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NAC1 modulates sensitivity of ovarian cancer cells to cisplatin by altering the HMGB1-mediated autophagic response.

Zhang Y, Cheng Y, Ren X, Zhang L, Yap KL, Wu H, Patel R, Liu D, Qin ZH, Shih IM, Yang JM - Oncogene (2011)

Bottom Line: Treatment with 3-methyladenosine, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin.Regulation of autophagy by NAC1 was mediated by the high-mobility group box 1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1.The results of our study not only revealed a new mechanism determining cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Penn State Hershey Cancer Institute, Pennsylvania State University College of Medicine and Milton S. Hershey Medical Center, Hershey, PA, USA.

ABSTRACT
Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to have important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We report here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of autophagy in ovarian cancer cell lines, A2780, OVCAR3 and SKOV3. We further demonstrated that knockdown of NAC1 by RNA interference or inactivation of NAC1 by inducing the expression of a NAC1 deletion mutant that contains only the BTB/POZ domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity. Moreover, inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was attributed to suppression of autophagy, we assessed the effects of the autophagy inhibitors 3-methyladenosine and chloroquine, and small interfering RNAs (siRNAs) targeting beclin 1 or Atg5 on the cytotoxicity of cisplatin. Treatment with 3-methyladenosine, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Regulation of autophagy by NAC1 was mediated by the high-mobility group box 1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1. The results of our study not only revealed a new mechanism determining cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1-mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.

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Cisplatin induces autophagy in ovarian cancer cells(A) A2780 and OVCAR3 cells were treated with the indicated concentrations of cisplatin for 24 h in the absence or presence of 10 nM of bafilomycin A1. At the end of treatment, cell lysates were prepared, resolved by SDS-PAGE, and subjected to Western blot analysis using anti-LC3, anti-p62 or anti-tubulin antibodies, respectively. Tubulin was used as a loading control. (B) A2780 and OVCAR3 cells were transfected with a GFP-LC3 plasmid, followed by treatment with the indicated concentrations of cisplatin for 24 h. At the end of treatment, the cells were inspected under a fluorescence microscope. Quantitation of the GFP-LC3 puncta was performed by counting 20 cells for each sample, and average numbers of puncta per cell were shown. The bars are the mean ± S.D. of triplicate determinations; results shown are the representative of three identical experiments. ** p < 0.01, t-test, cisplatin vs. vehicle.
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Figure 1: Cisplatin induces autophagy in ovarian cancer cells(A) A2780 and OVCAR3 cells were treated with the indicated concentrations of cisplatin for 24 h in the absence or presence of 10 nM of bafilomycin A1. At the end of treatment, cell lysates were prepared, resolved by SDS-PAGE, and subjected to Western blot analysis using anti-LC3, anti-p62 or anti-tubulin antibodies, respectively. Tubulin was used as a loading control. (B) A2780 and OVCAR3 cells were transfected with a GFP-LC3 plasmid, followed by treatment with the indicated concentrations of cisplatin for 24 h. At the end of treatment, the cells were inspected under a fluorescence microscope. Quantitation of the GFP-LC3 puncta was performed by counting 20 cells for each sample, and average numbers of puncta per cell were shown. The bars are the mean ± S.D. of triplicate determinations; results shown are the representative of three identical experiments. ** p < 0.01, t-test, cisplatin vs. vehicle.

Mentions: To determine the effect of cisplatin on autophagy and the role of autophagy in determining the sensitivity of cancer cells to this drug, we first examined the activity of autophagy in ovarian cancer cells treated with cisplatin. As shown in Fig. 1A, treatment of the human ovarian cancer cell lines A2780 and OVCAR3 with cisplatin caused a dose-dependent activation of autophagy, as evidenced by the increases in the amount of LC3 II and decreases in the amount of p62, two selective markers of autophagy. Fig. 1A also shows that LC3 II levels were further elevated in the presence of bafilomycin A1, an inhibitor of autophagosomelysosome fusion and LC3 II degradation, indicating an increase of autophagic flux in the cisplatin-treated cancer cells. The stimulative effect of cisplatin on autophagy was verified by a GFP-LC3 puncta formation assay, which demonstrated an increase in the number of GFP-LC3 puncta in the tumor cells treated with cisplatin (Fig. 1B). Next, we determined whether the cisplatin-stimulated autophagy played a protective or sensitizing role in tumor cells treated with this drug. We found that in comparison to treatment with cisplatin alone, co-treatment of ovarian cancer cells with cisplatin and inhibitors of autophagy, 3-MA or chloroquine, enhanced the cytotoxicity (Fig. 2A) and apoptosis (Fig. 2B and Fig. 2C) induced by cisplatin. To further prove the role of autophagy in determining sensitivity of tumor cells to cisplatin, we suppressed autophagy by silencing the expression of autophagy-related genes beclin 1 or Atg5, and then measured the effects of inhibiting autophagic activity on clonogenicity of the tumor cells. Fig. 2D shows that knockdown of beclin 1 or Atg5 in A2780 and OVCAR3 cells significantly reinforced the colony-inhibitory effect of cisplatin. These results suggest that cisplatin induces a canonical autophagy, and induction of autophagy plays a protective role in tumor cells subjected to the cytotoxicity of cisplatin.


NAC1 modulates sensitivity of ovarian cancer cells to cisplatin by altering the HMGB1-mediated autophagic response.

Zhang Y, Cheng Y, Ren X, Zhang L, Yap KL, Wu H, Patel R, Liu D, Qin ZH, Shih IM, Yang JM - Oncogene (2011)

Cisplatin induces autophagy in ovarian cancer cells(A) A2780 and OVCAR3 cells were treated with the indicated concentrations of cisplatin for 24 h in the absence or presence of 10 nM of bafilomycin A1. At the end of treatment, cell lysates were prepared, resolved by SDS-PAGE, and subjected to Western blot analysis using anti-LC3, anti-p62 or anti-tubulin antibodies, respectively. Tubulin was used as a loading control. (B) A2780 and OVCAR3 cells were transfected with a GFP-LC3 plasmid, followed by treatment with the indicated concentrations of cisplatin for 24 h. At the end of treatment, the cells were inspected under a fluorescence microscope. Quantitation of the GFP-LC3 puncta was performed by counting 20 cells for each sample, and average numbers of puncta per cell were shown. The bars are the mean ± S.D. of triplicate determinations; results shown are the representative of three identical experiments. ** p < 0.01, t-test, cisplatin vs. vehicle.
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Related In: Results  -  Collection

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Figure 1: Cisplatin induces autophagy in ovarian cancer cells(A) A2780 and OVCAR3 cells were treated with the indicated concentrations of cisplatin for 24 h in the absence or presence of 10 nM of bafilomycin A1. At the end of treatment, cell lysates were prepared, resolved by SDS-PAGE, and subjected to Western blot analysis using anti-LC3, anti-p62 or anti-tubulin antibodies, respectively. Tubulin was used as a loading control. (B) A2780 and OVCAR3 cells were transfected with a GFP-LC3 plasmid, followed by treatment with the indicated concentrations of cisplatin for 24 h. At the end of treatment, the cells were inspected under a fluorescence microscope. Quantitation of the GFP-LC3 puncta was performed by counting 20 cells for each sample, and average numbers of puncta per cell were shown. The bars are the mean ± S.D. of triplicate determinations; results shown are the representative of three identical experiments. ** p < 0.01, t-test, cisplatin vs. vehicle.
Mentions: To determine the effect of cisplatin on autophagy and the role of autophagy in determining the sensitivity of cancer cells to this drug, we first examined the activity of autophagy in ovarian cancer cells treated with cisplatin. As shown in Fig. 1A, treatment of the human ovarian cancer cell lines A2780 and OVCAR3 with cisplatin caused a dose-dependent activation of autophagy, as evidenced by the increases in the amount of LC3 II and decreases in the amount of p62, two selective markers of autophagy. Fig. 1A also shows that LC3 II levels were further elevated in the presence of bafilomycin A1, an inhibitor of autophagosomelysosome fusion and LC3 II degradation, indicating an increase of autophagic flux in the cisplatin-treated cancer cells. The stimulative effect of cisplatin on autophagy was verified by a GFP-LC3 puncta formation assay, which demonstrated an increase in the number of GFP-LC3 puncta in the tumor cells treated with cisplatin (Fig. 1B). Next, we determined whether the cisplatin-stimulated autophagy played a protective or sensitizing role in tumor cells treated with this drug. We found that in comparison to treatment with cisplatin alone, co-treatment of ovarian cancer cells with cisplatin and inhibitors of autophagy, 3-MA or chloroquine, enhanced the cytotoxicity (Fig. 2A) and apoptosis (Fig. 2B and Fig. 2C) induced by cisplatin. To further prove the role of autophagy in determining sensitivity of tumor cells to cisplatin, we suppressed autophagy by silencing the expression of autophagy-related genes beclin 1 or Atg5, and then measured the effects of inhibiting autophagic activity on clonogenicity of the tumor cells. Fig. 2D shows that knockdown of beclin 1 or Atg5 in A2780 and OVCAR3 cells significantly reinforced the colony-inhibitory effect of cisplatin. These results suggest that cisplatin induces a canonical autophagy, and induction of autophagy plays a protective role in tumor cells subjected to the cytotoxicity of cisplatin.

Bottom Line: Treatment with 3-methyladenosine, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin.Regulation of autophagy by NAC1 was mediated by the high-mobility group box 1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1.The results of our study not only revealed a new mechanism determining cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Penn State Hershey Cancer Institute, Pennsylvania State University College of Medicine and Milton S. Hershey Medical Center, Hershey, PA, USA.

ABSTRACT
Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to have important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We report here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of autophagy in ovarian cancer cell lines, A2780, OVCAR3 and SKOV3. We further demonstrated that knockdown of NAC1 by RNA interference or inactivation of NAC1 by inducing the expression of a NAC1 deletion mutant that contains only the BTB/POZ domain significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity. Moreover, inhibition of autophagy and sensitization to cisplatin by NAC1 knockdown or inactivation were accompanied by induction of apoptosis. To confirm that the sensitizing effect of NAC1 inhibition on the cytotoxicity of cisplatin was attributed to suppression of autophagy, we assessed the effects of the autophagy inhibitors 3-methyladenosine and chloroquine, and small interfering RNAs (siRNAs) targeting beclin 1 or Atg5 on the cytotoxicity of cisplatin. Treatment with 3-methyladenosine, chloroquine or beclin 1 and Atg5-targeted siRNA also enhanced the sensitivity of SKOV3, A2780 and OVCAR3 cells to cisplatin, indicating that suppression of autophagy indeed renders tumor cells more sensitive to cisplatin. Regulation of autophagy by NAC1 was mediated by the high-mobility group box 1 (HMGB1), as the functional status of NAC1 was associated with the expression, translocation and release of HMGB1. The results of our study not only revealed a new mechanism determining cisplatin sensitivity but also identified NAC1 as a novel regulator of autophagy. Thus, the NAC1-mediated autophagy may be exploited as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.

Show MeSH
Related in: MedlinePlus