**5. Conclusions**

The clinical e fficacy of the combined regimen of Dox and cyclophosphamide against breast cancer in vivo is probably related to their combined cytotoxicity as well as their ability to increase ROS production. One way to improve existing anti-cancer treatments is to reduce o ff-target adverse e ffects without reducing e fficacy against breast cancer. In the present study, the addition of γToc to Dox and 4-Cyc di fferentially and specifically reduced ROS levels after only 1 h in the ovarian granulosa cell line COV434 and maintained the percentages of condensed nuclei indicative of cell damage at the same levels as the DMSO control in the two ovarian cell lines expressing steroidogenic pathways, whereas γToc increased cell damage caused by the chemotherapeutics in the breast cancer T47D cell line. If the protective e ffects of γToc in the presence of Dox + 4-Cyc can be repeated in normal, non-cancerous, primary-derived granulosa cells and the chemotherapeutic enhancing e ffects of γToc can be demonstrated in primary-derived breast cancer tumour cells, this will confirm the potential for antioxidant γToc to be developed as an adjunct to existing breast cancer chemotherapy, which will improve fertility preservation for premenopausal breast cancer patients.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/1/51/s1, **Figure S1**: Comparison of DAPI and Crystal Violet Datasets: The numbers of human COV434 ( **A**), MCF7 (**B**), T47D ( **C**) and OVCAR ( **D**) cells per well that were determined using a crystal violet assay were divided by 100 to allow comparison with the DAPI dataset on the same axis. The crystal violet values show mean ± stdev (*n* = 3). The DAPI values are the sum of nuclei with 'condensed' + 'uncertain' + normal morphologies scored in images from three replicate experiments. Each group of apoptotic bodies were assumed to have been formed by the fragmentation of a single nucleus and were therefore given a score of '1'. These were added to the numbers of small, irregularly shaped DAPI-dense nuclei. Nuclei classified as 'uncertain' formed 8.9% (COV434), 2.4% (MCF7), 1.4% (OVCAR) and 1.2% (T47D) of the total numbers of nuclei in all assessed images, whereas the normal nuclei formed 85% (COV434), 95% (MCF7), 94% (OVCAR) and 97.2% (T47D). DAPI values shown as the mean ± stdev (*n* = 3). The crystal violet cell data were subjected to 1Way ANOVA with a Tukey post-test. Significant di fference from control. \*\* *p* > 001, \*\*\* *p* > 0.001, or a v b *p* > 0.05, a v c *p* > 0.001. **Figure S2**: Images of DAPI-stained cell nuclei obtained using an Olympus AX70 fluorescence microscope at ×20 magnification after cells were exposed to chemotherapeutics and tocopherols for 24 h. Dox—doxorubicin, Cyc—cyclophosphamide, Toc—tocopherol.

**Author Contributions:** Conceptualization, F.Y.; methodology, F.Y., D.F.G.; formal analysis, F.Y., D.F.G., investigation, D.F.G.; resources, F.Y.; data curation, F.Y., D.F.G.; writing—original draft preparation, D.F.G.; writing—review and editing, F.Y., D.F.G.; supervision, F.Y.; project administration, F.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Yvette DeGraaf and Pat Vilimas of the Flinders Microscopy and Cell Biology Facility gave much appreciated technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.
