Combination chemotherapy of oxaliplatin and 5-fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ovary: a potential treatment strategy

Abstract

Resistance of ovarian mucinous adenocarcinoma to standard chemotherapy with paclitaxel and carboplatin is associated with poor prognosis, and an effective treatment is needed. The present study aimed to identify an effective chemotherapy for ovarian mucinous adenocarcinoma. Five human ovarian mucinous adenocarcinoma cell lines (MN-1, OMC-1, RMUG-L, RMUG-S, TU-OM-1) were used in this study. Sensitivity of the cells to the anticancer agents was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and we assessed drug sensitivity by calculating the assay area under the curve for each agent. Protein expression was confirmed by Western blot analysis. We also examined the efficacy of combination chemotherapy on survival in a xenograft model of nude mice. The IC(50) to anticancer agents ranged widely. The assay area under the curve indicated that two of five cell lines (MN-1, TU-OM-1) were sensitive to oxaliplatin, 5-fluorouracil and etoposide, and only one (TU-OM-1) was sensitive to 7-ethyl-10-hydroxycamptothecin, which is an active metabolite of camptothecin. All cell lines were resistant to cisplatin and paclitaxel. The combination of oxaliplatin and 5-fluorouracil resulted in additive or synergistic effects on all cell lines. The combination of oxaliplatin and 5-fluorouracil significantly prolonged survival in a ovarian mucinous adenocarcinoma xenograft model of nude mice. Protein expression levels of the excision repair cross-complementation group 1 were lower in oxaliplatin sensitive cell lines. Exposure to 5-fluorouracil down-regulated cross-complementation group 1 expression in ovarian mucinous adenocarcinoma cells. We conclude that combination chemotherapy consisting of oxaliplatin and 5-fluorouracil was an effective treatment for ovarian mucinous adenocarcinoma and may be a pivotal candidate for a novel treatment strategy.

Figure 1

Effects of 5‐fluorouracil (5FU) are synergistic or additive with those of oxaliplatin (L‐OHP). L‐OHP was combined with 5FU or etoposide (VP‐16) at a fixed ratio that spanned the individual IC₅₀ of each drug. The combination effects of L‐OHP and 5FU were synergistic or additive in all MAC cell lines tested. Data were analyzed by the method of Chou and Talalay^{( 20 )} to determine the combination index values. The results shown are an average of at least two independent experiments.

Figure 2

The effects of oxaliplatin (L‐OHP) combined with 5‐fluorouracil (5FU) on mucinous adenocarcinoma (MAC) cell proliferation and apoptosis.(a) Five MAC cell lines were treated with 3.1 µM (L‐OHP) and/or 5.8 µM 5FU for 72 h compared with the control (phosphate‐buffered saline). Cell‐growth inhibition was determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐dyphenyltetrazolium bromide assay. Cell growth was suppressed more than 50% in four of five cell lines when L‐OHP was combined with 5FU at the same concentrations calculated by clinically achievable AUC. Points represent mean ± SD from six dishes. Treatment with L‐OHP combined with 5FU increased apoptotic cells. Cells were treated with 3.1 µM L‐OHP in the presence or absence of 5.8 µM 5FU for 24 h, then stained with Annexin V–PE. The number of apoptotic cells increased additively after treating with L‐OHP combined with 5FU in (b) MN‐1 cells and (c) TU‐OM‐1 cells. Similar results were obtained in the other three cell lines (data not shown). The results shown represent duplicate experiments.

Figure 3

Treatment with oxaliplatin (L‐OHP) combined with 5‐fluorouracil (5FU) prolongs survival in mice with implanted TU‐OM‐1 cells. Female nude mice (five per group) were given an intraperitoneal (i.p.) injection of 2 × 10⁶ TU‐OM‐1 cells followed by weekly i.p. injections of 250 µL PBS, 12.5 mg/kg L‐OHP, and/or 25 mg/kg 5FU for 3 weeks (days 5, 12, 19). Treatment with L‐OHP and 5FU prolonged survival relative to treatment with PBS, L‐OHP or 5FU (P < 0.005).

Figure 4

The high protein expression levels of ERCC1 in oxaliplatin‐resistant mucinous adenocarcinoma (MAC) cells. (a) Protein expression of the cell‐survival signaling pathways (Akt, MEK, Bcl2, and Bcl‐X_L) and (b) DNA repair (ERCC1, XPF, and XRCC1) in MAC cells was determined by Western blot analysis. The results shown represent duplicate experiments.

Figure 5

5‐fluolouracil (5FU) down‐regulates ERCC1 expression. (a) Each of the cell lines was treated by 5FU with or without oxaliplatin (L‐OHP) for 24 h. The cells were then collected and protein expressions of ERCC1 were determined by Western blot analysis. After treatment with 5FU, the protein expression levels of ERCC1 were down‐regulated. The results shown represent duplicate experiments. (b) Nude mice bearing TU‐OM‐1 were treated with phosphate‐buffered saline (PBS) or 5FU intrapenitoneally for 24 h. Then, the expression levels of ERCC1 protein were determined by Western blot analysis. ERCC1 proteins were down‐regulated only in tumors from mice treated with 5FU. The results shown represent duplicate experiments.