Polphylipoprotein-induced autophagy mechanism with high performance in photodynamic therapy.

Polphylipoprotein (PLP) is a recently developed nanoparticle with high biocompatibility and tumor selectivity, and which has demonstrated unprecedentedly high performance photosensitizer in photodynamic therapy (PDT) and photodynamic diagnosis. On the basis of these discoveries, PLP is anticipated to have a very high potential for PDT. However, the mechanism by which PLP kills cancer cells effectively has not been sufficiently clarified. To comprehensively understand the PLP-induced PDT processes, we conduct multifaceted experiments using both normal cells and cancer cells originating from the same sources, namely, RGM1, a rat gastric epithelial cell line, and RGK1, a rat gastric mucosa-derived cancer-like mutant. We reveal that PLP enables highly effective cancer treatment through PDT by employing a unique mechanism that utilizes the process of autophagy. The dynamics of PLP-accumulated phagosomes immediately after light irradiation are found to be completely different between normal cells and cancer cells, and it becomes clear that this difference results in the manifestation of the characteristic effect of PDT when using PLP. Since PLP is originally developed as a drug delivery agent, this study also suggests the potential for intracellular drug delivery processes through PLP-induced autophagy.

Combined adipose-derived mesenchymal stem cell and antibiotic therapy can effectively treat periprosthetic joint infection in rats.

Periprosthetic joint infection (PJI) is characterized by biofilm infection, which is difficult to alleviate while preserving implant integrity. Furthermore, long-term antibiotic therapy may increase the prevalence of drug-resistant bacterial strains, necessitating a non-antibacterial approach. Adipose-derived stem cells (ADSCs) exert antibacterial effects; however, their efficacy in PJI remains unclear. This study investigates the efficacy of combined intravenous ADSCs and antibiotic therapy in comparison to antibiotic monotherapy in a methicillin-sensitive Staphylococcus aureus (MSSA)-infected PJI rat model. The rats were randomly assigned and equally divided into 3 groups: no-treatment group, antibiotic group, ADSCs with antibiotic group. The ADSCs with antibiotic group exhibited the fastest recovery from weight loss, with lower bacterial counts (p = 0.013 vs. no-treatment group; p = 0.024 vs. antibiotic group) and less bone density loss around the implants (p = 0.015 vs. no-treatment group; p = 0.025 vs. antibiotic group). The modified Rissing score was used to evaluate localized infection on postoperative day 14 and was the lowest in the ADSCs with antibiotic group; however, no significant difference was observed between the antibiotic group and ADSCs with antibiotic group (p < 0.001 vs. no-treatment group; p = 0.359 vs. antibiotic group). Histological analysis revealed a clear, thin, and continuous bony envelope, a homogeneous bone marrow, and a defined, normal interface in the ADSCs with antibiotic group. Moreover, the expression of cathelicidin expression was significantly higher (p = 0.002 vs. no-treatment group; p = 0.049 vs. antibiotic group), whereas that of tumor necrosis factor (TNF)-α and interleukin(IL)-6 was lower in the ADSCs with antibiotic group than in the no-treatment group (TNF-α, p = 0.010 vs. no-treatment group; IL-6, p = 0.010 vs. no-treatment group). Thus, the combined intravenous ADSCs and antibiotic therapy induced a stronger antibacterial effect than antibiotic monotherapy in a MSSA-infected PJI rat model. This strong antibacterial effect may be related to the increased cathelicidin expression and decreased inflammatory cytokine expression at the site of infection.

Cell-Level Analysis Visualizing Photodynamic Therapy with Porphylipoprotein and Talaporphyrin Sodium.

We revealed the difference in the mechanism of photodynamic therapy (PDT) between two photosensitizers: porphylipoprotein (PLP), which has recently attracted attention for its potential to be highly effective in treating cancer, and talaporphyrin sodium (NPe6). (1) NPe6 accumulates in lysosomes, whereas PLP is incorporated into phagosomes formed by PLP injection. (2) PDT causes NPe6 to generate reactive oxygen species, thereby producing actin filaments and stress fibers. In the case of PLP, however, reactive oxygen species generated by PDT remain in the phagosomes until the phagosomal membrane is destroyed, which delays the initiation of RhoA activation and RhoA*/ROCK generation. (4) After the disruption of the phagosomal membrane, however, the outflow of various reactive oxygen species accelerates the production of actin filaments and stress fibers, and blebbing occurs earlier than in the case of NPe6. (5) PLP increases the elastic modulus of cells without RhoA activity in the early stage. This is because phagosomes are involved in polymerizing actin filaments and pseudopodia formation. Considering the high selectivity and uptake of PLP into cancer cells, a larger effect with PDT can be expected by skillfully combining the newly discovered characteristics, such as the appearance of a strong effect at an early stage.

Large intraosseous chronic expanding hematoma after total hip arthroplasty presenting with chronic disseminated intravascular coagulation: a case report and literature review.

BACKGROUND: A chronic expanding hematoma (CEH) is a rare complication caused by surgery or trauma; it mostly affects the soft tissues, such as those in the trunk or extremities. We present the first case of a large intraosseous CEH presenting with chronic disseminated intravascular coagulation (DIC), 22 years after total hip arthroplasty (THA); the CEH was treated with a single-stage excision and revision THA. CASE PRESENTATION: A 67-year-old man presented to our hospital with left thigh pain and an enlarging mass. He had no history of trauma, anticoagulant use, or a collagen vascular disorder. The patient initially declined surgery. Two years later, radiographs and computed tomography images revealed progressive osteolysis, marginal sclerosis, and calcification in the left femur, in addition to loosening of the femoral component. Laboratory data revealed anemia and chronic DIC of unknown causes. Magnetic resonance imaging revealed a "mosaic sign" on the mass, indicating a mix of low- and high-signal intensities on T2-weighted images. Needle biopsy prior to surgery revealed no infection or malignant findings. An intraosseous CEH was suspected due to extensive osteolysis and loosening of the femoral component. No other factors that could induce chronic DIC were identified, such as sepsis, leukemia, cancer, trauma, liver disease, aneurysms, or hemangiomas. Therefore, we speculated that the anemia and chronic DIC were caused by the large intraosseous CEH. A single-stage revision THA with surgical excision was performed to preserve the hip function and improve the chronic DIC. The postoperative histopathological findings were consistent with an intraosseous CEH. The anemia and chronic DIC improved after 7 days. There was no recurrence of intraosseous CEH or chronic DIC at the 6-month follow-up. The left thigh pain improved, and the patient could ambulate with the assistance of a walking frame. CONCLUSIONS: The loosening of the femoral component caused persistent movement, which may have caused intraosseous CEH growth, anemia, and chronic DIC. It is important to differentiate CEHs from malignant tumors with hematomas. Furthermore, the "mosaic sign" noted in this case has also been observed on magnetic resonance images in other cases of CEH.

Direct analysis of the actin-filament formation effect in photodynamic therapy.

Photodynamic therapy (PDT) is a method in which a photosensitizer is administered in vivo and irradiated with light to generate reactive oxygen species (ROS), thereby causing the selective death of cancer cells. Since PDT is a noninvasive cancer treatment method with few adverse effects, it has attracted considerable attention and is increasingly used. In PDT, there are two dominant processes based on the actin filament (A-filament) formation effect: the destruction of cells by necrosis and vascular shutdown. Despite the importance of its fine control, the mechanism of the reaction process from the generation of reactive oxygen by photoinduction inducing the formation of A-filament and its polymerization to form stress fibers (S-fibers) has not yet been clarified because, for example, it has been difficult to directly observe and quantify such processes in living cells by conventional methods. Here, we have combined atomic force microscopy (AFM) with other techniques to reveal the mechanism of the A-filament and S-fiber formation processes that underlie the cell death process due to PDT. First, it was confirmed that activation of the small G protein RhoA, which is a signal that induces an increase in A-filament production, begins immediately after PDT treatment. The production of A-filament did not increase with increasing light intensity when the amount of light was large. Namely, the activation of RhoA reached an equilibrium state in about 1 min: however, the production of A-filament and its polymerization continued. The observed process corresponds well with the change in the amount of phosphorylated myosin-light chains, which induce A-filament polymerization. The increase in the elastic modulus of cells following the formation of S-fiber was confirmed by AFM for the first time. The distribution of generated A-filament and S-fiber was consistent with the photosensitizer distribution. PDT increases A-filament production, and when the ROS concentration is high, blebbing occurs and cells die, but when it is low, cell death does not occur and S-fiber is formed. That is, it is expected that vascular shutdown can be controlled efficiently by adjusting the amount of photosensitizer and the light intensity.

Near-Infrared Light Irradiation of Porphyrin-Modified Gold Nanoparticles Promotes Cancer-Cell-Specific Cytotoxicity.

The use of nanoparticles has been investigated as a new cancer treatment. These can induce specific cytotoxicity in cancer cells. In particular, Au nanoparticles (AuNPs) have unique characteristics. The maximum absorption spectrum of AuNPs can be adjusted to modify their size or shape to absorb near-infrared light that can penetrate into tissue without photodamage. Thus, the combination of AuNPs and near-infrared light can be used to treat cancer in deep-seated organs. To obtain effective cancer-specific accumulation of AuNPs, we focused on porphyrin and synthesized a porphyrin-attached Au compound: Au-HpD. In this study, we investigated whether Au-HpD possesses cancer-specific accumulation and cytotoxicity. Intracellular Au-HpD accumulation was higher in cancer cells than in normal cells. In order to analyze the cytotoxicity induced by Au-HpD, cancer cells and normal cells were co-cultured in the presence of Au-HpD; then, they were subjected to 870 nm laser irradiation. We observed that, after laser irradiation, cancer cells showed significant morphological changes, such as chromatin condensation and nuclear fragmentation indicative of cell apoptosis. This strong effect was not observed when normal cells were irradiated. Moreover, cancer cells underwent cell apoptosis with combination therapy.

Dabigatran Etexilate Induces Cytotoxicity in Rat Gastric Epithelial Cell Line via Mitochondrial Reactive Oxygen Species Production.

Dabigatran is a novel oral anticoagulant that directly inhibits free and fibrin-bound thrombins and exerts rapid and predictable anticoagulant effects. While the use of this reagent has been associated with an increased risk of gastrointestinal bleeding, the reason why dabigatran use increases gastrointestinal bleeding risk remains unknown. We investigated the cytotoxicity of dabigatran etexilate and tartaric acid, the two primary components of dabigatran. The cytotoxicity of dabigatran etexilate and tartaric acid was measured in a cell viability assay. Intracellular mitochondrial reactive oxygen species (mitROS) production and lipid peroxidation were measured using fluorescence dyes. Cell membrane viscosity was measured using atomic force microscopy. The potential of ascorbic acid as an inhibitor of dabigatran cytotoxicity was also evaluated. The cytotoxicity of dabigatran etexilate was higher than that of tartaric acid. Dabigatran etexilate induced mitROS production and lipid peroxidation and altered the cell membrane viscosity. Ascorbic acid inhibited the cytotoxicity and mitROS production induced by dabigatran etexilate. Therefore, we attributed the cytotoxicity of dabigatran to dabigatran etexilate, and proposed that the cytotoxic effects of dabigatran etexilate are mediated via mitROS production. Additionally, we demonstrated that dabigatran cytotoxicity can be prevented via antioxidant treatment.

The cytotoxicity of cyclophosphamide is enhanced in combination with monascus pigment.

Monascus pigment is derived from red-mold rice fermented by monascus purpureus and utilized as a natural coloring agent and natural food additive in East Asia. Monascus pigment works as a radical scavenger. Some antioxidant combine cancer chemo-therapy to protect normal tissue because chemotherapy induce side effect for normal tissue. This combination therapy can attenuate the cytotoxicity of anti-cancer drugs by antioxidants effects. However, the effect of this combination therapy for cancer cells dose not investigate enough. In this study, we investigated the combination effect of anti-oxidants and anti-cancer drugs. We selected an anti-oxidant as monascus pigment and following four anti-cancer drugs: doxorubicin, tamoxifen, paclitaxicel, and cyclophosphamide. Combination treatment with monascus pigment and cyclophosphamide enhanced the cytotoxicity of cyclophosphamide. Moreover, this combination treatment accelerated apoptosis. The spot on TLC assay board of the monascus pigment and cyclophosphamide mixture is different from the spot of monascus pigment alone and cyclophosphamide alone. The interaction between monascus pigment and cyclo-phosphamide can produce some cytotoxicity compounds or accelerate intracellular cyclophosphamide accumulation. Hence, we concluded that the interaction of both cyclophosphamide and monascus pigment involved enhancement of cyclophosphamide cytotoxicity.

Factors Associated With Patient Satisfaction After Opening-Wedge High Tibial Osteotomy.

BACKGROUND: Opening-wedge high tibial osteotomy (OWHTO) is expected to result in higher patient satisfaction compared with knee arthroplasty due to joint preservation. However, patient satisfaction rates as well as factors associated with satisfaction after OWHTO remain unclear. PURPOSE: To evaluate patient subjective satisfaction after OWHTO and determine factors associated with patient satisfaction after OWHTO. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: This study enrolled 123 patients (123 knees) who underwent unilateral OWHTO. Clinical parameters, including range of motion (ROM), visual analog scale (VAS) score for pain, Knee injury and Osteoarthritis Outcome Score (KOOS), weightbearing line ratio (WBLR), and medial proximal tibial angle (MPTA), were assessed before surgery and at the final follow-up at a minimum of 2 years. Patient satisfaction was evaluated using a 5-point scale regarding (1) surgery, (2) pain relief, (3) knee mobility, (4) daily living function, and (5) lower extremity alignment. The mean overall satisfaction scores for the 5 questions were calculated, and the sample was divided into 2 main groups (satisfied or unsatisfied). Preoperative characteristics, physical activity level, patient expectations for surgery, ROM, and KOOS were compared between the groups. Cartilage regeneration was assessed at the time of plate removal, and WBLR and MPTA were also assessed. Factors associated with patient satisfaction were analyzed using multivariable logistic regression analysis. RESULTS: The mean ± SD follow-up was 54.6 ± 20.6 months. The mean WBLR significantly changed from 20.7% ± 11.8% preoperatively to 66.9% ± 10.2% at the final follow-up, and all KOOS subscale scores significantly improved after surgery. Of the 123 patients, 109 (88.6%) were graded as satisfied. Factors associated with patient satisfaction were expectations met (odds ratio, 17.4; P = .026), better postoperative KOOS Pain score (odds ratio, 1.30; P = .001), and better postoperative KOOS Activities of Daily Living score (odds ratio, 1.36; P = .002). CONCLUSION: OWHTO is an effective treatment in terms of subjective satisfactory outcomes. Patient expectations for surgery have a significant effect on patient satisfaction. Surgeons should consider patient expectations before OWHTO and provide patient education to improve patient satisfaction.

Nitric oxide regulates the expression of heme carrier protein-1 via hypoxia inducible factor-1α stabilization.

Photodynamic therapy (PDT) is a cancer therapy that capitalizes on cancer-specific porphyrin accumulation. We have investigated this phenomenon to propose the following three conclusions: 1) the mechanism underlying this phenomenon is closely related to both nitric oxide (NO) and heme carrier protein-1 (HCP-1), 2) NO inactivates ferrochelatase, and thus, the intracellular porphyrin levels in the cells are increased by the administration of an NO donor after 5-aminolevulinic acid treatment, 3) HCP-1 transports not only heme but also other porphyrins. Since NO stabilizes hypoxia-inducible factor (HIF)-1α, resulting in the upregulation of heme biosynthesis, HCP-1 expression can be increased by HIF-1α stabilization. In this study, we determined whether NO regulates HCP-1 expression by stabilizing HIF-1α expression. For this purpose, rat gastric cancer cell line RGK36 was treated with L-arginine or N6-(1-iminoethyl)-L-lysine (L-NIL). L-arginine treatment increased the intracellular NO concentration, and both HCP-1 and HIF-1α expression, while L-NIL treatment decreased them. Cytotoxicity of PDT was enhanced by L-arginine, following intracellular hemato-porphyrin dihydrochloride (HpD) accumulation. Both Cytotoxicity of PDT and HpD accumulation were decreased by L-NIL. The HCP-1 and HIF-1α expression, intracellular HpD accumulation and PDT cytotoxicity were decreased by 2-methoxyestradiol, which is a HIF-1α inhibitor. Moreover, these phenomena were not increased by a combination of both L-arginine and 2-Me. Thus, HCP-1 can be a downstream target of HIF-1α. These effects were also induced in the human gastric cancer cell line MKN45. Taken together, we conclude that HCP-1 expression is regulated by NO via HIF-1α stabilization.