Glyceollins Trigger Anti-Proliferative Effects in Hormone-Dependent Aromatase-Inhibitor-Resistant Breast Cancer Cells through the Induction of Apoptosis

Abstract

Aromatase inhibitors (AIs) are standard treatment for estrogen-dependent postmenopausal breast tumors; however, resistance develops leading to tumor relapse and metastasis. We previously demonstrated that glyceollin inhibits proliferation, survival, and migration of hormone-independent letrozole-resistant breast cancer. Since many AI-resistant tumors remain hormone-dependent, identifying distinctions between estrogen-receptor-positive (ER+) and ER-negative (ER-) AI-resistant tumor response to therapy is critical. We hypothesize that treating ER+ letrozole-resistant T47D breast cancer cells (T47DaromLR) with a combination of 10 μM glyceollin and 0.5 μM lapatinib (a dual EGFR/HER2 inhibitor) will decrease cell proliferation through induction of apoptosis. The T47DaromLR cells were found to overexpress HER2 and MAPK while maintaining aromatase and ER levels compared to their letrozole-sensitive (T47Darom) counterparts. In the absence of estrogen stimulation, glyceollin ± lapatinib had no effect on the proliferation of the T47Darom cells, while glyceollin treatment caused 46% reduction in the proliferation of T47DaromLR cells, which was further diminished when combined with lapatinib. While neither agent influenced cell migration, glyceollin and lapatinib reduced S and G2/M phase cell entry and exclusively induced apoptosis by 1.29-fold in the T47DaromLR cells. Taken together, these results suggest that glyceollins and lapatinib may have potential as a novel combination therapeutic approach for hormone-dependent, letrozole-resistant tumors.

Novel Therapeutic Combination Targets the Growth of Letrozole-Resistant Breast Cancer through Decreased Cyclin B1

“…Previous studies demonstrated that AI-resistant cells are sensitive to the inhibitory properties of glyceollins [ 23 , 26 ]. Since it is unlikely that monotherapy (i.e., a single phytochemical) can exhibit a durable, long-term anti-cancer effect on refractory breast cancer cells, we chose to examine the effects of combining a phytochemical with lapatinib.…”

“…Given the recent results from a Phase III clinical trial demonstrating dual HER2 blockade with lapatinib and trastuzumab in combination with an AI was associated with superior progression free survival compared to trastuzumab plus an AI in postmenopausal women with HER2-positive hormone-receptor-positive metastatic breast cancer [ 32 ], we were encouraged to study the combination of lapatinib plus glyceollin as AI-resistant cells overexpress HER2 and EGFR (i.e., which is the target of lapatinib). We were also interested in this therapeutic combination since our previous report demonstrated that glyceollin and lapatinib inhibited the proliferation of hormone-dependent AI-resistant T47D cells through induction of apoptosis [ 26 ]. Therefore, we chose to examine whether this combination would be effective in hormone-independent LTLT-Ca AI-resistant cells.…”

“…While these results were promising, given that the LTLT-Ca cells overexpressed HER2 and EGFR and that glyceollin inhibited EGFR, we were prompted to test whether the combination of lapatinib (a dual HER2/EGFR inhibitor) and glyceollin could act synergistically to inhibit the proliferation and motility of the LTLT-Ca cells. Because the LTLT-Ca cells express higher levels of HER2 and EGFR than the AC-1 cells, we expected these cells to be more sensitive to the anti-proliferative properties of lapatinib and glyceollin and behave much like what was previously demonstrated with the AI-resistant T47DaromLR cells [ 26 ]. However, this was not the case, as the AC-1 cells and the LTLT-Ca cells responded similarly.…”

“…When the cells were 70–80% confluent, they were treated with vehicle control (DMSO), 5 μM Lap, 10 μM Gly, and 5 μM Lap plus 10 μM Gly for 24 h. The cells were scraped and homogenized in cold RIPA buffer supplemented with a protease inhibitor (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein separation, membrane blotting, and antibody probing were conducted as previously described [ 26 ] using the following primary antibodies against cyclin B1 (Rb IgG, D5C10) (1:1000), cdk2 (Rabbit mAb, 78B2) (1:1000), p21 (Rabbit mAb, 78B2) (1:1000), p27 (Rabbit mAb, 78B2) (1:1000), β actin (Rabbit mAb, 13E5) (1:1000), or GAPDH (Rb mAb (14C10) (1:1000) (Cell Signaling Technology, Danvers, MA, USA), or against cdk1 (Rabbit mAb, YE324) (1:2000) (Abcam Waltham, Boston, MA, USA). Afterwards, they were then washed and incubated with anti-rabbit secondary antibody.…”

RNA-Seq Dissects Incomplete Activation of Phytoalexin Biosynthesis by the Soybean Transcription Factors GmMYB29A2 and GmNAC42-1

“…The most well-known phytoalexins that are clinical drugs include the anticancer metabolite taxol [ 8 ], the antidiabetic berberine [ 9 ], and the antimalarial artemisinin [ 10 ]. Many other phytoalexins are in (pre)clinical testing [ 5 , 11 , 12 ], including the glyceollins from soybean that inhibit the survival and proliferation of several cancer cell and tumor types by mechanisms that are distinct from conventional therapeutics [ 13 , 14 , 15 ]. Additionally, glyceollins hold promise as natural antimicrobials in the swine industry [ 16 , 17 ], yet exhibit neuroprotective activities in mammalian cells [ 18 ].…”