A Mechanistic Investigation on the Anticancer Properties of SYA013, a Homopiperazine Analogue of Haloperidol with Activity against Triple Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is one of the most malignant cancers associated with early metastasis, poor clinical prognosis, and high recurrence rate. TNBC is a distinct subtype of breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptors (HER2). Development of effective TNBC therapies has been limited partially due to the lack of specific molecular targets and chemotherapy involving different cytotoxic drugs suffers from significant side effects and drug-resistance development. Therefore, there is an unmet need for the development of novel and efficient therapeutic drugs with reduced side effects to treat TNBC. We have previously reported that certain analogues of haloperidol (a typical antipsychotic drug used for treating mental/mood disorders such as schizophrenia and bipolar disorder) suppress the viability of a variety of solid tumor cell lines, and we have identified 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluoro-phenyl)butan-1-one (SYA013) with such antiproliferative properties. Interestingly, unlike haloperidol, SYA013 shows moderate selectivity toward σ2 receptors. In this study, we explored the potential of SYA013 in modulating the important biological events associated with cell survival and progression as well as the mechanistic aspects of apoptosis in a representative TNBC cell line (MDA-MB-231). Our results indicate that SYA013 inhibits the proliferation of MDA-MB-231 cells in a concentration-dependent manner and suppresses cell migration and invasion. Apoptotic studies were also conducted in MDA-MB-468 cells (cells derived from a 51-year old Black female with metastatic adenocarcinoma of the breast.). In addition, we have demonstrated that SYA013 induces MDA-MB-231 cell death through the intrinsic apoptotic pathway and may suppress tumor progression and metastasis. Taken together, our study presents a mechanistic pathway of the anticancer properties of SYA013 against TNBC cell lines and suggests a potential for exploring SYA013 as a lead agent for development against TNBC.

Introduction

Breast cancer continues to be the most frequent solid tumor cancers affecting women worldwide, and the second leading cause of cancer-related death in women in the United States. In 2020, it was estimated that over 276,480 women and over 2620 men will be diagnosed with breast cancer in the United States with an estimated death of 42,170 and 520 of women and men, respectively.[1] Although breast cancer is approximately 100 times more common in women than in men, males tend to have poorer outcomes due to delays in diagnosis.[2] Global gene expression studies have revealed four molecular intrinsic subtypes of breast cancers, which include luminal A (ER+ and/or PR+, HER2−), luminal B (ER+ and/or PR+, HER2+), basal cell-like (ER–, PR–, HER2−), HER2-enriched (ER–, PR–, HER2+), and a normal breast-like group.[3−6] The basal-cell-like subgroup has high histological grade and high proliferation rates due to high frequency loss-of-function mutations of the p53 tumor suppressor protein and loss of retinoblastoma protein (RB-loss). They are also associated with breast cancer type 1 (BRCA1) gene mutation and poor prognosis and characterize 10–25% of breast cancers. Triple-negative breast cancer or TNBC is a subtype of breast cancer in which approximately 50–75% is characterized as basal cell-like cancer[7] and represents a heterogeneous group of breast cancers whose prognosis is poor and is deficient in the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2).[8,9] TNBC is known to have a propensity to act more aggressively among other breast cancers and accounts for 10–15% of all breast cancers.[10]

In the US, TNBC incidence and mortality rates vary by race and ethnicity. The incidence of TNBC is highest among women of African descent.[11] African–American women are likely to be diagnosed with TNBC up to three times more than Caucasians.[12] African–Americans with TNBC often have the worse outcome, with 5 year survival estimated at 70% as compared to over 80% for other subpopulations.[13] TNBC continues to be a major challenge in targeted therapy management due to its lack of hormone receptors[10] that serve as therapeutic targets in hormone receptor-positive breast cancers. Currently, there is a lack of targeted therapies for TNBC, and therapeutic agents used for other subtypes of breast cancers are not helpful due to the lack of target receptors. As a result, conventional chemotherapy is the mainstay despite the toxicity associated with them. Over the past few decades, a number of anticancer drugs were reported for TNBC each with different therapeutic interventions.[14] Anthracycline-taxane-based neoadjuvant therapy is recommended for early stage TNBC.[15] Progression of the disease to metastatic TNBC (mTNBC) presents an even greater challenge to therapy compared to other breast cancer subtypes. The mTNBC has a median overall survival of approximately 9–12 months with conventional cytotoxic chemotherapy.[16,17] With the poor outcomes and significant side-effects of currently used anticancer drugs, there is a dire need for novel therapeutic agents with improved efficacy and minimum side-effects for the treatment of TNBC.[18]

Haloperidol is a well-known/standard conventional antipsychotic agent used in the treatment of mental and mood disorders including schizophrenia, bipolar disorder, and acute and chronic psychosis.[19,20] Interestingly, in a study of patients suffering from schizophrenia who were under antipsychotic medications, it was observed that there was a reduction in the risk of cancer and it was thought that the antineoplastic effects of various antipsychotic drugs possibly contributed to such effect.[21] Such an observation led to the evaluation of haloperidol and its derivatives for their anticancer properties in various cell lines.[22−24] Incidentally, haloperidol was also demonstrated to interact with sigma receptors, and in our quest to identify new ligands for sigma receptors, we synthesized homopiperazine analogues of haloperidol and screened them against a variety of cancer cell lines.[25,26] One such compound, SYA013 (Figure ), was observed to demonstrate cytotoxic effects in various solid tumor cell lines including TNBC cell lines (MDA-MD-468 and MDA-MD-231), human alveolar basal epithelial adenocarcinoma cell line (A549), prostate cancer cell line (PC-3), and pancreatic cancer cell lines (MIA PaCa-2 and Panc-1). We have also examined the effect of SYA013 on the viability of nontumorigenic breast epithelial cells (MCF-10A). The results demonstrated that SYA013 has marked selective toxicity against MDA-MD-231 and MDA-MB-468 cell lines compared to the nontumorigenic breast epithelial MCF-10A cells.[25] Furthermore, SYA013 was more than 2-fold effective in suppressing the viability of the MDA-MB-231 cells compared to a standard anti-TNBC drug, Cisplatin.[25]

Figure 1

Structure of SYA013.

In this study, we have evaluated the anticancer properties, the mechanisms, and pathways by which SYA013 influences the biological events associated with TNBC cell survival, growth, and progression using MDA-MB-231 as a model TNBC cell line.

Results

SYA013 Inhibits Cell Proliferation and Colony Formation and Arrests Cell Cycle

Cell proliferation studies were carried out on a MDA-MB-231 cell line. Cells treated with SYA013 concentrations as low as 1 μM for 48 h demonstrated a decrease in cell proliferation, and the number of viable cells decreased with increasing concentrations of the drug. Cell proliferation was completely halted at 2 μM (Figure A). The ability of SYA013 to inhibit MDA-MB-231 cell survival and colony formation was also investigated. As shown in Figure B,C SYA013 significantly reduced the ability of treated cells to form colonies as shown by cell survival fractions.[27] The number of colonies formed relative to the density of cells seeded at 500 cells per well decreased by 19 and 43% at 5 and 10 μM, respectively. Further investigation of the effect of SYA013 on cell cycle indicated that it blocks MDA-MB-231 cell proliferation at the G0/G1 phase (Figure D,E).

Figure 2

SYA013 inhibits cell proliferation (A), represses colony formation (B, C), and arrests cell cycle (D, E) in MDA-MB-231 cells. Treatment of MDA-MB-231 cells with SYA013 for 48 h as described in the methods shows reduction in cell proliferation and decrease survival by 19 and 43% at 5 and 10 μM, respectively, and arrest cell cycle at the G0/G1 phase.

SYA013 Prevents the Formation of Spheroids and Disintegrates Preformed Spheroids

Culturing cells in 3D format helps to observe drug response characteristics in a system that more closely mimics the in vivo environment of tumors.[28,29] We therefore conducted a spheroid-based assay to determine the effect of SYA013 on spheroids formed using MDA-MB-231 cells. Our results indicate that SYA013 disintegrates preformed spheroids and prevents the formation of spheroids and are shown in Figure A,B.

Figure 3

SYA013 prevents the formation of spheroids, disintegrates preformed spheroids, and suppresses migration of MDA-MB-231 cells. Treatment with SYA013 for 48 h disintegrates preformed spheroids (A), prevents the formation of spheroids (B), and inhibits cell migration (C) as indicated by a significant decrease in number of migrated cells (D). MDA-MB-231 spheroids were treated with SYA013 for 48 h and stained with acridine orange/ethidium bromide (AO/EB), where green signifies live cells and red indicates dead cells. Each point represents the mean ± SEM of 4 determinations. *p <0.05, **p <0.01, and ***p <0.001 compared to the nontreated cells using one-way ANOVA analysis followed by the Dunnett’s test.

SYA013 Inhibits TNBC Cell Migration and Invasion

Cancer metastasis is a collective outcome of numerous changes in tumor cells and their microenvironment that support cellular migration and invasion into the host’s healthy tissue.[30] TNBC depends on migration and invasion to affect nearby and distant tissues. To determine the ability of a ligand to prevent the mobility of MDA-MB-231 cells, the migration assay was conducted. The results show that treatment with SYA013 for 12 and 24 h caused a significant decrease in cells moving into the wound area (Figure C,D) and as early as 12 h (Figure D) compared to the control group.

SYA013 (2, 5, and 10 μM) inhibited MDA-MB-231 cell migration by 55, 61, and 95%, respectively (Figure C,D). Also, the transwell matrigel invasion chamber assay was used to analyze the ability of MDA-MB-231 single cells (2D) to directionally respond to chemo-attractants[31] after 22 h treatment with the ligand. We report that treating MDA-MB-231 cells with SYA013 (0–20 μM) prevented the invasion of MDA-MB-231 cells in the 2D cell invasion assay (Figure A,B). It is important to note that these concentrations did not affect the cell viability within 24 h. Furthermore, SYA013 at 2, 5, 10, and 20 μM decreased the number of invaded cells by 22, 26, 49, and 80%, respectively, when compared to the control in the 3D spheroid invasion assay (Figure C,D).

Figure 4

SYA013 inhibits MDA-MB-231 cell invasion in the 2D assay (A, B) and the 3D spheroid invasion assay (C, D). SYA013 significantly reduced the number of cells that entered the invasion chamber compared to the control (A) and quantification of 2D cell invasion (B). Spheroid invasion assay showing the effect of SYA013 on MDA-MB-231 spheroids (C) and the quantification of invaded area through the matrigel as compared to the control (D). Each point represents the mean ± SEM of 4 determinations. *p <0.05, **p <0.01, and ***p <0.001 vs nontreated control cells were compared by the Dunnett’s multiple comparison test.

SYA013 Induces Apoptotic Cell Death

Certain morphological and biochemical events are indicated in the apoptosis-mediated cell death. DNA fragmentation is considered as one of the hallmarks of apoptotic cell death, which results in degradation of nuclear DNA into small nucleosomal units. To investigate the mode of cell death induced in MDA-MB-231 cells, the cells were treated with SYA013 and subsequently stained with the acridine orange/ethidium bromide (AO/EB) dye mixture. The results show the differential staining of the live and dead cells. Acridine orange stained the nuclei of live cells green, while the nuclei of dead cells were stained red by ethidium bromide. SYA013-treated cells showed red-stained nuclei indicating cell death due to potential apoptosis (Figure A).[32] The apoptotic effects of the SYA013 were also evaluated using flow cytometry analysis by staining the treated MDA-MB-231 cells with propidium iodide and Annexin V-FITC and analyzing the results with a flow cytometer. Flow cytometric data revealed that apoptosis was induced in a concentration-dependent manner (Figure B,C) with an increase in the population of late apoptosis being associated with a corresponding increase in the concentration of SYA013. Additionally, SYA013 was evaluated in MDA-MB-468 cells (derived from a 51-year-old Black female with metastatic adenocarcinoma of the breast) for apoptosis using AO/EB staining and flow cytometric analysis with propidium iodide/Annexin V-FITC staining. MDA-MB-468 cells treated with SYA013 at varying concentrations showed a similar concentration-dependent apoptosis pattern (Figures S1 and S2).

Figure 5

SYA013 causes cell death by inducing apoptosis. SYA013-induced death of MDA-MB-231 cells with acridine orange/ethidium bromide (AO/EB) dye staining, scale bar = 50 μm (A). The apoptotic markers were evaluated using the flow cytometry. MDA-MB-231 cells were treated with SYA013 (0–20 μM) for 48 h, and Annexin V and PI stainings were applied (B). The distribution of cells in flow cytometry analysis on the bar graph shows high population concentration at the late stage of apoptosis (C). *p <0.05, **p <0.01, and ***p <0.001. EA = early apoptosis; LA = late apoptosis

Caspases 8 and 9 are activated during cell apoptosis.[33,34] However, caspases 3 and 7 play a significant role in executing the cleavage that occurs during apoptosis.[35] Hence, we probed the contribution of these caspases in the SYA013-induced apoptosis in MDA-MB-231 cells using the Caspase GLO assay kit (Promega, Madison, WI). The results demonstrate that caspases 3/7 and 9 were activated in the MDA-MB-231 cells treated with SYA013 (1–5 μM) (Figure ).

Figure 6

SYA013 activates caspases 3/7 and 9 and induces apoptosis in MDA-MB-231 cells. The activation of caspases 3, 7, and 9 were determined after treating MDA-MB-231 cells with SYA013 for 48 h. Caspase Glo reagent was added and incubated at room temperature for 1 h, caspase activity was measured using the illuminometer plate reader, and the data plotted using GraphPad. *p <0.05, **p <0.01, and ***p <0.001.

SYA013 Induces Apoptosis by Activating Proapoptotic Proteins

Since SYA013 activated caspases in MDA-MB-231 cells as observed in the caspase Glo assay, it was of interest to know whether this was due to changes at the cellular levels of the caspases and other proteins. To achieve this, we conducted a western blotting analysis on MDA-MB-231 cell lysates after 48 h treatment with SYA013 and probed for the levels of key proapoptotic, prosurvival (anti-apoptotic), cell-cycle regulators, and death receptor proteins. We then examined the effect of the compound on caspases and poly(ADP-ribose) polymerase (PARP) activity to determine if the activation of caspases occurred during SYA013-induced apoptosis. Figure A,B shows that treatment of MDA-MB-231 cells with SYA013 appeared to suppress the levels of caspase 3 and PARP. It has been reported that cytochrome c (Cyt c) binds to apoptotic-protease-activating factor-1 (Apaf-1) and enables Apaf-1 to recruit and stimulate the inactive caspase 9 zymogen to its active form.[36,37] Then, the active caspase 9 triggers a cascade of caspase-activated events that lead to apoptosis.[35] During apoptosis, Cyt c enters the cytosol from mitochondria where it carries out apoptotic activities.[38−42] Since SYA013 activated caspase 9, we investigated whether it would affect SYA013-induced apoptosis associated with mitochondrial signaling. Therefore, the effect of the SYA013 on Cyt c, a protein associated with intrinsic apoptosis pathway was examined. The results in Figure reveal an increase in the levels of Cyt c after treatment of MDA-MB-231 cells with SYA013 (Figure A,B). We also found that the amount of antiapoptotic proteins decreased in cells treated with the SYA013 for 48 h compared to control (Figure C,D). However, SYA013-treated cells did not have a visible significant effect on death receptors (Figure E,F) and cell cycle regulators (Figure G,H).

Figure 7

Effect of SYA013 on proapoptotic (A, B), antiapoptotic (C, D), death receptors (E, F), and cell cycle proteins (G, H). Western blot analysis shows that SYA013 activates Bcl-2 family proapoptotic proteins and antagonize Bcl-2 family prosurvival proteins in MDA-MB-231 cells. Cells were treated for 48 h and lysed. Lysates containing 50 μg of protein were analyzed by western blotting, probing with the respective antibodies followed by chemiluminescent detection. Beta-actin was used as the internal control. *p <0.05, **p <0.01, and ***p <0.001.

Discussion

Numerous studies have shown that due to lack of hormone receptors (estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor 2 (HER2))[3] targeted therapies that are highly effective and improve response and survival in other types of cancers are not effective for patients with TNBC. Thus, currently, TNBC has no approved targeted therapies, and the best treatment option is chemotherapy, which is associated with high relapse rate within 1–4 years.[16] Currently, several available drugs used in conventional chemotherapy affect tumor cells as well as normal cells causing toxic side effects and imposing limitations on appropriate treatment doses as well as efficacy.[43] In this regard, we have evaluated the cytotoxicity potential of SYA013 against TNBC cell lines, MDA-MB-231 and MDA-MB-468 cells and successfully demonstrated it to be effective.[25] SYA013 was over 2-fold more effective than cisplatin, a neo-adjuvant for TNBC patients with BRCA gene mutation.[25] SYA013 exhibits selective toxicity on TNBC cells compared to MCF-10A cells which we hypothesize could be possibly due to its preferential binding toward σ2 receptors (σ2R). σ2 receptors are known to play key roles in preferentially targeting cancer cells to exhibit an intrinsic anticancer effect by σ2 receptor ligands (σ2RLs).[44,45] Such selective targeting of TNBC cells is important toward discovering novel therapeutic targets for TNBC.[46−51] However, several studies have implicated the role of σ2RLs in the treatment of TNBC in that they undergo receptor-mediated endocytosis, which provide a means of selectively transporting drugs into tumor cells[52−54] and then into the mitochondria, an important drug target site due to the regulatory processes of apoptosis and homeostasis.[55] In particular, sigma-2 receptor agonists have been reported to induce cell death through various pathways as they activate caspases 3, 8 (extrinsic), and 9 (intrinsic) and some have even proposed that conjugation of a σ2R agonist and cytotoxic warheads could be a viable approach to improve their potency.[52,55−57] In this study, we have shown that SYA013, an analogue of haloperidol with moderate selectivity toward σ2R, induces apoptosis in MDA-MB-231 and MDA-MB-468 cells as demonstrated by propidium iodide/Annexin V-FITC flow cytometric analysis and nuclei fragmentation seen with the acridine orange-ethidium bromide staining. The intrinsic and extrinsic apoptotic signalling pathways trigger the activation of caspases 3 and 7 leading to DNA fragmentation and subsequently apoptosis. The SYA013-induced upregulation of cells caspases 3 and 7 as well as DNA fragmentation in MDA-MB-231 cells suggest a clear indication that the mode of cell death is by apoptosis. SYA013-treated cells also expressed caspase 9 and cytochrome c and, thus, provide additional evidence for the intrinsic apoptotic pathway, which is consistent with previous reports.

We have further shown that SYA013 inhibits cell proliferation and arrests cell cycle at G0/G1 in a concentration-dependent manner. However, it did not show any significant effect on cell cycle regulatory proteins.[55] We also report that SYA013 induces caspase-mediated apoptosis and prevents cell migration and invasion with western blotting confirming apoptosis as a mechanism of cell death through the elevated levels of caspases 3 and 9 as well as PARP. In addition, we have observed an increase in the expression of the Bcl-2 family proapoptotic Bax protein while the levels of antiapoptotic proteins such as B-cell lymphoma 2 (Bcl-2), Myeloid cell leukemia 1 (Mcl-1), and Bcl-2 like protein X (Bcl-XL) were decreased.

The apoptosis induced by SYA013 in MDA-MB-231 cells has potential therapeutic implication given that the mitochondria is an important site of drug target due to regulatory processes of apoptosis and homeostasis.[55] It is worth noting that we have demonstrated in a previous study that SYA013 displays optimal selective toxicity against several solid tumor cell lines that include MDA-MB-468 when compared to the nontumorigenic epithelial MCF-10A cell line.[25] The identification of SYA013 as having the ability to induce substantial cell death in MDA-MB-468 cells derived from an African–American is considered significant because reports have indicated that, in the U.S, the rate of TNBC is higher in premenopausal women of African descent compared to non African–American patients, and this extends globally in young women of African background.[58,59] It is evident from the cell proliferation, colony formation, and cell cycle analysis data that SYA013 clearly produces the cytotoxic effects in tumorigenic MDA-MB-231 cells. Although SYA013 shows sigma receptor binding affinity with moderate σ2R selectivity, these antiproliferative effects of SYA013 cannot be attributed to the σ2R receptor route unless proven.[60] Investigation on the involvement of σ2Rs or other receptors is certainly necessary to establish the cellular target and possible route of action. Nevertheless, SYA013 may have the potential to be used as a monotherapeutic agent or could be combined with other agents for synergistic activity and/or to avert adverse side effects through dose reduction.

We have also evaluated the role of death receptor proteins and demonstrated that they did not have any significant effect, suggesting that the apoptosis induced by SYA013 was not by the extrinsic pathway. Moreover, the upregulation of cytochrome c in this SYA013-treated cells confirmed that the intrinsic pathway was involved in the apoptotic process. Based on the data from our current study, we propose a possible apoptotic pathway mediated by SYA013 and is shown in Figure . Briefly, SYA013-induced DNA fragmentation acts as the stimulus, which initiates the intrinsic apoptotic pathway system. Activation of several death stimuli causes Bax to translocate and localize on the mitochondrial surface causing conformational changes and formation of pores on the mitochondrial membrane. The ligand may also bind to and prevent B-cell leukemia-extra-large (BCL-XL), myeloid cell leukemia 1 (MCL-1), and B-cell-like protein 2 (BCL-2) prosurvival proteins from stopping the release of cytochrome C, and therefore, BCL-2, BCL-XL, and Mcl-1 would fail to sequester the proapoptotic BcL-2 family, Bax. These stimuli cause changes in the inner mitochondria resulting in the opening of the mitochondrial permeability transition (MPT) pore and the loss of mitochondrial transmembrane potential. The ensuing conformational changes trigger the release of the proapoptotic proteins, cytochrome C, and Smac/DIABLO to the cytosol from their usual location in the mitochondrial intermembrane space. In the cytosol, cytochrome c binds to and activates apoptotic protease activating factor 1 (APAF-1) and the cytochrome-APAF-1 cleaves procaspase 9 to form the active caspase 9 leading to the formation of an apoptosome. On the other hand, Smac/DIABLO promotes apoptosis by inhibiting the inhibitors of apoptosis proteins (IAP) while activated caspase 9 signals the cleavage of procaspases 3/7 to the active caspases 3/7. These events lead to apoptosis of the target cell.[61−74]

Figure 8

Proposed apoptotic pathway for the anticancer compound, SYA013.

Conclusions

We have shown that SYA013 inhibits crucial biological events associated with MDA-MB-231 cell survival and progression including cell proliferation, apoptosis, and colony formation. We have reported that SYA013 induces cell death by the intrinsic apoptotic pathway and its ability to disrupt the key functions of tumor cells makes it a potential candidate for further studies and may serve as a potential viable approach for the effective treatment of TNBC. Our future plan includes additional studies demonstrating the role of σ2Rs and identifying other cellular targets if any, involved in imparting cytotoxicity in MDA-MB-231 and MDA-MB-468 cells. We also intend to evaluate any synergism involving clinical cancer drugs such as doxorubicin, gemcitabine, cisplatin, and 5-fluorouracil. Additional studies involving animal models are necessary to explore the potential of SYA013 for use as a monotherapeutic agent or in a combination treatment with another agent for synergistic activity and/or to avert adverse side effects by dose reduction.

Materials and Methods

Human adenocarcinoma MDA-MB-231 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified eagle medium high glucose, GlutaMax (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin-neomycin antibiotic mixture (PSN) were purchased from Life Technologies (Grand Island, NY), and phosphate buffered saline was purchased from Genesee Scientific (San Diego, CA). Ibidi u-slide 8-well glass bottom and ibidi cell culture inserts 2 were purchased from ibidi (Madison, WI). Caspase Glo 3/7, caspase Glo 9, caspase Glo 8 were purchased from Promega Corporation (Madison, WI). Apoptosis antibody sampler kit, proapoptosis Bcl-2 family antibody sampler kit, and cytochrome c antibody were purchased from Cell Signaling Technology (Danvers, MA). Mini-PROTEAN TGX precast protein gel, Precision Plus protein dual color standards, Immun-Blot PVDF membrane, Laemmli premixed protein sample buffer for SDS-PAGE, Tris buffered saline, 10× Tris/Glycine premixed electrophoresis buffer, blotting-grade blocker nonfat dry milk for western blotting, and clarity western ECL substrate were purchased from Bio-Rad (Hercules, CA). Cisplatin was purchased from Santa Cruz biotechnology (Dallas, TX). SYA013 was synthesized and characterized in our lab as previously described,[75] demonstrated to bind to sigma −2 receptor (σ2Ki = 5.6 nM), and shown to be effective at inhibiting cancer cell viability.[25]

Cell Culture

MDA-MB-231 cells were cultured in Dulbecco’s modified eagle medium high glucose, GlutaMax (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), and PSN (100 U/mL penicillin, 50 μg/mL neomycin). The cells were incubated in a humidified incubator with an atmosphere of 95% carbon dioxide (CO2) at 37 °C and subcultured when approximately 80–90% confluent. Unless stated otherwise, assays were performed with experimental media containing 5% FBS. A stock solution was prepared by dissolving SYA013 in dimethyl sulfoxide (DMSO) and acetone at a ratio of 1:9.

Cell Proliferation Assay

MDA-MB-231 cells were plated at 20,000 cells/well in 24-well plates for 24 h. The initial or baseline number of cells was determined at the onset of treatments by washing, trypsinizing, and counting the cells in selected wells. The rest of the wells were treated with SYA013 (0–5 μM) for 48 h. Viable cells were counted in triplicate wells using a Countess II automated cell counter (Life Technology Corporation (Grand Island, NY. USA). The significance of effect was established using a GraphPad prism.

Colony Formation Assay

MDA-MB-231 cells were plated at a density of 1.5 × 105 cells/mL in T-25 flasks and allowed to attach overnight. The cells were treated with SYA013 (0–20 μM) for 48 h after which they were trypsinized, counted, and replated in 6-well plates at a density of 500–1000 cells per well and maintained in a complete growth medium for 12 days. They were then fixed with a solution of acetic acid in methanol (1:7) and stained with 1% crystal violet solution in methanol. Colonies consisting of 50 or more cells were counted using NIH ImageJ,[76] (http://rsb.info.nih.gov/ij/). The plating efficiency (PE) and number of colonies formed/number of cells seeded × 100 for the untreated wells were calculated. The surviving fraction (SF) was calculated using the following formula: EF = PE of treated samples / PE of control × 100.[27]

TNBC Spheroid Formation and Disintegration

MDA-MB-231 cells were seeded in 96 U Nuncleon Sphera round-bottom plate at a density of 2 × 104 cells/ml, immediately treated with varying concentrations of SYA013 (0–10 μM), and incubated at 37 °C/5% CO2. The treatment was repeated after 24 h, and the ability of cells to form spheroids was evaluated after 48 h exposure to SYA013. The spheroids were stained with 5 μg/mL of acridine orange/ethidium bromide (AO/EB) solution, and the images captured using a Nikon Ti Eclipse microscope at 4× magnification. In another experiment to evaluate the effect of SYA013 on pre-formed spheroids, cells were seeded at 5 × 103 cells/well in a 96 U Nuncleon Sphera plate and incubated at 37 °C/5% CO2. Spheroids were allowed to form for 3 days after which SYA013 (0–10 μM) was applied for 48 h. The spheroid size and morphology were monitored, replacing 50% of old media with new media in each well and taking pictures daily for 11 day. On the 11th day, the spheroids were stained with 5 μg/mL of acridine orange/ethidium bromide (AO/EB) solution and the images captured using the Nikon Ti Eclipse microscope at 4× magnification.

Apoptosis Assay and Caspase Activation Analysis

The mode of cell death induced by SYA013 was established using the modified acridine orange/ethidium bromide (AO/EB) staining method,[77] Annexin V/propidium iodide flow cytometry, and caspase activation analysis. Briefly, MDA-MB-231 cells (5 × 104 cells/ml) were seeded into a 96-wells plate and incubated at 37 °C in 5% CO2/95% humidified air. In the next day, these were treated with SYA013 (0–10 μM) followed by incubation. This treatment was repeated after 24 h. After 48 h of treatment, 10 μL of EB/AO (100 μg/ml) was added to each well and incubated for 10 min in the dark. The nuclei were observed for any changes after treatment, and images were captured using Nikon Eclipse Ti 100 inverted fluorescent microscope (Nikon Instruments, Inc., Melville, NY). Further determination of the cell death induced by SYA013 was performed using Annexin V/propidium iodide (AV/PI) staining using flow cytometry. MDA-MB-231 cells (5 × 105 cells/well) suspended in media supplemented with 5% FBS were plated into 6-well culture plates (Costar Corning, NY), incubated at 37 °C in 5% CO2/95% humidified air, and allowed to attach overnight. These were then treated with SYA013 (0–20 μM) for 48 h. The mode of cell death was evaluated according to the manufacturer’s protocol using the ApopNexin Fluorescein isothiocyanate (FITC) apoptosis detection kit (EMD Millipore, Temecula, CA). Briefly, the cells were harvested with accutase and centrifuged at 500×g for 5 min. The pellets were washed twice with 1× DPBS on ice and resuspended in 1× binding buffer. Annexin V FITC and PI were added to the cell suspensions and incubated for 10 min at room temperature in the dark. Analysis was done using the FACS Calibur with Cell Quest Pro software (BD Biosciences NJ).

Caspase activation in MDA-MB-231 cells after treatment with SYA013 was evaluated by measuring the intensity of the fluorescence using the caspase Glos 3/7, caspase Glos 8 and 9 assay Kits (Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells were suspended in 100 μL media with 5% FBS, seeded in 96-well plates (1.5 × 104 cells/well) for caspases 3/7 and (3 × 104) for caspases 8 and 9. The cells were incubated at 37 °C in 5% CO2/95% humidified air overnight to attach. Then, the cells were treated with SYA013 (0–5 μM) for 48 h and the prepared multiplexing reagent (caspase Glo Buffer and the lyophilized substrate) at 100 μL/well, protecting from light. The cells were incubated at room temperature for 1 h. The luminescence was measured by reading the fluorescence at 485/525 nm. The graph of the relative maximum fluorescence intensity was drawn using GraphPad Prism 5.

Cell Cycle Analysis

To determine the effect of SYA013 on the cell cycle, MDA-MB-231 cells (2 × 105 cells per well) in a medium supplemented with 10% FBS were seeded in 6-well plates and incubated at 37 °C in 5% CO2/95% humidified air overnight for attachment. Prior to treatment, the old medium was replaced with media containing 5% FBS, cells were treated with SYA013 (0–10 μM), and treatment was repeated after 24 h. Briefly, cells were harvested, washed with PBS, and centrifuged at 300×g for 5 min and the pellets resuspended in 100 μL of PBS and passed through a 28 5/8 needle to keep them single. To fix the cells, cold 70% ethanol was added drop wise while vortexing to fix and permeate the cells and stored at −20 °C overnight. The cells were then washed with PBS ×2 and centrifuged at 500×g for 5 min. Modified Vindelov’s reagent (ribonuclease A and propidium iodide in PBS)[78] was added to the cells, and the phase distribution of cells was determined using a Becton Dickinson FACSort flow cytometer with CellQuest software (Mansfield, MA). The percentage of cells in each phase was determined in the gated population of singlet cells, and the results plotted using GraphPad Prism 5.

TNBC Cell Migration Assay

The ability of SYA013 to inhibit migration and invasion was evaluated using the wound-healing technique. Cell-free zones (wounds) were obtained using ibidi culture inserts in ibidi u-slide 8-well glass bottom to generate two confluent monolayers of cells separated by the wounds. MDA-MB-231 cells (5 × 104 cells) were seeded in ibidi cell culture inserts, allowed to grow to confluence, and serum-starved for 24 h. After treatment with SYA013 (0–10 μM), wound closure was monitored by capturing images with the Nikon Eclipse Ti 100 inverted microscope (Nikon Instruments Inc., Melville, NY) at 0, 6, 12, and 24 h post-treatment and the images analyzed as previously described.[79] The number of cells that migrated into the previously cell-free area was counted and graphed using GraphPad Prism 5.

TNBC Cell Invasion Assay

The effect of SYA013 on cell invasion (2D) was determined using matrigel-coated 24-well plate with 8 μm pore inserts. The 24-well plate with Matrigel invasion inserts (Corning, Bedford, MA) was rehydrated with serum-free DMEM with glutamate for 2 h at 37 °C/5% CO2. MDA-MB-231 cells (2 × 105 cells/ml) suspended in 500 μL of media containing 0.1% FBS and SYA013 (0–10 μM) were added into the upper chamber of the inserts, while 750 μL media with 10% FBS was placed into the lower chambers to serve as chemoattractant. The plates were then incubated at 37 °C/5% CO2 for 22 h to allow the cells to invade from the upper chamber to the lower chamber through the Matrigel. The cells were rinsed with PBS, fixed with 4% formaldehyde in PBS, permeabilized with 100% methanol, and stained with 2% crystal violet for 1 h. The Olympus IX70 microscope was used to image the invading cells at 4× magnification. Invaded cells were counted using ImageJ[76] (http://rsb.info.nih.gov/ij/) and quantified using GraphPad Prism 5.

The ability of SYA013 to inhibit invasion of cells from a formed spheroid in 3D format was determined using a protocol for this assay adapted from Vinci et al.[80] Briefly, MDA-MB-231 cells were plated in 96-well U-Nuncleon Sphera plates (Thermo-Scientific, Waltham, MA) at a density of 5 × 104 cells/mL in complete growth media. Following the formation of compact spheroids, 850 μL of BD Matrigel (Corning, Bedford, MA) was pipetted into the 10 prechilled microcentrifuge tubes. SYA013 (3.4–34 μL) was added to each tube to yield final concentrations of 0–20 μM of SYA013 in the mixture. The mixture was stirred gently, and 100 μL of growth medium from each well containing the spheroid was carefully replaced with 100 μL of Matrigel/ligand mixture. The plate was incubated for 1 h to embed the spheroids while solidifying the Matrigel after which additional 100 μL of Matrigel-containing SYA013 was added to the corresponding wells (0–20 μM). The bright field images of Matrigel embedded spheroids were taken at 0 h and after 72 h using the Nikon Ti Eclipse microscope at 4× magnification. The area of invasion was quantified using NIS Element Software, and the data analyzed using GraphPad Prism 5.

Western Blot Analysis

In addition to caspases, the effect of SYA013 on the expression of other apoptotic, antiapoptotic (survival), death receptor, and cell cycle in MDA-MB-231 cells after treatment was evaluated by western blotting. Briefly, cells were plated in 6-well tissue culture plates at a density of 2 × 105 cells/well in media supplemented with 10% FBS and allowed to adhere to the plates overnight at 37 °C in 5% CO2/95% humidified air. Prior to treatment, the CGM was replaced with 5% supplemented media and treated with SYA013 (0–10 μM). The treatment was repeated after 24 h for a total time of exposure of 48 h. The cells were then washed with PBS and lysed with RIPA buffer supplemented with 1× protease inhibitor cocktail (Sigma, St. Louis, MO), and the protein concentration in the lysates was evaluated using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL). Lysates containing 50 μg of protein were mixed with Laemmli sample buffer and placed in a boiling water bath for 5 min. Aliquots of each sample were resolved on 10–20% gradient SDS-PAGE gel. Resolved proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane; the membranes were blocked with 5% fat-free milk (Sigma, St. Louis, MO) for 1 h at room temperature, immunoblotted using primary antibodies against different protein groups including angiogenesis, death receptors, apoptosis, and cell cycle purchased from Cell Signaling Technology (Danvers, MA), and then incubated overnight at 4 °C. The next day, membranes were incubated for 90 min with horseradish peroxidase-linked anti-rabbit IgG secondary antibodies from Santa-Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL) was used to develop the immunoblots using the ChemiDoc Imaging System (Bio-Rad, CA).

Statistical Analysis

The graphical and statistical analyses of data were performed using GraphPad Prism version 5.0 for Windows (San Diego, CA). The results were expressed as the means (± SEM). To obtain the concentration-response curves, the percent inhibition was plotted against the log of the inhibitor concentrations. Nonlinear regression plots were generated, and the concentrations that inhibit 50% of the activity (IC50) were obtained. Data were also analyzed using one-way ANOVA, and statistical differences between control and treated groups were determined by Dunnett’s post-test comparisons. Significance was defined as *p <0.05; **p < 0.01; and ***p < 0.001.