Title:Calotropis Procera Induced Caspase-Dependent Apoptosis and Impaired Akt/mTOR
Signaling in 4T1 Breast Cancer Cells
Volume: 22
Issue: 18
Author(s): Ana Carolina Silveira Rabelo*, Maria Angélica Miglino, Shirley Arbizu, Ana Cláudia O. Carreira, Antônio José Cantanhede Filho, Fernando José Costa Carneiro, Marjorie Anne A. Layosa and Giuliana Noratto
Affiliation:
- Laboratory of Stem Cell, University of São Paulo (USP), São Paulo, 05508 270, Brazil
Keywords:
stage IV human breast cancer, milkweed (Calotropis procera), MAPK/ERK1/2, medicinal plants, angiogenesis, metastasis.
Abstract:
Introduction: Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in
folk medicine to treat various diseases for more than 1500 years.
Aims: Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against
4T1 breast cancer cells and in BALB/c mice.
Methods: 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability
by 50% (IC50). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined
following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were
injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg
GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein
expression and histology.
Results: In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage
and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation
contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which
are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results
showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis
reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested
anti-invasive cancer activity of CphE.
Conclusion: CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential
for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE
anticancer and antimetastatic activity in vivo.
