergent saponins which reduce oil-water interfacial tensions causing disintegration of lipid membranes and causing cell lysis, creating an interfering variable. Here, we evaluate WYE at sub-lethal concentrations in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Quantification of saponins, membrane potential, lytic death and sub-lethal WYE changes in whole transcriptomic (WT) mRNA, miRNAs and biological parameters were evaluated. WYE caused 346 differentially expressed genes (DEGs) out of 48,226 transcripts tested; where up-regulated DEGS reflect immune stimulation, TNF signaling, COX2, cytokine release and cholesterol/steroid biosynthesis. Down-regulated DEGs reflect losses in cell division cycle (CDC), cyclins (CCN), cyclin-dependent kinases (CDKs), centromere proteins (CENP), kinesin family members (KIFs) and polo-like kinases (PLKs), which were in alignment with biological studies. Sub-lethal concentrations of WYE appear to evoke pro-inflammatory, steroid biosynthetic and cytostatic effects in TNBC cells.

S-Adenosylmethionine Inhibits Cell Growth and Migration of Triple Negative Breast Cancer Cells through Upregulating MiRNA-34c and MiRNA-449a.

Triple-negative breast cancer (TNBC) is one of the most common malignancies worldwide and shows maximum invasiveness and a high risk of metastasis. Recently, many natural compounds have been highlighted as a valuable source of new and less toxic drugs to enhance breast cancer therapy. Among them, S-adenosyl-L-methionine (AdoMet) has emerged as a promising anti-cancer agent. MicroRNA (miRNA or miR)-based gene therapy provides an interesting antitumor approach to integrated cancer therapy. In this study, we evaluated AdoMet-induced modulation of miRNA-34c and miRNA-449a expression in MDA-MB-231 and MDA-MB-468 TNBC cells. We demonstrated that AdoMet upregulates miR-34c and miR-449a expression in both cell lines. We found that the combination of AdoMet with miR-34c or miR-449a mimic strongly potentiated the pro-apoptotic effect of the sulfonium compound by a caspase-dependent mechanism. For the first time, by video time-lapse microscopy, we showed that AdoMet inhibited the in vitro migration of MDA-MB-231 and MDA-MB-468 cells and that the combination with miR-34c or miR-449a mimic strengthened the effect of the sulfonium compound through the modulation of β-catenin and Small Mother Against Decapentaplegic (SMAD) signaling pathways. Our results furnished the first evidence that AdoMet exerts its antitumor effects in TNBC cells through upregulating the expression of miR-34c and miR-449a.

Metformin and oxyphotodynamic therapy as a novel treatment approach for triple-negative breast cancer.

BackgroundTreatment for triple-negative breast cancer (TNBC) remains a significant challenge due to a lack of targeted therapies. While photodynamic therapy (PDT) has been utilized as a treatment approach for several types of cancer, oxyphotodynamic therapy (OPDT) is a novel method that improves treatment efficacy by increasing local oxygen concentration. Metformin (MET) has been demonstrated utility as an anti-tumor agent by acting through the adenosine monophosphate-activated protein kinase (AMPK) pathway. We hypothesized that MET in combination with heme, a byproduct of 5-aminolevulinic acid (ALA), may increase cytotoxicity for cancer treatment. This study aimed to investigate the synergistic effect of MET and ALA with PDT or OPDT on TNBC tumorigenic cells.MethodsThe treatment efficacy and phototoxicity of PDT or OPDT were determined using a cell viability assay. PDT/OPDT experiments were carried out in nine groups based on different combinations and concentrations of ALA and/or MET. To calculate the synergistic effect by compuSyn soft for different groups, cells were incubated with ALA and/or MET at the following concentrations (0, 0.25, 0.5,1, 2, 4, 8, 16, 24, and 32 mM). The fluorescence of ALA-induced protoporphyrin IX (PpIX) and MitoTracker Green were observed under a confocal microscope.ResultsThe optimized therapeutic concentration ratio of ALA and MET was determined to be 1:1. The inhibition of cancer growth (IC50) for each group was 14.03, 10.62, 7.71, 18.27, 22.09, 23.96, 4.57, 10.20, and 8.18 mM, respectively. The combination index (CI) values (fa =0.5) of the last three combination groups (groups 7, 8, and 9) were 0.44, 1.70, and 1.47, respectively. PpIX fluorescence intensity of group 9 (ALA-MET-OPDT group) remained the highest among all groups, indicating an enhanced therapeutic effect.ConclusionsThis study introduces OPDT as a novel anti-tumor therapy for TNBC. Furthermore, the combined use of ALA and MET had a synergistic anti-tumor effect in TNBC cells when combined with OPDT.

Abstract 548: Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis

Abstract Background: Apoptosis resistance is a hallmark of cancer, and apoptosis regulators have been targeted for cancer treatment. A class of such regulators is inhibitor of apoptosis proteins (IAPs), which suppress caspase activity by inducing degradation of active caspases or blocking interaction of caspases with their substrates. Dysregulated IAP expression has been reported in many cancers, including breast cancer, and is involved in chemoresistance. IAPs have therefore become attractive targets for cancer treatment. Birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspase (SMAC, an endogenous antagonist of IAPs), has been shown to effectively activate apoptotic signaling by targeting IAPs and has potential application for treatment of multiple cancers. In addition, birinapant showed synergistic anti-tumor effects with several widely used chemotherapeutic agents in various cancers. Here, we assessed whether birinapant synergizes with commonly used anti-cancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), everolimus (mTOR inhibitor), and gemcitabine, in triple-negative breast cancer (TNBC). Among these drugs, gemcitabine strongly synergized with birinapant (combination index [CI] <1). We hypothesize that birinapant enhances anti-tumor efficacy of gemcitabine in TNBC by inducing intrinsic pathway-dependent apoptosis. Methods: The in vitro anti-tumor efficacy of birinapant, gemcitabine, and their combination was determined using CellTiter-Blue viability and soft agar colony formation assays. The CI (CalcuSyn software) was used to quantify the synergy. The effects of drug treatment on cell cycle distribution were analyzed by flow cytometry; the effects on apoptosis were assessed by annexin V-PE and 7-AAD staining, and induction of apoptosis was further confirmed by pan-caspase inhibitor treatment; and the effects on IAP degradation were examined by Western blotting. The in vivo antitumor efficacy was determined using TNBC xenograft mouse models. Results: Birinapant as a single agent had varying anti-proliferation effects in TNBC cells (IC50 = 0.21 to >20 µM). It had no synergistic effects with entinostat, cisplatin, paclitaxel, voxtalisib, dasatinib, or everolimus but strongly synergized with gemcitabine (CI <1 µM) in tested TNBC cells. Birinapant and gemcitabine synergistically inhibited the growth of TNBC cells by activating the intrinsic apoptosis pathway, accomplished by inducing degradation of cIAP2 and XIAP, which led to apoptotic cell death. Furthermore, birinapant significantly enhanced anti-tumor effectiveness of gemcitabine in TNBC xenograft mouse models (n = 10; P < 0.05) by inducing apoptosis. Conclusion: Our findings demonstrate the therapeutic potential of birinapant for enhancing the anti-tumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins. Citation Format: Xuemei Xie, Jangsoon Lee, Troy Pearson, Alexander Y. Lu, Debu Tripathy, Gayathri R. Devi, Chandra Bartholomeusz, Naoto T. Ueno. Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 548.

Curcumol enhances the anti-tumor effects of metformin via suppressing epithelial-mesenchymal transition in triple-negative breast cancer.

BackgroundTriple-negative breast cancer (TNBC) is a severe disease with a high mortality rate. Metformin has been found to possess anti-tumor properties. Curcumol, an active ingredient extracted from curcuma, exerts the protective effect in TNBC cells through inducing apoptosis. However, the effects of curcumol combined with metformin on the treatment of TNBC have yet to be fully established.MethodsTNBC cells MDA-MB-231 and MDA-MB-468 cells were used in the study. TNBC cells were treated with curcumol and metformin alone or treated with curcumol combined with metformin. Cell viability was determined using Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The levels of proteins were measured using Western blot. Wound healing assay and Transwell invasion assays were used to determine cell migration and invasion ability, respectively. A xenograft model was established to investigate the tumor growth ability. Immunohistochemistry was performed to determine the expression of Ki-67 and Vascular endothelial growth factor (VEGF).ResultsIn the study, the administration of curcumol alone had no significant effects on the TNBC cells. However, the anti-proliferation, anti-metastasis, and anti-epithelial-mesenchymal transition (EMT) effects of metformin were enhanced by the addition of curcumol. Further, curcumol reversed TNBC cell proliferation, migration, invasion, and EMT induced by rucaparib, and enhanced the effect of metformin on rucaparib-induced TNBC cells. The combination of curcumol and metformin also suppressed tumor growth, EMT marker expression, and the activation of Wnt2/β-Catenin signaling during in vivo experiments.ConclusionsThe combination of curcumol and metformin enhances the anti-tumor effects of metformin on TNBC via inhibiting EMT. Curcumol combined with metformin may hold promise as a therapeutic strategy for TNBC.