Optimization of 3D hydrogel microenvironment for enhanced hepatic functionality of primary human hepatocytes.

Although primary human hepatocytes (PHHs) are the gold standard in drug efficacy and metabolism studies, long-term survival of PHHs and maintenance of their hepatic function are still challenging. In this study, we focused on the effect of the initial microenvironment on upregulation and long-term preservation of hepatic function of PHHs encapsulated within biodegradable hydrogel systems. PHHs were encapsulated in RGD-functionalized hybrid hydrogels with various degrees of degradability, and their hepatic functionality was analyzed. Regardless of the hydrogel elastic modulus, the combination with nondegradable hydrogels had a predominantly negative effect on the prompt engraftment of PHHs, whereas a degradable hydrogel with intermediate initial degradability was most effective in maintaining hepatic function. Efficient network formation by PHHs and cocultured cells, along with the control of hydrogel degradation, governed the hepatic functionality at an early stage and upon long-term cultivation. Under optimized conditions, expression of genes involved in biological processes such as focal adhesions, cell survival, cytoskeleton formation, and extracellular matrix interactions was significantly higher than that in a control with relatively delayed initial degradation. Thus, we suggest that the orchestrated control of initial cellular remodeling may play an important role in the maintenance of hepatic function in a three-dimensional PHH culture.

Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1.

The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration and its dysregulation is widely implicated in various cancers. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We further identified that the anaphase-promoting complex/cyclosome (APC/C)Cdh1 E3 ubiquitin ligase complex, which plays a key role governing eukaryotic cell cycle progression, intrinsically regulates Hippo signaling activities. CDH1 recognizes LATS kinases to promote their degradation and, hence, YAP/TAZ regulation by LATS phosphorylation is under cell cycle control. As a result, YAP/TAZ activities peak in G1 phase. Furthermore, we show in Drosophila eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation.

Cationic amino acid transporter PQLC2 is a potential therapeutic target in gastric cancer.

Tumor cells overexpress amino acid transporters to meet the increased demand for amino acids. PQ loop repeat-containing (PQLC)2 is a cationic amino acid transporter that might be involved in cancer progression. Here, we show that upregulation of PQLC2 is critical to gastric cancer (GC) development in vitro and in vivo. Both PQLC2 mRNA and protein were overexpressed in GC tissues, especially of the diffuse type. Overexpression of PQLC2 promoted cell growth, anchorage independence, and tumor formation in nude mice. This was due to activation of MEK/ERK1/2 and PI3K/AKT signaling. Conversely, PQLC2 knockdown caused growth arrest and cell death of cancer cells and suppressed tumor growth in a mouse xenograft model. These results suggest that targeting PQLC2 is an effective strategy for GC treatment.

Elasticity-based development of functionally enhanced multicellular 3D liver encapsulated in hybrid hydrogel.

Current in vitro liver models provide three-dimensional (3-D) microenvironments in combination with tissue engineering technology and can perform more accurate in vivo mimicry than two-dimensional models. However, a human cell-based, functionally mature liver model is still desired, which would provide an alternative to animal experiments and resolve low-prediction issues on species differences. Here, we prepared hybrid hydrogels of varying elasticity and compared them with a normal liver, to develop a more mature liver model that preserves liver properties in vitro. We encapsulated HepaRG cells, either alone or with supporting cells, in a biodegradable hybrid hydrogel. The elastic modulus of the 3D liver dynamically changed during culture due to the combined effects of prolonged degradation of hydrogel and extracellular matrix formation provided by the supporting cells. As a result, when the elastic modulus of the 3D liver model converges close to that of the in vivo liver (≅ 2.3 to 5.9 kPa), both phenotypic and functional maturation of the 3D liver were realized, while hepatic gene expression, albumin secretion, cytochrome p450-3A4 activity, and drug metabolism were enhanced. Finally, the 3D liver model was expanded to applications with embryonic stem cell-derived hepatocytes and primary human hepatocytes, and it supported prolonged hepatocyte survival and functionality in long-term culture. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. STATEMENT OF SIGNIFICANCE: We provide a functionally improved 3D liver model that recapitulates in vivo liver stiffness. We have experimentally addressed the issues of orchestrated effects of mechanical compliance, controlled matrix formation by stromal cells in conjunction with hepatic differentiation, and functional maturation of hepatocytes in a dynamic 3D microenvironment. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. Additionally, recent advances in the stem-cell technologies have made the development of 3D organoid possible, and thus, our study also provides further contribution to the development of physiologically relevant stem-cell-based 3D tissues that provide an elasticity-based predefined biomimetic 3D microenvironment.

Shikonin induces apoptosis of lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling antagonized by p300.

Shikonin derivatives exert powerful cytotoxic effects including induction of apoptosis. Here, we demonstrate the cytotoxic efficacy of shikonin in vivo in xenograft models, which did not affect body weight as well as its reduction of cell viability in vitro using several non-small cell lung cancer (NSCLC) cell lines. We found that inhibition of AKT by shikonin activated the forkhead box (FOX)O3a/early growth response protein (EGR)1 signaling cascade and enhanced the expression of the target gene Bim, leading to apoptosis in lung cancer cells. Overexpression of wild-type or a constitutively active mutant of FOXO3a enhanced shikonin-induced Bim expression. The NAD+-dependent histone deacetylase sirtuin (SIRT)1 amplified the pro-apoptotic effect by deacetylating FOXO3a, which induced EGR1 binding to the Bim promoter and activated Bim expression. Meanwhile, PI3K/AKT activity was enhanced, whereas that of FOXO3a was reduced and p300 was upregulated by treatment with a sublethal dose of shikonin. FOXO3a acetylation was enhanced by p300 overexpression, while shikonin-induced Bim expression was suppressed by p300 overexpression, which promoted cell survival. FOXO3a acetylation was increased by p300 overexpression and treatment with SIRT1 inhibitor, improving cell survival. In addition, shikonin-induced FOXO3a nuclear localization was blocked by AKT activation and SIRT1 inhibition, which blocked Bim expression and conferred resistance to the cytotoxic effects of shikonin. The EGR1 increase induced by shikonin was restored by pretreatment with SIRT1 inhibitor. These results suggest that shikonin induces apoptosis in some lung cancer cells via activation of FOXO3a/EGR1/SIRT1 signaling, and that AKT and p300 negatively regulate this process via Bim upregulation.