Increased Oxidative Stress Induced by Bioactive Compounds Induce Apoptotic Cell Death in Human Breast Cancer Cells
Authors: Blassan P George
Heidi Abrahamse
Journal: Oxidative Medicine and Cellular Longevity (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Bioactive compounds; Berries; Oxidative stress; Apoptosis; Breast cancer cells
Affiliations: Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, Doornfontein, 2028 Johannesburg, South Africa.

Abstract:  Bioactive compounds from plants represent good candidate drugs for the prevention and treatment of various forms of cancer. Berries are rich sources of bioactive compounds, and there has been an increasing interest in the study of therapeutic action of wild berries. Oxidants are generated continuously in biological system as a result of physiological process. When there is an imbalance between oxidants and antioxidants, it leads to a condition called oxidative stress. Natural compounds as inducers of oxidative stress are able to modulate the physiological functions of cancer cells leading to cell death or survival. The aim of this study was to evaluate the induction of apoptosis by isolated bioactive compounds (1-(2-hydroxyphenyl)-4-methylpentan-1-one (C1) and 2-[(3-methylbutoxy) carbonyl] benzoic acid (C2)) from Rubus fairholmianus against MCF-7 breast cancer cells. The exposure of C1 and C2 reduced viability (IC50 of C1: 4.69; C2: 8.36 μg/mL) and proliferation. Cytochrome c release from mitochondria and changes in mitochondrial membrane potential of treated cells supported the intrinsic apoptotic cell death. Reactive oxygen species (ROS) production after treatment with C1 and C2 was found to be higher and induced nuclear damage. Expression of apoptotic proteins after the treatments was significantly upregulated as indicated using immunofluorescence (caspase 9, p53, and Bax), western blotting (p53, cleaved PARP, cytochrome c, and Bax), and ELISA (caspase 9) analysis. Overall, C1 was more cytotoxic, increased the ROS production in dichlorodihydrofluorescein diacetate assay, and induced apoptosis in breast cancer cells. These results illustrate that berry bioactive compounds have strong chemopreventive potential. In this article, we provide information on prooxidant and anticancer activities of Rubus bioactive compounds. Natural products have always demonstrated a significant contribution to the development of several cancer chemotherapeutic drugs. Most of these compounds are known to affect the redox state of the cell; and studies on these compounds have focused on their antioxidant property instead of prooxidant properties. 

Retracted: No

PubPeer comments:
 Would the authors kindly post high quality original images of Figure 10b? The images I can access online are pixelated, making image analysis difficult. Thank you. *** ![file] , 
 Colleague, the western blot analysis is a qualitative assessment of the expression of proteins. The images provided in this paper is as it was performed, was not manipulated in any fashion and clearly acceptable to the reviewers and the journal especially in light of the fact that quantitative assays supported the expression of these proteins. Kindly read the paper in its entirety to appreciate the significance of this specific assay. , 
 #2 For western blot, we first stained the SDS gels with coomassie blue to check the protein separation. After confirming the presence of proteins, we ran a new gel for the separation and transfer to membrane. The membrane was then stained with ponceau stain to check the presence of protein and the location of target protein. Once this was done, we cut the membranes (only the target protein region and used it for antigen detection). The reason why we cut the membrane into small areas was to analyse different proteins with different molecular weight in the same gel. This is the reason why we donu2019t have the full membrane picture with us. We have the image that is been used for publication. We do have SDS gel with full protein transfer and we also have the ponceau stained membrane before we trimmed it. The PARP cleavage can be seen prominently after antibody reaction, the less intense bands were thick brown bands after antibody detection. Most of the proteins are visible from the gel itself as light and dark blue bands. We have provided the best images and data available on western blot including the gel image shown below. This experiment was done to confirm the presence of these proteins and these results were supported by immunofluorescence, cytochrome c release and mitochondrial membrane potential assays. !![file] , 
 #3 I appreciate the two replies by the authors. My request was for a higher quality image of this figure as the published version shows only u201cdotsu201d when viewed closely (see inverse image below). If I understand your response, it sounds as if you *do* have access to the image used in the published manuscript, and perhaps your original image has a higher resolution than the one published. *** ![file] ,