Preclinical evaluation of exemestane as a novel chemotherapy for gastric cancer

Abstract

CYP19A1/aromatase (Ar) is a prognostic biomarker of gastric cancer (GCa). Ar is a critical enzyme for converting androstenedione to oestradiol in the steroidogenesis cascade. For decades, Ar has been targeted with Ar inhibitors (ARIs) in gynaecologic malignancies; however, it is unexplored in GCa. A single-cohort tissue microarray examination was conducted to study the association between Ar expression and disease outcome in Asian patients with GCa. The results revealed that Ar was a prognostic promoter. Bioinformatics analyses conducted on a Caucasian-based cDNA microarray databank showed Ar to be positively associated with GCa prognosis for multiple clinical modalities, including surgery, 5-Fluorouracil (5-FU) for adjuvant chemotherapy, or HER2 positivity. These findings imply that targeting Ar expression exhibits a potential for fulfilling unmet medical needs. Hence, Ar-targeting compounds were tested, and the results showed that exemestane exhibited superior cancer-suppressing efficacy to other ARIs. In addition, exemestane down-regulated Ar expression. Ablating Ar abundance with short hairpin (sh)Ar could also suppress GCa cell growth, and adding 5-FU could facilitate this effect. Notably, adding oestradiol could not prevent exemestane or shAr effects, implicating a nonenzymatic mechanism of Ar in cancer growth. Regarding translational research, treatment with exemestane alone exhibited tumour suppression efficacy in a dose-dependent manner. Combining subminimal doses of 5-FU and exemestane exerted an excellent tumour suppression effect without influencing bodyweight. This study validated the therapeutic potentials of exemestane in GCa. Combination of metronomic 5-FU and exemestane for GCa therapy is recommended.

Keywords: aromatase; exemestane; gastric cancer.

Figure 1

Ar expression is the gatekeeper of gastric cancer (GCa) prognosis. A, OS curve associated with Ar expression status in general patients with GCa. The red line indicates high expression, and black line indicates low expression. At the initial time‐point (0 mo), 581 patients had high Ar expression levels and 295 had low Ar expression levels. At the last time‐point (150 mo), one patient had high Ar expression and none of the patients had low Ar expression. The corresponding HR was 1.98, and P‐value was 3.6e‐12. B, OS curve associated with Ar expression status in GCa patients with surgery. The corresponding HR was 1.73, and P‐value was .00028. C, OS associated with Ar expression status in patients with GCa who underwent 5‐FU therapy. The corresponding HR was 2.22, and P‐value was 4.1e‐10. D, PFS (after therapy; 150 mo) after associated with Ar expression status in patients with GCa who underwent 5‐FU therapy. The corresponding HR was 1.51, and P‐value was .019. E, OS associated with Ar expression status in patients with GCa who were HER2 negative. The corresponding HR was 2.07 and P‐value was 2.6e‐07. F, OS associated with Ar expression status in patients with GCa who were HER2 positive. The corresponding HR was 1.94, and P‐value was 4.7e‐06

Figure 2

Differential cytotoxic effects of ARIs on gastric cancer (GCa) cells. A, The cytotoxic effect of ARIs was determined using WST‐1 cytotoxicity assay conducted on GCa cells (AGS, SCM‐1 and MKN45). The mean absorbance (450 nm) showed the viability of GCa cells treated with increasing concentrations (0, 20, 40, 60, 80 and 100 μmol/L) of various ARIs for 48 h (type I: anastrozole and letrozole; type II: exemestane). B, IC50 values indicating the cytotoxic efficacy of exemestane against GCa cells were calculated using CalcuSyn software. C, Cell growth suppression effect of ARIs was measured using a colony formation assay, and the results showed that the ARIs exhibited different efficacy levels in suppressing GCa cell growth. Long‐term (2 wk) and low‐dose (20 μmol/L) treatment showed various inhibitory efficacy levels. D, Down‐regulation of Ar by treating GCa cells with exemestane. GCa cells were treated with exemestane (0, 5, 10 and 15 μmol/L) for 48 h, and then, Ar mRNA was analysed using qRT‐PCR. *, ** and *** indicate significant differences with P‐values <.05, .01 and .001, respectively

Figure 3

Expression, but not catalytic activity, of Ar affects gastric cancer (GCa) cell growth. A, Knockdown efficacy of Ar shRNA in AGS cells. Upper band densitometry results represent the PCR product of Ar cDNA, and lower bar chart represents the quantitation of RT‐PCR results. B, Cell growth of shAr compared with shLuc on AGS cells. The upper image shows the representative wells of the colony formation assay. The lower bar chart represents the quantitation result of the colony formation assay. C, Cytotoxicity of 5‐FU against shLuc and shAr AGS cells. D, Cytotoxicity of 5‐FU and/or oestradiol (E2; 10 nmol/L) against shLuc and shAr AGS cells. E, Colony‐forming ability of shLuc and shAr AGS cells treated with 5‐FU. F, Colony‐forming ability of shLuc and shAr AGS cells treated with 5‐FU and/or oestradiol (E2, 10 nmol/L)

Figure 4

Combination treatment of exemestane and 5‐FU suppresses gastric cancer (GCa) cell growth in vitro. A, Cytotoxic efficacy of 5‐FU against GCa cells. Upper panel: mean absorbance (450 nm) was evaluated to determine the viability of GCa cells treated with 5‐FU (0, 20, 40, 60, 80, 100 μmol/L) for 48 h. Lower panel: 5‐FU cytotoxic IC50 values were calculated using CalcuSyn software. B, Cytotoxic efficacy of 5‐FU (10 μmol/L) and in combination with exemestane (20, 40, 60, 80 and 100 μmol/L) against GCa cells. C, Colony suppression efficacy of treatment with 5‐FU alone (10 μmol/L), treatment with exemestane alone (20 μmol/L) and combination treatment against GCa cells

Figure 5

Combination treatment of exemestane and 5‐FU suppresses gastric cancer (GCa) tumour growth in vivo. A, MKN45 xenograft mouse model was used for testing tumour suppression effect of exemestane. The placebo (PBS, con, black line) or exemestane (low dose, 10 mg/kg, EXE‐10, red line; medium dose, 20 mg/kg, EXE‐20, green line) was intraperitoneally injected when the tumour size reached 200 mm³ three times per week for four consecutive weeks. The red arrow indicates the time of initial drug injection. B, The same GCa tumour model and treatment procedure were used to test the effect of combination treatment of exemestane and 5‐FU. Results for the placebo (PBS, con, black line), low‐dose 5‐FU (5‐FU, 5 mg/kg, grey line) or combination treatment of 5‐FU and exemestane (low dose, 10 mg/kg, EXE‐10, red line; medium dose, 20 mg/kg, EXE‐20, green line) are presented. C, Ar Immunoblot of xenografted tumour from placebo and exemestane (10 mg/kg) treated mice. Left‐handed side image is representative tumours, and the right‐handed side bar graft is the quantitation of five pairs of tumours. C#1 represented control group number 1 tumour; where E#1 represented exemestane # 1 tumour. D, Bodyweight, tumour weight and tumour weight‐to‐bodyweight ratio of GCa mice at the time of killing. # are the P‐value comparing groups with Veh using t test. *, ** and *** indicate significant differences for P‐values <.05, .01 and .001, respectively