IC 10 ( µµµµ g/ml)Cell type
B. Bcl-xL blocks apoptosis but not mitotic cell death induced by doxorubicin
1. Bcl-xL overexpression blocks doxorubicin-induced apoptosis in Huh-7 hepatoma cells
While apoptosis was induced in Huh-7 cells treated with 10 µg/ml of doxorubicin,
doxorubicin-induced apoptosis was blocked in Bcl-xL-overexpressing cells as described in part A (Fig 2).
2. Bcl-xL delays LD doxorubicin-induced mitotic cell death
Bcl-xL is well-known to be protective against apoptosis induced by various stimuli (Kirkin V et al., 2004). But, it has not been clarified whether Bcl-xL affects senescence or mitotic cell death induced by anticancer drugs of low doses. Our previous study demonstrated that LD doxorubicin induces mitotic cell death in human hepatoma cells. Therefore, we investigated whether Bcl-xL overexpression affects LD doxorubicin-induced mitotic cell death. First, we examined whether LD doxorubicin-induced changes in cellular morphologies are modulated by Bcl-xL overexpression. Huh-7 cells or Bcl-xL-overexpressing Huh-7 cells were treated with 50 ng/ml doxorubicin for the indicated time points and cells were stained with DAPI and Texas-Red-conjugated phalloidin to detect the nuclei and actin expression (Fig.
10). Cells were enlarged and multinucleation was significantly induced after treatment with LD doxorubicin in both Huh-7 and Bcl-xL-overexpressing cells.
Since SLP was observed in Huh-7 cells during the progression of LD doxorubicin-induced mitotic cell death, we investigated whether Bcl-xL overexpression could inhibit SLP. Huh-7 or Bcl-xL overexpressing Huh-7 cells were treated with 50 ng/ml doxorubicin for the indicated time periods and assay for SA-β-galactosidase activity, a biomarker for cellular senescence, were performed (Fig. 11). SA-β-gal activities in both cell lines were gradually increased following LD doxorubicin treatment in incubation time-dependent manner, although the increase in SA-β-gal activity was
slightly delayed in Bcl-xL-overexpressing cells. These results suggest that Bcl-xL overexpression does not block the induction of SLP, a preceding event of LD doxorubicin-induced mitotic cell death. Next, we investigated whether Bcl-xL overexpression could inhibit LD doxorubicin-induced mitotic cell death. Cellular viability was slowly decreased in Huh-7 cells treated with 50 ng/ml doxorubicin, resulting in 50% at 9 days (Fig. 12). In Bcl-xL-overexpressing cells treated with 50 ng/ml doxorubicin, the viability was also decreased in time-dependent manner, although reduction of cellular viability in these cells was slightly delayed. Therefore, Bcl-xL overexpression cannot block LD doxorubicin-induced mitotic cell death.
3. Bcl-xL does not inhibit LD doxorubicin-induced aberrant cell cycle progression and downregulation of cell cycle regulators.
Next, we examined the cell cycle progression of Huh-7 or Bcl-xL-overexpressing Huh-7 cells treated with LD doxorubicin, monitoring the changes in DNA contents by FACS analysis (Fig. 13A). In Huh-7 cells treated with LD doxorubicin, the G0/G1
cell populations were decreased at 1 day and then cells were transiently arrested at G2/M phase at 3 day. From this time, hyperploid cells (>4n DNA cells) appeared and gradually increased thereafter. While cellular viability was decreased by 50% at 9 days of doxorubicin treatment, the percentage of sub-G1 (apoptotic) population was not significantly increased, suggesting that the major mode of cell death by LD doxorubicin was not apoptosis. This aberrant cell cycle progression was also observed in Bcl-xL overexpressing cells treated with LD doxorubicin. We further examined the protein levels of several regulators of mitosis, Cdc2, Cdc25c, Cyclin A, Cyclin B, MAD2 and CENP-A in Huh-7 or Bcl-xL overexpressing Huh-7 cells treated with LD doxorubicin (Fig. 13B). All tested cell cycle regulators were downregulated in both cell lines treated with LD doxorubicin in time-dependent manner, suggesting that Bcl-xL overexpression does not inhibit LD doxorubicin-induced aberrant cell cycle progression.
4. Missegregation of chromosomes and loss in the integrity of nuclear membranes are observed in both Huh-7 and Bcl-xL overexpressing Huh-7 cells treated with LD doxorubicin
Next, we examined the changes of nuclear morphologies and microtubule structure during LD doxorubicin-induced mitotic cell death by immunocytochemistry using DAPI and anti-α-tubulin antibody (Fig. 14). In a few untreated Huh-7 or Bcl-xL-overexpressing Huh-7 cells, possibly cells undergoing mitosis, bipolar spindle structure and equally segregated chromosomes into opposite poles were observed.
However, cells with tri- or quadric-polar spindle structures were frequently observed both in Huh-7 and Bcl-xL-overexpressing cells after treatment with LD doxorubicin.
These aberrant spindle structures may contribute to abnormal mitosis via missegregation of chromosomes and subsequent formation of anueploid cells. Next, we analyzed the changes in the integrity of nuclear membrane by LD doxorubicin treatment. Huh-7 or Bcl-xL overexpressing Huh-7 cells were treated LD doxorubicin for the indicated time periods were stained with the specific antibody for Lamin B, a component of nuclear envelope (Fig.15A). Untreated Huh-7 or Bcl-xL-overexpressing Huh-7 cells had intact nuclear membrane structure. However, disrupted nuclear membrane boundaries were frequently observed both types of cells treated with LD doxorubicin. Lamin B protein levels were also downregulated in both cells (Fig. 15B). These results suggest that Bcl-xL overexpression does not block aberrant formation of spindle structures and loss of nuclear membrane integrity induced by LD doxorubicin.
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Fig. 10. Bcl-xL did not block LD doxorubicin-induced multinucleation. Huh-7 and Bcl-xL-overexpressing Huh-7 cells were treated with LD doxorubicin for the indicated time periods. Immunocytochemistry using DAPI and Texas-Red-conjugated phalloidin was performed to detect the changes in the nuclei and actin structure. Cells were visualized by confocal laser microscopy (magnification 1200X).