Effects of protein binding and low-dose radiation on anti-cancer activities of 4-hydroxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid in leukemic K562 and multidrug resistant K562/Dox cell lines

Abstract

Leukemia is the hematologic malignancy and common treatment for this disease is chemotherapy. Multidrug resistance (MDR) is the major impediment in treating with chemotherapeutic drugs to cancer patients. P-glycoprotein which uses adenosine triphosphate (ATP) as energy to pump out the anticancer drugs to the extracellular space is usually overexpressed in the membrane of MDR cancer cells which is one of the main causes of MDR. Plant-based products have diverse pharmacological activities draw attention to develop as new MDR inhibitors. Phenolic acid derivatives, 4-hydroxybenzoic acid (4-HBA) and 4-hydroxy-3-methoxybenzoic acid (Vanillic acid, VA), have antioxidant and anticancer activities. However, the efficacy of phenolic acid depends on protein binding. In addition, low-dose radiation (LDR) can give beneficial effects such as hormetic effect and adaptive response to normal cells which lead to adapted cellular response to subsequent exposure of challenging radiation doses. On the other hand, LDR can cause cancer cells death. This work studied on the effects of protein binding and low-dose radiation on anticancer activities of 4-HBA and VA in leukemic K562 and multidrug resistant K562/Dox cells. First objective was to investigate the binding mechanisms of 4-HBA and VA with human serum albumin (HSA). The results indicated that both 4-HBA and VA could interact with HSA. According to the results of fluorescence quenching process, static quenching mechanism was occurred in the HSA-4-HBA system whereas a dynamic quenching mechanism was the main process in the HSA-VA system. Two main interaction forces (hydrogen bonds and Van der Waals forces) were involved in binding of HSA to 4-HBA or VA. Second objective was to investigate the anti-proliferative activities of 4-HBA and VA in K562 and K562/Dox cancer cells and their possible mechanisms. The results showed that 4-HBA and VA had decreased the cell viability in both dosage and time dependency. The co-treatment of pirarubicin (Pira) with 4-HBA and VA (0.01. 0.1, 1, and 10 mM) could decrease the IC50 value of Pira in K562 and K562/Dox cells at 48 and 72 hours. The succinate dehydrogenase (SDH) activity, mitochondrial membrane potential (ΔΨm), and adenosine triphosphate (ATP) levels were significantly reduced in K562, and K562/Dox cells treated with 4-HBA and VA (0.01. 0.1, 1, and 10 mM). The results of the drug kinetic uptake study stated that the intracellular Pira concentration was increased in both 4-HBA and VA treated K562/Dox cells. Last objective was to investigate the effect of LDR on anticancer activities of 4-HBA and VA in K562 and K562/Dox cancer cells. The results suggested that pre-exposure to LDR followed by 4-HBA or VA treatment could enhance the impairment of cellular energetic state in both K562 and K562/Dox cells and the inhibition of P-glycoprotein-mediated efflux Pira in K562/Dox cells. Moreover, 4-HBA and VA (0.01, 0.1, 1, and 5 mM) showed non-competitive inhibition of Pira efflux in K562/Dox cells. In conclusion, 4-HBA and VA could interact with HSA. Two main interaction forces (hydrogen bonds and Van der Waals forces) were involved in interaction. 4-HBA and VA had induced cell death in both K562 and K562/Dox cells via impairment of cellular energetic state. In addition, 4-HBA and VA could modulate P-gp function by non-competitive inhibition and damage energetic state of the K562/Dox cancer cells. Pre-exposure to low-dose radiation followed by 4-HBA and VA treatment could enhance the impairment of energetic state of the K562 and K562/Dox cancer cells and inhibit the P-gp pumping function of Pira to the extracellular space in K562/Dox cells.