Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4

Abstract

Background: Adenylate kinase is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. An isoform of this enzyme, adenylate kinase 4 (AK4), is localized in the mitochondrial matrix and is believed to be involved in stress, drug resistance, malignant transformation in cancer, and ATP regulation. However, the molecular basis for the AK4 functions remained to be determined.

Methods: HeLa cells were transiently transfected with an AK4 small interfering RNA (siRNA), an AK4 short hairpin RNA (shRNA) plasmid, a control shRNA plasmid, an AK4 expression vector, and a control expression vector to examine the effect of the AK4 expression on cell proliferation, sensitivity to anti-cancer drug, metabolome, gene expression, and mitochondrial activity.

Results: AK4 knockdown cells treated with short hairpin RNA increased ATP production and showed greater sensitivity to hypoxia and anti-cancer drug, cis-diamminedichloro-platinum (II) (CDDP). Subcutaneous grafting AK4 knockdown cells into nude mice revealed that the grafted cells exhibited both slower proliferation and reduced the tumor sizes in response to CDDP. AK4 knockdown cell showed a increased oxygen consumption rate with FCCP treatment, while AK4 overexpression lowered it. Metabolome analysis showed the increased levels of the tricarboxylic acid cycle intermediates, fumarate and malate in AK4 knockdown cells, while AK4 overexpression lowered them. Electron microscopy detected the increased mitochondrial numbers in AK4 knockdown cells. Microarray analysis detected the increased gene expression of two key enzymes in TCA cycle, succinate dehydrogenase A (SDHA) and oxoglutarate dehydrogenease L (OGDHL), which are components of SDH complex and OGDH complex, supporting the metabolomic results.

Conclusions: We found that AK4 was involved in hypoxia tolerance, resistance to anti-tumor drug, and the regulation of mitochondrial activity. These findings provide a new potential target for efficient anticancer therapies by controlling AK4 expression.

Keywords: Adenylate kinase; Drug resistance; Energy metabolism; Flux analysis; Hypoxia; Metabolome; Mitochondria.

Fig. 1

Evaluation of adenylate kinase 4 (AK4) expression and intracellular localization in HeLa cells. a Left panel: western blotting evaluation of AK4 (25 kDa) and alpha-tubulin (55 kDa) expression at the indicated time-points under hypoxic conditions (1 % O₂). Right panel: band densities were quantified using Image Lab (BioRad) and normalized to the internal control (alpha-tubulin). *P < 0.05, **P < 0.01. b Left panel: western blotting analysis of AK4 expression after deferoxamine (DFO) treatment. Right panel: quantified data normalized by internal control alpha-tubulin were conducted by using Image Lab (BioRad). *P < 0.05, **P < 0.01. c Left panel: immunohistochemical evaluation of the intracellular localization of AK4. Upper panel: normoxia; lower: hypoxia (1 % Day 2); right panel: subcellular localization of AK4. HK2 (100 kDa) was an hypoxic marker, ATP5a (54 kDa) was a mitochondrial marker, and alpha-tubulin (55 kDa) was the loading control. d AK4 expression in a stable transformant containing AK4 tagged with GFP on its C terminus. In addition to internal AK4 (25 kDa), overexpressed AK4:GFP (50 kDa) signaling was also detected. VDAC or β-Actin is used for mitochondrial loading control or total protein loading control, respectively. e Intracellular localization of the AK4GFP stable transformant. Top: GFP, center: MitoTracker Red, bottom: merged

Fig. 2

Decreased drug tolerance due to adenylate kinase 4 (AK4) knockdown in HeLa cells. a Left panel: western immunoblotting showing AK4 knockdown in cells expressing AK4 short hairpin (sh)RNA. AK4, 25 kDa; phosphorylated 5΄ AMP-activated protein kinase (p-AMPK), 64 kDa; β-actin, 42 kDa. Right panel: band densities were quantified using Image Lab (BioRad) and normalized to the internal control, alpha-tubulin *P < 0.05, **P < 0.01. b proliferation curves for AK4 shRNA cells and control shRNA cells, evaluated by xCELLigence (Real Time Cell Analysis System). Cells were evaluated after seeding at 5000 cells/well. The data are shown as mean ± standard deviation (SD) Cell Index (CI) values. c Cell numbers were counted 3 days after seeding under normoxic and hypoxic (1 % O₂) conditions *P < 0.05, **P < 0.01. d Evaluation of drug sensitivity after AK4 knockdown showing the half maximal inhibitory concentration (IC50) for cis-diamminedichloro-platinum(II) (CDDP) and doxorubicin. IC50 values were determined by MTS assays. **P < 0.01. e Images of tumors resulting from HeLa cells grafted subcutaneously in nude mice on day 40 after grafting. f Graph showing the volume of HeLa cell grafts in nude mice at the indicated time-points. The data are shown as mean tumor volume (cm³) ± SD, **P < 0.01. g Western blotting analysis of HeLa cells 3 days after siRNA treatment. AK4, VDAC, Bax, or Bcl2 were detected at 25 kDa, 39 kDa, 21 kDa, or 26 kDa, respectively

Fig. 3

Oxygen consumption rate (OCR) and metabolite analysis in HeLa cells expressing adenylate kinase 4 (AK4) or control short hairpin (sh)RNA. a Relative ATP level per cell. 10,000 cells were seeded in 96 wells, and measured 3 days after siRNA treatment. ATP level was normalized by cell number deduced from cyquant assay. *p < 0.05. b Flux analyzer OCR measurements were performed in triplicate in cells harboring AK4 shRNA or control shRNA before and after administration of oligomycin, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), antimycin, and rotenone, as indicated. *P < 0.05, **P < 0.01. c OCR of AK4-overexpressing and control cells before and after administration of oligomycin, FCCP, antimycin, and rotenone, as indicated. d Representative metabolomics analysis of compounds involved in the tricarboxylic acid (TCA) cycle. Left: comparison of control vector (green) and AK4 overexpression (red). Right: comparison of control shRNA (green) and AK4 shRNA (blue). *P < 0.05, **P < 0.01

Fig. 4

Alteration of mitochondria by adenylate kinase 4 (AK4) knockdown. a Representative electron microscope images of cells in normoxic condition. Left panel: HeLa cells transfected with control short hairpin (sh)RNA. Right panel: HeLa cells expressing adenylate kinase 4 (AK4) shRNA. The arrows indicate mitochondria. Right: The graphs show the relative mitochondrial counts and cross-sectional area, as indicated. Mitochondrial numbers were counted in ten random pictures taken at a magnification of 10,000. Mitochondrial size was determined using BZ-II analyzer (Keyence) software. We examined 70 mitochondria in control shRNA cells and 100 mitochondria for in AK4 shRNA cells. b Relative mitochondrial (mt)DNA copy number under the conditions indicated. mtDNA was measured by real-time PCR using the Human mtDNA Monitoring Primer Set (Takara Japan). Briefly, we measured mtDNA (ND1 and ND5), then normalized to the nuclear DNA level (SLCO2B1 and SERPINA1). The mtDNA content index was the ratio of mtDNA/nuclear DNA, calculated by dividing the ND1 and ND5 signals by the SLCO2B1 and SERPINA1 signals. P < 0.05, **P < 0.01

Fig. 5

A proposed role of adenylate kinase 4 (AK4) in mitochondria. a Proposed adenine nucleotide metabolism in the presence of AK4. ADP that returns to the mitochondria is converted to ATP by ATP synthase. AK4 forms complexes with hexokinase 2 (HK2), voltage-dependent anion channel (VDAC), and adenine nucleotide translocase (ANT) for the efficient recycling of ADP. This interaction with the mitochondrial inner membrane protein, ANT, is important for AK4-mediated protection from oxidative stress. AK3 is thought to be important for regeneration of GDP, which is necessary for the tricarboxylic acid (TCA) cycle. I, II, III, IV, and V indicate complexes I, II, III, IV and V (ATP synthase), respectively. AK4 may also cause competitive inhibition of AK3, which recycles the GDP required for the TCA cycle. b Proposed adenine nucleotide metabolism in the absence of AK4. HK2, VDAC, and ANT do not recycle ADP efficiently in the absence of AK4. The ATP concentration decreases focally in the cytosol, leading to 5΄ AMP-activated protein kinase (AMPK) phosphorylation and mitochondrial activation. AK4 knockdown may promote AK3 activity by canceling its inhibition to AK3. This proposed hypothesis remains to be tested. Dotted lines indicate reduced efficiency of ADP recycling