2-Amino-3-Chlorobenzoic Acid from Streptomyces coelicolor: A Cancer Antagonist Targeting PI3K/AKT Markers via miRNA Modulation

Abstract

Background/Objectives: Actinomycetes, particularly species within the Streptomyces genus, are renowned for their ability to produce a wide array of bioactive molecules with therapeutic potential. This study aimed to comprehensively investigate the antimicrobial and anticancer properties of Streptomyces coelicolor ERI-15, with a particular focus on a purified compound, 2-amino-3-chlorobenzoic acid (2A3CB), and its efficacy against microbial pathogens and breast cancer cell lines. Methods: Antimicrobial compounds were produced through fermentation techniques and isolated via column chromatography. Bioassay-guided fractionation was conducted against Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli (ATCC 25922), and Bacillus subtilis (ATCC 441). Major fractions were further purified using preparative thin-layer chromatography (TLC). The structures of active compounds were elucidated using spectral analyses including IR, mass spectrometry, and ¹H/¹³C NMR. The compound 2A3CB (m/z 171) was tested against MDA-MB-231 and 3T3 cell lines. Cytotoxicity was assessed by the MTT assay, and apoptotic mechanisms were explored via cell proliferation assays, dual fluorescent staining, migration and invasion assays, and analysis of apoptotic markers at mRNA and protein levels. Results: 2A3CB exhibited strong cytotoxic effects on MDA-MB-231 cells, with IC₅₀ values of 26 µM, 5 µM, and 7.2 µM at 24, 48, and 72 h, respectively. It significantly inhibited cell proliferation and migration, and induced apoptosis via caspase-mediated pathways. Expression levels of PTEN, PCNA, BAX, and STAT3 were downregulated, suggesting inhibition of metastasis through the suppression of invasion and migration. Conclusions: The results demonstrate that 2A3CB, derived from S. coelicolor ERI-15, possesses potent antimicrobial and anticancer properties. Its ability to inhibit growth and induce apoptosis in MDA-MB-231 breast cancer cells highlights its potential as a natural therapeutic candidate for targeted cancer treatment, particularly in breast cancer progression.

Keywords: PCNA; Streptomyces; apoptosis; bioautography; column chromatography.

Figure 1

Bioautography and Thin Layer Chromatography (TLC) of Streptomyces coelicolor ERI-15 metabolites against methicillin-resistant staphylococcus aureus (MRSA). (A) Bioautography of major active fractions (7, 8, and 9) against methicillin-resistant Staphylococcus aureus (MRSA). This image displays an agar plate that has been uniformly inoculated with MRSA. Labeled paper strips (7, 8, and 9), each containing different fractions of metabolites extracted from S. coelicolor ERI-15, are placed on the surface of the agar. The presence of clear, circular areas surrounding these paper strips, known as zones of inhibition, indicates that the metabolites within fractions 7, 8, and 9 exhibit antimicrobial activity, effectively inhibiting the growth of MRSA. Control discs, labeled with numbers other than 7, 8, and 9, demonstrate either no inhibition or only minimal inhibition of bacterial growth. (B). TLC profile of Compound-3 of S. coelicolor ERI-15 a: Crude metabolites; b: Purified compound (solvent system: 4:6 Hexane-Ethyl acetate).

Figure 2

Chemical characterization of the compound-3 (2A2CB) of S. coelicolor. ERI-15. (A). UV Vis spectral data of pooled fraction-7–9, (B,C). FTIR spectral data analysis of Fractions 7–9. (D,E). ¹H NMR spectrum of Compound-3 of Streptomyces sp. ERI-15. (F). Structural formula and name of the active molecule formula C₇H₂ClN₀₂. Derived structure details from ¹H and ¹³C NMR values. Its IUPAC name is 2-amino-3-chlorobenzoic acid (2A3CB).

Figure 3

(A): Assay of alkaline phosphatase activity. This figure illustrates the alkaline phosphatase (ALP) activity in control and DAS-treated MDA-MB-231 cell lines. ALP levels are expressed as mU/mg protein, with values representing the mean of three independent experiments. (B): assay of Lactate Dehydrogenase activity. This figure presents the lactate dehydrogenase (LDH) activity in control and treated MDA-MB-231 cells. LDH units are expressed as µM of pyruvate liberated per minute per mg of protein. The values represent the mean of three independent experiments, converted to percentages of the control. Statistically significant differences compared to control cells are indicated by * p < 0.05, ** p < 0.01. (C) The results highlight the impact of 2A3CB treatment on ALP activity in both cell lines, demonstrating the potential effects of the tested compounds on enzyme activity.

Figure 4

Scratch closures with tested S. coelicolor ERI-15 crude and 2A3CB treatment. (A). fold values was calculated using imageJ values between untreated and treated groups. (B). Scratch wound of remaining 5 upper images show S. coelicolor ERI-15 crude and 2A3CB treatment cells, monolayers were mechanically wounded with a 20–200 μL sterile pipette tip following treatment with S. coelicolor ERI-15 crude and 2A3CB treatment. Bar = 200 μm. Statistically significant differences compared to control cells are indicated by * p < 0.05 and ** p < 0.01 indicate statistically significant differences from control group.

Figure 5

Enzymatic Caspase-9, and caspase-3 in MDA-Mb-231 treated with various concentrations of S. coelicolor ERI-15 crude and 2A3CB; C: control cells treated with DMSO 0.01% (vehicle). Data were shown as the mean ± SD from three independent experiments. One-way ANOVA followed by Tuckey’s multiple comparison tests were used to determine mean differences between groups. Statistical significance is shown in the figure as follows: by * p < 0.05 and ** p < 0.01 compared to control.

Figure 6

2A3CB activate the cellular nuclear damage in MDA-Mb-231 breast cancer cells lines. The DNA damage and formation linear strand was excited and showed yellow-orange fluorescence. Live and active cells emit green fluorescence, but apoptotic and necrotic cells emit total red-orange fluorescence evenly. (A). 2A3CB increased the double strand damage and apoptotic formation in ref orange colour. (B). DNA damage was expressed in yellow and orange colour quantification using imageJ software tool and valued revealed in fold change. Data are shown as mean ± SD from representative experiment studied in triplicate. * p < 0.05 and ** p < 0.01 compared to control., treated groups were compared with the DMSO group.

Figure 7

2A3CB abrogates tumorigenic properties. (A). 2A3CB treated cells breast cancer cells was examined for mRNA expression using real time PCR. The mRNA target of Pten, P13K, and AKT markers were quantified. (B). mRNA-specific miRNAs 21, 23, 142 and 221 were quantified using miRNA specific primers and real time PCR. Data are shown as mean ± SD from representative experiment studied in triplicate. Significant differences compared to the control group (DMSO) are denoted as follows: * p < 0.05 and ** p < 0.01.