Tadashi Yoshizawa

Involvement of c-Myc in the proliferation of MCF-7 human breast cancer cells induced by bHLH transcription factor DEC2

Differentiated embryonic chondrocyte expressed gene 1 (DEC1; BHLHE40/Stra13/Sharp2) and differentiated embryonic chondrocyte expressed gene 2 (DEC2; BHLHE41/Sharp1) are basic helix-loop-helix (bHLH) transcriptional factors that are involved in the regulation of cell differentiation, circadian rhythms, response to hypoxia and carcinogenesis. Previous studies have demonstrated that the expression of DECs is induced under hypoxic conditions in various normal and cancer cell lines. In the present study, using RT-qPCR and western blot analysis, we demonstrated that hypoxia induced the expression of DEC1 and DEC2 in MCF-7 human breast cancer cells; their expression levels reached a peak at different time points. In particular, we found that the expression pattern of the hypoxia-inducible factor (HIF)-1α protein was similar to DEC1, and that of the HIF-2α protein was identical to that of DEC2. The knockdown of HIF-2α using siRNA suppressed the phosphorylation of Akt, as well as the expression of DEC2 and c-Myc. Hypoxia failed to affect the expression of DEC2 and c-Myc when the PI3K/Akt signaling pathway was blocked. In addition, the overexpression of DEC1 and DEC2 was induced by transfecting the cells with a pcDNA vector. The overexpression of DEC2, but not that of DEC1, increased the proliferation of the MCF-7 cells under both normoxic and hypoxic conditions. Concomitantly, the expression of c-Myc was upregulated by exposure to hypoxia and by the overexpression of DEC2. In conclusion, DEC2 participates in hypoxia-induced cell proliferation by functioning as a target gene of the PI3K/Akt signaling pathway and regulating the expression of c-Myc.

Neuropathology of PARK14 is identical to idiopathic Parkinson's disease

Phospholipase A2, group IV (PLA2G6) is the causative gene for PARK14-linked parkinsonism (PARK14) and neurodegeneration with brain iron accumulation 2 (NBIA2). NBIA2 is an infantor childhood-onset psychomotor disorder with truncal hypotonia, pyramidal signs, cerebellar ataxia, optic atrophy, and dystonia. Patients with PARK14 are clinically distinguished from those with NBIA2 by adultonset dystoniaor dyskinesia-parkinsonism of longer symptom duration. There have been 7 autopsy cases of NBIA2 in which postmortem examination revealed severe brain atrophy with numerous axonal swellings, Lewy bodies, and tau pathology in addition to excess iron deposition in the globus pallidus. Although Klein and colleagues reported a female patient with mutations of PLA2G6, who presented with prolonged course of parkinsonism, pathologic findings of the patient was similar to those of NBIA2. Here, we report a 48-year-old Japanese woman with PARK14 in whom the pathological features 23 years after onset were identical to those of idiopathic Parkinson’s disease (PD). This female patient had developed bradykinesia of her right upper and lower extremities at the age of 25. Her parents were first cousins, and her older brother had also developed dystonia-parkinsonism at the age of 39. At 4 months after the start of L-dopa treatment, the patient showed wearing-off phenomenon and dyskinesia. In addition, she exhibited the symptoms of typical parkinsonism, including tremor, postural instability, rigidity, and autonomic dysfunction. Pyramidal signs, cerebellar ataxia, dystonia, and dementia were not observed. Single-photon emission computerized tomography dopaminergic imaging with N-x-fluoropropyl-2b-carbomethoxy-3b-(4-[I]iodophenyl)nortopane revealed diffusely reduced bilateral uptake in the striatum. Cardiac uptake of meta-iodobenzylguanidine, a physiological analog of norepinephrine, was reduced. Genetic analysis of these siblings revealed a homozygous mutation of p.A499T (c. 1495G>A), as reported previously. The patient died of sepsis at the age of 48. Postmortem examination revealed no atrophy of the brain, which weighed 1330 g before fixation. Marked depigmentation of the substantia nigra and locus ceruleus was evident (Fig. 1A). Microscopically, the substantia nigra and locus ceruleus showed severe loss of neurons with gliosis. Many Lewy bodies and Lewy neurites were found in the dorsal vagal nucleus, locus ceruleus, and substantia nigra (Fig. 1B). Several Lewy bodies were also observed in the temporal mesocortex (Braak stage 4; Fig. 1C). Accumulation of phosphorylated asynuclein was seen in nerve cell bodies and processes in the myenteric plexus of the gastrointestinal tract, adrenal gland, and cardiac sympathetic nerves (Fig. 1D, E). There were no iron deposition and spheroid formation throughout the brain. PARK14 and NBIA2 with PLA2G6 mutations cause Lewy body pathology, including typical PD. The present study indicated that long-term follow-up does not necessarily cause overlap between neuropathology of NBIA2 and PD. Recently, in 250 patients with idiopathic PD, 4 mutations of PLA2G6 were reported, and 3 of them affected the enzyme activities of PLA2G6. The impairment of PLA2G6-dependent Ca signaling is a common upstream feature in both PARK14 and idiopathic PD, triggering the dysfunction of autophagy and loss of dopaminergic neurons. These findings suggest that mutations of PLA2G6 could be associated with a proportion of cases of idiopathic PD.

Invasive micropapillary carcinoma of the extrahepatic bile duct and its malignant potential

Invasive micropapillary carcinoma (IMPC) was originally described as a distinctive type of invasive carcinoma in the breast, but it has not been recognized as a histological type of the extrahepatic bile duct cancer. The present study demonstrated clinicopathological features and patient prognosis of IMPC. We examined histological reviews of 93 consecutive cases of the extrahepatic bile duct cancer and identified 13 cases which included IMPC component. The component of IMPC ranged from 5 to 60% of the primary tumor tissue, which was mainly detected at the invasive front of the tumor. Of the 13 cases, 12 (92.3%) carcinomas with IMPC showed lymph node metastasis more frequently compared to conventional adenocarcinoma (39.2%, P<0.001). Presence of IMPC component was significantly associated with poor overall survival (P=0.003). In conclusion, extrahepatic bile duct carcinoma with IMPC component showed significant lymphatic invasion, lymph node metastasis, and resulted in poor prognosis.

Basic helix-loop-helix transcription factor DEC2 functions as an anti-apoptotic factor during paclitaxel-induced apoptosis in human prostate cancer cells.

The functions of basic helix-loop-helix (bHLH) transcription factor-differentiated embryonic chondrocyte (DEC)1 (BHLHE40) and 2 (BHLHE41) are involved in various fields such as circadian rhythms, immune responses, cell proliferation, hypoxia reaction as well as malignant tumors. Previous findings showed that DEC served as apoptosis regulators of various cancer cell lines. However, little is known regarding the expression of DEC1 and DEC2 in prostate cancer cells. The present study aimed to examine the roles of DEC1 and DEC2 in human prostate cancer DU145 and PC-3 cells that were treated with paclitaxel. The expression of DEC1 and DEC2 was decreased in DU145 cells but was increased in PC-3 cells when treated with paclitaxel. DU145 cells were more sensitive to paclitaxel than PC-3 cells since the amount of cleaved poly(ADP-ribose) polymerase (PARP) reached its peak at 50 μM of paclitaxel in DU145 cells but at 100 μM in PC-3 cells. In addition, the amount of cleaved PARP was decreased by DEC1 siRNA, while it was increased by DEC2 siRNA in the presence of paclitaxel. Although DEC2 overexpression slightly inhibited cleaved PARP in the two cell lines, the effects of DEC1 overexpression on apoptosis remain to be determined. In conclusion, DEC1, at least partly, exerted a pro-apoptotic effect, whereas DEC2 exerted an anti-apoptotic effect in paclitaxel-induced apoptosis of human prostate cancer cells.

DEC1 promotes hypoxia-induced epithelial-mesenchymal transition (EMT) in human hepatocellular carcinoma cells

Differentiated embryonic chondrocyte (DEC) 1 has been reported to be involved in cell differentiation, hypoxia response, and cancer progression. Recent studies have demonstrated that hypoxia-inducible factor (HIF)-1α induces epithelial-mesenchymal transition (EMT) in carcinoma cells to facilitate cell invasiveness and metastasis. However, it remains unclear whether DEC1 participates in hypoxia-mediated EMT processes. In the present study, we reported that hypoxia induced DEC1 expression in hepatocellular carcinoma (HCC) HepG2 cells, and DEC1 negatively regulated expression of HIF-1α and E-cadherin in transcriptional/translational levels. Cell morphological changes were evaluated with hematoxylin and eosin (H-E) staining. Exposure to hypoxia caused spindle-like shape in some of the HepG2 cells, and DEC1 overexpression furthered these changes. In conclusions, DEC1 is involved in hypoxia-induced EMT processes via negatively regulating E-cadherin expression in HepG2 cells.

Podoplanin-mediated TGF-β-induced epithelial-mesenchymal transition and its correlation with bHLH transcription factor DEC in TE-11 cells

Podoplanin is reported involved in the collective cell invasion, another tumor invasion style which is distinct from the single cell invasion, so-called epithelial-mesenchymal transition (EMT). In this study, we investigated the correlation between podoplanin and EMT-related markers in esophageal squamous cell carcinoma (ESCC), and evaluated its linkage with the basic helix-loop-helix (bHLH) transcription factor differentiated embryonic chondrocyte (DEC) 1 and DEC2. Three ESCC cell lines and human squamous cell carcinoma A431 cells were subjected to western blot analyses for podoplanin and EMT markers, as well as the expression of DEC1 and DEC2. By RT-qPCR and western blotting, we found that TGF-β increased the expression of podoplanin and mensenchymal markers (e.g., N-cadherin and vimentin), while decreased the expression of epithelial markers (e.g., Claudin-4 and E-cadherin), accompanied by Smad2 phosphorylation and slug activation. Moreover, TGF-β has different effects on the expression of DEC1 and DEC2, that is, it upregulates DEC1, but downregulates DEC2. Capability of cell proliferation, invasion and migration were further analyzed using CCK-8 assay, Matrigel-invasion assay, and the wound-healing assay, respectively. The proliferation, invasion and migration ability were significantly lost in podoplanin-knockdown cells when compared with the scrambled siRNA group. In addition to these changes, the expression of Claudin-4, but not that of Claudin-1 or E-cadherin, was induced by the siRNA against podoplanin. On the contrary, overexpression of DEC1 and DEC2 exhibits opposite effects on podoplanin, but only slight effect on Claudin-4 was detected. These data indicated that podoplanin is significantly associated with EMT of TE-11 cells, and may be directly or indirectly regulated by bHLH transcription factors DEC1 and DEC2.

Expression of cancer-associated fibroblast markers in advanced colorectal cancer

Colorectal cancer is one of the most common causes of mortality from cancer worldwide. Previous studies have demonstrated that cancer-associated fibroblasts (CAFs) promote neoangiogenesis and tumor growth for various tumors. The present study analyzed CAF markers, including α-smooth muscle actin (α-SMA), collagen I, platelet-derived growth factor receptor-β (PDGFR-β), and D2-40 (antibody recognizing podoplanin), and vessel markers, including cluster of differentiation (CD)31 and CD34, for 121 advanced colorectal cancer cases using a digital image analyzing technique. The association between CAF markers and vessel markers with clinicopathological factors was investigated. Furthermore, the association between CAF markers with each other, and their association with vessel markers was analyzed. Mean/median expression area of stromal and vessel markers in tumors were collagen I, 26.787%; D2-40, 1.372%; PDGFR-β, 11.646%; α-SMA-positive and desmin-negative myofibroblasts (α-SMA subtraction), 15.372%; CD31, 3.635%; and CD34, 2.226%. The expression area of α-SMA subtraction was significantly correlated with collagen I (P<0.001, correlation rho=0.509). High levels of α-SMA subtraction (P=0.002), collagen I (P=0.040), and PDGFR-β (P=0.040) expressions tended to be associated with high venous invasion. D2-40 did not correlate with other CAF and vessel markers. These results indicated that individual CAFs may have different expression patterns, and different strength effects for venous invasion in advanced colorectal cancer stroma.

Development of Hepatocellular Carcinoma in Chronic Hepatitis C Patients 20 Years after Achieving a Sustained Virological Response with Interferon Therapy: A Report of Two Cases

Advances in interferon (IFN)‐based therapy for chronic hepatitis C have led to a high rate of sustained virological response (SVR), which means viral clearance. However, some cases have been reported to develop hepatocellular carcinoma (HCC) over 10 years after achieving the SVR. Here, we report two patients who developed HCC 20 years after SVR with IFN therapy. Both of the patients were male and achieved SVR at the age of 46 years and 61 years, respectively. These cases suggest the need for long‐term follow‐up in patients with chronic hepatitis C even if SVR is achieved.

Neoangiogenesis of gastric submucosa‑invasive adenocarcinoma

Early gastric cancer may be defined as mucosal or submucosal invasive carcinoma, and exhibits a good prognosis: 90% of patients survive >10 years. Early gastric cancer infrequently exhibits lymph node metastasis, although submucosal invasion, the presence of vascular invasion and/or lymphatic permeation are independent risk factors for lymph node metastasis in early gastric cancer. The analysis of tumor lymphangiogenesis and angiogenesis are important to determine the extent of invasive progression and metastasis in patients. Previously, the presence of vessels expressing the D2-40 antibody and the factor-VIII protein has been identified immunohistochemically. The vessels that are immunoreactive for D2-40 and factor-VIII are morphologically similar to lymphatic vessels or small-size veins, also termed venules. In the present study, the association between tumor invasion and neoangiogenesis in early gastric cancer was examined. The D2-40/factor-VIII double-stained vessel (DSV) density was analyzed, in addition to lymphatic and blood vessel (vein and artery) density, using 46 submucosa-invasive and 50 mucosal carcinomas, and 20 non-neoplastic gastric tissues. The lymphatic density and DSV density of submucosa beneath the carcinoma and submucosa of the surrounding region in submucosa-invasive carcinoma were significantly increased (P<0.001) in comparison with those in mucosal carcinoma or non-neoplastic gastric tissue. No significant difference was observed in blood vessel density between non-neoplastic gastric, mucosal carcinoma and submucosa-invasive carcinoma tissues other than that of mucosa. The present study suggests the potential for the presence of D2-40/factor-VIII DSV and the importance of this vessel for neoangiogenesis in early gastric cancer.

Colocalization of Bunina bodies and TDP-43 inclusions in a case of sporadic amyotrophic lateral sclerosis with Lewy body-like hyaline inclusions: Possible origin of Bunina bodies

Sporadic amyotrophic lateral sclerosis (sALS) is characterized pathologically by loss of upper and lower motor neurons with occurrence of transactivation response DNA‐binding protein 43 kDa (TDP‐43)‐immunoreactive skein‐like and round hyaline inclusions. Lewy body‐like hyaline inclusions (LBHIs) are also found in a small proportion of sALS cases as well as in individuals with familial ALS with mutations in the Cu/Zu superoxide dismutase (SOD1) gene. LBHIs in sALS are immunopositive for TDP‐43, but not for SOD1. The occurrence of Bunina bodies (BBs) is another key pathological feature of sALS. BBs are immunonegative for TDP‐43 but immunopositive for cystatin C, transferrin, peripherin and sortilin‐related receptor CNS expressed 2 (SorCS2). Despite differences between BBs and TDP‐43 inclusions in terms of protein constituents and ultrastructure, the two inclusions are known to be linked. We recently encountered a case of sALS of 10 months duration in which many round hyaline inclusions, LBHIs and BBs were found in the anterior horn cells of the spinal cord. Our immunohistochemical and ultrastructural examinations revealed the presence of BBs within the skein‐like and round hyaline inclusions, and in the LBHIs. Colocalization of BB‐related proteins (cystatin C, transferrin and SorCS2) and TDP‐43 was also confirmed in the halo of LBHIs as well as in the marginal portion of the skein‐like and round hyaline inclusions. These findings suggest that there is some relationship between BBs and TDP‐43‐immunoreactive inclusions in terms of their formation processes.