Satoko Morohashi

Anti‐apoptotic effect of the basic helix‐loop‐helix (bHLH) transcription factor DEC2 in human breast cancer cells

DEC1 (BHLHB2/Stra13/Sharp2) and DEC2 (BHLHB3/Sharp1) are basic helix‐loop‐helix (bHLH) transcription factors that are involved in circadian rhythms, differentiation and the responses to hypoxia. We examined whether DEC1 and DEC2 are involved in apoptosis regulation, in human breast cancer MCF‐7 cells. We found that siRNA‐mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with that in control cells transfected with nonspecific siRNA. However, knockdown of DEC1 by siRNA did not affect cell survival. Knockdown of DEC2 affected the expression of mRNA or proteins related to apoptosis, such as Fas, c‐Myc, caspase‐8, poly (ADP‐ribose) polymerase (PARP) and Bax. We also showed that tumor necrosis factor‐α (TNF‐α) up‐regulates the expression of DEC1 and DEC2. DEC2 over‐expression caused by the transfection of an expression vector reduced the amounts of cleaved PARP and caspase‐8 induced by TNF‐α treatment, whereas DEC1 over‐expression increased it. Finally, we revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase‐8 induced by DEC2 siRNA with or without TNF‐α. These data indicate that DEC2 has an anti‐apoptotic effect, whereas DEC1 has a pro‐apoptotic effect, which are involved in the balance of survival of human breast cancer MCF‐7 cells.

The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer

DEC1 (BHLHE40/Stra13/Sharp2) is a basic helix-loop-helix (bHLH) transcription factor that is involved in the regulation of apoptosis and cell proliferation and the response to hypoxia. Epithelial-mesenchymal transition (EMT) is an important step leading to invasion and migration of various tumor cells, and TGF-β treatment has been shown to induce cancer cells to undergo EMT. However, the significance of DEC1 in TGF-β-induced EMT remains unknown. We examined the role of DEC1 in EMT of PANC-1 cells, a human pancreatic cancer cell line. As a result, we found that DEC1 was upregulated by TGF-β in PANC-1 cells, and regulated the expression and the levels of nuclear, cytoplasmic or membrane localization of EMT-related factors, including phosphorylated Smad3 (pSmad3), snail, claudin-4 and N-cadherin. In the presence of TGF-β, DEC1 knockdown by siRNA inhibited morphological changes during EMT processes, while TGF-β induced PANC-1 cells to taken on a spindle-shaped morphology. Furthermore, a combination treatment of DEC1 expression with TGF-β was closely linked to the migration and invasion of PANC-1 cells. Immunohistochemically, DEC1 and pSmad3 were detected within pancreatic cancer tissues, whereas claudin-4 expression was weaker in the cancer tissues compared with the adjacent non-cancer pancreatic tissues. These findings suggest that DEC1 plays an important role in the regulation of these EMT-related factors in pancreatic cancer.

L1 Cell adhesion molecule (L1CAM) expression at the cancer invasive front is a novel prognostic marker of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most extremely aggressive cancers with a poor prognosis after curative resection. L1 cell adhesion molecule (L1CAM) is a 200–220 kDa type I transmembrane glycoprotein of the immunoglobulin superfamily, which has been shown to affect the prognosis of several cancers. No clinicopathological significance of L1CAM expression has been examined at the invasive front of PDAC. In this study, we examined the relationship between L1CAM expression and clinicopathological features in PDAC by immunohistochemistry.

PERIOD1 is an Anti-apoptotic Factor in Human Pancreatic and Hepatic Cancer Cells

PERIOD1 (PER1) is a clock gene. We examined the effect of knockdown of PER1 on apoptosis in pancreatic cancer (MIA PaCa-2 and PANC-1) and hepatocellular carcinoma (HepG2) cells. Transfection of siRNA against PER1 into these cells increased the cleaved forms of caspases and poly-ADP-ribose-polymerase and induced apoptosis in all three cell lines. In the two pancreatic cancer cell lines, PER1 knockdown resulted in upregulation of Bax and downregulation of Bcl-2. Expression of p53 was not altered in the two pancreatic cancer cell lines containing mutated p53, but was upregulated in the HepG2 cells containing wild-type p53. Cell proliferation of MIA PaCa-2 and HepG2 was inhibited by PER1 knockdown. We also examined, by immunohistochemical staining, the expression of PER1 in pancreatic cancer tissue and found that PER1 was strongly expressed in pancreatic cancer cells. These results indicate that PER1 acts as an anti-apoptotic factor in pancreatic cancer cells.

PERIOD1 (PER1) has anti-apoptotic effects, and PER3 has pro-apoptotic effects during cisplatin (CDDP) treatment in human gingival cancer CA9-22 cells

PERIOD (PER) proteins are transcriptional regulators that are involved in circadian rhythms, sleep homeostasis, cell proliferation and tumour progression. We previously showed that the expression of PER1 was related to the regulation of apoptosis in human pancreatic cancer and hepatocellular carcinoma cells. However, the significance of PER in oral cancer has not been reported, and the detailed molecular mechanisms by which anti-tumour drug induces apoptosis in gingival cancer cells are not well understood. We examined whether PER1 and PER3 are involved in the regulation of apoptosis in human gingival cancer CA9-22 cells. The expression of PER1 and PER3 was upregulated and downregulated, respectively, by cis-diamminedichloroplatinum (II) (cisplatin: CDDP) treatment in CA9-22 cells, whereas CDDP treatment had little effects on the expression of PER1 and PER3 in human gingival fibroblasts (HGF-1). We found that short interference RNA (siRNA)-mediated knockdown of PER1 enhanced apoptosis of CA9-22 cells, and that PER1 regulated the amount of Bim, an apoptosis-related molecule. On the other hand, PER3 knockdown had an inhibitory effect on the apoptosis of CA9-22 cells induced by CDDP treatment. These results suggest that the alternation of expression of PER1 and PER3 was related to the apoptosis of CA9-22 cells. Furthermore, PER1 was intensely stained in the gingival cancer tissues, whereas PER3 was significantly stained in the non-tumour tissues by immunohistochemistry. These findings suggest that PER1 and PER3 have anti-apoptotic and pro-apoptotic effects in human gingival cancer CA9-22 cells, respectively. The balance of PER1 and PER3 may modulate apoptotic reactions in gingival cancer cells.

Anti-apoptotic effect of claudin-1 in tamoxifen-treated human breast cancer MCF-7 cells

BackgroundClaudin-1 is a membrane protein of tight junctions, and is associated with the development of various cancers. However, the significance of claudin-1 expression in cancer cells is not well understood. Here, we showed for the first time the anti-apoptotic effect of claudin-1 in human breast cancer MCF-7 cells.MethodsHuman breast cancer MCF-7 and T47 D cells were treated with or without tamoxifen, siRNA against claudin-1, or tamoxifen and claudin-1 siRNA. The samples were analyzed by RT-PCR, Western blotting or immunofluorescent staining.ResultsThe expression of claudin-1 was upregulated in tamoxifen-treated MCF-7 cells, whereas the expression of claudin-1 was not altered in tamoxifen-treated T47 D cells. Knockdown of claudin-1 by siRNA increased the amount of poly (ADP-ribose) polymerase (PARP) regardless of tamoxifen treatment in MCF-7 cells, but not T47 D cells. In the cell membranes of the MCF-7 cells, tamoxifen treatment increased the amount of claudin-1, but decreased the amount of β-catenin. Claudin-1 siRNA increased the amount of E-cadherin in the cytoplasm of the MCF-7 cells as well as the amount of β-catenin in their cell membranes.ConclusionThese results indicate that claudin-1 has anti-apoptotic effects, and is involved in the regulation of the expression and subcellular localization of β-catenin and E-cadherin in MCF-7, but not T47 D cells.

Incidental prostate 18F-FDG uptake without calcification indicates the possibility of prostate cancer

Incidental 18F-fluorodeoxyglucose (18F-FDG) uptake in the prostate is often experienced in clinical practice; however, it is difficult to determine whether incidental uptake is indicative of a malignancy or benign state based on the maximum standardized uptake value (SUVmax). In the present study, we investigated the clinical significance of incidental prostate uptake by 18F-FDG positron emission tomography (PET)/CT, and examined the differences between malignant and benign uptake from a clinicopathological viewpoint. We reviewed 3,236 male subjects who underwent 18F-FDG PET/CT scans at Hirosaki University Hospital (Japan) from 2008 to 2012 in order to identify cases of incidental prostate FDG uptake. The final diagnosis was made by serum prostate-specific antigen (PSA) levels, biopsy, imaging studies and clinical follow-up with PET findings. Incidental FDG uptake of the prostate was observed in 53 cases (2%). Four cases were excluded due to insufficient clinical data, and 49 cases were included in the present study. Of the 49 cases, 8 (16%) had prostate cancer, while 41 (84%) were benign. All 8 malignant cases had high uptake areas, e.g. in the prostate peripheral zone, where there was no coexistence of calcification or FDG uptake. Of the 41 benign cases, 19 had high uptake in the inner zone, 17 in the peripheral zone, and 5 in both the inner and peripheral zones. Of the 41 cases, 18 (44%) showed FDG uptake coexisting with prostatic calcification. Incidental prostate 18F-FDG uptake infrequently signifies prostate cancer; however, FDG uptake not coexisting with calcification indicates the possibility of prostate cancer and should be included in the differential diagnosis for performing other clinical examinations.

Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver.

Smads are intracellular signaling mediators. Complexes of Smad2 and Smad3 with Smad4 transmit transforming growth factor-beta (TGF-β) receptor-induced signaling. Snail plays important roles in mesoderm formation, gastrulation, neural crest development, and epithelial mesenchymal transition. However, it remains unknown whether Smad3 and Snail expression is circadian rhythm-dependent. Here, we showed for the first time that Smad3 and Snail show circadian expression in human gingival fibroblasts (HGF-1) and human mesenchymal stem cells (MSC) after serum shock. They also showed circadian expression in the mouse liver. We confirmed that BMAL1/2, DEC1/2, VEGF, and PER1/2/3 also show circadian expression in both HGF-1 and MSC. The mRNA peaks and phases in circadian expression of these genes differed between HGF-1 and MSC. In a luciferase assay, Smad3 promoter activity was upregulated by CLOCK/BMAL1. These findings suggest that Smad3 and Snail have circadian rhythm in vitro and vivo, and that circadian expression of Smad3 depends on CLOCK/BMAL1.

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.