The present results demonstrated that the recombinant pPIC3.5K-hTopI plasmid, which contained a copy of the human DNA topoisomerase I (hTopI), was successfully constructed. The Pichia transformants or recombinant yeast (GS115-pPIC3.5K-hTopI), which contained a multi-copy number of hTopI, was also successfully produced via an in vivo strategy. The cell density of GS115-pPIC3.5K-hTopI was likely to be unaffected by the copy number of hTopI. However, the total protein expression and the target enzyme activity of the recombinant yeast were increased in accordance with the increased copy number of hTopI in the host, whereby the yeast that was able to survive at the highest concentration of Geneticin expressed the highest level of total protein and had the highest activity of the enzyme. The recombinant yeast was able to differentiate the growth inhibitory activity of quercetin and F1, indicating that the growth inhibitory activity of pure and plant substances could be predicted by yeast-based screening assay and further confirmed using MTT assay. Quercetin induced cell cycle arrest at S phase only in MDA-MB-231, but the study found F1 induced cell cycle arrest at both S and G2/M phases. Quercetin induced apoptosis in MDA-MB-231, but F1 did not. The F1 showed lower mRNA expression levels of CYP1A1 and CYP1B1 (carcinogenicity). However, the study found a higher mRNA expression level of CYP2S1 (cytotoxicity) in quercetin-treated MDA-MB-231 following 72 h of treatment.
Pichia is a widely used host system for the expression of heterologous proteins [23]. In addition to the popularity factors described in the Introduction, this system also offers the strong and highly regulated alcohol oxidase promoter, stable integration events in the host chromosomal DNA and efficient techniques for high-density cultivation to express the protein of interest [24]. Therefore, this study utilised this yeast system to express hTopI, whereby the gene encoding the protein of interest is approximately 2,298 bp. The expression using recombinant yeast containing a single copy number of the target gene was disappointingly low; indeed, the multi-copy number of the gene expression cassette has been one of the most effective strategies to increase the expression of the GOI [9, 25–27]. The recombinant yeast was successfully constructed using the pPIC3.5K vector in this study via the in vivo strategy to determine the effects of gene copy number on cell density, the expression of total protein, and the target enzyme activity in Pichia.
His + Pichia transformants with multi-copy inserts (recombinant yeast) resistant to various concentrations of Geneticin were also successfully selected in this study. However, the selected clones' cell density was likely not affected by the copy number of the target gene in the host, which may interpret as not affect the downstream metabolic activity of the cells. According to previous studies, gene expression induction resulted in excessive plasmid replication that consequently increased the plasmid copy number in the transformants [28–30]. However, this phenomenon contributed also to the host cell metabolic burden [28, 31]. As a result, the metabolic activity was strongly impaired in the cells, indicated by the decelerated increase in biomass and OD. For the effect of the in vivo strategy, the study found the highest expression level of total protein (as much as 1.76 mg/ml) in GS115-pPIC3.5K-hTopI resistant to 1.00 mg/ml Geneticin at 48 h of incubation. However, normalisation of the total protein level per hour and per cell density in each transformant was statistically insignificant compared to the total protein level in control. Therefore, the study is continued by investigating the target enzyme activity, whereby the study found the increment of gene copy number to increase the enzyme activity of hTopI produced in GS115-pPIC3.5K-hTopI.
A transformant or clone with two identical copies of a gene under the control of an identical promoter, in theory, should produce twice as much protein. However, in practice, increasing the gene dosage does not necessarily increase protein expression. In some cases, e.g., human trypsinogen [32] and Na-ASPI [33], increased the gene dosage reduced the protein expression. Therefore, an optimal level rather than a maximal copy number should be considered due to other possible protein expression bottlenecks, e.g., protein translation, secretion or degradation [32, 34–36]. Furthermore, an increased copy number of foreign genes may result in the alteration of normal metabolism in Pichia, leading to a negative influence on the normal cell physiology of multiple-copy recombinant yeast, especially in the case of secretory expression, which includes a reduction in methanol consumption capacity and specific growth rate, decreased cell viability, increased instability of integrated foreign genes or diminished cell secretory ability [37]. For this reason, it is suggested to test the transformants with increasing gene copy numbers and later identify the optimal gene copy number for maximum protein production [34, 38]. Although the strategy used in this study did not significantly change the expression of total protein per cell density in each clone, the ability of hTopI expressed by GS115-pPIC3.5K-hTopI resistant to various Geneticin concentrations was increased as the resistance towards Geneticin was increased. This event also further showed that the hTopI expression ability in this study was able to relax supercoiled DNA, and the enzyme activity increased with increasing target gene copy number.
Incubation of the recombinant yeast (GS115-pPIC3.5K-hTopI) with quercetin and F1 also showed that both substances had different growth inhibitory activity to reduce the growth of the recombinant yeast, which could be further confirmed when MDA-MB-231 was used for the screening. MDA-MB-231 is a poorly differentiated, highly aggressive and invasive breast cancer cell type. It is a model representing triple-negative breast cancer, which is characterised by the lack of oestrogen receptor, progesterone receptor, E-cadherin and HER2 growth factor receptor, but presenting with mutated p53 gene expression [39]. Therefore, MDA-MB-231 is the ideal cell model for investigating the effectiveness of newly developed chemotherapeutic agents. In this study, quercetin and F1 were found to reduce the proliferation of MDA-MB-231 by inducing different cell cycle arrest profiles. Regulation of the cell cycle is crucial for the development of healthy cells. Nevertheless, cancerous cells exhibit uncontrolled cell proliferation and evasion of apoptosis resulting from dysfunction of the cell cycle's checkpoint and destruction [40]. Uncontrolled cell growth and apoptosis resistance are the major defects in cancer cells; thus, discovering potential compounds targeting cell cycle mechanisms and apoptotic machinery could be effective against uncontrolled cell proliferation in neoplasia. This study also explored the mechanism by which quercetin and F1 inhibited MDA-MB-231 cell proliferation in different manners.
Quercetin and F1 were also found to exhibit different cell cycle arrest profiles. Red onion peel is known to contain high concentrations of quercetin [41–42] and is able to induce cell cycle arrest and apoptosis in different cancer cells [43], whereas F1 is able to obstruct cell cycle progression in both S and G2/M phases, suggesting that different comprehensive effects of quercetin and F1 in MDA-MB-231. One possible explanation is the presence of different compound compositions in F1, which gives rise to different molecular mechanisms of cell cycle regulation induced by F1. Nguyen's study reported that 20 µM quercetin induced cell cycle arrest at the S and G2/M phases in MDA-MB-231 at 48 h of treatment [44]. This finding was linked to the increased signalling activities of p21 and GADD45, which contributed to G1/S and G2/M phase arrest, respectively, regulated by p53 [44]. Another study indicated that cell cycle arrest was observed at G2/M phase after treatment of MDA-MB-231 with 100 µM quercetin for 24 and 48 h [45]. Nevertheless, Rivera’s study showed cell cycle arrest at G2/M phase in MDA-MB-231 after 48 h of treatment with 15 µM quercetin [46]. In this study, cell cycle arrest was observed at S phase only after treatment with quercetin for 48 h, which is slightly in contrast to previously published findings. This difference may be due to variation in the cell treatment concentration, where a higher concentration of quercetin was utilised for the experiments. For apoptosis analysis, the results showed that an apoptotic effect was observed in MDA-MB-231 treated with quercetin, but F1 did not induce apoptosis in MDA-MB-231. The induction of apoptotic effects in MBA-MD-231 cells in this study was consistent with other findings using the same cell line [44–47]. This result suggests that cell proliferation inhibition by F1 in MDA-MB-231 occurred through mechanisms other than the apoptosis pathway.
CYP genes have been extensively confirmed to be involved in the metabolism of pro-carcinogenic compounds [48]. Other studies have also shown that the genes encoding these proteins are linked to other cell signalling pathways critical for cell cycle regulation [49]. For instance, the aryl hydrocarbon receptor (AHR), responsible for activating CYP genes' transcription, is a protein that affects cell cycle regulation [50]. Several findings have shown that dietary flavonoids play a role as AHR ligands with either antagonist or agonist activity to inhibit cancer cell growth [51–52]. Additionally, flavonoids may also undergo CYP1-mediated oxidative metabolism to become anti-proliferative products [51]. A study has demonstrated anti-proliferative and cytostatic effects of a flavonoid lipid molecule, eupatorine, in breast cancer cells due to the involvement of CYP1-mediated metabolism [53]. The studies showed that cell cycle arrest at G2/M phase induced by eupatorine could be reversed when MDA-MB-468 cells were coincubated with the CYP1 inhibitor acacetin. Another finding by Atherton confirmed that metabolites produced from the isoflavones daidzein and genistein via CYP1A1, CYP1A2 and CYP1B1 metabolism induced an anti-proliferative response in MCF-7 cells [54].
In this study, the analysis of CYP genes showed that quercetin and F1 induced mRNA expression of CYP1A1 and CYP1B1, with the highest level was observed at 48 h of treatment. This phenomenon corresponded to the initiation of cell cycle arrest at S phase by quercetin in MDA-MB-231. These results also corresponded to the profound changes in cell cycle progression, which was also observed at 48 h of treatment with further induction of cell cycle arrest at G2/M phase by F1. The finding is supported by numerous studies that revealed the role of quercetin in cancer proliferation in relation to its interaction with CYP family enzymes. For instance, quercetin was shown to be an agonist of CYP1A1 in breast cancer cells [55]. A study by Ciolino also showed that quercetin increases CYP1A1 mRNA through mediation by the AHR receptor [56]. Furthermore, the metabolism of quercetin by the CYP1 enzyme, particularly CYP1A1 and CYP1B1, intensifies their anti-proliferative effects in breast cancer cells [55]. Hence, these results suggested that the anti-proliferative effect of F1 on MDA-MB-231 might be due to the metabolic activities of these CYPs resulting in the production of active metabolites, which indirectly modulate the cell cycle progression and survival of MDA-MB-231. For CYP2S1 gene expression analysis, F1 (but not quercetin) induced significantly high gene expression levels in MDA-MB-231 at 24 and 48 h of treatment. The selective expression of CYP2S1 in MDA-MB-231 treated with F1 suggested that CYP2S1 likely plays a role in the regulation of F1 anticancer activity, which is likely regulated by AHR [57].
In conclusion, the recombinant yeast produced in this study can provide preliminary information on the growth inhibitory activity of quercetin and F1, which can then be further confirmed using the MTT assay. The study also explored the basic mechanism by which quercetin and F1 inhibited cell proliferation in MDA-MB-231 via different manners.