Gemcitabine intercellular diffusion mediated by gap junctions: new implications for cancer therapy

Abstract

Background: Solid tumors are often poorly vascularized, with cells that can be 100 microm away from blood vessels. These distant cells get less oxygen and nutrients and are exposed to lower doses of chemotherapeutic agents. As gap junctions allow the passage of small molecules between cells, we tested the possibility that the chemotherapeutic agent gemcitabine can diffuse through gap junctions in solid tumors.

Results: We first showed with a dye transfer assay that the glioblastoma and the osteosarcoma cells used in this study have functional gap junctions. These cells were genetically engineered to express the herpes simplex virus thymidine kinase (TK), and induced a "bystander effect" as demonstrated by the killing of TK-negative cells in presence of the nucleoside analogue ganciclovir (GCV). The ability of gemcitabine to induce a similar bystander effect was then tested by mixing cells treated with 3 microM gemcitabine for 24 hours with untreated cells at different ratios. In all cell lines tested, bystander cells were killed with ratios containing as low as 5% treated cells, and this toxic effect was reduced in presence of alpha-glycyrrhetinic acid (AGA), a specific gap junction inhibitor. We also showed that a 2- or a 24-hour gemcitabine treatment was more efficient to inhibit the growth of spheroids with functional gap junctions as compared to the same treatment made in presence of AGA. Finally, after a 24-hour gemcitabine treatment, the cell viability in spheroids was reduced by 92% as opposed to 51% in presence of AGA.

Conclusion: These results indicate that gemcitabine-mediated toxicity can diffuse through gap junctions, and they suggest that gemcitabine treatment could be more efficient for treating solid tumors that display gap junctions. The presence of these cellular channels could be used to predict the responsiveness to this nucleoside analogue therapy.

Figure 1

Cx43 expression and functionality in glioblastoma and osteosarcoma cell lines. A. Immunofluorescence of Cx43 shown by confocal microscopy; scale bar: 20 μm: B. Intercellular communication measured by double dye flow cytometry. The percentage of communicating cells represents the percentage of DiI-stained cells that picked up calcein from calcein-loaded cells in presence (open bars) or not of AGA (red bars). Each value is the mean ± s.d. of triplicates of at least three separate experiments. Statistical significance between untreated and AGA-treated cells was evaluated by a Student t-test (*, p < 0.05; **, p < 0.01). Bystander effect of the thymidine kinase/ganciclovir strategy: C. In SKI-1 glioma cells. D. In MNNG/HOS osteosarcoma cells. Data are the means ± S.D. of five replicates of three separate experiments.

Figure 2

Gemcitabine cytotoxicity on glioblastoma and osteosarcoma cells. Cell survival was measured 4 days after a 24-hour gemcitabine treatment. Cell survival was expressed in comparison to untreated cells. Each value is the mean ± S.D. of five replicates.

Figure 3

Inhibiton of dye transfer by gemcitabine. The transfer of calcein between cells was evaluated after a 24-hour treatment with 3 μM gemcitabine. Data are the means ± S.D. of three independent experiments. Statistical significance for the inhibition of gap junction was evaluated by a Student t-test (*, p < 0.05; **, p < 0.01).

Figure 4

Bystander effect of gemcitabine in glioblastoma and osteosarcoma cell lines. A. Experimental design of the bystander effect assay: B. Bystander effect of gemcitabine with (blue bars) or without AGA (red bars). Data are the means ± S.D. of five replicates of at least three separate experiments. Statistical significance between untreated and AGA-treated cells was evaluated by a Student t-test (*, p < 0.05; **, p < 0.01): C. Absence of cisplatine and temozolomide bystander effect in SKI-1 and MNNG/HOS, respectively.

Figure 5

Dye transfer in multicellular spheroids. The transfer of calcein was measured in SKI-1 and MG-63 spheroids in absence (grey area) or presence (black line) of AGA. The plot presented for each cell line is representative of an experiment performed three times.

Figure 6

Gemcitabine cytotoxic effect on spheroids. A. Spheroid diameters were measured 6 days after gemcitabine treatment for 2 hours or 24 hours with (blue bars) or without AGA (red bars). Spheroid diameters are expressed as percentage of untreated spheroids with or without AGA. Data are the means of five replicates ± S.D. of one representative experiment performed twice. Statistical significance between untreated and gemcitabine-treated cells was evaluated by a Student t-test (**, p < 0.01; NS, p > 0.05): B. SKI-1 viability in spheroids was measured 6 days after gemcitabine treatment for 24 hours. Spheroid viability is expressed as percentage of untreated spheroids with or without AGA (-). Individual data of eight replicates are displayed for each condition. Means are presented as horizontal bars. Statistical significance between untreated (-) and AGA-treated cells was evaluated by a Student t-test (***, p < 0.0001).