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, 15 (4), 826-837
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Repression of Hexokinases II-Mediated Glycolysis Contributes to Piperlongumine-Induced Tumor Suppression in Non-Small Cell Lung Cancer Cells

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Repression of Hexokinases II-Mediated Glycolysis Contributes to Piperlongumine-Induced Tumor Suppression in Non-Small Cell Lung Cancer Cells

Li Zhou et al. Int J Biol Sci.

Abstract

Deregulation of glycolysis is a common phenomenon in human non-small cell lung cancer (NSCLC). In the present study, we reported the natural compound, piperlongumine, has a profound anti-tumor effect on NSCLC via regulation of glycolysis. Piperlongumine suppressed the proliferation, colony formation and HK2-mediated glycolysis in NSCLC cells. We demonstrated that exposure to piperlongumine disrupted the interaction between HK2 and VDAC1, induced the activation of the intrinsic apoptosis signaling pathway. Moreover, our results revealed that piperlongumine down-regulated the Akt signaling, exogenous overexpression of constitutively activated Akt1 in HCC827 and H1975 cells significantly rescued piperlongumine-induced glycolysis suppression and apoptosis. The xenograft mouse model data demonstrated the pivotal role of suppression of Akt activation and HK2-mediated glycolysis in mediating the in vivo antitumor effects of piperlongumine. The expression of HK2 was higher in malignant NSCLC tissues than that of the paired adjacent tissues, and was positively correlated with poor survival time. Our results suggest that HK2 could be used as a potential predictor of survival and targeting HK2 appears to be a new approach for clinical NSCLC prevention or treatment.

Keywords: Akt; Hexokinases II; glycolysis; non-small cell lung cancer; piperlongumine.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Inhibitory effects of piperlongumine on NSCLC cells. A, the chemical structure of piperlongumine. B, piperlongumine inhibits anchorage-dependent growth in a panel of human lung cancer cells, including H23 (left), HCC827 (middle) and H1975 (right). Cell proliferation assay was performed as described in the “Material and Methods” section. Data shown are the proliferation ability of human lung cancer cells treated with different concentrations of piperlongumine compared with the dimethyl sulfoxide-treated group. C, the effect of piperlongumine on anchorage-dependent growth of HBE cells. D, A colony formation assay was performed as described in the “Material and Methods” section. Data shown are the colony formation ability of H23 (top), HCC827 (middle) and H1975 (bottom) cells treated with different concentrations of piperlongumine compared with the dimethyl sulfoxide-treated group. The average colony number was calculated from three separate experiments. Asterisk, significant (*p<0.01, **p<0.01, ***p<0.001) suppression of colony formation by piperlongumine compared with DMSO treated group. PL, Piperlongumine.
Figure 2
Figure 2
Piperlongumine regulates glycolysis in NSCLC cells. NSCLC cells, including H23 (A), HCC827 (B) and H1975 (C) were treated with different concentrations of piperlongumine for 24 h. Western blotting was performed to detect HK2 expression (left). The levels of glucose consumption (middle) and lactate production (right) were examined in these cells. Columns, mean of the number of colonies as determined from three independent experiments; bars, standard deviation; asterisk, significant (*p<0.05, **p<0.01, ***p<0.001) suppression of HK2 expression or glycolysis by piperlongumine compared with DMSO treated group. PL, Piperlongumine.
Figure 3
Figure 3
Piperlongumine disrupts the interaction of HK2 and VDAC1 and induces apoptosis. A, HCC827 (left) and H1975 (right) cells were treated with piperlongumine for 24 h, co-immunoprecipitation was conducted to detect the interaction between HK2 and VDAC1. B, HCC827 cells were treated with piperlongumine for 24 h, co-immunoprecipitation and western blot were performed to identify the phosphorylation of HK2. C, HK2-WT or HK2-T473D was transfected into HCC827 cells, the co-immunoprecipitation and western blot were conducted to determine the interaction between HK2 and VDAC1 as indicated. D, HCC827 (left) and H1975 (right) cells were treated with piperlongumine for 24 h, cytosolic fractions and mitochondrial fractions were isolated, western blot was conducted to detect the target proteins as indicated. E, HCC827 (left) and H1975 (right) cells were treated with piperlongumine for 24 h, the whole cell extract was subjected to western blot analysis as indicated. F, HCC827 (left) and H1975 (right) cells were treated with piperlongumine for 24 h, the flow cytometry was conducted for apoptosis analysis. PL, Piperlongumine. G and H, HCC827 (G) and H1975 (H) stable cells were treated with piperlongumine for 48 h, western blot and MTS assay were conducted. Asterisk, significant (*p<0.05, **p<0.01) difference between groups as indicated. ns, not statistically significant.
Figure 4
Figure 4
Down-regulation of Akt signaling is required for piperlongumine-mediated glycolysis suppression. A, HCC827 (left), H1975 (middle) and H23 cells were treated with piperlongumine for 24 h, western blot was conducted to detect the target proteins as indicated. B and C, HCC827 (B) and H1975 (C) cells were treated with piperlongumine or wortmannin for 24 h, the whole cell extract was subjected to western blot analysis as indicated, glucose consumption and lactate production were measured as described in the “Material and Methods” section. D and E, HCC827 (D) and H1975 (E) cells were Transient transfected with Myr-Akt1 or vector, 24 h later, the transfected cells were treated with piperlongumine for another 24 h. Then the cells were seeded into 6-well plate at the concentration of 5×105/well, and cultured for another 12 h. The western blot was conducted to detect the target proteins, and glucose consumption and lactate production were measured as indicated. F, HCC827 cells were treated as described in Figure 4D, and western blot was conducted to detect the cleaved-PARP and -caspase 3. G, Real-time RT-PCR analysis of total HK2 mRNA levels in HCC827 and H1975 cells. H, HCC827 cells were treated with piperlongumine, western blot was conducted to detect the target proteins as indicated. H, HCC827 cells were transfected with c-Myc and treated with piperlongumine for 24 h, western blot was performed to identify the target proteins as indicated. Asterisk, significant (*p<0.01, **p<0.01, ***p<0.001) suppression of glycolysis by piperlongumine compared with DMSO treated group or Akt transfected group. PL, Piperlongumine.
Figure 5
Figure 5
Piperlongumine inhibits tumor growth in NSCLC xenograft mouse models. A and B, the tumor volumes of HCC827 (A) and H1975 (B) xenograft were measured twice a week. C and D, at the treatment endpoint, the HCC827 (C) and H1975 (D) tumor-bearing mice were sacrificed, and tumors were removed, photographed and weighed. E and F, during the treatment period, the body weight of HCC827 (E) and H1975 (F) tumor-bearing mice were measured twice a week to determine the toxicity of piperlongumine. For A-E, data are shown as mean values ± S.D. obtained from 6 mice in each group. E. Immunohistochemical staining detection of Ki67, p-Akt, p-S6, VDAC1, and HK2 in tumor sections from Piperlongumine or vehicle-treated group mice. All panels are of the same magnification. The integrated optical density (IOD) was evaluated using the Image-Pro Plus software (version 6.2) program. Data are shown as mean values ± S.D. Asterisk, significant (*p<0.05, **p<0.01, ***p<0.001,) suppression of tumor growth between piperlongumine- and vehicle-treated group. PL, Piperlongumine.
Figure 6
Figure 6
HK2 is over-expressed in NSCLC and positively correlated with poor patient outcomes. A, Immunohistochemical staining was performed on a lung cancer tissue array using the HK2 antibody. The intensity was evaluated using Image-Pro PLUS (v.6) and Image J (NIH) computer software. The representative image (left) and density of each sample (right) are shown. The asterisk indicates a significant difference (***p < 0.001, Mann-Whitney U-test) between tumor and adjacent tissue as indicated. B-D, Kaplan-Meier survival analysis for the relationship between survival time and HK2 signature in NSCLC was performed by using the online tool (http://kmplot.com/analysis/). B, OS, Overall Survival. C, FP, First Progression Survival. D, PPS, Post Progression Survival. p < 0.05 was considered to be a statistically significant difference.

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