Cucurbitacin B Causes Increased Radiation Sensitivity of Human Breast Cancer Cells via G2/M Cell Cycle Arrest

Purpose. To explore the effects of cucurbitacin B on the radiation survival of human breast cancer cells and to elucidate the cellular mechanism of radiosensitization if any. Materials and Methods. Human breast carcinoma cell lines were treated with cucurbitacin B before irradiation with 0–10 Gy of 137Cs gamma rays. The effect of cucurbitacin B on cell-survival following irradiation was evaluated by colony-forming assay. Cell cycle distributions were investigated using flow cytometry. Real-time PCR and western blots were performed to investigate the expression of cell cycle checkpoints. Results. Cucurbitacin B inhibited breast cancer cell proliferation in a dose-dependent manner. Only MDA-MB-231 and MCF7:5C cells but not SKBR-3 cells were radiosensitized by cucurbitacin B. Flow cytometric analysis for DNA content indicated that cucurbitacin B resulted in G2/M arrest in MDA-MB-231 and MCF7:5C but not SKBR-3 cells. Moreover, Real-time PCR and western blot analysis demonstrated upregulated p21 expression before irradiation, a likely cause of the cell cycle arrest. Conclusion. Taken together, these findings suggest that cucurbitacin B causes radiosensitization of some breast cancer cells, and that cucurbitacin B induced G2/M arrest is an important mechanism. Therefore, combinations of cucurbitacin B with radiotherapy may be appropriate for experimental breast cancer treatment

Functional Analysis of Familial Asp67Glu and Thr1051Ser BRCA1 Mutations in Breast/Ovarian Carcinogenesis

Estrogen is believed to be pre-initiator in the risk of breast cancer. The BRCA1 is a tumor suppressor gene associated with breast and ovarian cancer risk. This report describes functional analysis of two BRCA1 missense mutations (Asp67Glu and Thr1051Ser) observed in the familial breast/ovarian cancer patients in Thailand. Levels of luciferase activity of the two mutations were relatively lower than in the wild-type BRCA1. It is indicated that mutants may fail to promote the estrogen receptor dependent functions. It is presumed that estrogen and insulin/IGF-1 regulate c-Myc and cyclin D1 during breast cancer cell proliferation. It is also likely to affect ubiquitination mechanism. Since three affected cancer families carry the Asp67Glu mutation, it is believed that this type of mutation could have some effect on breast/ovarian cancer progression

Antiproliferative Effects of Cucurbitacin B in Breast Cancer Cells: Down-Regulation of the c-Myc/hTERT/Telomerase Pathway and Obstruction of the Cell Cycle

Naturally occurring cucurbitacins have been shown to have anticancer, antimicrobial and anti-inflammatory activities. In this study, we determined the effects of cucurbitacin B extracted from the Thai herb Trichosanthes cucumerina L. on telomerase regulation in three human breast cancer cell lines (T47D, SKBR-3, and MCF-7) and a mammary epithelium cell line (HBL-100). Cell viability after treatment with cucurbitacin B, which is an active ingredient of this herb, was assessed. Telomeric Repeat Amplification Protocol (TRAP) assays and RT-PCR (qualitative and realtime) were performed to investigate activity of telomerase as well as expression of human telomerase reverse transcriptase (hTERT) and c-Myc. The c-Myc protein level was also determined in SKBR-3 and HBL-100 cells. Our results show that the cucurbitacin B inhibits growth and telomerase activity in the three breast cancer cell lines and exerts an obvious inhibitory effect in the estrogen receptor (ER)-negative breast cancer SKBR-3 cells. The expression of hTERT and c-Myc were also inhibited by cucurbitacin B, In addition, a clear reduction of c-Myc protein was observed after treatment in SKBR-3 cells even with a concentration of cucurbitacin B that was ten-times lower compared to the concentration used for HBL-100. Our findings imply that cucurbitacin B exerts an anticancer effect by inhibiting telomerase via down regulating both the hTERT and c-Myc expression in breast cancer cells

The effectiveness of cucurbitacin B in BRCA1 defective breast cancer cells.

Cucurbitacin B (CuB) is one of the potential agents for long term anticancer chemoprevention. Cumulative evidences has shown that cucurbitacin B provides potent cellular biological activities such as hepatoprotective, anti-inflammatory and antimicrobial effects, but the precise mechanism of this agent is not clearly understood. We examine the biological effects on cancer cells of cucurbitacin B extracted from a Thai herb, Trichosanthes cucumerina L. The wild type (wt) BRCA1, mutant BRCA1, BRCA1 knocked-down and BRCA1 overexpressed breast cancer cells were treated with the cucurbitacin B and determined for the inhibitory effects on the cell proliferation, migration, invasion, anchorage-independent growth. The gene expressions in the treated cells were analyzed for p21/(Waf1), p27(Kip1) and survivin. Our previous study revealed that loss of BRCA1 expression leads to an increase in survivin expression, which is responsible for a reduction in sensitivity to paclitaxel. In this work, we showed that cucurbitacin B obviously inhibited knocked-down and mutant BRCA1 breast cancer cells rather than the wild type BRCA1 breast cancer cells in regards to the cellular proliferation, migration, invasion and anchorage-independent growth. Furthermore, forcing the cells to overexpress wild type BRCA1 significantly reduced effectiveness of cucurbitacin B on growth inhibition of the endogenous mutant BRCA1 cells. Interestingly, cucurbitacin B promotes the expression of p21/(Waf1) and p27(Kip1) but inhibit the expression of survivin. We suggest that survivin could be an important target of cucurbitacin B in BRCA1 defective breast cancer cells

The clonal anchorage-independent growth, cell migration and invasion after treatment with 12 µg/ml cucurbitacin B.

<p>(A) and (B), Anchorage-independent growth with or without cucurbitacin B treatment in each group of the MCF-7 and MDA-MB-231 cells. (C) and (D), The capability of cell migration in the MCF-7 and MDA-MB-231. (E) and (F), The invasive capability of the MCF-7 and MDA-MB-231, respectively. (* <i>p</i><0.01).</p

Cell proliferations of MCF-7 (A) and MDA-MB-231 (B) after treatment with 12 µg/ml cucurbitacin B into the culture medium in the parental cells, transfected control (shRNA-scrambled) cells and shRNA-BRCA1 knocked-down cells.

<p>Each experiment was performed in triplicate. ShRNA-BRCA1 knocked-down cells showed significant highly sensitive to cucurbitacin B when compared to the BRCA1 parental cells, (* <i>p</i><0.01).</p