Protein kinase C delta enhances the diagnostic performance of hepatocellular carcinoma.

BACKGROUND: The conventional markers for hepatocellular carcinoma (HCC), α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP), have several limitations; both have low sensitivity in patients with early-stage HCC; low sensitivity for AFP with HCC after eliminating hepatitis C virus (HCV); low specificity for DCP in patients with non-viral HCC, which is increasing worldwide; low specificity for AFP in patients with liver injury; and low specificity for DCP in patients treated with warfarin. To overcome these issues, the identification of novel biomarkers is an unmet need. OBJECTIVE: This study aimed to assess the usefulness of serum protein kinase C delta (PKCδ) for detecting these HCCs. METHODS: PKCδ levels were measured using a sandwich enzyme-linked immunosorbent assay in 363 chronic liver disease (CLD) patients with and without HCC. RESULTS: In both viral and non-viral CLD, PKCδ can detect HCCs with high sensitivity and specificity, particularly in the very early stages. Notably, the value and sensitivity of PKCδ were not modified by HCV elimination status. Liver injury and warfarin administration, which are known to cause false-positive results for conventional markers, did not modify PKCδ levels. CONCLUSIONS: PKCδ is an enhanced biomarker for the diagnosis of HCC that compensates for the drawbacks of conventional markers.

Low geriatric nutritional risk index predicts poor prognosis in patients with cirrhosis: a retrospective study.

Aim: Malnutrition, which increases the risk of liver disease-related events and mortality, is a serious complication in cirrhosis. This study aimed to investigate whether the geriatric nutritional risk index (GNRI) could predict the long-term prognosis in patients with cirrhosis. Methods: We retrospectively evaluated 266 patients with cirrhosis and classified them into two groups based on baseline GNRI scores: risk (≤98, n = 104) and no-risk groups (>98, n = 162). The cumulative survival rates were compared between the two groups in patients with compensated and decompensated cirrhosis, respectively. Cox proportional hazards regression analysis was used to identify significant and independent factors associated with mortality. Results: The median observation period was 54.9 (33.6-61.7) months and 65 (24.4%) liver disease-related deaths occurred during the follow-up period. The GNRI scores significantly and inversely correlated with Child-Pugh score (r = -0.579), model for end-stage liver disease score (r = -0.286), and Mac-2 binding protein glycosylation isomer (r = -0.494). Multivariate analysis identified low GNRI as a significant and independent factor associated with mortality [overall cohort: hazard ratio (HR), 0.926; p < 0.001; compensated cirrhosis: HR, 0.947; p = 0.003; decompensated cirrhosis: HR, 0.923; p < 0.001]. The risk group demonstrated significantly lower cumulative survival rates than the no-risk group in overall cohort, and patients with compensated and decompensated cirrhosis (p < 0.001, <0.001, and = 0.013, respectively). Conclusion: Low GNRI was associated with poor long-term prognosis in both patients with compensated and decompensated cirrhosis. Therefore, the GNRI is a simple and useful tool for predicting prognosis and modifying the nutritional status in patients with cirrhosis.

Protein Kinase C Delta Is a Novel Biomarker for Hepatocellular Carcinoma.

Backgrounds and Aims: Hepatocellular carcinoma (HCC) is the most common cancer with a poor prognosis. Identification of an alternative biomarker that can detect early-stage and conventional tumor marker-negative HCC is urgently needed. We found that protein kinase C delta (PKCδ) is specifically secreted from HCC cell lines into extracellular space and contributes to tumor development and that its serum levels were elevated in HCC patients. This study aimed to assess the practical usefulness of serum PKCδ for detecting HCC in chronic liver disease (CLD) patients. Methods: Serum PKCδ levels in 313 CLD patients with and without HCC (n = 187 and 126, respectively) were measured using a sandwich enzyme-linked immunosorbent assay. The diagnostic performance of PKCδ for HCC was evaluated using the receiver operating characteristic curve analysis and was compared with that of conventional markers, α-fetoprotein (AFP), and des-γ-carboxy prothrombin (DCP). Results: Serum PKCδ levels in HCC patients were significantly higher than those in CLD patients without HCC. PKCδ distinguished HCC patients from CLD patients without HCC, with high sensitivity and specificity. Subgroup analyses revealed that the diagnostic performance of PKCδ for HCC was comparable to that of AFP and DCP, and that approximately 40% of AFP/DCP double-negative HCC patients were positive for PKCδ. PKCδ yielded better diagnostic performance for detecting solitary small-sized (ie, very early stage) HCC than AFP and DCP. There was no significant correlation between serum PKCδ and AFP/DCP levels. Conclusion: Serum PKCδ is a novel HCC biomarker, which is independent of and complementary to conventional markers. Specifically, PKCδ may be useful for detecting very early-stage or AFP/DCP double-negative HCC.

Safety and efficacy of pentazocine-midazolam combination for pain and anxiety relief in radiofrequency ablation therapy for hepatocellular carcinoma.

Background and Aim: Radiofrequency ablation (RFA) therapy is frequently used as first-line treatment for small hepatocellular carcinoma (HCC). RFA is often associated with pain; however, no definitive solution has been established for its relief. We retrospectively analyzed the safety and efficacy of the combination of pentazocine and midazolam to relieve pain experienced by HCC patients undergoing RFA. Methods: We studied 77 patients with 98 HCCs treated with RFA between January 2015 and August 2019. Patients were divided into two groups: the sedative-free group, which included those who received pentazocine alone, and the pentazocine-midazolam group, which included those who received a combination of pentazocine and midazolam. The degrees of analgesia and sedation were evaluated using the numerical rating scale (NRS) and the Richmond Agitation-Sedation Scale (RASS), respectively. Other parameters such as treatment time, awakening time, midazolam dosage, vital signs, local recurrence rate, and time to recurrence were also examined. Results: The median NRS score and RASS score were significantly lower in the pentazocine-midazolam group. Ninety-five percent of patients in the pentazocine-midazolam group had no memory of the RFA session. The treatment time and awakening time were prolonged for the pentazocine-midazolam group. No significant differences in oxygen saturation, recurrence rates, and time to local recurrence were observed between groups. Conclusion: A combination of pentazocine and midazolam is safe and effective for pain and anxiety relief experienced by patients undergoing RFA for local treatment of HCC.

Real-time breath ammonia measurement using a novel cuprous bromide sensor device in patients with chronic liver disease: a feasibility and pilot study.

We developed a small portable sensor device using a p-type semiconductor cuprous bromide (CuBr) thin film to measure breath ammonia in real time with highsensitivity and selectivity. Breath ammonia is reportedly associated with chronic liver disease (CLD). We aimed to assess the practical utility of the novel CuBr sensor device for exhaled breath ammonia and the correlation between breath and blood ammonia in CLD patients. This was a feasibility and pilot clinical study of 21 CLD patients and 18 healthy volunteers. Breath ammonia was directly and quickly measured using the novel CuBr sensor device and compared with blood ammonia measured at the same time. CLD patients had significantly higher breath ammonia levels than healthy subjects (p = 1.51 × 10-3), with the level of significance being similar to that for blood ammonia levels (p= 0.024). Significant differences were found in breath and blood ammonia between the healthy and cirrhosis groups (p = 2.97 × 10-3 and 3.76 × 10-3, respectively). Significant, positive correlations between breath and blood ammonia were noted in the CLD group (R = 0.747, p = 1.00 × 10-4), healthy/CLD group (R = 0.741, p = 6.75 × 10-8), and cirrhosis group (R = 0.744, p = 9.52 × 10-4). In conclusion, the newly developed, easy-to-use, and small portable CuBr sensor device was able to non-invasively measure breath ammonia in real time. Breath ammonia measured using the device was correlated with blood ammonia and the presence of liver cirrhosis, and might be an alternative surrogate biomarker to blood ammonia.

Unconventional Secretion of PKCδ Exerts Tumorigenic Function via Stimulation of ERK1/2 Signaling in Liver Cancer.

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi-independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer-related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer. SIGNIFICANCE: PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

Groundwater oxygen anomaly related to the 2016 Kumamoto earthquake in Southwest Japan.

Here, we report the groundwater oxygen isotope anomalies caused by the 2016 Kumamoto earthquake (MJMA7.3) that occurred in Southwest Japan on April 16, 2016. One hundred and seventeen groundwater samples were collected from a deep well located 3 km to the southeast of the epicenter in Mifune Town, Kumamoto Prefecture; they were drinking water packed in PET bottles and distributed in the area between April 2015 and March 2018. Further, the oxygen and hydrogen isotopes were evaluated via cavity ring-down spectroscopy without performing any pretreatment. An anomalous increase was observed with respect to the δ18O value (up to 0.51‰) soon after the earthquake along with a precursory increase of 0.38‰ in January 2016 before the earthquake. During these periods, there was no noticeable change in the hydrogen isotopic ratios. Rapid crustal deformation related to the earthquake may have enhanced the microfracturing of the aquifer rocks and the production of new surfaces, inducing δ18O enrichment via oxygen isotopic exchange between rock and porewater without changing δ2H.

Live-cell imaging of HP1α throughout the cell cycle of mouse C3H10T1/2 cells and rhythmical flickering of heterochromatin dots in interphase.

Heterochromatin protein 1 alpha (HP1α) localizes to heterochromatin in interphase and shows dynamic molecular behavior in living cells. We previously reported that during mitosis, the majority of HP1α diffused into the cytoplasm but some remained in centromere heterochromatin. Here, we further characterize the molecular behavior of HP1α throughout the cell cycle. Time-lapse imaging of DsRed-HP1α through two successive cell divisions indicated that interphase can be divided into four phases. HP1α forms heterochromatin dots in early G1, which are maintained without any apparent changes (Phase 1). However, the HP1α dots begin to diffuse into the nucleoplasm and start flickering with a rhythmical cycle (Phase 2). Then, the HP1α dots diffuse further towards the periphery of the nucleus (Phase 3), and uniformly diffuse throughout the entire nucleus (Phase 4). Rhythmical flickering of HP1α dots in the middle of interphase may be useful for following cell cycle progression in mouse living cells.

A rapid and simple method of evaluating the dimeric tendency of fluorescent proteins in living cells using a truncated protein of importin α as fusion tag.

Enhanced green fluorescent protein (EGFP) and its yellow variant (Venus) are weakly dimeric under physiological conditions. We designed a simple method to evaluate the dimeric tendency of fluorescent proteins in living mammalian cells. A novel single mutation, A206L, interfering with the hydrophobic interactions of the dimer interface in Venus, contributed to its monomerization, and was as effective as the A206K mutation in this assay.

Improvement of a Venus-based bimolecular fluorescence complementation assay to visualize bFos-bJun interaction in living cells.

Bimolecular fluorescence complementation (BiFC) assay makes it possible to visualize protein-protein interactions in living cells. In this assay, Venus, a bright-yellow variant of green fluorescent protein, is known to produce fluorescent backgrounds due to non-specific interactions. In this study we found that the V150A mutation increased by 8.6-fold the signal-to-noise ratio in the BiFC assay of bFos-bJun interaction.