Previous preclinical studies have shown a significant cytotoxic eribulin activity in the nanomolar range in several tumour cell lines. In this study, IC50 ranged from 1.58nM to 0.7nM after 24h exposure time which is within the values reported in the literature [3, 16, 23, 24].
We demonstrate a significant extra cell killing after irradiation combined with eribulin at IC50 concentrations as compared with the irradiation alone ranging from 17–32% and 18–34% for HeLa and FaDu cells respectively. Our results are in accordance with those found in a recent study  where they reported an increase in growth inhibition of 29% and 37% for irradiated H446 and H841 small lung cancer cell lines respectively exposed to 0.625nM eribulin.
The present study demonstrates the in vitro radiosensitizing effect of eribulin in the two human cell lines tested in a dose-dependent manner. We show an enhancement of radiation response in pretreated cells with a SER up to 2.71 and 2.32 for HeLa and FaDu cells respectively which means that combined treatment is more than twice as effective as irradiation alone. To further examine the eribulin induced radiosensitization we have calculated the extra cell killing of combined treatment at a clinically relevant fraction dose of 2Gy. At the IC50 concentrations, the ratio of the SF2 between untreated and treated cells were 1.36 and 1.56 for Hela and FaDu respectively, which correspond to an increase of 36% and 56% in tumour cell death after each fraction of 2Gy. This effect would be magnified in a conventional radiotherapy treatment of 25–30 fractions and could have a significant effect on local tumour control. Our results demonstrate a true synergistic interaction between irradiation and eribulin with a combination index of 0.82 and 0.76 for Hela and FaDu cells respectively, both below 1. We can affirm that radiosensitizing effect is even more pronounced in head and neck line. Our results agree with those previously published regarding the radiosensitizing potential of eribulin. Helfrich et al.  found a remarkable enhancement of irradiation response by eribulin in lung cancer cell lines. The same results were obtained by Miki et al.  using U87MG glioma cells in a clonogenic assay.
The eribulin concentrations required to radiosensitization in our study are in the nanomolar range which can be reached in the plasma of patients treated with eribulin.The plasma Cmax after eribulin i.v. bolus at the maximum tolerated dose (MTD:1.4 mg/m2) is 1µM, higher than the concentration required for radiosensitization . This indirect data suggests that the use of eribulin in radiochemotherapy regimens is clinically feasible and should be evaluated in clinical trials. Moreover, the prolonged half-life of the drug provides sustained plasma concentrations above the concentrations needed to attain cytotoxicity for one week, which could result in a treatment benefit with daily radiotherapy fractions.
To identify the exact mechanisms of the interaction between irradiation and drug is challenging. A modification of radiation cell survival curves in presence of drug is key to demonstrate a true radiosensitization effect. In this study we show a clear modification of radiation cell survival curve shape with an increase in α parameter and a decrease in β parameter (Fig. 1, Table 1). These changes could suggest drug interference with radiation-induced DNA damage repair mechanisms. Prior study , reported an over-expression of histone H2AX, a marker of DNA damage, evident after combined treatment in glioma cells respect to each agent separately.
It is well known that G2/M is the most radiosensitive cell cycle phase. Our results show that eribulin induces a marked accumulation of cells in the G2/M phase of the cell cycle in both cell lines. Our work is also supported by others that also demonstrate an increase in G2/M phase cells after drug treatment [3, 16, 17].
As shown in Fig. 2, the G2/M arrest is greater in the FaDu cell line at 24h exposure time, which could explain the greater radiosensitizing effect in this line as compared to HeLa cells. Based on the results of our study we can conclude that drug-induced cell cycle changes constitute the main mechanism of radiosensitization by eribulin.
Our results demonstrate that eribulin induces marked apoptosis in HeLa and FaDu cells (Fig. 3,4). Furthermore, we found that in HeLa cells treated with 3nM eribulin and irradiation, the apoptosis percentage is 8.93% higher than the sum of both agents separately, which shows that this drug induces an increase in radiation-induced apoptosis. Although the difference does not reach statistical significance due to the small sample size, this effect is considered relevant in the mechanisms of action of this drug. A similar finding was reported in other study with docetaxel whose radiosensitization mechanisms are similar to eribulin, in which an increased radiation-induced apoptosis is described with high drug concentrations . We suggest that radiation-induced apoptosis is involved in the mechanism of eribulin radiosensitization depending on the concentration of the drug and the type of cell line tested.
Recent data  confirm that eribulin induces the apoptotic caspases − 3/7 in all cell lines tested and enhances the radiation-induced apoptotic cells. Another study  reports an increase of cleaved caspase-3 after 10nM eribulin exposure but suggests that combined treatment induces cell death through a caspase-independent mechanism.