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Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation.

Huang H, Shah K, Bradbury NA, Li C, White C - Cell Death Dis (2014)

Bottom Line: In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca(2+) uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation.Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration.These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca(2+)-dependent ROS production.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, Rosalind Franklin University of Medicine & Science, North Chicago, IL, USA.

ABSTRACT
Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca(2+) uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca(2+)-dependent ROS production.

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Mcl-1 does not affect cell proliferation in lung cancer cells. (a) Western blot showing efficient knockdown of Mcl-1 by shRNA-transfected stable cell lines from A549, H1299 and H460 cells. Bcl-xL and VDAC levels are unaffected. (b) Summary of proliferation in control Mcl-1 WT and Mcl-1 knockdown lung cancer cells (mean±S.E.; P>0.05; student's t-test)
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fig5: Mcl-1 does not affect cell proliferation in lung cancer cells. (a) Western blot showing efficient knockdown of Mcl-1 by shRNA-transfected stable cell lines from A549, H1299 and H460 cells. Bcl-xL and VDAC levels are unaffected. (b) Summary of proliferation in control Mcl-1 WT and Mcl-1 knockdown lung cancer cells (mean±S.E.; P>0.05; student's t-test)

Mentions: In addition to Mcl-1, both Bcl-2 and Bcl-xL are known to interact with VDAC, raising the possibility of some functional redundancy among the Bcl-2 proteins. Given the difference in apparent binding affinity (Figure 1), however, we hypothesized that in cancer cells overexpressing Bcl-2 proteins, especially those addicted to Mcl-1, Mcl-1/VDAC interactions would dominate. To help define the phenotypes specifically dependent on a Mcl-1/VDAC interaction, we selected three different NSCLC lines (A549, H1299 and H460) previously demonstrated to be similar, with respect to having high Mcl-1 expression,26,27 but different in their relative levels of Bcl-xL (Figure 4a). Mitochondrially generated ROS have been defined as a physiological signal that promotes cancer cell migration and proliferation.12,31, 32, 33 We employed a scratch wound-healing assay to examine the dependence of Mcl-1 expression in NSCLC cells on these processes. The NSCLC cell lines A549, H1299 and H460 were treated with either control or Mcl-1 siRNAs and the knockdown effectiveness was confirmed by western blot (Figure 4b; see Figure 2c for A549 western blot). The wound area was measured at 0 and 24 h, as depicted for A549 (Figure 4c). In all three cell lines the loss of Mcl-1 expression significantly slowed the rate of wound closure (Figure 4d). This assay, however, does not effectively distinguish between differences in proliferation and migration; therefore, the effect of Mcl-1 knockdown on proliferation was assessed in the three NSCLC cell lines using a microplate assay based on measurement of cellular DNA content. The need to track proliferation across several days in control and Mcl-1 knockdown cells necessitated the generation of stable Mcl-1 knockdowns using shRNAs and antibiotic selection. Western blots confirmed that Mcl-1 was successfully knocked down in A549, H1299 and H460 cell lines without affecting either VDAC1/3 or Bcl-xL expression levels (Figure 5a). Mcl-1 knockdown had no significant effect on cell proliferation (Figure 5b), suggesting that Mcl-1 promotes migration but not proliferation in NSCLC cell lines.


Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation.

Huang H, Shah K, Bradbury NA, Li C, White C - Cell Death Dis (2014)

Mcl-1 does not affect cell proliferation in lung cancer cells. (a) Western blot showing efficient knockdown of Mcl-1 by shRNA-transfected stable cell lines from A549, H1299 and H460 cells. Bcl-xL and VDAC levels are unaffected. (b) Summary of proliferation in control Mcl-1 WT and Mcl-1 knockdown lung cancer cells (mean±S.E.; P>0.05; student's t-test)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237246&req=5

fig5: Mcl-1 does not affect cell proliferation in lung cancer cells. (a) Western blot showing efficient knockdown of Mcl-1 by shRNA-transfected stable cell lines from A549, H1299 and H460 cells. Bcl-xL and VDAC levels are unaffected. (b) Summary of proliferation in control Mcl-1 WT and Mcl-1 knockdown lung cancer cells (mean±S.E.; P>0.05; student's t-test)
Mentions: In addition to Mcl-1, both Bcl-2 and Bcl-xL are known to interact with VDAC, raising the possibility of some functional redundancy among the Bcl-2 proteins. Given the difference in apparent binding affinity (Figure 1), however, we hypothesized that in cancer cells overexpressing Bcl-2 proteins, especially those addicted to Mcl-1, Mcl-1/VDAC interactions would dominate. To help define the phenotypes specifically dependent on a Mcl-1/VDAC interaction, we selected three different NSCLC lines (A549, H1299 and H460) previously demonstrated to be similar, with respect to having high Mcl-1 expression,26,27 but different in their relative levels of Bcl-xL (Figure 4a). Mitochondrially generated ROS have been defined as a physiological signal that promotes cancer cell migration and proliferation.12,31, 32, 33 We employed a scratch wound-healing assay to examine the dependence of Mcl-1 expression in NSCLC cells on these processes. The NSCLC cell lines A549, H1299 and H460 were treated with either control or Mcl-1 siRNAs and the knockdown effectiveness was confirmed by western blot (Figure 4b; see Figure 2c for A549 western blot). The wound area was measured at 0 and 24 h, as depicted for A549 (Figure 4c). In all three cell lines the loss of Mcl-1 expression significantly slowed the rate of wound closure (Figure 4d). This assay, however, does not effectively distinguish between differences in proliferation and migration; therefore, the effect of Mcl-1 knockdown on proliferation was assessed in the three NSCLC cell lines using a microplate assay based on measurement of cellular DNA content. The need to track proliferation across several days in control and Mcl-1 knockdown cells necessitated the generation of stable Mcl-1 knockdowns using shRNAs and antibiotic selection. Western blots confirmed that Mcl-1 was successfully knocked down in A549, H1299 and H460 cell lines without affecting either VDAC1/3 or Bcl-xL expression levels (Figure 5a). Mcl-1 knockdown had no significant effect on cell proliferation (Figure 5b), suggesting that Mcl-1 promotes migration but not proliferation in NSCLC cell lines.

Bottom Line: In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca(2+) uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation.Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration.These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca(2+)-dependent ROS production.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology & Biophysics, Rosalind Franklin University of Medicine & Science, North Chicago, IL, USA.

ABSTRACT
Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca(2+) uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca(2+)-dependent ROS production.

Show MeSH
Related in: MedlinePlus