Effect of chemotherapeutics on ovarian function in women with breast cancer

Author: Daniela Figueroa Gonzalez

Figueroa Gonzalez, Daniela, 2020 Effect of chemotherapeutics on ovarian function in women with breast cancer, Flinders University, College of Medicine and Public Health

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This PhD thesis evaluated the effect of a clinically relevant combination of chemotherapeutics; Doxorubicin (Dox) and 4-Cyc on cytotoxicity and ROS production by human breast and ovarian cancer-derived cell lines in vitro.

Breast cancer is the number one malignancy among women accounting for more than 400,000 deaths each year globally. The incidence of breast cancer is higher in western societies and in postmenopausal women, but in Asian countries is increasingly reported in young premenopausal women; around 25% of all breast cancer patients in Asia are premenopausal women ≤ 35 years old.

The combination of Dox and cyclophosphamide (AC) is one of the most widespread anthracycline-based adjuvant regimens used for early-stage and advanced breast cancer in both premenopausal and postmenopausal women. In early trials treatment with AC resulted in improved survival and recurrence compared to CMF; Furthermore, AC was considered less toxic than CMF which justifies the use of AC regimen in premenopausal patients. Nonetheless, AC-induced adverse effects such as peripheral neuropathy, anaemia and cardiac failure are described for breast cancer patients. Moreover, AC induces premature ovarian failure in young breast cancer patients. Approximately 35% of breast cancer patients treated with AC report amenorrhea a year after completion of chemotherapy. Studies suggest that both Dox and cyclophosphamide target proliferating cells in the ovary by interfering with DNA replication and by inducing ROS production, but there are currently no reports describing the cytotoxic effects on ovarian granulosa cells of the combined Dox and cyclophosphamide regimen used to treat breast cancer patients.

The administration of Vitamin E lowered Dox-induced ROS damage in animal studies and reduced Dox toxicity without reducing its effectiveness as chemotherapeutic agent. Therefore, it was hypothesized that the addition of alpha and gamma tocopherol to the AC regimen would decrease chemotherapeutic-induced ROS and therefore, will reduce cytotoxicity on ovarian granulosa cells. But due to the anticancer effect that gamma tocopherol demonstrated against breast cancer cells in both in vitro and in vivo studies, it was hypothesised that γToc, but not Toc, would potentiate the cytotoxic activity of the combined Dox and 4-Cyc regimen in vitro.

The cytotoxic effects of clinical relevant doses of Dox and 4-Cyc as single agents and as combined AC regimen were tested on breast cancer (MCF-7) and ovarian granulosa (KGN) cells. Dox was cytotoxic to both MCF-7 and KGN cells, but no effect on KGN cell viability was observed after exposure to 4-Cyc. Only a long exposure (72h) at the highest concentration (2.5μM) of 4-Cyc was able to significantly reduced the numbers of viable MCF-7 cells. The combination of Dox and 4-Cyc caused the same cytotoxicity to MCF-7 breast cancer cells in vitro as Dox alone, suggesting that the concentration of 4-Cyc chosen for this study was too low for this in vitro study.

The numbers of MCF-7 or KGN cell viability were not affected after exposure to αToc, but γ-Toc was significantly more cytotoxic to MCF-7 cells than to KGN cells. Similarly γToc combined to chemotherapeutics increased the cytotoxicity of the combined Dox and 4-Cyc regiment against MCF-7 cells but did not increase nor reduce the cytotoxicity of Dox and 4-cyc towards the KGN cells. This study showed that the addition of γToc to the combined of Dox and 4-Cyc regimen has the potential to increase the in vitro efficacy against breast cancer cells without affecting viable ovarian granulosa cells.

The effective concentration of chemotherapeutics and γToc that killed 25% (EC25) of MCF-7 breast cancer cells were determined to evaluate the potential synergistic effect between Dox and 4-Cyc. T47D breast cancer cells, OVCAR-3 ovarian cancer cells and COV434 granulosa cancer cells were exposed to MCF-7 derived EC25 values of Dox and 4-Cyc. 4-Cyc significantly decreased the cell viability of T47D, OVCAR-3 and COV434 to 71±2.2%, 60±14% and 45±8.6% of the media controls, respectively. Similarly, MCF-7 EC25 values of Dox significantly reduced T47D, OVCAR-3 and COV434 cell viability to 66±5.6%, 74±5% and 57±1.9% respectively. MCF-7 derived EC25 values of γToc significantly decreased breast cancer cell viability, but had no effect on ovarian granulosa cells. Combining EC25 values of Dox with EC25 values of 4-Cyc had no synergistic effect. No significant differences in cell viability were observed between cells exposed to Doxorubicin or 4-Cyc and cells exposed to the combination of Doxorubicin and 4-Cyc, in any of the cell lines tested. Similarly, EC25 values of γToc did not affect cytotoxicity of the combined Doxorubicin and 4-Cyc in any of the cell lines.

The effect of chemotherapeutics and antioxidants on ROS production by human breast and ovarian cancer-derived cell lines was tested. Exposure to 4-Cyc induced more ROS faster than exposure to Dox, except in COV434 cells, in which both agents generated the same response. Similar to the results from the cell viability assays, exposure to the combination of Doxorubicin and 4-Cyc did not increase the amount of ROS compared to each agent alone, except in COV434 cells where levels of ROS were slightly higher than after exposure to each chemotherapeutic alone. Exposure to γToc did not stimulate ROS generation by human breast and ovarian cancer-derived cell lines; but the addition of γToc to the combination of Doxorubicin and 4-Cyc significantly decreased the amount of ROS produced in COV434 granulosa cancer cells, even though the numbers of viable cells were not affected.

Apoptosis induction was measured in human breast and ovarian cancer-derived cell lines. Dox caused apoptosis, as observed by TUNEL and DAPI positive results, in 14±2% and 11±3% in MCF-7 and T47D cells, respectively. Similarly, 4-Cyc – related apoptosis percentages were between 10-25% in all cell lines. No caspase -3, -7 activity was observed in the MCF-7 human breast cancer cell line which was expected as MCF-7 cells are deficient in caspase -3 expression; however the absence of caspase -3, -7 activity in T47D cells after 24h exposure to Dox or 4-Cyc suggest that that the optimal time period to measure caspase -3, -7 in T47D cells is different than for the ovarian cell lines.

In ovarian cancer-derived cells, the combination of Dox and 4-Cyc caused a significant caspase -3, -7 activation, but luminescence values were not significantly different compared to the exposure to 4-Cyc alone. Similarly, percentage of apoptotic cells after exposure to the combination of Dox and 4-Cyc was not significantly different to the percentage of dead cells after treatment with either Dox or 4-Cyc as single agents as shown by both TUNEL and DAPI results. Adding α or γToc to the combination of Dox and 4-Cyc had no effect on Caspase -3, -7 activity or apoptosis induction in any of the cell lines tested compared to the combination of Dox and 4-Cyc.

From the results of PhD project it can be concluded that the combination of Dox and 4-yc did not potentiate the in vitro cytotoxic effect of each agent alone against breast cancer cells. γToc was found to be cytotoxic towards the breast cancer cell lines but had no effect against COV434 or KGN granulosa cells. It was also concluded d that the antioxidant properties of γToc reduced chemotherapy-related ROS generation and probably supported estrogen hormone production by the KGN cells, suggesting that γToc may have the potential to be developed into a fertility preservation therapy for premenopausal breast cancer patient.

Keywords: Breast cancer, chemotherapeutics, Doxorubicin, cyclophosphamide, Ovarian failure, early menopause, Vitamin E, ROS

Subject: Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Medicine and Public Health
Supervisor: Fiona Young