Proteomic Identification and Quantification of Secretory Proteins in Human Dermal Fibroblast-Conditioned Medium for Wound Repair and Hair Regeneration.

Background: Human dermal fibroblasts secrete numerous growth factors and proteins that have been suggested to promote wound repair and hair regeneration. Methods: Human dermal fibroblast-conditioned medium (DFCM) was prepared, and proteomic analysis was performed. Secretory proteins in DFCM were identified using 1-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis, in-gel trypsin protein digestion, and quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS). Identified proteins were analyzed using bioinformatic methods for the classification and evaluation of protein-protein interactions. Results: Using LC-MS/MS, 337 proteins were identified in DFCM. Among them, 160 proteins were associated with wound repair, and 57 proteins were associated with hair regeneration. Protein-protein interaction network analysis of 160 DFCM proteins for wound repair at the highest confidence score (0.9) revealed that 110 proteins were grouped into seven distinctive interaction networks. Additionally, protein-protein interaction network analysis of 57 proteins for hair regeneration at the highest confidence score revealed that 29 proteins were grouped into five distinctive interaction networks. The identified DFCM proteins were associated with several pathways for wound repair and hair regeneration, including epidermal growth factor receptor, fibroblast growth factor, integrin, Wnt, cadherin, and transforming growth factor-ß signaling pathways. Conclusion: DFCM contains numerous secretory proteins that comprise groups of protein-protein interaction networks that regulate wound repair and hair regeneration.

TGF-ß1 upregulates Sar1a expression and induces procollagen-I secretion in hypertrophic scarring fibroblasts.

Hypertrophic scarring (HTS) is a common fibroproliferative disorder that typically follows thermal and other injuries involving the deep dermis. The underlying pathogenic mechanisms are regulated by transforming growth factor-ß (TGF-ß); however, the exact mechanisms in HTS have not been elucidated. We conducted this study to explore the cellular signaling mechanisms for expression of Sar1a, a coat protein complex II-associated small GTPase, in HTS fibroblasts (HTSF). We found that Sar1a was upregulated in HTSF as compared to that in normal fibroblasts. Furthermore, stimulation of TGF-ß1 increased the expression of Sar1a in HTSF, and small interfering RNA for Sar1a suppressed procollagen-I (PC-I) secretion. Next we investigated the signaling mechanism from TGF-ß1 to Sar1a expression and its association with PC-I secretion. In the presence of TGF-ß-activated kinase 1 (TAK1), c-Jun N-terminal kinase, or p38 inhibitors, the effect of TGF-ß1 on Sar1a expression and PC-I secretion significantly decreased; however, it had no effect on collagen-1A (Col-1A) expression. Further, the inhibitors of Smad3 or extracellular signal-regulated kinases inhibited TGF-ß1-induced Col-1A expression but had no effect on PC-I secretion and Sar1a expression. Taken together, our results suggested that TGF-ß1 induces Sar1a expression through TAK1 signaling and this signaling event regulates PC-I secretion in HTSF.

Sequential delivery of long-pulsed 755-nm alexandrite laser and long-pulsed 1,064-nm neodymium:yttrium-aluminum-garnet laser treatment for pigmented disorders.

BACKGROUND: Long-pulsed (LP) lasers at pulse durations of 1-300 ms have been used to destroy nests of nevi cells by selectively targeting pigment chromophores. OBJECTIVE: To evaluate dual-wavelength LP laser-induced tissue reactions. METHODS: The patterns of LP 755-nm alexandrite (Alex) and/or 1064-nm neodymium (Nd):yttrium-aluminum-garnet (YAG) laser-induced tissue reactions were macroscopically evaluated using a tattoo-embedded phantom. Additionally, a pilot in vivo human study was performed for common acquired melanocytic nevus, of which dermoscopic images and high-speed cinematographs were obtained. RESULTS: Combinations of Nd:YAG and Alex laser treatments at interpulse intervals of 10 or 20 ms generated round to oval zones of photothermal and photoacoustic injury in two distinctive areas containing disintegrated tattoo particles. Treatment at interpulse intervals of 10 or 20 ms between Alex and Nd:YAG pulses elicited lesser degrees of thermal damage to surrounding tissues, compared to treatment at 100 or 200 ms. Immediately after combined LP laser treatment of human nevus lesions in vivo, Nd:YAG-Alex treatment at a 20-ms interpulse interval exhibited more remarkable crusting and erosive appearances than Alex-Nd:YAG treatment. CONCLUSION: For treating pigmented disorders, sequential delivery of LP Nd:YAG and Alex pulses at short interpulse intervals of 10-20 ms can effectively destroy nests of pigment chromophores.

Interactive thermal tissue reactions of 7-MHz intense focused ultrasound and 1-MHz and 6-MHz radiofrequency on cadaveric skin.

BACKGROUND: Intense focused ultrasound (IFU) and radiofrequency (RF) systems generate thermal tissue reactions in multiple zones in the skin, with the microscopic features thereof varying according to energy sources and treatment parameters. OBJECTIVE: To evaluate interactive thermal tissue reactions of IFU and RF in cadaveric skin. METHODS: Thermal reaction patterns generated by IFU, invasive bipolar RF, and non-invasive monopolar RF treatments were analyzed in cadaveric skin of the inner thigh. Additionally, combination treatment, including IFU and invasive bipolar RF, IFU and non-invasive monopolar RF, invasive bipolar RF and IFU, and non-invasive monopolar RF and IFU, was delivered to cadaveric skin and microscopically evaluated. RESULTS: Combination treatment with 1.5-mm IFU followed by 1.5-mm invasive RF elicited multiple thermal injury zones of coagulation and ablation in the mid to lower dermis. Therein, IFU-induced thermal reactions were indistinguishable from RF-induced thermal reactions. Non-invasive RF treatment on IFU-pretreated cadaveric tissue specimens exhibited greater degrees of thermal injury, with wider and deeper penetration, compared to non-invasive RF treatment alone. Furthermore, RF-pretreated tissues showed marked differences in the patterns of IFU-induced thermal tissue reactions. CONCLUSION: Our data suggest that combination treatments with IFU and RF elicit various patterns of interactive thermal tissue reactions.

Pattern analysis of laser-tattoo interactions for picosecond- and nanosecond-domain 1,064-nm neodymium-doped yttrium-aluminum-garnet lasers in tissue-mimicking phantom.

During laser treatment for tattoo removal, pigment chromophores absorb laser energy, resulting in fragmentation of the ink particles via selective photothermolysis. The present study aimed to outline macroscopic laser-tattoo interactions in tissue-mimicking (TM) phantoms treated with picosecond- and nanosecond-domain lasers. Additionally, high-speed cinematographs were captured to visualize time-dependent tattoo-tissue interactions, from laser irradiation to the formation of photothermal and photoacoustic injury zones (PIZs). In all experimental settings using the nanosecond or picosecond laser, tattoo pigments fragmented into coarse particles after a single laser pulse, and further disintegrated into smaller particles that dispersed toward the boundaries of PIZs after repetitive delivery of laser energy. Particles fractured by picosecond treatment were more evenly dispersed throughout PIZs than those fractured by nanosecond treatment. Additionally, picosecond-then-picosecond laser treatment (5-pass-picosecond treatment + 5-pass-picosecond treatment) induced greater disintegration of tattoo particles within PIZs than picosecond-then-nanosecond laser treatment (5-pass-picosecond treatment + 5-pass-nanosecond treatment). High-speed cinematography recorded the formation of PIZs after repeated reflection and propagation of acoustic waves over hundreds of microseconds to a few milliseconds. The present data may be of use in predicting three-dimensional laser-tattoo interactions and associated reactions in surrounding tissue.

Tolerogenic dendritic cells show gene expression profiles that are different from those of immunogenic dendritic cells in DBA/1 mice.

Tolerogenic dendritic cells (tDCs) play an important role in inducing peripheral tolerance; however, few tDC-specific markers have been identified. The aims of this study were to examine whether tDCs show a different gene expression profile from that of immunogenic DCs and identify specific gene markers of each cell type, in DBA/1 mice. tDCs were generated by treating immature DCs (imDCs) with TNF-α and type II collagen. The gene expression profiles of mature (m)DCs and tDCs were then investigated by microarray analysis and candidate markers were validated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Supervised selection identified 75 gene signatures, 63 of which were consistently upregulated in mDCs and 12 of which were upregulated only in tDCs. Additionally, 10 genes were overexpressed or equally expressed in both tDCs and mDCs. Scin (tDC-specific genes) and Orm1, Pdlim4 and Enpp2 (mDC-specific genes) were validated by real-time qRT-PCR. Taken together, these results clearly show that tDCs and mDCs can be identified according to their expression of specific gene markers.

Serum reactivity against herpes simplex virus type 1 UL48 protein in Behçet's disease patients and a Behçet's disease-like mouse model.

Herpes simplex virus (HSV) infection is a possible pathogenic factor in Behçet's disease (BD). Using proteomics analysis, this study detected a target HSV protein. Serum IgA and IgG reactivities against the identified protein were evaluated in patients with BD and in BD-like mice. A total of 4 protein bands generated by immunoprecipitation were analysed by proteomics, and HSV UL48 was commonly found in both IgA- and IgG-reactive protein bands. Compared with controls, patients with BD and BD-like mice exhibited higher titres of IgA reacting with recombinant HSV UL48 protein. Further proteomics analysis revealed that human heat shock cognate 71 kDa protein (Hsc71) is a cross-reacting target antigen against anti-HSV UL48 antibody. In addition, our data demonstrated a very strong association between serum IgG reactivity against recombinant human Hsc71 and recombinant HSV UL48 in patients with BD. We suggest that HSV infection and impaired human Hsc71 activity may be associated with the activation of autoreactive lymphocytes.

Protein-protein interaction between caveolin-1 and SHP-2 is dependent on the N-SH2 domain of SHP-2.

Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) is known to protect neurons from neurodegeneration during ischemia/reperfusion injury. We recently reported that ROS-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 in astrocytes and complex formation between caveolin-1 and SHP-2 in response to oxidative stress. To examine the region of SHP-2 participating in complex formation with caveolin-1, we generated three deletion mutant constructs and six point mutation constructs of SHP-2. Compared with wild-type SHP-2, binding of the N-SH2 domain deletion mutant of SHP-2 to p-caveolin-1 was reduced greatly, using flow cytometric competitive binding assays and surface plasmon resonance (SPR). Moreover, deletion of the N-SH2 domain of SHP-2 affected H2O2-mediated ERK phosphorylation and Src phosphorylation at Tyr 419 in primary astrocytes, suggesting that N-SH2 domain of SHP-2 is responsible for the binding of caveolin-1 and contributes to the regulation of Src phosphorylation and activation following ROS-induced oxidative stress in brain astrocytes.

Decreased tissue and serum expression of galectin-7 in patients with hypertrophic scars.

Hypertrophic scars (HS) result from an imbalance between collagen biosynthesis and matrix degradation during wound healing. In this study a proteomics approach was used to compare the protein profiles of skin tissue obtained from patients with HS and healthy controls. One of the epidermal proteins, galectin-7 was markedly down-regulated in HS. Serum levels of galectin-7 in 27 patients with HS were less than 1/3 of those in 15 healthy controls. Tissue protein expression was subsequently evaluated using immunohistochemical staining on HS tissue and on serially-obtained control tissue during wound healing. Weaker galectin-7 immunoreactivity was detected along the cytoplasmic membrane of basal and suprabasal cells in samples from HS. In addition, galectin-7 was stained in the extracellular space of the upper papillary dermis in HS tissue. Ablative laser treatment, used to induce wound healing of healthy control tissue, demonstrated marked galectin-7 expression at the cytoplasmic membrane on days 3, 5, 14 and 21. Pronounced galectin-7 staining at the upper papillary dermis was detected on days 1, 3 and 10. These results suggest that the differences in galectin-7 expression and subcellular and extracellular distribution may be crucially involved in the pathogenic process of HS.

Anti-cyclic citrullinated peptide antibodies and joint involvement in Behçet's disease.

PURPOSE: We aimed to determine the prevalence of anti-cyclic citrullinated peptide (anti-CCP) antibodies in a large group of Korean patients with Behçet's disease (BD), with and without joint involvement, and to compare these findings with the prevalences of anti-CCP antibodies in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). MATERIALS AND METHODS: We tested 189 patients with BD, 105 with RA, and 36 with SLE for anti-CCP antibodies and IgM rheumatoid factor in serum. We reviewed the medical records of patients with BD to investigate their personal and clinical characteristics as well as their laboratory test results. RESULTS: Anti-CCP antibodies were detected in seven of the 189 BD patients (3.7%), at a mean titer of 30.6±44.4 U/mL, in 86 of the 105 RA patients (81.9%) with a mean titer of 198.8±205.7 U/mL, and in nine of the 36 SLE patients (25%) with a mean titer of 180.4±113.9 U/mL. One of the seven anti-CCP-positive BD patients fulfilled the diagnostic criteria for both BD and RA. Five of the seven anti-CCP-positive BD patients (71.4%) had polyarticular joint involvement, and the other two patients (28.6%) had oligoarticular involvement. CONCLUSION: We determined the prevalence of anti-CCP antibodies in a large group of Korean BD patients with and without joint involvement. Negative anti-CCP test in patients with BD may help to differentiate BD from RA and SLE, all of which present with similar clinical features.

The chaperone activity of α-synuclein: Utilizing deletion mutants to map its interaction with target proteins.

α-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, α-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. α-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. α-Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61-140 [α-syn(61-140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments α-syn(1-60) and α-syn(96-140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar α-synuclein had greater chaperone activity than nonfibrillar α-synuclein. It is concluded that the amyloidogenic NAC region (residues 61-95) contains the chaperone-binding site which is optimized for target protein binding as a result of its ß-sheet formation and/or ordered aggregation by α-synuclein. On the other hand, the first 60 residues of α-synuclein modulate the protein's chaperone-active site, while at the same time protecting α-synuclein from fibrillation. On its own, however, this fragment [α-syn(1-60)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of α-synuclein in its chaperone activity have been delineated.

Identification of HnRNP-A2/B1 as a target antigen of anti-endothelial cell IgA antibody in Behçet's disease.

Behçet's disease (BD) is a chronic, multisystemic vasculitis that theoretically affects all sizes and types of blood vessels. Although pathogenesis remains enigmatic, endothelial cells are believed to be the primary target in this disease. We detected the target protein using western blotting and immunoprecipitation and determined the amino-acid sequence of the peptide by liquid chromatography-matrix assisted laser desorption/ionization-tandem time-of-flight analysis (LC-MALDI-TOF/TOF). Serum reactivity against the recombinant target protein was analyzed by immunoblotting. Serum reactivity against streptococcal 65-kD heat shock protein (hsp-65) and the recombinant target protein was investigated by ELISA. The 36-40-kD protein band that was obtained from immunoprecipitation, which was analyzed by LC-MALDI-TOF/TOF, exhibited the amino-acid sequences of heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP-A2/B1). Reactivity of serum IgA against human recombinant hnRNP-A2/B1 was detected in 25 of 30 BD patients (83.3%), 4 of 30 systemic lupus erythematosus patients (13.3%), 8 of 30 rheumatoid arthritis patients (26.7%), 9 of 30 Takayasu's arteritis patients (30%), 6 of 30 healthy controls (20%), and none of 30 IgA nephropathy patients. Optical densities obtained from ELISAs against the recombinant human hnRNP-A2/B1 were correlated with those against the recombinant streptococcal hsp-65.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.

Expression of Pro-inflammatory Protein S100A12 (EN-RAGE) in Behçet's Disease and Its Association with Disease Activity: A Pilot Study.

BACKGROUND: S100A12 is a member of the S100 family of calcium-binding proteins and is secreted either in inflamed tissues or in the bloodstream by activated neutrophils. Expression of S100A12 has been reported in various diseases, especially non-infectious inflammatory diseases, such as Kawasaki disease, giant cell arteritis and inflammatory bowel disease. OBJECTIVE: This study was conducted to determine both the tissue expression and the serum levels of S100A12 in Behçet's disease (BD) patients and the correlation of the S100A12 serum level with disease activity of BD. METHODS: We included in this study ten BD patients who fulfilled the criteria for diagnosis, according to the International Study Group for BD. The activity of BD was calculated using the BD Current Activity Form. The serum concentrations of both S100A12 and interleukin-8 were measured by the enzyme-linked immunosorbent assay, before and after treatment. Immunohistochemical studies were also performed to detect S100A12 expression in the skin. RESULTS: The serum S100A12 level was significantly increased in the active BD period (p<0.001), in the inactive BD period (p=0.041) and in patients with active Kawasaki disease (p=0.028), compared with the serum level in the healthy controls. The serum S100A12 level decreased significantly from baseline, compared to post-treatment (p=0.017). The activity score of BD was significantly correlated with the serum level of S100A12 (Spearman's coefficient=0.464, p=0.039). Immunohistochemical studies showed that S100A12 was strongly expressed in the erythema nodosum-like skin lesions of patients. CONCLUSION: S100A12 contributes to the pathogenesis of BD related to neutrophil hyperactivity and reflects the disease activity in BD patients.

The insect peptide coprisin prevents Clostridium difficile-mediated acute inflammation and mucosal damage through selective antimicrobial activity.

Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the α-helical region of it (LLCIALRKK) that had antimicrobial activity (J.-S. Hwang et al., Int. J. Pept., 2009, doi:10.1155/2009/136284). Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.

Downregulation of erythropoietin receptor by overexpression of phospholipase C-gamma 1 is critical for decrease on focal adhesion in transformed cells.

BACKGROUND: Phospholipase C-γl (PLC-γl) is known to play a critical role in cell adhesion and migration and is highly expressed in metastatic tumors. In the current study, we found that cells transformed by PLC overexpression (PLC-γl cells) exhibited a marked decrease in expression of the Epo receptor (EpoR). Here, we assessed the role of EpoR-dependent signaling pathways in PLC-γl-dependent regulation of cell adhesion and migration. METHODS: Expression and phosphorylation of EpoR and its functional role in PLC-γl cells were evaluated by immunoblot analysis or cell adhesion assay. The mechanism for PLC-γ1-induced EpoR downregulation was analyzed by blockage of proteosomal degradation with MG132. EpoR expression was also confirmed in colorectal cancer tissues in which PLC-γl was highly expressed. RESULTS: EpoR was present on rat fibroblasts, where it functionally active and capable of increasing cell adhesion and migratory activity. However, PLC-γl cells significantly decreased the Epo-dependent effects via ubiquitination-proteosomal degradation of EpoR. A marked decrease of EpoR expression was confirmed in colorectal cancer tissues that showed high-level of PLC-γl expression. CONCLUSION: The Epo/EpoR complex plays a critical role in the adhesion and migration of rat fibroblasts, and its functional inactivation is associated with PLC-γl-dependent reduction of cell-matrix adhesion and this also affects cell migration.

Identification of streptococcal proteins reacting with sera from Behçet's disease and rheumatic disorders.

OBJECTIVES: We evaluated the reactivity of sera from Behçet's disease (BD), systemic lupus erythematosus (SLE), dermatomyositis (DM), rheumatoid arthritis (RA), and Takayasu's arteritis (TA) patients against human α-enolase and streptococcal α-enolase, and identified additional streptococcal antigens. METHODS: Enzyme-linked immunosorbent assay (ELISA) and immunoblotting were performed using sera from patients with BD, SLE, DM, RA, and TA and healthy volunteers (control) against human α-enolase and streptococcal α-enolase. Immunoblot analysis and matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry were used to identify and recombine other streptococcal antigens. RESULTS: Specific positive signals against recombinant human α-enolase were detected by IgM ELISA of serum samples from 50% of BD, 14.3% of SLE, 57.1% of DM, 42.9% of RA, and 57.1% of TA patients. Specific positive signals against streptococcal α-enolase were detected from 42.9% of BD, 14.3% of DM, and 14.3% of TA patients. No SLE and RA sera reacted against streptococcal α-enolase antigen. Streptococcal proteins reacting with sera were identified as hypothetical protein (HP) for SLE and DM patients, acid phosphatase (AP) for RA patients, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for TA patients. CONCLUSIONS: We observed that RA patients did not present serum reactivity against either HP or GAPDH though BD, SLE, DM, and TA patients did. Also, AP reacted with sera from BD, SLE, DM, RA, and TA patients.

Clostridium difficile toxin A decreases acetylation of tubulin, leading to microtubule depolymerization through activation of histone deacetylase 6, and this mediates acute inflammation.

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2',3'-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.

Evaluation of the role of hexokinase type II in cellular proliferation and apoptosis using human hepatocellular carcinoma cell lines.

UNLABELLED: The (18)F-FDG uptake pattern on PET could be an indicator of the prognosis and aggressiveness of various tumors, including hepatocellular carcinoma (HCC). Hexokinase, especially hexokinase type II (HKII), plays a critical role in (18)F-FDG uptake in rapidly growing tumors. We established a stable cell line overexpressing HKII by the transfection of full DNA of HKII to HCC cells (SNU449) that express low levels of HKII and investigated how (18)F-FDG uptake mechanisms, especially overexpression of HKII, are linked to tumor proliferation mechanisms. METHODS: The HKII gene was stably transfected to SNU449 cells with an expression vector. HKII expression in the cells was verified by reverse-transcriptase polymerase chain reaction, Western blot analysis, adenosine triphosphate (ATP) and lactate production, (18)F-FDG uptake measurement, and confocal microscopy. Cellular proliferation activity and response to the anticancer drug cisplatin were evaluated by cell counting using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. For the evaluation of molecular pathways involved in tumor proliferation, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was investigated. RESULTS: The stable cell line produced HKII effectively, but expression of other enzymes or transporters for glycolysis, such as glucose-6-phosphatase (G6Pase), HKI and III, and glucose transporter type 1 and 2 (Glut-1 and Glut-2), did not show any changes. (18)F-FDG uptake was significantly increased after transfection. ATP and lactate production was also increased after transfection. Overexpressed HKII was associated with mitochondria on confocal microscopy. Cells with overexpression of HKII, compared with the nontransfected cell line, showed 1.5- to 2-fold higher cell survival and resistance to the anticancer agent cisplatin (2- to 8-fold). In the molecular study, the activated form of Akt was increased after transfection, and PI3K inhibitor dissociated the mitochondrial HKII to the cytoplasm. In addition, the adenosine monophosphate-activated protein kinase (AMPK) pathway is also involved in Akt signaling. CONCLUSION: HKII plays an important role in (18)F-FDG uptake and tumor proliferation by both the PI3K-dependent and the PI3K-independent Akt signal pathways; therefore, the (18)F-FDG uptake pattern on a PET scan can be a surrogate marker of prognosis in HCC.