Abstract
BackgroundCell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated.MethodsIn this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069–09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress.ResultsPLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids).ConclusionThus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects.Graphical abstract
Highlights
Cell culture is an essential tool in drug discovery
We evaluated the effects of PLN on HepG2/C3A cells based on parameters of cytotoxicity, genotoxicity, proliferation, cell death, oxidative stress, and mitochondrial potential
Our results showed that the reduction in the viability of PLN treatments was related to the reduction of cell proliferation due to the cell cycle arrest of the cells in the G2 and mitosis phases and by the induction of cell death by apoptosis
Summary
Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. A clear understanding of the mechanisms of cell death induction by candidate chemotherapeutic compounds is essential for the development of specific and effective anticancer therapies [1]. Specific target drugs are developed only after studying the molecular mechanisms of cell death and proliferation and by understanding the differences in each type of cancer [2, 3]. The regulation of apoptosis and autophagy comprise the most well-known molecular process, modulating the activity of BCL-2 family of proteins-one of the main mechanisms of induction of death [7,8,9]. The BCL-2 family comprises a group of genes encoding pro or anti-apoptotic proteins, and the imbalance between the amounts of these proteins within the cell can dissipate the signaling of death by one of the pathways of apoptosis (intrinsic pathway). About 27 genes (ATG genes) are involved in the execution of autophagic pathways, while the other 40 genes related to other pathways may be involved in autophagy [12, 13]
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