Calcium wave propagation during cell extrusion.

Oncogenically transformed or apoptotic cells are removed from epithelial sheets by cell-cell communication between the transformed/apoptotic cells (extruding cells) and the nearest neighboring cells. Cell extrusion is driven by actomyosin contraction and lamellipodial crawling of the nearest neighboring cells. Recent studies have found that distal cell communication also plays a role in cell extrusion. Specifically, distal cells located 3-16 cells away from the extruding cell are coordinated by calcium waves and collectively migrate toward the extruding cell to initiate cell extrusion. Here, I describe how calcium waves are generated and contribute to the extrusion of cells in mammals and zebrafish.

Safety and efficacy of brilliant blue g250 (BBG) for lens capsular staining: a phase III physician-initiated multicenter clinical trial.

PURPOSE: To evaluate the safety and efficacy of BBG (Brilliant Blue G250) for lens capsular staining during cataract surgery with continuous curvilinear capsulorhexis. STUDY DESIGN: Prospective clinical study. METHODS: This clinical trial enrolled 30 eyes of 30 patients who underwent cataract surgery with BBG (0.25 mg/mL Brilliant Blue G250) for capsular staining. Visualization of the lens capsule and the ease of capsulorhexis with BBG staining were evaluated in five grades (grade 0 to 4) by the Independent Data Monitoring Committee and the surgeons. The safety of BBG was also evaluated in terms of ocular and systemic tolerance for 7 days after surgery. RESULTS: The use of BBG improved visualization of the lens capsule and complete capsulorhexis was performed in all patients. The major endpoint (Independent Data Monitoring Committee evaluation) showed that use of BBG improved visualization of the lens capsule and the ease of capsulorhexis (grades 2 to 4); the committee's grading results were similar to those of the surgeons. Frequent complications observed in more than two eyes were conjunctival injection, corneal edema and intraocular pressure elevation. No severe complications were observed in ocular and systemic evaluations. CONCLUSION: BBG staining contributed to improved visualization of the lens capsule and aided in the completion of capsulorhexis during cataract surgery. The use of BBG for capsular staining also exhibited favorable safety results.

Targeting E3 Ubiquitin Ligases and Deubiquitinases in Ciliopathy and Cancer.

Cilia are antenna-like structures present in many vertebrate cells. These organelles detect extracellular cues, transduce signals into the cell, and play an essential role in ensuring correct cell proliferation, migration, and differentiation in a spatiotemporal manner. Not surprisingly, dysregulation of cilia can cause various diseases, including cancer and ciliopathies, which are complex disorders caused by mutations in genes regulating ciliary function. The structure and function of cilia are dynamically regulated through various mechanisms, among which E3 ubiquitin ligases and deubiquitinases play crucial roles. These enzymes regulate the degradation and stabilization of ciliary proteins through the ubiquitin-proteasome system. In this review, we briefly highlight the role of cilia in ciliopathy and cancer; describe the roles of E3 ubiquitin ligases and deubiquitinases in ciliogenesis, ciliopathy, and cancer; and highlight some of the E3 ubiquitin ligases and deubiquitinases that are potential therapeutic targets for these disorders.

Relationship between surrounding tissue morphology and directional collective migration of the posterior lateral line primordium in zebrafish.

Collective cell migration, in which cells assemble and move together, is an essential process in embryonic development, wound healing and cancer metastasis. Chemokine signaling guides cell assemblies to their destinations. In zebrafish posterior lateral line primordium (PLLP), a model system for collective cell migration, it has been proposed that the chemokine ligand Cxcl12a secreted from muscle pioneer cells (MPs) and muscle fast fibers (MFFs), which are distributed along with the horizontal midline, binds to the receptor Cxcr4b in PLLP and that Cxcl12a-Cxcr4b signaling guides the anterior-to-posterior migration of PLLP along the horizontal midline. However, how the surrounding tissues affect PLLP migration remains to be elucidated. Here, we investigated the relationship between the PLLP and the surrounding tissues and found that a furrow between the dorsal and ventral myotomes is generated by Sonic hedgehog (Shh) signaling-dependent MP and MFF differentiation and that the PLLP migrates in this furrow. When transient inhibition of Shh signaling impaired both the furrow formation and differentiation of cxcl12a-expressing MPs/MFFs, directional PLLP migration was severely perturbed. Furthermore, when differentiated MPs and MFFs were ablated by femtosecond laser irradiations, the furrow remained and PLLP migration was relatively unaffected. These results suggest that the furrow formation between the dorsal and ventral myotomes is associated with the migratory behavior of PLLP.

Calcium Wave Promotes Cell Extrusion.

When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.

Shootins mediate collective cell migration and organogenesis of the zebrafish posterior lateral line system.

The zebrafish sensory posterior lateral line is an excellent model system to study collective cell migration and organogenesis. Shootin1 is a cytoplasmic protein involved in neuronal polarization and axon guidance. Previous studies have shown that shootin1 couples actin filament retrograde flow with extracellular adhesive substrates at the leading edge of axonal growth cones, thereby producing mechanical force for the migration and guidance of axonal growth cones. However, the functions of shootin in peripheral cells remain unknown. Here we identified two novel shootin family members, shootin2 and shootin3. In zebrafish, shootin1 and shootin3 are expressed in the posterior lateral line primordium (PLLP) and neuromasts during embryonic development. A shootin1 mutant displayed a reduced speed of PLLP migration, while shootin1;shootin3 double mutation inhibited cell proliferation in the PLLP. Furthermore, our results suggest that shootin1 and shootin3 positively regulate the number of neuromasts and the number of cells in deposited neuromasts. Our study demonstrates that shootins mediate collective cell migration of the posterior lateral line primordium and formation of neuromasts in zebrafish.

Wide-field laser ophthalmoscopy for imaging of gas-filled eyes after macular hole surgery.

BACKGROUND AND OBJECTIVE: Existing ophthalmoscopy methods are unable to obtain clear fundus autofluorescence (FAF) images in gas-filled eyes. The purpose of this study was to evaluate the capability of wide-field laser ophthalmoscopy (Optos) in obtaining FAF images in gas-filled eyes for the assessment of macular hole (MH) closure after surgery. METHODS: This was an interventional case series. Eighteen consecutive patients with unilateral MH underwent vitrectomy with internal limiting membrane peeling and 20% sulfur hexafluoride gas tamponade. FAF images using Optos were recorded preoperatively and postoperatively (days 1, 2, and 7). RESULTS: On postoperative days 1, 2, and 7, FAF images were obtained from 11/18 (61.1%), 9/18 (50.0%), and 17/18 eyes (94.4%), respectively, using Optos. The quality of FAF images using Optos was sufficient to determine MH closure in 9/18 (50.0%) of gas-filled eyes postoperatively. Quantitative analysis of FAF images was helpful in determining complete or partial closure of the MH. CONCLUSION: FAF imaging using Optos might be a useful adjunct to optical coherence tomography as a supportive method to guide the release from facedown posturing in some cases of MH.

Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle formation in zebrafish.

Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called "collective cell migration," is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as "cell collectivity," remain largely unknown. During the formation of Kupffer's vesicle (KV, an organ of laterality in zebrafish), KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent findings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration.

Magnetic resonance imaging and thallium-201 scintigraphy for the diagnosis of localized pigmented villonodular synovitis arising from the elbow: A case report and review of the literature.

Pigmented villonodular synovitis (PVNS) arising from the elbow joint is extremely rare; only 24 cases have been reported. It is extremely difficult to differentiate PVNS from other soft tissue tumors on the basis of imaging findings alone. Therefore, a biopsy is required for definitive diagnosis. A 20-year-old female reported a mass on her right elbow. Physical examination revealed a tumor measuring 3.0x3.0 cm. Magnetic resonance imaging (MRI) revealed that the signal intensity of the tumor was isointense to muscle on T1-weighted images; however, it was hyper- or isointense to muscle on T2-weighted images. In images obtained by gadolinium-enhanced MRI, the margin of the tumor was well-contrasted. Thallium (Tl)-201 scintigrams revealed an abnormal accumulation in the area of the mass in the early and delayed phases. On the basis of clinical findings, imaging characteristics and incision biopsy results, localized PVNS was diagnosed and marginal excision was performed. We thus identified an extremely rare case of PVNS arising from the elbow joint. When interpreting Tl-201 images for the assessment of bone and soft tissue lesions, it is important to recognize PVNS as a condition that simulates malignant tumors. Furthermore, PVNS should be considered in the differential diagnosis when increased Tl-201 activity is closely related to the joint. MRI aids in the differentiation by demonstrating features of hemosiderin degradation products. These findings are likely to be extremely helpful in the differential diagnosis of bone and soft tissue tumors.

Clinical evaluation and feasibility of changing intraoperative visibility with a novel viewing filter system for human eye.

PURPOSE: The study was conducted to develop a new viewing system as a clinical prototype that enables visibility during surgery. METHODS: The system was composed of several filters attached to the microscope. This nonrandomized, retrospective, observational case series study involved 33 eyes from 32 patients who presented with various diseases and underwent surgery. The authors evaluated the changes in visualization focusing on controlling intraoperative visibility under air infusion and enhancing Brilliant Blue G staining focusing a sharp-cut filter Y (SCY). Visibility was compared under various surgical conditions, including cataract surgery, both with and without this system. Quantitative analysis of changes in intraoperative reflection including halation under air infusion and Brilliant Blue G intensity was carried out using the International Commission on Illumination 1976 (L*, a*, b*) color space method. RESULTS: A SCY reduced the reflection and halation by a maximum of 69.6%, when compared with use of no filter under air infusion (P < 0.01). The color difference between Brilliant Blue G-stained and nonstained areas was improved by 127.8% relative to values with no filter and using SCY (P < 0.01) in macular hole cases. Furthermore, in cataract surgery with corneal opacity, improvement of visibility was observed by SCY insertion. CONCLUSION: The system improved intraoperative visibility under air infusion and the Brilliant Blue G staining intensity by use of SCY during vitrectomy.

Quantitative analysis of vitreous and plasma concentrations of brilliant blue g after use as a surgical adjuvant in chromovitrectomy.

PURPOSE: To measure the concentration of brilliant blue G (BBG) in vitreous and plasma after use as a surgical adjuvant for staining and peeling of the internal limiting membrane to determine potential systemic adverse effects. METHODS: This study was designed as a prospective, interventional, clinical, case series. Five eyes from five patients with macular hole or epiretinal membrane underwent BBG-assisted internal limiting membrane and epiretinal membrane removal. The vitreous samples were obtained and stored at the end of surgery in all five cases. The plasma specimens were extracted and stored at the end of the operation, after 4 hours, and after 7 days post operation. For BBG analysis of plasma and vitreous, high-performance liquid chromatography coupled with tandem mass spectrometric detection was used. RESULTS: Brilliant blue G was not detected in plasma from all five cases at the three points of measurement. The mean vitreous BBG concentration was 34.5 ± 23.7 ng/mL (range, 11.3-70.9 ng/mL). Postoperative progress was good, and adverse effects were not observed in any of the five cases. CONCLUSION: Brilliant blue G, which remained at low levels in the vitreous cavity, was not found in the systemic blood flow after the operation. Thus, any adverse effects of systemic BBG would be avoided.

Zoledronic acid significantly enhances radiation­induced apoptosis against human fibrosarcoma cells by inhibiting radioadaptive signaling.

Zoledronic acid (ZOL), a third-generation bisphosphonate, inhibits bone resorption, as well as exhibiting direct antitumor activity. To date, however, the combined effects of ZOL and ionizing radiation (IR) have not been assessed in patients with soft tissue sarcoma. We have, therefore, assessed the combined effects of ZOL and IR in fibrosarcoma cells. HT1080 fibrosarcoma cells were treated with ZOL and/or IR, together or sequentially and the antitumor effects were assessed. We found that ZOL significantly enhanced IR-induced apoptosis, especially when cells were treated with ZOL followed by IR. We, therefore, assessed the detailed mechanism of sequential treatment with ZOL and IR. Cells in G2 and M phases, the most radiosensitive phases of the cell cycle, were not increased by low concentrations of ZOL. However, the levels of expression of Akt, ERK1/2 and NF-κB proteins, all of which are related to radioadaptive resistance, were increased within a short time after irradiation with 3 Gy, and this expression was inhibited by a low concentration of ZOL, which blocked the prenylation of small GTPases. This sequential treatment also increased the generation of reactive oxygen species (ROS). These results suggest that the combination of ZOL with IR may be beneficial in treating patients with soft tissue sarcoma.

Effectiveness of sulforaphane as a radiosensitizer for murine osteosarcoma cells.

Sulforaphane (SFN), a naturally occurring member of the isothiocyanate family, is effective against various types of malignant tumor cells. We studied whether the combination of SFN and radiation would be more effective against osteosarcoma cells when compared to these treatments alone. LM8 murine osteosarcoma cells were cultured with various concentrations of SFN for 24 h and/or 2 Gy X-irradiation. The effects of individual and combination treatments on the number of cells, the cell cycle, cell proliferation-related factors and apoptosis were analyzed. The combination of SFN plus radiation had significantly greater antitumor effects than either treatment alone. Exposure to SFN increased the population of cells in the G2/M phase. Combination treatment resulted in a higher percentage of cells being in sub-G1 than did SFN alone. In addition, the combination of SFN and radiation effectively induced nuclear fragmentation and apoptotic bodies, as shown by DAPI staining. The combination of SFN and 2 Gy radiation increased the cleavage and activation of caspase-3 compared with SFN or radiation alone, as shown by western blotting. Although radiation alone increased the phosphorylation of ERK and Akt proteins, the combination of SFN and radiation induced suppression of ERK and Akt phosphorylation when compared with radiation alone. We found that SFN enhanced the radiosensitivity of LM8 murine osteosarcoma cells by inducing apoptosis through G2/M-phase arrest and by inhibiting ERK and Akt activation. These findings suggest that SFN can be used as a radiosensitizer for osteosarcomas.

Effects of low-intensity pulsed ultrasound on osteosarcoma and cancer cells.

Metastatic bone tumors cause pain and pathological fractures due to bone destruction. If we could enhance new osteogenic activities and prevent progression of osteolytic change by malignant cells, patients could achieve satisfactory activity of daily living. Low-intensity pulsed ultrasound (LIPUS), which leads to bone formation by osteoblasts, has been used for the treatment of fractures. LIPUS has been reported to enhance the effect of an anticancer drug on lymphoma and liver cancer cells. However, there have been no reports of proliferation, vascularization and migration effects on cancer cells. In this study, we investigated the effects of LIPUS treatment on cancer and osteosarcoma cells and specifically whether it promotes bone formation without accelerating proliferation of tumor cells. We used MC3T3-E1 cells, a mouse osteoblast cell line, LM8, a mouse osteosarcoma cell line, SaOS2, a human osteosarcoma cell line, 786-O, a human renal cancer cell line, PC-3, a human prostate cancer cell line, and A549, a human lung cancer cell line. The expression of extracellular signal-regulated kinase (ERK1/2), Akt, ß-catenin, vascular endothelial growth factor (VEGF), and cell migration were analyzed. LIPUS stimulation did not affect proliferation of all the cells examined. The phosphorylation of ERK1/2 and Akt was induced by LIPUS stimulation in MC3T3-E1, LM8, SaOS2 and A549 cells, but not in PC-3 and 786-O cells. LIPUS stimulation did not significantly increase ß-catenin. VEGF protein levels and cell migration were significantly increased only in MC3T3-E1 cells. It may be concluded that LIPUS stimulation on metastatic bone tumors induces differentiation of osteoblasts without proliferation of tumor cells. Our study suggests that LIPUS may be a new method of treatment without surgery for metastatic bone tumors.

High Efficacy of Preoperative Low-Dose Radiotherapy with Sanazole (AK-2123) for Extraskeletal Ewing's Sarcoma: A Case Report.

Extraskeletal Ewing's sarcoma is a rare soft tissue tumor that is morphologically indistinguishable from Ewing's sarcoma of bone. We report a case of extraskeletal Ewing's sarcoma with several systemic problems. A 69-year-old man presented with a 5-month history of a rapidly enlarging mass in the right thigh. Because preoperative radiotherapy with sanazole (AK-2123) contributed the tumor mass reduction down to 40% in size, the tumor was successfully resected with clear surgical margins and repaired with a musculocutaneous flap. The high efficacy of pre-operative low-dose radiotherapy with sanazole was histologically confirmed that the resected tumor specimen involved no viable tumor cells and showed 100% necrosis. Based on clinical outcomes in this case, the combined modality of pre-operative low-dose radiotherapy with hypoxic cell radiosensitizer and adequate surgical resection might provide for the useful clinical application of extraskeletal Ewing's sarcoma treatment.

Combined effects of bisphosphonate and radiation on osteosarcoma cells.

UNLABELLED: Antitumour effects of third-generation bisphosphonates (BPs), such as zoledronic acid (ZOL), and the combined effects of ZOL with other anticancer agents against osteosarcoma cells have been reported previously. The aim of this study was to identify further combined antitumour effects using BPs and radiation in osteosarcoma cell lines. MATERIALS AND METHODS: Cell proliferation, cell cycle analysis, and nuclear morphology were examined in each osteosarcoma cell line divided into three groups (ZOL alone, radiation alone and the ZOL/radiation combination). RESULTS: Combined therapy (low-concentration ZOL and low-dose radiation) had significant growth inhibitory effects compared to the use of ZOL or radiation individually. Flow cytometric analysis revealed an increase in cells in the sub-G(1) phase by combined treatment, and apoptotic cells were also observed. CONCLUSION: These findings suggest that combination therapy using BPs and radiation may be a promising therapy for osteosarcoma, producing fewer side effects and complications in the near future.

A novel MEK1/2 inhibitor induces G1/S cell cycle arrest in human fibrosarcoma cells.

Blockade of the ERK pathway has antitumor effects against malignant tumor cells. In this study, we investigated the antitumor activity of JTP-70902, a novel specific MEK inhibitor, against human fibrosarcoma cells in which the ERK pathway is constitutively activated. JTP-70902 was synthesized at Japan Tabacco. Human fibrosarcoma HT1080 cells were cultured. JTP-70902 was added at various concentrations. The number of viable cells was counted employing a trypan blue dye exclusion test. Unsynchronized cells were exposed to JTP-70902 for 24 h. The nuclei were stained with propidium iodide. The DNA content was measured using a FACSCalibur flow cytometer. Protein extraction and Western blot analysis were performed. (1) A dose-dependent inhibition of cell growth was observed at concentrations of 10 nM or more. Forty-eight hours after the treatment, the growth of HT1080 cells was completely inhibited by 200 nM JTP-70902. (2) FACS analysis revealed that a 24-h exposure to JTP-70902 increased the population of G1/S phase cells in a dose-dependent manner. (3) The phosphorylation of ERK was inhibited by JTP-70902. Furthermore, after the treatment with JTP-70902, p21WAF1/CIP1 and p27KIP1 protein expression increased and the phosphorylation of RB was reduced. Our results showed that JTP-70902 inhibits cell growth and induces cell cycle arrest in human Ras mutant fibrosarcoma cells. These results indicate that JTP-70902 might be an attractive compound for molecular-targeting chemotherapy for malignant soft tissue tumors with the activation of the Ras-MEK-ERK pathway.

Zoledronic acid inhibits proliferation of human fibrosarcoma cells with induction of apoptosis, and shows combined effects with other anticancer agents.

Third-generation bisphosphonates are known to inhibit bone resorption and also appear to exhibit direct anti-tumour activity. We previously reported that third-generation bisphosphonates such as zoledronic acid (ZOL) have a direct antitumour effect, and synergistically augment the effects of antitumor agents in osteosarcoma cells. There has been no report on the antitumor effect of ZOL against soft tissue sarcoma. The aim of this study was to evaluate the antitumor effect of this drug on a human fibrosarcoma cell line, in terms of proliferation and apoptosis, and, moreover, to evaluate the combined effects of ZOL with other antitumor drugs against the human fibrosarcoma cell line. HT1080 cells were treated with ZOL at various concentrations up to 10 microM, and then cell proliferation, cell cycle, nuclear morphology, and Western blot analyses were performed to study the antitumor effects of ZOL alone, and, moreover, HT1080 cells were treated with ZOL and other anticancer drugs such as paclitaxel, docetaxel, doxorubicin, etoposide, 5-fluorouracil, gemcitabine, cisplatin, or methotrexate to investigate the combined effects using proliferation and cell cycle analyses. We found that ZOL strongly inhibited in vitro proliferation, arrested the cell cycle between S and G2/M phases, and induced the apoptosis of human fibrosarcoma cells. Moreover, ZOL augmented the effect of antitumor agents when administered concurrently with paclitaxel, docetaxel, doxorubicin, etoposide, 5-fluorouracil, gemcitabine, and cisplatin in human fibrosarcoma cells. The treatment of fibrosarcoma with ordinary antitumor drugs is not fully effective. These findings suggest that ZOL directly affects the proliferation and survival of fibrosarcoma cells, and that the combined administration of ZOL with other antitumor agents may improve the efficacy of fibrosarcoma treatment. These results support the possibility that their combined use could be beneficial in the treatment of patients not only with various types of cancer or osteosarcoma, but also with soft tissue sarcoma.

Histone deacetylase inhibitors and 15-deoxy-Delta12,14-prostaglandin J2 synergistically induce apoptosis.

PURPOSE: The clinically relevant histone deacetylase inhibitors (HDI) valproic acid (VPA) and suberoylanilide hydroxamic acid exert variable antitumor activities but increase therapeutic efficacy when combined with other agents. The natural endogenous ligand of peroxisome proliferator-activated receptor gamma 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a potent antineoplastic agent. Therefore, we investigated whether these HDIs in combination with 15d-PGJ(2) could show synergistic antitumor activity in colon cancer DLD-1 cells. EXPERIMENTAL DESIGN: Cell viability was determined using a Cell Counting Kit-8 assay. Apoptosis and reactive oxygen species (ROS) generation were determined using flow cytometry analysis. Western blotting and real-time reverse transcription-PCR analysis were carried out to investigate the expression of apoptosis-related molecules. Mice bearing DLD-1 xenograft were divided into four groups (n = 5) and injected everyday (i.p.) with diluent, VPA (100 mg/kg), 15d-PGJ(2) (5 mg/kg), or a combination for 25 days. RESULTS: HDI/15d-PGJ(2) cotreatments synergistically induced cell death through caspase-dependent apoptosis in DLD-1 cells. Moreover, HDIs/15d-PGJ(2) caused histone deacetylase inhibition, leading to subsequent ROS generation and endoplasmic reticulum stress to decrease the expression of antiapoptotic molecules Bcl-X(L) and XIAP and to increase that of proapoptotic molecules CAAT/enhancer binding protein homologous protein and death receptor 5. Additionally, VPA/15d-PGJ(2) cotreatment induced ROS-dependent apoptosis in other malignant tumor cells and was more effective than a VPA or 15d-PGJ(2) monotherapy in vivo. CONCLUSIONS: Cotreatments with the clinically relevant HDIs and the endogenous peroxisome proliferator-activated receptor gamma ligand 15d-PGJ(2) are promising for the treatment of a broad spectrum of malignant tumors.