Anticancer effects of sanguinarine in triple-negative breast cancer cells via apoptosis induction and cell cycle arrest

DOI: https://doi.org/10.21203/rs.3.rs-1602051/v1

Abstract

Patients with triple-negative breast cancer (TNBC) are typically treated with non-targeted chemotherapeutic agents, and the late disease stages are known to be resistant to chemotherapy. Therefore, it is necessary to develop novel therapeutic agents that are safer and more effective for enhancing the outcomes of chemotherapeutic agents. The natural alkaloid sanguinarine (SANG) is a quaternary benzophenanthridine alkaloid that has demonstrated synergistic therapeutic effects when combined with various chemotherapeutic drugs. SANG can also induce cell cycle arrest and trigger apoptosis in various cancer cells. Therefore, in this study, we investigated the molecular mechanism underlying SANG activity in MDA-MB-231 and MDA-MB-468 cells, as two genetically different models of TNBC. SANG decreased the viability of both cell lines, but MDA-MB-468 cells were more sensitive to SANG than MDA-MB-231 cells. SANG affected cell cycle progression in both cell lines. Further, S-phase cell cycle arrest-mediated apoptosis was found to be the primary contributor to cell growth inhibition in MDA-MB-231 cells. SANG-treated TNBC cells showed significantly upregulated mRNA expression of 18 genes associated with apoptosis, including eight members of the TNF receptor superfamily (TNFRSF), three members of the BCL2 family, and two members of the caspase (CASP) family in MDA-MB-468 cells; in MDA-MB-231 cells, two members of the TNF superfamily and four members of the BCL2 family were affected. In conclusion, these results indicate that SANG shows markedly different anticancer effects and apoptosis-related gene expression changes in the two cell lines. Thus, SANG demonstrates potent anticancer effects in TNBC cell lines, suggesting its potential as a single or adjunct therapeutic agent against TNBC.

1. Introduction

In the United States, breast cancer (BC) is the most common malignancy and the second leading cause of death among women aged 20–59 years 1. Gene expression is controlled by molecular signal transduction, and alterations in transcriptional settings are characteristic of cancer cells 2. BC is a heterogeneous cancer type categorized based on its molecular features. Triple-negative breast cancer (TNBC) that affects approximately 15% of patients with BC 2. However, the incidence of this subtype is two to three times higher in African American (AA) women than in other ethnic groups 3,4. TNBC is the most aggressive BC, and has a poor outcome compared to other BC subtypes 3. Based on immunohistochemical characteristics, TNBC cells lack the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) 3,5. Currently, chemotherapy is the primary treatment for patients with TNBC. However, disease recurrence can manifest within the first two years with an aggressive metastatic pattern 6. Hence, the continuous pursuit of novel agents is necessary and novel drugs with the potential to induce cell cycle arrest and apoptosis are being pursued for cancer therapy 7.

In cancer, the characteristic mechanism of tumors to overcome DNA damage is a high proliferation rate, boosted by rapid cell cycle progression 8. The signaling pathways controlling these events affect cancer cells significantly 9. Indeed, retracting the cell cycle checkpoints preceding DNA repair can promote apoptotic signaling, leading to transcriptional suppression and cell death 10. Apoptosis is a controlled process that maintains cellular homeostasis and is mediated by extrinsic (death receptor (DR)-mediated) and intrinsic (mitochondrial or BCL2-dependent) pathways. The nature of the stimulus is the main contributing factor leading to the activation of either or both apoptotic pathways 11-15. The extrinsic apoptotic pathway is initiated upon binding of pro-apoptotic ligands, such as tumor necrosis factor-alpha (TNF-α), and FAS ligand with their respective transmembrane receptors, which activate proteases known as caspases, leading to cell death. Induction of the intrinsic apoptotic pathway occurs instantaneously with increasing mitochondrial membrane permeability and the release of different pro-apoptotic proteins, such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF), which disrupts the balance between anti-apoptotic and pro-apoptotic proteins, leading to caspase-dependent and/or independent cell death 16,17. Therefore, induction of cell cycle arrest and apoptosis has been considered a promising approach for treating BC 15,18,19

Sanguinarine (SANG), a benzophenanthridine alkaloid derived from the rhizomes of Sanguinaria canadensis plants, has remarkable biological activity and anticancer potential 17. The anticancer effect of SANG has been strongly linked with its ability to induce cell death via the extrinsic and intrinsic apoptotic pathways 17,20, as well as to induce DNA fragmentation 21-24. SANG has been reported to induce apoptosis in prostate cancer 25-47 and BC cells 48-52. SANG can also induce apoptosis via free radical initiation and mitochondrial dysfunction 31,32. These mechanisms affect different proteins, including signal transducer and activator of transcription 3 (STAT3), p53, B-cell lymphoma 2 (BCL2) family members, caspases, inhibitor of apoptosis family (IAP), and extracellular signal-regulated kinase 1/2 (ERK1/2) 17,53. In MDA-MB-231 TNBC cells, SANG has demonstrated apoptotic effects by upregulating apoptosis-mediated proteins (p27, Cyt-c, and truncated BH3 interacting domain death agonist (tBID)), while inhibiting others (cyclin D1, STAT3, x-link IAP; XIAP, cIAP-1, cIAP-2, and BCL2; CASP8 and FADD-like apoptosis regulator; and c-FLIP/CFLAR) 49,51-54.

Resistance to chemotherapy-induced cytotoxicity is a major challenge in cancer therapy 17. Therefore, drug combinations are crucial to achieve significant synergistic therapeutic effects 55. In various chemotherapeutic drug-resistant cancer cells, SANG has been shown to enhance the anticancer effects of multiple drugs 56,57, including doxorubicin 58 and paclitaxel 27. In MDA-MB-231 BC cells, SANG can synergize with TNF-related apoptosis-inducing ligand (TRAIL)-linked apoptosis 52. Further, the combination of SANG with a sub-lethal dose of digitonin induces an increased cytotoxic effect in MCF-7 BC cells 59.

Although the anti-apoptotic effects of SANG in the MDA-MB-231 TNBC cell model have been reported, its effects on MDA-MB-468 cells, a phenotypically distinct TNBC cell line, have not yet been studied. Therefore, this comparative study aimed to determine the link between phenotype-related gene expression and sensitivity to SANG in these two TNBC models. We also analyzed the impact of SANG on genes mediating apoptosis in both models under identical experimental settings using a human apoptosis gene expression array.

2. Materials And Methods

2.1 Cell culture and media

The human TNBC cell lines MDA-MB-231 (ATCC® HTB-26™) and MDA-MB-468 (ATCC® HTB-132™) were purchased from ATCC (Manassas, VA, USA). MDA-MB-231 and MDA-MB-468 cells were derived from Caucasian American (CA) and African American (AA) individuals, respectively. Cell culture media and supplements were purchased from ATCC (VWR International, Radnor, PA, USA), Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and Thermo Fisher Scientific (Waltham, MA, USA). Both cell lines were grown as monolayers in tissue culture flasks and maintained at 37°C with 5% CO2 in a humidified environment. The TNBC cell lines were cultured in Dulbecco's modified Eagle’s medium (DMEM) containing 4 mM L-glutamine, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (1% P-S). The media were changed as required after washing the cells with Dulbecco's phosphate-buffered saline (DPBS), and the cells were sub-cultured using phenol-free trypsin/ethylenediaminetetraacetic acid (EDTA, 0.25%). Experimental media supplemented with 2.5% heat inactivated FBS were used for all assays.

2.2. Cell viability assay

The effect of SANG on the viability MDA-MB-231 and MDA-MB-468 cell viability was determined using Alamar Blue® (AB®, Sigma-Aldrich, St. Louis, MO, USA) as previously described 60. The alkaloid compound sanguinarine chloride hydrate ≥ 98% (HPLC) was purchased from Sigma-Aldrich and reconstituted at a concentration of 15 mM in dimethyl sulfoxide (DMSO, ATCC), aliquoted, and stored at -20°C for later use. TNBC cells were seeded in quintuplicate at 5 × 104 cells/100 µL/well in 96-well plates and incubated overnight at 37°C in a humidified atmosphere with 5% CO2. TNBC cells were then treated with DMSO (≤ 0.1%) or graded concentrations of SANG (0–5 µM). After 24 h of incubation, 20 µL of AB® resazurin solution (0.5 mg/ml in sterile phenol red-free HBSS) was added to each well followed by further incubation for 4 h at 37°C. The fluorescence intensity, indicating the reduction of resazurin by metabolically active TNBC cells, was measured at an excitation/emission wavelength of 530/590 nm using a Synergy HTX Multi-Mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The data obtained from the cell viability analysis were presented as the average of three independent experiments.

2.3. Cell proliferation assay

The effect of SANG on cell proliferation was evaluated in TNBC cell lines, MDA-MB-231 and MDA-MB-468, using the same protocol as the cell viability assay with essential modifications 60. Briefly, the experiment was initiated with seeding 1 × 104 cells/100 µL/well. The cells were then treated for 48–96 h with SANG at concentrations ranging from 0–2.0 µM. The control wells were treated with DMSO at the highest concentrations (< 0.1%).

2.4. Flow cytometry assays

2.4.1. Cell cycle analysis

Cell cycle evaluation of SANG-treated TNBC cells was performed using a previously described protocol 60. Briefly, cells seeded at 1.5 × 106 cells/4 ml/T25 cell culture flask were incubated overnight under the same incubation conditions. SANG was added in another 2 ml at four concentrations (0–1.5 µM) to each cell line. Cells corresponding to the control wells were treated with DMSO at the highest concentration of SANG (< 0.1%). After 24 h, cells from each treatment group were harvested, centrifuged at 1,000 rpm for 5 min, washed in DPBS, fixed in cold 70% ethanol, and kept in the refrigerator for at least four hours. The suspended cells were gently vortexed, pelleted, and washed twice with Dulbecco’s phosphate-buffered saline. Finally, the cells were resuspended in 200 µl 1X propidium iodide (PI) with RNase staining solution (Abcam, Cambridge, MA, USA) and incubated for 30 min at 37°C in the dark. The cell distributions across the cell cycle were established using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The data presented were generated from three independent analyses for each cell line.

2.4.2. Apoptosis assay

To establish the apoptotic effect of SANG in TNBC cells, we followed a previously described protocol 60 using the Annexin V-FITC (Ann FITC) Apoptosis Detection Kit Plus (Ray-Biotech, Norcross, GA, USA). Briefly, MDA-MB-231 and MDA-MB-468 cells were seeded at 5 × 105 cells/2 ml/well in 6-well plates and incubated overnight. The cells were treated for 24 h with optimized doses of SANG ranging from to 0–4.5 µM for MDA-MB-231 cells and 0–4.0 µM for MDA-MB-468 cells. The corresponding control wells were exposed to DMSO at a concentration equivalent to that in the highest tested dose (< 0.1%). After the pre-designated experimental period, cells from each well were collected, centrifuged, and washed in DPBS. In another set of tubes, cell pellets were resuspended in 500 µL of 1X Annexin V binding buffer, and sequentially labeled for 10 min with 5 µL each of Ann FITC and PI. A flow cytometer was used to analyze apoptosis in 1×104 events/sample. Unstained samples indicated live cells. Annexin V-stained cells were considered apoptotic, whereas cells in the late apoptotic or necrotic phases were positive for both Annexin-V and PI. Cell-Quest software was used to determine the percentage of apoptotic and necrotic cells in each treatment.

2.5. Gene expression analyses

2.5.1. Treatment of TNBC cells

Two T75 flasks seeded with 10 × 106 cells/10 ml for each line were designated as either DMSO-or SANG-treated cells. Each cell line was treated with the compound at the specified concentration corresponding to its IC50 value obtained from the viability study (3.5 µM in MDA-MB-231 cells and 2.6 µM in MDA-MB-468 cells) 61, 62, following overnight incubation. Control cells were treated with DMSO at a concentration equivalent to that in the tested SANG concentration (˂ 0.1% DMSO). After 24 h of treatment with or without SANG, the cells from each flask were collected in a fresh tube, pelleted, washed with DPBS, and stored at -80°C for later use.

2.5.2. RNA extraction

According to the manufacturer’s guidelines, RNA was extracted from previously frozen cell samples in 1 ml of TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and homogenized briefly for 20 s. Then, 0.2 ml of chloroform (Sigma-Aldrich) was added to each vial for phase separation, vortexed, and incubated for 3 min at room temperature. All samples were then centrifuged for 15 min at 10,000 × g and 8°C. The RNA-rich upper layer was transferred to a fresh tube with 0.5 ml of 2-propanol to precipitate the RNA. The obtained pellets were washed with 75% ethanol, air-dried for 10 min, and reconstituted in 50 µL nuclease-free water.

2.5.3. Complementary DNA (cDNA) synthesis

The purity and concentration of RNA from each sample were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific, Inc.). Next, 5 µg/ml of RNA was incubated with a 1X DNase cocktail using a DNA-free kit ((Thermo Fisher Scientific) for 30 min at 37°C. The reaction was terminated by adding a DNase inactivator. All samples were then centrifuged at 9,000 rpm for 3 min to precipitate the unwanted cellular DNA and the DNA-free supernatant was collected for cDNA synthesis by reverse transcription (RT). cDNA was generated using the iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Each well of 96-well PCR plates was loaded with 5 µL of DNA-free RNA, 9 µL of nuclease-free water, and 6 µL of advanced reaction mix reverse transcriptase cocktail. The RT reaction was performed as follows: RT for 20 min at 46°C and RT inactivation for 1 min at 95°C. The cDNA obtained from each sample was stored at -80°C for the PCR assay.

2.5.4. Quantitative reverse transcriptionpolymerase chain reaction (qRTPCR) apoptosis array

A 96-well human apoptosis array (SAB Target List, cat #10034106, Bio-Rad) was loaded with 10 µL of cDNA (2.3 ng) and Sso Advanced™ Universal SYBR® Green Supermix (Bio-Rad) for a total volume of 20 µL/well. The plate was briefly shaken for 5 min and centrifuged at 1,000 × g; fluorescence quantification was performed using the Bio-Rad CFX96 Real-Time System (Bio-Rad). cDNA was amplified through 39 cycles of denaturation beginning with 30 s of activation at 95°C, 10 s of denaturation at 95°C, and 20 s of annealing at 60°C. The final extension step was completed at 65°C for 31 s. The qRT-PCR data were confirmed for each cell line using at least three independent experiments.

2.6. Statistical analysis.

The data obtained from this study were analyzed using GraphPad Prism 6.2 software (GraphPad Software, Inc., San Diego, CA, USA). Data are expressed as mean ± SEM from three biological replicates. The IC50 values were calculated using a nonlinear regression model of log (inhibitor) vs. the normalized response-variable slope in the software with the R2 best fit and the lowest 95% confidence interval. An Excel spreadsheet was used to calculate the IC50 ± SEM average of biological replicates. Cell cycle distribution and apoptosis data were analyzed using CellQuest software (BD Biosciences, San Jose, CA, USA). CFX 3.1 Manager software (Bio-Rad) was used to quantify gene expression in the apoptosis arrays. The significance of differences was determined using analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. Unpaired Student’s t-test was used to analyze two datasets. One-way analysis of variance was used to compare more than two groups. Differences were considered significant at P < 0.05 (as mentioned in the figures and legends).

3. Results

3.1 Sanguinarine cytotoxic effects

The cytotoxic effects of SANG in MDA-MB-231 and MDA-MB-468 TNBC models were examined using AB® assays to detect metabolically active cells. Figure 1 shows the dose-dependent inhibition of cell viability in both cells following 24 h exposure to SANG. The IC50 values indicate that MDA-MB-468 cells are more sensitive to SANG (IC50 = 2.60 µM) than MDA-MB-231 cells (IC50 = 3.56 µM). MDA-MB-468 cells showed a highly significant decrease of 20% (P < 0.0001) in cell viability at all tested concentrations, starting with 1µM µM SANG.

The same effect was noted at 2.5 µM in MDA-MB-231 cells. This finding confirmed a 2.5-fold greater sensitivity in MDA-MB-468 cells than in MDA-MB-231 cells.

3.2. Sanguinarine inhibits the proliferation of triple-negative breast cancer cells

The antiproliferative effect of SANG was evaluated in both MDA-MB-231 and MDA-MB-468 TNBC cells using the AB® assay to determine the effect of the compound on the cellular metabolic activities. In both cell lines, SANG inhibited proliferation in a dose- and time-dependent manner compared with that in DMSO-treated control cells (Fig. 2a and b). Inhibition of cell proliferation was validated by the decrease in IC50 values at different periods of exposure (48–96 h). The effect of SANG at different exposure periods vs. the control indicated a similar response in both cell lines, with MDA-MB-468 being more sensitive. Indeed, a highly significant reduction (P < 0.0001) in cell proliferation was found with 0.25 µM in MDA-MB-468 cells and with 0.75 µM in MDA-MB-231 cells. Furthermore, a highly significant difference (P < 0.0001) was also observed between different exposure periods, except for a non-significant difference between the 48 h vs. 72 h treatment periods in MDA-MB-468 cells (NS; Fig. 2b).

3.3. Sanguinarine affects cell cycle progression in triple-negative breast cancer cells

To test the hypothesis that cell cycle blockade mediates SANG-induced cytotoxic and antiproliferative effects, we performed flow cytometric analysis using the fluorescent probe PI. Following 24 h exposure, the response of the SANG-treated cells was compared with that that of DMSO-control cells (Fig. 3a and b). The figures showed only a slight change in cell distribution

among the three phases of the cell cycle in the MDA-MB-468 cells (Fig. 3b). In contrast, significant dose-dependent changes (P < 0.05–P < 0.0001, Fig. 3a) were observed in the SANG-treated MDA-MB-231 cells. At the highest tested concentration of 1.5 µM, the DNA histogram exhibited a sub-G1 phase (~ 25%) in MDA-MB-231 cells (Fig. 3a), which suggested the presence of dead cells, but this was not detected in MDA-MB-468 cells (Fig. 3b). A significant decrease in the G0/G1 phase (20%; P < 0.05–P < 0.001) was observed in MDA-MB-231 cells compared with only a minor reduction (~ 6%; P < 0.01–P < 0.001) in MDA-MB-468 cells. Consequently, MDA-MB-231 cells arrested at the S phase and G2/M phase increased by approximately 15% and 5%, respectively (P < 0.001-P < 0.0001; Fig. 3a). Meanwhile, lower concentrations of SANG (0.5 and 1.0 µM) induced less than 10% increase in the S-phase, accompanied with a minor but significant accumulation (P < 0.01, Fig. 3b) in the G2/M phase of up to 7%, at 1.5 µM SANG. Thus, these data implicate cell cycle arrest in the SANG-induced antiproliferative effects observed in MDA-MB-231 cells. However, in MDA-MB-468 cells, this is not the leading mechanism, and other mechanisms may be involved.

3.4. Sanguinarine triggers apoptosis in triple-negative breast cancer cells

Flow cytometric analysis was performed to determine whether apoptosis was involved in the reduced in cell viability and proliferation rate by SANG. For this assay, TNBC cells were treated with SANG at various doses (2.5–4.5 µM in MDA-MB-231 cells and 1–4 µM in MDA-MB-468 cells). A significant increase in apoptotic cells was observed in SAN-treated cells compared to the control (P < 0.001–P < 0.0001; Fig. 4a and b). Notably, a slow rise in apoptosis across the tested concentrations was found from 3.0–4.5 µM SANG in MDA-MB-231 cells and 2.0–4.0 µM SANG in MDA-MB-468 cells. The AA model (MDA-MB-468) cells were approximately 3-fold more sensitive to SANG than MDA-MB-231 cells. At the lowest tested concentration (2.5 µM), SANG induced apoptosis in 30% of the treated MDA-MB-231 cells compared with the control (Fig. 4a), whereas 80% of the analyzed MDA-MB-468 cells were in the apoptotic phase following exposure to 2.0 µM SANG (Fig. 4b). Notably, at 4 µM of SANG, a higher percentage of necrotic cells was detected in MDA-MB-231 cells than in MDA-MB-468 cells (27% vs.8%, respectively), indicating the tendency of CA TNBC cells to undergo necrosis rather than apoptosis. Overall, the data suggest that apoptosis is a leading mechanism that mediates cell death in SANG-treated MDA-MB-468 cells.

3.5. Gene expression profile in sanguinarine-treated triple-negative breast cancer cells

The mechanism underlying the profound apoptotic effects in SANG-treated TNBC cells was further investigated using qRT-PCR. Following a previously described protocol 61,62, cells were treated for 24 h with a specific dose of SANG equivalent to their IC50 values (3.5 µM for MDA-MB-231 cells and 2.6 µM for MDA-MB-468 cells; Fig. 1). An overview of the normalized apoptosis-related gene expression provided insight into the impact of SANG on various genes that regulate the apoptotic pathway (Fig. 5a and b). In the figure, the red dots indicate the upregulated genes in both cell lines. The green-colored dots representing the downregulated genes were observed only in MDA-MB-231 cells (Fig. 5a). The black dots in the middle panel indicate the range of unchanged gene expression in both cell line

Table I. Fold-change of mRNA gene expressions after 24h exposure to the alkaloid compound sanguinarine (SANG) at the corresponding IC50 s of 3.5 µM in MDA-MB-231 and 2.6 µM in MDA-MB-468 TNBC cells.

Control vs. treated MDA-MB-231 cells

  

Control vs. treated MDA-MB-468 cells

Target gene

Fold (+/-)

p-value

  

Target gene

Fold (+)

p-value

LTA

+ 15.0

0.0122

  

TNFRSF11B

+ 4.41

0.0317

BAG3

+ 9.22

0.0114

  

TNFRSF25

+ 3.30

0.0023

BCL2L11

+ 4.67

0.0404

  

HRK

+ 3.21

0.0289

GADD45A

+ 4.17

0.0324

  

BCL2L11

+ 3.17

0.0047

BCL2A1

LTBR

TP53

AKT1

CASP6

BCL2L1

GUSB

BIRC5

+ 3.65

− 11.0

− 3.74

− 3.35

− 3.23

− 3.13

− 3.10

− 1.85

0.0007

0.0227

0.0196

0.0126

0.0031

0.0028

0.0187

0.0152

  

TNFRSF10B

DFFA

NOD1

TNFRSF10A

TNFRSF21

BIRC3

BAX

DAPK1

CFLAR

CASP10

FADD

TRADD

TRAF2

CASP1

+ 2.80

+ 2.57

+ 2.56

+ 2.45

+ 2.42

+ 2.39

+ 2.28

+ 2.22

+ 2.20

+ 2.14

+ 2.13

+ 1.96

+ 1.95

+ 1.85

0.0070

0.0082

0.0272

0.0015

0.0091

0.0047

0.0008

0.0112

0.0054

0.0105

0.0141

0.0002

0.0114

0.0384

MDA-MB-231 cells (left panel) showed mixed alterations in their mRNAs. The upregulated mRNAs: lymphotoxin alpha (LTA), BCL2-associated athanogene 3 (BAG3), BCL2 like protein 11 (BCL2L11), the growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BCL2-related protein A1 (BCL2A1) and the repressed mRNAs, including lymphotoxin beta receptor cells, (LTBR), tumor protein p53 (TP53), BCL2 like protein 1 (BCL2L1), AKT serine/threonine kinase 1 (AKT1), CASP6, glucuronidase beta (GUSB), and baculoviral inhibitor of apoptosis family (IAP) repeat-containing 5 (BIRC5). In contrast, MDA-MB-468 cells gene expression analysis (right panel), showed only a significant upregulation in the mRNA of 18 genes, including TNF (tumor necrosis factor) receptor superfamily (TNFRSF) 11B and 25, harakiri BCL2 Interacting Protein (HRK), B-cell lymphoma 2 (BCL2) Like protein 11 (BCL2L11), TNFRSF10B, deoxyribonucleic acid (DNA) fragmentation factor subunit alpha (DFFA), nucleotide-binding oligomerization domain-containing protein 1 (NOD1), TNFRSF10A /21, baculoviral (IAP) repeat-containing 3 (BIRC3), BCL2 associated X-protein (BAX), death-associated protein kinase 1 (DAPK1), CASP8 and FADD-like apoptosis regulator (CFLAR), ), caspase (CASP)10, FAS-associated via death domain (FADD), TNFRSF1A associated via death domain (TRADD), TNF receptor associated factor 2 (TRAF2), and CASP1.

Several upregulated genes with significant roles in apoptosis were identified in MDA-MB-468 cells. This finding supports our previous flow cytometry data that indicate apoptosis as the leading mechanism activated by SANG to inhibit cell proliferation in this AA model. Indeed, after 24 h of exposure to 2.6 µM SANG, a total of 18 genes were significantly upregulated in the MDA-MB-468 cell model (P < 0.05–P < 0.001) with a 1.85-4.41-fold increase in their transcriptomic levels (Fig. 6a-d and Table 1). These augmented genes belonged to different protein families, as shown in Fig. 6. Eight members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) were identified, including TNFRSF11B with the highest fold increase (+ 4.41), TNFRSF25, TNFRSF10B/A, TNFRSF21, FAS-associated via death domain (FADD), TNFRSF1A associated via death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2) (Fig. 6a). Further, two members of the caspase (CASP) family, CASP1 and 10 were upregulated in MDA-MB-468 cells (Fig. 6b), in addition to three members of the BCL2 family: BCL2 like protein 11 (BCL2L11), harakiri BCL2 interacting protein (HRK), and BCL2 associated X-protein (BAX) (Fig. 6c). More than two-fold increase was observed in five other genes, including the deoxyribonucleic acid (DNA) fragmentation factor subunit alpha (DFFA), baculoviral IAP repeat-containing 3 (BIRC3), nucleotide-binding oligomerization domain-containing protein 1 (NOD1), CFLAR, and death-associated protein kinase 1 (DAPK1) (Fig. 6d).

In contrast, the response of MDA-MB-231 cells to the compound was different. Fewer apoptosis-related genes were activated (Fig. 7a and b), with a significantly higher fold increase than in MDA-MB-468 cells. When exposed to 3.5 µM of SANG, the mRNA of five genes was remarkably augmented by 3.65–15.0-fold (Fig. 7a). Lymphotoxin alpha (LTA) was the most abundant gene (15-fold), followed by BCL2-associated athanogene 3 (BAG3), BCL2L11, growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BCL2-related protein A1 (BCL2A1). BCL2L11 was the only commonly upregulated gene in both cell models, but its upregulation was higher in MDA-MB-231 cells than in MDA-MB-468 cells (4.67 vs. 3.17-fold).

In contrast to MDA-MB-468 cells, the other seven genes were significantly downregulated in MDA-MB-231 cells (Fig. 7b), with lymphotoxin beta receptor cells (LTBR) being the most profoundly attenuated (-11.0-fold). A less than 4-fold decrease was observed in four genes, including the tumor protein p53 (TP53), AKT sereness/threonine kinase 1 (AKT1), CASP6, BCL2

like 1(BCL2L1), and glucuronidase beta (GUSB), in addition to the least repressed gene, baculoviral (IAP) repeat-containing 5 (BIRC5, -1.85-fold).

MDA-MB-231 cells (left panel) show mixed mRNA alterations. The upregulated mRNAs were lymphotoxin alpha (LTA), BCL2-associated athanogene 3 (BAG3), BCL2 like protein 11 (BCL2L11), growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BCL2-related protein A1 (BCL2A1), and the repressed mRNAs were lymphotoxin beta receptor cells (LTBR), tumor protein p53 (TP53), BCL2 like protein 1 (BCL2L1), AKT serine/threonine kinase 1 (AKT1), CASP6, glucuronidase beta (GUSB), and baculoviral inhibitor of apoptosis family (IAP) repeat-containing 5 (BIRC5). In contrast, MDA-MB-468 cells (right panel) only showed significant upregulation of 18 genes, including TNF (tumor necrosis factor) receptor superfamily (TNFRSF) 11B, TNFRSF 25, harakiri BCL2 Interacting Protein (HRK), B-cell lymphoma 2 (BCL2)-like protein 11 (BCL2L11), TNFRSF10B, deoxyribonucleic acid (DNA) fragmentation factor subunit alpha (DFFA), nucleotide-binding oligomerization domain-containing protein 1 (NOD1), TNFRSF10A /21, baculoviral (IAP) repeat-containing 3 (BIRC3), BCL2 associated X-protein (BAX), death-associated protein kinase 1 (DAPK1), CASP8, FADD-like apoptosis regulator (CFLAR), caspase (CASP)10, FAS-associated via death domain (FADD), TNFRSF1A associated via death domain (TRADD), TNF receptor-associated factor 2 (TRAF2), and CASP1.

4. Discussion

Naturally occurring compounds are promising therapeutic agents for managing different cancer types, including BC. The current study was designed to investigate the mechanism underlying the anticancer effect of the natural alkaloid SANG in two genetically different models of TNBC cells (Fig. 8). A panel of assays was performed to define the mechanism employed by SANG in these two cell lines. Consistent with other studies, our data strongly support the potency of 0–10 µM SANG in decreasing cell viability, proliferation rate, as well as cell cycle arrest and apoptosis induction in various cell models 21,25,26,30,39,42,54,63−67. Remarkably, the obtained data indicate a higher cytotoxic potency in MDA-MB-468 cells than in MDA-MB-231 cells (Fig. 1). The compound showed substantial potential to decrease the proliferation rate (Fig. 2) in MDA-MB-468 cells compared to that in MDA-MB-231 cells. The difference in cell density is suggested as the main contributing factor leading to the obtained inhibition of cell proliferation meanwhile not affecting cell viability. Furthermore, SANG induced different patterns of cell cycle arrest in both cells, which manifested as high cell cycle interruption and a tendency to cause necrosis in MDA-MB-231 cells whereas MDA-MB-468 cells showed mild cell cycle arrest without necrosis. Indeed, further protein studies are required to interpret the effect of the compound on different phases. SANG can alter expression of various genes, orchestrating both intrinsic and extrinsic apoptosis pathways by employing different mechanisms in the two cell lines. The phenotypic differences in these TNBC models could provide a rationale for the different mechanisms and potency outcomes that favor the MDA-MB-468 model. MDA-MB-231 and MDA-MB-468 cells are classified as TNBC cells; however, they have a different molecular profile. Generally, the incidence of TNBC is higher in AA compared with CA. The claudin-low MDA-MB-231 cells are triple-negative/basal-B mammary carcinoma, while the MDA-MB-468 cells are triple-negative/basal-A mammary carcinoma. Also, MDA-MB-231 cells are characterized by activating KRAS mutations and the protooncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog B (BRAF). Compared with CA, mutation of KRAS and BRAF are not found in MDA-MB-468 cells 68. The AA women exhibit epidermal growth factor receptor (EGFR) amplification and mutated phosphatase tensin homolog (PTEN), with higher somatic copy number alterations (CNA) segments and TP53 mutation, as well as a higher expression of the proliferative marker Ki-67 69,70. Meanwhile, a lower proportion of Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) and DNA methylation levels is revealed in AA than in CA 71–74. A less frequency of BRCA1, the tumor suppressor gene-mediating DNA repair, the mutation is found among AA women compared with its counterpart CA 75,76.

The ultimate goal of chemotherapeutic agents is to control cell cycle progression and enhance apoptosis 10. Indeed, a close association between apoptosis and the cell cycle has been established 77. Moreover, our cell cycle distribution analyses (Fig. 3) indicate a relatively discrete response in the two TNBC cell lines with respect to apoptosis (Fig. 4). In MDA-MB-468 cells, the minor response in the three phases indicates that cell cycle arrest was not the principal mechanism underlying the profound apoptotic effects in SANG-treated MDA-MB-468 cells. Therefore, we suggest the involvement of another apoptotic mechanism activated by SANG. In contrast, cell cycle arrest-mediated apoptosis was detected in MDA-MB-231 cells as evidenced in all phases, particularly the S-phase, and the most distinctive appearance of the sub-G1 peak, which is considered a biomarker for DNA damage-mediated apoptosis. Also, S-phase arrest is concord with a decreased G1/G0 and G2/M phases, reduced DNA synthesis, and it is the lead of reduced proliferation and cell viability 78,79.

In our study, transcript analysis of apoptosis-related genes in SANG-treated TNBC cells indicated that SANG has the potential to impact various genes by modulating both intrinsic and extrinsic pathways (Figs. 5–7). The mRNA expression profile showed a greater number of altered genes in MDA-MB-468 cells and confirmed the higher vulnerability of this cell model compared to MDA-MB-231 cells. The data showed that the 18 most significantly affected genes in MDA-MB-468 cells were upregulated. Meanwhile, SANG-treated MDA-MB-231 cells exhibited significant upregulation in the mRNA expression of five genes, and downregulation of seven genes, revealing the existence of two different mechanisms underlying the apoptotic pathway. Therefore, our results suggest a close association between cell genotype and gene expression changes in SANG-treated TNBC cells.

In the TNBC models investigated, SANG altered the expression of three caspase family members. In MDA-MB-468 cells, an almost 2-fold increase in the mRNA levels of CASP1 and CASP10 was observed. In contrast, only one caspase (CASP6) was affected in MDA-MB-231 cells and its expression was significantly downregulated. Caspases are cysteine-related aspartate proteases that are expressed in immune and non-immune cells 80. Under normal physiological conditions, caspases mediate both the intrinsic and extrinsic apoptosis pathways to sustain cellular homeostasis 12,81. According to their cellular functions, caspases are classified as initiator, effector, or inflammatory caspases 82. CASP1 is a member of the inflammatory caspase family and has a unique function that is distinct from other apoptotic caspases 80. In response to infection, CASP1 triggers pyroptosis, a type of cell death, as an innate immune mechanism that activates IL-1β and IL-18 82. Distinct from normal tissues, low CASP1 expression has been detected in various types of cancer cells, including BC. Inhibiting CASP1 expression was previously found to decrease apoptosis and promote proliferation, invasion, and progression in MDA-MB-231 cells 83. In contrast, an elevated level of CASP1 in fibroblasts is known to induce apoptosis and cell death 84. In MCF7 BC cells, the initiator caspase CASP10 sensitizes cells to TRAIL-induced apoptosis 85. Previous reports have also suggested the anticipated role of CASP1 with CASP10 in inducing intrinsic apoptotic pathways by activating BID and increasing the mitochondrial release of Cyt-c 86–89. In contrast, repression of CASP6 in MDA-MB-231 cells indicated resistance to apoptosis (Fig. 7b). Indeed, CASP6 is a downstream effector and executioner caspase that enhances apoptosis by activating various cellular proteins 90. These findings support the role of CASP1 and CASP10 in promoting apoptosis in SANG-treated MDA-MB-468 cells and explain the weaker apoptotic response exhibited by MDA-MB-231 cells.

Three members of the BCL2 family were found to be upregulated in the TNBC cells in this study. The mRNA of BCL2L11 was significantly increased in both TNBC cell models (Fig. 6c and 7a), whereas upregulated HRK and BAX were exclusive to MDA-MB-468 cells (Fig. 6c). The BCL2 family regulates the intrinsic apoptotic pathway through two main groups of proteins: pro- and anti-apoptotic proteins 13. A balance between these two groups is essential for maintaining mitochondrial membrane integrity 91. As a typical mechanism for resisting apoptosis, cancer cells upregulate anti-apoptotic proteins and downregulate pro-apoptotic proteins 92. Previous studies using different cell lines have attributed the antiproliferative and proapoptotic effects of SANG to the imbalance between pro- and anti-apoptotic proteins 42.

In agreement with these findings, we suggest that SANG induces intrinsic apoptosis by altering the expression of various BcL-2 family members. One of the different mechanisms employed by cancer cells is the suppression of BCL2L11 (also known as BIM), which leads to tumor growth, metastasis, and drug resistance 93,94. In contrast, forced upregulation of this gene in BC cells is known to alter the balance of BCL2 proteins to the apoptotic phenotype and the release of Cyt-c and Smac/DIABLO, which activate the caspase cascade 95. Indeed, the pro-apoptotic gene BCL2L11 contains BH3, a crucial factor in apoptosis induction 96. Moreover, the dual function of BCL2L11 in regulating autophagy and apoptosis may overcome the challenge of chemotherapeutic drug resistance 97,98. Therefore, in treating BC subtypes, chemotherapeutic drugs such as doxorubicin and paclitaxel regulate BCL2L11 expression and its signaling pathways as a potential mechanism apoptotic cell death 93,99. Upregulated expression of HRK selectively abolishes the function of anti-apoptotic proteins and stimulates intrinsic mitochondrial apoptosis 100,101. The role of overexpressed HRK in inducing both intrinsic and extrinsic apoptosis pathways has been demonstrated in various cancer cell types, including BC cells 102–106, and is strongly linked to the existence of BH3 96. Furthermore, the ability of SANG to upregulate the effector pro-apoptotic protein BAX has been demonstrated in various cell models 31,33,34,37,42,107,108. The upregulation of BAX mRNA has been demonstrated to antagonize the anti-apoptotic role of other BCL2 family members, leading to an increase in mitochondrial membrane permeability and cytochrome c release preceding caspase activation and apoptosis 109. Arguably, we suggest that SANG induces intrinsic apoptosis by upregulating the expression of various proapoptotic members of the BCL2 family.

In MDA-MB-231 cells, two anti-apoptotic genes, BCL2L1 and BCL2A1, were inversely altered (Fig. 7a and b), in addition to the significantly upregulated binding protein, BAG (Fig. 7a). Meanwhile, the mRNA of BCL2L1 was downregulated, which is consistent with previously reported studies 40,42, and surprisingly, BCL2A1 mRNA was significantly increased. In various cancer types, including TNBC, highly upregulated BCL2A1 and BCL2L1 (also known as BCL-XL) are closely associated with resistance to targeted agents and chemotherapeutic drugs 110–115. Compared with normal breast tissues, upregulated BCL2L1 levels are linked with BC initiation and progression, particularly at a higher grade of the disease, and are most likely associated with migration and metastasis 116–118. Furthermore, various mechanisms are associated with apoptosis induction in BC, including BCL2L1 pathway modulation 119, changing the BAX/BCL2L1 ratio 120, and using BCL2L1 antisense oligonucleotide 121. Moreover, BAG3 protein is considered a standard biomarker in specific cancer cells 122. Overexpression of BAG3 in various cancer types, including BC 123–127, inhibits apoptosis 128 while promoting proliferation 129 and chemotherapy resistance 130. Thus, inhibiting BAG3 expression is suggested as a promising strategy for cancer therapy 122. Regulation of this gene in MDA-MB-231 cells could weaken the apoptotic effect of SANG in this model. Thus, we collectively suggest the implication of BCL2A1 and BAG3 upregulation in the relative resistance of MDA-MB-231 cells to SANG-induced intrinsic apoptosis compared with that in MDA-MB-468 cells.

Distinct from MDA-MB-231 cells, eight members of TNFRSF were significantly upregulated in SANG-treated MDA-MB-468 cells, including four death receptors (DRs): TNFRSF25 (DR3), TNFRSF10A (DR4), TNFRSF10B (DR5), and TNFRSF21 (DR6), as well as TNFRSF11B, FADD, TRADD, and TRAF2 (Fig. 6a). These death receptors on the cell surface transmit apoptotic signals once they bind to their specific death ligands 131. Simultaneously, the advantage of inducing a selective apoptotic effect in cancer cells without harming healthy cells has led to the TRAIL-mediated apoptotic pathway being considered as a promising approach in cancer therapy 132. Understanding the involvement of various TNFRSF family members in apoptosis has elucidated the prospects of modulating these proteins in cancer treatment 133. Moreover, in BC, as well as in various other cancer cells, the adaptor molecules, FADD and TRADD, were previously found to interact with upregulated TNFRSF25 and TNFRSF10A/B to enhance apoptosis by triggering TRAIL-mediated apoptosis 132,134−138. These mechanisms induce various cellular signaling pathways, such as caspase activation, MAPK, and NF-κB 132,139−141. Augmentation of all these TNFRSF genes in MDA-MB-468 cells in our study strongly supports the previous findings. Therefore, we suggest that SANG induces the extrinsic apoptosis pathway via TRAIL-binding to its death receptor TNFRSF10A/B in SANG-treated MDA-MB-468 cells. In line with other findings in BC 142, upregulation of FADD mRNA in our study could also mediate the apoptotic effect observed in MDA-MB-468 cells. The ability of TNFRSF21 to induce apoptosis 143, probably through the mitochondria-mediated intrinsic pathway and BAX interaction 144, suggest TNFRSF21 and BAX upregulation as one of the mechanisms underlying apoptosis induction in SANG-treated MDA-MB-468 cells.

Our mRNA analysis also indicated upregulation of TRAF2 and TNFRSF11B mRNAs in MDA-MB-468 cells (Fig. 6a). Recent studies have demonstrated the involvement of dually functional TRAF2 in both pro- and anti-apoptotic signals 145,146. TNFRSF11B (also known as osteoprotegerin, OPG) is a well-known prognostic marker for BC 147,148. Overexpression of this gene induces apoptosis resistance and enhances cancer cell viability, invasion, metastasis, and indicates poor prognosis 149,150. Similarly, upregulated TRAF2 induces apoptosis resistance by enhancing TNF-alpha-mediated activation of several pathways such as MAPK8/JNK and NF-ƙB. Hence, the transcriptomic upregulation of both TNFRSF11B and TRAF2 could weaken the apoptotic potency of SANG in MDA-MB-468 cells. However, further investigation of the exact mechanism involving TRAF2 in SANG-treated MDA-MB-468 cells is warranted.

In SANG-treated MDA-MB-468 cells, more than 2-fold upregulation was observed in the apoptosis-mediated gene, CFLAR (Fig. 6d). The role of this gene as an apoptosis inducer or inhibitor 151,152 is controversial, most likely because of its various isoforms. CFLAR has demonstrated the potency to inhibit the DR-induced apoptosis pathway, which inhibits CASP8 stimulation 152–154. Therefore, the role of CFLAR as an anti-apoptotic gene was anticipated in our study, mainly with unchanged expression of CASP8 in SANG-treated MDA-MB-468 cells.

Two members of the IAP family, BIRC3 (cellular IAP2) and BIRC5 (survivin) 155,156, were inversely altered in SANG-treated TNBC cells (Fig. 6d and 7b). SANG treatment upregulated the expression of BIRC3 mRNA in MDA-MB-468 cells and downregulated BIRC5 levels in MDA-MB-231 cells. The IAP family is known to regulate the intrinsic and extrinsic apoptotic pathways, in addition to playing a minor role in the execution phase of apoptosis 156. A recent study on invasive breast carcinomas negated the role of BIRC3 in regulating any of these apoptotic pathways 155. Two pro-oncogenic proteins 156, characterized by baculoviral IAP repeat (BIR) domains 155, are known to be involved in various signaling pathways that regulate cell viability, proliferation, differentiation, and apoptosis 157. Elevated expression of these genes was previously detected in MDA-MB-231 and MDA-MB-468 TNBC cells, compared with that in normal breast cells 158–160 and was closely associated with resistance to apoptosis induction and chemotherapeutic efficacy 156,161−163. Therefore, the obscure role of BIRC3 upregulation in SANG-treated MDA-MB-468 cells require further investigation, even though BIRC5 suppression in MDA-MB-231 cells could mediate apoptosis.

In MDA-MB-468 cells, only the pro-apoptotic gene DAPK1 was significantly upregulated by SANG (Fig. 6d). DAPK1 is involved in cell proliferation, autophagy, and immune response 164–166. This gene is characterized by both the kinase domain and C-terminal death domain 167. Low levels of DAPK1 have been determined in various cancer types compared to control cells 168. In addition, forced DAPK1 upregulation through TNF-α or INF-γ 167 inhibits anti-apoptotic proteins 168 and ultimately induces apoptosis 169. However, a recent study demonstrated a higher expression of DAPK1 in BC cells, notably, in the most aggressive and metastatic TNBC cells with mutated p53, compared to that in healthy breast tissues 170,171. Here, we suggest that DAPK1 upregulation mediates apoptosis in MDA-MB-468 cells, particularly with unchanged anti-apoptotic genes.

Two other pro-apoptotic mRNAs, DFFA and NOD1, were found to be upregulated in SANG-treated MDA-MB-468 cells (Fig. 6d). These genes are crucial for caspase-dependent apoptotic pathways 172–174. In cancer, DFFA (also known as DFF45, DFF1, or ICAD) acts as a substrate for caspase 3, triggering DNA fragmentation during apoptosis 172,173. Reduced expression of DFFA has been measured in various cancer types as a part of an apoptosis-resistance mechanism for enhanced cancer progression 175. Similarly, the protein NOD1-mediates apoptosis pathways by recruiting caspases through its characteristic domains 174 or directly through a RIPK2-dependent mechanism 176. The abolished expression of NOD1 in MCF-7 BC cells is closely associated with an increased estrogen-induced proliferation rate and failure to undergo NOD1-mediated apoptosis, as its overexpression significantly decreased cell proliferation 177–179. Thus, our findings suggest DFFA and NOD1 upregulation is one of the underlying mechanisms downstream of the apoptosis pathway in SANG-treated MDA-MB-468 cells.

In SANG-treated MDA-MB-231 cells, LTBR (also known as TNFRSF3) and its ligand LTA, were inversely and highly altered at -11-fold and + 15-fold, respectively (Fig. 7a, Table I). Analogous to other TNFSF members, these two proteins activate NF-κB signaling, mediating various cellular mechanisms including viability, proliferation, immune response, and apoptosis 180. The dual role of LTA and LTBR in enhancing and suppressing tumor growth has been previously reported in an in vivo model 181–185. Previous studies have demonstrated a crucial role of LTA transcriptomic upregulation in triggering apoptosis 186. In contrast, other studies have suggested that repression of various signaling pathways leads to uncontrolled cancer cell proliferation 187. This perplexing response of LTA and LTBR necessitates further investigation to identify the mechanism of LTA upregulation in MDA-MB-231 cells undergoing apoptosis.

The alkaloid compound SANG upregulates the expression of GADD45A by approximately 4-fold in MDA-MB-231 cells (Fig. 7a, Table I). In TNBC cells, low expression of GADD45A, together with p53 and DNA damage response genes, is linked with the lack of ER, PR, and HER2 expression 188. This inducible stress gene regulates various cellular processes such as the cell cycle, DNA repair, and apoptosis 189 by activating multiple signaling pathways such as the c-Jun amino-terminal kinase (JNK), NF-қB, and p38 MAPK signaling pathways 190–194. Hence, the impact of SANG in MDA-MB-231 cells (Figs. 2 and 4) matches and agrees with the previously reported potential of GADD45A to induce anti-proliferative effects and S-phase cell cycle arrest 189.

Significant inhibition of TP53 mRNA expression by SANG was detected only in MDA-MB-231 cells (Fig. 7b, Table I). Under normal conditions, TP53 responds to various stresses by inducing different cellular processes such as cell cycle arrest, DNA repair, and apoptosis 195. The main function of TP53 is to prevent tumorigenesis and maintain genomic integrity 196. However, mutated TP53 loses its tumor-suppressor function and acquires oncogenic properties that enhance tumor progression 197. Almost 80% of MDA-MB-231 and MDA-MB-468 TNBC patients are diagnosed with mutated TP53. This high level of mutated proteins is closely associated with poor prognosis and resistance to chemotherapy 198,199. Although a previous study revealed an association between the antiproliferative and proapoptotic effects of SANG with the decreased levels of TP5342, others highlighted the strong antiproliferative potential SANG regardless of TP53 status 40. Therefore, TP53 repression could be a significant contributor to apoptosis in the MDA-MB-231 cell model.

Further, SANG showed the potential to attenuate the mRNA expression of GUSB and AKT1 in MDA-MB-231 cells. Upregulated expression of GUSB has been implicated in an increased risk of cancer 200. The chemopreventive effect of GUSB inhibitors has been validated by reduced cell proliferation and apoptosis induction in various cancer types, including BC 200–202. Similarly, a tumor size reduction was found in in vivo models of BC upon combining GUSB inhibitors with the anticancer drug, irinotecan 203. The multifunctional gene, AKT1, is a protein kinase B (AKT) isoform and the downstream effector of phosphatidylinositol 3-kinase (PI3K), which promotes cell growth by phosphorylating and controlling mammalian target of rapamycin (mTOR) signaling, as well as many targets 204–206. Upregulation of AKT1 in MDA-MB-231 cells and in patients with BC promotes proliferation and is closely associated with the aggressive nature of the disease 207–210. The significance of targeting AKT1 has been highlighted in other studies. For example, reduced AKT1 expression delays metastasis by inhibiting the Erb-B2 receptor tyrosine kinase 2 (ErbB2) pathway 209. Silencing AKT1 decreases lung colonization of TNBC cells mediated by apoptosis induction 211. More importantly, knockdown of AKT1 in MDA-MB-231 and MDA-MB-468 TNBC cells can enhance the expression of BCL2L11, a known promoter of apoptosis 211. Therefore, our results strongly support these previous findings, as they suggest an association between AKT1 attenuation and BCL2L11 upregulation in SANG-treated MDA-MB-231 cells (Fig. 7a). Hence, inhibition of AKT1 and GUSB expression in MDA-MB-231 cells 203 could be one of the key mechanistic factors involved in SANG-induced apoptosis.

5. Conclusions

The current study provides insights into the anticancer effects and the underlying molecular mechanism of the natural alkaloid SANG in two genetically different models of TNBC cells: the mesenchymal, MDA-MB-231, and the basal-like 1, MDA-MB-468 cells. Indeed, the variation in molecular profiles between the two cell models including protein and gene expression, somatic DNA copy number alteration (CNA), somatic mutations, and DNA methylation patterns 74, could explain the different responses obtained in the treated cells. SANG significantly decreased the proliferation rate in both TNBC cell lines. In MDA-MB-468 cells, a profound apoptotic effect with very weak cell cycle arrest suggested the involvement of cell cycle arrest-independent apoptosis. Apoptosis-related gene arrays indicated that SANG alters the transcriptomic levels of various genes. Few studies have examined the clinical effect of SANG against cancer, even though herbal beverages with small doses of SANG have been used in folk medicine for treating many respiratory and heart diseases 212,213. The current study highlights the need for further investigation in other TNBC models, in vivo and translational studies on SANG using the obtained data as the basis for developing molecularly targeted therapies to enhance the clinical outcomes of currently used chemotherapeutic agents in patients with TNBC.

Declarations

Author Contributions: Conceptualization, S.S.M. and K.F.A.S.; methodology, S.S.M.; validation, S.S.M., S.N., and T.W.; formal analysis, S.S.M.; investigation, S.S.M., S.N., and T.W.; resources, K.F.A.S.; data curation, S.S.M.; writing—original draft preparation, S.S.M.; writing—review and editing, S.S.M., N.O.Z., and K.F.A.S.; visualization, S.S.M.; supervision, S.S.M. and K.F.A.S.; project administration, S.S.M. and K.F.A.S.; funding acquisition, K.F.A.S. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by a grant from the National Institute on Minority Health and Health Disparities (NIMHD) (U54 MD007582).

Acknowledgments: The authors are grateful for the assistance of Ramesh Badisa, College of Pharmacy and Pharmaceutical Science, Florida A&M University.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Conflicts of Interest: The authors declare no conflicts of interest.

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