Real-time assessment of platinum sensitivity of primary culture from a patient with ovarian cancer with extensive metastasis and the platinum sensitivity enhancing effect by metformin

Abstract

The aim of the present study was to perform a rapid evaluation of the efficiency of commonly used platinum-based chemotherapy regimens for patients with ovarian cancer with extensive metastases using an in vitro method combined with culturing primary cells and real-time monitoring, and to further explore the enhanced effect of metformin on susceptibility of ovarian cancer cells to platinum-based chemotherapy. The primary omental metastatic (OM) cells were isolated from the omentum metastasis of a surgical patient with stage IIIc ovarian carcinoma. Drug sensitivity was evaluated using the xCELLigence system, and screening of the most effective platinum chemotherapy was performed through analysis of cell susceptibility to cisplatin, carboplatin, nedaplatin and paclitaxel or docetaxel alone or in combination. At the same time, this system was used to determine whether metformin was able to increase the sensitivity of cancer cells to platinum chemotherapy. The results revealed that nedaplatin exhibited the most marked cytotoxic effect on the OM cells, followed by those of carboplatin and cisplatin. The addition of docetaxel enhanced the cytotoxic effect, and the combination of platinum and paclitaxel also enhanced the effect. Metformin rapidly increased the sensitivity of cells to platinum-based chemotherapy, and this effect was dose-dependent. The sensitivity of OM cells to different platinum-based regimens was varied. The effect of metformin on chemotherapeutic sensitization of cancer cells is clear in vitro, and the real-time cell analyzer assay has the potential to assist in determining individualized drug regimens for patients with metastatic ovarian cancer.

Keywords: omentum metastases; ovarian cancer; platinum; primary cell culture; real-time cell analyzer system; sensitivity.

Figure 1.

Morphology of ovarian cancer cells generated from the primary tumor and omentum metastases in a female patient aged 69 years with ovarian cancer who had extensive peritoneal metastasis. Typical phase-contrast images at day 4 are presented. (A) Ovarian cancer cells from the primary tumor; cancer cells were surrounded by fibroblasts (magnification, ×200). (B) Cells generated from omental metastatic cells (magnification, ×100).

Figure 2.

Real-time cell analysis of the cytotoxic effect of single chemotherapy drugs on OM cells. OM cells were seeded into the E-Plate and the CI was determined. (A) The normalized CI of OM cells treated with PTX (30 nM), DTX (50 nM), DDP (15 µM), CBP (330 µM) or NDP (95 µM). The drugs were added at ~20 h as indicated by the black line. The normalized CI was generated by normalizing CIs to the CI at the time of drug addition. (B) Slopes of CI curves in (A) at 0–20 (pretreatment), 20–40, 40–60 and 60–80 h. (C) The normalized CI was calculated at various time points. The results are expressed as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001, compared with DDP; (D) The inhibitory rate of single chemotherapy drugs on OM cells was calculated on the basis of cell viability analyzed using an alamarBlue assay. The results are expressed as the mean ± SD. *P<0.05, **P<0.01. OM, omental metastatic; CI, cell index; CBP, carboplatin; PTX, paclitaxel; DDP, cisplatin; DTX, docetaxel; NDP, nedaplatin; SD, standard deviation.

Figure 3.

Real-time cell analysis of the cytotoxic effect of chemotherapy drug combinations on the OM cells. OM cells were seeded into the E-Plate and the CI was recorded. (A) The normalized CI of OM cells treated with DDP (15 µM) + PTX (30 nM), CBP (330 µM) + DTX, (50 nM), or NDP (95 µM) + DTX (50 nM), with or without 8 mM MTF. The drugs were added at ~20 h as indicated by the black line. The normalized CI was generated by normalizing CIs to the CI at the time of drug addition. The CI of non-treated cells is presented in the inset as the CI reached 3.0 at the end of experiment. (B) The slope of CI curves in (A) at 0–20 (pretreatment), 20–40, 40–60, 60–80 and 80–93 h. (C) The normalized CI following the addition of drugs were calculated (C-1: DDP+PTX vs. CBP+DTX vs. NDP+DTX; C-2: DDP+PTX vs. DDP+PTX+MTF; C-3: CBP+DTX vs. CBP+DTX+MTF; C-4: NDP+DTX vs. NDP+DTX+MTF). The results are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001; C-1, compared with DDP+PTX; C-2 to C-4, compared with drug combinations without MTF respectively. OM, omental metastatic; CI, cell index; CBP, carboplatin; PTX, paclitaxel; DDP, cisplatin; DTX, docetaxel; NDP, nedaplatin; MTF, metformin.

Figure 4.

Real-time cell analysis demonstrates that the chemotherapy sensitization effect of MTF was dose-dependent. In total, 5,000 OM cells were seeded into the E-Plate and the CI was recorded. (A) The normalized CI of OM cells treated with NDP (95 µM) + DTX (50 nM) with 4, 8 and 16 mM MTF. The drugs were added at ~14 h as indicated by the black line. The normalized CI was generated by normalizing CIs to the CI at the time of drug addition. The CI of non-treated cells is presented in the inset as the CI reached 3.5 at the end of the experiment. (B) The slope of CI curves in (A) at 0–14 (pre-treatment), 14–24, 24–34, 34–44, 44–54 and 54–63 h. (C) The normalized CI was calculated at various time points. The results are expressed as the mean ± standard deviation. *P<0.05 and **P<0.01. OM, omental metastatic; CI, cell index; DTX, docetaxel; NDP, nedaplatin; MTF, metformin.

Figure 5.

MTF-enhanced NDP induces apoptosis. (A) MTF increased the caspase-3 and PARP expression in omental metastatic cells. GAPDH was used as the loading control. Relative cell expression of (B) caspase-3 and (C) PARP. (D) Cleaved caspase-3 and (E) PARP were also increased when MTF (8 mM) was added. The results are expressed as the mean ± standard deviation for experiments performed in triplicate. **P<0.01 and ***P<0.001. NDP, nedaplatin; MTF, metformin.