Phosphorylation of apoptosis repressor with caspase recruitment domain by protein kinase CK2 contributes to chemotherapy resistance by inhibiting doxorubicin induced apoptosis

Abstract

The development of cancer resistance to chemotherapy is the major obstacle to cancer therapy. Here, we identified that the phosphorylation of apoptosis repressor with caspase recruitment domain (ARC) at threonine 149 was essential to inhibit doxorubicin (DOX) induced apoptosis and mitochondrial fission in cancer cells. Our further study showed that casein kinase II (CK2) inhibitors could decrease the phosphorylation levels of ARC and make cancer cells sensitive to undergoing apoptosis. Furthermore, CK2α and CK2α', catalytic subunits of CK2, were observed to translocate into nuclear in cancer cells with the treatment of DOX. Finally, the synergistically therapeutic effect by combining DOX and CK2 inhibitor was confirmed in tumor xenograft model. Taken together, our results revealed that CK2-mediated phosphorylation of ARC contributed to chemotherapy resistance by inhibiting DOX induced apoptosis and combining DOX with CK2 inhibitor could induce apoptosis of cancer cells synergistically by down-regulating the phosphorylation of ARC. Therefore, development of new therapeutic strategies based on ARC and CK2, is promising for overcoming cancer resistance to chemotherapy.

Keywords: ARC; CK2; apoptosis; chemotherapy resistance; doxorubicin.

Figure 1. ARC is phosphorylated and localizes to mitochondria in cancer cells

A. Analysis of total ARC and phosphorylated ARC (p-ARC) expression levels in cancer cells including HeLa and SGC-7901 cells. HEK-293 cells served as a negative control. ARC and p-ARC were detected by immunoblot. A representative result of three independent experiments is shown. B. Distribution of ARC was detected in HeLa cells by immunofluorescence. HeLa cells labeled with MitoTracker (red), stained with anti-ARC antibody, and monitored by FITC-labeled secondary antibody (green), and DAPI (blue). C. Distribution of ARC was detected by immunofluorescence in SGC-7901 cells which were analyzed as described for B. D. HeLa and SGC-7901 cells were harvested for the detection of ARC and p-ARC in the cytosol (top) and mitochondria-enriched HM (bottom). A representative blot of three independent experiments is shown. E. Immunoblot of ARC and p-ARC proteins in clinical samples with one normal gastric tissue (N) and four gastric tumor tissues (T).

Figure 2. ARC requires to be phosphorylated to inhibit DOX induced apoptosis and mitochondria fission

A–B. ARC and p-ARC levels were detected in HeLa (A) and SGC-7901 (B) cells treated with DOX (2 μM) at the indicated time. C–D. wtARC inhibited DOX-induced cell death. HeLa (C) and SGC-7901 (D) cells were infected with adenovirus ARC, ARC T149A mutant adenovirus (ARC T149A) or adenovirus β-gal. 24 hours after infection they were treated with DOX (2 μM) for 36 hours. Cell death was assessed by trypan blue exclusion. *p < 0.05 vs DOX alone. E–F. wtARC but not ARCT149A prevented DOX-induced mitochondrial fission. HeLa (E) or SGC-7901 (F) was infected with adenovirus ARC or ARC T149A. 24 hours after infection they were treated with DOX (2 μM). 12 hours after treatment, mitochondrial fission was detected. *p < 0.05 vs DOX alone. G–H. Caspase-3 activities were detection in HeLa (G) or SGC-7901 (H) cells which were treated as in C and D. *p < 0.05 vs DOX alone. I. Analysis of cytochrome C (Cyt-C) release in HeLa cells treated as described in C. Data are expressed as the mean ± SD of 3 independent experiments.

Figure 3. Inhibition endogenous ARC phosphorylation by CK2 inhibitor sensitizes cells to undergoing apoptosis

A–B. CK2 inhibitor DRB was able to inhibit endogenous ARC phosphorylation. HeLa (A) and SGC-7901 (B) were treated with DRB as indicated concentration for 36 hours, total ARC and p-ARC were detected by immunoblotting. C–D. CK2 inhibitor TBB was able to inhibit endogenous ARC phosphorylation in HeLa (C) and SGC-7901 (D). E–F. CK2 inhibitor prevents ARC accumulations in mitochondria. HeLa cells (E) and SGC-7901 cells (F) were treated with DRB or TBB for 36 hours. Cells were harvested for the immunoblot analysis of ARC and p-ARC in the cytosol and mitochondria-enriched HM. G–H. DRB sensitizes DOX to induce cell death. HeLa (G) and SGC-7901 (H) were administrated with indicated concentration of DRB for 36 hours and then treated with DOX (0.2 μM). Cell death was analyzed 36 hours after treatment. *p < 0.05 vs DOX alone. I–J. caspase-3 activity was boosted by combination of CK2 inhibitor and DOX at low dose of 0.2 μmol/L in HeLa (I) or SGC-7901cells (J) *p < 0.05 vs DOX alone. Data are expressed as the mean ± SD of 3 independent experiments.

Figure 4. CK2 regulates apoptosis through targeting ARC

A–D. ARC-attenuated DOX induced cells death is abolished by CK2 inhibitor. HeLa (A and C) or SGC-7901 (B and D) cells were infected with adenovirus ARC, and treated with DRB (A and B) or TBB (C and D) for 36 hours. Cells were exposed to DOX (2 μM). Cell death was analyzed by trypan blue exclusion. *p < 0.05 vs DOX +ARC. E. The binding of ARC to endogenous CK2α is reduced in Hela cells exposed to DOX. HeLa cells were treated with DOX (2 μM) for 6 hours and the association between ARC and CK2α was analyzed by immunoprecipitation (IP) followed by immunoblot (IB). Data are expressed as the mean ± SD of 3 independent experiments.

Figure 5. CK2α translates to nuclear in cancer cells exposed to DOX

A. Analysis of CK2 subunits protein levels in HeLa cells treated with DOX (2 μM) as the indicated time. CK2α, CK2α' and CK2β were detected by immunoblot. B. DOX induces CK2α and CK2α' accumulated in nuclear from cytosol. HeLa cells were treated with 2 μM DOX and harvested at the indicated time after treatment for immunoblot analysis of CK2 subunits in the cytosol and nuclear. C–D. Distributions of CK2α and CK2α' were detected by immunofluorescence. HeLa cells were labeled with DAPI (blue), stained with anti-CK2α (C) or anti-CK2α' (D) antibody and then monitored by FITC-labeled secondary antibody (green) and DAPI (blue). Bar = 20 μm. E. The distribution of ARC in mitochondrial is reduced in HeLa cells exposed to DOX. HeLa cells were treated with DOX (2 μM) for 6 hours and harvested for the immunoblot analysis of ARC in the cytosol and mitochondria-enriched HM. A representative result of three independent experiments is shown.

Figure 6. DOX combines with CK2 inhibitor enhance chemotherapeutic effect in vivo

A. A total of 1 × 10⁷ HeLa cells were injected subcutaneously into BALB/c nude mice. When tumors reached 250–300 mm³, therapeutic treatment as indicated was given every other day. Tumor volumes were monitored during 2-week therapy (n = 6 each group) (top). At the end of this experiment, tumors were dissected and photographed. Representative images of tumors were shown (bottom), scale bar = 1 cm. B. Body weight of the tumor bearing mice described in A was measured during two-week therapeutic treatment (n = 6 each group). Error bars represent SD. C. Immunoblot analysis as indicated in the xenograft tumors described in A. A representative result of three independent experiments is shown. D. Apoptosis detected in sections of xenograft tumors described in A by TUNEL assay, n = 6 each group. *P < 0.05 compared with DOX (1 mg/kg) alone. Error bars represent S.D.