Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs. r

Silk fibroin sol-gel transitions were studied by monitoring the process under various physicochemical conditions with optical spectroscopy at 550 nm. The secondary structural change of the fibroin from a disordered state in solution to a beta-sheet-rich conformation in the gel state was assessed by FTIR and CD over a range of fibroin concentrations, temperatures, and pH values. The structural changes were correlated to the degree of gelation based on changes in optical density at 550 nm. No detectable changes in the protein secondary structure (FTIR, CD) were found up to about 15% gelation (at 550 nm), indicating that these early stages of gelation are not accompanied by the formation of beta-sheets. Above 15%, the fraction of beta-sheet linearly increased with the degree of gelation. A pH dependency of gelation time was found with correlation to the predominant acidic side chains in the silk. Electrostatic interactions were related to the rate of gelation above neutral pH. The overall independencies of processing parameters including concentration, temperature, and pH on gel formation and protein structure can be related to primary sequence-specific features in the molecular organization of the fibroin protein. These findings clarify aspects of the self-assembly of this unique family of proteins as a route to gain control of material properties, as well as for new insight into the design of synthetic silk-biomimetic polymers with predictable solution and assembly properties.

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Aim:To investigate the status of Phosphatidylinositol 3-kinase (PI3K)/PTEN/AKT/mammalian target of rapamycin (mTOR) pathway and its correlation with clinicopathological features and matrix metalloproteinase-2, -9 (MMP-2, 9) in human hepatocellular carcinoma (HCC).Methods: PTEN, Phosphorylated AKT (p-AKT), Phosphorylated mTOR (p-mTOR), MMP-2, MMP-9 and Ki-67 expression levels were evaluated by immunohistochemistry on tissue microarrays containing 200 HCCs with paired adjacent noncancerous liver tissues. PTEN, MMP-2 and MMP-9 mRNA levels were determined by real-time RT-PCR in 36 HCCs. The relationships between PI3K/PTEN/AKT/mTOR pathway and clinicopathological factors and MMP-2, 9 were analyzed in HCC.Results: In HCC, PTEN loss and overexpression of p-AKT and p-mTOR were associated with tumor grade, intrahepatic metastasis, vascular invasion, TNM stage and high Ki-67 labeling index (P < 0.05). PTEN loss was correlated with p-AKT, p-mTOR and MMP-9 overexpression. Furthermore, PTEN and MMP-2, 9 mRNA levels were down-regulated and up-regulated in HCC compared with paired non-cancerous liver tissues, respectively (P < 0.01). PTEN, MMP-2 and MMP-9 mRNA levels were correlated with tumor stage and metastasis. There was an inverse correlation between PTEN and MMP-9 mRNA expression. However, PI3K/PTEN/AKT/mTOR pathway was not correlated with MMP-2.Conclusions: PI3K/PTEN/AKT/mTOR pathway, which is activated in HCC, is involved in invasion and metastasis through up-regulating MMP-9 in HCC.

Adhesion, spreading, proliferation, and collagen matrix production of human bone marrow stromal cells (BMSCs) on an RGD-modified silk matrix was studied. Anterior cruciate ligament fibroblasts (ACLFs) were used as a control cell source. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the modified silk matrices support improved BMSC and ACLF attachment and show higher cell density over 14 days in culture when compared with the non-RGD-modified matrices. Collagen type I transcript levels (at day 7) and content (at day 14) was significantly higher on the RGD-modified substrate than on the nonmodified group. The ability of RGD-coupled silk matrices to support BMSC attachment, which leads to higher cell density and collagen matrix production in vitro, combined with mechanical, fatigue, and biocompatibility properties of the silk protein matrix, suggest potential for use of this biomaterial for tissue engineering.

The aberrant activation of sonic hedgehog (SHH) pathway contributes to initiation and progression of various malignancies. However, the roles and underlying mechanisms of SHH signaling pathway in invasion and metastasis of liver cancer have not been well understood. In this study, we found that SHH signaling was activated and correlated with invasion and metastasis in hepatocellular carcinoma (HCC). Enhanced SHH signaling by recombinant human SHH N-terminal peptide (rSHH-N) promoted hepatoma cell adhesion, migration and invasion, whereas blockade of SHH signaling with SHH neutralizing antibody or cyclopamine suppressed hepatoma cell adhesion, migration and invasion. Furthermore, matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities were upregulated and downregulated by rSHH-N and SHH signaling inhibitor, respectively. The rSHH-N-mediated hepatoma cell migration and invasion was blocked by MMP-specific inhibitors or neutralizing antibodies to MMP-2 and MMP-9. In addition, phosphorylations of AKT and focal adhesion kinase (FAK) were increased and decreased by rSHH-N and SHH signaling inhibitor, respectively. Further investigations showed that activation of AKT and FAK were required for rSHH-N-mediated upregulation of MMP-2 and MMP-9, cell migration and invasion. Finally, we found that SHH protein expression was positively correlated with phosphorylatd FAK Tyr397, phosphorylatd AKT Ser473, MMP-2 and MMP-9 protein expressions in HCC samples. Taken together, our findings suggest that SHH pathway induces cell migration and invasion through FAK/AKT signaling-mediated MMP-2 and MMP-9 production and activation in liver cancer.

Aim: Recent studies have underlined causative links between microRNA (miRNA) deregulation and cancer development. However, the relevance of abnormally expressed miRNA to tumor biology has not been well understood in hepatocellular carcinoma (HCC).Methods: A bead-based miRNA expression profiling method was performed on 20 pairs of surgically removed HCC and adjacent non-tumorous tissue (NT). Special miR-338 downregulations and miR-338 associated with clinical characteristics was validated in an extended samples set of 36 paired HCC and adjacent non-tumorous liver tissues by real-time reverse transcription polymerase chain reaction (RT-PCR) analysis.Results: Out of our bead-based microarray data, 12 upregulated and 19 downregulated miRNA were found to be associated with HCC. Further characterization of miRNA-338, in which 20 pairs of the samples were clustered clearly into two groups according to expression of miR-338, revealed that the level of miR-338 expression can be associated with clinical aggressiveness, such as, tumor size, tumor-node-metastasis stage, vascular invasion and intrahepatic metastasis. These results were validated by real-time RT-PCR analysis. Conclusion:Our study suggests that miRNA expression could have relevance to the clinical behavior of HCC and that the bead-based miRNA expression profiling method might be a suitable system to assay miRNA expression in large-scale diagnostic trails.

MicroRNAs are involved in human carcinogenesis and cancer progression. Our previous study has shown that loss of miR-338-3p expression is associated with clinical aggressiveness of hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of miR-338-3p remain unknown in HCC. To determine whether and how miR-338-3p influences liver cancer cell invasion, we studied miR-338-3p in the liver cancer cell lines, and we found that miR-338-3p is down-regulated in treated cells. Forced expression of miR-338-3p in SK-HEP-1 cells suppressed cell migration and invasion, whereas inhibition of miR-338-3p in SMMC-7721 cells induced cell migration and invasion. Furthermore, smoothened (SMO) was identified as a direct target of miR-338-3p. Forced expression of miR-338-3p down-regulated SMO and matrix metalloproteinase (MMP)-9 expression, but inhibition of miR-338-3p up-regulated SMO and MMP9 expression. However, small interfering RNA targeted SMO reversed the effects induced by blockade of miR-338-3p. SMO and MMP9 were overexpressed and associated with invasion and metastasis in HCC tissues. These data indicate that miR-338-3p suppresses cell invasion by targeting the smoothened gene in liver cancer in vitro and miR-338-3p might be a novel potential strategy for liver cancer treatment.