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.
Bimolecular fluorescence complementation (BiFC) assay makes it possible to visualize protein-protein interactions in living cells. In this assay, Venus, a brightyellow 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.
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.
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