Kv3.1 and Kv3.4, Are Involved in Cancer Cell Migration and Invasion

Figures

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  Figure 1. The three cell confluency conditions of A549, MDA-MB-231, and HT-29 cells. A549, MDA-MB-231, and HT-29 cells were grown until they reached the appropriate cell densities. Low cell density was defined as approximately 20~30% cell confluency, medium density as approximately 40~60% cell confluency, and high density as approximately 80~90% cell confluency. Representative images of each cell condition are shown in the figure.

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  Figure 2. Increased pericellular hypoxia and ROS levels according to the increase in cell density. (A) Western blot data demonstrate that HIF-1α expression, which was considered to be induced by pericellular hypoxia in our experiments, was significantly higher in high-density than in low-density in A549 and MDA-MB-231 cells, whereas HIF-1α expression was not increased in high-density compared with that in low-density in HT-29 cells. (B) Representative images show increased ROS signals (green) according to the increase in cell density. The graphs show the quantitative analysis of the ROS data. ROS levels were significantly increased with an increase in cell density in A549 and MDA-MB-231 cells, whereas ROS levels in HT-29 cells were significantly increased only when the cells were cultured at a high density. All experiments were performed in triplicate, and the data represent the mean ± standard error. * p < 0.05 and ** p < 0.01 versus the low-density value.

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  Figure 3. Changes in mRNA and protein expression of Kv3.1, Kv3.3, and Kv3.4 according to cell density. (A) RT-PCR data demonstrating that Kv3.1, Kv3.3, and Kv3.4 mRNA was expressed in A549, MDA-MB-231, and HT-29 cells. (B) The protein expression levels of Kv3.1, Kv3.3, and Kv3.4 were analyzed by Western blot. Kv3.1 and Kv3.4 were increased in A549 cells dependent on the cell density, whereas Kv3.3 was not altered according to the cell density. (C,D) Kv3.1 and Kv3.4 protein expression levels were analyzed in MDA-MB-231 and HT-29 cells by Western blot. Kv3.1 and Kv3.4 were increased in MDA-MB-231 cells according to the increase in cell density. Only Kv3.1 was significantly increased in high-density HT-29 cells compared to that in low-density HT-29 cells. Kv3.4 expression was not significantly increased in HT-29 cells as the cell density increased. All experiments were performed in triplicate, and the data represent the mean ± standard error. * p < 0.05 and ** p < 0.01 versus the low-density value.

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  Figure 4. Effect of BDS-II on cell proliferation, migration, and invasion. (A) Representative Hemacolor® rapid staining images demonstrate that 24 h of BDS-II (500 nM) treatment did not affect the proliferation of A549, MDA-MB-231, and HT-29 cells. The MTT data also demonstrated that BDS-II did not affect cell proliferation in the three cell lines. (B) Representative images demonstrate that 500 nM BDS-II significantly inhibited migration by almost half in A549, MDA-MB-231, and HT-29 cells. (C) Hemacolor® rapid staining images demonstrate that the number of cells that migrated through the membrane was reduced in A549 and MDA-MB-231 cells by 500 nM BDS-II treatment. All experiments were performed in triplicate or quadruplicate, and the data represent the mean ± standard error. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the control value.

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  Figure 5. The expression of Kv3.1, Kv3.3, and Kv3.4 and the effect of BDS-II on L-132 cells. (A) RT-PCR data demonstrate that L-132 cells express all of the 8 oxygen-sensitive Kv channels at the mRNA level. (B) The protein expression levels of Kv3.1, Kv3.3, and Kv3.4 were analyzed by Western blot in L-132 cells. The expression of all three Kv channels was not significantly altered according to the cell density. (C) Representative images demonstrate that 500 nM BDS-II did not affect L-132 cell migration. (D) Hemacolor® rapid staining images demonstrate that the number of L-132 cells that migrated through the membrane was not altered by 500 nM BDS-II treatment. All experiments were performed in quadruplicate, and the data represent the mean ± standard error.

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  Figure 6. Acridine orange staining represents alterations in the diameter of acidic compartments in A549 and L-132 cells according to cell density. The orange-red fluorescence of acridine orange staining demonstrates the acidic compartments, including the intracellular organelles, and the yellow-green color indicates a slightly acidic or neutral pH. (A) The number of small (0.4–0.6 μm) acidic compartments in A549 cells was significantly increased and the number of large (1–1.2 μm) acidic compartments in A549 cells was decreased when the cells reached a high density compared to that of A549 cells at a low density. (B) When A549 cells were incubated with 500 nM BDS-II for 30 h, BDS-II had no effect on A549 cells cultured at a low density (n = 13), whereas the same concentration of BDS-II decreased the number of small (0.4–0.6 and 0.8–1.0 μm) acidic compartments and increased the number of large (1–1.2 μm) acidic compartments in A549 cells cultured at a high density (n = 17). (C) The number of small acidic compartments in L-132 cells was also significantly increased when the cells reached a high density compared with that of L-132 cells cultured at a low density. (D) L-132 cells were also…

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  Figure 7. Effect of BDS-II on ERK activation according to the increase in cell density. ERK activation according to the increase in cell density was analyzed in A549 and L-132 cells using the ratio of phosphorylated ERK (pERK) to total ERK (tERK) protein expression. (A) Quantitative data demonstrate that ERK was activated in A549 cells cultured at a high density compared with A549 cells cultured at a low density. ERK activation in medium-density A549 cells did not significantly differ from that in low-density A549 cells. (B) ERK activation was not altered according to the cell density in L-132 cells. When the cells were incubated with 500 nM BDS-II for 30 h, ERK activation in the high-density A549 or L-132 cells was inhibited. BDS-II had no effect on ERK activation in A549 or L-132 cells cultured at either a low or medium density. All experiments were performed in triplicate, and the data represent the mean ± standard error. * p < 0.05 and ** p < 0.01 versus the low-density or control value.