EFFECT OF PIPERINE ON MUTANT p53 (R248Q) AGGREGATION IN NON-SOLID TUMORS

INTRODUCTION AND OBJECTIVES: p53 plays an important role in tumor suppression, either by inducing cell cycle arrest and repairing cellular DNA damage, or by promoting cell apoptosis. However, several types of cancer are characterized by a high incidence of TP53 gene mutation, which in turn is capable of altering p53 conformation. This can trigger p53 intracellular aggregation, causing loss of p53 normal functions, or even generating a gain in p53 oncogenic functions. Piperine, a bioactive compound present in black pepper, presents an important chemopreventive and chemotherapeutic action in in vitro and in vivo studies. However, the mechanisms of action of this compound have not been fully elucidated, especially regarding the possible involvement of p53 (wild-type or mutant form). Thus, the aim of this work is to investigate the effect of piperine on cells that do not express p53, in comparison with cells that express wild-type or mutant p53 (R248Q). MATERIAL AND METHODS: HL60 (acute promyelocytic leukemia cell line that does not express p53), REH (acute lymphoblastic leukemia cell line expressing wild-type p53), and Namalwa (lymphoma cell line expressing mutant p53 R248Q) were used in the experiments. Initially, mitochondrial metabolism was evaluated by the MTT reduction method. Then, immunocytochemistry experiments were performed under a fluorescence microscope. Subsequently, a p53 Seprion-ELISA analysis was performed. Finally, analysis were carried out by fluorescence spectroscopy. RESULTS AND CONCLUSION: MTT reduction assay demonstrated that piperine promoted a dose- and time-dependent effect on HL60, REH, and Namalwa cell lines. However, it had a greater cytotoxic effect on HL60 cells and a lesser effect on Namalwa cells, suggesting a possible p53-independent mechanism. The immunocytochemistry experiments indicated the presence of amyloid-like aggregates in HL60, REH and Namalwa cell lines. However these aggregates only co-localized with p53 in Namalwa cells. suggesting the existence of amyloid-like p53 aggregates in this lymphoma cell line, whereas piperine has shown to increase these p53 aggregates. Then, by p53-seprion-ELISA, we verified that the cell extract of Namalwa had the highest amount of p53 in the amyloid fraction, even when compared to the positive control (MDA-MB-231). In addition, piperine further increased these p53 aggregates in Namalwa cells. Finally, the data obtained by fluorescence spectroscopy suggest that piperine was able to induce the formation of mutant p53 (R248Q) aggregates in vitro. Together, our results suggest that Namalwa cells could be more resistant to piperine action, possibly due to the increased aggregation of mutant p53 (R248Q). Thus, the investigation of the involvement of p53 in the action of compounds, such as piperine, may be important to elucidate the mechanisms of action in cancer cells.