HMGB2 Promotes De Novo Lipogenesis to Accelerate Hepatocyte Proliferation During Liver Regeneration.

Liver regeneration is a well-orchestrated compensatory process that is regulated by multiple factors. We recently reported the importance of the chromatin protein, a high-mobility group box 2 (HMGB2) in mouse liver regeneration. However, the molecular mechanism remains unclear. In this study, we aimed to study how HMGB2 regulates hepatocyte proliferation during liver regeneration. Seventy-percent partial hepatectomy (PHx) was performed in wild-type (WT) and HMGB2-knockout (KO) mice, and the liver tissues were used for microarray, immunohistochemistry, quantitative polymerase chain reaction (qPCR), and Western blotting analyses. In the WT mice, HMGB2-positive hepatocytes colocalized with cell proliferation markers. In the HMGB2-KO mice, hepatocyte proliferation was significantly decreased. Oil Red O staining revealed the transient accumulation of lipid droplets at 12-24 hr after PHx in the WT mouse livers. In contrast, decreased amount of lipid droplets were found in HMGB2-KO mouse livers, and it was preserved until 36 hr. The microarray, immunohistochemistry, and qPCR results demonstrated that the expression of lipid metabolism-related genes was significantly decreased in the HMGB2-KO mouse livers. The in vitro experiments demonstrated that a decrease in the amount of lipid droplets correlated with decreased cell proliferation activity in HMGB2-knockdown cells. HMGB2 promotes de novo lipogenesis to accelerate hepatocyte proliferation during liver regeneration.

The crucial role of SETDB1 in structural and functional transformation of epithelial cells during regeneration after intestinal ischemia reperfusion injury.

Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75 min of ischemia followed by 3, 24, and 48 h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24 h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

Protective role of estrogen through G-protein coupled receptor 30 in a colitis mouse model.

Estrogen and its receptors are involved in the pathogenesis of gastrointestinal diseases such as colitis. However, the role of the membrane estrogen receptor G-protein-coupled receptor 30 (GPR30) in colitis is poorly understood. We therefore investigated the effect of estrogen in dextran sulfate sodium (DSS)-induced colitis. Male C57BL/6 mice were administered 1.5% DSS for 5 days and treated with 17ß-estradiol (E2), GPR30 agonist (G1), or GPR30 antagonist (G15) for 8 days. Inflammation grade was evaluated by disease activity index (DAI) and histomorphological score. Colon tissues were immunohistochemically analyzed and revealed high expression of membrane GPR30, histone 3 lysine 36 dimethylation, and lysine 79 trimethylation in normal mouse colon epithelial cells but significantly decreased expression in DSS-treated mice, whereas the expression was partially preserved after treatment with E2 or G1. Colon shortening and DAI were significantly lower in E2- and G1-treated mice compared to DSS-treated mice. Caudal type homeobox 2 (CDX2) expression and cell proliferation differed in normal colon epithelial cells but overlapped in those of DSS-treated mice. Administration of E2 and G1 reduced CDX2 expression and cell proliferation. Altered expression of claudin-2 and occludin were observed in the colonic epithelium of DSS-treated mice, and these changes were significantly lower in the colon of E2- and G1-treated mice. These results indicate that estrogen regulates histone modification, cell proliferation, and CDX2 expression through GPR30, which affects intestinal epithelial barrier function. We conclude that estrogen protects against intestinal epithelial damage through GPR30 by enhancing intestinal epithelial barrier function in DSS-induced colitis in mice.

Tmem161a regulates bone formation and bone strength through the P38 MAPK pathway.

Bone remodeling is an extraordinarily complex process involving a variety of factors, such as genetic, metabolic, and environmental components. Although genetic factors play a particularly important role, many have not been identified. In this study, we investigated the role of transmembrane 161a (Tmem161a) in bone structure and function using wild-type (WT) and Tmem161a-depleted (Tmem161aGT/GT) mice. Mice femurs were examined by histological, morphological, and bone strength analyses. Osteoblast differentiation and mineral deposition were examined in Tmem161a-overexpressed, -knockdown and -knockout MC3T3-e1 cells. In WT mice, Tmem161a was expressed in osteoblasts of femurs; however, it was depleted in Tmem161aGT/GT mice. Cortical bone mineral density, thickness, and bone strength were significantly increased in Tmem161aGT/GT mice femurs. In MC3T3-e1 cells, decreased expression of alkaline phosphatase (ALP) and Osterix were found in Tmem161a overexpression, and these findings were reversed in Tmem161a-knockdown or -knockout cells. Microarray and western blot analyses revealed upregulation of the P38 MAPK pathway in Tmem161a-knockout cells, which referred as stress-activated protein kinases. ALP and flow cytometry analyses revealed that Tmem161a-knockout cells were resistant to oxidative stress. In summary, Tmem161a is an important regulator of P38 MAPK signaling, and depletion of Tmem161a induces thicker and stronger bones in mice.

Clec4A4 Acts as a Negative Immune Checkpoint Regulator to Suppress Antitumor Immunity.

Clec4A4 is a C-type lectin receptor (CLR) exclusively expressed on murine conventional dendritic cells (cDC) to regulate their activation status. However, the functional role of murine Clec4A4 (mClec4A4) in antitumor immunity remains unclear. Here, we show that mClec4A4 serves as a negative immune checkpoint regulator to impair antitumor immune responses. Deficiency of mClec4A4 lead to a reduction in tumor development, accompanied by enhanced antitumor immune responses and amelioration of the immunosuppressive tumor microenvironment (TME) mediated through the enforced activation of cDCs in tumor-bearing mice. Furthermore, antagonistic mAb to human CLEC4A (hCLEC4A), which is the functional orthologue of mClec4A4, exerted protection against established tumors without any apparent signs of immune-related adverse events in hCLEC4A-transgenic mice. Thus, our findings highlight the critical role of mClec4A4 expressed on cDCs as a negative immune checkpoint molecule in the control of tumor progression and provide support for hCLEC4A as a potential target for immune checkpoint blockade in tumor immunotherapy.

A Preliminary Pathological Evaluation of Extracellular Volume Fraction with Contrast-enhanced Computed Tomography as a Novel Quantitative Parameter of Pancreatic Fibrosis.

Objective The extracellular volume (ECV) calculated based on contrast-enhanced computed tomography (CT) has been reported as a novel imaging parameter reflecting the morphological change of fibrosis in several parenchymal organs. Our retrospective study assessed the validity of the ECV fraction for diagnosing pancreatic fibrosis and the appropriate imaging condition as the "equilibrium phase". Methods In 27 patients undergoing multiphasic CT and subsequent pancreaticoduodenectomy, we investigated pathological fibrotic changes related to the ECV fraction and conducted analyses using the value obtained by subtracting the equilibrium CT value of the portal vein from that of the abdominal aorta (Ao-PVequilibrium) to estimate eligibility of the equilibrium phase. Results In all patients, the ECV fraction showed a weak positive correlation with the collagenous compartment ratio (r=0.388, p=0.045). All patients were divided into two groups - the high-Ao-PVequilibrium group and low-Ao-PVequilibrium group - based on the median value. No significant correlation was found in the high-Ao-PVequilibrium group, whereas a significant correlation was observed in the low-Ao-PVequilibrium group (r=0.566, p=0.035). Conclusion The ECV fraction is a possible predictive factor for histopathological pancreatic fibrosis. In its clinical application, the eligibility of the "equilibrium phase" may affect the diagnostic capability. It will be necessary to verify the imaging conditions in order to improve the accuracy of the diagnosis.

Gut dysbiosis promotes the breakdown of oral tolerance mediated through dysfunction of mucosal dendritic cells.

While dysbiosis in the gut is implicated in the impaired induction of oral tolerance generated in mesenteric lymph nodes (MesLNs), how dysbiosis affects this process remains unclear. Here, we describe that antibiotic-driven gut dysbiosis causes the dysfunction of CD11c+CD103+ conventional dendritic cells (cDCs) in MesLNs, preventing the establishment of oral tolerance. Deficiency of CD11c+CD103+ cDCs abrogates the generation of regulatory T cells in MesLNs to establish oral tolerance. Antibiotic treatment triggers the intestinal dysbiosis linked to the impaired generation of colony-stimulating factor 2 (Csf2)-producing group 3 innate lymphoid cells (ILC3s) for regulating the tolerogenesis of CD11c+CD103+ cDCs and the reduced expression of tumor necrosis factor (TNF)-like ligand 1A (TL1A) on CD11c+CD103+ cDCs for generating Csf2-producing ILC3s. Thus, antibiotic-driven intestinal dysbiosis leads to the breakdown of crosstalk between CD11c+CD103+ cDCs and ILC3s for maintaining the tolerogenesis of CD11c+CD103+ cDCs in MesLNs, responsible for the failed establishment of oral tolerance.

Extracellular Volume Fraction Calculated Using Contrast-Enhanced Computed Tomography as a Biomarker of Oxaliplatin-Induced Sinusoidal Obstruction Syndrome: A Preliminary Histopathological Analysis.

Background: Oxaliplatin (OX)-based chemotherapy induces sinusoidal obstruction syndrome (SOS) in the nontumorous liver parenchyma, which can increase the risk of liver resection due to colorectal liver metastasis (CRLM). The extracellular volume (ECV) calculated from contrast-enhanced computed tomography (CT) has been reported to reflect the morphological change of hepatic fibrosis. The present retrospective study aimed to evaluate the ECV fraction as a predictive factor for OX-induced SOS. Methods: Our study included 26 patients who underwent liver resection for CRLM after OX-based chemotherapy with a preoperative dynamic CT of appropriate quality. We investigated the relationship between the pathological SOS grade and the ECV fraction. Results: Overall, 26 specimens from the patients were graded with the SOS classification of Rubbia-Brandt et al. as follows: grade 0, n = 17 (65.4%); grade 1, n = 4 (15.4%); and grade 2, n = 5 (19.2%). No specimens showed grade 3 SOS. In a univariate analysis, the ECV fraction in grade 0 SOS was significantly lower than that in grade 1 + 2 SOS (26.3 ± 3.4% vs. 30.6 ± 7.0%; P = 0.025). The cutoff value and AUC value of the ECV fraction to distinguish between grades 0 and 1 + 2 were 27.5% and 0.771, respectively. Conclusions: Measurement of the ECV fraction was found to be a potential noninvasive diagnostic method for determining early-stage histopathological sinusoidal injury induced by OX-based chemotherapy.

Pivotal role of High-Mobility Group Box 2 in ovarian folliculogenesis and fertility.

BACKGROUND: High-Mobility Group Box 1 (HMGB1) and HMGB2 are chromatin-associated proteins that belong to the HMG protein family, and are involved in the regulation of DNA transcription during cell differentiation, proliferation and regeneration in various tissues. However, the role of HMGB2 in ovarian folliculogenesis is largely unknown. METHODS: We investigated the functional role of HMGB1 and HMGB2 in ovarian folliculogenesis and fertilization using C57BL/6 wild type (WT) and HMGB2-knockout (KO) mice. Ovarian tissues were obtained from WT and HMGB2-KO mice at postnatal days 0, 3, 7, and 2, 6 months of age, then performed immunohistochemistry, qPCR and Western blotting analyses. Oocyte fertilization capability was examined by natural breeding and in vitro fertilization experiments. RESULTS: In HMGB2-KO mice, ovary weight was decreased due to reduced numbers of oocytes and follicles. Natural breeding and in vitro fertilization results indicated that HMGB2-KO mice are subfertile, but not sterile. Immunohistochemistry showed that oocytes expressed HMGB2, but not HMGB1, in neonatal and adult WT ovaries. Interestingly, in HMGB2-KO ovaries, a compensatory increase in HMGB1 was found in oocyte nuclei of neonatal and 2-month-old mice; however, this was lost at 6 months of age. CONCLUSIONS: The depletion of HMGB2 led to alterations in ovarian morphology and function, suggesting that HMGB2 plays an essential role in ovarian development, folliculogenesis and fertilization.

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.

EPLINß Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINß. In this study, we investigated the role of EPLINß in osteoblasts using EPLINß-deficient (EPLINßGT/GT ) mice. The skeletal phenotype of EPLINßGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINßGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINßGT/GT mice, suggesting aberrant cell adhesion. In EPLINßGT/GT osteoblasts, α- and ß-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINß knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINß knockdown cells, suggesting an important role for EPLINß in osteoblast formation. In conclusion, we propose that EPLINß is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

Spatiotemporal expression of HMGB2 regulates cell proliferation and hepatocyte size during liver regeneration.

Liver regeneration is an extraordinarily complex process involving a variety of factors; however, the role of chromatin protein in hepatocyte proliferation is largely unknown. In this study, we investigated the functional role of high-mobility group box 2 (HMGB2), a chromatin protein in liver regeneration using wild-type and HMGB2-knockout (KO) mice. Liver tissues were sampled after 70% partial hepatectomy (PHx), and analyzed by immunohistochemistry, western blotting and flow cytometry using various markers of cell proliferation. In WT mice, hepatocyte proliferation was strongly correlated with the spatiotemporal expression of HMGB2; however, cell proliferation was significantly delayed in hepatocytes of HMGB2-KO mice. Quantitative PCR demonstrated that cyclin D1 and cyclin B1 mRNAs were significantly decreased in HMGB2-KO mice livers. Interestingly, hepatocyte size was significantly larger in HMGB2-KO mice at 36-72 h after PHx, and these results suggest that hepatocyte hypertrophy appeared in parallel with delayed cell proliferation. In vitro experiments demonstrated that cell proliferation was significantly decreased in HMGB2-KO cells. A significant delay in cell proliferation was also found in HMGB2-siRNA transfected cells. In summary, spatiotemporal expression of HMGB2 is important for regulation of hepatocyte proliferation and cell size during liver regeneration.

Crucial role of high-mobility group box 2 in mouse ovarian follicular development through estrogen receptor beta.

High-mobility group box 2 (HMGB2) is a chromatin-associated protein that is an important regulator of gene transcription, recombination, and repair processes. The functional importance of HMGB2 has been reported in various organs, including the testis, heart, and cartilage. However, its role in the ovary is largely unknown. In this study, ovary tissues from wild-type (WT) and HMGB2-knock-out (KO) mice were examined by histopathological staining and immunohistochemistry. The ovary size and weight were significantly lower in HMGB2-KO mice than in age-matched WT littermates. Histopathological analysis revealed ovarian atrophy and progressive fibrosis in 10-month-old HMGB2-KO mouse ovaries. Compared to age-matched WT mice, the numbers of oocytes and developing follicles were significantly decreased at 2 months of age and were completely depleted at 10 months of age in HMGB2-KO mice. Immunohistochemistry revealed the expression of HMGB2 in the granulosa cells of developing follicles, oocytes, some corpora lutea, and stromal cells. Importantly, HMGB2-positive cells were co-localized with estrogen receptor beta (ERß), but not ERα. Estrogen response element-binding activity was demonstrated by southwestern histochemistry, and it was decreased in HMGB2-KO mouse ovaries. Cell proliferation activity was also decreased in HMGB2-KO mouse ovaries in parallel with the decreased folliculogenesis. These results indicated that the depletion of HMGB2 induced ovarian atrophy that was characterized by a decreased ovarian size and weight, progressive fibrosis, as well as decreased oocytes and folliculogenesis. In conclusion, we demonstrated the crucial role of HMGB2 in mouse ovarian folliculogenesis through ERß expression.

Depletion of high-mobility group box 2 causes seminiferous tubule atrophy via aberrant expression of androgen and estrogen receptors in mouse testis†.

High-mobility group box 2, a chromatin-associated protein that interacts with deoxyribonucleic acid, is implicated in multiple biological processes, including gene transcription, replication, and repair. High-mobility group box 2 is expressed in several tissues, including the testis; however, its functional role is largely unknown. Here, we elucidated the role of high-mobility group box 2 in spermatogenesis. Paraffin-embedded testicular tissues were obtained from 8-week-old and 1-year-old wild-type and knock-out mice. Testis weight and number of seminiferous tubules were decreased, whereas atrophic tubules were increased in high-mobility group box 2-depleted mice. Immunohistochemistry revealed that atrophic tubules contained Sertoli cells, but not germ cells. Moreover, decreased cell proliferation and increased apoptosis were demonstrated in high-mobility group box 2-depleted mouse testis. To elucidate the cause of tubule atrophy, we examined the expression of androgen and estrogen receptors, and the results indicated aberrant expression of androgen receptor and estrogen receptor alpha in Sertoli and Leydig cells. Southwestern histochemistry detected decreased estrogen response element-binding sites in high-mobility group box 2-depleted mouse testis. High-mobility group box 1, which has highly similar structure and function as high-mobility group box 2, was examined by immunohistochemistry and western blotting, which indicated increased expression in testis. These findings indicate a compensatory increase in high-mobility group box 1 expression in high-mobility group box 2 knock-out mouse testis. In summary, depletion of high-mobility group box 2 induced aberrant expression of androgen receptor and estrogen receptor alpha, leading to decreased germ cell proliferation and increased apoptosis which resulted in focal seminiferous tubule atrophy.

Congenital Deficiency of Conventional Dendritic Cells Promotes the Development of Atopic Dermatitis-Like Inflammation.

Atopic dermatitis (AD) is a common pruritic inflammatory skin disease characterized by impaired epidermal barrier function and dysregulation of Thelper-2 (TH2)-biased immune responses. While the lineage of conventional dendritic cells (cDCs) are implicated to play decisive roles in T-cell immune responses, their requirement for the development of AD remains elusive. Here, we describe the impact of the constitutive loss of cDCs on the progression of AD-like inflammation by using binary transgenic (Tg) mice that constitutively lacked CD11chi cDCs. Unexpectedly, the congenital deficiency of cDCs not only exacerbates the pathogenesis of AD-like inflammation but also elicits immune abnormalities with the increased composition and function of granulocytes and group 2 innate lymphoid cells (ILC2) as well as B cells possibly mediated through the breakdown of the Fms-related tyrosine kinase 3 ligand (Flt3L)-mediated homeostatic feedback loop. Furthermore, the constitutive loss of cDCs accelerates skin colonization of Staphylococcus aureus (S. aureus), that associated with disease flare. Thus, cDCs maintains immune homeostasis to prevent the occurrence of immune abnormalities to maintain the functional skin barrier for mitigating AD flare.

In situ strategy for biomedical target localization via nanogold nucleation and secondary growth.

Immunocytochemistry visualizes the exact spatial location of target molecules. The most common strategy for ultrastructural immunocytochemistry is the conjugation of nanogold particles to antibodies as probes. However, conventional nanogold labelling requires time-consuming nanogold probe preparation and ultrathin sectioning of cell/tissue samples. Here, we introduce an in situ strategy involving nanogold nucleation in immunoenzymatic products on universal paraffin/cryostat sections and provide unique insight into nanogold development under hot-humid air conditions. Nanogold particles were specifically localized on kidney podocytes to target synaptopodin. Transmission electron microscopy revealed secondary growth and self-assembly that could be experimentally controlled by bovine serum albumin stabilization and phosphate-buffered saline acceleration. Valuable retrospective nanogold labelling for gastric H+/K+-ATPase was achieved on vintage immunoenzymatic deposits after a long lapse of 15 years (i.e., 15-year-old deposits). The present in situ nanogold labelling is anticipated to fill the gap between light and electron microscopy to correlate cell/tissue structure and function.

Essential role of submandibular lymph node dendritic cells in protective sublingual immunotherapy against murine allergy.

While sublingual immunotherapy (SLIT) is known as an allergen-specific treatment for type-1 allergies, how it controls allergic pathogenesis remains unclear. Here, we show the prerequisite role of conventional dendritic cells in submandibular lymph nodes (ManLNs) in the effectiveness of SLIT for the treatment of allergic disorders in mice. Deficiency of conventional dendritic cells or CD4+Foxp3+ regulatory T (Treg) cells abrogates the protective effect of SLIT against allergic disorders. Furthermore, sublingual antigenic application primarily induces antigen-specific CD4+Foxp3+ Treg cells in draining ManLNs, in which it is severely impaired in the absence of cDCs. In ManLNs, migratory CD11b+ cDCs are superior to other conventional dendritic cell subsets for the generation of antigen-specific CD4+Foxp3+ Treg cells, which is reflected by their dominancy in the tolerogenic features to favor this program. Thus, ManLNs are privileged sites in triggering mucosal tolerance mediating protect effect of SLIT on allergic disorders that requires a tolerogenesis of migratory CD11b+ conventional dendritic cells.

Author Correction: Pivotal role of CD103 in the development of psoriasiform dermatitis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Photodynamic Therapy Using a Novel Phosphorus Tetraphenylporphyrin Induces an Anticancer Effect via Bax/Bcl-xL-related Mitochondrial Apoptosis in Biliary Cancer Cells.

Photodynamic therapy (PDT) uses photosensitizer activation by light of a specific wavelength, and is a promising treatment for various cancers; however, the detailed mechanism of PDT remains unclear. Therefore, we investigated the anticancer effect of PDT using a novel phosphorus tetraphenylporphyrin (Ptpp) in combination with light emitting diodes (Ptpp-PDT) in the NOZ human biliary cancer cell line. Cell viability and apoptosis were examined by MTT assay, flow cytometry and TUNEL assay for 24 hr after Ptpp-PDT. MitoTracker and JC-1 were used as markers of mitochondrial localization and membrane potential. The levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes, Bcl-2 family proteins, cytochrome c and cleaved caspase-3 were examined by western blotting and immunohistochemistry. The results revealed that Ptpp localized to mitochondria, and that Ptpp-PDT efficiently decreased cell viability in a dose- and time-dependent manner. JC-1 and OXPHOS complexes decreased, but apoptotic cells increased from 6 to 24 hr after Ptpp-PDT. A decrease in Bcl-xL and increases in Bax, cytochrome c and cleaved caspase-3 were also found from 6 to 24 hr after Ptpp-PDT. Based on these results, we conclude that Ptpp-PDT induces anticancer effects via the mitochondrial apoptotic pathway by altering the Bax/Bcl-xL ratio, and could be an effective treatment for human biliary cancer.