**2.4 Cucurbitacin E (4′)**

The inhibition of breast cancer metastasis in mouse models by CuE was reported. To evaluate the effect of CuE on the proliferation and apoptosis of inoculated 4T1 and MDA-MB231 cells in vivo, the expression of proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 was tested by immunohistochemical analysis [18]. CuE targets the dissemination of breast cancer cells from the primary tumor but not the outgrowth of established micrometastases in target organs (lung, liver, between others). CuE exerts no significant effect on tumor cell apoptosis or proliferation in vivo [19].

CuE demonstrated cytotoxic activity against human oral squamous cell carcinoma SAS cells with an IC50 of 3.69 μM and induced the apoptosis of SAS cells after 24 h of treatment, but not MRC-5 or HS68 cells, which showed a dose-dependent reduction. Microscopic examination showed that following exposure to CuE (2.5 μM) for 6–24 h, the cells displayed a remarkable change in their morphology, and CuE induced the death of cancer cells [20].

 The inhibitory effect of CuE on the proliferation of Bcap37 and MDA-MB-231 cells was assessed by the MTT assay. Breast cancer cells were treated with various concentrations (0, 0.1, 1, 10, and 100 μM) of CuE or DMSO as a control for 24, 48, and 72 h. The MTT method was then used to determine the number of viable cells. The data indicated that CuE inhibited cell growth in a concentration- and time-dependent manner (ANOVA, p < 0.05). After treatment with 0.1 μM CuE for

#### *Cytotoxic and Antitumoral Activities of Compounds Isolated from Cucurbitaceae Plants DOI: http://dx.doi.org/10.5772/intechopen.82213*

24 h, the growth of Bcap37 and MB-231 cells was significantly inhibited. At a CuE concentration of 100 μM, most of the cancer cells detached from the dish [21].

 Additionally, CuE was evaluated on the chondrosarcoma SW 1353 cancer cell line, and the IC50 values indicated higher toxicity in this cell line than in the previously test lines (MTT assay). The amount of CuE that induced a mortality of 50% was calculated after 6, 12, and 24 h of treatment, and the results were 13.55, 12.65, and 9.16 μM, respectively [22]. The cytotoxic effect of CuE was tested on HeLa and U2OS cells, and the IC50 values were 6.43 and 15.07 nM, respectively. The inhibition of tubulin polymerization in vitro had an IC50 of 566.91 nM [13].

 The effects of CuE from *E. elaterium* fruit on the expression of the BAX, caspase-3, LC3, and VEGF and c-MYC genes in the AGS cell line were investigated. The sub-G1 accumulation of AGS cells treated with CuE was increased compared to that of untreated cells. Moreover, the treatment of AGS cells with CuE-induced cell death. Additionally, the effects of CuE on the mRNA expression levels of the LC3, VEGF, BAX, caspase-3, and c-MYC genes were evaluated using qRT-PCR. LC3 mRNA levels were increased approximately 20-fold after treatment with CuE at a concentration of 0.1 μg/mL for 24 h. However, BAX, caspase-3, and c-MYC mRNA levels at these concentrations were not changed by the treatment [23, 24].
