Abstract
Background/Aim: Chemotherapy resistance in triple-negative breast cancer (TNBC) cells is well documented. Therefore, it is necessary to develop safer and more effective therapeutic agents to enhance the outcomes of chemotherapeutic agents. The natural alkaloid sanguinarine (SANG) has demonstrated therapeutic synergy when coupled with chemotherapeutic agents. SANG can also induce cell cycle arrest and trigger apoptosis in various cancer cells. Materials and Methods: In this study, we investigated the molecular mechanism underlying SANG activity in MDA-MB-231 and MDA-MB-468 cells as two genetically different models of TNBC. We employed various assays including Alamar Blue to measure the effect of SANG on cell viability and proliferation rate, flow cytometry analysis to study the potential of the compound to induce apoptosis and cell cycle arrest, quantitative qRT PCR apoptosis array to measure the expression of different genes mediating apoptosis, and the western system was used to analyze the impact of the compound on AKT protein expression. Results: SANG lowered cell viability and disrupted cell cycle progression in both cell lines. Furthermore, S-phase cell cycle arrest-mediated apoptosis was found to be the primary contributor to cell growth inhibition in MDA-MB-231 cells. SANG-treated TNBC cells showed significantly up-regulated mRNA expression of 18 genes associated with apoptosis, including eight TNF receptor superfamily (TNFRSF), three members of the BCL2 family, and two members of the caspase (CASP) family in MDA-MB-468 cells. In MDA-MB-231 cells, two members of the TNF superfamily and four members of the BCL2 family were affected. The western study data showed the inhibition of AKT protein expression in both cell lines concurrent with up-regulated BCL2L11 gene. Our results point to the AKT/PI3K signaling pathway as one of the key mechanisms behind SANG-induced cell cycle arrest and death. Conclusion: SANG shows anticancer properties and apoptosis-related gene expression changes in the two TNBC cell lines and suggests AKT/PI3K pathway implication in apoptosis induction and cell cycle arrest. Thus, we propose SANG’s potential as a solitary or supplementary treatment agent against TNBC.
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 distinguish cancer cells (2). BC is a heterogeneous cancer type categorized based on its molecular features. Triple-negative breast cancer (TNBC) affects approximately 15% of patients with BC (2). However, the incidence of this subtype is two to three folds 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 different BC subtypes (3, 5). Based on immunohistochemical characteristics, the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are missing in TNBC cells (3, 6). Currently, chemotherapy is the principal treatment for patients with TNBC. However, disease recurrence can manifest within the first two years with an aggressive metastatic pattern (7). Hence, the continuous pursuit of novel agents is necessary, and novel drugs with the potential to arrest the cell cycle and induce apoptosis are being pursued in cancer therapy (8).
In cancer, the characteristic mechanism of tumors to overcome DNA damage is rapid cell proliferation, boosted by continuous cell cycle progression (9). The signaling pathways controlling these events affect cancer cells significantly (10). Indeed, retracting the cell cycle checkpoints preceding DNA repair can promote apoptotic signaling, leading to transcriptional suppression and cell death (11). Apoptosis is a controlled process that maintains cellular homeostasis and is mediated by two well-controlled mechanisms: 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 initiation of either or both apoptotic pathways (12-16). The extrinsic apoptotic signaling pathway is activated upon proapoptotic ligands, such as tumor necrosis factor-alpha (TNF-α), and FAS ligands with their respective transmembrane receptors, which activate a protease family caspases, leading to cell death. Induction of the intrinsic apoptotic pathway occurs instantaneously with increasing mitochondrial membrane flexibility and the passage of different proapoptotic proteins, such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF), which disturbs the balance between antiapoptotic and proapoptotic proteins, leading to caspase-mediated and/or simultaneous cell death (17, 18). Therefore, triggering cell cycle arrest and apoptosis has been considered a promising approach for treating BC (16, 19, 20).
Sanguinarine (SANG), a benzo phenanthridine alkaloid extracted from the rhizomes of Sanguinaria canadensis plants, has remarkable biological activity and anticancer potential (18). The anticancer effect of SANG has been strongly linked with its ability to promote cell death via the extrinsic and intrinsic apoptotic signaling pathways (18, 21) and to induce DNA fragmentation (22-25). SANG has been reported to induce apoptosis in BC cells (26-30). SANG can 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, an inhibitor of apoptosis family (IAP), and extracellular signal-regulated kinase 1/2 (ERK1/2) (18, 33). In MDA-MB-231 cells, SANG has demonstrated apoptotic effects by up-regulating apoptosis-mediated proteins while inhibiting others (27, 29, 30, 33, 34).
Resistance to chemotherapeutic agents-mediated cytotoxicity is a significant challenge in cancer therapy (18). Therefore, drug combinations are crucial to achieving significant synergistic therapeutic effects (35). In various chemotherapeutic drug-resistant cancer cells, SANG has been presented to enhance the anticancer impacts of multiple medicines (36, 37), including doxorubicin (38) and paclitaxel (39). In MDA-MB-231 cells, SANG can synergize with TNF-related apoptosis-inducing ligand (TRAIL)-linked apoptosis (30). Further, combining SANG with a sub-lethal dose of digitonin induces an increased cytotoxic effect in MCF-7 BC cells (40).
Molecular-directed therapy has also been incorporated into clinical trials to manage various human tumors (41). AKT is believed to be a promising target therapy in cancer management (42). Akt signaling is significantly activated in cancer cells (43), and up-regulation of AKT isoforms without gene augmentation has been found in BC (44). AKT activation also enhances tumor progress and resistance to chemotherapy treatments (45). Meanwhile, inhibition of Akt increased tamoxifen-stimulated apoptosis (46).
Although the antiapoptotic effects of the compound in MDA-MB-231 cells have been reported, its effects on the MDA-MB-468 cell line, a phenotypically distinct TNBC cell line, have not yet been studied. Thus, this comparative study intended to establish 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 using a human apoptosis gene expression array.
Materials and Methods
Cell culture and media. The TNBC cells MDA-MB-231 (ATCC® HTB-26™) and MDA-MB-468 (ATCC® HTB-132™) were purchased from ATCC (Manassas, VA, USA). The immortalized cell lines MDA-MB-231 and MDA-MB-468 were isolated and immortalized from Caucasian American (CA) and African American (AA) breast tumors, 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 kept at 37°C with 5% CO2 in a humidified environment. The TNBC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 4 mM L-glutamine, in addition to 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (1% P-S). The media were frequently changed 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 with 2.5% heat-inactivated FBS was used for all experiments.
Cell viability assay. The effect of SANG on MDA-MB-231 and MDA-MB-468 cell viability was established using Alamar Blue® (AB®, Sigma-Aldrich, St. Louis, MO, USA) as previously described (47). 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 maintained overnight at 37°C in a humidified incubator with 5% CO2. TNBC cells were then treated with DMSO (≤0.1%) or graded doses of SANG (0-5 μM). After 24 h, 20 μl of AB® resazurin solution (0.5 mg/ml in sterile HBSS) was added to each well, followed by 4 h incubation. The fluorescence intensity, indicating resazurin reduction by metabolically active TNBC cells, was quantified at an excitation/emission wavelength of 530/590 nm using a Synergy H.T.X. 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.
Cell proliferation assay. The influence 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 (47). Briefly, the experiment was initiated by seeding 1×104 cells/100 μl/well. The cells were then exposed for 48-96 h with SANG at gradual concentrations ranging from 0-2.0 μM. The control wells were exposed to DMSO at the highest concentrations (<0.1%).
Flow cytometry assays. Cell cycle analysis. Cell cycle evaluation of SANG-treated TNBC cells was performed using a previously described protocol (47). Cells seeded at 1.5×106 cells/T25 cell culture flask were incubated overnight under the same incubation conditions. SANG was added to each cell line at four concentrations (0-1.5 μM). Cells corresponding to the control were treated with DMSO at <0.1%. After 24h, cells from each treatment group were pelleted, washed in DPBS, fixed in cold 70% ethanol, and kept in the refrigerator for at least four hours. Again, the suspended cells were pelleted and washed with DPBS. Finally, the cells were resuspended in 1X propidium iodide (PI) with RNase staining solution (Abcam, Cambridge, MA, USA) and kept 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 for each cell line is generated from three independent studies.
Apoptosis assay. To study the apoptotic effect of SANG in TNBC cells, we followed a previously described protocol (47) using the Annexin V-FITC (Ann FITC) Apoptosis Detection Kit Plus (Ray-Biotech, Norcross, GA, USA). Briefly, cells under investigation were seeded in 6-well plates and incubated overnight. The cells were treated for 24 h with optimized doses of SANG ranging from 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 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 P.I. Cell-Quest software was used to determine the distribution of apoptotic and necrotic cells in each treatment.
Gene expression analyses. Treatment of TNBC cells. Two T-75 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) (48, 49), following overnight incubation. Control cells were treated with DMSO at <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 saved at −80°C.
RNA extraction. Following the manufacturer’s guidelines, RNA was extracted from previously frozen cell samples in 1 ml of TRIzol reagent (Thermo Fisher Scientific, Inc.) and homogenized briefly for 20 s. Then, 200 μl of chloroform (Sigma-Aldrich) was added to each sample, vortexed, and incubated for 3 min at room temperature. All vials 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 pellets were washed with 75% ethanol, air-dried for 10 min, and reconstituted in 50 μl nuclease-free water.
Complementary DNA (cDNA) synthesis. The RNA purity and concentration of each sample were evaluated using a NanoDrop spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific, Inc.). Next, 5 μg/ml of RNA was combined 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 centrifuged at 9,000 rpm for 3 min to precipitate the unwanted cellular DNA. The DNA-free supernatant was collected for cDNA synthesis by reverse transcription (RT). cDNA was obtained using the iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). In PCR 96-well plates, each well was loaded with 5 μl of DNA-free R.N.A., 9 μl of nuclease-free water, and 6 μl of advanced reaction mix reverse transcriptase cocktail. The recommended RT reaction was performed as follows: RT for 20 min at 46°C and RT inactivation for 1 min at 95°C. The cDNA presenting each sample was stored at −80°C for the PCR assay.
Quantitative qRT-PCR 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 and centrifuged at 1,000×g; fluorescence quantification was completed 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 last extension phase was completed at 65°C for 31 s. The qRT-PCR data were confirmed for each cell line using at least three independent experiments.
Western blot analysis. Western System (ver.6.0 Protein Simple, San Jose, CA, USA) was employed to determine the protein expression using a 12-230 kDa Wes Separation Module-25 capillary cartridge and its compatible Anti-Rabbit Detection Module kit. The primary and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Following the previously mentioned protocol (50), cells were treated and collected, and the cell pellets were lysated using a cocktail of lysis buffer and protease inhibitor. Protein quantification in cell lysates was measured using a Pierce™ B.C.A. protein assay kit (Thermo Scientific, Rockford, IL, USA). For band immunodetection, we used the primary antibodies AKT (9272S), the endogenous control antibody GAPDH (14C10) Rabbit mAb #2118S, and the secondary antibody Anti-rabbit IgG, HRP-linked antibody (7074S). A preliminary optimization study was established to detect the ideal concentrations for the cell lysates and antibodies under investigation. For both cell lines, 0.5 mg/ml of the cell lysates was applied as an optimum concentration. For AKT primary antibody, the ideal dilution factor was 1:50 and 1:100 for MDA-MB-231 and MDA-MB-468 cell lysate, respectively. The data generated from three independent studies were normalized using GAPDH and analyzed using Protein Simple Compass software and Prism-GraphPad software.
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 calculated 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. A 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).
Results
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 1 μM 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.
Cytotoxic effects of SANG in MDA-MB-231 and MDA-MB-468 cell lines. The two cell lines were seeded and treated for 24 h with 0-5 μM of SANG. Cell viability data are expressed as percentages of cell survival compared to the control. Each point represents the average±standard error of the mean (SEM) of three biological and six technical replicates. The IC50±SEM value was averaged from the three biological replicates. The significance of the difference between the control vs. treated groups was analyzed using a one-way analysis of variance (ANOVA) and was validated using Bonferroni’s multiple comparisons test. The difference was considered significant at **p=0.01 and ****p=0.0001. SANG: Sanguinarine.
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 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 (Figure 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 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; Figure 2b).
Effect of SANG on the proliferation rate of (a) MDA-MB-231 and (b) MDA-MB-468 TNBC cells. Both cell lines were consistently seeded, incubated overnight, and treated with SANG for 48-96 h with 0-2.0 μM SANG. Each data point presents the average±SEM of three biological replicates of six technical replicates each. The IC50±SEM value was averaged from three biological replicates using excel software. ANOVA (One and Two ways), followed by Bonferroni’s multiple comparisons tests, were used to calculate the p-values for the difference between control vs. treated cells at each exposure period (*) or between the different incubation periods (#), respectively. ****/####p=0.0001 indicates a statistically significant difference. SANG: Sanguinarine.
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 of DMSO-control cells (Figure 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 (Figure 3b). In contrast, significant dose-dependent changes (p=0.05, p=0.0001, Figure 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 (Figure 3a), which suggested the presence of dead cells, but this was not detected in MDA-MB-468 cells (Figure 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; Figure 3a). Meanwhile, lower concentrations of SANG (0.5 and 1.0 μM) induced less than a 10% increase in the S-phase, accompanied by a minor but significant accumulation (p=0.01, Figure 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 means may be involved.
Effect of SANG on cell cycle progression in (a) MDA-MB-231 and (b) MDA-MB-468 TNBC cells. The growing cells were seeded and treated for 24 h with SANG at three different doses, 0-1.5 μM. Flow cytometry using the fluorescent probe PI was used to measure the changes in the three phases of cell cycle. For each cell line, the data were confirmed in three biological replicates with three technical replicates. FACSCalibur was used to analyze the percentage of cells in different phases among SANG-treated samples vs. control. One-way ANOVA for multiple comparisons followed by Bonferroni’s test was used to obtain the p-values. The difference between control and treated samples across the three phases was considered significant at *p=0.05, **p=0.01, ***p=0.001, and ****p=0.0001. SANG: Sanguinarine.
Sanguinarine triggers apoptosis in triple-negative breast cancer cells. Flow cytometric analysis was performed to determine whether apoptosis was involved in reducing 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; Figure 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 (Figure 4a), whereas 80% of the analyzed MDA-MB-468 cells were in the apoptotic phase following exposure to 2.0 μM SANG (Figure 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.
Apoptotic effects of SANG in (a) MDA-MB-231 and (b) MDA-MB-468 TNBC cell lines. Both cell lines were exposed to the alkaloid compound SANG for 24 h at six different concentrations ranging=0-4.5 μM in MDA-MB-231 cells and 0-4.0 μM in MDA-MB-468 cells, while control cells were exposed to <0.1% DMSO. Both treated and control cells were labeled for 10 min with Ann FITC/PI mixture. Each bar in the graph represents the mean±combined percentage of early and late apoptotic cells and the SEM of three biological replicates of three technical replicates. The significance of difference between various treatments vs. control was evaluated using one-way ANOVA for multiple comparisons, followed by Bonferroni’s test to determine the p-values. ***p=0.001 and ****p=0.0001 indicate a significant difference. Ann Fitc, Annexin V-FITC; PI, propidium iodide, SANG: sanguinarine.
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 (48, 49), 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; Figure 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 (Figure 5a and b). In the figure, the red dots indicate the up-regulated genes in both cell lines. The green-colored dots representing the down-regulated genes were observed only in MDA-MB-231 cells (Figure 5a). The black dots in the middle panel indicate the range of unchanged gene expression in both cell lines.
Categorization of apoptosis-related gene expression induced by SANG in (a) MDA-MB-231 and (b) MDA-MB-468 TNBC cells. A volcano plot illustrates the altered genes in both cell lines following 24-h exposure to the compound at 3.5 μM for MDA-MB-231 cells and 2.6 μM for MDA-MB-468 cells. The augmented mRNAs are indicated in red, and the repressed genes are indicated in green; unchanged gene expression appears as black dots. The number of up-regulated genes was highly recognizable in MDA-MB-468 cells; the attenuated mRNAs were only evident in MDA-MB-231 cells. SANG: Sanguinarine.
Several up-regulated 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 up-regulated 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 (Figure 6a-d and Table I). These augmented genes belonged to different protein families, as shown in Figure 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) (Figure 6a). Further, two members of the caspase (CASP) family, CASP1 and 10 were up-regulated in MDA-MB-468 cells (Figure 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) (Figure 6c). More than a 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) (Figure 6d).
Gene expression quantification in SANG-treated MDA-MB-468 cells. Cells were exposed for 24 h to 2.6 μM of SANG. The transcriptomic levels of normalized mRNAs indicated a significant increase in 18 genes. (a) Tumor necrosis factor (TNF) receptor superfamily (TNFRSF): 11B, 25,10B/A and 21, FAS-associated via death domain (FADD), TNFRSF1A associated via death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2). (b) Caspase (CASP) 1 and 10. (c) B-cell lymphoma 2 (BCL2) like protein 11 (BCL2L11), harakiri BCL2 interacting protein (HRK), and BCL2-associated X-protein (BAX). (d) DNA fragmentation factor subunit alpha (DFFA); a baculoviral inhibitor of apoptosis (IAP) repeat-containing 3 (BIRC3); nucleotide-binding oligomerization domain-containing protein 1 (NOD1); CASP8 and FADD-like apoptosis regulator (CFLAR), and death-associated protein kinase 1 (DAPK1). The graphs represent the mean±SEM of three biological replicates. The significance of the difference between the DMSO-treated vs. SANG-treated MDA-MB-468 cells was determined using an unpaired t-test. The difference between control and treated cells was considered significant at *p=0.05, **p=0.01, and ***p=0.001. SANG: Sanguinarine.
Fold-change of mRNA gene expressions after 24 h exposure to the alkaloid compound SANG at the corresponding IC 50 s of 3.5 μM in MDA-MB-231 and 2.6 μM in MDA-MB-468 TNBC cells.
In contrast, the response of MDA-MB-231 cells to the compound was different. Fewer apoptosis-related genes were activated (Figure 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 (Figure 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 up-regulated gene in both cell models, but its up-regulation was higher in MDA-MB-231 cells than in MDA-MB-468 cells (4.67 vs. 3.17-fold).
Gene expression quantification in SANG-treated MDA-MB-231 cells. Cells were treated for 24h with 3.5 μM of the alkaloid compound SANG. The transcriptomic levels of normalized mRNAs indicated a significant mixed fold-change in twelve genes. (a) The up-regulated mRNAs included 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). (b) The repressed mRNAs included 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). The graphs represent the mean±SEM of three independent biological replicates. The significance of the difference between the DMSO-treated vs. SANG-treated MDA-MB-231 cells was determined using an unpaired t-test. The difference between control and treated cells was significant at *p=0.05, **p=0.01, and ***p=0.001. SANG: Sanguinarine.
In MDA-MB-468 cells, the other seven genes were significantly down-regulated in MDA-MB-231 cells (Figure 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).
SANG modulates protein expression of AKT signaling pathway mediating apoptosis in TNBC cells. Although the mRNA of AKT1 was only inhibited in MDA-MB-231 cells, data obtained from the protein expression study indicated the ability of SANG to repress the protein expression of AKT in both cell models. Compared with control cells and as demonstrated in both western bands (Figure 8a and b) and Compass software data analyses, SANG induced more than 40% inhibition in AKT for MDA-MB-231 cell lysates (p=0.0001). In comparison, AKT protein was attenuated by 25% in SANG-treated MDA-MB-468 cells (p=0.01).
Effect of SANG on AKT protein expression in MDA-MB-231 and MDA-MB-468 TNBC cells. The automated, simple western system with its Compass software was used for band immunodetection in control vs. treated cells. Quantitative analysis of GAPDH-normalized data was established for MDA-MB-231 (a) and MDA-MB-468 (b) cells. The data generated from three independent experiments (n=9) are presented as the mean±SEM. The significance of the difference was analyzed using an unpaired t-test. ****p=0.0001 and **p=0.01 are considered significant difference. SANG: Sanguinarine.
Discussion
Naturally occurring compounds are promising therapeutic agents for managing different cancer types, including BC. This study was designed to investigate the mechanism underlying the anticancer effect of the natural alkaloid compound SANG in two genetically different TNBC cell lines.
SANG has been proven to induce several biological activities, such as antioxidant, anti-inflammatory, and antimicrobial properties (51). Also, SANG consistently showed safe effects (52). 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 and proliferation rate, together with cell cycle arrest and apoptosis induction (23, 53). Remarkably, the obtained data indicate a higher cytotoxic potency in MDA-MB-468 cells than in MDA-MB-231 cells (Figure 1). The compound showed substantial potential to decrease the proliferation rate (Figure 2) in MDA-MB-468 cells compared to that in MDA-MB-231 cells.
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 the expression of various genes, orchestrating both intrinsic and extrinsic apoptosis pathways by employing other 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 another molecular profile.
The ultimate target of chemotherapeutic drugs is to control cell cycle progression and induce apoptosis (11). Furthermore, our cell cycle distribution analyses (Figure 3) indicate a relatively discrete response in the two TNBC cell lines with respect to apoptosis (Figure 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 decreased G1/G0 and G2/M phases and reduced DNA synthesis, and it leads to reduced proliferation and cell viability (54, 55).
In our study, transcript analysis of apoptosis-mediated genes in SANG-treated TNBC cells indicated that SANG has the potential to impact various genes by modulating both intrinsic and extrinsic signaling pathways (Figure 5, Figure 6, and Figure 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 augmented. Meanwhile, SANG-treated MDA-MB-231 cells exhibited significant up-regulation in the mRNA expression of five genes and down-regulation 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 up-regulation 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 down-regulated. The inflammatory caspase CASP1 has a unique function that is distinct from other apoptotic caspases (56). Different 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 (57). In contrast, an elevated level of CASP1 in fibroblasts is known to provoke apoptosis and cell death (58). In MCF7 BC cells, the initiator caspase CASP10 sensitizes cells to TRAIL-induced apoptosis (59). 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 (60-63). In contrast, repression of CASP6 in MDA-MB-231 cells indicated resistance to apoptosis (Figure 7b). Indeed, CASP6 is a downstream effector and executioner caspase that enhances apoptosis by activating various cellular proteins (64). 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 genes of the BCL2 family were found to be up-regulated in the TNBC cells under investigation. The mRNA of BCL2L11 was significantly up-regulated in both TNBC cell models (Figure 6c and Figure 7a), whereas up-regulated HRK and BAX were exclusive to MDA-MB-468 cells (Figure 6c). The BCL2 family controls the intrinsic apoptotic pathway through two main groups of proteins: pro- and antiapoptotic proteins (14). In agreement with these findings, we suggest that SANG enhances intrinsic apoptosis by modifying the expression of some BcL-2 family members.
Furthermore, the potential of SANG to up-regulate the effector proapoptotic protein BAX has been exhibited in various cell lines (32, 65). The up-regulation of BAX mRNA has been shown to neutralize the antiapoptotic function of other BCL2 family members, increasing the mitochondrial membrane permeability and cytochrome c release preceding caspase activation and apoptosis (66). Arguably, we suggest that SANG induces intrinsic apoptosis by up-regulating the expression of various proapoptotic members of the BCL2 family.
In MDA-MB-231 cells, two antiapoptotic genes, BCL2L1 and BCL2A1, were inversely altered (Figure 7a and b), in addition to the significantly up-regulated binding protein, BAG (Figure 7a). In various cancer types, including TNBC, highly up-regulated BCL2A1 and BCL2L1 (also known as BCL-XL) are closely associated with resistance to targeted agents and chemotherapeutic drugs (67-72). Furthermore, various mechanisms are associated with apoptosis induction in BC, including BCL2L1 pathway modulation (73) and changing the BAX/BCL2L1 ratio (74). Overexpression of BAG3 in various cancer types, including BC (75, 76), inhibits apoptosis (77) while promoting proliferation (78) and chemotherapy resistance (79). Thus, inhibiting BAG3 expression is suggested as a promising approach for cancer therapy (80). 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 up-regulation in the relative resistance of MDA-MB-231 cells to SANG-stimulated intrinsic apoptosis compared with that in MDA-MB-468 cells.
Distinct from MDA-MB-231 cells, eight members of TNFRSF were significantly up-regulated 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 (Figure 6a). These death receptors on the cell surface transmit apoptotic signals once they bind to their specific death ligands (81). In BC, as well as in various other cancer cells, the adaptor molecules, FADD and TRADD, were previously found to interact with up-regulated TNFRSF25 and TNFRSF10A/B to enhance apoptosis by triggering TRAIL-mediated apoptosis (82-87). Augmentation of all these TNFRSF genes in MDA-MB-468 cells in our study strongly supports the previous findings. The potential of TNFRSF21 to induce apoptosis (88), probably via the mitochondria-mediated intrinsic pathway and BAX interaction (89), suggests TNFRSF21 and BAX up-regulation as one of the mechanisms underlying apoptosis induction in SANG-treated MDA-MB-468 cells.
Our mRNA analysis also indicated up-regulation of TRAF2 and TNFRSF11B mRNAs in MDA-MB-468 cells (Figure 6a). Recent studies have demonstrated the involvement of dually functional TRAF2 in both pro- and antiapoptotic signals (90, 91). Also, TNFRSF11B’s overexpression induces apoptosis resistance, enhances cancer cell viability, invasion, and metastasis, and indicates poor prognosis (92, 93). Hence, the transcriptomic up-regulation 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 up-regulation was observed in the apoptosis-mediated gene CFLAR (Figure 6d). CFLAR has demonstrated the potency to inhibit the DR-induced apoptosis pathway, which inhibits CASP8 stimulation (94-96). Therefore, the role of CFLAR as an antiapoptotic gene was anticipated in our study, mainly with an unchanged expression of CASP8 in SANG-treated MDA-MB-468 cells.
Two members of the IAP family, BIRC3 (cellular IAP2) and BIRC5 (survivin) (97, 98), were inversely regulated in SANG-treated TNBC cells (Figure 6d and Figure 7b). SANG treatment up-regulated the expression of BIRC3 mRNA in MDA-MB-468 cells and down-regulated BIRC5 levels in MDA-MB-231 cells. The IAP family is known to regulate the intrinsic and extrinsic apoptotic pathways and play a minor role in the execution phase of apoptosis (98). 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 (99-101) and was closely associated with resistance to apoptosis induction and chemotherapeutic efficacy (98, 102-104). Therefore, the obscure role of BIRC3 up-regulation in SANG-treated MDA-MB-468 cells requires further investigation, whereas BIRC5 suppression in MDA-MB-231 cells could mediate apoptosis.
In MDA-MB-468 cells, only the proapoptotic gene DAPK1 was significantly up-regulated by SANG (Figure 6d). Forced DAPK1 up-regulation inhibits antiapoptotic proteins (105) and ultimately induces apoptosis (106). Here, we suggest that DAPK1 up-regulation mediates apoptosis in MDA-MB-468 cells, particularly with unchanged antiapoptotic genes.
Two other proapoptotic mRNAs, DFFA and NOD1, were up-regulated in SANG-treated MDA-MB-468 cells (Figure 6d). These genes are crucial for caspase-dependent apoptotic pathways (107, 108). Thus, our findings suggest DFFA and NOD1 up-regulation is one of the fundamental 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 (Figure 7a and Table I). Previous findings have demonstrated the vital role of LTA transcriptomic up-regulation in triggering apoptosis (109). In contrast, other studies have suggested that repression of various signaling pathways leads to uncontrolled cancer cell proliferation (110). This perplexing response of LTA and LTBR necessitates further investigation to identify the mechanism of LTA up-regulation in MDA-MB-231 cells undergoing apoptosis.
The alkaloid compound SANG up-regulated the expression of GADD45A approximately 4-fold in MDA-MB-231 cells (Figure 7a and Table I). In TNBC cells, low expression of GADD45A and p53 and DNA damage response genes are linked with the absence of ER, PR, and HER2 expression (111). This inducible stress gene regulates various cellular processes such as the cell cycle, DNA repair, and apoptosis (112). Hence, the impact of SANG in MDA-MB-231 cells (Figure 2 and Figure 4) matches and agrees with the previously reported potential of GADD45A to induce antiproliferative effects and S-phase cell cycle arrest (112).
Significant inhibition of TP53 mRNA expression by SANG was detected only in MDA-MB-231 cells (Figure 7b and Table I). Almost 80% of MDA-MB-231 and MDA-MB-468 TNBC patients are diagnosed with mutated TP53. This elevated level of mutated proteins is closely connected with poor prognosis and resistance to chemotherapy (113). Therefore, TP53 repression could be a significant contributor to apoptosis in the MDA-MB-231 cell model.
Among all altered genes examined, BCL2L11 was the only one that was up-regulated in both cell models. More importantly, many studies revealed the association between AKT1 and BCL2L11. The knockdown of AKT1 in MDA-MB-231 and MDA-MB-468 TNBC cells can enhance the expression of BCL2L11, a known promoter of apoptosis (114). In addition, it was found that the silencing of AKT1 decreases the colonization of TNBC cells mediated by apoptosis induction (114). Consistent with these results, a previous study has found that AKT1 sustains the survival of tumor-initiating cells by decreasing the expression of BCL2L11 (115). 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 (116-118). The up-regulation of AKT1 in MDA-MB-231 cells and patients with BC promotes proliferation and is closely associated with the disease’s aggressive nature (119). The significance of targeting AKT1 has been highlighted in other studies (120). Meanwhile, SANG showed the potential to attenuate the mRNA expression of GUSB and AKT1 in MDA-MB-231 cells. The western study proved the ability of SANG to inhibit the protein expression of AKT in both cell models. Our results strongly support these previous findings (114, 115) and suggest that AKT inhibition (Figure 8a and b) synchronized with the up-regulated level of BCL2L11 expression in both cell lines could be one of the factors involved in SANG-induced apoptosis in both cell models. The up-regulated expression of GUSB has been implicated in increasing the risk of cancer (121). Also, the chemopreventive effect of GUSB inhibitors has been validated by reduced cell proliferation and apoptosis induction in various cancer types, including BC (121-123).
Conclusion
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 (124), 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. The western study also suggested that targeting AKT/PI3K is an anticipated mechanism for SANG-induced apoptosis. Few studies have investigated 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 (125, 126). The current study highlights the need for further investigation in other TNBC models, in vivo and translational studies on SANG using the obtained data to develop molecularly targeted therapies to enhance the clinical outcomes of currently used chemotherapeutic agents in patients with TNBC.
Funding
This research was funded by a grant from the National Institute on Minority Health and Health Disparities (NIMHD), grant number U54 MD007582.
Acknowledgements
The Authors are grateful for the assistance of Dr. Ramesh Badisa, College of Pharmacy and Pharmaceutical Science, Florida A&M University.
Footnotes
Conflicts of Interest
The Authors declare no conflicts of interest.
Authors’ Contributions
Conceptualization, SSM, and KFAS; methodology, SSM; validation, SSM, SN, and TW; formal analysis, SSM; investigation, SSM, SN, and TW; resources, KFAS; data curation, SSM; writing—original draft preparation, SSM; writing—review and editing, SSM, NOZ, LML, and KFAS; visualization, SSM; supervision, SSM, LML, and KFAS; project administration, SSM, and KFAS; funding acquisition, KFAS. All Authors have read and agreed to the published version of the manuscript.
- Received February 12, 2023.
- Revision received March 27, 2023.
- Accepted April 5, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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