Nitroxoline induces cell apoptosis by inducing MDM2 degradation in small-cell lung cancer

Abstract

The proto-oncogene MDM2 is a nuclear-localized E3 ubiquitin ligase, which promotes tumor formation by targeting tumor suppressor proteins, such as p53, for proteasomal degradation. In this study, the anti-infective drug nitroxoline (NXQ) was screened out to effectively inhibit cell survival of small-cell lung cancer (SCLC) cells, and induce SCLC cell apoptosis by suppressing antiapoptotic proteins (such as Bcl-2 and MCL1) and upregulating proapoptotic protein Bim. In the mechanistic study, NXQ was found to downregulate MDM2 expression by inducing its proteasomal degradation, and thus upregulated p53 expression, which was a substrate protein of MDM2. Moreover, overexpression of MDM2 decreased the cytotoxicity of NXQ on SCLC cells. These results demonstrated that NXQ displayed anti-SCLC activity by suppressing MDM2 expression, which suggested that anti-infective NXQ had potential for SCLC treatment by targeting the MDM2/p53 axis.

Keywords: MDM2; cell apoptosis; nitroxoline; p53; small-cell lung cancer.

Figure 1

Nitroxoline (NXQ) was screened out to inhibit cell survival in small‐cell lung cancer (SCLC) cells. A, H446 cells were treated with 96 drugs or dimethylsulfoxide (DMSO) for 24 hours, followed by Cell Counting Kit‐8 (CCK‐8) staining. B, The chemical structure of NXQ. C, SCLC cells were treated with indicated concentrations of NXQ for 24 hours, and cell viability was measured by CCK‐8 staining

Figure 2

Nitroxoline (NXQ) induces cell apoptosis in small‐cell lung cancer (SCLC) cells. A, H446 cells were treated with increasing concentrations of NXQ for indicated time, followed by Cell Counting Kit‐8 (CCK‐8) staining. B, H1882 cells were treated with increasing concentrations of NXQ for indicated time, followed by CCK‐8 staining. C, H446, H1882, and H1417 cells were treated with increasing concentrations of NXQ for 24 hours, followed by immunoblotting against PARP. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. D, H446, H1882, and H1417 cells were treated with increasing concentrations of NXQ for 24 hours, followed by immunoblotting against Bcl‐2, MCL1, Bim, and GAPDH

Figure 3

Nitroxoline (NXQ) downregulates MDM2 expression in small‐cell lung cancer (SCLC) cells. A, H446, H1688, and H1882 cells were treated with 0 or 20 μM NXQ for 24 hours, followed by immunoblotting against MDM2 and p53. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. B, H446 and H1882 cells were treated with increasing concentrations of NXQ for 24 hours, and then cells were prepared for immunoblotting against MDM2, p53, and GAPDH. C, H446 and H1882 cells were treated with 10 μM NXQ for indicated time, and then cells were prepared for immunoblotting against MDM2 and p53. GAPDH was used as a loading control

Figure 4

Overexpression of MDM2 decreases the cytotoxicity of nitroxoline (NXQ) on small‐cell lung cancer (SCLC) cells. A, H446 cells transfected with empty vector (EV) or Myc‐MDM2 were treated with increasing concentrations of NXQ for 24 hours, followed by Cell Counting Kit‐8 (CCK‐8) staining. B, H446 cells transfected with EV or Myc‐MDM2 were treated with increasing concentrations of NXQ. Twenty‐four hours later, cells were lysed and prepared for immunoblotting against p53, Myc, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). C, H1882 cells transfected with EV or Myc‐MDM2 were treated with increasing concentrations of NXQ for 24 hours, followed by CCK‐8 staining. D, H1882 cells transfected with EV or Myc‐MDM2 were treated with increasing concentrations of NXQ. Twenty‐four hours later, cells were lysed and prepared for immunoblotting against p53 and Myc. GAPDH was used as a loading control

Figure 5

Nitroxoline (NXQ) induces MDM2 degradation via the proteasomal pathway. A, H446 cells were treated with NXQ at indicated concentrations for 24 hours, followed by quantitative real‐time polymerase chain reaction (qRT‐PCR) against MDM2 and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). B, H1882 cells were treated with NXQ at indicated concentrations for 24 hours, followed by qRT‐PCR against MDM2 and GAPDH. C, H446 cells were treated with NXQ alone or with MG132 or chloroquine (CHQ) for 12 hours, followed by immunoblotting against MDM2. GAPDH was used as an internal control. D, Statistical analysis for MDM2/GAPDH of (C). Abbreviation: n.s., nonsense