Development of Potent Forchlorfenuron Analogs and Their Cytotoxic Effect in Cancer Cell Lines

Abstract

Forchlorfenuron (FCF) is a synthetic plant cytokinin widely used in agriculture to promote fruit size, that paradoxically inhibits proliferation, migration, and invasion in human cancer cell lines. FCF has also been shown to affect HIF-1α and HER2, which are both known to play a crucial role in cancer cell survival. In this study, we have developed potent FCF analogs through structural modification of FCF, coined UR214-1, UR214-7, and UR214-9. Compared to parental FCF, these analogs are more effective in decreasing viability and proliferation in both ovarian and endometrial cancer cell lines. These FCF analogs also suppress HER2 expression at a concentration lower than that of FCF. In addition, we found that treatment with either FCF or its analogs decreases the expression of human epididymis protein 4 (HE4), which is commonly upregulated in ovarian and endometrial cancers. Given the association between cancer behavior and HE4 production in gynecologic cancers, our findings may provide insight useful in the development of new treatment strategies for gynecologic cancers.

Figure 1

The effect of FCF on the viability of ovarian and endometrial cancer cell lines. Cancer cell lines were treated with FCF (0, 100, 300 µM) for either 24 h or 72 h, after which the cell viability was measured by MTS assay.

Figure 2

The effect of FCF analogs on the viability of ovarian and endometrial cancer cell lines. (A) Cell lines were treated with a fixed concentration (100 µM) of FCF or FCF analog for 24 h. After treatment, cell viability was determined by MTS assay. (N = 3, see Suppl. Fig. A for mean values and SEM) (B) ECC-1 cells were treated with UR214-7 or UR214-9 at the listed concentrations for 48 h. After treatment, cell viability was determined by MTS assay (left). Cell proliferation was measured by BrdU incorporation (right). (C) ECC-1 cells were treated with vehicle, UR214-7, or UR214-9 at the indicative concentrations for 24 or 72 h. Cell viability was determined by MTS assay. (D) ECC-1 cells were treated with UR214-7 or UR214-9 at 33 µM for 48 h. Cells undergoing apoptosis were measured by caspase-3/7 activity assay (E) Each indicated cells were treated with various concentrations of FCF analogs for 72 h. After treatment, cell viability was determined by MTS assay.

Figure 3

The effect of FCF analogs on HER2 expression. (A) ECC-1 or HCH-1 cells were incubated with UR214-7 (33 µM), UR214-9 (33 µM) or FCF (300 µM) for 24 h. The levels of each protein were determined by Western blot analysis. (B) HCH-1 cells were incubated with UR14-9 (10 µM) or FCF (100 µM) for 48 h (top) or transfected with septin-2 targeting siRNA or non-targeting control siRNA for 24 or 48 h (bottom). Cell lysates were collected. The cellular levels of HER2 were determined by human HER2 enzyme-linked immunosorbent assay. (C) The cells were transfected with septin-2 targeting siRNA or non-targeting control siRNA. At 48 h post transfection, cell population and relative expression of septin-2 were determined by sulforhodamine B assay (top) and Western blot analysis (bottom), respectively.

Figure 4

The effect of FCF analogs on the expression of HE4. (A) HE4 overexpression clones (OVCAR8-C5) were treated with or without FCF (300 µM) for 7 h, after which the cell lysates and matching culture media were collected and analyzed for the levels of HE4 using enzyme immunoassays. The amount of secreted HE4 was normalized to the protein concentration of respective cell lysate. (B) ECC-1 and HCH-1 cells were treated with the indicated concentrations of FCF for 5 h and the secreted levels of HE4 were measured. (C) ECC-1 cells were treated with either UR214-1, UR214-7 or FCF at the concentrations indicated for 5 h at which point the secreted levels of HE4 were determined. (D) ECC-1 cells were washed and incubated with FCF (300 µM) for 5 h in basal media. Relative HE4 expression was determined by real time PCR (normalized to B2M). (E) ECC-1 cells were transfected with septin-2 targeting siRNA or non-targeting control siRNA for 48 h (bottom) or for 72 h (top). Relative gene expression of HE4 was determined by real time PCR (normalized to TBP; bottom). The intracellular levels of HE4 were measured using enzyme immunoassays (top).

Figure 5

(A) Correlation of septins with survival rates of endometrial cancer patients was analyzed using the TCGA dataset. (p < 0.05); x-axis: days; y-axis: survival fractions. (B) Relative expression of septin-2, -3 and -7 in endometrial cancers (TCGA dataset). (C) Relative gene expression of various septins was analyzed from the dataset (Wong-77-MAS5.0-u133p2), which contains gene expression profiling of microdissected cancer stroma samples from high grade serous ovarian cancer patients (red) and those from normal ovarian stroma (blue). (**p < 0.01, Student’s t-test vs. microdissected normal ovarian stroma).