Hexokinase II-derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells

Abstract

Overexpression of mitochondria-bound hexokinase II (HKII) in cancer cells plays an important role in their metabolic reprogramming and protects them against apoptosis, thereby facilitating their growth and proliferation. Here, we show that covalently coupling a peptide corresponding to the mitochondrial membrane-binding N-terminal 15 aa of HKII (pHK) to a short, penetration-accelerating sequence (PAS) enhances the cellular uptake, mitochondrial localization, and cytotoxicity of the peptide in HeLa cells. Further analysis revealed that pHK-PAS depolarized mitochondrial membrane potential, inhibited mitochondrial respiration and glycolysis, and depleted intracellular ATP levels. The effects of pHK-PAS were correlated with dissociation of endogenous full-length HKII from mitochondria and release of cytochrome c Of significance, pHK-PAS treatment of noncancerous HEK293 cells resulted in substantially lower cytotoxicity. Thus, pHK-PAS effectively disrupted the mitochondria-HKII association in cancer cells, which led to mitochondrial dysfunction and, finally, apoptosis. Our results demonstrate the potential of the pHK-PAS cell-penetrating peptide as a novel therapeutic strategy in cancer.-Woldetsadik, A. D., Vogel, M. C., Rabeh, W. M., Magzoub, M. Hexokinase II-derived cell-penetrating peptide targets mitochondria and triggers apoptosis in cancer cells.

Keywords: ATP; cytochrome c; cytotoxicity; glycolysis; oxidative phosphorylation.

Figure 1.

Quantification of cellular uptake of pHK and pHK-PAS. A) Time course of peptide internalization. HeLa cells were incubated with 25 µM pHK_A488 (left) or pHK-PAS_A488 (middle) in serum-free DMEM for 30–180 min. B) Dose dependence of peptide internalization. HeLa cells were incubated with 5–50 µM pHK_A488 (left) or pHK-PAS_A488 (middle) in serum-free DMEM for 2 h. After peptide incubation, cells were washed 3 times with ice-cold PBS, trypsinized, centrifuged, and resuspended in ice-cold PBS with 10% FBS, and fluorescence was measured by FACS. Peptide internalization was determined by subtraction of background signal (cells treated with vehicle alone) from fluorescence intensities of peptide-treated cells and plotted relative to the maximum fluorescence intensity observed (right). (Error bars lie within the symbol for some data points.) ns, nonsignificant (P > 0.05). *P < 0.01, **P < 0.001, ***P < 0.0001 compared with pHK.

Figure 2.

Role of cell-surface proteoglycans in cellular uptake pHK and pHK-PAS. A, B) Wild-type CHO-K1 (left) and proteoglycan-deficient pgsA-745 (middle) cells were incubated with 5–50 µM pHK_A488 (A) or pHK-PAS_A488 (B) in serum-free DMEM for 2 h. C) Effect of free heparin on peptide internalization. HeLa cells were pretreated for 30 min at 37°C in serum-free DMEM with extracellular heparin (0–25 μg/ml), then treated with 25 µM pHK_A488 (left) or pHK-PAS_A488 (middle) and maintained for 2 h at 37°C in the presence of inhibitor and peptide. After peptide incubation, cells were washed 3 times with ice-cold PBS, trypsinized, centrifuged, and resuspended in ice-cold PBS with 10% FBS, and fluorescence was measured by FACS. Peptide internalization was determined by subtraction of background signal (cells treated with vehicle alone) from fluorescence intensities of peptide-treated cells and plotted relative to the maximum fluorescence intensity observed (right). (Error bars lie within the symbol for some data points.) ns, nonsignificant (P > 0.05). *P < 0.01, ***P < 0.0001 compared with controls.

Figure 3.

Determination of cellular uptake mechanisms of pHK and pHK-PAS. A) Effects of low temperature and energy depletion on peptide internalization. HeLa cells were preincubated for 1 h at 4°C in serum-free DMEM or pretreated for 1 h at 37°C with 10 mM sodium azide and 6 mM 2-deoxy-d-glucose in serum- and glucose-free DMEM to deplete cellular ATP. pHK_A488 (25 µM; left) or pHK-PAS_A488 (25 µM; middle) was then added, and cells were maintained for 2 h at 4°C or in the presence of sodium azide/2-deoxy-d-glucose at 37°C. B) Effects of endocytosis inhibitors on peptide internalization. HeLa cells were treated for 30 min at 37°C in serum-free DMEM with the following: 10 µM chlorpromazine (Chlor; clathrin-dependent endocytosis), 5 mM methyl-β-cyclodextrin (MβCD; lipid raft–mediated endocytosis), 4 µM filipin (Filip; caveolae-dependent endocytosis), 10 µM nocodazole (Nocod; microtubule polymerization), or 10 µM cytochalasin D (Cyto D; macropinocytosis). Cells were then treated with 25 µM pHK_A488 (left) or pHK-PAS_A488 (middle) and maintained for 2 h at 37°C in the presence of inhibitors and peptides. Thereafter, cells were washed 3 times with ice-cold PBS, trypsinized, centrifuged, and resuspended in ice-cold PBS with 10% FBS, and fluorescence was measured by FACS. Cells that were treated with peptide without inhibitors at 37°C were used as control, and cells that were treated with vehicle alone served as background. Uptake efficiencies (right) were determined from the ratio of fluorescence of cells treated with peptide under different inhibition conditions to control cells. ns, nonsignificant (P > 0.05). **P < 0.001, ***P < 0.0001 compared with controls at 37°C.

Figure 4.

Intracellular localization of pHK and pHK-PAS. A, B) Colocalization of pHK_A488 (A) and pHK-PAS_A488 (B) with mitochondria in HeLa (top), CHO-K1 (middle), and pgsA-745 (lower) cells. Cells were incubated with 25 µM pHK_A488 or pHK-PAS_A488 in phenol red– and serum-free medium for 2 h, then stained with organelle markers (5 μg/ml Hoechst 33342 and 50 nM MitoTracker Red) for 30 min before confocal imaging. Scale bars, 10 µM. C) Pearson’s correlation coefficient for pHK_A488 (red bars) and pHK-PAS_A488 (blue bars) in HeLa, CHO-K1, and pgsA-745 cells, respectively. Pearson’s correlation coefficient measures pixel-by-pixel covariance in the signal level of 2 images and is a useful means of evaluating colocalization (72). ***P < 0.0001 compared with the correlation coefficient of pHK_A488 in the same cell line.

Figure 5.

Complementary measures of the cytotoxic effects of pHK and pHK-PAS. A, B) Dose- and time-dependent inhibition of MTS reduction. HeLa (A) and HEK293 (B) cells were treated with the indicated concentrations of pHK (left) or pHK-PAS (right) for the indicated durations. Cells that were treated with peptide-free carrier were used as control. The percent viability was determined form the ratio of the absorbance of treated cells to control cells. (Error bars lie withing the symbol for some data points.) C, D) Detection of apoptosis and necrosis. C) FACS analysis of annexin V/PI staining of HeLa cells that were either untreated (control; left) or treated with 50 μM pHK (middle) or pHK-PAS (right) for 24 h. The bottom left quadrant (annexin V⁻/PI⁻) represents live cells; bottom right (annexin V⁺/PI⁻), early apoptotic cells; top right (annexin V⁺/PI⁺), late apoptotic cells; and top left (annexin V⁻/PI⁺), necrotic cells. D) A summary of the incidence of early and late apoptosis and necrosis in HeLa cells that were treated with pHK (left bars) or pHK-PAS (right bars) determined from FACS analysis of annexin V/PI staining in panel C. ns, nonsignificant (P > 0.05). *P < 0.01, **P < 0.001 compared with pHK.

Figure 6.

Effects of pHK and pHK-PAS on intracellular ATP levels and mitochondrial membrane potential. A) Dose- and time-dependent decrease in intracellular ATP levels. HeLa cells were treated with pHK (red bars) or pHK-PAS (blue bars) for the indicated durations. Two peptide concentrations, 25 (open bars) or 50 µM (filled bars), were used. ATP levels were then measured by using the CTG assay. Cells that were treated with peptide-free carrier were used as control. Relative ATP levels were determined from the ratio of the luminescence of treated cells to control cells. ns, nonsignificant (P > 0.05). **P < 0.001, ***P < 0.0001 compared with controls. B) ΔΨ_m depolarization of the inner mitochondrial membrane. HeLa cells were treated with 25 or 50 µM pHK (left) or pHK-PAS (middle) for 24 h, and fluorescence of the ΔΨ_m probe TMRM was measured by using FACS. Cells that were treated with the uncoupling agent, FCCP (10 µM), were used as positive controls for ΔΨ_m dissipation, and cells that were treated with vehicle alone served as negative controls. TMRM fluorescence intensity is plotted as a percentage of controls (right). **P < 0.001 compared with pHK.

Figure 7.

Effects of pHK and pHK-PAS on cellular metabolic activities. A) Changes in mitochondrial bioenergetic function. HeLa cells were treated with 25 μM pHK (red bars) or pHK-PAS (blue bars) for 24 h at 37°C. Cells were then subjected to sequential addition of 1.0 μM FCCP and a mixture of 1.0 μM rotenone and 1.0 μM antimycin A, and the OCR was measured on a Seahorse XFp Extracellular Flux Analyzer. Spare respiratory capacity (right bars) was determined from the change in OCR from the baseline rate in response to FCCP, and maximal respiration (left bars) from the difference between the FCCP-induced OCR and the value after addition of rotenone and antimycin A. B) Changes in glycolytic function. HeLa cells were treated with 25 μM pHK (red bars) or pHK-PAS (blue bars) for 24 h at 37°C, followed by sequential addition of 10 mM glucose and 1.0 μM oligomycin, and the ECAR was measured on a Seahorse XFp Extracellular Flux Analyzer. Glycolysis (left bars) and glycolytic capacity (right bars) were determined from changes in ECAR from the baseline rate in response to glucose and oligomycin, respectively. ns, nonsignificant (P > 0.05). *P < 0.05, **P < 0.001.

Figure 8.

Effects of pHK-PAS on HKII and cytochrome c localization. HeLa and HEK293 cells were treated with 50 µM pHK-PAS for 24–72 h. Thereafter, cells were harvested and mitochondria-enriched, and cytosolic fractions were isolated. Samples were normalized for protein content and electrophoresed on a 10–12% SDS polyacrylamide gel, then transferred to a nitrocellulose membrane; incubated overnight with mouse anti-human HK II, VDAC, or cytochrome c Abs, followed by 3 h with horseradish peroxidase–conjugated mouse IgG Ab; and finally visualized by using SuperSignal West Pico Chemiluminescent Substrate. A, C) Immunoblots of HKII, VDAC1, and cytochrome c (Cyt-c) in the mitochondria-enriched (left) and cytosolic fractions (right) of control (t = 0) and pHK-PAS-treated (24–72 h) HeLa (A) and HEK293 (C) cells. VDAC and β-actin were used as loading controls for the mitochondria-enriched and cytosolic fractions, respectively. B, D) Changes in the HKII and cytochrome c content of the cytosolic fraction of control and pHK-PAS–treated HeLa (B) and HEK293 (D) cells determined by densitometric quantification of the band intensities in panels A and C, respectively. ***P < 0.0001 compared with controls.

Figure 9.

Proposed model of pHK-PAS action. Coupling of PAS to pHK enhances the peptide’s cellular uptake. This uptake occurs by both macropinocytosis and energy-independent mechanisms (translocation across the plasma membrane). In the case of uptake by macropinocytosis, the PAS sequence facilitates escape of the peptide from macropinosomes to the cytosol, whereas translocation provides the peptide with direct access to the cytosol. Once in the cytosol, pHK-PAS accumulates at the mitochondrial membrane, where it binds to VDAC and displaces endogenous full-length HKII in the process. The disruption of the HKII–VDAC interaction leads to ΔΨ_m depolarization, inhibition of mitochondrial respiration and glycolysis, depletion of intracellular ATP levels, release of cytochrome c, and, finally, apoptosis.