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.