Endoprosthetic reconstruction is widely applied for pathological fractures of the proximal humerus; however, functional impairment is usually unsatisfactory. The aims of the current study are to evaluate the reliability of interlocking intramedullary (IM) nailing with cement augmentation as a fixation method in proximal humeral lesions and to assess functional outcomes.
We reviewed 32 patients with pathological fractures of the proximal humerus who underwent interlocking IM nailing and cement augmentation. Functional scores and pain relief were assessed as outcomes.
The mean follow-up period was 14.2 months. The mean Musculoskeletal Tumor Society functional score and Karnofsky performance status scale score were 27.7 and 75.6, respectively. Improvement of pain assessed using the visual analogue scale was 6.2 on average. Thirty-one patients (97%) experienced no pain after surgery. The mean ranges of forward flexion and abduction were 115° and 112.6°, respectively. All patients achieved stability and had no local recurrence without failure of fixation until the last follow-up.
Proximal interlocking IM nailing with cement augmentation appears to be a reliable treatment option for pathological or impending fractures of the proximal humerus in selected patients with metastatic tumors, even with extensive bone destruction.
Humerus; Metastasis; Intrameullary nailing; Pathologic fracture
Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes.
Materials and Methods
The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined.
CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors.
EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.
Lung cancer; RET oncogene; tyrosine kinase inhibitor; resistance; epidermal growth factor
Mesenchymal stem cells (MSCs) are clinically useful due to their capacity for self-renewal, their immunomodulatory properties and tissue regenerative potential. These cells can be isolated from various tissues and exhibit different potential for clinical applications according to their origin, and thus comparative studies on MSCs from different tissues are essential. In this study, we investigated the immunophenotype, proliferative potential, multilineage differentiation and immunomodulatory capacity of MSCs derived from different tissue sources, namely bone marrow, adipose tissue, the placenta and umbilical cord blood. The gene expression profiles of stemness-related genes [octamer-binding transcription factor 4 (OCT4), sex determining region Y-box (SOX)2, MYC, Krüppel-like factor 4 (KLF4), NANOG, LIN28 and REX1] and lineage-related and differentiation stage-related genes [B4GALNT1 (GM2/GS2 synthase), inhibin, beta A (INHBA), distal-less homeobox 5 (DLX5), runt-related transcription factor 2 (RUNX2), proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein alpha (C/EBPA), bone morphogenetic protein 7 (BMP7) and SOX9] were compared using RT-PCR. No significant differences in growth rate, colony-forming efficiency and immunophenotype were observed. Our results demonstrated that MSCs derived from bone marrow and adipose tissue shared not only in vitro trilineage differentiation potential, but also gene expression profiles. While there was considerable interdonor variation in DLX5 expression between MSCs derived from different tissues, its expression appears to be associated with the osteogenic potential of MSCs. Bone marrow-derived MSCs (BM-MSCs) significantly inhibited allogeneic T cell proliferation possibly via the high levels of the immunosuppressive cytokines, IL10 and TGFB1. Although MSCs derived from different tissues and fibroblasts share many characteristics, some of the marker genes, such as B4GALNT1 and DLX5 may be useful for the characterization of MSCs derived from different tissue sources. Collectively, our results suggest that, based on their tri-lineage differentiation potential and immunomodulatory effects, BM-MSCs and adipose tissue-derived MSCs (A-MSCs) represent the optimal stem cell source for tissue engineering and regenerative medicine.
distal-less homeobox 5; mesenchymal stem cells; multipotency
We aimed to describe the clinical characteristics and outcomes of unplanned excisions of synovial sarcomas.
In total, 90 patients with synovial sarcomas in the extremities were retrospectively reviewed. Patients were divided into unplanned excision (n = 38) and planned excision (n = 52) groups. The average follow-up period was 6 years. The clinicopathological characteristics and oncologic outcomes were compared.
The unplanned excision group showed longer duration of symptoms before diagnosis (p = 0.023), smaller lesion dimensions (p = 0.001), superficial location (p = 0.049), and predilection in the upper extremities (p = 0.037). Synovial sarcomas were most commonly misdiagnosed as neurogenic tumors (56%) in the upper extremities or as cystic masses (47%) in the lower extremities. Oncological outcomes, including disease-specific survival, metastasis-free survival, or local recurrence were not significantly different between the 2 groups (p = 0.159, p = 0.444, and p = 0.335, respectively). Repeated unplanned excision (p = 0.012) and delayed re-excision (p = 0.038) were significant risk factors for local recurrence in the unplanned excision group.
Synovial sarcomas treated with unplanned excision had distinct characteristics. These findings are important for developing diagnostic and therapeutic strategies for synovial sarcoma.
Synovial sarcoma; Oncologic outcomes; Magnetic resonance imaging
Vaccine strategies utilizing dendritic cells (DCs) to elicit anti-tumor immunity are the subject of intense research. Although we have shown that DCs pulsed with heat-treated tumor lysate (HTL) induced more potent anti-tumor immunity than DCs pulsed with conventional tumor lysate (TL), the underlying molecular mechanism is unclear. In order to explore the molecular basis of this approach and to identify potential antigenic peptides from pancreatic cancer, we analyzed and compared the major histocompatibility complex (MHC) ligands derived from TL- and HTL-pulsed dendritic cells by mass spectrophotometry.
Materials and Methods
Human monocyte-derived dendritic cells were pulsed with TL or HTL prior to maturation induction. To delineate differences of MHC-bound peptide repertoire eluted from DCs pulsed with TL or HTL, nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) was employed.
HTL, but not TL, significantly induced DC function, assessed by phenotypic maturation, allostimulation capacity and IFN-γ secretion by stimulated allogeneic T cells. DCs pulsed with TL or HTL displayed pancreas or pancreatic cancer-related peptides in context of MHC class I and II molecules. Some of the identified peptides had not been previously reported as expressed in pancreatic cancer or cancer of other tissue types.
Our partial lists of MHC-associated peptides revealed the differences between peptide profiles eluted from HTL-and TL-loaded DCs, implying that induced heat shock proteins in HTL chaperone tumor-derived peptides enhanced their delivery to DCs and promoted cross-presentation by DC. These findings may aid in identifying novel tumor antigens or biomarkers and in designing future vaccination strategies.
Tumor lysate; heat shock proteins; dendritic cells; tumor antigens; nanoflow LC-MS-MS
Transplantation of mesenchymal stem cells (MSCs) was reported to improve functional outcomes in a rat model of ischemic stroke, and subsequent studies suggest that MSC-derived microvesicles (MVs) can replace the beneficial effects of MSCs. Here, we evaluated three different MSC-derived MVs, including MVs from untreated MSCs (MSC-MVs), MVs from MSCs treated with normal rat brain extract (NBE-MSC-MVs), and MVs from MSCs treated with stroke-injured rat brain extract (SBE-MSC-MVs), and tested their effects on ischemic brain injury induced by permanent middle cerebral artery occlusion (pMCAO) in rats. NBE-MSC-MVs and SBE-MSC-MVs had significantly greater efficacy than MSC-MVs for ameliorating ischemic brain injury with improved functional recovery. We found similar profiles of key signalling proteins in NBE-MSC-MVs and SBE-MSC-MVs, which account for their similar therapeutic efficacies. Immunohistochemical analyses suggest that brain-extract—treated MSC-MVs reduce inflammation, enhance angiogenesis, and increase endogenous neurogenesis in the rat brain. We performed mass spectrometry proteomic analyses and found that the total proteomes of brain-extract—treated MSC-MVs are highly enriched for known vesicular proteins. Notably, MSC-MV proteins upregulated by brain extracts tend to be modular for tissue repair pathways. We suggest that MSC-MV proteins stimulated by the brain microenvironment are paracrine effectors that enhance MSC therapy for stroke injury.
We present here the oncological and functional outcomes of limb salvage with or without reconstruction for primary sarcomas in the pelvic bone.
Forty-four patients who underwent pelvic resection for primary sarcomas involving the pelvis were reviewed. The average follow-up period was 39 months (range, 0 to 146 months). Chondrosarcoma (n = 17) and osteosarcoma (n = 10) were the most common diagnoses. Various clinicopathologic factors were analyzed in relation to the oncological outcomes of overall survival and local recurrence. The Musculoskeletal Tumor Society functional scores and complications were compared according to the tumor location, pelvic continuity and the type of resection.
The overall 5-year survival rate was 40%. Metastasis at the time of diagnosis, the surgical margin and the histologic grade were the independent prognostic factors for survival. The surgical margin was an independent prognostic factor for local recurrence. An ischiopubic location of the tumor, restoration of pelvic continuity and hip joint reconstruction with total hip replacement arthroplasty were related with higher functional scores. Complications occurred in 50% of the patients and the complications varied according to the tumor location with infection being the most common complication.
Achieving an adequate surgical margin is necessary for improving the oncological outcome of pelvic sarcomas. Restoration of the pelvic continuity and the hip joint improves the functional outcome. However, complications are common and so careful selection of the reconstruction method is needed.
Pelvis; Sarcoma; Reconstruction; Survival; Function
We compared indomethacin therapy with the more aggressive approaches of anti-cancer chemotherapy and surgery in the treatment of isolated Langerhans cell histiocytosis (LCH) of bone in children.
Comparisons were made with respect to healing of the lesion without recurrence, time to radiological healing of the lesion, time to functional recovery, and complications related to treatment.
Complete radiologic healing of the lesion (mean, 15.3 months) and functional recovery (mean, 5.6 months) were observed in all patients treated with either approach. No significant differences were noted in the time to complete radiologic healing or the time to functional recovery between the two groups. There were no recurrences with either approach until the last follow-up (mean, 56 months). Complications were common with anti-cancer chemotherapy, but indomethacin was well-tolerated.
Indomethacin seems to be effective for treating isolated LCH of bone in children. Hence, morbidities associated with aggressive treatment approaches such as anti-cancer chemotherapy or surgery can be avoided.
Langerhans cell histiocytosis; Indomethacin; Children
The purpose of this study is to report on the incidence and the experience in management of radiation-induced sarcoma (RIS) at a large single center in Korea for 15 years.
Materials and Methods
We retrospectively reviewed the sarcoma registry of a large institution from January 2000 to April 2014.
Out of the 3,674 patients listed in the registry, 33 patients (0.9%) diagnosed with RIS were identified. The median latency of RIS was 12.1 years. The number of cases of RIS increased from four cases in the years 2000-2003 to 14 cases in the years 2012-2014. The most common histology was osteosarcoma (36.4%). The median follow-up period was 23.1 months, the median overall survival (OS) of all patients was 2.9 years, and their 5-year survival rate was 44.7%. Univariate and multivariate analyses showed association of the age at diagnosis (p=0.01) and the treatment aim (p=0.001) with the OS. The median OS and the 5-year survival rate of patients treated with curative surgery (n=19) were 9.6 years and 65%, respectively, and of the conservatively treated patients, 0.7 years and 0% (n=14). Re-irradiation was delivered to nine patients, and radiation toxicity was observed in five patients.
In this study, RIS accounted for 0.9% of the cases of sarcoma, with increasing incidence. Despite the association of curative resection with increased survival, it could be applied to only 58% of the patients. Considering the limited treatment options for RIS, conduct of a genetic study to identify the underlying mechanism of RIS is needed.
Radiation-induced neoplasms; Sarcoma; Survival
The prognostic factors of chondrosarcoma remain uncertain as only a few large studies with long-term follow-up have been reported. The aim of this study was to analyze oncological outcomes and prognostic factors.
Materials and Methods
A retrospective review of oncological outcomes and prognostic factors was performed on 125 consecutive chondrosarcoma patients who underwent surgery at our institution.
Overall survival was 91.6%±2.5%, 84.1%±3.8%, and 84.1%±3.8% at 5, 10, and 15 years respectively. Among the histological types, dedifferentiated type showed the worst survival (p < 0.001). As for conventional type chondrosarcoma, histologic grade and anatomical location predicted outcome, with high-grade with axial location having the worst outcome (p < 0.001). In contrast, low-grade chondrosarcoma of appendicular skeleton could be treated safely by intralesional curettage.
Histological type was significantly associated with the outcome of chondrosarcoma. For the conventional type, histologic grade and anatomical location predicted outcome, with high-grade with axial location having the worst outcome.
Chondrosarcoma; Prognosis; Recurrence; Pelvis; Histology
We conducted a nationwide study to assess the incidence and treatment patterns of extremity soft tissue sarcoma (STS) in South Korea.
Materials and Methods
The nationwide incidence and treatment patterns of extremity STS were assessed using two nationwide databases, the Korea National Cancer Incidence (KNCI) database and the Health Insurance Review and Assessment Service (HIRA) database.
A total of 1,236 patients were newly diagnosed with extremity STS during the 3-year study period, from 2009 to 2011. The annual incidence of extremity STS in the Korean population was approximately 0.9 per 100,000 people with a male bias that increased with age and was especially pronounced amongst individuals aged > 80 years. Approximately 7% of patients did not receive any treatment, and surgical excision was performed for 85% of those who were treated.
This is the first nationwide study of the incidence and treatment patterns of extremity STS in Korea using two national databases (KNCI and HIRA), which include the entire Korean population. The results of this study may be useful for future planning and management of STS, at the national level.
Sarcoma; Incidence; Registries; Korea; Therapy
Unplanned excision of extremity soft tissue sarcoma (STS) is common and has detrimental effects not only on patients’ oncologic outcomes but also on functional and economic issues. However, no study has analyzed a nationwide population-based database. To estimate the incidence and treatment pattern of unplanned excision in extremity STS in the Korean population, a nationwide epidemiologic study was performed using the Korean Health Insurance Review and Assessment Service database, a centralized nationwide healthcare claims registry of Korea that covers the entire Korean population. Among 1,517 patients with extremity STS in the 4-year study period, 553 (36.5%) underwent unplanned excision (unplanned group). About 80% of unplanned excisions were performed in tertiary or general hospitals. Of the unplanned group, 240 (43.4%) underwent re-excision with or without radiation therapy and/or chemotherapy, and 51 (9.2%) did not undergo re-excision but were treated with radiation therapy and/or chemotherapy; whereas, 262 (47.4%) did not undergo any further treatment following unplanned excision. This study is the first nationwide population-based study on the unplanned excision of extremity STS. The results may have implications in establishing preventive or therapeutic measures to reduce the burden of unplanned excision of extremity STS.
Tumorigenic potential of human pluripotent stem cells (hPSCs) is an important issue in clinical applications. Despite many efforts, PSC-derived neural precursor cells (NPCs) have repeatedly induced tumors in animal models even though pluripotent cells were not detected. We found that polysialic acid-neural cell adhesion molecule (PSA-NCAM)− cells among the early NPCs caused tumors, whereas PSA-NCAM+ cells were nontumorigenic. Molecular profiling, global gene analysis, and multilineage differentiation of PSA-NCAM− cells confirm that they are multipotent neural crest stem cells (NCSCs) that could differentiate into both ectodermal and mesodermal lineages. Transplantation of PSA-NCAM− cells in a gradient manner mixed with PSA-NCAM+ cells proportionally increased mesodermal tumor formation and unwanted grafts such as PERIPHERIN+ cells or pigmented cells in the rat brain. Therefore, we suggest that NCSCs are a critical target for tumor prevention in hPSC-derived NPCs, and removal of PSA-NCAM− cells eliminates the tumorigenic potential originating from NCSCs after transplantation.
•PSA-NCAM− cells isolated from neural rosettes are classified as multipotent NCSCs•NCSCs are a potential target for tumor prevention in hPSC-derived-NPC-based therapy•Removal of PSA-NCAM− cells prevents the introduction of mesodermal tumor in the CNS•Removal of PSA-NCAM− cells prevents the introduction of unwanted grafts in the CNS
By PSA-NCAM-targeted cell sorting from hPSC-derived neural rosettes, D.-W. Kim, D.-S. Kim, and colleagues show that PSA-NCAM− cells are multipotent NCSCs with the capacity to differentiate into both ectodermal and mesodermal lineages. They also demonstrate that NCSCs are responsible for mesodermal tumor and unwanted graft formations in the CNS. These results suggest that removal of PSA-NCAM− cells eliminates the tumorigenic potential originating from NCSCs.
Comorbidity has not been examined as an independent prognostic factor in soft tissue sarcoma (STS). We examined the prognostic impact of comorbidity on oncologic outcome in STS with an adjustment for possible confounding factors.
A retrospective review was performed on 349 patients who had undergone surgery for high-grade localized STS of extremity at our institute. Conditions known to alter the risk of mortality, as defined in the Charlson comorbidity index, were classified as comorbidities and 43 patients (12%) had at least one comorbidity at the time of surgery. The association of comorbidity and oncologic outcomes of local recurrence-free survival (LRFS) and disease-specific survival (DSS) were tested with adjustment for confounding factors.
Comorbidity was associated with old age, high tumor grade, and large tumor size. The presence of comorbidity was independently associated with poor LRFS and DSS, even after adjusting for confounding factors including age and treatment variables.
Our data suggest that the presence of comorbidity is an independent prognostic factor for extremity STS.
Sarcoma; Comorbidity; Prognosis; Age of onset; Treatment outcome
Recently, we reported the properties of CD31-expressing cells in healthy individuals. However, the characteristics of CD31-expressing cells derived from coronary artery disease (CAD) patients remain unknown. This study aimed to investigate the relationship between circulating CD31+ cells and CAD as well as their biological characteristics. Analysis with flow cytometry revealed that CD31+ cells (C-CD31) from the peripheral blood (PB) of CAD patients exhibited low levels of T-cell marker and high levels of macrophage marker compared with the PB-CD31+ cells from healthy individuals (H-CD31). In addition, the expression levels of multiple pro-angiogenic and chemokine genes were significantly down-regulated in C-CD31. However, inflammatory gene IL-1α was highly up-regulated in C-CD31. Patients with unstable angina (UA) had significantly more CD31+ cells in the PB than healthy control group (P < 0.001). Moreover, there were significant correlations between the number of CD31+ cells and cardiovascular (CV) disease activity (R = 0.318, P = 0.006) and the number of diseased coronaries (R = 0.312, P = 0.005). For the diagnostic category of UA, the area under curve was 0.803 (P < 0.001). In conclusion, C-CD31 have impaired angiogenic potential and the number of circulating CD31+ cells were correlated with CV risk. These findings may contribute to the understanding of the pathogenesis of CAD.
angina pectoris; angiogenesis effect; coronary artery disease; CD31 antigen; inflammation
Most of default mode network (DMN) studies in patient with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD) were based only on the comparing two groups, namely patients and controls. Information derived from comparing the three groups, normal, aMCI, and AD group, simultaneously may lead us to better understand the progression of dementia. The purpose of this study was to evaluate functional connectivity of DMN in the continuum from normal through aMCI to AD. Differences in functional connectivity were compared between the three groups using independent component analysis. The relationship between the functional connectivity and disease progression was investigated using multiple regression analysis with Mini-Mental State Examination (MMSE) scores. The results revealed differences throughout the left posterior cingulate cortex (PCC), left middle temporal gyrus (MTG), right middle frontal gyrus (MFG), and bilateral parahippocampal gyrus (PHG). Both patients with aMCI and AD showed decreased connectivity in the left PCC and left PHG compared with healthy subjects. Furthermore, patient with AD also showed decreased connectivity in the left MTG and right PHG. Increased functional connectivity was observed in the right MFG of patients with AD compared with other groups. MMSE scores exhibited significant positive and negative correlations with functional connectivity in PCC, MTG and MFG regions. Taken together, an increased functional connectivity in the MFG for AD patients might compensate for the loss of function in the PCC, MTG via compensatory mechanisms in cortico-cortical connections.
Resting-state; Functional magnetic resonance imaging; Alzheimer’s Disease; Mild Cognitive Impairment; Default Mode Network
Red blood cell (RBC) supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs) from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells via in vitro culture. Among them, human cord blood (CB) and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB) are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showed in vitro RBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. This study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. The results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials in in vitro culture systems.
Non-spine bone metastasis accounts for approximately 20% of all skeletal metastases, but little data have been published that focused on bone metastasis to the pelvis and extremities as an initial manifestation of cancer. We determined 1) clinicopathologic characteristics of patients who presented with non-spine bone metastasis of unknown primary malignancy, and 2) process by which the diagnosis of primary cancer was made. We retrospectively reviewed 84 patients with bone metastasis of unknown primary cancer site at the time of presentation. The study population consisted of 56 men and 28 women, with a mean age of 59.1 yr (17.5-85.6 yr). The average follow-up period was 20.8 months (1-120 mo). Primary cancer site was identified in 79 patients (94.0%), and was determined to be the lung (46.4%), kidney (13.1%), liver (9.5%), thyroid (8.3%), and prostate (4.8%). Five-year overall survival rate was 28.0%. Multiple bone metastases, distant organ metastasis, and multiple bone with organ metastases were the significant prognostic factors in univariate analysis. Multiple bone metastases remained significant after multivariate analysis (P = 0.008). Lung cancer is the most common site of primary cancer, and patients with multiple bone metastases have a poor prognosis, possibly due to disseminated cancer and a greater tumor burden.
Neoplasm Metastasis; Neoplasms; Non-Spine; Diagnosis
Osteosarcoma is the most common malignant bone tumor in children and adolescents. This aggressive cancer mostly occurs in the long bones. Therefore, novel therapeutic approaches, such as biological therapies and gene therapy, are required to efficiently treat osteosarcoma. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) has been demonstrated to inhibit the growth of several types of cancer cells and a number of studies have shown that osteosarcoma may be vulnerable to biological therapies. However, little is known regarding the therapeutic effects of capsaicin on osteosarcoma. This study investigated the effects of capsaicin on MG63 human osteosarcoma cells, in addition to elucidating the regulatory signaling pathways underlying the effects of capsaicin, the caspase cascade and the antioxidant enzyme system. The MG63 cell line was treated with various concentrations of capsaicin. Cells were analyzed using MTT and flow cytometry, and the presence of DNA fragmentation was evaluated using TUNEL assay. Results showed capsaicin induced apoptosis in MG63 cells. Thus, capsaicin exhibited an anticancer effect in osteosarcoma cells.
capsaicin; MG63 human osteosarcoma cell; apoptosis; caspase cascade; antioxidant enzyme system
X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene that encodes the peroxisomal ATP-binding cassette (ABC) transporter subfamily D member 1 protein (ABCD1), which is referred to as the adrenoleukodystrophy protein (ALDP). Induction of the ABCD2 gene, the closest homolog of ABCD1, has been mentioned as a possible therapeutic option for the defective ABCD1 protein in X-ALD. However, little is known about the transcriptional regulation of ABCD2 gene expression. Here, through in silico analysis, we found two putative TCF-4 binding elements between nucleotide positions −360 and −260 of the promoter region of the ABCD2 gene. The transcriptional activity of the ABCD2 promoter was strongly increased by ectopic expression of β-catenin and TCF-4. In addition, mutation of either or both TCF-4 binding elements by site-directed mutagenesis decreased promoter activity. This was further validated by the finding that β-catenin and the promoter of the ABCD2 gene were pulled down with a β-catenin antibody in a chromatin immunoprecipitation assay. Moreover, real-time PCR analysis revealed that β-catenin and TCF-4 increased mRNA levels of ABCD2 in both a hepatocellular carcinoma cell line and primary fibroblasts from an X-ALD patient. Interestingly, we found that the levels of very long chain fatty acids were decreased by ectopic expression of ABCD2-GFP as well as β-catenin and TCF-4. Taken together, our results demonstrate for the first time the direct regulation of ABCD2 by β-catenin and TCF-4.
Cell transplantation of myelin-producing exogenous cells is being extensively explored as a means of remyelinating axons in X-linked adrenoleukodystrophy. We determined whether 3,3',5-Triiodo-L-thyronine (T3) overexpresses the ABCD2 gene in the polysialylated (PSA) form of neural cell adhesion molecule (NCAM)-positive cells and promotes cell proliferation and favors oligodendrocyte lineage differentiation.
Materials and Methods
PSA-NCAM+ cells from newborn Sprague-Dawley rats were grown for five days on uncoated dishes in defined medium with or without supplementation of basic fibroblast growth factor (bFGF) and/or T3. Then, PSA-NCAM+ spheres were prepared in single cells and transferred to polyornithine/fibronectin-coated glass coverslips for five days to determine the fate of the cells according to the supplementation of these molecules. T3 responsiveness of ABCD2 was analyzed using real-time quantitative polymerase chain reaction, the growth and fate of cells were determined using 5-bromo-2-deoxyuridine incorporation and immunocytochemistry, respectively.
Results demonstrated that T3 induces overexpression of the ABCD2 gene in PSA-NCAM+ cells, and can enhance PSA-NCAM+ cell growth in the presence of bFGF, favoring an oligodendrocyte fate.
These results may provide new insights into investigation of PSA-NCAM+ cells for therapeutic application to X-linked adrenoleukodystrophy.
X-linked adrenoleukodystrophy; polysialylated form of the neural cell adhesion molecule cells; 3,3',5-Triiodo-L-thyronine; ABCD2 gene
Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPCPSA-NCAM+) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPCPSA-NCAM+ extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80∼98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPCPSA-NCAM+ showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPCPSA-NCAM+ differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPCPSA-NCAM+ compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.
The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.
drug evaluation, preclinical; drug screening; induced pluripotent stem cells; models, biological; tissue therapy
Unplanned excision of a soft tissue sarcoma generally requires reexcision to achieve an adequate surgical margin. Many surgeons assume delay of definitive surgery adversely affects patient survival and local recurrence. However, no clear evidence of this assumption can be found in the literature.
We asked whether delay in reexcision affects patient survival and local recurrence in reexcision after unplanned excision for soft tissue sarcoma.
Patients and Methods
We retrospectively reviewed 104 patients who underwent definitive surgery after unplanned excision of a localized soft tissue sarcoma. The average age of the patients was 44 years (range, 5–81 years). The most common diagnoses were malignant fibrous histiocytoma (36) and synovial sarcoma (22). Locations of the tumors were the lower extremity (62), upper extremity (32), and trunk (10). The median interval to definitive surgery was 32 days (interquartile range, 22–50 days). The minimum followup was 0.2 years (median, 4.7 years; range, 0.2–16.7 years).
The 5-year disease-specific survival was 88% and 5-year local control rate was 74%. We found no difference in disease-specific survival or local recurrence according to the time until definitive surgery. Higher histologic grade and larger tumor size independently predicted disease-specific survival whereas a positive margin at reexcision and larger tumor size independently predicted local control.
The data suggest any influence of delayed definitive surgery is likely to be of minor clinical importance.
Level of Evidence
Level III, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.