Structure of helenalin, BHM, and BHG.

Inhibition abilities and chemosensitizing effects of helenalin, BHM, and BHG on ABC transporters in ABCB1 or ABCG2 overexpressing cells. (a,b) The intracellular calcein and hoechst33342 fluorescence were increased under helenalin, BHM, or BHG 30 min treatments in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (c–e) The synergistic cytotoxicity of helenalin, BHM, or BHG co-treated with docetaxel and doxorubicin indicated that helenalin, BHM, or BHG increased sensitivity to the chemotherapeutics docetaxel and doxorubicin in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (f–g) The MDR preventive effects of helenalin, BHM, and BHG in MDA/doc cells. Synergy assessment of helenalin, BHM, and BHG in combination with doxorubicin or docetaxel in ABCB1/HEK293 (h) and ABCG2/HEK293 (i) cell lines. Synergy scores were interpreted as follows: <−10, antagonism; −10 to 10, additive effect; >10, synergism. Statistical differences were evaluated by the ANOVA followed by post hoc analysis (Tukey’s test). * denotes p < 0.05 as compared to doxorubicin or docetaxel alone treatments. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

Inhibition abilities and chemosensitizing effects of helenalin, BHM, and BHG on ABC transporters in ABCB1 or ABCG2 overexpressing cells. (a,b) The intracellular calcein and hoechst33342 fluorescence were increased under helenalin, BHM, or BHG 30 min treatments in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (c–e) The synergistic cytotoxicity of helenalin, BHM, or BHG co-treated with docetaxel and doxorubicin indicated that helenalin, BHM, or BHG increased sensitivity to the chemotherapeutics docetaxel and doxorubicin in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (f–g) The MDR preventive effects of helenalin, BHM, and BHG in MDA/doc cells. Synergy assessment of helenalin, BHM, and BHG in combination with doxorubicin or docetaxel in ABCB1/HEK293 (h) and ABCG2/HEK293 (i) cell lines. Synergy scores were interpreted as follows: <−10, antagonism; −10 to 10, additive effect; >10, synergism. Statistical differences were evaluated by the ANOVA followed by post hoc analysis (Tukey’s test). * denotes p < 0.05 as compared to doxorubicin or docetaxel alone treatments. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

Inhibition abilities and chemosensitizing effects of helenalin, BHM, and BHG on ABC transporters in ABCB1 or ABCG2 overexpressing cells. (a,b) The intracellular calcein and hoechst33342 fluorescence were increased under helenalin, BHM, or BHG 30 min treatments in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (c–e) The synergistic cytotoxicity of helenalin, BHM, or BHG co-treated with docetaxel and doxorubicin indicated that helenalin, BHM, or BHG increased sensitivity to the chemotherapeutics docetaxel and doxorubicin in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (f–g) The MDR preventive effects of helenalin, BHM, and BHG in MDA/doc cells. Synergy assessment of helenalin, BHM, and BHG in combination with doxorubicin or docetaxel in ABCB1/HEK293 (h) and ABCG2/HEK293 (i) cell lines. Synergy scores were interpreted as follows: <−10, antagonism; −10 to 10, additive effect; >10, synergism. Statistical differences were evaluated by the ANOVA followed by post hoc analysis (Tukey’s test). * denotes p < 0.05 as compared to doxorubicin or docetaxel alone treatments. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

Inhibition abilities and chemosensitizing effects of helenalin, BHM, and BHG on ABC transporters in ABCB1 or ABCG2 overexpressing cells. (a,b) The intracellular calcein and hoechst33342 fluorescence were increased under helenalin, BHM, or BHG 30 min treatments in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (c–e) The synergistic cytotoxicity of helenalin, BHM, or BHG co-treated with docetaxel and doxorubicin indicated that helenalin, BHM, or BHG increased sensitivity to the chemotherapeutics docetaxel and doxorubicin in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (f–g) The MDR preventive effects of helenalin, BHM, and BHG in MDA/doc cells. Synergy assessment of helenalin, BHM, and BHG in combination with doxorubicin or docetaxel in ABCB1/HEK293 (h) and ABCG2/HEK293 (i) cell lines. Synergy scores were interpreted as follows: <−10, antagonism; −10 to 10, additive effect; >10, synergism. Statistical differences were evaluated by the ANOVA followed by post hoc analysis (Tukey’s test). * denotes p < 0.05 as compared to doxorubicin or docetaxel alone treatments. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

Inhibition abilities and chemosensitizing effects of helenalin, BHM, and BHG on ABC transporters in ABCB1 or ABCG2 overexpressing cells. (a,b) The intracellular calcein and hoechst33342 fluorescence were increased under helenalin, BHM, or BHG 30 min treatments in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (c–e) The synergistic cytotoxicity of helenalin, BHM, or BHG co-treated with docetaxel and doxorubicin indicated that helenalin, BHM, or BHG increased sensitivity to the chemotherapeutics docetaxel and doxorubicin in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (f–g) The MDR preventive effects of helenalin, BHM, and BHG in MDA/doc cells. Synergy assessment of helenalin, BHM, and BHG in combination with doxorubicin or docetaxel in ABCB1/HEK293 (h) and ABCG2/HEK293 (i) cell lines. Synergy scores were interpreted as follows: <−10, antagonism; −10 to 10, additive effect; >10, synergism. Statistical differences were evaluated by the ANOVA followed by post hoc analysis (Tukey’s test). * denotes p < 0.05 as compared to doxorubicin or docetaxel alone treatments. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

Inhibition abilities and chemosensitizing effects of helenalin, BHM, and BHG on ABC transporters in ABCB1 or ABCG2 overexpressing cells. (a,b) The intracellular calcein and hoechst33342 fluorescence were increased under helenalin, BHM, or BHG 30 min treatments in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (c–e) The synergistic cytotoxicity of helenalin, BHM, or BHG co-treated with docetaxel and doxorubicin indicated that helenalin, BHM, or BHG increased sensitivity to the chemotherapeutics docetaxel and doxorubicin in ABCB1/HEK293 cells and ABCG2/HEK293 cells. (f–g) The MDR preventive effects of helenalin, BHM, and BHG in MDA/doc cells. Synergy assessment of helenalin, BHM, and BHG in combination with doxorubicin or docetaxel in ABCB1/HEK293 (h) and ABCG2/HEK293 (i) cell lines. Synergy scores were interpreted as follows: <−10, antagonism; −10 to 10, additive effect; >10, synergism. Statistical differences were evaluated by the ANOVA followed by post hoc analysis (Tukey’s test). * denotes p < 0.05 as compared to doxorubicin or docetaxel alone treatments. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

The effects of helenalin, (helenalinyl)malonate (BHM), and bis(helenalinyl)glutarate (BHG) on cell proliferation of MDA-MB-231 through MYC-dependent pathway. (a) MYC expression under helenalin, BHM, and BHG 72 h treatments in MDA-MB-231 cells was determined by enzyme-linked immunosorbent assay (ELISA). (b) Relative MYC levels in MDA-MB-231 cells transfected with scrambled siRNA (negative control) or MYC siRNA by western blotting. The uncropped blots are shown in the Supplemental Materials. (c) The cytotoxic effect of helenalin, BHM, and BHG in MDA-MB-231 cells with or without MYC siRNA transfection. Cytotoxicity was detected after a 72-h treatment. JQ1 was used as a positive control of MYC inhibitor. Statistical differences were evaluated by Student’s t-test. * denoted p < 0.05 as compared to untreated control in (b) and as compared to MDA-MB-231 cell line in (c). Data presented as mean ± standard error of at least two experiments, each in duplicate.

The effects of helenalin, (helenalinyl)malonate (BHM), and bis(helenalinyl)glutarate (BHG) on cell proliferation of MDA-MB-231 through MYC-dependent pathway. (a) MYC expression under helenalin, BHM, and BHG 72 h treatments in MDA-MB-231 cells was determined by enzyme-linked immunosorbent assay (ELISA). (b) Relative MYC levels in MDA-MB-231 cells transfected with scrambled siRNA (negative control) or MYC siRNA by western blotting. The uncropped blots are shown in the Supplemental Materials. (c) The cytotoxic effect of helenalin, BHM, and BHG in MDA-MB-231 cells with or without MYC siRNA transfection. Cytotoxicity was detected after a 72-h treatment. JQ1 was used as a positive control of MYC inhibitor. Statistical differences were evaluated by Student’s t-test. * denoted p < 0.05 as compared to untreated control in (b) and as compared to MDA-MB-231 cell line in (c). Data presented as mean ± standard error of at least two experiments, each in duplicate.

The effects of helenalin, BHM, and BHG on cell cycle in MDA-MB-231 cells. Helenalin, BHM, and BHG significantly induced MDA-MB-231 cell arrest in the G2/M phase and subG1 phase, respectively. * represented p < 0.05 as compared to untreated control.

The effects of BHM, and BHG on MDA-MB-231 cells apoptosis. All three test compounds increased apoptotic cells in MDA-MB-231. * represented p < 0.05 as compared to untreated control.

The underlying mechanism of apoptosis and G2/M arrest was determined by ROS assay and ELISA. The apoptotic cell-induced effects of helenalin, BHM, and BHG were detected by increasing ROS levels (a) and p53 expression (b). Helenalin, BHM, and BHG suppressed STAT3-MYC-CDC25-CDK1 signaling and increased p21 to induce G2/M arrest in MDA-MB-231 cells (c–f). * represented p < 0.05 as compared to untreated control. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

The underlying mechanism of apoptosis and G2/M arrest was determined by ROS assay and ELISA. The apoptotic cell-induced effects of helenalin, BHM, and BHG were detected by increasing ROS levels (a) and p53 expression (b). Helenalin, BHM, and BHG suppressed STAT3-MYC-CDC25-CDK1 signaling and increased p21 to induce G2/M arrest in MDA-MB-231 cells (c–f). * represented p < 0.05 as compared to untreated control. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

The underlying mechanism of apoptosis and G2/M arrest was determined by ROS assay and ELISA. The apoptotic cell-induced effects of helenalin, BHM, and BHG were detected by increasing ROS levels (a) and p53 expression (b). Helenalin, BHM, and BHG suppressed STAT3-MYC-CDC25-CDK1 signaling and increased p21 to induce G2/M arrest in MDA-MB-231 cells (c–f). * represented p < 0.05 as compared to untreated control. Each data is expressed as the mean ± standard error of at least two experiments, each performed in duplicate.

Bioinformatics analysis of the expression of MYC and its regulating pathway in the TNBC patients. (a) The oncoprint analysis of MYC amplification or mRNA upregulation in the TNBC cohort and non-TNBC cohort from TCGA and METABRIC breast cancer datasets. (b) MYC mRNA expression was detected in breast cancer according to the 3-gene classifier subtype. (c) The oncoprint analysis of STAT3, MYC, CDC25, and CDK1 in the TNBC cohort and non-TNBC cohort from METABRIC breast cancer datasets. (d) Kaplan–Meier survival analysis of patients with alterations in query genes. (e) Pearson’s correlation of STAT3 and MYC expression in the TNBC cohort.

Bioinformatics analysis of the expression of MYC and its regulating pathway in the TNBC patients. (a) The oncoprint analysis of MYC amplification or mRNA upregulation in the TNBC cohort and non-TNBC cohort from TCGA and METABRIC breast cancer datasets. (b) MYC mRNA expression was detected in breast cancer according to the 3-gene classifier subtype. (c) The oncoprint analysis of STAT3, MYC, CDC25, and CDK1 in the TNBC cohort and non-TNBC cohort from METABRIC breast cancer datasets. (d) Kaplan–Meier survival analysis of patients with alterations in query genes. (e) Pearson’s correlation of STAT3 and MYC expression in the TNBC cohort.

Helenalin, BHM, and BHG suppressed TNBC tumor growth in the xenotransplantation model. (a) The intensity of fluorescence was proportional to tumor size. There was no observable toxicity at 48 hpi after each compound treatment. P denoted as paclitaxel (positive control). (b) The MDA-MB-231 xenotransplantation model was treated in the presence and absence of test compounds, and helenalin, BHM, and BHG significantly inhibited tumor growth after 48 h of treatment. # p < 0.05 compared with the control; * p < 0.05 compared with control cells treated with test compounds. hpi: hours post-treatment or post-injection.

Helenalin, BHM, and BHG suppressed TNBC tumor growth in the xenotransplantation model. (a) The intensity of fluorescence was proportional to tumor size. There was no observable toxicity at 48 hpi after each compound treatment. P denoted as paclitaxel (positive control). (b) The MDA-MB-231 xenotransplantation model was treated in the presence and absence of test compounds, and helenalin, BHM, and BHG significantly inhibited tumor growth after 48 h of treatment. # p < 0.05 compared with the control; * p < 0.05 compared with control cells treated with test compounds. hpi: hours post-treatment or post-injection.