Hyaluronic Acid Improves Hydrogen Peroxide Modulatory Effects on Calcium Channel and Sodium-Potassium Pump in 4T1 Breast Cancer Cell Line

Abstract

Maintaining homeostasis of ion concentrations is critical in cancer cells. Under hypoxia, the levels of channels and pumps in cancer cells are more active than normal cells suggesting ion channels as a suitable therapeutic target. One of the contemporary ways for cancer therapy is oxidative stress. However, the effective concentration of oxidative stress on tumor cells has been reported to be toxic for normal cells as well. In this study, we benefited from the modifying effects of hyaluronic acid (HA) on H₂O₂, as a free radical source, to make a gradual release of oxidative stress on cancer cells while preventing/decreasing damage to normal cells under normoxia and hypoxic conditions. To do so, we initially investigated the optimal concentration of HA antioxidant capacity by the DPPH test. In the next step, we found optimum H₂O₂ dose by treating the 4T1 breast cancer cell line with increasing concentrations (0, 10, 20, 50,100, 200, 500, and 1000 μM) of H₂O₂ alone or H₂O₂ + HA (83%) for 24 hrs. The calcium channel and the sodium-potassium pumps were then evaluated by measuring the levels of calcium, sodium, and potassium ions using an atomic absorption flame spectrophotometer. The results revealed that treatment with H₂O₂ or H₂O₂+ HA led to an intracellular increase of calcium, sodium, and potassium in the normoxic and hypoxic circumstances in a dose-dependent manner. It is noteworthy that H₂O₂ + HA treatment had more favorable and controllable effects compared with H₂O₂ alone. Moreover, HA optimizes the antitumor effect of oxidative stress exerted by H₂O₂ making H₂O₂ + HA suitable for clinical use in cancer treatment along with chemotherapy.

Figure 1

Schematic illustration of how H₂O₂ or H₂O₂ + HA functions on 4T1 cell ion channels/pumps. (a) The cell is normal and the channels function well to maintain the homeostasis of cell ions. (b) The membranous channels/pumps of cells exposed to H₂O₂ are disrupted, and the balance of ions is disrupted within the cell causing a severe imbalance due to an increase in intracellular Ca²⁺, K⁺, and Na⁺. Intracellular Ca²⁺ accumulation induces apoptosis in the cell. (c) HA softens oxidative effects of H₂O₂, modifies the harsh release of oxidants from H₂O₂, controls cell proliferation, and gently directs the cell toward apoptosis.

Figure 2

Characterization of the hypoxic condition by analysis of PDL-1 expression on 4T1 cancer cell membrane after 24 hrs of incubation (a). Different rate of PDL-1 expression on 4T1 cancer cell membrane under normoxic or hypoxic condition after 24 hrs of incubation (b). (A) Normoxia, (B) hypoxia, and (C) merge of (A) and (B).

Figure 3

Scavenging of DPPH radical by hyaluronic acid: hyaluronic acid was prepared at concentrations of (0, 0.25, 0.5, 0.75, 1%), and then, the modifying capacity of hyaluronic acid on oxidative stress was investigated with the DPPH assay. Data are expressed as mean ± SD of three independent experiments; undisclosed SDs fall within respective symbols.

Figure 4

Effect of H₂O₂ or H₂O₂ + HA on the intracellular Ca²⁺ levels in breast cancer cells treated under normoxic or hypoxic conditions after 24 hrs of treatment: (a) (a1) Cells under normoxia and treated with H₂O₂. (a2) Cells under normoxia and treated withH₂O₂ + HA. (a3) Cells under hypoxia and treated with H₂O₂. (a4) Cells under hypoxia and treated with H₂O₂ + HA. Data are shown as mean ± SD (n = 3) (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001vs. respective control). (b) Comparison of intracellular Ca²⁺ levels in 4T1 cancer cells after 24 h of treatments with different concentrations in normoxia and hypoxia conditions determined by scratch assay. Data are shown as mean ± SD (n = 3) (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001vs. similar concentrations in different conditions. Each index is represented with assigned color in the legend).

Figure 5

Changes in Na⁺ and K⁺ levels in 4T1 cancer cells treated with different concentrations of H₂O₂ or H₂O₂ + HA under normoxia and hypoxia for 24 hrs. (a) Cells under normoxia and treated with H₂O₂. (b) Cells under normoxia and treated with H₂O₂ + HA. (c) Cells under hypoxia and treated with H₂O₂. (d) Cells under hypoxia and treated with H₂O₂ + HA. Data are expressed as mean ± SD from three independent experiments (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001vs. respective control).