Biochemical Journal

Research article

Aldo-keto reductase family 1, member B10 is secreted through a lysosome-mediated non-classical pathway

Di-xian Luo , Mei C. Huang , Jun Ma , Zachary Gao , Duan-fang Liao , Deliang Cao


AKR1B10 (aldo-keto reductase family 1, member B10) protein is primarily expressed in normal human small intestine and colon, but overexpressed in several types of human cancers and considered as a tumour marker. In the present study, we found that AKR1B10 protein is secreted from normal intestinal epithelium and cultured cancer cells, as detected by a newly developed sandwich ELISA and Western blotting. The secretion of AKR1B10 was not affected by the protein-synthesis inhibitor cycloheximide and the classical protein-secretion pathway inhibitor brefeldin A, but was stimulated by temperature, ATP, Ca2+ and the Ca2+ carrier ionomycin, lysosomotropic NH4Cl, the G-protein activator GTPγS and the G-protein coupling receptor N-formylmethionyl-leucyl-phenylalanine. The ADP-ribosylation factor inhibitor 2-(4-fluorobenzoylamino)-benzoic acid methyl ester and the phospholipase C inhibitor U73122 inhibited the secretion of AKR1B10. In cultured cells, AKR1B10 was present in lysosomes and was secreted with cathepsin D, a lysosomal marker. In the intestine, AKR1B10 was specifically expressed in mature epithelial cells and secreted into the lumen at 188.6–535.7 ng/ml of ileal fluids (mean=298.1 ng/ml, n=11). Taken together, our results demonstrate that AKR1B10 is a new secretory protein belonging to a lysosome-mediated non-classical protein-secretion pathway and is a potential serum marker.

  • aldo-keto reductase family 1
  • member B10 (AKR1B10)
  • ATP-binding-cassette transporter (ABC transporter)
  • calcium signalling
  • cancer marker
  • lysosomal exocytosis
  • non-classical protein-secretion pathway


AKR1B10 (aldo-keto reductase family 1, member B10), also named ARL-1 (aldose reductase-like-1), is a protein identified from human hepatocellular carcinomas [1]. AKR1B10 belongs to the AKR superfamily composed of >100 proteins that are structurally and/or functionally conserved in the hierarchy of organisms, from bacteria to humans [25]; these proteins are widely implicated in carbonyl detoxification, procarcinogen activation, lipid metabolism, and cell carcinogenesis and cancer therapy [69]. AKR1B10 is a monomeric enzyme with NADPH as a co-enzyme, and its enzyme activity is regulated by S-thiolation at the protein level [1,10]. AKR1B10 can efficiently catalyse the reduction of dietary and cellular aromatic and aliphatic aldehydes and ketones, including highly electrophilic α,β-unsaturated aldehydes and antitumour drugs containing carbonyl groups [9,1116]. The electrophilic carbonyls constantly produced by lipid peroxidation are highly cytotoxic and can cause protein dysfunction and DNA damage (breaks and mutations) by interacting with nucleophiles in these macromolecules, resulting in mutagenesis, carcinogenesis or apoptosis [12,1721]. Therefore AKR1B10 may protect host cells from carbonyl lesions. AKR1B10 may also affect cell growth and differentiation by modulating the levels of the signalling molecule retinoic acid through reducing all-trans-retinal, 9-cis-retinal and 13-cis-retinal to their corresponding retinols [2224]. In human mammary epithelial cells, AKR1B10 is up-regulated with cell transformation and blocks the ubiquitin-dependent degradation of ACCA (acetyl-CoA carboxylase-α), a rate-limiting enzyme in de novo fatty acid synthesis, promoting fatty acid and lipid synthesis [25,26]. Therefore AKR1B10 also affects cell growth and survival by modulating lipid synthesis and bio-membrane function. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small interfering RNA-induced AKR1B10 silencing results in apoptotic cell death due to reduced lipid, particularly phospholipid, synthesis, mitochondrial dysfunction and subsequent oxidative stress, cytochrome c efflux and caspase 3 cleavage [25]. Therefore AKR1B10 may be an important protein in cell growth and proliferation.

AKR1B10 is normally expressed in the small intestine and colon with lower levels in the liver, but overexpressed in several human tumours, such as liver, lung, cervical and endometrial cancers [1,27,28]. The relationship between AKR1B10 and cigarette-smoke-related lung cancer is well documented. AKR1B10 is overexpressed in NSCLC (non-small cell lung cancer) in smokers [28], and cigarette smoke up-regulates AKR1B10 expression in the airway epithelium of healthy smokers with no evidence of lung cancer and activates procarcinogens in smoke, such as polycyclic aromatic hydrocarbons [2931]. Therefore AKR1B10 is considered to be an early-process protein and a potential biomarker in cigarette smoke-induced lung cancer [28,32]. This encouraged us to explore the potential of AKR1B10 as a serum marker for human cancer.

AKR1B7 [also named MVDP (mouse vas deferens protein)] and AKR1C [also known as DDH (dihydrodiol dehydrogenase)] are secreted, although the secretory mechanism (pathway) is unclear [33,34]. As an AKR protein, AKR1B10 is identical with AKR1B7 and AKR1C in amino acid sequence and stereostructure and thus may be a secretory protein. Therefore in the present study, we detected AKR1B10 in culture medium from cancer cells that express this protein and found that AKR1B10 is secreted. This result provids crucial evidence to compel a clinical study for AKR1B10 as a serum marker.


Procurement of ileal fluids and paraffin sections

Ileal fluids and paraffin sections were collected from individuals visiting the gastrointestinal clinic of the Memorial Medical Center (Springfield, IL, U.S.A.) using an IRB (institutional review board) protocol approved by the SCRIHS (Springfield Committee for Research Involving Human Subjects). Informed written consent was obtained from all donors. Specimens from the donors who were diagnosed without any gastrointestinal diseases were used for the present study. After collection, ileal fluids were immediately centrifuged at 600 g to remove cells and debris, followed by centrifugation at 10000 g for 20 min to collect clear supernatants for sandwich ELISA and Western blot analyses. Paraffin sections were subjected to immunohistochemistry (see below).

Cell cultures

Human colorectal adenocarcinoma cell lines HCT-8, HCT116, HT29 and RKO, human alveolar basal epithelial adenocarcinoma cell lines NCI-H460 and A549, human breast carcinoma cell lines MDA-MB-468, MCF-7 and BT-20, and the HEK-293T (human embryonic kidney cells expressing the large T-antigen of simian virus 40) cell line were maintained, as recommended by the A.T.C.C., in RPMI 1640, DMEM (Dulbecco's modified Eagle's medium) or F-12K medium supplemented with 10% FBS (fetal bovine serum), 2 mM glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen) at 37 °C, 5% CO2. As indicated, the medium was collected, centrifuged at 600 g for 10 min at 4 °C to remove cells and debris, and then subjected to analysis.

Sandwich ELISA and AKR1B10 measurements

An ELISA was developed to test AKR1B10 in culture medium and body fluids. In this method, a polyclonal antibody raised in a goat against the whole AKR1B10 protein was used as a capture antibody, and a rabbit polyclonal antibody against an AKR1B10-specific peptide was used as a detection antibody [26]. The sandwich ELISA was conducted as follows. High-binding 96-well plates were coated with 100 μl of 7 μg/ml capture antibody in a coating buffer and incubated at 4 °C overnight. After being washed three times with PBS, the wells were blocked with 250 μl of blocking buffer (Alpha Diagnostic) at 37 °C for 2 h. Samples (100 μl each) were added into the wells in duplicate. Plates were incubated at 37 °C for 1 h, washed five times with PBST (PBS containing 0.05% Tween 20), and incubated at 37 °C for 1 h with 100 μl per well of biotin-labelled detection antibody diluted 1:500 with antibody diluent. After being washed five times with PBST, plates were incubated at 37 °C for 30 min with 100 μl per well of streptavidin–HRP (horseradish peroxidase) conjugates diluted 1:5000 with antibody diluent. Specific binding was detected at 37 °C for 20 min with 100 μl of TMB (HRP Substrate; Thermo Scientific). Reactions were stopped with 50 μl of stop solution (Alpha Diagnostic) and A450 values were read within 30 min with A620 nm as a reference. Purified recombinant AKR1B10 protein was used for a standard curve for concentration calculation.

Lysosome isolation and proteinase K protection

Lysosomes were isolated as described previously [35]. Briefly, 5×107 cells were washed three times with PBS, resuspended in 2 ml of PBS containing 10 μg/ml leupeptin and 0.5 mM PMSF and disrupted using a Dounce homogenizer. Debris and nuclei were discharged at 1200 g, followed by 50000 g for 10 min at 4 °C. Supernatants were collected; pellets (lysosomes) were washed three times with PBS and suspended in 150 μl of PBS. For protection assays, pellets and supernatants (50 μl of each) were exposed on ice to 0.0125 mg/ml proteinase K for 30 min, with or without 0.5% Triton X-100, and then subjected to Western blot analysis for AKR1B10, cathepsin D and β-actin.

Western blot

Proteins were separated by SDS/PAGE (8–12% gel) and blotted on to nitrocellulose membranes. Anti-AKR1B10 (generated in our laboratory), anti-cathepsin D (Cell Signaling Technology), anti-vimentin and anti-β-actin (Sigma-Aldrich) antibodies were probed and detected as described previously [26].

Fluorescent protein protection

NCI-H460 cells (4×105) were transfected with EGFP (enhanced green fluorescent protein)–AKR1B10 expression vector and seeded into six-well plates. After incubation for 36 h, cells were stained with 100 nM LysoTracker® Red DND-99 in serum-free medium for 30 min, followed by staining with 0.5 μg/ml Hoechst stain for 5 min. Cells were switched to 1 ml of warm HBSS (Hank's balanced salt solution), and images were taken immediately with excitation and emission at 577 nm and 590 nm for DND-99, 488 nm and 509 nm for EGFP and 365nm and 480nm for Hoechst stain respectively. For protection assays, 1 ml of 2× digitonin (20 μM final concentration) in warm HBSS was added to the cells. After incubation at room temperature (20–22 °C) for 5 min, cells were exposed to trypsin (100 μg/ml final concentration) and images were captured immediately.

AKR1B10 activity

The enzyme activity of AKR1B10 in the medium was detected as follows. Cells (5×105 in a 60 mm-diameter dish) were incubated overnight in medium containing 10% FBS. After washing once with PBS, cells were fed with 3 ml of serum-free medium for 30 min. The medium was collected, centrifuged at 600 g for 10 min to remove cells and debris, concentrated 5-fold with a dialysis column (Millipore) and then subjected to enzyme activity assays in 500 μl reaction mixtures containing 20 mM DLglyceraldehyde, 135 mM sodium phosphate (pH 7.0), 0.2 mM NADPH, 50 mM KCl and 200 μl of concentrated medium. Reactions were conducted at 35 °C for 30min. Oxidized NADPH was measured at A340 to indicate enzymatic activity. Purified AKR1B10 recombinant protein was used as a positive control, and fresh serum-free medium was a blank control. Enzymatic activity was expressed as nmol oxidized NADPH/ml of medium per h.


AKR1B10 in small intestine and colon biopsies was examined by immunohistochemistry. Briefly, after being deparaffinized and hydrated, tissue slides were immersed into preheated citric acid buffer (pH 6.5) at 90–95 °C for 20 min with microwaving. After being blocked with 5% horse serum for 30 min, the slides were incubated with a specific rabbit anti-AKR1B10 antibody (diluted 1:50) [26] at 4 °C in a humid box overnight. Thereafter the slides were washed three times and then incubated with HRP-conjugated secondary antibody (diluted 1:800; Pierce) at room temperature for 1 h. Enhanced DAB (diaminobenzidine) solution (Pierce) was used to visualize signals.

Statistical analysis

Results are means±S.D. of three independent experiments. Statistical analysis was performed using a Student's t test or Chi square tests, as appropriate, with the INSTAT statistical analysis package (GraphPad Software) to determine statistical significance (P<0.05).


AKR1B10 is secreted into the culture medium

Using the developed sandwich ELISA (see Supplementary Figure S1 at, we detected AKR1B10 in culture medium. As shown in Figure 1A, AKR1B10 was detected in the medium of HCT-8, NCI-H460, A549, MDA-MB-468 and BT-20 cells, but not in that of HCT116, HT29, RKO and MCF-7 cells. Western blotting confirmed the presence of AKR1B10 in the medium and the correlation with its expression levels inside the cells. In addition, ectopically expressed EGFP–AKR1B10 in HEK-293T cells was also secreted (Figure 1A). Of note, to exclude the possibility that AKR1B10 in the medium was derived from dead cells, the cultured cells were washed with PBS and fresh medium was applied for 30 min. After collection, the medium was centrifuged at 600 g to remove cells and debris. In addition, β-actin, a non-secretory protein, in the medium was also examined to exclude the medium proteins from dead cells. The results suggest that AKR1B10 in the medium is secreted by cancer cells.

Figure 1 AKR1B10 secretion from tumour cells

(A) AKR1B10 in the medium from the cells indicated. Cells (2.5×105 of each cell line) were cultured overnight in six-well plates and then fed with 1.5 ml of serum-free medium for 30 min. The medium was harvested for sandwich ELISA (upper panel) and Western blotting (lower panel) as described in the Materials and methods section. Cell lysates were prepared to check the endogenous AKR1B10 levels; HEK-293T cells were transfected with EGFP or EGFP–AKR1B10 vector. (B) Kinetics of AKR1B10 release. HCT-8 cells (2.5×105) were cultured in a six-well plate. On the next day, cells were fed with 2 ml of fresh medium containing 0, 1 or 10% normal or dialysed FBS. FBS dialysis was conducted with a 5.0 kDa dialysis membrane. At the indicated time points, 200 μl of medium was collected and processed for sandwich ELISA and Western blotting. *P<0.01 and #P<0.05 compared with serum-free (0% FBS) medium. (C) Effect of cell density on AKR1B10 secretion. HCT-8 and NCI-460 cells as indicated were incubated in six-well plates overnight and then fed with 1 ml of serum-free medium. After 30 min, medium was collected for ELISA and Western blotting. Bars indicate AKR1B10 in the medium (ng/ml); curves denote AKR1B10 secretory efficiency (ng/million cells). (D) AKR1B10 activity. Cells (2.5×105) as indicated were seeded into six-well plates overnight and then fed with 1 ml of serum-free medium for 30 min. Medium was collected and subjected to an enzyme activity assay as described in the Materials and methods section.

The secretion of AKR1B10 was stimulated by serum (Figure 1B). In the presence of 10% FBS, AKR1B10 in the freshly fed medium of HCT-8 cells was detected at 0.5 min and peaked within 2 min (Figure 1B, i); AKR1B10 secretion was less efficient in serum-free or low serum conditions. Serum dialysed with 5.0 kDa cut-off filters had less stimulatory activity at early time points, but the same plateau was eventually reached within 1 h (Figure 1B, ii). This result suggests that certain small molecules (such as ions) in the serum were responsible for the immediate early stimulation, but large molecules (>5.0 kDa) participated in the later phase of stimulation, bringing the secretion to the same plateau. Cell density also affected the secretion of AKR1B10. As shown in Figure 1(C), the levels of AKR1B10 in the medium increased with cell number, but the secretory rate per million cells appeared to decrease inversely.

AKR1B10 is a reductase with activity to aldehydes [1]. To understand the functionality of the secreted AKR1B10, we tested its enzyme activity to DL-glyceraldehyde and the results showed that AKR1B10 in the medium was enzymatically active (Figure 1D).

AKR1B10 is secreted by a non-classical protein-secretion pathway

Soluble proteins are secreted by either classical or non-classical pathways [36]. In the classical protein-secretion pathway, a secretory protein is translocated by an N-terminal signal peptide into the ER (endoplasmic reticulum) at the time of synthesis and then secreted via the Golgi complex; therefore this secretion is affected by protein synthesis and transport from the ER to the Golgi. To identify the secretory pathway of AKR1B10, we analysed its amino acid sequence using the SignalP 3.0 server (, and results indicated that AKR1B10 does not contain a signal peptide (P=0.000; see Supplementary Figure S2 at Exposing cells to 15 μg/ml of CHX (cycloheximide), a protein synthesis inhibitor, did not affect the secretion of AKR1B10 for up to 8 h (Figure 2A), nor did the classical secretion pathway inhibitor brefeldin A, which blocks protein transport from the ER to the Golgi. As a positive control, brefeldin A efficiently blocked the secretion of vimentin through the classical ER/Golgi pathway [37] (Figure 2B, lower panel). These results suggested that AKR1B10 is secreted through a non-classical protein-secretion pathway.

Figure 2 AKR1B10 secretion through a non-ER/Golgi pathway

(A) CHX. HCT-8 cells (1.25×105) were seeded in 12-well plates overnight and then exposed to 15 μg/ml of the protein synthesis inhibitor CHX in fresh serum-free medium. At 30 min before the indicated time points, the medium was replaced with fresh serum-free medium and collected for sandwich ELISA (upper panel) and Western blotting (lower panel) as described in the Materials and methods section. *P<0.01 compared with AKR1B10 in medium at 0, 4 and 8 h. (B) Brefeldin A. HCT-8 and NCI-460 cells (1.25×105) were cultured in 12-well plates overnight and then exposed to the ER-to-Golgi protein transport inhibitor brefeldin A at 0, 2 or 10 μg/ml in fresh serum-free medium for 8 h. Medium was collected for sandwich ELISA and Western blotting. Vimentin was detected by Western blotting as a positive control.

Lysosomes are involved in AKR1B10 secretion

Lysosome-related protein secretion is a common and well-known non-classical mechanism [36,38] and was tested for AKR1B10 secretion. Lysosomes were isolated by ultracentrifugation. Western blotting showed that AKR1B10 was present inside the lysosomes, as indicated by cathepsin D, and was protected from proteinase K (0.0125 mg/ml) digestion (Figure 3A). This protection was abolished when 0.5% Triton X-100 was added to break down the biomembrane. AKR1B10 in lysosomes was confirmed by a fluorescent protease protection assay in living cells. EGFP–AKR1B10 fusion protein was expressed in NCI-H460 cells, and lysosomes were tracked by LysoTracker® Red DND-99. After free cytosolic proteins were digested by trypsin, leftover EGFP–AKR1B10 in organelles was examined and merged with LysoTracker® Red DND-99. As shown in Figure 3(B), protease-protected EGFP–AKR1B10 was located in lysosomes. The lysosome-mediated AKR1B10 was further confirmed with lysosome-specific protein markers. Cathepsin D, a luminal protein of lysosomes, was detected together with AKR1B10 in the medium of HCT-8 and NCI-H460 cells, whereas LAMP-1 (lysosome-associated membrane protein 1), a lysosomal transmembrane protein, was not (Figure 3C). Non-secretory β-actin was not detected either, excluding the protein sources from dead cells. Taken together, these results suggest that AKR1B10 is secreted through lysosomal exocytosis.

Figure 3 Lysosome-mediated AKR1B10 secretion

(A) Lysosome isolation and proteinase K protection. NCI-H460 cells (5×107) were disrupted using a Dounce homogenizer and lysosomes were isolated for proteinase K protection assay as described in the Materials and methods section. Triton X-100 (0.5%) was added to a sample to destroy biomembranes. (B) Fluorescent protein protection. NCI-H460 cells (4×105) were transfected with an EGFP–AKR1B10 fusion-protein expression vector. After incubation for 36 h, cells were stained with 100 nM LysoTracker® Red DND-99 in serum-free medium for 30 min, followed by Hoechst stain at 0.5 μg/ml for 5 min. Cytosolic proteins were digested by trypsin (100 μg/ml) in the presence of digitonin at 20 μM in warm HBSS. Images were captured as described in the Materials and methods. (C) HCT-8 and NCI-460 cells (1.25×105) were incubated in 12-well plates overnight and then fed with 1 ml of serum-free medium. After 30 min, medium and cell lysate were collected for Western blotting.

Secretion of AKR1B10 is affected by temperature, ATP, Ca2+ and NH4Cl

Serum stimulation indicated that AKR1B10 secretion was affected by small molecules (Figure 1B, i and ii). We examined the effect on AKR1B10 secretion of small molecules and factors that influence lysosomal exocytosis, such as temperature, ATP, Ca2+ and NH4Cl [39,40]. Results showed that in both HCT-8 and H460 cells, the secretion of AKR1B10 was blocked at 4 °C, but stimulated at 42 °C (Figure 4A), demonstrating a temperature-dependent manner. Similarly, ATP stimulated AKR1B10 secretion, but in contrast, Mg2+ (2 mM) blocked lysosome-mediated AKR1B10 secretion (Figure 4B), probably by chelating ATP4−, an active form of ATP [35,41]. EDTA acted as an antagonist of Mg2+ inhibition of AKR1B10 secretion (Figure 4B). Ca2+ plays a critical role in the merging of lysosomes with the cell membrane, promoting lysosome exocytosis [42]. The present study demonstrated that the secretion of AKR1B10 was significantly stimulated by Ca2+ ions (100 μM) or ionomycin (1–2 μM), a chemical carrier of Ca2+ [43] (Figure 4C).

Figure 4 Regulatory factors for AKR1B10 secretion

(A) Temperature. HCT-8 and NCI-460 cells (1.25×105 of each) were seeded into 12-well plates overnight and then exposed to the temperature indicated for 30 min. The cells were fed with fresh serum-free medium at the same temperature for 30 min, and medium was collected for sandwich ELISA (upper panel) and Western blotting (lower panel) as described in the Materials and methods. *P<0.01 and #P<0.05 compared with cells at 37 °C. (B) ATP. HCT-8 cells (1.25×105) were incubated in 12-well plates overnight and then exposed to 1 mM EDTA or 2 mM Mg2+ in serum-free medium for 2 h. At 30 min before harvest, ATP (1 mM) was added and the medium was subjected to sandwich ELISA and Western blot analysis. *P<0.01 compared with control cells without treatment; #P<0.01 compared with Mg2+ alone or P<0.05 compared with Mg2+ plus ATP. (C) Ca2+. HCT-8 cells (1.25×105) were seeded into 12-well plates and exposed to Ca2+ ions (100 μM) (i) or ionomycin at 1 or 2 μM (ii) in serum-free medium for 30 min and the medium was harvested for sandwich ELISA and Western blotting. *P<0.05 compared with control; **P<0.01 compared with ATP or Ca2+ alone; #P<0.05 and ##P<0.01 compared with blank control (Con) and DMSO vehicle. (D) NH4Cl. HCT-8 cells (1.25×105) were seeded into 12-well plates overnight and then divided into two groups. One group was pre-treated with 50 mM NH4Cl for 2 h to exhaust lysosomal exocytosis and then fed with fresh serum-free medium; the other was exposed to 50 mM NH4Cl in fresh serum-free medium. After 30 min, medium was harvested for sandwich ELISA and Western blotting. *P<0.01 and #P<0.05 compared with no-NH4Cl control.

Protein translocation and lysosomal exocytosis are affected by the luminal pH of lysosomes [44]. An increase in luminal pH stimulates exocytosis, but the resultant decrease in ΔpH (cytosolic pH minus lysosomal luminal pH) between the cytosol and the lysosomal lumen blocks protein translocation into lysosomes. NH4Cl is a lysosomotropic chemical that can increase lysosomal pH and plays a dual role in lysosome-mediated protein secretion. By increasing lysosomal pH, NH4Cl stimulates lysosomal exocytosis and the release of proteins that are already in lysosomes. Meanwhile, due to the decrease of ΔpH between lysosomes and cytosol, NH4Cl blocks the translocation of proteins and eventually exhausts the protein secretion [35,45]. In the present study, we found that exposing cells to NH4Cl (50 mM) exhausted lysosomal exocytosis, leading to a significant decrease of AKR1B10 in freshly fed medium; in contrast, AKR1B10 in the medium was increased when NH4Cl (50 mM) was added simultaneously with the fresh testing medium (Figure 4D).

Secretion of AKR1B10 is regulated by lysosome exocytosis signalling

Lysosome exocytosis is a Ca2-dependent process, and several signalling proteins regulate intracellular Ca2+ concentrations and thus are implicated in secretory lysosome exocytosis [4650]. PLC (phospholipase C) hydrolyses PIP2 (phosphatidylinositol 4,5-bisphosphate) to produce two second messengers: IP3 (myo-inositol 1,4,5-trisphosphate) and DAG (diacylglycerol) [51]. IP3 releases Ca2 from intracellular storage pools and raises cytosolic Ca2 whereas ARF (ADP-ribosylation factor) induces lysosome exocytosis by increasing PIP2 production [52]. Exo-1 [2-(4-fluorobenzoylamino)-benzoic acid methyl ester] is an ARF inhibitor [53] and U73122 is a PLC inhibitor. Our results showed that Exo-1 and U73122 both significantly inhibited the secretion of AKR1B10 in a dose-dependent manner (Figure 5). On the other hand, PLC activity is regulated by G-proteins, and our results showed that the G-protein activator GTPγS and the G-protein coupling receptor activator fMLP (N-formylmethionyl-leucyl-phenylalanine) dose-dependently enhanced AKR1B10 release (Figure 5). Cathepsin D release was altered consistently with that of AKR1B10 in response to these inhibitors and activators. The results suggest that the secretion of AKR1B10 is regulated by lysosome exocytosis signalling, confirming its lysosome-mediated secretion mechanism.

Figure 5 Effect of Ca2+ signalling on AKR1B10 secretion

HCT-8 cells (1.25×105) were incubated in 12-well plates overnight and then pretreated with the indicated concentrations of Exo-1, U73122, GTPγS or fMLP for 2 h. Cells were then fed with fresh serum-free medium supplemented with the same concentrations of these agents for 30 min. Medium and cell lysate were harvested and subjected to sandwich ELISA (upper panel) and Western blot analyses (lower panel) as described in the Materials and methods section. Western blot data indicate the cell samples treated with 10−4 M Exo-1, 10−6 M U73122, 10−4 M GTPγS, or 10−6 M fMLP.

AKR1B10 is translocated into lysosomes through ABC (ATP-binding cassette) transporters

Proteins that are secreted through the lysosome-mediated pathway are often translocated into lysosomes by ABC transporters [54]. To understand the transmembrane mechanism of AKR1B10, we exposed cells to the ABC transporter inhibitors Glib (glibenclamide) and DIDS (4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid). Results showed that Glib and DIDS both suppressed AKR1B10 secretion (Figure 6), suggesting the role of ABC transporters in the entry of AKR1B10 into lysosomes.

Figure 6 AKR1B10 translocation into lysosomal lumen by ABC transporters

HCT-8 cells (1.25×105) were cultured in 12-well plates overnight and then exposed to the ABCA1 inhibitors Glib or DIDS in serum-free medium for 2 h. ATP (1 mM) was added 30 min before harvest. Medium, isolated lysosomes and whole cell lysates were subjected to sandwich ELISA (upper panel) or Western blotting (lower panel). * and #P<0.05 compared with the relevant controls; ** and ##P<0.01 compared with ATP alone.

AKR1B10 is specifically expressed in mature intestinal epithelium and secreted into the lumen

AKR1B10 mRNA is abundant primarily in the colon and small intestine [1]. The present study showed that AKR1B10 protein is specifically expressed in the mature epithelium of the colon and small intestine (Figure 7A). To understand its secretory behaviour in vivo, we examined AKR1B10 in ileal fluids from 11 human subjects by ELISA. Results showed that AKR1B10 was secreted into the lumen at 188.6–535.7 ng/ml of ileal fluids (mean=298.1 ng/ml) (Figure 7B). Representative specimens were subjected to Western blot analysis. As shown in Figure 7(C), AKR1B10 and cathepsin D, but not the non-secretory protein LAMP-1, were detected in these tested ileal fluids, thereby indicating that AKR1B10 in the normal intestine is secretory through the lysosome exocytosis, consistent with that in cultured cancer cells.

Figure 7 AKR1B10 cellular distribution and secretion in human colon and small intestine

(A) Immunohistochemistry. Paraffin-embedded small intestine and colon biopsies were stained with AKR1B10-specific antibody to demonstrate its cellular distributions. The images show AKR1B10 expression in the epithelium of normal small intestine (i) and colon (ii). (B) AKR1B10 concentrations (ng/ml) in ileal fluids of 11 subjects, measured by sandwich ELISA as described in the Material and methods section. (C) Western blots of representative ileal fluid samples (a–d) showing AKR1B10, secretory lysosomal cathepsin D, and non-secretory lysosomal LAMP-1 proteins.


The present study demonstrated AKR1B10 as a novel secretory protein of the AKR superfamily. Soluble proteins are secreted through either the classical (ER/Golgi-dependent) or the non-classical (ER/Golgi-independent) protein-secretion pathway. In the classical pathway, target proteins typically contain an N-terminal signal peptide that directs the protein into the ER, and the protein is secreted through the Golgi complex [36,55,56]. In contrast, non-classical protein export features the lack of a conventional signal peptide at the N-terminus and resistance to the ER/Golgi-dependent protein-secretion inhibitor brefeldin A. Proteins secreted via the non-classical pathway usually have a low molecular mass at 12–45 kDa, and several mechanisms are described, including a lysosome-mediated secretory pathway, exosome release, specific membrane transporters and plasma membrane exocytosis [5759]. Among them, the lysosome-mediated pathway is the most common and well investigated, and many proteins are secreted by this pathway, such as IL-1β (interleukin-1β), HSP70 (heat-shock protein 70), sTNF (soluble tumour necrosis factor), ferritin and HMGB1 (high-mobility group B, protein 1) [36,38,39,6063].

AKR1B10 is secreted through a lysosome-mediated non-classical pathway, as evidenced by three lines of experimental data: (i) AKR1B10 is present in lysosomal compartments, as indicated by lysosomal isolation, protease protection and fluorescent co-localization; (ii) AKR1B10 is secreted together with cathepsin D, a lysosome luminal marker, but not LAMP-1, a lysosomal transmembrane protein; and (iii) AKR1B10 secretion is not affected by the protein synthesis inhibitor CHX and the classical protein-secretion pathway inhibitor brefeldin A, but is stimulated by temperature, ATP, Ca2+ and pH, the factors that influence lysosomal exocytosis [39,40]. Ca2+ is a central player of lysosome exocytosis [42,64,65]. ATP activates G-protein-coupled P2 receptors (P2Y and P2X receptors) and mobilizes Ca2 from intracellular storage pools [66,67]. The signal proteins ARF, PLC and G-proteins regulate secretory lysosome exocytosis by modulating intracellular Ca2+ concentrations [4648,5052]. PLC hydrolyses PIP2, producing IP3 that triggers Ca2+ mobilization [51]. PIP2 is produced by the lipid kinases PI4K (phosphatidylinositol 4-kinase) and PIP5K (PIP 5-kinase), which catalyse sequentially the phosphorylation of PI (phosphatidylinositol) to PIP2 at the plasma membrane [47,48]. PI is synthesized with PA (phosphatidic acid) as a precursor and the PA is produced by PLD (phospholipase D), which hydrolyses membrane PC (phosphatidylcholine). ARF-GTP activates PLD to control the synthesis of PIP2 [4749]. Our results showed that an ARF inhibitor Exo-1 and a PLC inhibitor U73122 can significantly inhibit AKR1B10 secretion in a dose-dependent manner, whereas a G-protein agonist GTPγS and a G-protein coupling receptor ligand fMLP stimulated AKR1B10 release (Figure 5). Taken together, our results suggests that AKR1B10 is secreted through a lysosome-mediated pathway. Of note, several non-classical protein-secretion pathways have been identified as described above, and a protein may be exported via multiple non-classical secretory pathways. For instance, IL-1β is released via (i) secretory lysosome exocytosis, (ii) membrane microvesicle release, (iii) exosome release and (iv) specific membrane transporters [68]. The present study shows the secretion of AKR1B10 through the lysosome-mediated non-classical pathway, but other mechanisms are not yet excluded. Further study is warranted to clarify this issue.

Transporters on lysosomal membranes are implicated in the entry of proteins into its compartments, such as the ABC family and LAMP-1/2 [6971]; ABC transporters are most frequently involved in the influx of secretory proteins, such as IL-1β [54]. Similarly, the present study revealed that the ABC transporters also contribute to the entry of AKR1B10 into lysosomes, as evidenced by the specific inhibitors Glib and DIDS.

The lysosome-mediated secretion pathway is often implicated in inflammatory and immune responses. For example, HMGB1 and IL-1β release from monocytes and macrophages is induced by lipopolysaccharide [54,60,62]. This pathway is also used for secretion of proteins by tumour cells without inflammatory stimuli. For instance, HSP70 is secreted from many tumour cells, such as human prostate carcinoma cells (PC-3 and LNCaP), human squamous carcinoma cells (A431) and rat mammary adenocarcinoma cells (RBA) [72]. Lysosomal enzymes, such as cathepsin D, are also secreted into the tumour cell microenvironment by lysosome exocytosis [73,74]. The present study demonstrated that AKR1B10 is secreted by colorectal adenocarcinoma, lung cancer and breast cancer cells, as well as by normal intestinal epithelium, advancing the understanding of lysosomes as a secretory organelle. In the AKR superfamily, MVDP and DDH are also secretory proteins, but the secretory mechanism is unclear [33,34]. In view of the structural identity of the AKR proteins, it is possible that these MVDP and DDH proteins may also be secreted by the same mechanism as AKR1B10.

Misfolded or denatured proteins are often directed into and degraded in lysosomes. AKR1B10 secreted into the medium through lysosome exocytosis, however, is enzymatically functional with the same molecular mass as that in cytosol (results not shown), indicating that AKR1B10 secretion is an active process, although the biological function remains unclear. Several housekeeping reductases are secretory, such as TrxR (thioredoxin reductase), MVDP and DDH [33,75,76]. AKR1B10 is an important host-protection reductase, detoxifying electrophilic carbonyls and their glutathione conjugates at physiological levels [13], modulating the levels of cellular signalling molecule retinoic acid [77] and inactivating cytostatic anticancer agents such as daunorubicin [14]. AKR1B10 is secreted as an enzymatically active protein, suggesting its potential role as a carbonyl scavenger or retinoic acid modulator in cell spaces or far-distant organs, particularly in inflammatory tissues with enhanced oxidative stress and lipid peroxidation [78]. It is needless to say that due to overexpression in human tumours, the secretory feature of AKR1B10 may render it a serum marker for cancer.

In summary, the present study has identified AKR1B10 as a novel secretory protein through a lysosome-mediated non-classical pathway. In view of its overexpression in human tumours, this finding may indicate AKR1B10 as a potential serum marker for human malignant diseases.


Deliang Cao designed the research, obtained grant support and co-wrote the manuscript. Di-xian Luo performed all of the experiments (except immunohistochemistry and some cell culture) and co-wrote the manuscript. Mei Huang obtained patient consent and collected biopsies and ileal fluid. Jun Ma performed the immunohistochemistry experiments. Zachary Gao performed cell culture. Duan-fang Liao was involved in discussion of the results and manuscript revision prior to submission.


This work was supported in part by the National Cancer Institute [grant number CA122622] and the Department of Defense Breast Cancer Research Program [grant number BC083555].

Abbreviations: ACCA, acetyl-CoA carboxylase-α; ABC, ATP-binding cassette; AKR1B10, aldo-keto reductase family 1, member B10; ARF, ADP-ribosylation factor; CHX, cycloheximide; DDH, dihydrodiol dehydrogenase; DIDS, 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; Exo-1, 2-(4-fluorobenzoylamino)-benzoic acid methyl ester; FBS, fetal bovine serum; fMLP, N-formylmethionyl-leucyl-phenylalanine; Glib, glibenclamide; HBSS, Hank's balanced salt solution; HEK-293T, human embryonic kidney cells expressing the large T-antigen of simian virus 40; HMGB, high-mobility group B; HRP, horseradish peroxidase; HSP, heat-shock protein; IL, interleukin; IP3, myo-inositol 1,4,5-trisphosphate; LAMP, lysosome-associated membrane protein; MVDP, mouse vas deferens protein; PA, phosphatidic acid; PBST, PBS containing 0.05% Tween 20; PC, phosphatidylcholine; PI, phosphatidylinositol; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PLD, phospholipase D


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