GTN has been reviewed as a potent cytotoxic agent with the induction of apoptosis in many cancer cells lines (13). The promising anticancer properties of GTN have been studied in several breast cancer cells including SK-BR-3, MDA-MB-231 and MCF-7 (21, 25–28). In SK-BR-3 cells, GTN-induced apoptosis was associated with autophagy via p-p38 and p-JNK1/2 upregulation and Akt downregulation (25). Whereas, the non-apoptotic cell death mechanisms namely necroptosis and anoikis induced by GTN were reported in human invasive breast cancer cells MDA-MB-231 (26). Not only limited to its pure compound, research on the effects of GTN’s derivatives as well as its potential combination with other biomaterials were also reported. A study conducted by Boonmuen et al., 2016 found that 5-acetyl goniothalamin (5GTN), a natural derivative of GTN was more potent than GTN in mediating the toxicity towards MCF-7 and MDA-MB-2 breast cancer cells (27). Besides, a fluorescent 2,1,3-benzothiadiazole-containing goniothalamin derivative, BTD − GTN (1) hybrid, was successfully synthesised in an investigation to gain insights into the subcellular localisation and mechanism of action of MDA-MB-231 cells, which might involve a cascade of events, starting with their interaction with mitochondria (29). In a recent study, a polymeric nanosystem, in which the racemic mixture of GTN (rac-GTN) was encapsulated in pH-responsive acetalated dextran (Ac-Dex) nanoparticles (NPs), has been developed to improve the pharmacokinetic behaviour and selectivity of GTN against cancer cells including MCF-7 and MDA-MB-231 cells (30).
Multiple polymeric nanoparticles, liposomes, and micelles have demonstrated great potential in drug delivery. However, the potentials of these materials are challenged by their poor bioavailability and biodegradability, instability in the circulation and inadequate distribution in the tissue (31). On the other hand, sol-gel bioactive glass is known to be bioactive, biocompatible and degradable; therefore, there is no requirement for a second surgical procedure to remove the material from the body. Bioactive glass (BG) with controlled diameter serves as an ideal carrier for delivery of anticancer in the body. One of the approaches proposed is by loading the anticancer drugs directly into the MBG to produce local chemotherapeutic effects. The potential development of BG as a drug carrier has been reported for several anticancer drugs including doxorubicin, imatinib and 5-Fluorouracil (11, 12, 32, 33). The third drug has been extensively used clinically to treat various types of cancer. Despite its efficacy in killing the cancer cells, the only drawback is that it is easily metabolized due to the short biological lifespan. A study done by El Kadi et. al (2015) revealed the potential use of bioactive glass nanoparticles (SiO2-CaO-P2O5) as a delivery system for 5-Fluorouracil (33). Their findings demonstrate that the BG was able to sustain the release of 5-Fluorouracil for more than 32 days, which could prevent cancer recurrence after resection. Another approach is through the modification of the BG surface by incorporating the anticancer metals to improve its specificity towards the receptors of cancer cells. The examples include terbium (Tb), holmium (Ho), 153Sm-ethylenediaminetetrame thylphosphonic acid (153Sm-EDTMP) and yttrium. Interestingly, a recent research studied the combined effects of the copper (Cu)-doped BGs, the surface-modified BG, which exhibited both photothermal and chemotherapeutic activities towards bone tumours (8). Apart from these approaches, the nanoscale HA-based biomaterials have been found capable of inhibiting proliferation and inducing apoptosis in various cancer cells including breast, colon, gastric, osteosarcoma and liver cancer cells (34–40).
The present study was designed to examine the combined effects of GTN and BG (GTN-BG) in MCF-7 cells in comparison with a single GTN treatment to postulate whether the combination could enhance the antitumor effects. The BG was successfully prepared using sol-gel method and the characterisation was performed to understand its properties and predict its biological performance in this study. The synthesised BG 45S5 is classified as fine powder with high surface area that enables high dissolution rate. Moreover, it was reported that the use of fine powders has enhanced the deposition level of Ca-P layer on the glass surface and material degradation and resorption rates (41). Besides, the synthesised BG 45S5 comprises the rough surface, and mainly consists of mesopores that formed as a result of gel formation. Due to the mesoporous texture and high surface area, which can adsorb a range of substances including proteins and cells, the sol-gel method has become an option for several biomedical applications (42).
The cellular response was first evaluated by looking at the inhibitory effects of GTN-BG in MCF-7 cells. We have demonstrated that the combination of GTN-BG was more potent than GTN in inhibiting the proliferation of MCF-7 cells, while the control cells of HMSC were not affected. The analysis of cell cycle progression indicated the cell cycle arrest occurred at similar phases in both GTN and GTN-BG-treated cells, which was at G0/G1 and G2/M phases. Concurrently, there was a reduction in the percentage of cells at S phase. Other studies have demonstrated that GTN arrested cell cycle at G0/G1 in MCF-7(28), G2/M in MDA-MB-231 (21) and S phase in Hela cells (43). The relative contribution of G1 and G2/M arrests may vary according to the cell line, treatment time and dosage. The G1 arrest was reported to be associated with the reduced expression of cyclin D1 and CDK4 mRNA, in addition to the downregulation of CDK2. Meanwhile, the G2/M phase arrest was believed to be related with the downregulation of CCNB1, CCNB2 and CDK1 (27).
The mode of cell death was determined by annexin V-FITC assay. We have demonstrated that both GTN and GTN-BG treatments induced apoptosis in MCF-7, as the percentages of both early and late apoptotic cells were significantly increased after the treatments. In a comparison with GTN, the cells treated with GTN-BG cells produced lower number of viable cells, but higher number of late apoptotic cells than that of GTN. Although the percentage of necrotic cells increased in both treatments, apoptosis was considered as the primary mode of cell death as the total percentage of apoptotic cells was higher than the necrotic cells.
Activation of caspases is the key event in apoptosis, since the caspases initiate irreversible processes of cell death (44). Cells undergo apoptosis through two major pathways, namely the extrinsic (death receptor pathway) and intrinsic (the mitochondrial pathway) pathways; both pathways end with the execution phase, which is regarded as the final pathway of apoptosis (44, 45). Activation of caspases namely the caspase-8, caspase-9 and caspase-3/7 were examined in this study. Caspase-8 is involved in extrinsic pathway of apoptosis, while the other two caspases are involved in intrinsic pathway of apoptosis. In this study, we have found that GTN-BG significantly activated caspase-8 and caspase-9 that are responsible for extrinsic and intrinsic pathway of apoptosis, respectively. The caspase cascade was further triggered through the activation of caspase-3/7. Theoretically, the activation of caspase-8 is mediated by cell surface death receptors, such as Fas, tumour necrosis factor receptor, and TRAIL receptors. Cell death ligand triggers oligomerization of the receptors and recruitment of the adaptor protein, Fas-associated death domain (FADD) and caspase-8 to form death-inducing signalling complex (DISC) (45). Whereas, the activation of caspase-9 is initiated by the release of cytosolic cytochrome from the mitochondria to the cytoplasm and its binding to the apoptosis protease-activating factor 1 (Apaf-1) and procaspase-9; the binding generates an intracellular DISC-like complex known as “apoptosome”, which later activates caspase-9 (45). The culmination of both extrinsic and intrinsic pathways ends at the point of the execution phase, which is considered as the final pathway of apoptosis. Caspase-3, caspase-6, and caspase-7 function as an effector or “executioner” caspases, cleaving various substrates that drive the terminal events of the programmed cell death (44, 46).
The present study demonstrated the relatively high inhibitory effects of GTN-BG towards the proliferation of MCF-7 cells and activation of caspase-8,9 and 3/7 in comparison to that of GTN. It is important to note that both treatments of GTN-BG and GTN have activated the same cellular death program known as apoptosis, which resulted in the cell cycle arrest at G0/G1 and G2/M phases. Unfortunately, the understanding of mechanism of BG’s action in enhancing the antitumor effect of GTN in MCF-7 remains unclear. However, we strongly believe that such enhancement might be due to the physicochemical properties of BG. The porous structure with high surface areas of the sol-gel-derived BG makes entrapment of molecules or drug inside the pores more effective than other materials, which enhance the delivery of the drug to the cells (47). Upon contact with biological fluids, dissolution of BG does not only trigger the release of drugs but also ions. Consequently, BG dissolves gradually and the released ions would stimulate the growth of hydroxyapatite (HA) layer on its surface (48–52). Some studies have shown that nanoscale HA-based biomaterials can inhibit proliferation and induce apoptosis in several types of cancer cells including breast cancer cells (34–40). The production of intracellular reactive oxygen species and activation of p53 that are responsible for the DNA damage and apoptosis were reported in MCF-7 cells co-cultured with HA nanoparticles (40).
The release of ionic dissolution products from the BG such as Ca2+, Na+, PO43−, and Si4+ has been shown to result in the rise of pH and osmotic pressure in its vicinity, which created an efficient antibacterial effect (53, 54). The role of extracellular pH in treating cancer cells has been emphasised in many studies. It is known that the extracellular pH of cancer cells is more acidic as compared to the normal cells, due to the excess in anaerobic glycolysis. Thus, increasing the extracellular pH may be a good strategy to inhibit the progression of tumour cells (55–57).