**2.5 Cucurbitacin R (5′)**

 One additional cucurbitacin was discovered in the roots of *C. tayuya* and was identified as cucurbitacin R (CCR) (**5′**). This compound was experimentally assessed in isogenic colon cancer cell lines HCT116 and Hke-3 by the MTT assay and induced the apoptosis of both HCT116 and Hke-3 cells with IC50 values 37 and 27 μM, respectively [15].

#### **2.6 Cucurbitacin I (6′)**

CuI, also known as elatericin B or JSI 124, has been isolated from different plants, such as *M. balsamina* L. (balsam pear), *C. tayuya* (tayuya), *Cucurbita andreana* (winter squash), and *C. colocynthis* (bitter cucumber). This compound was tested in isogenic colon cancer cell lines HCT116 and Hke-3 and was found to induce apoptosis in both lines, with IC50 values of 0.29 and 0.09 μM, respectively [15].

The cytotoxicity IC50 values of CuI in SW 1353 cells after 6, 12, and 24 h of treatment were 7.93, 8.31, and 5.06 μM, respectively [22]. The cytotoxic activity of CuI against SW-480 human colon cancer cells was tested. In this case, CuI diminished cell proliferation in a concentration-dependent manner and increased apoptosis, enhancing cycle arrest at the G2/M phase [25].

 The cytotoxicity of CuI was tested in HeLa and U2OS cells, and the IC50 values were 44.77 and 23.47 nM, respectively. The inhibition of tubulin polymerization in vitro had an IC50 > 1 mM [13]. The effects of CuI purified 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 CuI was increased compared to that of untreated cells. Moreover, treatment of AGS cells with CuI-induced cell death. Additionally, the effects of CuI on the mRNA expression levels of the LC3, VEGF, BAX, caspase-3, and c-MYC genes were evaluated using qRT-PCR. After treatment, at a concentration of 0.5 μg/mL for 24 h, LC3 mRNA levels were increased approximately 25-fold, and VEGF mRNA levels were increased approximately 4.4-fold. However, BAX, caspase-3, and c-MYC mRNA levels were not considerably changed after treatment with CuI [23, 24].
