Anticancer Potential of Pyridoxine-Based Doxorubicin Derivatives: An In Vitro Study

Abstract

Doxorubicin (DOX) is a prevalent anticancer agent; however, it is unfortunately characterized by high cardiotoxicity, myelosuppression, and multiple other side effects. To overcome DOX limitations, two novel pyridoxine-derived doxorubicin derivatives were synthesized (DOX-1 and DOX-2). In the present study, their antitumor activity and mechanism of action were investigated. Of these two compounds, DOX-2, in which the pyridoxine fragment is attached to the doxorubicin molecule via a C3 linker, revealed higher selectivity against specific cancer cell types compared to doxorubicin and a promising safety profile for conditionally normal cells. However, the compound with a C1 linker (DOX-1) was not characterized by selectivity of antitumor action. It was revealed that DOX-2 obstructs cell cycle progression, induces apoptosis via the mitochondrial pathway without the development of necrosis, and showcases antioxidant capabilities, underlining its cell-regulatory roles. In contrast to doxorubicin's DNA-centric mechanism, DOX-2 does not interact with nuclear DNA. Given these findings, DOX-2 presents a new promising direction in cancer therapeutics, which is deserving of further in vivo exploration.

Keywords: DNA intercalation; anticancer agents; antioxidants; apoptosis induction; cell-cycle arrest; doxorubicin; doxorubicin derivatives; pyridoxine; vitamin B6.

Scheme 1

The synthesis of pyridoxine and doxorubicin derivatives linked by a peptide bond. Reagents and reaction conditions: (i) Me₂CO, HCl; (ii) KMnO₄, H₂O/Me₂CO; (iii) doxorubicin, DIPEA, HATU, DCM; (iv) MnO₂, CH₂Cl₂; (v) (2-Ethoxy-2-oxoethyl)triphenylphosphonium chloride, Et₃N, DCM; (vi) K₂CO₃, MeOH/H₂O.

Figure 1

The chemical structure of Doxorubicin (DOX) and its derivatives DOX-1 and DOX-2. Bonds in bold black represent the pyridoxine moiety in the compounds; bonds in bold red represent a linker.

Figure 2

The distribution of MCF-7 tumor cells across cell cycle phases ((a) G0/G1 phase, (b) S phase, (c) G2/M phase) when treated with doxorubicin (blue columns) and its derivatives DOX-2 (red columns) for 72 h using Hoechst 33342 dye as an indicator. p > 0.05 (ns), 0.05–0.01 (*), 0.01–0.001 (**), 0.001–0.0001 (***), <0.0001 (****). The experiment was independently repeated three times in duplicate. This figure displays data from the most representative experiment.

Figure 2

The distribution of MCF-7 tumor cells across cell cycle phases ((a) G0/G1 phase, (b) S phase, (c) G2/M phase) when treated with doxorubicin (blue columns) and its derivatives DOX-2 (red columns) for 72 h using Hoechst 33342 dye as an indicator. p > 0.05 (ns), 0.05–0.01 (*), 0.01–0.001 (**), 0.001–0.0001 (***), <0.0001 (****). The experiment was independently repeated three times in duplicate. This figure displays data from the most representative experiment.

Figure 3

The distribution of MCF-7 cells into apoptosis phases after DOX-2 treatment for 72 h according to Annexin V-FITC/DAPI staining. Treatment with Triton X-100—24 h. The figures present the average data derived from two independent experiments conducted in duplicate.

Figure 4

Percentage of cells with reduced mitochondrial potential ΔΨ (cells with low Rh123 fluorescence) when treated with doxorubicin (red columns) and its derivatives (DOX-1 ((a), green columns) and DOX-2 ((b), blue columns)) for 72 h. p > 0.05 (ns), 0.05–0.01 (*), 0.01–0.001 (**), 0.001–0.0001 (***), <0.0001 (****). The figures present the average data derived from two independent experiments conducted in triplicate.

Figure 5

MCF-7 (A) and HCT-116 (B) tumor cells treated with doxorubicin and its derivatives DOX-1 and DOX-2, as well as with Hoechst 33342 dye for nucleus visualization. DOX-1 and DOX-2—5 μM, 24 h incubation; DOX—50 μM, 2 h incubation. The experiment was independently repeated three times in triplicate. The figures display data from the most representative experiment.

Figure 5

MCF-7 (A) and HCT-116 (B) tumor cells treated with doxorubicin and its derivatives DOX-1 and DOX-2, as well as with Hoechst 33342 dye for nucleus visualization. DOX-1 and DOX-2—5 μM, 24 h incubation; DOX—50 μM, 2 h incubation. The experiment was independently repeated three times in triplicate. The figures display data from the most representative experiment.

Figure 6

Absorption spectra of doxorubicin (a) and its derivatives DOX-1 (b) and DOX-2 (c) when interacting with chicken erythrocyte genomic DNA. The experiment was independently repeated two times in duplicate. The figures display averaged spectra.

Figure 6

Absorption spectra of doxorubicin (a) and its derivatives DOX-1 (b) and DOX-2 (c) when interacting with chicken erythrocyte genomic DNA. The experiment was independently repeated two times in duplicate. The figures display averaged spectra.

Figure 7

Electrophoregram of genomic DNA compositions with doxorubicin (DOX) and its derivatives DOX-1 (a) and DOX-2 (b). Red fluorescence corresponds to doxorubicin (emission at 532 nm) and green represents DNA stained with SYBR Green (emission at 473 nm). The experiment was independently repeated two times.

Figure 8

Tumor cells, intact and treated with H₂O₂ 1 mM (20 min), DOX (0.5 and 1.1 μM, 72 h), DOX-1 (6.1 and 11.3 μM, 72 h), DOX-2 (10 and 20 μM, 72 h). Agarose gel electrophoresis, SYBR Green I staining. The experiment was independently repeated two times in triplicate.

Figure 9

Polymerization kinetics of highly purified porcine neuronal tubulin in the presence of doxorubicin and its derivatives DOX-1 and DOX-2, negative (vinblastine) and positive (paclitaxel) controls. The experiment was independently repeated two times. The figures display data from the most representative experiment.