Baicalein was purchased from the China National Institutes for Food and Drug Control, (Beijing, China). Cisplatin, anti-haemagglutinin (HA) mouse IgG, sulforhodamine B, trichloroacetic acid, acetic acid, Tris, dimethylsulfoxide (DMSO) and oleamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture reagents were purchased from Gibco-BRL (Grand Island, NY, USA) and calcein acetoxymethyl ester (AM) was purchased from Invitrogen Life Technologies (Calrsbad, CA, USA). G418, hygromycin, and doxycycline were purchased from Calbiochem (San Diego, CA, USA). Secondary antibodies for western blotting were purchased from Amersham Biosciences (Piscataway, NJ, USA). All other reagents were purchased from Sigma-Aldrich unless otherwise stated.

The HeLa cell line expressing Cx26 was previously described and characterized (14). In this cell line, obtained via transfection, Cx26 expression is under the control of a single bidirectional tetracycline-inducible promoter. Cx26 has a thrombin-cleavable C-terminal epitope tag (3.2 kDa) that includes an HA epitope. Cx26-expressing HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10% newborn bovine serum, 100 μg/ml G418 sulfate, and 200 μg/ml hygromycin B at 37°C in a 5% CO₂ humidified incubator. Connexin expression was induced by incubation with 1 μg/ml doxycycline for 48 h prior to all experiments.

The SRB colorimetric assay was used to measure the toxic effect of baicalein on cell viability (15). Cells were seeded onto 96-well plates for 24 h, followed by incubation with different concentrations of baicalein for 24 h, and were cultured for an additional 12 h. The culture medium was then removed and the cells were fixed with 50 μl of ice-cold 50% trichloroacetic acid solution at 4°C for 60 min, rinsed five times with tap water and dried at room temperature overnight. Next, 100 μl of 0.4% SRB solution was added to each well and incubated at room temperature for 30 min. Unbound dye was removed by washing five times with 1% acetic acid solution and drying at room temperature. A total of 10 mM Tris base solution (pH 10.5) was used to dissolve the protein-bound dye, and the plate was placed on a plate shaker for 15 min. The optical density (OD)_{570 nm} was measured using a 96-well MRX plate reader (Dynex Technologies, Chantilly, VA, USA).

Functional GJs were examined as described by Goldberg et al (16). Cells were grown to confluence in 12-well plates. Donor cells from one well were incubated with growth medium supplemented with a freshly made solution of 5 μmol/l calcein AM for 30 min at 37°C. Calcein AM is intracellularly converted into the GJ-permeable dye calcein. After three consecutive washes with phosphate-buffered saline (PBS) to remove excess dye, the donor cells were trypsinized and seeded onto the receiver cells at a 1:150 donor/receiver ratio. They were allowed to form GJs for 4 h at 37°C and then examined under a fluorescence microscope (IX71; Olympus, Tokyo, Japan). Images were captured of ~20 cells per well (magnification, ×400) to count receiver cells containing calcein per donor cell. The average number of receiver cells containing calcein per donor cell was used as a measure of the GJIC.

Following three washes with ice-cold PBS, cells were lysed using lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM Na₃VO₄, 1 mM β-glycerophosphate, 1:1,000 protease inhibitors). The cell lysate was sonicated and centrifuged at 14,167 × g for 30 min at 4°C. The DC protein assay kit was used to determine the protein concentration (Bio-Rad, Hercules, CA, USA). A total of 20 μg of protein from each sample was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose blotting membranes, followed by immunoblotting. Monoclonal antibodies against HA IgG (1:1,000) or β-actin (1:10,000) were used. Immunopositive bands were visualized using the Amersham ECL™ Plus Western Blotting Detection kit (GE Healthcare).

All experiments of cell exposure to cisplatin were performed in the dark at 37°C. Baicalein was dissolved in DMSO. When combined treatment with cisplatin and baicalein was performed, baicalein was added to the cells 3 h prior to cisplatin addition. The GJ inhibitor oleamide was added to the cell culture medium at a 50 μM concentration 3 h prior to the exposure to cisplatin.

Cisplatin toxicity was assessed by a standard colony forming assay as described by Jenser et al (5) with a few modifications. Briefly, cells were cultured at low (100 cells/cm²) or high density (30,000 cells/cm²), corresponding to conditions permissive of gap junction formation or not, respectively. For the high-density condition, cells were grown to confluence prior to cisplatin exposure. Following treatment with cisplatin for 1 h, cells were washed with PBS, harvested by trypsinization, counted, and seeded onto 6-well dishes at a 500 cells/well density. The cells were incubated for an additional 7-day period, and next fixed and stained with 1% crystal violet in ethanol. Colonies containing ≥50 cells were counted. Colony formation rates were normalized to the efficiency of colony formation of cells not treated with drugs (control). For the low-density condition, cells were seeded onto 6-well plates at a 100 cells/ml density. After 4 h adherence, cells were exposed to cisplatin and then replenished with fresh medium. They were rinsed and assessed for colony formation as described above. Cells in this condition were unable to form GJs, since there is no possibility to contact each other at such low density.

To avoid discrepancy in results caused by the fact that cells are at different cell cycle phases, we incubated the cells in serum-free medium for 24 h prior to the addition of cisplatin, to ensure cell synchronicity at the G1 phase.

Data from the experiments of different treatments were analyzed by unpaired Student’s t-tests using the SigmaPlot 10.0 software (Systat Software Inc., San Jose, CA, USA). Data are presented as mean ± SEM. P<0.05 was considered to indicate statistically significant differences.

GJ channels are formed by the end-to-end docking of two hemichannels in adjacent cells; GJIC occurs only when cells contact each other. As an initial experiment to determine the effect of GJIC on cisplatin toxicity, Cx26-expressing HeLa cells were cultured under conditions where GJ formation was possible (high density; 30,000 cells/cm²) or not (low density; 100 cells/cm²). In both conditions, cisplatin caused cell death in a concentration-dependent manner. However, the toxic effect of cisplatin was substantially greater at high-density compared to low-density cultures (Fig. 1). This indicates that cell culture density might have an effect on cisplatin cytotoxicity. The concentration of cisplatin used in the experiment was within the therapeutic range used during chemotherapy (17).

The formation of GJs is only one of the numerous differences between cells cultured at low and high density. To determine whether the effects of high cell density were due to GJIC, GJ coupling was examined in the cultures under different conditions of connexin expression and chemical inhibition.

First, connexin expression was studied in Cx26-expressing HeLa cells. Connexin expression was induced by incubation with 1 μg/ml doxycycline for 48 h (see Materials and methods). Fig. 2A and B show the expression of connexin and GJ dye coupling 48 h following exposure to doxycycline.

The cells treated with doxycycline (expressing connexin, GJs formed) showed a markedly higher sensitivity to cisplatin at high compared to low density growth conditions. The surviving fraction decreased from 0.78±0.04 to 0.53±0.05, respectively. Notably, the inhibition of cell survival by cisplatin was not affected by addition of doxycycline in low density cultures (Fig. 3).

To further assess the role of GJIC on cell density-dependent cisplatin sensitivity, oleamide, a GJ channel inhibitor (18), was used to inhibit GJIC in HeLa cells, and its effect was assessed by the GJ dye-coupling assay (Fig. 4A and B). In low-density cultures (without GJIC), treatment with oleamide did not affect cisplatin toxicity. However, in high-density cultures (with GJIC), cisplatin toxicity was attenuated upon inhibition of GJIC by oleamide, manifested as a significant increase of the cell surviving fraction from 0.57±0.04 to 0.73±0.06 (Fig. 5). Thus, inhibition of GJIC during the 1 h of exposure to cisplatin reversed the effect of high density cell culture on cisplatin toxicity.

These results indicated that GJIC enhances cisplatin toxicity and fully accounts for the observed density-dependent effect of cisplatin toxicity.

The formation of GJs composed of Cx26 affected the cytotoxicity of cisplatin. This observation was consistent with our hypothesis that enhancement of the GJ function would increase the cytotoxic action of cisplatin, and thus increase cisplatin sensitization. Would baicalein affect the GJ function and cisplatin cytotoxicity? To determine this, we first examined the cytotoxic effect of baicalein using the SRB assay.

As shown in Fig. 6, baicalein had no effect on cell viability until the concentration reached 100 μM. Thus, the concentrations of baicalein used in the following experiments (≤0.1 μM) had no cytotoxic effect on HeLa cells.

The effect of baicalein on dye coupling among cultured cells was assayed by the parachute assay. Donor cells were labeled with the junction-permeable dye calcein and then seeded onto unlabeled receiver cells with different concentrations of baicalein for 4 h. GJ intercellular communication was expressed as the number of receiver cells receiving calcein from a labeled cell, normalized to that of control cells (non-treated). As shown in Fig. 7A and B, baicalein markedly increased the dye spread from donor cells to receiver cells in a dose-dependent manner. Fig. 8A and B show that the dye spread through GJs treated with 0.1 μM baicalein was markedly decreased from 4 to 24 h in Cx26-transfected HeLa cells. These results indicated that baicalein can enhance GJIC in Cx26-expressing HeLa cells.

The effects of baicalein on cisplatin-induced cytotoxicity were examined in HeLa cells expressing Cx26. Cells seeded at high or low cell density were treated with baicalein (0.1 μM) for 3 h, followed by exposure to 0.5 μM cisplatin + baicalein for 1 h. The clonogenic survival of HeLa cells was assessed 7 days following exposure to cisplatin and baicalein. Baicalein enhanced cisplatin cytotoxicity in high-density cultures but had no effect in low-density cultures; the surviving fraction substantially decreased from 0.57±0.04 in low-density cultures to 0.34±0.04 in high-density cultures. This effect of baicalein was only observed in conditions permissive of GJ formation.

To further assess the role of GJIC in the improvement of cisplatin sensitivity induced by baicalein, oleamide was used to inhibit GJIC in HeLa cells (Fig. 9A and B). Consistent with our previous results, treatment with oleamide and baicalein did not affect cisplatin toxicity in low-density cell cultures (without GJIC). However, the improvement of cisplatin toxicity induced by baicalein was attenuated upon inhibition of GJIC by oleamide in high-density cultures (with GJIC). An increase of the surviving fraction, from 0.34±0.04 to 0.58±0.07, was observed (Fig. 10). These results showed that oleamide can inhibit the improvement of the dye spread through GJs induced by baicalein, and thus reduce cisplatin cytotoxicity improved by baicalein at high cell density. Therefore, baicalein improves cisplatin cytotoxicity by enhancing GJIC in Cx26-expressing HeLa cells.

To determine whether baicalein affects the expression of connexin, the level of Cx26 was assessed by western blotting in cells induced by doxycycline and exposed to baicalein. Fig. 11 shows that treatment with baicalein (0.0125–0.1 μM) for 4 h did not affect Cx26 expression. Fig. 12 shows that treatment with baicalein (0.1 μM) for 4 to 24 h did not change Cx26 expression. These results suggested that the mechanism of baicalein-induced enhancement of GJIC does not involve an increase in the expression of connexin.