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Figure 1. Scheme of synthesis: (A) synthesis of acetylated conjugates SAB-1, -3, -5, -7, -9, -11, -13, -15, -17, and -19 from halogenoquinones with tetra-O-acetyl-6-mercaptoglucose; (B) synthesis of the acetylated conjugates SAB-21, -23, and -25 from juglone derivatives and naphthazarin; and (C), synthesis of the deacetylated (unprotected) conjugates SAB-2, -4, -6, -8, -10, -12, -14, -16, -18, -20, -22, -24, and -26 from the corresponding acetylated conjugates.
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Figure 2. Cytotoxicity and selectivity of the synthesized compounds. (A) The Western blot examination of GLUT-1 expression in human prostate cancer PC-3 versus human prostate non-cancer PNT2 cells and its quantification. β-actin was used as a loading control (mean ± SEM; n = 3; Student′s t-test). (B) Ratio of IC50 (PNT2)/IC50 (PC-3) indicated selective cytotoxicity towards human prostate cancer cells versus human prostate non-cancer cells. “#”, IC50 towards one of the tested cell line was >100 µM; “##”, IC50s (inhibition concentrations 50%) towards both tested cell line were >100 µM. (C) Pooled selectivity index (SI) value of acetylated derivatives (Ac-Glc) vs. non-acetylated derivatives (free-Glc) (mean ± SEM; n ≥ 10; * p < 0.05, Student′s t-test). For each compound, the SI was calculated as IC50 (PNT2)/IC50 (PC-3). (D) Structures of compounds SAB-13 and -14. (E) The mean cytotoxicity of the most promising compounds SAB-13 and -14 in five prostate cancer (LNCaP, 22Rv1, VCaP, PC-3, and DU145) vs. five non-cancer (PNT2, RWPE-1, HEK 293T, MRC-9, and HUVEC) cell lines (mean ± SEM; n = 5; Student′s t-test). Cells were treated for 48 h. The viability was measured by MTT assay.
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Figure 3. Cytotoxicity of the compounds is related to the Warburg effect. (A) The expression of the glucose transporter 1 mRNA (GLUT-1) in five human prostate cancer cell lines (LNCaP, 22Rv1, VCaP, PC-3, and DU145) versus four human non-cancer cell lines (PNT2, RWPE-1, HEK 293T, MRC-9, and HUVEC) (mean ± SEM; n = 5; Student′s t-test). The expression was measured by qPCR. (B) The viability of PC-3 cells incubated with SAB-13 and -14 in glucose-free (−Glc) or in glucose-containing media (+Glc, 2 g/L). Cytotoxic activity was measured using MTT test following 24 h of treatment. (C,D) Concurrent inhibition of glucose uptake by the SAB-13 and -14. PC-3 cells were treated with the compounds for 24 h and then the glucose uptake was measured using 2-NBDG-based assay either in cell culture by plate reader (C) or in single cells using flow cytometry technique (D) and then normalized to cell viability (mean ± SEM; n = 3; * p < 0.05, one-way ANOVA test). Apigenin (Apig), phloretin (Plt), and cytochalasin B (Ccl-B) were used as positive controls. The viability was measured by MTS assay (B,C) or by flow cytometry using PI staining (D).
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Figure 4. Pro-apoptotic activity of SAB-13 and SAB-14. (A–C,E,F), FACS analysis of the cells after 48 h treatment. Analysis of apoptosis induction in 22Rv1 (A) and PC-3 cells (B) using Annexin-V-FITC/propidium iodide (PI) double staining. PC-3 cells were pre-treated with 100 µM of pan-caspase inhibitor z-VAD(OMe)-fmk (zVAD) for 1 h and then treated with indicated concentrations of the drugs for 48 h (B). Viable cells (Annexin-V-FITC(–)/PI(–), LL quadrant) or early apoptotic cells (Annexin-V-FITC(+)/PI(–), LR quadrant) were quantified using the Cell Quest Pro software (C) (mean ± SEM; n = 3; * p < 0.05, Student′s t-test). (D) Western blotting analysis of the protein expression in 22Rv1 cells after 48 h of treatment. β-actin was used as a loading control (mean ± SEM; n = 3; one-way ANOVA test). Anisomycin (Aniso; treatment with 10 µM for 48 h) was used as a positive control. (E,F) Cell cycle analysis of 22Rv1 cells using PI staining, apoptotic cells were detected as sub-G1 population (E) (mean ± SEM; n = 3; * p < 0.05, one-way ANOVA test).
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Figure 5. The effect of SAB-13 on proteome of prostate cancer cells. 22Rv1 cells were treated with SAB-13 (2 µM, 48 h). The changes in proteome identified by LC-MS/MS and later analyzed using Ingenuity Pathway Analysis (IPA) software and z-score algorithm. (A) Hypothetical protein interaction networks between regulated proteins and the proteins predicted to be involved in interactions (constructed using IPA software). (B) Some kinases predicted to be affected under the treatment were further validated by Western blotting (marked by red circle). The activation was observed 2 h after the treatment. β-actin was used as a loading control. The expression phospho-kinases was normalized to the non-phospho-kinases expression levels (mean ± SEM; n = 3; * p < 0.05; one-way ANOVA test). (C) Gene ontology analysis. (D) Top upstream targets and the calculated z-score of the effect (activation/inhibition), predicted by IPA. p-values of overlap is indicated on the graph (significance: p < 0.05, Fischer′s exact test). (E) Top canonical pathways, predicted by IPA. The target/process is expected to be activated if z-score > 0 (red area) or suppressed if z-score < 0 (green area) (significance: p < 0.05, Fischer′s exact test). (F) Representative scheme of oxidative phosphorylation. Affected proteins discovered by proteome analysis are marked with color.
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Figure 6. Effect of SAB-13 and SAB-14 on mitochondrial membrane potential and ROS production. (A,B) Effect of the drugs on mitochondrial membrane potential (MMP, ΔΨm). 22Rv1 cells were treated with the SAB-13 or SAB-14 for indicated time, harvested with trypsin, stained with JC-1, and measured by FACS (A), and the cells containing depolarized mitochondria were quantified using the Cell Quest Pro software (B) (mean ± SEM; n = 3; * p < 0.05, one-way ANOVA test). CCCP (50 µM) was used as a positive control. (C,D) Effect on ROS production in 22Rv1 cells after 2 h of treatment. Cells were stained with CM-H2DCFDA, treated with the drugs, harvested, and analyzed by FACS (C), and the ROS level was quantified using the Cell Quest Pro software (D) (mean ± SEM; n = 3; * p < 0.05, one-way ANOVA test). H2O2 (200 µM) was used as a positive control. (E) 22Rv1 cells were pre-treated with 1 mM NaC (N-acetyl-l-cysteine) for 1 h and then co-treated with the investigated drugs for 48 h FBS- and glucose-free media. Cell viability was measured by MTT assay, treatment time was 48 h. (F,G), Western blotting analysis of cleaved caspase-3 and -9 levels in 22Rv1 cells treated with SAB-13 for 2 h (F) or 48 h (G). Anisomycin (Aniso) was used as a positive control. β-actin was used as a loading control (mean ± SEM; n = 3; one-way ANOVA test).
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Figure 7. Quantification of the proteins in mitochondrial, cytoplasmic, and total fractions. 22Rv1 cells were treated with the drugs for 48 h, the proteins were extracted and fractionated using Cell Fractionation Kit (abcam). The mitochondrial (Mito), cytoplasmatic (Cyto) or total (Total) fractions were concentrated and analyzed by Western blotting. Cells treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 50 µM) were used as a positive control. α-tubulin was used as a loading control, the protein expression was normalized to α-tubulin expression levels either in cytosolic fraction or in the total protein lysate (mean ± SEM; n = 3; * p < 0.05; one-way ANOVA test).
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Figure 8. Suggested mechanism of anticancer activity in vitro.
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