Hydrogen Sulfide-Releasing Carbonic Anhydrase Inhibitors Effectively Suppress Cancer Cell Growth

Abstract

This study proposes a novel therapeutic strategy for cancer management by combining the antitumor effects of hydrogen sulfide (H₂S) and inhibition of carbonic anhydrases (CAs; EC 4.2.1.1), specifically isoforms IV, IX, and XII. H₂S has demonstrated cytotoxicity against various cancers at high concentrations. The inhibition of tumor-associated CAs leads to lethal intracellular alkalinization and acidification of the extracellular tumor microenvironment and restores tumor responsiveness to the immune system, chemotherapy, and radiotherapy. The study proposes H₂S donor-CA inhibitor (CAI) hybrids for tumor management. These compounds effectively inhibit the target CAs, release H₂S consistently, and exhibit potent antitumor effects against MDA-MB-231, HCT-116, and A549 cancer cell lines. Notably, some compounds display high cytotoxicity across all investigated cell lines. Derivative 30 shows a 2-fold increase in cytotoxicity (0.93 ± 0.02 µM) under chemically induced hypoxia in HCT-116 cells. These compounds also disturb the cell cycle, leading to a reduction in cell populations in G₀/G₁ and S phases, with a notable increase in G₂/M and Sub-G₁. This disruption is correlated with induced apoptosis, with fold increases of 37.2, 24.5, and 32.9 against HCT-116 cells and 14.2, 13.1, and 19.9 against A549 cells compared to untreated cells. These findings suggest the potential of H₂S releaser-CAI hybrids as effective and versatile tools in cancer treatment.

Keywords: antitumor; cancer; carbonic anhydrase; donor; hybrid; hydrogen sulfide; in vitro; inhibitor; multitarget; tumor.

Figure 1

Schematic representation of a hypoxic tumor cell and proteins/processes implicated in its pH regulation. The transmembrane CA IX, XII, and CA IV (omitted), with an extracellular active site, catalyze the CO₂ hydration to bicarbonate and proton. Other involved proteins include (a) aquaporins, (b) anion exchangers (AE1–AE3 isoforms), (c) Na⁺/HCO₃⁻ cotransporters, (d) mono-carboxylate carrier (MTC1 and MTC4), (e) glucose transporter (GLUT1 and GLUT3), (f) ATP-dependent Na⁺/K⁺ antiporter, (g) Na⁺/H⁺ antiporter, (h) plasma membrane proton pump H⁺/ATPase, and (i) H⁺ channels.

Figure 2

Rational design of H₂S releaser–CAI hybrid derivatives as antitumor agents.

Scheme 1

Synthesis of H₂S releaser-CAI derivatives 19–33. Reagents and conditions: (a) SOCl₂, dry EtOH, 0 → 60 °C, 6 h; (b) S₈, 135 → 220 °C, 6–8 h; (c) H₂SO₄ 9M, CH₃COOH, 100 °C, 4 h; (d) RCHO, dry MeOH, reflux, 4 h; (e) NaBH₄, dry MeOH, reflux, 0.5–2 h; (f) R-X, dry DMF, r.t or 60 °C, 0.5–6 h; (g) DIPEA, PyBOP, dry DMF, r.t, o.n. [58].

Figure 3

H₂S-releasing profile of compounds 3, 22, 23, 26, 29, and 30 (150 µM). Compounds were incubated in rat liver homogenate at 37 °C. H₂S concentration was determined spectrophotometrically at timed intervals. Data are expressed as mean ± SEM of three independent assays.

Figure 4

Effects of compounds 23, 26, and 30 on the cell cycle phases of HCT-116 and A549 cancer cells.

Figure 5

Effects of compounds 23, 26, and 30 on the percentage of annexin V-FITC-positive staining in HCT-116 and A549 cancer cells. The experiments were conducted in triplicates. The four quadrants are identified as LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.