Calotropis procera Selectively Impaired the 4T1 Breast Cancer Cells Growth by Preferentially Blocking Akt/mTOR Signaling
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Abstract
Objectives
To investigate the mechanisms underlying the anticancer activity of Calotropis procera crude phenolics extract (CphE).
Methods
CphE were obtained from leaves homogenized with ethanol (1g:150mL), followed by filtration and evaporation using a rotary evaporator. Quercetin was used as a positive control since is one of the major flavonoids in C. procera. 4T1 cells were treated with CphE (31500g gallic acid equivalent (GAE)/mL), quercetin (Q) (0.63g/mL) or DMSO (control) to assess cell viability using resazurin kit and reactive oxygen species (ROS) using the Carboxy-H2DFFDA probe (Sigma-Aldrich, St Louis, MO). Protein and mRNA expression were investigated using standard procedures and cell migration by wound healing assay.
Results
4T1 cell viability was inhibited by CphE (within 31125g GAE/mL) and Q (0.63g/mL) in a dose-dependent manner, with IC50=49.6g GAE/mL and 1,75g/mL, respectively. However, ROS levels were decreased in cells treated with CphE (down to 0.7-fold of control) while Q induced ROS (up to 1.5-fold of control). These results suggest a contrasting response from 4T1 breast cancer cells to individual phenolics present in CphE. The CphE-induced caspase and PARP-dependent apoptosis and cell viability suppression were mediated by CphE-mediated oxidative stress reduction consistent with phospho-ERK1/2 downregulation (down to 0.4-fold of control). Conversely, Q apoptotic and cell viability suppression mechanisms are mediated by induction of ROS-phospho-ERK1/2 (up to 1.6-fold of control) axis. The Akt/mTOR/CREB pathway was downregulated at a similar extend by CphE and Q, consistent with cell migration (suppressed by 40% and 20% by CphE and Q, respectively) and with protein levels of phospho-Src (downregulated to 0.2-fold and 0.4-fold of control) and phospho-CREB (0.7-fold and 0.6-fold of control) by CphE and Q, respectively.
Conclusions
CphE inhibited cell viability, induced apoptosis and reduced cell migration. These effects were the result of the modulation of proteins that play an important role in epithelial-mesenchymal transition and cell invasion. These findings provide new insights into the anti-cancer mechanisms of C. procera as a promising herb used in folk medicine for breast cancer treatment.
Funding Sources
Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES); Universidade de So Paulo (USP).
