Development of a novel Hsp90 inhibitor NCT-50 as a potential anticancer agent for the treatment of non-small cell lung cancer

Abstract

Despite the development of advanced therapeutic regimens such as molecular targeted therapy and immunotherapy, the 5-year survival of patients with lung cancer is still less than 20%, suggesting the need to develop additional treatment strategies. The molecular chaperone heat shock protein 90 (Hsp90) plays important roles in the maturation of oncogenic proteins and thus has been considered as an anticancer therapeutic target. Here we show the efficacy and biological mechanism of a Hsp90 inhibitor NCT-50, a novobiocin-deguelin analog hybridizing the pharmacophores of these known Hsp90 inhibitors. NCT-50 exhibited significant inhibitory effects on the viability and colony formation of non-small cell lung cancer (NSCLC) cells and those carrying resistance to chemotherapy. In contrast, NCT-50 showed minimal effects on the viability of normal cells. NCT-50 induced apoptosis in NSCLC cells, inhibited the expression and activity of several Hsp90 clients including hypoxia-inducible factor (HIF)-1α, and suppressed pro-angiogenic effects of NSCLC cells. Further biochemical and in silico studies revealed that NCT-50 downregulated Hsp90 function by interacting with the C-terminal ATP-binding pocket of Hsp90, leading to decrease in the interaction with Hsp90 client proteins. These results suggest the potential of NCT-50 as an anticancer Hsp90 inhibitor.

Figure 1

Synthesis of NCT-50. (a) The chemical structures of novobiocin, deguelin, and NCT-50. (b) Synthetic scheme of NCT-50.

Figure 2

Effects of NCT-50 on the viability and colony formation of NSCLC cells. (a) NSCLC (H1299, H460, A549 and H226B) cells and those carrying resistance to cisplatin (H1299/CsR), pemetrexed (H1299/PmR), and paclitaxel (H226B/PcR) were treated with increasing concentrations of NCT-50 (a) for 3 days. Cell viability was determined by the MTT assay. (b,c) The anchorage-dependent (b) and -independent (c) colony formation of NSCLC cells treated with increasing concentrations of NCT-50 was determined as described in Methods. (d) H460 cells were treated with increasing concentrations of ganetespib (Gane) or PU-H71 (PU) for 3 days. Cell viability was determined by the MTT assay. The bars represent the mean ± SD. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test compared with vehicle-treated control group.

Figure 3

Association of apoptosis with NCT-50-induced cell death. (a–d) H1299 and H460 cells were treated with NCT-50 for 2 days. (a) The distribution of cells in each phase of the cell cycle was analyzed by flow cytometry. (b) Condensed, fragmented or degraded nuclei was analyzed by Hoechst 33258 staining and counted. (c) The level of cleaved PARP expression was analyzed by Western blot analysis. (d) The annexin V-positive cell population was determined by flow cytometry as described in Methods. The bars represent the mean ± SD. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test compared with vehicle-treated control group.

Figure 4

Improved safety of NCT-50 compared with known Hsp90 inhibitors and deguelin. (a) Various normal cells were treated with vehicle (DMSO) or NCT-50 (0.1, 1, and 10 μM) for 3 days. Cell viability was determined by the MTT assay. (b) BEAS-2B cells were treated with increasing concentrations of Hsp90 inhibitors [ganetespib (Gane) or PU-H71 (PU)] for 2 days. Cell viability was determined by the MTT assay. (c) Body weight changes between vehicle- (control) and NCT-50-treated mice. (d) The level of GOT, GPT, and BUN in the serum was determined as described in Methods and expressed as a percentage of vehicle-treated control group. (e) The histopathological changes in liver, lung, brain, and kidney from mice treated with vehicle or NCT-50 were evaluated by H&E-stained section of the tissues. The representative images were shown. (f) Spectrophotometric analysis of NADH dehydrogenase activity using mitochondria-enriched fractions was performed as described in Methods. (g) HT-22 cells were treated with various concentrations of deguelin or NCT-50 for 2 days. Cell viability was determined by the MTT assay. (h) Representative images showing tyrosine hydroxylase immunoreactivity in the midbrain from vehicle, deguelin, or NCT-50-treated mice. Right. Quantitative analysis of tyrosine hydroxylase immunoreactivity in each group, expressed as a percentage of vehicle-treated control group. The bars represent the mean ± SD. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test compared with vehicle-treated control group.

Figure 5

Downregulation of Hsp90 function by treatment with NCT-50. (a,b) H1299 and H460 cells were treated with NCT-50 for 24 h under hypoxic (a) or normoxic (b) conditions. The level of Hsp90 client proteins such as HIF-1α (a), EGFR, IGF1R, Akt, and MEK1/2 (b) was determined by Western blot analysis. (c,d) H1299 and H460 cells were treated with NCT-50 for 24 h. The mRNA expression of HIF-1α target genes (VEGF, TGFB3, and PDGFB) (c) or non-HIF-1α target genes (DDIT3, PPP1R15A, and TRIB3) (d) were determined by real-time PCR analysis. (e,f) HUVECs were treated with CM obtained from NSCLC cells treated with vehicle or NCT-50 (5 or 10 μM). The proliferation (e) and tube formation (f) of HUVECs were determined as described in Methods. (g) H460 cells were treated with NCT-50, ganetespib (Gane) or PU-H71 (PU) for 24 h under hypoxic conditions. The level of HIF-1α expression was determined by Western blot analysis. The bars represent the mean ± SD. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test compared with vehicle-treated control group.

Figure 6

NCT-50-mediated inhibition of Hsp90 function by binding to the ATP binding pocket in the C-terminal domain of Hsp90. (a) H1299 and H460 cells were treated with NCT-50 (5 μM) for 1 h, followed by hypoxic incubation for 4 h. Total cell lysates were prepared and immunoprecipitated with anti-Hsp90 antibodies. The interaction between HIF-1α and Hsp90 was analyzed by Western blot analysis. (b) Recombinant Hsp90 protein was incubated with ATP-agarose in the presence or absence of NCT-50. The protein bound to the ATP-agarose beads was determined by Western blot analysis. (c) The binding of NCT-50 (5 μM) to full-length (FL) and truncated domains (N: N-terminal; M: middle; C: C-terminal) of recombinant Hsp90 proteins was determined by pull-down assay using ATP-agarose beads. Agarose beads without ATP were used to determine specific binding of Hsp90 proteins to ATP. (d) Competition between NCT-50 and cisplatin for the binding to the N and C domains of Hsp90 was determined by pull-down assay using biotinylated NCT-50. (e) Left. Binding site for NCT-50 in the dimerization interface of open state hHsp90. Chain A is rendered in orange ribbon, and chain B is blue ribbon. The active site is shown as electrostatic property surface map. Red, blue, and white colored regions correspond to negatively charged, positively charged, and neutral areas, respectively. Right. Docked pose of NCT-50 (carbon in yellow). Key amino acid residues within the binding site are rendered in grey capped stick. Hydrogen bonding interactions are depicted as yellow dashed lines and pi-cation interaction is depicted as green dashed lines. A and B indicate chain names.