Breast Cancer Cell Death Research Articles

Variations in the Anticancer Activity of Free and Bound Phenolics of Finger Millet (Eleusine coracana (L) Gaertn; Variety KMR-301) Seeds

• Procyanidin B1, 3-O-methyl quercetin and Epigallocatechin are the major phenolics of FM-FP. • FM-BP is rich in Protocatecheuic acid, Ferulic acid and Vanillic acid. • FM-FP exhibited better cytotoxicity against breast cancer cell lines compared to FM-BP. • FM-FP induced fragmentation of DNA and apoptotic cell death in breast cancer cells. Finger millet (FM, Eleusine coracana (L) Gaertn; also known as “ragi” in Kannada language) is one of the widely used millets especially in south India and parts of Africa. FM is consumed in various forms that include porridge, mudde (ball), dosa (a pan cake), idly (a savory rice cake) and biscuits/cookies. FM is also used in the form of malted drink known as “malted ragi or ragi malt”. FM is a rich source of dietary fiber, tannins, phenolic compounds and calcium. Prior studies have shown health beneficial properties such as anti-diabetic and anti-inflammatory effects of FM, and ascribed those properties to the presence of dietary fiber and phenolic compounds. But, very minimal information is available pertaining to the effect of FM phenolic compounds on cell lines representing carcinomas of colon and rectum; and breast. Hence, we have studied the effect of FM phenolic compounds on colorectal carcinoma and breast cancer cell lines. Sequentially extracting the seeds by 70% ethanol and 10% alkali generated free (FM-FP) and bound (FM-BP) phenolic compounds respectively. UPLC-QTOF-MS was used to determine the phytochemical composition. Antioxidant potential was determined by ferric reducing antioxidant power assay (FRAP) and radical scavenging activity (DPPH assay). Effect of FM-FP and FM-BP on cellular proliferation was determined by sulforhodamine-B assay. Staining the untreated and treated cells with acridine orange and ethidium bromide followed by analyzing the stained cells using fluorescence microscope yielded key information about impact of extracts on cell death. Effect on cell cycle was determined by staining the cells with DAPI followed by analyzing the stained cells using NC-3000. Analysis of the UPLC-QTOF -MS data showed the presence of phenolics and phenolic acid derivatives, flavonoids and aminoacids in the FM-FP and FM-BP fractions. Both fractions exhibited ferric ion reduction ability and DPPH radical scavenging potential. But, the effect of FM-FP and FM-BP on cell proliferation varied significantly from cell line to cell line. FM-FP exhibited better cytotoxic potential compared to FM-BP when tested against breast cancer cell lines. Cytotoxic FM-FP induced G0/G1 or G2/M arrest in a cell line dependent fashion and increased the fragmentation of DNA leading to accumulation of cells in Sub-G1 phase. In summary, results of our study demonstrated the strength of finger millet free- and bound phenolic compounds for exhibiting antioxidant property, and the potential to modulate the proliferative potential of breast and colorectal cancer cells.

A GSH-depleted platinum(IV) prodrug triggers ferroptotic cell death in breast cancer

Cisplatin is the first-line drug for treatment of various solid tumors including breast cancer due to the broad anti-tumor spectrum and strong anti-tumor effect. However, serious side effects and long-term medication of reduced sensitivity by high GSH in tumor cells have severely restricted its further clinical application. Herein, a GSH-depleted Pt(IV) prodrug (Platin B) based on cisplatin and 4-carboxylphenylboronic acid pinacol ester was prepared to solve the problems. As an excellent GSH scavenger, 4-carboxylphenylboronic acid pinacol ester could be activated by intracellular redox reactions to release quinone methide, thereby amplifying oxidative stress and leading to breast cancer ferroptosis therapy. Interestingly, the consumption of GSH can also reduce cisplatin inactivation, enhance the sensitivity of tumor cells to cisplatin and efficiently induce apoptosis/ferroptosis. This work highlights the use of GSH scavenger for triggering ferroptotic cell death in breast cancer. A GSH-depleted platium(IV) prodrug was successfully synthesized by oxidation and esterification reaction, which exerts an efficient tumor ferroptosis therapy.

PKHB1, a thrombospondin-1 peptide mimic, induces anti-tumor effect through immunogenic cell death induction in breast cancer cells

ABSTRACT Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide. Recent advances in the field of immuno-oncology demonstrate the beneficial immunostimulatory effects of the induction of immunogenic cell death (ICD). ICD increases tumor infiltration by T cells and is associated with improved prognosis in patients affected by triple negative breast cancer (TNBC) with residual disease. The aim of this study was to evaluate the antitumoral effect of PKHB1, a thrombospondin-1 peptide mimic, against breast cancer cells, and the immunogenicity of the cell death induced by PKHB1 in vitro, ex vivo, and in vivo. Our results showed that PKHB1 induces mitochondrial alterations, ROS production, intracellular Ca2+ accumulation, as well calcium-dependent cell death in breast cancer cells, including triple negative subtypes. PKHB1 has antitumor effect in vivo leading to a reduction of tumor volume and weight and promotes intratumoral CD8 + T cell infiltration. Furthermore, in vitro, PKHB1 induces calreticulin (CALR), HSP70, and HSP90 exposure and release of ATP and HMGB1. Additionally, the killed cells obtained after treatment with PKHB1 (PKHB1-KC) induced dendritic cell maturation, and T cell antitumor responses, ex vivo. Moreover, PKHB1-KC in vivo were able to induce an antitumor response against breast cancer cells in a prophylactic application, whereas in a therapeutic setting, PKHB1-KC induced tumor regression; both applications induced a long-term antitumor response. Altogether our data shows that PKHB1, a thrombospondin-1 peptide mimic, has in vivo antitumor effect and induce immune system activation through immunogenic cell death induction in breast cancer cells.

Robust delivery of RIG-I agonists using extracellular vesicles for anti-cancer immunotherapy.

The RIG‐I pathway can be activated by RNA containing 5′ triphosphate, leading to type I interferon release and immune activation. Hence, RIG‐I agonists have been used to induce immune responses against cancer as potential immunotherapy. However, delivery of 5′ triphosphorylated RNA molecules as RIG‐I agonists to tumour cells in vivo is challenging due to the susceptibility of these molecules to degradation. In this study, we demonstrate the use of extracellular vesicles (EVs) from red blood cells (RBCs), which are highly amenable for RNA loading and taken up robustly by cancer cells, for RIG‐I agonist delivery. We evaluate the anti‐cancer activity of two novel RIG‐I agonists, the immunomodulatory RNA (immRNA) with a unique secondary structure for efficient RIG‐I activation, and a 5′ triphosphorylated antisense oligonucleotide with dual function of RIG‐I activation and miR‐125b inhibition (3p‐125b‐ASO). We find that RBCEV‐delivered immRNA and 3p‐125b‐ASO trigger the RIG‐I pathway, and induce cell death in both mouse and human breast cancer cells. Furthermore, we observe a significant suppression of tumour growth coupled with increased immune cell infiltration mediated by the activation of RIG‐I cascade after multiple intratumoral injections of RBCEVs loaded with immRNA or 3p‐125b‐ASO. Targeted delivery of immRNA using RBCEVs with EGFR‐binding nanobody administrated via intrapulmonary delivery facilitates the accumulation of RBCEVs in metastatic cancer cells, leading to potent tumour‐specific CD8+ T cells immune response. This contributes to prominent suppression of breast cancer metastasis in the lung. Hence, this study provides a new strategy for efficient RIG‐I agonist delivery using RBCEVs for immunotherapy against cancer and cancer metastasis.

Resveratrol sensitizes breast cancer to PARP inhibitor, talazoparib through dual inhibition of AKT and autophagy flux

The efficacy of poly (ADP-ribose) polymerase inhibitors (PARPi) is largely limited to the homologous recombination (HR) deficient cancers. Therefore, there is a necessity to explore novel drug combinations with PARPi to enhance its anti-cancer activity in HR-proficient cancers. By analysing the patient data in cBioPortal, we found copy number amplification of PARP1 in ∼ 22.8% of breast cancers. PARP1 upregulation has been correlated with unfavourable outcome with PARPi treatment. To overcome this adversity, we explored the effect of resveratrol, a natural molecule chemosensitizer, in enhancing the effects of the third generation PARPi, talazoparib (BMN673), against breast adenocarcinoma. Our results show that resveratrol effectively sensitized talazoparib induced cell death in HR proficient and BRCA wild-type breast cancer cells in vitro. Mechanistically, resveratrol caused dysregulation of cell cycle and enhanced talazoparib-induced double strand breaks (DSBs), leading to abnormal mitotic progression culminating in mitotic catastrophe. Intriguingly, our results showed potential of resveratrol in dual-inhibition of AKT-signalling and autophagy flux to impair HR-mediated DSB-repair in breast cancer cells. By using EGFP-LC3 and tf-LC3 (mRFP-EGFP-LC3) expressing breast cancer cells, we found that resveratrol attenuates fusion of autophagosome and lysosome though induction of lysosomal-membrane-permeabilization (LMP). The combination of resveratrol and talazoparib effectively reduced cell proliferation in the high-density cell proliferation assay and also led to tumour volume reduction in vivo pre-clinical SCID-mice model. The combination caused no or minimal cytotoxicity in three different normal cell lines in vitro. Taken together, our work proposes the usage of resveratrol as a chemosensitizer along with talazoparib for targeting HR-proficient breast cancers in clinical settings.

Hydrogen phosphate selectively induces MDA MB 231 triple negative breast cancer cell death in vitro

Phosphate ions are the most abundant anions inside the cells, and they are increasingly gaining attention as key modulators of cellular function and gene expression. However, little is known about the effect of inorganic phosphate ions on cancer cells, particularly breast cancer cells. Here, we investigated the toxicity of different phosphate compounds to triple-negative human breast cancer cells, particularly, MDA-MB-231, and compared it to that of human monocytes, THP-1. We found that, unlike dihydrogen phosphate (H2PO4−), hydrogen phosphate (HPO42−) at 20 mM or lower concentrations induced breast cancer cell death more than immune cell death, mainly via apoptosis. We correlate this effect to the fact that phosphate in the form of HPO42− raises pH levels to alkaline levels which are not optimum for transport of phosphate into cancer cells. The results in this study highlight the importance of further exploring hydrogen phosphate (HPO42−) as a potential therapeutic for the treatment of breast cancer.

Zerumbone-induced reactive oxygen species-mediated oxidative stress re-sensitizes breast cancer cells to paclitaxel.

Chemotherapy is an effective approach for cancer therapy when plant-derived sensitizers are combined with chemotherapeutics. Zerumbone, a natural phytochemical, has been documented to have various pharmacological roles. Here, we evaluated the chemosensitization potential of zerumbone in a breast cancer cell line in vitro. Zerumbone-induced cytotoxicity in MCF-7 cells was assessed by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT)-based metabolic analysis. Reactive oxygen species (ROS)-mediated mitochondrial membrane alterations, DNA damage, and apoptotic morphological changes were measured by fluorescence microscopy methods. A biochemical assay was employed to analyze Thiobarbituric acid reactive substances (TBARS) and antioxidant levels. Apoptotic marker expression levels were investigated by immunoblotting. MTT assay revealed that zerumbone significantly enhanced paclitaxel (PTX)-induced cell death in breast cancer cells in a concentration-dependent manner. Furthermore, our study demonstrated that zerumbone (15 μM) significantly enhanced ROS when combined with PTX (1 μM) treatment. Additionally, we observed that zerumbone enhanced the impairment of mitochondrial membrane potential and oxidative DNA damage, thereby inducing apoptosis in combination with PTX. Western blot analysis indicated that zerumbone significantly upregulated BAX, caspase-7, and caspase-9 expression and decreased BCL-2 expression, thereby inducing proapoptotic protein-mediated cell death combined with PTX. The prooxidant properties of zerumbone potentially resensitize breast cancer cells to PTX by enhancing intracellular ROS-mediated oxidative stress.

Combinatorial Effects of the Natural Products Arctigenin, Chlorogenic Acid, and Cinnamaldehyde Commit Oxidation Assassination on Breast Cancer Cells.

Major obstacles in current breast cancer treatment efficacy include the ability of breast cancer cells to develop resistance to chemotherapeutic drugs and the off-target cytotoxicity of these drugs on normal cells, leading to debilitating side effects. One major difference between cancer and normal cells is their metabolism, as cancer cells acquire glycolytic and mitochondrial metabolism alterations throughout tumorigenesis. In this study, we sought to exploit this metabolic difference by investigating alternative breast cancer treatment options based on the application of phytochemicals. Herein, we investigated three phytochemicals, namely cinnamaldehyde (CA), chlorogenic acid (CGA), and arctigenin (Arc), regarding their anti-breast-cancer properties. These phytochemicals were administered alone or in combination to MCF-7, MDA-MB-231, and HCC1419 breast cancer or normal MCF-10A and MCF-12F breast cells. Overall, our results indicated that the combination treatments showed stronger inhibitory effects on breast cancer cells versus single treatments. However, only treatments with CA (35 μM), CGA (250 μg/mL), and the combination of CA + CGA (35 μM + 250 μg/mL) showed no significant cytotoxic effects on normal mammary epithelial cells, suggesting that Arc was the driver of normal cell cytotoxicity in all other treatments. CA + CGA and, to a lesser extent, CGA alone effectively induced breast cancer cell death accompanied by decreases in mitochondrial membrane potential, increased mitochondrial superoxide, reduced mitochondrial and glycolytic ATP production, and led to significant changes in cellular and mitochondrial morphology. Altogether, the combination of CA + CGA was determined as the best anti-breast-cancer treatment strategy due to its strong anti-breast-cancer effects without strong adverse effects on normal mammary epithelial cells. This study provides evidence that targeting the mitochondria may be an effective anticancer treatment, and that using phytochemicals or combinations thereof offers new approaches in treating breast cancer that significantly reduce off-target effects on normal cells.

Anticancer potential of Phoenix dactylifera L. seed extract in human cancer cells and pro-apoptotic effects mediated through caspase-3 dependent pathway in human breast cancer MDA-MB-231 cells: an in vitro and in silico investigation

BackgroundPhoenix dactylifera L. has a diverse set of pharmacological properties due to its distinct phytochemical profile. The purpose of this study was to investigate the anticancer potential of Phoenix dactylifera seed extract (PDSE) in human breast cancer MDA-MB-231 and MCF-7 cells, as well as liver cancer HepG2 cells, and to investigate the anticancer efficacy in triple-negative MDA-MB-231 cells, followed by in silico validation of the molecular interaction between active components of PDSE and caspase-3, an apoptosis executioner protein .MethodsIn this study, human cancer cell lines were cultured and subsequently treated with 10 to 100 μg/mL of PDSE. MTT test was performed to determine the cell viability, MMP was measured using fluorescent probe JC-1, nuclear condensation was determined by Hoechst 33258 dye, Annexin V-FITC & PI staining and cell cycle analysis were evaluated through flow cytometer, and apoptotic markers were detected using western blotting. The bioactive agents in PDSE were identified using high-performance liquid chromatography (HPLC) analysis. The binding affinity was validated using molecular docking tools AutoDock Vina and iGEMDOCK v2.1.ResultsCell viability data indicated that PDSE inhibited cell proliferation in both breast cancer cells and liver cancer cells. MDA-MB-231 cells showed maximum growth inhibition with an IC50 value of 85.86 μg/mL for PDSE. However, PDSE did not show any significant toxicity against the normal Vero cell line. PDSE induced MMP loss and formation of apoptotic bodies, enhanced late apoptosis at high doses and arrested cells in the S phase of cell cycle. PDSE activated the enzymatic activity of cleaved caspase-3 and caused the cleavage of poly-ADB ribose polymerase (PARP) protein. PDSE upregulated pro-apoptotic Bax protein markedly but no significant effect on tumor suppressor protein p53, while it downregulated the anti-apoptotic Bcl-2 protein expression. HPLC analysis showed the presence of rutin and quercetin bioactive flavonols in ethanolic extract of PDS. Interestingly, both active components revealed a strong binding interaction with amino acid residues of caspase-3 (PDB ID: 2XYP; Hetero 4-mer - A2B2) protein.ConclusionPDS could serve as a potential medicinal source for apoptotic cell death in human breast cancer cells and, thus, could be used as a promising and crucial candidate in anticancer drug development. This study warrants further in vivo research, followed by clinical investigation.

BCL2A1 regulates Canady Helios Cold Plasma-induced cell death in triple-negative breast cancer

Breast cancer is the leading cause of cancer death among women. Triple-negative breast cancer (TNBC) has a poor prognosis and frequently relapses early compared with other subtypes. The Cold Atmospheric Plasma (CAP) is a promising therapy for prognostically poor breast cancer such as TNBC. The Canady Helios Cold Plasma (CHCP) induces cell death in the TNBC cell line without thermal damage, however, the mechanism of cell death by CAP treatment is ambiguous and the mechanism of resistance to cell death in some subset of cells has not been addressed. We investigate the expression profile of 48 apoptotic and 35 oxidative gene markers after CHCP treatment in six different types of breast cancer cell lines including luminal A (ER+ PR+/−HER2−), luminal B (ER+PR+/−HER2+), (ER−PR−HER2+), basal-like: ER−PR−HER2− cells were tested with CHCP at different power settings and at 4 different incubation time. The expression levels of the gene markers were determined at 4 different intervals after the treatment. The protein expression of BCL2A1 was only induced after CHCP treatment in TNBC cell lines (p < 0.01), whereas the HER2-positive and ER, PR positive cell lines showed little or no expression of BCL2A1. The BCL2A1 and TNF-alpha expression levels showed a significant correlation within TNBC cell lines (p < 0.01). Silencing BCL2A1 mRNA by siRNA increased the potency of the CHCP treatment. A Combination of CHCP and CPI203, a BET bromodomain inhibitor, and a BCL2A1 antagonist increased the CHCP-induced cell death (p < 0.05). Our results revealed that BCL2A1 is a key gene for resistance during CHCP induced cell death. This resistance in TNBCs could be reversed with a combination of siRNA or BCL2A1 antagonist-CHCP therapy.

Exploration of novel VEGFR2 tyrosine kinase inhibitors via design and synthesis of new alkylated indolyl-triazole Schiff bases for targeting breast cancer

According to the World Health Organization (WHO) statistics: In 2020, there were 2.3 million women diagnosed with breast cancer and 685,000 deaths globally. Therefore, searching for new leads for fighting this type of cancer is necessary. VEGFR-2 kinase plays a crucial role in the proliferation, migration, and survival of breast cancer cells so, identifying novel inhibitors for VEGFR-2 could be effective in breast cancer treatment. Accordingly, novel heterocyclic compounds containing indole, 1,2,4-triazole, and glycosyl or allyl moieties were synthesized. The synthesized compounds were evaluated for their cytotoxic and apoptotic activities towards breast cancer cell lines (MCF7). In this regard, compounds 6, 17, and 18 exhibited promising cytotoxic activity against MCF-7 cells with IC50 values of 3.06, 1.18, and 3.02 μM compared to Sorafenib (IC50 = 2.13 μM). Interestingly, among the identified lead molecules, compound 17 displayed remarkable VEGFR2 inhibition activity with IC50 value of 19.8 nM compared to Sunitinib (IC50 = 75 nM) and Sorafenib (IC50 = 30 nM). Moreover, it is significantly stimulated apoptotic breast cancer cell death; it induced apoptosis by 17.4 %, arresting the cell cycle phases at G1 and S-phases. Additionally, in vivo (Xenograft model) study validated the anticancer activity of the hit compound 17, which showed a tumor inhibition ratio of 54.2 % compared to 5-FU (49.5%) with an improvement of hematological and biochemical parameters. The results disclosed that the identified hit compound 17 is validated for impeding cell proliferation and migration through apoptosis activation and VEGFR2 inhibition.

Evaluation of apoptotic and anti-cancer efficacy of microwave assisted (ZnO-Ag2O) hybrid nanoparticles in MCF-7 breast cancer cell lines

The current study presents the synthesis and evaluation of zinc oxide and silver oxide hybrid nanoparticles for their potential apoptotic and anti-cancer activity. Owing to their small particle size, high surface area, and synergistic effects of embedded biomolecules, microwave-assisted synthesised nanoparticle-based drug delivery systems have significant potential in the field of biopharmaceutics and medicine. The higher activity of the hybrid nanoparticles may be attributed to their spherical shape, crystalline nature and lower particle size of 10–100 nm, as shown by different characterization methods as UV, FTIR, Zeta potential, particle size analysis, XRD, SEM, TEM, ICP-OES examination. The cell cycle analysis, cell death, and apoptosis in breast cancer cells (MCF-7) treated with synthesized hybrid NPs were evaluated in this work. Investigation of cell cycle analysis using flow cytometry has been utilized to analyse different phases of cell division and proliferation. The cytotoxic effect of microwave-assisted synthesised hybrid nanoparticles on MCF-7 cell line was studied using MTT assay and found to have significant cytotoxic activity with an IC50 value of 24.31 µg/ml when compared to standard drug Doxorubicin. Moreover, in vivo toxicity performed in Wistar rats reveals safety of developed hybrid nanoparticles. The findings demonstrate the powerful therapeutic efficacy of synthesized hybrid nanoparticles as well as the potential for development of future anticancer medication.

Hexadecanoic acid-enriched extract of Halymenia durvillei induces apoptotic and autophagic death of human triple-negative breast cancer cells by upregulating ER stress

Objective: To investigate the effect of the hexane solvent fraction of Halymenia durvillei (HDHE) on triple-negative breast cancer. Methods: The phytochemical profile of HDHE was investigated by GC-MS. The cytotoxicity of HDHE against MDA-MB-231 cells was determined. The apoptotic and autophagic effects of HDHE were analyzed. The expression of molecular markers controlling apoptosis, autophagy, DNA damage, and endoplasmic reticulum (ER) stress was determined. Results: HDHE contains a mixture of fatty acids, mainly hexadecanoic acid. HDHE at a cytotoxic concentration induced apoptotic death of MDA-MB-231 cells through mitochondrial membrane dysfunction, and induction of apoptosis markers, and increased the expression of proteins related to DNA damage response. HDHE also induced the expression of LC-3, a marker of autophagic cell death at a cytotoxic concentration. Moreover, HDHE modulated the expression of ER stress genes. Conclusions: The hexadecanoic acid-enriched extract of Halymenia durvillei promotes apoptosis and autophagy of human triple-negative breast cancer cells. This extract may be further explored as an anticancer agent for triple-negative breast cancer.

The phloroglucinol calcitrinone A, a novel mitochondria-targeting agent, induces cell death in breast cancer cells

Breast cancer is the most common cancer and the leading cause of cancer-related mortality among females worldwide. From the leaves of Callistemon citrinus, we have isolated a novel phloroglucinol dimer, calcitrinone A, and analyzed its potential anticancer activity using the triple-negative breast cancer cell line MDA-MB-231. Calcitrinone A decreased the total intracellular ATP levels, inhibited proliferation, and induced apoptosis in MDA-MB-231 cells, but was less toxic to peripheral blood mononuclear cells. The antiproliferative and apoptosis-inducing effects of calcitrinone A were confirmed in vivo using breast cancer xenografts grown on chick chorioallantoic membranes. Mechanistic analysis showed mitochondrial membrane-potential dissipation and interference with energy-yielding processes resulting in cell accumulation in the S phase of the cell cycle. Seahorse assay analysis revealed an early inhibition of mitochondrial oxidative phosphorylation (OXPHOS). At the molecular level, calcitrinone A inhibited activity of the succinate-coenzyme Q reductase (SQR) (mitochondrial complex II). In silico docking identified the coenzyme Q binding pocket as a possible high affinity binding site for calcitrinone A in SQR. Inhibition of complex II was accompanied by strong elevation of mitochondrial superoxide and cytoplasmic ROS. Calcitrinone A might be a promising anticancer lead compound acting through the interference with the mitochondrial complex II activity.

Abstract GS1-09: Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by lack of ER, PR, and Her2. Up to 85% of TNBCs have a p53 mutation as their oncogenic driver. TP53 is a tumor suppressor known as the “guardian of the genome” for its roles in regulating growth and death after genomic insult. Due to this high frequency of p53 mutations in TNBCs, targeting p53 mutants in a clinical setting is highly attractive. However, there are currently no FDA-approved drugs that can directly target p53 mutant TNBCs. We propose pathways exist that, when inhibited, will induce the specific death of p53-mutant breast cancer cells, but not p53-wild type breast cells. To investigate this hypothesis, we performed high-throughput drug screening to identify drugs that induce death in p53-mutant TNBCs. We then characterized the identified drugs for mechanism of death induction and in vivo drug effect. Methods: In vitro and in silico drug screens were conducted with small-molecule libraries of drugs with known protein targets, and screens were integrated and common drugs identified. P53-wild type and mutant cells were treated with identified drugs and stained with DAPI and DRAQ7 to identify growth-suppressive and death-inductive effects. One of these drugs, the GPX4 inhibitor ML-162, was selected for further study. Mechanism of death induction by ML-162 was determined with AnnexinV/PI, caspase cleavage, and ferroptosis assays. To confirm these results, GPX4 was knocked out and replicated the effect of ML-162. To determine the reliance of this phenotype on p53 mutational status, a series of inducible p53-mutant cell lines were created in ER+ and TNBC cell lines and then treated with small molecule inhibitors. ML-162 in vivo effect was demonstrated by treating p53-mutant TNBC xenografts in nude mice. Results: Integration of in vitro and in silico screens identified 6 common drugs, representing cell cycle inhibitors, cell division inhibitors, a proteasome inhibitor, and a peroxidase inhibitor. Identified drugs were demonstrated to reduce growth or induce death of p53-mutant TNBC cell lines. The GPX4 inhibitor ML-162 was further characterized, and found to induce death through ferroptosis, and not apoptosis or necroptosis. GPX4 knockout in p53-mutant TNBC cell lines induced ferroptosis and can be blocked with an anti-ferroptosis drug. Inducible p53-mutations in ER+ and TNBC cell lines were treated with ML-162. P53 mutations in TNBC, and not ER+, cell lines showed increased sensitivity to ML-162. To demonstrate in vivo effect of ML-162, TNBC cell line xenografts were grown in nude mice and treated with ML-162. This treatment significantly reduced tumor volume and also induced lipid peroxidation, a hallmark of ferroptosis. Conclusion: Our high-throughput screening demonstrated several of the identified drugs suppress growth or induce death preferentially in p53-mutant breast cancers. One of these drugs, ML-162, induces death of p53-mutant triple-negative breast cancer cells through induction of ferroptosis, both in vitro and in vivo. These studies provide the basic science foundation to further develop ferroptosis inducers for the targeted treatment of p53-mutant breast cancers. This work was funded by the John Charles Cain Endowment. Citation Format: William M Tahaney, Jing Qian, Reid Powell, Cassandra L Moyer, Yanxia Ma, Nghi Nguyen, Jamal Hill, Clifford Stephan, Abhijit Mazumdar, Peter JA Davies, Powel H Brown. Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-09.

Abstract P1-08-22: The role of DAXX as a critical growth regulator of triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is a heterogeneous disease that frequently presents at a metastatic stage or upon initial early stage diagnosis, and may recur quickly. Treatment options are limited outside of cytotoxic chemotherapy in the adjuvant or metastatic setting and there is an immediate need to identify and validate novel biomarkers that predict sensitivity to chemotherapy. Previously, it was shown that the pro-apoptotic protein DAXX is required for paclitaxel-mediated breast cancer cell death. Based on these data and our recent discoveries that DAXX is a potent inhibitor of breast cancer stem cells in ER+ breast cancer (Peiffer et al., Cancer Res. 2019; Peiffer et al., NPJ Breast Cancer. 2020), we next explored the role of DAXX in TNBC. We found that high DAXX expression in TNBC cells regulates cell proliferation and activities of PARP-1 and JNK; two pathways required for cell death. The goals of the current work were to investigate the mechanism by which DAXX regulates proliferation at the level of PARP-1 and/or JNK and test if DAXX expression predicts sensitivity to carboplatin, paclitaxel, doxorubicin, the PARP-1 inhibitor olaparib, or the JNK inhibitor SP600125 in TNBC cells. Methods: Proliferation and cell cycle analysis were performed in three TNBC cell lines (MDA-MB-231, BT549, and MDA-MB-468) under DAXX expression and depletion conditions using an siRNA approach. Sensitivity to carboplatin, paclitaxel, and doxorubicin was determined by measuring proliferation in a dose-dependent manner. PARP-1 activity was assessed by detecting global protein PARylation levels using Western blotting. JNK activation was analyzed by measuring phosphorylated JNK levels. A JNK inhibitor (SP600125) was used to determine if JNK signaling was responsible for restricting cell proliferation and if DAXX was required for the elevated JNK activity. Olaparib was used to assess the role of PARP-1 activation in DAXX-depleted cells. Results: The TNBC cell lines expressed high levels of DAXX. Depletion of DAXX increased cell proliferation by promoting cell cycle progression through the S-phase. The consequence of this increased cell proliferation was decreased sensitivity to carboplatin and paclitaxel, but not to doxorubicin. Mechanistically, at least one chemotherapeutic agent, carboplatin induced JNK activation and DAXX was required for the increased JNK activity. The JNK inhibitor (SP600125) partially reversed resistance to carboplatin in DAXX-depleted cells. Additionally, genetic knockdown of DAXX resulted in elevated PARP-1 activity as measured by global PARylation of proteins. PARP-1 blockade using olaparib also partially reversed resistance to carboplatin in DAXX-depleted cells. Conclusions: These results suggest that DAXX is a critical growth regulator and predictor of response to carboplatin and paclitaxel in triple negative breast cancer cells. DAXX is necessary to activate the JNK pathway in response to carboplatin and it is this carboplatin-mediated JNK signaling that inhibits cell proliferation. Furthermore, DAXX limits PARP-1 activity and is necessary for increased sensitivity to carboplatin or paclitaxel. Data herein support assessing DAXX expression in triple negative tumors to determine if high DAXX expression predicts increased sensitivity to carboplatin, paclitaxel, and if DAXX-low expressing TNBC cells require addition of a JNK or PARP-1 inhibitor. Citation Format: Clodia Osipo, Debra Wyatt, Michelle Fernandez, Daniel S Peiffer, Kathy S Albain. The role of DAXX as a critical growth regulator of triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-22.

Short Communication: Terpenoids isolated from Lansium domesticum fruit peel induce apoptosis and cell cycle arrest in T-47D cell lines

Abstract. Fadhilah K, Wahyuono S, Astuti P. 2022. Short Communication: Terpenoids isolated from Lansium domesticum fruit peel induce apoptosis and cell cycle arrest in T-47D cell lines. Biodiversitas 23: 1167-1171. One of the characteristics of cancer cells is uncontrolled proliferation. This can be caused by aberrant in the proteins that play a role in the process of apoptosis and cell cycle. Lamesticumin A and 2-ethyl,3-(1’-hydroxy-2’-menthene) propenal have been isolated from ethyl acetate extract of L. domesticum fruit peel had cytotoxicity against T-47D cells. However, the study on how these terpenoids induce cell death is still unknown. Flow cytometry analysis was carried out to evaluate the effect of the compounds in cell cycle modulation and apoptotic induction. The terpenoid compounds induced apoptosis and cell cycle arrest in S phase in a dose dependent manner. Compound 1 synergized the effect of doxorubicin in inducing cell death through mechanism involving the cell cycle. Compound 1 revealed better activity in killing T-47D cells rather than compound 2. Further studies are needed to explore the potential of these compounds in promoting cell death of breast cancer.

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

SRT1720 induces SIRT1-independent cell death in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops after a long period of human T-cell leukemia virus (HTLV)-1 infection and is associated with host aging in addition to genetic abnormalities in HTLV-1 infected cells. SIRT1 is a histone deacetylase involved in cell cycle and apoptosis. We previously showed the high expression of SIRT1 protein in peripheral blood mononuclear cells from patients with ATL. There have been many reports that SIRT1 inhibitors show tumor-suppressive effects. On the other hand, SIRT1 activator SRT1720 induces the cell death of multiple myeloma and breast cancer cells. However, the effect of SRT1720 on ATL is unknown. This study aimed to evaluate the effect of SRT1720 on cell death in leukemic cell lines in vitro and freshly isolated ATL cells ex vivo and in an ATL in vivo mouse model. SRT1720 reduced cell viability in vitro and ex vivo. Additionally, SRT1720 increased the number of apoptotic cells, as shown by annexin V positive cells, cleaved poly (ADP-ribose) polymerase 1, cleaved caspase-3, and fragmented DNA. SRT1720 also induced mitochondrial outer membrane permeabilization with the generation of mitochondrial reactive oxygen species and autophagy. However, SIRT1 knockdown did not attenuate SRT1720-induced cell death in leukemic cell lines. Finally, SRT1720 treatment decreased the growth of human ATL tumor xenografts in immunodeficient mice. Our study shows that while SRT1720 does not target SIRT1, it induces cell death in ATL cells via apoptosis and autophagy and has antitumor activity.

Induction of Paraptotic Cell Death in Breast Cancer Cells by a Novel Pyrazolo[3,4-h]quinoline Derivative through ROS Production and Endoplasmic Reticulum Stress.

Chemotherapy has been a standard intervention for a variety of cancers to impede tumor growth, mainly by inducing apoptosis. However, development of resistance to this regimen has led to a growing interest and demand for drugs targeting alternative cell death modes, such as paraptosis. Here, we designed and synthesized a novel derivative of a pyrazolo[3,4-h]quinoline scaffold (YRL1091), evaluated its cytotoxic effect, and elucidated the underlying molecular mechanisms of cell death in MDA-MB-231 and MCF-7 breast cancer (BC) cells. We found that YRL1091 induced cytotoxicity in these cells with numerous cytoplasmic vacuoles, one of the distinct characteristics of paraptosis. YRL1091-treated BC cells displayed several other distinguishing features of paraptosis, excluding autophagy or apoptosis. Briefly, YRL1091-induced cell death was associated with upregulation of microtubule-associated protein 1 light chain 3B, downregulation of multifunctional adapter protein Alix, and activation of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase. Furthermore, the production of reactive oxygen species (ROS) and newly synthesized proteins were also observed, subsequently causing ubiquitinated protein accumulation and endoplasmic reticulum (ER) stress. Collectively, these results indicate that YRL1091 induces paraptosis in BC cells through ROS generation and ER stress. Therefore, YRL1091 can serve as a potential candidate for the development of a novel anticancer drug triggering paraptosis, which may provide benefit for the treatment of cancers resistant to conventional chemotherapy.