Figure 1.
Identification of CuE as a potent anti-cancer compound in different cancer cell lines, including TNBC cell lines.
A. Four different cancer cell lines were treated with 12 different cucurbitacins (10 µM) for 48 h. Cell viability was measured with the SRB assay. DMSO was used as the negative control. 1. Jinfushanencin B; 2. Cucurbitacin B; 3. 23, 24-dihydrocucurbitacin B; 4. 23,24-dihydrocucurbitacin D; 5. Cucurbitacin E; 6.22, 23-Dihydrocucurbitacin E; 7. 25-acetoxy-23,24-dihydrocucurbitacin F; 8. 23,24-dihydrocucurbitacin F; 9. Cucurbitacin L; 10. Kinoin B; 11. Endecaphyllacin A; 12. Elaeocarpucin C. Five potent anti-cancer compounds were labeled with underline. B. Chemical structure of CuE. C. Five different TNBC breast cancer cell lines treated with different concentrations (0–100 nM)) of CuE. Cell viability was measured with the SRB assay.
Table 1.
IC50 of five cucurbitane compounds in six cancer cell lines.
Figure 2.
CuE suppresses DNA synthesis in MDA-MB-468 and SW527 TNBC cell lines.
A. CuE (0–50–100–200 nM) was used to treat MDA-MB-468 and SW527 cells for 24 h. DNA synthesis was measured by the EdU assay. B. Quantitative data of MDA-MB-468. Percentage of EdU positive proliferating cells vs. total cells is shown. *** p<0.001, student t-test. C. Quantitative data of SW527. Percentage of EdU positive proliferating cells vs. total cells is shown. ** p<0.01, *** p<0.001, student t-test.
Figure 3.
CuE induces cell cycle G2/M arrest in MDA-MB-468 and SW527 TNBC cell lines.
A. MDA-MB-468 and SW527 cells were treated with CuE (0–50–100–200 nM) for 24 h. Cells were stained with PI and analyzed by flow cytometry. The cell cycle graph was analyzed by FlowJo software (version 7.6). B. CuE significantly increased the percentage of G2/M phase MDA-MB-468 cells compared to DMSO. ** p<0.01, *** p<0.001, student t-test. C. CuE significantly increased the percentage of G2/M phase SW527 cells compared to DMSO. ** p<0.01, *** p<0.001, student t-test.
Figure 4.
CuE induces apoptosis in MDA-MB-468 and SW527 TNBC cell lines.
A. Cell morphology changed dramatically when MDA-MB-468 and SW527 cells were treated with CuE (200 nM) for 24 h. B. MDA-MB-468 and SW527 cells were treated with CuE (0–50–100–200 nM) for 24 h. Cells were stained with Annexin V/7AAD and analyzed by flow cytometry. Doxorubicin was used as a positive control. C. Quantitative data of panel A. Percentage of Annexin V-positive cells is shown. * p<0.05, ** p<0.01, *** p<0.001, student t-test.
Figure 5.
CuE modulates the expression levels of multiple cell cycle, apoptosis, and signaling regulators in MDA-MB-468 and SW527 TNBC cell lines.
A. MDA-MB-468 and SW527 cells were treated with CuE (0–50–100–200 nM) for 24 h. Cell lysate were collected for WB, to detect PARP, cleaved Caspase-3, Survivin, Mcl-1, XIAP, Bcl-2, Bcl-xL, Cyclin B1, Cyclin D1, Cyclin E1, p21, and p27. β-actin and GAPDH were used as loading controls. B. MDA-MB-468 and SW527 cells were treated with CuE (0–50–100–200 nM) for 24 h. pSTAT3, total STAT3, pAKT, total AKT, pJNK, total JNK, p-c-Jun, total c-Jun, pERK and total ERK were examined by WB. GAPDH was used as a loading control.