Heat shock protein 22 (HSPB8) reduces the migration of hepatocellular carcinoma cells through the suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway.

Small heat shock proteins (HSPs) regulate a variety of cell functions. Among them, HSP22 and HSP20 are recognized to be ubiquitously expressed in various tissues. With regard to hepatocellular carcinoma (HCC) cells, we previously reported that phosphorylated HSP20 plays a suppressive role in transforming growth factor (TGF)-α-induced cell migration and invasion. In the present study, we investigated whether or not HSP22 is implicated in HCC cell migration. We detected HSP22 protein expression both in human HCC tumor (189.9±68.4ng/mg protein) and the adjacent non-tumor liver tissues (167.9±94.6ng/mg protein). The cases of low-quantity HSP22 protein level group (88.3≧ng/mg protein, the optimum cut-off value of HSP22) were increased in tumor tissues compared with the adjacent non-tumor tissues. The migration of human HCC-derived HuH-7 cells stimulated by TGF-α or hepatocyte growth factor (HGF) was significantly enhanced by the knockdown of HSP22 expression. Down-regulation of HSP22 protein in the cells markedly strengthened the AKT phosphorylation induced by TGF-α or HGF. Inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathway, which suppressed the TGF-α-induced migration, significantly reduced the amplification by HSP22 knockdown. PI3K but not AKT was coimmunoprecipitated with HSP22 in HuH-7 cells. In addition, in human HCC tissues, a significantly lower HSP22 protein level in tumor tissues than in adjacent non-tumor tissues was observed more frequently in cases of moderately or poorly differentiated HCC than well-differentiated HCC. Taken together, our results strongly suggest that HSP22 represses HCC progression, especially HCC cell migration, by the down-regulation of the PI3K/AKT signaling pathway.

Heat shock protein 20 (HSPB6) regulates TNF-α-induced intracellular signaling pathway in human hepatocellular carcinoma cells.

We previously demonstrated that the expression of HSP20, a small heat shock protein, is inversely correlated with the progression of HCC. Inflammation is associated with HCC, and numerous cytokines, including TNF-α, act as key mediators in the progression of HCC. In the present study, we investigated whether HSP20 is implicated in the TNF-α-stimulated intracellular signaling in HCC using human HCC-derived HuH7 cells in the presence of TNF-α. In HSP20-overexpressing HCC cells, the cell growth was retarded compared with that in the control cells under long-term exposure of TNF-α. Because NF-κB pathway is the main intracellular signaling system activated by TNF-α, we investigated the effects of HSP20-overexpression of this pathway. The protein levels of IKK-α, but not IKK-ß, in the HSP20-overexpressing cells were decreased. Short-term exposure to TNF-α-induced phosphorylation and degradation of IκB, and the phosphorylation and transactivational activity of NF-κB were suppressed in the HSP20-overexpressing HCC cells. Furthermore, the increase in IKK-α levels was accompanied by a decrease in the HSP20 levels in human HCC tissues. These findings strongly suggest that HSP20 might decrease the IKK-α protein level and that it down-regulates the TNF-α-stimulated intracellular signaling in HCC, thus resulting in the suppression of HCC progression.

Utility of real-time shear wave elastography for assessing liver fibrosis in patients with chronic hepatitis C infection without cirrhosis: Comparison of liver fibrosis indices.

AIM: To clarify the diagnostic impact of liver fibrosis except for cirrhosis identified using shear wave elastography (SWE) in chronic hepatitis C (CHC) patients, and to compare the performance in diagnosing liver fibrosis among SWE and liver fibrosis indices. METHODS: A total of 55 CHC patients who underwent liver biopsy were analyzed. The diagnostic performance for identifying significant liver fibrosis (F2-F3) for SWE, FIB-4 index, aspartate aminotransferase-to-platelet ratio index (APRI) and Forns' index was assessed using receiver-operator curve (ROC) analysis. RESULTS: The median SWE elasticity value, FIB-4 index, APRI and Forns' index in the F0-F1 and F2-F3 groups were 6.3 kPa and 13.1 kPa; 1.52 and 4.45; 0.41 and 1.43; and 7.69 and 8.85, respectively (P < 0.001 for all four methods). Multivariate analysis showed that SWE was independently associated with the presence of significant liver fibrosis (odds ratio, 2.52; 95% confidence interval, 1.49-4.28; P < 0.001). The area under the ROC curve for SWE in diagnosing significant liver fibrosis was 0.94, indicating high diagnostic value, compared with 0.86, 0.88 and 0.83, for the FIB-4 index, APRI and Forns' index, respectively, which corresponds to moderate diagnostic value. The accuracy of SWE, FIB-4 index, APRI and Forns' index for diagnosing significant liver fibrosis was 90.9%, 76.4%, 74.5% and 67.2%, respectively. CONCLUSION: SWE has excellent ability for diagnosing significant liver fibrosis in CHC even when patients with cirrhosis are excluded. The diagnostic performance of SWE is superior to that of three liver fibrosis indices.

Intra-individual comparison of liver stiffness measurements by magnetic resonance elastography and two-dimensional shear-wave elastography in 888 patients.

PURPOSE: Quantitative elastography methods, such as ultrasound two-dimensional shear-wave elastography (2D-SWE) and magnetic resonance elastography (MRE), are used to diagnose liver fibrosis. The present study compared liver stiffness determined by 2D-SWE and MRE within individuals and analyzed the degree of agreement between the two techniques. METHODS: In total, 888 patients who underwent 2D-SWE and MRE were analyzed. Bland-Altman analysis was performed after both types of measurements were log-transformed to a normal distribution and converted to a common set of units using linear regression analysis for differing scales. The expected limit of agreement (LoA) was defined as the square root of the sum of the squares of 2D-SWE and MRE precision. The percentage difference was expressed as (2D-SWEMRE)/ mean of the two methods×100. RESULTS: A Bland-Altman plot showed that the bias and upper and lower LoAs (ULoA and LLoA) were 0.0002 (95% confidence interval [CI], -0.0057 to 0.0061), 0.1747 (95% CI, 0.1646 to 0.1847), and -0.1743 (95% CI, -0.1843 to -0.1642), respectively. In terms of percentage difference, the mean, ULoA, and LLoA were -0.5944%, 19.8950%, and -21.0838%, respectively. The calculated expected LoA was 17.1178% (95% CI, 16.6353% to 17.6002%), and 789 of 888 patients (88.9%) had a percentage difference within the expected LoA. The intraclass correlation coefficient of the two methods indicated an almost perfect correlation (0.8231; 95% CI, 0.8006 to 0.8432; P<0.001). CONCLUSION: Bland-Altman analysis demonstrated that 2D-SWE and MRE were interchangeable within a clinically acceptable range.

Intra-individual Comparisons of the Ultrasound-Guided Attenuation Parameter and the Magnetic Resonance Imaging-Based Proton Density Fat Fraction Using Bias and Precision Statistics.

Ultrasound-based techniques using the attenuation coefficient, including the ultrasound-guided attenuation parameter (UGAP), have been developed for the quantification of hepatic steatosis. The magnetic resonance imaging-based proton density fat fraction (MRI-PDFF) is considered to be more accurate than liver biopsy for liver fat quantification. The aim of this study was to perform intra-individual comparisons of UGAP and MRI-PDFF for determining hepatic steatosis grade. The study enrolled 309 patients who underwent UGAP and MRI-PDFF measurements. Bland-Altman analysis was conducted after transforming MRI-PDFF values to a normal distribution and converted to a common set of units using linear regression analysis for differing scales. The expected limits of agreement (LOA) was defined as the square root of the sum of the squares of UGAP and MRI-PDFF precision. A Bland-Altman plot revealed that the bias and upper and lower LOAs (ULOA and LLOA) were -0.0047, 0.1160 and -0.1255, respectively. The percentage difference indicated that the mean, ULOA and LLOA were -1.1434%, 18.1723% and -20.4590%, respectively. The calculated expected LOA was 18.5449%, and 283 of 309 patients (91.6%) had a percentage difference within 18.5449%. Bland-Altman analysis revealed that UGAP and MRI-PDFF were interchangeable within a clinically acceptable range.

Changing Methods of Education During a Pandemic: Questionnaire Survey about Examinations for Nuclear Medicine Technology at Educational Institutions in Japan.

Coronavirus disease 2019 (COVID-19) has spread around the world. Its effects go far beyond health care: education has to be conducted so as to prevent infection among students and faculty. Accordingly, changes have occurred in Japan's educational institutions, including methods of preparing students for examinations for nuclear medicine. To assess the quality of training for radiologic technologists, we investigated the related changes undertaken at educational institutions. We investigated the lecture format for teaching nuclear medicine technology at Japanese institutions during COVID-19 and efforts to ensure the quality of conventional education. Methods: We sent a questionnaire to 19 Japanese institutions. It addressed the lecture format and initiatives in examinations for nuclear medicine technology in the first and second semesters of 2020. Results: We obtained responses from 17 institutions. In the first semester of 2020, the lecture format for nuclear medicine technology included remote, hybrid (combination of remote and face-to-face), and video-on-demand lectures. To reinforce the effect of the new teaching formats, institutions adopted various methods, such as enhancing the possibility of allowing students to ask questions, increasing the number of quizzes during lectures, delivering lectures to YouTube, and introducing an e-learning system. In the second semester of 2020, the lecture format included face-to-face, remote, hybrid, and video-on-demand lectures. In that second semester, the number of institutions providing face-to-face lectures while taking thorough measures against infection showed a marked increase. Conclusion: The institutions introduced various educational techniques and initiatives. They prioritized students' understanding of lecture content and applied what they considered the best teaching methods. Sharing information about the changes adopted at different institutions should help promote good radiologic technologists-even during a pandemic.

Suppression by heat shock protein 20 of hepatocellular carcinoma cell proliferation via inhibition of the mitogen-activated protein kinases and AKT pathways.

Heat shock protein (HSP) 20, one of the low-molecular weight HSPs, is known to have versatile functions, such as vasorelaxation. However, its precise role in cancer proliferation remains to be elucidated. While HSP20 is constitutively expressed in various tissues including the liver, we have previously reported that HSP20 protein levels in human hepatocellular carcinoma (HCC) cells inversely correlate with the progression of HCC. In this study, we investigated the role of HSP20 in HCC proliferation. The activities of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and AKT were negatively correlated with the HSP20 protein levels in human HCC tissues. Since HSP20 proteins were hardly detected in HCC-derived cell lines, the effects of HSP20 expression were evaluated using human HCC-derived HuH7 cells that were stably transfected with wild-type human HSP20 (HSP20 overexpressing cells). In HSP20 overexpressing cells, cell proliferation was retarded, and the activation of the mitogen-activated protein kinases (MAPKs) signaling pathways, including the ERK and JNK, and AKT pathways, as well as cyclin D1 accumulation induced by either transforming growth factor-α (TGFα) or hepatocyte growth factor, were significantly suppressed compared with the empty vector-transfected cells (control cells). Taken together, our findings strongly suggest that HSP20 suppresses the growth of HCC cells via the MAPKs and AKT signaling pathways, thus suggesting that the HSP20 could be a new therapeutic target for HCC.

Educational Effect of Remote Lectures for Students Aiming to Become Radiologic Technologists: Questionnaire on Nuclear Medicine Examinations.

In the latter half of 2019, coronavirus disease 2019 (COVID-19) began spreading worldwide. To prevent COVID-19 infection, all teaching at Suzuka University of Medical Sciences from April to June 2020 took place as remote lectures, not in the face-to-face format. This study analyzed postlecture questionnaire responses regarding face-to-face and remote teaching on the subject of nuclear medicine technology examinations. We examined the educational effect of using remote lectures. Methods: We conducted a questionnaire survey among students by means of a 5-point evaluation scale about satisfaction, comprehension, concentration, preparation, reviewing, and the question environment for face-to-face and remote lectures. Results: We present the results as means and SDs. Satisfaction results for face-to-face and remote lectures were 3.30 ± 0.72 and 3.36 ± 0.88, respectively. Comprehension results for face-to-face and remote lectures were 3.30 ± 0.71 and 3.30 ± 0.83, respectively. Concentration results for face-to-face and remote lectures were 3.50 ± 0.69 and 3.05 ± 0.90, respectively. The preparation results for face-to-face and remote lectures were 2.57 ± 0.88 and 2.67 ± 0.94, respectively. The reviewing results for face-to-face and remote lectures were 2.84 ± 0.85 and 3.39 ± 0.89, respectively. The question environment results for face-to-face and remote lectures lessons were 2.94 ± 0.90 and 3.43 ± 0.84, respectively. There were no significant differences between face-to-face and remote lectures in terms of satisfaction, comprehension, or preparation. There were significant differences between face-to-face and remote lectures in terms of concentration, reviewing, and the questioning environment (P < 0.001). Conclusion: This comparative analysis of the postlecture questionnaire responses for face-to-face and remote formats in nuclear medicine technology examinations showed that remote lectures have a strong educational effect. We believe that, in future, remote lectures should be considered a tool in student education.

Liver fibrosis assessment using 99mTc-GSA SPECT/CT fusion imaging.

PURPOSE: To determine the utility of mean standardized uptake value (SUVmean) of whole liver measured by 99mTc-GSA SPECT/CT fusion imaging, for evaluation of liver fibrosis. MATERIALS AND METHODS: Eighty-six patients who underwent hepatectomy were enrolled, and were classified into the non-fibrosis or fibrosis group based on the pathological findings in the resected liver specimen. Univariate and multivariate analyses were performed between the two groups on four blood biochemical indices (albumin, total bilirubin, platelet count, and prothrombin time activity) and two 99mTc-GSA scintigraphy-derived liver function indices (LHL15 and SUVmean) to evaluate the independent predictive value for severe fibrosis. The diagnostic value of the index for severe fibrosis was assessed by calculating the area under the receiver operating characteristic curve. RESULTS: Multivariate analysis showed that prothrombin time activity [odds ratio (OR) 0.519], LHL15 (OR 0.513), and SUVmean (OR 0.168) significantly correlated with liver fibrosis. SUVmean showed the largest area under the curve, with value of 0.804, 0.730 for platelet count, 0.717 for LHL15, and 0.668 for prothrombin time activity. The optimal cut-off value for SUVmean was 6.7, which yielded 62.9% sensitivity and 96.9% specificity. CONCLUSIONS: SUVmean measured by 99mTc-GSA SPECT/CT fusion imaging enables highly accurate prediction of severe liver fibrosis.

Negative regulation by p70 S6 kinase of FGF-2-stimulated VEGF release through stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts.

UNLABELLED: To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2-stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts. INTRODUCTION: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2-activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known. MATERIALS AND METHODS: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase. RESULTS: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2-stimulated VEGF release and VEGF mRNA expression. The FGF-2-induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2-stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2-stimulated VEGF release and phosphorylation of SAPK/JNK. CONCLUSIONS: These results strongly suggest that p70 S6 kinase limits FGF-2-stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts.

Expression levels of heat shock protein 20 decrease in parallel with tumor progression in patients with hepatocellular carcinoma.

Heat shock protein (HSP) 20, a low-molecular-weight HSP, is constitutively expressed in various tissues, such as smooth muscle, skeletal muscle, and liver. However, the characteristics and function of HSP20 have not been precisely understood. In the present study, we investigated correlations of expression levels of HSP20 in hepatocellular carcinoma (HCC) tissues and the surrounding tissues with clinical and pathologic characteristics in 53 resected HCC specimens. Although HSP20 was detected in all 53 HCC tissues, the expression levels were reduced compared with those in the adjacent non-tumor tissues. The expression levels of HSP20 were inversely correlated with tumor stage by TNM classification (p<0.01), presence of microvascular invasion (p<0.05), and tumor size (p<0.05). Our findings strongly suggest that HSP20 may play a role against the progression of human HCC.

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma.

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.

Comparison Between Prone and Upright Imaging of the Inferior Wall Using 201TlCl Myocardial Perfusion SPECT.

Because it suppresses attenuation artifacts from the diaphragm, prone SPECT is suitable for evaluating the cardiac inferior wall. A solid-state dedicated cardiac camera allows upright imaging, which can also be used to avoid attenuation artifacts from the diaphragm. We compared prone and upright imaging for inferior wall evaluation using 201TlCl myocardial perfusion SPECT (MPS). Methods: The study targeted 45 patients. The prone imaging group included 23 subjects who underwent additional prone imaging because supine imaging indicated that the inferior wall had reduced uptake. The upright imaging group included 22 subjects who, in the past, had shown reduced uptake in the inferior wall during supine imaging. Using the MPS stress images and analysis software, we created a polar map showing the incorporation of the radioisotope throughout the whole of the myocardium; this polar map was then classified into 17 segments. The percentage uptake ratios of the inferior/anterior wall were calculated for the prone and upright acquisitions. These ratios were used as the ratio of percentage uptake in each segment of the anterior wall to percentage uptake in each segment of the inferior wall. In addition, 6 reviewers visually evaluated the uniformity within the inferior wall for both the prone and the upright imaging. Results: There was a significant difference in percentage uptake ratios between the prone and upright images in segments 4/1 (basal inferior/basal anterior; P < 0.05), 11/12 (mid inferolateral/mid anterolateral; P < 0.001), and 15/13 (apical inferior/apical anterior; P < 0.05). There were no significant differences between the prone and upright images in visual evaluations of uniformity within the inferior wall. Conclusion: In comparison with upright imaging, prone imaging has a higher rate of suppression of attenuation artifacts from the diaphragm. However, this difference does not seem to affect the images visually. Therefore, upright and prone imaging can be used interchangeably to evaluate the inferior wall.

Criteria for the addition of prone imaging to myocardial perfusion single-photon emission computed tomography for inferior wall.

OBJECTIVE: Myocardial perfusion single-photon emission computed tomography (SPECT) is occasionally suspected to generate images that represent either ischemia or infarction for the inferior wall [right coronary artery (RCA) disease] or attenuation artifacts because of the diaphragm. We often encounter this. The application of prone imaging is advantageous in the differentiation of RCA disease because of attenuation artifacts. If decreased accumulation of radioisotopes is observed at the site with either RCA disease or attenuation artifacts, then a criterion that enables the addition of prone imaging should be implemented. Then, we evaluated sites where RCA disease and attenuation artifacts would likely appear and investigated the threshold of decreased accumulation that enables utilization of prone imaging. PATIENTS AND METHODS: The patients in this study were divided into two groups: group A (20 patients) suspected to have attenuation artifacts because of the diaphragm and group B (14 patients) with RCA disease. Additional evaluation by prone imaging was performed in all patients. We utilized a 20-segment quantitative perfusion SPECT polar map in the supine and prone positions to compare the percentage increase in Thallium chloride (Tl) in both groups. We then investigated the percent uptake (%uptake) value of decreased accumulation in the inferior wall for the addition of prone imaging. RESULTS: The highest %uptake was present in segments 3, 4, 5, and 10 in group A after the prone imaging. Detection of attenuation artifacts from the diaphragm was easy in segments 3, 4, 5, and 10, and we set the %uptake threshold at 62, 61, 71, and 76%, respectively, in the supine position for the addition of prone imaging. CONCLUSION: A decrease of the %uptake in segments 3, 4, 5, and 10 after supine imaging is presumed to result from attenuation artifact or RCA disease. We established evaluation criteria for the addition of prone imaging in patients with decreased accumulation in the inferior wall during supine imaging.

Prostaglandin D2 induces the phosphorylation of HSP27 in osteoblasts: function of the MAP kinase superfamily.

We previously reported that prostaglandin D(2) (PGD(2)) stimulates the induction of heat shock protein 27 (HSP27) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether PGD(2) stimulates the phosphorylation of HSP27 in MC3T3-E1 cells exposed to heat shock. In the cultured MC3T3-E1 cells, PGD(2) markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85 in a time-dependent manner. Among the mitogen-activated protein (MAP) kinase superfamily, p44/p42 MAP kinase and p38 MAP kinase were phosphorylated by PGD(2) which had little effect on the phosphorylation of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). The PGD(2)-induced phosphorylation of HSP27 was attenuated by PD169316, an inhibitor of p38 MAP kinase or PD98059, a MEK inhibitor. SP600125, a SAPK/JNK inhibitor did not affect the HSP27 phosphorylation. In addition, PD169316 suppressed the PGD(2)-induced phosphorylation of MAPKAP kinase 2. These results strongly suggest that PGD(2) stimulates HSP27 phosphorylation via p44/p42 MAP kinase and p38 MAP kinase but not SAPK/JNK in osteoblasts.

Up-regulation by zinc of FGF-2-induced VEGF release through enhancing p44/p42 MAP kinase activation in osteoblasts.

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether zinc affects the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ZnSO(4) but not Na(2)SO(4). The enhancing effect of ZnSO(4) was dose-dependent between 1 and 100 muM. ZnSO(4) markedly enhanced the FGF-2-induced phosphorylation of p44/p42 MAP kinase while having little effect on the SAPK/JNK phosphorylation. PD98059 significantly reduced the amplification by ZnSO(4) of the FGF-2-stimulated VEGF release. Taken together, our findings strongly suggest that zinc enhances FGF-2-stimulated VEGF release resulting from up-regulating activation of p44/p42 MAP kinase in osteoblasts.

Phosphorylated Heat Shock Protein 20 (HSPB6) Regulates Transforming Growth Factor-α-Induced Migration and Invasion of Hepatocellular Carcinoma Cells.

Human hepatocellular carcinoma (HCC) is one of the major malignancies in the world. Small heat shock proteins (HSPs) are reported to play an important role in the regulation of a variety of cancer cell functions, and the functions of small HSPs are regulated by post-translational modifications such as phosphorylation. We previously reported that protein levels of a small HSP, HSP20 (HSPB6), decrease in vascular invasion positive HCC compared with those in the negative vascular invasion. Therefore, in the present study, we investigated whether HSP20 is implicated in HCC cell migration and the invasion using human HCC-derived HuH7 cells. The transforming growth factor (TGF)-α-induced migration and invasion were suppressed in the wild-type-HSP20 overexpressed cells in which phosphorylated HSP20 was detected. Phospho-mimic-HSP20 overexpression reduced the migration and invasion compared with unphosphorylated HSP20 overexpression. Dibutyryl cAMP, which enhanced the phosphorylation of wild-type-HSP20, significantly reduced the TGF-α-induced cell migration of wild-type HSP20 overexpressed cells. The TGF-α-induced cell migration was inhibited by SP600125, a c-Jun N-terminal kinases (JNK) inhibitor. In phospho-mimic-HSP20 overexpressed HuH7 cells, TGF-α-stimulated JNK phosphorylation was suppressed compared with the unphosphorylated HSP20 overexpressed cells. Moreover, the level of phospho-HSP20 protein in human HCC tissues was significantly correlated with tumor invasion. Taken together, our findings strongly suggest that phosphorylated HSP20 inhibits TGF-α-induced HCC cell migration and invasion via suppression of the JNK signaling pathway.

Direct association of heat shock protein 20 (HSPB6) with phosphoinositide 3-kinase (PI3K) in human hepatocellular carcinoma: regulation of the PI3K activity.

HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-α stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC.

Evaluation for clinical utility of GPC3, measured by a commercially available ELISA kit with Glypican-3 (GPC3) antibody, as a serological and histological marker for hepatocellular carcinoma.

AIMS: We evaluated the clinical utility of glypican-3 (GPC3), which has been proposed as a potential novel tumor marker for hepatocellular carcinoma (HCC), as a serological and histological marker for HCC. METHODS: The serum GPC3 level was compared between 200 patients with HCC and 200 patients with chronic liver disease (CLD). In addition, the expression of GPC3 was examined with immunohistochemistry on 38 resected specimens from patients with HCC. A commercially available GPC3 antibody was used for these analyses. RESULTS: The median values of serum GPC3 in patients with HCC and with CLD were 924.8 pg/mL and 1161.6 pg/mL, respectively. We found no elevation of serum GPC3 level in patients with HCC in comparison with those with CLD; rather the level was higher in patients with CLD (P < 0.0001). In immunohistochemical analysis, 14 of 38 (36.9%) HCC tissues were positive for GPC3, whereas no corresponding non-cancerous tissue was positive. The positivity for GPC3 tended to increase with pathologic decreased differentiation of HCC. CONCLUSIONS: We did not find serum GPC3 level, measured by a commercially available ELISA kit with GPC3 antibody, to be useful in the diagnosis of HCC. However, we did observe increased GPC3 staining in HCC tissue with moderate or poor differentiation, suggesting that GPC3 is produced by HCC tumors. This lack of utility could have been due to the measuring procedure used in the present study. Further evaluation of GPC3 in HCC with other measuring procedures is needed.