In this study, ZnO NPs were successfully synthesized using the extract obtained from Amycolatopsis roodepoortensis strain EA7. The absorption peak of ZnO NPs was confirmed within the range of 509 to 449 cm–1. TEM and SEM images illustrated the spherical shape of the ZnO NPs.TEM images confirmed the crystalline metal nanoparticles withsize 2.98 ± 0.69 nm. Previous studies on ZnO NPs biosynthesis using microbes and plants have reported absorption peaks within different ranges. For instance, ZnO NPs synthesized from Streptomyces sp. showed an absorption peak at 364 nm, with TEM micrographs indicating NP sizes around 20 nm [27]. Kalaba et al. [9] observed absorption peaks of ZnO NPs synthesized from Streptomyces in the range of 349–351 cm–1, with TEM analysis revealing spherical shapes and an average size of 21.72 ± 4.27 nm. This variation in absorption spectra across studies may be attributed to differences in NP shape and size, as well as the dielectric constant of the particles and the surrounding medium [20]. Compounds present in the biomass play a crucial role in the stabilization and stability of ZnO NPs, as evidenced by the various functional groups identified in the FT-IR spectra. The difference in particle size in DLS and TEM may be due to the polydispersity of the synthesized nanoparticles. The particle size in DLS does not only depend on the core of metal nanoparticles but also affects the materials adsorbed on the nanoparticle's surfaces such as stabilizers [10, 27]. Another reason for the size change is the non-homogeneous distribution of samples [10]. The significant value of zeta potential in this study indicates particle repulsion, higher Brownian motion, and lower aggregation tendency, all of which lead to long-term stability [27]. In the current study, ZnONPs synthesized showed antimicrobial effects concentration-dependent against the clinical and standard strains in all three investigated methods: Well Diffusion agar, MIC, and MBC. In the Well Diffusion Agar method, antimicrobial activity at concentrations of 2000–5000 µg/ml for both clinical and standard strains showed effectiveness. The minimum inhibitory concentration against clinical and standard strains of S. aureus was 250 and 125 µg/ml, respectively. while for clinical and standard strains of P. aeruginosa, was 500 µg/ml. Additionally, the values of MBC were obtained for both clinical and standard strains of P. aeruginosa and S. aureus 2000 and 1000 µg/ml, respectively. Abdo et al. [20] observed the antibacterial effect of ZnO NPs synthesized with P. aeruginosa bacteria using the well diffusion agar method at higher concentrations (250 and 150 mg/l) for S. aureus. They did not observe an antimicrobial effect for P. aeruginosa. Belly et al. [28] reported the values of minimum inhibitory concentration (MIC) of ZnO NPs synthesized from Arthrospira platensis algae in the MIC method, at concentrations of 50 mg/L for S.aureus and 25 mg/L for P. aeruginosa. It is not consistent with the results of our study. Additionally, ZnO NPs showed the greatest effect at a concentration of 500 µg/ml (P < 0.0001), 250, 125, 62.5, and 31.250 µg/ml (P < 0.001) in the cell line HT-29. Baskabadi et al. [26 ]. reported the effect of ZnO NPs synthesized from Angoza plant extract on the HT-29 colon cancer cell line(P < 0.001) with different concentrations of ZnO NPs (250,125, 62.5, and 31.2µg/ml) and the reduction of Bcl2 gene expression. Other studies have also observed the toxicity of ZnO NPs synthesized from plant leaves in a dose-dependent manner against various colon cancer cell lines, with cell survival decreasing as nanoparticle concentration increases [11]. It is consistent with the results of our study. In this research, the expression level of ATR, ATM, CHK1, and CHK2 genes effective in apoptosis increased significantly compared to the reference gene GAPDH, which indicates the induction of apoptosis in HT29 cells. However, there was no significant correlation in the reduction of the expression of MMP9 and BCL2 genes compared to the control sample. These synthesized nanoparticles could not inhibit MMP9 gene expression and prevent cell migration. Also, flow cytometry results showed, 46.74%( P < 0.0001) of the cells HT-29 entered the apoptosis stage compared to the control sample (GAPDH gene). In the cell cycle analysis, the synthesized nanoparticles caused a significant increase in the cell population in the sub-G1 phase, which indicates the induction of apoptosis. On the other hand, a significant decrease was observed in G1. These nanoparticles can also arrest cell cycle in G0/G1 phase. The increased expression of ATR, ATM, CHK1, and CHK2 genes confirms these results. ATR and ATM are responsible for the phosphorylation of checkpoint kinases 1 and 2 (CHK1,2). Increasing the expression level of CHK1 and CHK2 genes leads to activation of cell cycle checkpoint response, cell cycle arrest, DNA repair, and possibly cell death. ATM is initially activated in response to DNA double-strand breaks[29]. ATR is a key regulator of DDR(DNA damage response), and this finding emphasizes the relevance of DDR as a novel therapeutic target in cancer therapy[29, 30]. Recent reports have shown apoptotic cell death and cell cycle arrest due to exposure to nanoparticles synthesized with plant and microbial extracts, consistent with the results of our study[31]. Also, in the study of Tan et al.[ 16] a decrease in Bcl2 gene expression was observed after treatment with chitosan-iron oxide nanoparticles compared to the control sample. However, no significant difference was observed in MMP9 gene expression in HT-29 cells compared to the control sample. The toxicity of nanoparticles is caused by the interaction of the physicochemical properties of nanoparticles with body cells. Characteristics such as size, chemical composition, shape(spherical shape), surface structure(the smaller surface area), stability, surface charge, hydrophilicity or hydrophobicity, and accumulation of chemical functional groups affect the interaction of nanoparticles with body cells. The size of synthesized nanoparticles facilitates their penetration through cell membranes, resulting in damage to membrane permeability [10, 27]. This superior activity can be attributed to three primary mechanisms: (1) the release of toxic ions (Zn2+) inside the cell; (2) electrostatic attraction between ZnO NPs and the cell wall; and (3) the production of reactive oxygen species (ROS) [11, 20].
Through this interaction, ZnO NPs infiltrate the cell, distorting plasma membrane structure and compromising cell wall integrity, resulting in the release of cellular components [9, 20]. Also, the greater toxicity of ZnO NPs can be attributed to its high solubility in the extracellular region, which in turn increases the toxicity of ZN+ 2 intracellularly, or it may be due to direct entry into the cell and increases the level of ZN+ 2 be intracellular. ROS production induces high oxidative stress, resulting in cell death or damage [9, 20, 27]. The toxic effects resulting from ROS formation are attributed to the generation of various reactive species such as O2– (superoxide anion), OH– (hydroxyl ion), and H2O2 (hydrogen peroxide). While, O2– and OH– remain on the surface of the cell due to their negative charge and cannot penetrate the cell membrane, the final product H2O2 can penetrate the cell wall and membrane to interact with lipids, proteins, and nucleic acids, ultimately leading to cell death[10, 32]. The significant effect of nanoparticles on HT-29 cancer cells can be attributed to their direct impact on the cell's respiratory system in the mitochondria[33, 34]. Given the high mitochondrial activity in cancer cells compared to normal cells during the respiration process, ZnO NPs find a suitable substrate to target cancer cells effectively[11]. Additionally, differences in membrane morphology and pore size between cancer cells and normal cells, contribute to nanoparticle toxicity in cancer cells [16, 35]. Recent studies demonstrate the essential role of Chk1 and Chk2 in the network of genome-wide regulatory pathways that coordinate cell cycle progression with DNA repair and cell survival or death [29, 36]. The aim of anticancer drugs is to induce cell cycle arrest in phase M and initiate apoptosis. Apoptosis repair as an inhibitory anticancer strategy has been the main approach in the development of cancer treatments targeting the Bcl-2 family [16, 33]. Evidence suggests that DNA damage response signaling pathways in cancer therapy may be attractive targets for cancer therapy. Therefore, maintaining a sufficient antioxidant level in the body to maintain the optimal oxidant/antioxidant balance is an important strategy in preventing carcinogenesis [16, 35]. Nanoparticles exhibit unique characteristics due to their small size and large surface-to-volume ratio, leading to improvements in mechanical, magnetic, optical, and catalytic properties, thus enhancing their potential medicinal applications [10, 11]. It is suggested that more studies be conducted on the anti-cancer properties of metal nanoparticles to determine the medical importance of these nanoparticles. In short, in the present research, ZnO NPs were synthesized for the first time using bioactive compounds of Amycolatopsis roodepoortensis bacteria.The results showed the toxic effects of this nanoparticle on colon cancer cells (HT-29) .This nanoparticle induced apoptosis, which was confirmed by two methods, flow cytometry and Real Time PCR.