Karen A. Corleto

Many risk-eligible women refuse tamoxifen for primary prevention of breast cancer due to concerns about common side effects such as vasomotor symptoms. Tamoxifen may also induce or worsen insulin resistance and hypertriglyceridemia, especially in women with obesity. The combination of bazedoxifene and conjugated estrogens (BZA/CE) reduces vasomotor symptoms and is currently undergoing evaluation for breast cancer risk reduction. However, the impact of BZA/CE on insulin resistance and metabolic health, particularly in those with excess adiposity, is understudied. Here, we examined the effects of obesity on response to BZA/CE in a rat model of breast cancer risk using older ovary-intact rats. Female Wistar rats received carcinogen to increase mammary cancer risk and were fed a high-fat diet to promote obesity. Lean and obese rats were selected based on adiposity, and then randomized to BZA/CE or vehicle for 8 weeks. BZA/CE reduced adiposity, enriched small (insulin-sensitive) mammary adipocytes, increased the abundance of beneficial metabolic gut microbes ( Faecalbaculum rodentium and Odoribacter laneus ), and reversed obesity-associated changes in lipids and adipokines. BZA/CE also reversed obesity-induced mammary enrichment of cell proliferation pathways, consistent with risk-reducing effects. Together, these data support the use of BZA/CE to improve metabolic health and reduce breast cancer risk in individuals with obesity.

Background Women with obesity and/or type-II-diabetes have an increased breast cancer risk, increased metastasis, and poorer prognosis, especially after menopause. In a rat model of high-fat-diet and menopause-induced weight gain, we previously reported that treatment with the anti-diabetic drug metformin for 8-weeks after ovariectomy (OVX; modeling menopause) reduced growth of existing mammary tumors and inhibited new tumor formation. This identified the menopause transition as a potential window-of-opportunity for interventions to decrease obesity-associated breast cancer incidence and disease progression. Here, we extend these findings to determine if limiting metformin to the peak window of OVX-induced weight gain would have similar anti-cancer effects. Findings Metformin during the first four weeks following OVX is critical to reducing tumor burden, as rats treated with metformin early (weeks0-4-postOVX) had reduced tumor burden. Conversely, initiating metformin later in the postOVX period (weeks 4-8postOVX) did not reduce cancer burden. Despite improved tumor outcomes, metformin withdrawal after the early postOVX time had detrimental metabolic effects, including weight gain and increased adiposity, insulin, IGF1, and HOMA-IR, which correlate with increased cancer risk. Conclusions These data reveal early-postmenopause as a critical window when metformin decreases progression of existing disease and highlights the importance of maintaining treatment to prevent metabolic dysregulation, which could promote secondary tumors/metastasis. These findings also help explain the disconnect between epidemiological studies reporting anticancer benefits of metformin and more recent clinical trials that failed to see similar efficacy, potentially due to issues of timing and/or inclusion of women outside the early postmenopausal window and/or without underlying metabolic dysfunction.

Over the last several decades, a growing body of research has investigated the potential to repurpose the anti-diabetic drug metformin for breast cancer prevention and/or treatment. Observational studies in the early 2000s demonstrated that patients with diabetes taking metformin had decreased cancer risk, providing the first evidence supporting the potential role of metformin as an anti-cancer agent. Despite substantial efforts, two decades later, the exact mechanisms and clinical efficacy of metformin for breast cancer remain ambiguous. Here, we have summarized key findings from studies examining the effect of metformin on breast cancer across the translational spectrum including in vitro, in vivo, and human studies. Importantly, we discuss critical factors that may help explain the significant heterogeneity in study outcomes, highlighting how metformin dose, underlying metabolic health, menopausal status, tumor subtype, membrane transporter expression, diet, and other factors may play a role in modulating metformin’s anti-cancer effects. We hope that these insights will help with interpreting data from completed studies, improve the design of future studies, and aid in the identification of patient subsets with breast cancer or at high risk for the disease who are most likely to benefit from metformin treatment.

The proposed analytical method reports the separation and quantification of 21 amino acids including l-citrulline from fresh vegetables and commercial juices using a C8 column. Optimal separation conditions for amino acids analysis were obtained with 20 mM sodium acetate (solvent A) and water with organic modifier acetonitrile and methanol (solvent B; 18/50/32 V/V). The ideal pH and column temperature were found to be 5.40 and 35 °C, respectively. The LOD and LOQ values were obtained in the range of 0.02-0.19 ng/mL and 0.04-0.39 ng/mL for all amino acids respectively. Relative standard deviations (RSD) of intraday and interday analysis were found to be <2.7% and 7.9%, respectively. The recovery of amino acids were found be satisfactory for all the tested crops. The developed method was successfully used for the quantification of amino acids in six fresh vegetable juices including watermelon, cucumber, celery, calabaza squash, zucchini squash, yellow squash and commercial juices. Multivariate analysis was used to determine the significant differences in the amino acids profiles. l-citrulline content was highest in fresh watermelon juice (716.57 ± 24.80 μg/mL) and commercial watermelon lime juice (826.48 ± 34.48 μg/mL). The optimized analytical method is rapid, sensitive, accurate and reproducible for analysis of free amino acids including l-citrulline from different vegetable juices and other food products. To the best of our knowledge, this is the first report to separate OPA derivatives of amino acids using C8 column from watermelon, cucumber, zucchini squash, yellow squash, calabaza squash, and celery in a HPLC-FLD system.

Nitrate and polyphenols from the diet may enhance the production and bioavailability of nitric oxide, a radical signaling molecule critical for cardiovascular health. Understanding the stability of these bioactives in beetroot and arugula juices is important for their functions. In this study, the stability of nitrate and phenolics in beetroot and arugula juices was measured for 32 days at different temperatures (25, 4, −20, and −80 °C). The levels of nitrate were measured by reversed‐phase HPLC and initial levels were found to be 4965.34 ± 72.69 μg/mL for beetroot and 6310.20 ± 24.79 μg/mL for arugula. Interestingly, nitrate degradation started within 24 hr at 25 °C and after 4 days at 4 °C. At −20 °C and −80 °C, nitrate levels remained stable for one month. Total phenolics and free radical scavenging activity varied significantly during storage conditions. Beetroot juice at 25 °C, significant decrease in total phenolics and antioxidant activity was observed, whereas at 4, −20 and −80 °C, the levels remained relatively stable. By contrast, arugula juice at 25 and 4 °C, an increase in total phenolics and antioxidant activity were observed after one month. Furthermore, UPLC‐HR‐QTOF‐MS analysis demonstrated that flavonoid glucosides were converted to their aglycones and lower phenolics, resulting in higher total phenolics and antioxidant activity during storage. In conclusion, beetroot and arugula juices required frozen conditions for long‐term storage to prevent degradation of nitrate and to maintain their nutritional value. Practical Application: Beetroot and arugula juices have health‐beneficial compounds such as nitrate and phenolics. Understanding the proper storage conditions can allow consumers to make informed choices that can help fresh juices to maintain their health promoting properties.