In this research, cell cycle G0/G1 phase arrest and the increase of cell apoptosis rates were found with the increased of the SBA treatment levels. The proteomic measurements showed that a numbers of differentially expressed proteins, caused by SBA treatment, were mainly enriched in the DNA replication, base excision repair, nucleus excision repair, mismatch repair, ubiquitin-mediated proteolysis pathway, cell structural-proteins, and structures and functions of mitochondria. Moreover, the differential expressed proteins enriched in AMP-activated protein kinase (AMPK) pathway and the process of synthesis and metabolism of proteins were only found in 2.0 mg/mL SBA treatment.
The cell cycle progression regulates the condition of cell proliferation, which consists of three prominent phases to maintain DNA integrity [12]. In the herein study, there were increases in the percentage of cells at G0/G1 phase in SBA treatments, and the G1-phase cells that reflect the higher number of cells in the DNA repairing process. Bakke-McKellep, et al. [13] also found the same trends in Atlantic Salmon. In addition to FCM analysis, WB was performed to detect the expression of cell cycle key regulatory proteins (p21 and cyclin D1). The p21 protein belongs to the Cip/Kip family of CDK (cyclin-dependent kinase) inhibitors. Such protein is an important cell cycle regulator [14]. Loss of functional p21 along with p53 may lead to un-restrained cell cycle progression to the S phase, despite the presence of DNA damage [15]. Cyclin D1 is involved in the G1/S cell cycle progression, and the role of cyclin D1 seems to be opposite to p21 [16]. Cyclin D1 plays two opposing roles in cell proliferation [14], the cell cycle arrest, or both biological processes [17]. In addition, there is a relationship between these cell cycle regulatory proteins. For example, Cayrol, et al. [18] and Ando, et al. [19] suggested that p21 interacts with PCNA and blocks cell cycle progression. Cazzalini, et al. [20] indicated that p21 can prevent or replace the binding of polymerase δ to PCNA at the G1/S phase transition. Sheng et al. [21] showed that PCNA-mediated degradation of p21 can coordinate the DNA damage response and cell cycle regulation in individual cells.
In the herein research, cell apoptotic rate was determined using FCM and cell apoptotic regulatory proteins (Bcl-2 and Bax) that were tested using WB. The results showed that the apoptosis induction was achieved by Casp-3 and Casp-9 activations that lead to decrease Bcl-2 expression and increase Bax expression. Yang et al. [22] reported that the abnormal expression of the anti-apoptotic members (Bcl-2) and pro-apoptotic members (Bax) induces the apoptosis. The apoptosome (protein formed during the apoptosis) activates Casp-9, which triggers the activation of Casp-3 [23, 24]. The latter activation initiates proteolytic action which leads to cell death [23, 24]. In this study, SBA down-regulated the Bcl-2, up-regulated the Bax, and increased the level of cleaved caspases, including Casp-9 and Casp-3 activity. Such findings suggested the destruction of the outer mitochondrial membrane. Combined with GO annotation results in proteomics, the involved proteins in mitochondrial energy metabolism such as NADH dehydrogenase (F1RGE3), FAD-dependent oxidoreductase (F1S6H4) were decreased in both 0.5 and 2.0 mg/mL SBA treatments. This result indicates a low potential of the inner mitochondrial membrane [25]. Therefore, the potential destruction of homeostasis of inner and outer mitochondrial membranes occurred in the SBA treatments.
Interestingly, in 2.0 mg/mL SBA treatment, the differential expressed proteins were also enriched in ribosome structural and functional proteins, such as 40S ribosomal protein (P46405), 60S ribosomal protein (A0A287AE76), N(alpha)-acetyltransferase 10 (NAA 10, F1RZU5), NUFIP2, and FMR1 interacting protein 2 (FIP2, F1RN89). These proteins play significant roles in ribosome assembly, protein synthesis, and cell apoptosis [26]. For example, some ribosomal proteins mediate cell cycle and cell apoptosis [27]. The knockdown of ribosomal protein S15A induces human glioblastoma cell apoptosis [28]. Also, NAA 10 inhibits apoptosis [29], and the deficiency of NAA10 expression can induce cell cycle arrest and apoptosis [30]. The cytoplasmic FMR1-interacting protein 2 (CYFIP2) is involved in cell adhesion and apoptosis [31]. In addition, differential expressed proteins were also enriched in amide biosynthetic process, peptide biosynthetic and metabolic process, pep, tRNA methyltransferase activity, structural constituent of ribosome. These results may suggest that, with increasing SBA concentration, many biological processes have been affected, including that involved in the structural and functional biological processes of mitochondria and ribosome, as well as other processes related to protein synthesis and protein metabolism.
In the herein study, after being treated with SBA, the expressions of many proteins were significantly reduced, including PCNA, POLD1, POLA1, and RPA. These reduced proteins play crucial roles in different pathways such as DNA replication, base excision repair, nucleotide excision repair, and mismatch repair. Moreover, the expressions of these four proteins were markedly decreased in the 2.0 mg/mL SBA treatment. The related research showed that the expression of PCNA (known as cyclin) is necessary for cell proliferation and DNA synthesis [32], which reached its maximum level during the S-phase. The PCNA acts as an auxiliary protein for POLD1 in DNA synthesis [33]. POLD1 represents one of the three B polymerase families in eukaryotes. This family possesses a crucial role in leading- and lagging-strand synthesis [34–36]. Furthermore, POLD1 acts in several aspects of DNA synthesis and DNA-repair processes [37, 38]. The POLD1 complex coordinately interacts with a number of proteins that enable its function, such as DNA replication factor C (RFC) and PCNA [39]. POLA1 protein family plays essential roles in pyrimidine or purine metabolism. POLA1 together with PCNA, are key players in DNA replication during S phase of the cell cycle. The down-regulation of these proteins suppresses the cell cycle, especially DNA replication [40]. RPA, the laxative that keeping DNA regular can dynamically regulate mono-ubiquitination of PCNA [41, 42]. In addition to the above four proteins, Lig, MCM 2, MCM 4, MCM 5, and MSH2 were specially significantly enriched in 2.0 mg/mL SBA treatment.
In addition to DNA replication, both SBA treatments also affected many cellular processes, including base excision repair, nucleus excision repair, and mismatch repair pathways, though the ubiquitin-mediated proteolysis pathway. Ubiquitin-mediated proteolysis system acts in broad array of basic cellular processes, including regulation of the cell cycle, differentiation and development, the cellular response to extracellular effectors and stress, modulation of cell surface receptors and ion channels, DNA repair, regulation of the immune and inflammatory responses, and biogenesis of organelles [43]. Ubiquitin is linked to the target protein by a series of ubiquitin promoter enzymes. Ubiquitin promoter includes E1 ubiquitin activating enzyme, E2 ubiquitin binding enzyme, and E3 ubiquitin ligase enzymes. Ubiquitination is initiated by E1 which activates and transfers ubiquitin to E2. This E2 passes the ubiquitin to the corresponding E3 [43]. In the present research, 0.5 mg/mL SBA treatment lead to down-regulation of E1 only, while 2.0 mg/mL SBA treatment lead to down-regulation of both the E1 and E2 to affect the ubiquitin mediated proteolysis process, and finally could regulate many process such as cell cycle and apoptosis, etc.
AMP-activated protein kinase (AMPK), a central energy sensor, plays an important role in regulating cellular metabolism, and preserving cellular energy homeostasis. This sensor is involved in many cellular processes, including cell apoptosis [44]. In the present research, down-regulated proteins (SIRT1, PP2A, FASN, SCD, ACC, eEF2) and up-regulated protein (CPT1) were enriched in AMPK signal pathway in 2.0 mg/mL SBA treatment compared to control. SIRT1 is a key energy-sensing molecule in regulating mitochondrial biogenesis and has a mutual regulation with AMPK [45]. The inhibition of SIRT1 induces growth arrest and apoptosis in several types of cancer cells [46]. Park, et al. [47] indicated that AMPK is negatively regulated by PP2A, which participates in regulating many important physiological processes, such as cell cycle, growth, apoptosis, and signal transduction, G1-S transition, DNA synthesis, and mitotic initiation [48]. FASN plays an important role in regulating many cell processes, and some related reports indicated that mitochondrial dysfunction is participated in FASN inhibition, and consequently induces apoptosis [49]. AMPK activation decreases protein synthesis through inhibition of eEF2, which plays a major role in protein synthesis and cell survival [50]. Additionally, silencing of eEF2 expression increases mitochondrial elongation, cellular autophagy, and cisplatin sensitivity [51]. Thus, the effect of SBA on cell cycle, apoptosis, and mitochondrial pathway was confirmed again.
Cell morphology is important in controlling cell size, and regulating cell biological functions [52]. There are key structural proteins that play an important role in regulating cell morphology, including extracellular matrix (ECM) composition and cytoskeleton. The ECM composition can influence the cell morphology and differentiation [53]. Cytoskeleton acts a crucial role in maintaining cell morphology, cell migration, and the stabilization, and regulation of membrane proteins [54]. In the current research, down-regulated proteins such as FN1, GMFB, COL18A1 belonging to extracellular structure proteins were enriched in 0.5 mg/mL SBA treatment. These proteins were important in preserving cell morphology. For example, synthesis and surface expression of fibronectin (FN) are correlated with cell morphology and adhesiveness [55]. Glia maturation factor (GMF) induces characteristic changes of cell morphology in glioma cells [56]. Nguyen et al. [57] showed that exogenous COL18A1 possesses ability to restore morphology. In addition to overlapping proteins (FN1, and GMFB), there were other cytoskeleton proteins enriched in 2.0 mg/mL SBA treatment. These proteins are involved in down-regulation process and including LOC100624785, ACTBL2, LOC100158003, LASP1, CFL1, TAGLN, MAPRE1, ACTR3B, ACTN2, and AP4. The related trials have shown that microtubules are involved in maintaining cell morphology [58]. Actin plays critical roles in shaping and maintaining cell morphology. The change of cell morphology in budding yeast was mediated by polarization of the actin cytoskeleton [59, 60]. A critical role of Cofilin-1 (CFL1) in cell morphology and epithelial tissue organization was identified [61]. Alpha-actinin (ACTN) shows significant roles in other cellular processes, including transcriptional regulation, and maintain cytoskeletal architecture to maintain cell morphology [62, 63]. Transgelin (TAGLN) regulates cell morphology and motility, but the over-expression of this protein can affect the cell morphology [64, 65]. LASP-1 is involved directly or indirectly in cell morphology and cytokine secretion. Therefore, SBA could lead to cellular morphological changes by down-regulating the expression of the structural proteins.
Interestingly, after SBA treatments not all the expressions of the structural proteins related to cell morphology were decreased. There were enriched proteins in 0.5 mg/mL SBA treatment such as the up-regulated protein belonging to extracellular structure proteins (MUC4) and up-regulated protein (KIF23) belonging to cytoskeleton. Up-regulating proteins such as LAMB3, LAMA3, COL7A1 are belonging to extracellular structure proteins, while another up-regulation protein (KIF14) belonging to cytoskeleton was enriched in 2.0 mg/mL SBA treatment. The up-regulation of these proteins plays a key role in maintaining cell morphology and inhibiting apoptosis. For examples, laminins (LN) possess critical contribution to cell adhesion, proliferation, differentiation, and cell morphology and promotion of tissue survival [66]. The up-regulation of laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis [67]. MUC4/Y is anchored on the cytomembrane and can affect cell morphology and cell cycle, up-regulation of the potentiates proliferation, and suppresses apoptosis in pancreatic cancer cell line [68]. In general, with increasing the concentration of SBA, the expressions of laminins like LAMB3, LAMA3, and other proteins (MUC4, KIF23, and COL7A1) were up-regulated. Also, this increasing in SBA concentration may initiate feedback regulation to prevent excessive damage to cell morphology and apoptosis.