Scutellaria barbata D.Don extract regulates Ezrin-mediated triple negative breast cancer progress via suppressing the RhoA /ROCK1 signaling

Abstract

Background: Triple-negative breast cancer (TNBC) lacks effective therapeutic targets. Scutellaria barbata D.Don (SB) has been revealed to have anti-breast cancer (BC) effect, but the effect of SB extract in TNBC is still unclear. Herein, this research delves into the underlying mechanism.

Methods: SB was extracted by solvent extraction, and the main components were identified using an Agilent 6,520 HPLC-Chip/Q-TOF (Chip/Q-TOF) MS system. In vitro cell experiments were conducted. The effects of SB extract alone, SB extract plus EGF, GSK alone, GSK plus Ezrin overexpression, or SB extract plus Ezrin overexpression on cell viability, invasion, migration, and apoptosis were examined by cell function experiments. The apoptosis- and RhoA/ROCK1 pathway-related protein levels were analyzed by western blot assay.

Results: Mass spectrometry analysis exhibited that SB extract mainly contains long-chain fatty acids and ursolic acid. SB extract mitigated TNBC cell biological phenotypes, apoptosis- and RhoA/ROCK1 pathway-related marker expressions, which were reversed by EGF. The further results found that GSK obviously weakens TNBC cell biological behaviors, apoptosis- and RhoA/ROCK1 signaling-related protein levels, while oe-Ezrin treatment reverses the effect of GSK on TNBC cells. Moreover, SB extract regulated Ezrin-mediated function of TNBC cells by impeding the RhoA/ROCK1 pathway.

Conclusion: Our findings demonstrated that SB extract regulated Ezrin-mediated proliferation, migration, invasion, and apoptosis of TNBC cells via suppressing the RhoA /ROCK1 signaling. Our results offer the experimental foundation for further investigation of the anti-cancer role of SB in TNBC cells.

Highlights: SB extract inhibits the biological phenotypes of TNBC cells.SB extract inhibits the biological behaviors of TNBC cells through the RhoA/ROCK1 pathway.SB extract modulates Ezrin-mediated TNBC cell proliferation, migration, invasion, and apoptosis via restraining the RhoA/ROCK1 signaling.

Keywords: Ezrin; ROCK1; RhoA; scutellaria barbata D.Don extract; triple negative breast cancer.

Fig. 1

SB extract inhibited the biological phenotypes of TNBC cells. A) After cell cultures were treated with different dosage SB extract (0, 100, 200, 300, 400, and 500 μg/mL) for 24 h, the cell viability was detected by CCK-8. B) Clone formation was analyzed by colony formation assay. C) The numbers of migration and invasion cells were examined by transwell assay. D) The apoptosis cells were tested by flow cytometric. E) The protein expression levels of Bax, Bcl-2, and C-Capase3 were tested by western blot. n = 3. Data were displayed as mean ± SD. ^*P < 0.05, ^**P < 0.01.

Fig. 2

SB extract restrained the activation of the RhoA/ROCK1 pathway. A and B) After TNBC cells were subjected to 500 μg/mL SB extract for 24 h, and then treated with or without 50 ng/mL RhoA/ROCK1 pathway activator, EGF for 24 h, Western blot was selected to examine the levels of RhoA, p-RhoA, ROCK1, and p-ROCK1 in MDA-MB-231 and BT20 cells. n = 3. Data were presented as mean ± SD. ^*P < 0.05, ^**P < 0.01.

Fig. 3

SB extract regulated TNBC cell function by weakening the RhoA/ROCK1 pathway. TNBC cells were subjected to 500 μg/mL SB extract for 24 h and then treated with or without 50 ng/mL RhoA/ROCK1 pathway activator, EGF for 24 h. A) CCK-8 detected the cell viability of TNBC cells. B) The colony formation assay determined the clone formation of TNBC cells. C) Transwell assay analyzed the numbers of cell migration and invasion. D) The flow cytometric was used to assess the apoptosis of TNBC cells. E) The protein levels of Bax, Bcl-2 and C-Capase3 were measured by western blot. n = 3. Data were presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 4

RhoA/ROCK1 pathway regulated Ezrin-mediated function of TNBC cells. After TNBC cells transfected with or without oe-NC/oe-Ezrin were intervened with or without 1 μM RhoA/ROCK1 pathway inhibitor, GSK429286A for 24 h. A) The cell viability was evaluated by CCK-8. B) Clone formation was measured by the colony formation assay. C) The numbers of cell migration and invasion were tested utilizing transwell assay. D) The numbers of apoptotic cells were tested with flow cytometric. E) The protein levels of Bax, Bcl-2 and C-Capase3 were tested with western blot. F) The levels of RhoA, p-RhoA, ROCK1, p-ROCK1, and Ezrin were tested with western blot. n = 3. Data were manifested as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 4

RhoA/ROCK1 pathway regulated Ezrin-mediated function of TNBC cells. After TNBC cells transfected with or without oe-NC/oe-Ezrin were intervened with or without 1 μM RhoA/ROCK1 pathway inhibitor, GSK429286A for 24 h. A) The cell viability was evaluated by CCK-8. B) Clone formation was measured by the colony formation assay. C) The numbers of cell migration and invasion were tested utilizing transwell assay. D) The numbers of apoptotic cells were tested with flow cytometric. E) The protein levels of Bax, Bcl-2 and C-Capase3 were tested with western blot. F) The levels of RhoA, p-RhoA, ROCK1, p-ROCK1, and Ezrin were tested with western blot. n = 3. Data were manifested as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 5

SB extract regulated Ezrin-mediated function of TNBC cells by impeding the RhoA/ROCK1 pathway. After TNBC cells transfected with or without oe-NC/oe-Ezrin were treated with or without 500 μg/mL SB extract for 24 h. A) The cell viability was determined utilizing CCK-8. B) Clone formation was measured using the colony formation assay. C) The migration and invasion numbers of TNBC cells were tested with transwell assay. D) The numbers of apoptotic cells were tested with flow cytometric. E) The levels of Bax, Bcl-2, and C-Capase3 were tested with western blot. F) The levels of RhoA, p-RhoA, ROCK1, p-ROCK1, and Ezrin were assessed with western blot. n = 3. Data were exhibited as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 5

SB extract regulated Ezrin-mediated function of TNBC cells by impeding the RhoA/ROCK1 pathway. After TNBC cells transfected with or without oe-NC/oe-Ezrin were treated with or without 500 μg/mL SB extract for 24 h. A) The cell viability was determined utilizing CCK-8. B) Clone formation was measured using the colony formation assay. C) The migration and invasion numbers of TNBC cells were tested with transwell assay. D) The numbers of apoptotic cells were tested with flow cytometric. E) The levels of Bax, Bcl-2, and C-Capase3 were tested with western blot. F) The levels of RhoA, p-RhoA, ROCK1, p-ROCK1, and Ezrin were assessed with western blot. n = 3. Data were exhibited as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.