Chloroquine alleviates etoposide-induced centrosome amplification by inhibiting CDK2 in adrenocortical tumor cells

Abstract

The antitumor drug etoposide (ETO) is widely used in treating several cancers, including adrenocortical tumor (ACT). However, when used at sublethal doses, tumor cells still survive and are more susceptible to the recurring tumor due to centrosome amplification. Here, we checked the effect of sublethal dose of ETO in ACT cells. Sublethal dose of ETO treatment did not induce cell death but arrested the ACT cells in G2/M phase. This resulted in centrosome amplification and aberrant mitotic spindle formation leading to genomic instability and cellular senescence. Under such conditions, Chk2, cyclin A/CDK2 and ERK1/2 were aberrantly activated. Pharmacological inactivation of Chk2, CDK2 or ERK1/2 or depletion of CDK2 or Chk2 inhibited the centrosome amplification in ETO-treated ACT cells. In addition, autophagy was activated by ETO and was required for ACT cell survival. Chloroquine, the autophagy inhibitor, reduced ACT cell growth and inhibited ETO-induced centrosome amplification. Chloroquine alleviated CDK2 and ERK, but not Chk2, activation and thus inhibited centrosome amplification in either ETO- or hydroxyurea-treated ACT cells. In addition, chloroquine also inhibited centrosome amplification in osteosarcoma U2OS cell lines when treated with ETO or hydroxyurea. In summary, we have demonstrated that chloroquine inhibited ACT cell growth and alleviated DNA damage-induced centrosome amplification by inhibiting CDK2 and ERK activity, thus preventing genomic instability and recurrence of ACT.

Figure 1

ETO inhibits ACT cell growth. (a–d) Treatment of ETO (10 μM) for 24 h induces DNA damage response. ETO-treated H295 cells were co-stained with DNA dye (DAPI) with antibodies against (a) phospho-Ser139 of H2AX (γ-H2AX) or (b) phospho-Ser15 of p53 (p-p53). CTL: control (DMSO) treatment. Extracts of ETO-treated H295 cells were analyzed with antibodies against (c) phospho-Ser139 of H2AX (γ-H2AX) and GAPDH, or (d) p53, phospho-Ser15 of p53 (p-p53) and Ku70. (e and f) ETO inhibits ACT cell growth. The numbers of ACT H295 (e) and Y1 (f) cells were quantified in scramble control (CTL) or ETO treatment at different concentrations for different time points. n.s., no significance, **P<0.01, ***P<0.001.

Figure 2

Sublethal dose of ETO treatment disturbs cell cycle profile. (a) ETO treatment (10 μM, 72 h) disturbs the cell cycle profile. Quantification of different cell cycle stages in adrenocortical Y1 tumor cells in the presence or absence of ETO. (b and c) EdU incorporation and mitotic index are reduced in ETO-treated (10 μM, 24 h) adrenocortical H295 and Y1 tumor cell lines. Quantification of EdU incorporation (b) or mitotic index (c) in scramble control (CTL) or ETO-treated H295 and Y1 cell lines. These results are mean +/−s.d. from three independent experiments; more than 1000 cells were counted in each individual group. **P<0.01, ***P<0.001.

Figure 3

Long-term sublethal dose of ETO treatment induces genomic instability and cellular senescence. (a and b) Enlarged nuclei are observed in ETO-treated (10 μM, 72 h) adrenocortical Y1 tumor cells. (a) Examination of nuclear size by DAPI staining. The scale bar is 5 μm. (b) Quantitation of nuclear areas. The areas of nuclei from at least 100 Y1 cells in (a) were counted and compared in three independent experiments. (c and d) Cellular senescence are observed in ETO-treated adrenocortical Y1 tumor cells. (c) Cellular senescence is shown by beta-galatosidase activity after ETO treatment for 72 h. (d) Quantitation of cells with beta-galatosidase activity. These results are mean +/−s.d. from three independent experiments; more than 100 cells were counted in each individual group. Scale bar is 5 μM. **P<0.01, ***P<0.001.

Figure 4

ETO treatment induces centrosome amplification in ACT cells. (a, b) ETO treatment (10 μM, 24 h) induces aberrant mitotic apparatus. (a) Immunofluorescence examination of mitotic cells by staining with DNA (DAPI, blue) and mitotic spindle poles (γ-tubulin, red) in Y1 cells. (b) Quantitation of mitotic Y1 cells with aberrant mitotic spindle poles. (c, d) ETO treatment induces centrosome amplification. (c) Immunostaining of centrosome with antibody against γ-tubulin. DNA is stained by DAPI. Scale bar is 5 μm. (d) Quantitation of Y1 cells with multiple centrosomes (>2 centrosomes) during interphase. (e) Upregulation of cyclin A and activation of CDK2 upon ETO treatment (10 μM, 24 h) in adrenocortical Y1 tumor cells. Extracts of ETO-treated Y1 cell lysates are analyzed by immunoblotting with antibodies against cyclin A, CDK2, phospho-CDK2 on Thr160, and actin. (f) Inactivation of CDK2 inhibits ETO-induced centrosome amplification in Y1 cells. Quantitation of Y1 cells containing multiple centrosomes in the presence or absence of ETO with or without CDK2 inhibitor, roscovitine. All results are expressed as the mean+/−s.d. from at least three independent experiments. **P<0.01, ***P<0.001.

Figure 5

Inhibition of ERK1/2 inhibits ETO-induced centrosome amplification. (a) ERK1/2 is activated upon ETO treatment (10 μM, 24 h) in adrenocortical Y1 tumor cells. Extracts of ETO-treated Y1 cell lysates are analyzed by immunoblotting with antibodies against phospho-ERK1/2 on Thr202/Tyr204, ERK1/2 and GAPDH. (b) Inactivation of ERK inhibits ETO-induced centrosome amplification. Quantitation of Y1 cells containing multiple centrosomes in the presence or absence of ETO with ERK inhibitor, U0126. All results are expressed as the mean+/−s.d. from at least three independent experiments. **P<0.01, ***P<0.001.

Figure 6

Chloroquine inhibits DNA damage-induced centrosome amplification. (a, b) ETO treatment (10 μM, 24 h) induces autophagy in adrenocortical Y1 tumor cells. (a) Immunostaining of autophagosomes with antibody against LC3 in the presence or absence of ETO. DNA is stained with DAPI. (b) Extracts of ETO-treated Y1 cell lysates are analyzed by immunoblotting with antibodies against LC3, γ-H2AX and GAPDH. (c, d) Chloroquine inhibits ETO-induced centrosome amplification (c) and senescence (d) in adrenocortical Y1 tumor cells. Quantitation of Y1 cells containing multiple centrosomes (c) or senescence (d) in the presence or absence of ETO with or without chloroquine. (e) Chloroquine inhibits ETO-induced centrosome amplification in osteosarcoma U2OS cells. Quantitation of U2OS cells containing multiple centrosomes in the presence or absence of ETO with or without chloroquine. All results are expressed as the mean+/−s.d. from at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Figure 7

Chloroquine alleviates hydroxyurea-induced centrosome amplification. (a, b) Chloroquine inhibits hydorxyurea-induced (2 mM, 72 h) centrosome amplification in adrenocortical Y1 tumor (a) and osteosarcoma U2OS (b) cell lines. Quantitation of cells containing multiple centrosomes in the presence or absence of hydroxyurea with or without chloroquine. All results are expressed as the mean+/−s.d. from at least three independent experiments. n.s., no significance, *P<0.05, **P<0.01, ***P<0.001.

Figure 8

Chloroquine inhibits ETO-activated CDK2 and ERK1/2 in adrenocortical Y1 tumor cells. (a, b) Extracts of ETO-treated adrenocortical Y1 tumor cell lysates in the absence or presence of chloroquine (CQ) are analyzed by immunoblotting with antibodies against (a) cyclin A, actin, phospho-CDK2 on Thr160, GAPDH or (b) phospho-ERK on Thr202/Tyr204, ERK and actin.