PS9, Derived from an Aquatic Fungus Virulent Protein, Glycosyl Hydrolase, Arrests MCF-7 Proliferation by Regulating Intracellular Reactive Oxygen Species and Apoptotic Pathways

Abstract

One of the most common diseases in women is breast cancer, which has the highest death globally. Surgery, chemotherapy, hormone treatments, and radiation are the current treatment options for breast cancer. However, these options have several adverse side effects. Recently, peptide-based drugs have gained attention as anticancer therapy. Studies report that peptides from biological toxins such as venom and virulent pathogenic molecules have potential therapeutic effects against multiple diseases, including cancers. This study reports on the in vitro anticancer effect of a short peptide, PS9, derived from a virulent protein, glycosyl hydrolase, of an aquatic fungus, Aphanomyces invadans. This peptide arrests MCF-7 proliferation by regulating intercellular reactive oxygen species (ROS) and apoptotic pathways. Based on the potential for the anticancer effect of PS9, from the in silico analysis, in vitro analyses using MCF-7 cells were executed. PS9 showed a dose-dependent activity; its IC₅₀ value was 25.27-43.28 μM at 24 h. The acridine orange/ethidium bromide (AO/EtBr) staining, to establish the status of apoptosis in MCF-7 cells, showed morphologies for early and late apoptosis and necrotic cell death. The 2,7-dichlorodihydrofluorescein diacetate (DCFDA) staining and biochemical analyses showed a significant increase in reactive oxygen species (ROS). Besides, PS9 has been shown to regulate the caspase-mediated apoptotic pathway. PS9 is nontoxic, in vitro, and in vivo zebrafish larvae. Together, PS9 may have an anticancer effect in vitro.

Figure 1

In silico investigation of glycosyl hydrolase protein and its derived PS9. (A) 3D structure of the glycosyl hydrolase protein. (B) 3D structure of the PS9 peptide. (C) Hydrophobicity analysis in the helical wheel analysis.

Figure 2

Anticancer potency of PS9 due to neutral red uptake assay. The results were compared with the control, and the data were expressed as mean ± SD. The asterisk (*) represents the statistical significance at p < 0.05.

Figure 3

Total LDH release due to the PS9 treatment on MCF-7 cells. The data were compared with the untreated control. The data showed a significant difference at p < 0.05, which was indicated by an asterisk (*). The data were presented as the mean of three replicates ± SD.

Figure 4

Morphological analysis of PS9-treated cells at 20× magnifications in an inverted phase-contrast microscope. Both the circles and arrows represent the abnormal morphology of the cells.

Figure 5

Acridine orange/ethidium bromide (AO/EtBr) staining on MCF-7 cells to analyze the apoptotic stages. V, viable cells; E, early apoptotic cells; L, late apoptotic cells; and N, necrotic cells.

Figure 6

DCFDA staining to measure the ROS level on MCF-7 cells. PS9 treatment generates ROS compared to the untreated control.

Figure 7

Mitochondrial membrane potentiality of PS9. The PS9 treatment showed a dose-dependent activity on MCF-7 cells compared to the untreated control.

Figure 8

Gene expression analysis of PS9 on MCF-7 cells. The expression was calculated after the data were normalized with GAPDH. The data were expressed as mean ± SD of three independent experiments. The asterisk (*) denotes the significance level at p < 0.05 compared to the control.

Figure 9

Toxicity study on zebrafish larvae between 0 and 72 hpf. The larvae were treated with four different concentrations of PS9. The untreated larvae were considered the control, and the larvae treated with 1 mM of H₂O₂ were considered the positive control. The positive control showed abnormal morphologies such as a bent spine (BS).