HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

<p>HSPA5/GRP78/BiP plays an important role in cell survival or tumor progression. For these reasons, HSPA5 is an emerging therapeutic target in cancer development. Here we report that HSPA5 contributes to head and neck cancer (HNC) survival via maintenance of lysosomal activity; however, a nonthermal plasma (NTP, considered as a next-generation cancer therapy)-treated solution (NTS) inhibits HNC progression through HSPA5-dependent alteration of lysosomal activity. HSPA5 prevents NTS-induced lysosome inhibition through lysosomal-related proteins or regulation of gene expression. However, NTS-induced MUL1/MULAN/GIDE/MAPL (mitochondrial ubiquitin ligase activator of NFKB 1) leads to downregulation of HSPA5 via K48-linked ubiquitination at the lysine 446 (K446) residue. <i>MUL1</i> knockdown hinders NTS-induced lysosome inhibition or cytotoxicity through the reduction of HSPA5 ubiquitination in HNC cells. While MUL1 was suppressed, HSPA5 was overexpressed in tissues of HNC patients. NTS strongly inhibited HNC progression via alterations of expression of MUL1 and HSPA5, in vivo in a xenograft model. However, NTS did not induce inhibition of tumor progression or HSPA5 reduction in <i>MUL1</i> knockout (KO) HNC cells which were generated by CRISPR/Cas9 system. The data provide compelling evidence to support the idea that the regulation of the MUL1-HSPA5 axis can be a novel strategy for the treatment of HNC.</p

Non-Thermal Atmospheric Pressure Plasma Inhibits Thyroid Papillary Cancer Cell Invasion via Cytoskeletal Modulation, Altered MMP-2/-9/uPA Activity

<div><p>Plasma, the fourth state of matter, is defined as a partially or completely ionized gas that includes a mixture of electrons and ions. Advances in plasma physics have made it possible to use non-thermal atmospheric pressure plasma (NTP) in cancer research. However, previous studies have focused mainly on apoptotic cancer cell death mediated by NTP as a potential cancer therapy. In this study, we investigated the effect of NTP on invasion or metastasis, as well as the mechanism by which plasma induces anti-migration and anti-invasion properties in human thyroid papillary cancer cell lines (BHP10-3 and TPC1). Wound healing, pull-down, and Transwell assays demonstrated that NTP reduced cell migration and invasion. In addition, NTP induced morphological changes and cytoskeletal rearrangements, as detected by scanning electron microscopy and immunocytochemistry. We also examined matrix metalloproteinase (MMP)-2/-9 and urokinase-type plasminogen activator (uPA) activity using gelatin zymography, uPA assays and RT-PCR. FAK, Src, and paxillin expression was detected using Western blot analyses and immunocytochemistry. NTP decreased FAK, Src, and paxillin expression as well as MMP/uPA activity. In conclusion, NTP inhibited the invasion and metastasis of BHP10-3 and TPC1 cells by decreasing MMP-2/-9 and uPA activities and rearranging the cytoskeleton, which is regulated by the FAK/Src complex. These findings suggest novel actions for NTP and may aid in the development of new therapeutic strategies for locally invasive and metastatic cancers.</p></div

Invasion assays.

<p>(A) BHP10-3 and TPC1 cells were seeded on filters (pore size, 8 µm) coated with Matrigel in the upper compartment and exposed to He+O₂ gas only, 2 or 4 kV of NTP for 1 s. After 24 h, the cells in the pores or cells attached to undersurface of the membrane were counted, and those cells attached to the lower section were stained with H&E and counted using light microscopy (200×). Scale bar = 100 µm. (B) Quantification of the invasion assay data. NTP treatment significantly reduced the number of BHP10-3 (<i>P</i><0.001) and TPC1 (<i>P</i><0.001) cells that penetrated the membrane. The data represent the mean ± S.D. of three independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Wound healing assays.

<p>(A) BHP10-3 and TPC1 cells were plated in 12-well plates, grown to confluency, and the monolayer was wounded with a pipette tip. Wound healing was documented by photography after 24 h of incubation. Scale bar = 200 µm. (B) Quantification of cell migration. The NTP significantly inhibited the migration of BHP10-3 (<i>P</i><0.001) and TPC1 (<i>P</i><0.001) cells across the denuded zone. The data represent the mean ± S.D. of three independent experiments. NS, not significant; **<i>P</i><0.01, ***<i>P</i><0.001.</p

NTP decreases MMP-2/-9 and uPA activity in BHP10-3 and TPC1 cells.

<p>(A) Gelatin zymography for MMP-2/-9. NTP attenuated MMP-2/-9 enzymatic activity in both cell lines. (B) RT-PCR for MMP-2/-9. The mRNA levels of MMP-2/-9 decreased after NTP treatment in both cells. Each figure was representative of three experiments with triplicates. (C) uPA assays. NTP treatment significantly decreased uPA activity. The data represent the mean ± S.D. of three independent experiments. NS, not significant, **<i>P</i><0.01, ***<i>P</i><0.001. (D) Western blotting. ERK and Akt expression was significantly decreased after NTP treatment in an intensity-dependent manner. Each figure was representative of three experiments with triplicates.</p

Pull-down assays.

<p>Rho family (Rho, Rac1, and Cdc42) activity was evaluated using pull-down detection kits after exposure to gas (He+O₂) only, 2 or 4 kV of NTP for 1 s. NTP treatment of BHP10-3 and TPC1 cells significantly reduced the expression of GTP-RhoA and GTP-Rac1 (the active forms of these proteins). Each figure was representative of three experiments with triplicates.</p

NTP treatment altered the expression of FAK, Src, and paxillin.

<p>(A) Western blotting for p-FAK, p-Src, and p-paxillin. Immunocytochemical assay for (B) p-FAK and (C) p-paxillin. In the control and gas treated-cells, FAK was localized in small adhesion structures at the cell periphery. After NTP treatment, FAK focal accumulation was significantly decreased in both cell lines. Moreover, p-paxillin staining was co-localized with p-FAK staining. NTP treatment also decreased p-paxillin expression. Scale bar = 50 µm. Each figure was representative of three experiments with triplicates.</p

Non-thermal atmospheric pressure plasma (NTP) generating system used in this study (A) Photograph of our spray-type plasma generating system.

<p>(B) Optical emission spectra of the He and O₂ gas mixture plasma according to the electric intensity (2 or 4 kV) in the range of 280–920 nm. (C) Image of the “torch with spray-type” plasma jet with He and O₂. The visible plasma had a length of approximately 2.5 cm that varied with the gas flow and voltage.</p

Biofunctionalized Ceramic with Self-Assembled Networks of Nanochannels

Nature designs circulatory systems with hierarchically organized networks of gradually tapered channels ranging from micrometer to nanometer in diameter. In most hard tissues in biological systems, fluid, gases, nutrients and wastes are constantly exchanged through such networks. Here, we developed a biologically inspired, hierarchically organized structure in ceramic to achieve effective permeation with minimum void region, using fabrication methods that create a long-range, highly interconnected nanochannel system in a ceramic biomaterial. This design of a synthetic model-material was implemented through a novel pressurized sintering process formulated to induce a gradual tapering in channel diameter based on pressure-dependent polymer agglomeration. The resulting system allows long-range, efficient transport of fluid and nutrients into sites and interfaces that conventional fluid conduction cannot reach without external force. We demonstrate the ability of mammalian bone-forming cells placed at the distal transport termination of the nanochannel system to proliferate in a manner dependent solely upon the supply of media by the self-powering nanochannels. This approach mimics the significant contribution that nanochannel transport plays in maintaining living hard tissues by providing nutrient supply that facilitates cell growth and differentiation, and thereby makes the ceramic composite “alive”