Crystallization of Star-Shaped Poly(l-lactide)s with Arm Chains Aligned in the Same Direction in Two-Dimensional Crystals in a Langmuir Monolayer.

Polylactide (PLA) crystallizes to form extended-chain crystals in a Langmuir monolayer because crystallization is accelerated on the water surface. This is a unique situation where chain packing can be analyzed by simply measuring the lamellar thickness. Herein, star-shaped poly(l-lactide)s (PLLAs) with 2-12 arms were synthesized through the polymerization of l-lactide with various polyols as initiators, and their crystallization behavior in a monolayer was studied via atomic force microscopy. The PLLAs comprising 2-4 arms crystallized with all arms aligned in the same direction and being folded at the central polyol unit. Meanwhile, the PLLAs comprising 6 and 12 arms crystallized with both halves of the arms extended from the center to the opposite directions, most likely due to the steric hindrance of the crowded arms. Considering that the PLLAs crystallized from a once-formed condensed amorphous state during compression, they have a strong tendency to crystallize with the arms aligned in the same direction. The crystallization rate of star-shaped PLAs is known to reduce compared with that of a linear PLA even if the number of arms is as few as 2. This should be closely related to the unique crystallization behavior of the star-shaped PLLAs with the arms aligned in the same direction.

Gasdermin D-mediated release of IL-33 from senescent hepatic stellate cells promotes obesity-associated hepatocellular carcinoma.

Long-term senescent cells exhibit a secretome termed the senescence-associated secretory phenotype (SASP). Although the mechanisms of SASP factor induction have been intensively studied, the release mechanism and how SASP factors influence tumorigenesis in the biological context remain unclear. In this study, using a mouse model of obesity-induced hepatocellular carcinoma (HCC), we identified the release mechanism of SASP factors, which include interleukin-1ß (IL-1ß)- and IL-1ß-dependent IL-33, from senescent hepatic stellate cells (HSCs) via gasdermin D (GSDMD) amino-terminal-mediated pore. We found that IL-33 was highly induced in senescent HSCs in an IL-1ß-dependent manner in the tumor microenvironment. The release of both IL-33 and IL-1ß was triggered by lipoteichoic acid (LTA), a cell wall component of gut microbiota that was transferred and accumulated in the liver tissue of high-fat diet-fed mice, and the release of these factors was mediated through cell membrane pores formed by the GSDMD amino terminus, which was cleaved by LTA-induced caspase-11. We demonstrated that IL-33 release from HSCs promoted HCC development via the activation of ST2-positive Treg cells in the liver tumor microenvironment. The accumulation of GSDMD amino terminus was also detected in HSCs from human NASH-associated HCC patients, suggesting that similar mechanism could be involved in a certain type of human HCC. These results uncover a release mechanism for SASP factors from sensitized senescent HSCs in the tumor microenvironment, thereby facilitating obesity-associated HCC progression. Furthermore, our findings highlight the therapeutic potential of inhibitors of GSDMD-mediated pore formation for HCC treatment.

THz irradiation inhibits cell division by affecting actin dynamics.

Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

Relationship between podoplanin-expressing cancer-associated fibroblasts and the immune microenvironment of early lung squamous cell carcinoma.

AIM: Cancer-associated fibroblasts (CAFs) expressing podoplanin (PDPN) harbor a fibrous tumor microenvironment that promotes cancer progression in lung adenocarcinoma. In this study, we investigated whether tumor-promoting PDPN+ CAFs contribute to the immunosuppressive microenvironment in lung squamous cell carcinoma (SqCC). M&M: The gene expression profiles of immunosuppressive cytokines were compared using The Cancer Genome Atlas (TCGA) microarray lung SqCC data (n = 484) between a PDPN-high group and a PDPN-low group. Further, using patient-derived CAFs from surgically resected lung SqCC, the PDPN+ fraction was sorted and gene and protein expressions were analyzed. Finally, immunohistochemical staining was conducted on 131 surgically resected lung SqCC; CD8+ and FOXP3+ tumor infiltrating lymphocytes (TILs), and CD204+ tumor-associated macrophages (TAMs) were evaluated in cases with PDPN+ and PDPN- CAFs. RESULTS: Analysis of TCGA database revealed that the PDPN-high group exhibited significantly higher expression of interleukin (IL)-1A, IL-1B, IL-6, IL-10, monocyte chemoattractant protein-1 (CCL2), colony stimulating factor 1 (CSF1), fibroblast growth factor 2 (FGF2), galectin 1 (LGALS1), platelet derived growth factor subunit A (PDGFA), PDGFB, and transforming growth factor-ß1 (TGFB1) than those in the PDPN-low group. Among them, it was found that TGFB1 expression was higher in patient-derived PDPN+ CAFs. Immunohistochemical analyses revealed that more CD204+ TAMs infiltrated the tumor tissues in cases with PDPN+ CAFs than in cases with PDPN- CAFs (P <  0.03), while CD8+ and FOXP3+ TILs did not. Furthermore, in the same tumor, CD204+ TAMs infiltrated more in PDPN+ CAF-rich areas (P =  0.005). CONCLUSION: PDPN+ CAFs showed higher expression of TGFB1 and were associated with CD204+ TAM infiltration in stage-I lung SqCC, suggesting that PDPN+ CAFs were associated with the immunosuppressive tumor microenvironment.

Tumor-Infiltrating T Cells Concurrently Overexpress CD200R with Immune Checkpoints PD-1, CTLA-4, and TIM-3 in Non-Small-Cell Lung Cancer.

INTRODUCTION: CD200R has been reported to be the receptor for the immune checkpoint molecule CD200 and can transduce immune-suppressive signals. In this study, we mainly focused on the expression level of CD200R in T cells in pulmonary artery (PA) blood and non-small-cell lung cancer (NSCLC) tumor tissue. METHODS: Immune cells were isolated from dissected tumor samples and PA blood of NSCLC patients and analyzed with multiparameter flow cytometry. The co-expression of CD200R with other immune checkpoints, including programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), was also investigated. RESULTS: CD200R expression was observed on the surface of approximately 75% of T cells among tumor-infiltrating leukocytes (TILs). Compared to T cells extracted from TILs, only 55% of T cells extracted from PA blood exhibited CD200R expression. Moreover, with higher expression of CD200R, the expression of other immune checkpoints, including PD-1, CTLA-4, and TIM-3, was also increased in tumor-infiltrating T cells compared to T cells in PA blood. CONCLUSIONS: Our results showed that those tumors were dominated by T cells expressing CD200R together with other checkpoints, which suggests a phenotypic change after T cell infiltration into the tumor, such as T cell exhaustion.

Propagation of THz irradiation energy through aqueous layers: Demolition of actin filaments in living cells.

The effect of terahertz (THz) radiation on deep tissues of human body has been considered negligible due to strong absorption by water molecules. However, we observed that the energy of THz pulses transmits a millimeter thick in the aqueous solution, possibly as a shockwave, and demolishes actin filaments. Collapse of actin filament induced by THz irradiation was also observed in the living cells under an aqueous medium. We also confirmed that the viability of the cell was not affected under the exposure of THz pulses. The potential of THz waves as an invasive method to alter protein structure in the living cells is demonstrated.

The Actin-Family Protein Arp4 Is a Novel Suppressor for the Formation and Functions of Nuclear F-Actin.

The crosstalk between actin and actin-related proteins (Arps), namely Arp2 and Arp3, plays a central role in facilitating actin polymerization in the cytoplasm and also in the nucleus. Nuclear F-actin is required for transcriptional regulation, double-strand break repair, and nuclear organization. The formation of nuclear F-actin is highly dynamic, suggesting the involvement of positive and negative regulators for nuclear actin polymerization. While actin assembly factors for nuclear F-actin have been recently described, information about inhibitory factors is still limited. The actin-related protein Arp4 which is predominantly localized in the nucleus, has been previously identified as an integral subunit of multiple chromatin modulation complexes, where it forms a heterodimer with monomeric actin. Therefore, we tested whether Arp4 functions as a suppressor of nuclear F-actin formation. The knockdown of Arp4 (Arp4 KD) led to an increase in nuclear F-actin formation in NIH3T3 cells, and purified Arp4 potently inhibited F-actin formation in mouse nuclei transplanted into Xenopus laevis oocytes. Consistently, Arp4 KD facilitated F-actin-inducible gene expression (e.g., OCT4) and DNA damage repair. Our results suggest that Arp4 has a critical role in the formation and functions of nuclear F-actin.

Uptake of collagen type I via macropinocytosis cause mTOR activation and anti-cancer drug resistance.

Collagen type I (Col I) is one of the major extracellular matrix proteins in the cancer tissue. Previously, we have reported that Col I induces epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance by mTOR activation through Akt and ERK1/2 independent pathway. In this study, we aimed to elucidate the molecular mechanism of Col I induced EGFR-TKI resistance. First, we demonstrated the uptake of fluorescently labeled Col I by EGFR-mutated lung cancer cell line PC-9 cells using confocal microscopy and flow cytometry. Metabolome analysis revealed that the metabolic profiles of PC-9 cells was influenced by Col I treatment. Uptake of Col I into PC-9 cells was not inhibited by MMP inhibitor, GM6001, and endocytosis inhibitors, Pitstop2 and Dyngo4a; however, macropinocytosis inhibitor EIPA prevented its uptake. Moreover, the combination of EIPA and EGFR-TKI abrogated Col I-induced EGFR-TKI resistance in PC-9 cells. Inhibition of Rac1, which is essential for micropinocytosis, also decreased the uptake of Col I in PC-9 cells and restored their sensitivity to EGFR-TKI. Thus, EGFR mutated lung cancer cells could develop EGFR-TKI resistance by Col I uptake by macropinocytosis route.

Interaction between cancer cells and cancer-associated fibroblasts after cisplatin treatment promotes cancer cell regrowth.

Regrowth of cancer cells following chemotherapy is a significant problem for cancer patients. This study examined whether cancer-associated fibroblasts (CAFs), a major component of a tumor microenvironment, promote cancer cell regrowth after chemotherapy. First, we treated human lung adenocarcinoma cell line A549 and CAFs from four patients with cisplatin. Cisplatin treatment inhibited the viable cell number of A549 cells and induced epithelial-mesenchymal transition. After cisplatin was removed, A549 cells continued to manifest the mesenchymal phenotype and proliferated 2.2-fold in 4 days (regrowth of A549 cells). Cisplatin treatment inhibited the viable cell number of CAFs from four patients also. The CM (derived from cisplatin-pretreated CAFs from two patients) significantly enhanced the regrowth of cisplatin-pretreated A549 cells, and the CM derived from cisplatin-naïve CAFs marginally enhanced A549 regrowth. By contrast, the CM derived from either cisplatin-pretreated CAFs or cisplatin-naïve CAFs failed to enhance the growth of cisplatin-naïve A549 cells. The CM derived from cisplatin-pretreated CAFs did not enhance the proliferation of A549 cells in which epithelial-mesenchymal transition was induced by TGFß-1. Our findings indicate the possibility that humoral factors from cisplatin-pretreated CAFs promote the regrowth of cisplatin-pretreated A549 cells. These results suggest that interactions between cancer cells and CAFs may significantly enhance cancer cell regrowth within the tumor microenvironment after cisplatin treatment.

Secretion of high amounts of hepatocyte growth factor is a characteristic feature of cancer-associated fibroblasts with EGFR-TKI resistance-promoting phenotype: A study of 18 cases of cancer-associated fibroblasts.

Humoral factors from cancer-associated fibroblasts (CAFs) reportedly affect epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance in cancer cells with EGFR mutations. The aim of this study was to identify the robust humoral factors secreted from CAFs that induce the primary resistance to EGFR-TKI. We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell line (PC-9) treated with condition media (CM) from 18 cases of CAFs and matched non-cancerous-tissue-associated fibroblasts (NCAFs). We measured the expression levels of hepatocyte growth factor (HGF), interleukin-6, fibroblast growth factor-2, insulin-like growth factor-1, and vascular endothelial growth factor-A in CAFs and NCAFs. We examined whether HGF neutralizing antibody could annul the EGFR-TKI resistance induced by CM from CAFs. Compared to CM from NCAFs, CM from CAFs increased the resistance of PC-9 cells to EGFR-TKI in five out of 18 cases. Relative expression ratio of HGF messenger RNA was significantly higher in these five CAFs compared to others (P = 0.0013), whereas other cytokines were not. In four of these five cases, the addition of HGF neutralizing antibody significantly decreased the survival ratio of PC-9 cells. This study suggests that the secretion of higher amounts of HGF is the robust feature of EGFR-TKI resistance-promoting CAFs.

Effect of mycalolides isolated from a marine sponge Mycale aff. nullarosette on actin in living cells.

Discovery of novel bioactive compounds is important not only for therapeutic purposes but also for understanding the mechanisms of biological processes. To screen bioactive compounds that affect nuclear morphology in marine organism extracts, we employed a microscopy-based assay using DNA staining of human cancer cells. A crude extract from a marine sponge Mycale aff. nullarosette, collected from the east coast of Japan, induced cellular binucleation. Fractionation of the extract led to the isolation of mycalolides A and B, and 38-hydroxymycalolide B as the active components. Mycalolides have been identified as marine toxins that induce depolymerization of the actin filament. Live cell imaging revealed that low concentrations of mycalolide A produce binucleated cells by inhibiting the completion of cytokinesis. At higher concentrations, however, mycalolide A causes immediate disruption of actin filaments and changes in cell morphology, yielding rounded cells. These results suggest that the completion of cytokinesis is a process requiring high actin polymerization activity. Furthermore, luciferase reporter assays with mycalolide A treatments support the view that the level of globular actin can affect transcription of a serum response gene.

Actin polymerization is activated by terahertz irradiation.

Polymerization of monomeric actin into filaments has pivotal roles in cell motility, growth, differentiation, and gene expression. Therefore, techniques of manipulating actin polymerization, including actin-binding chemicals, have been developed for understanding and regulating multiple biological functions. Here, we demonstrate that irradiation with terahertz (THz) waves is a novel method of modulating actin polymerization. When actin polymerization reaction is performed under irradiation with 0.46 THz waves generated by a Gyrotron, actin polymerization was observed to be activated by monitoring the fluorescence of pyrene actin fluorophores. We also observed the number of actin filaments under a fluorescence microscope using the polymerized actin probe SiR-actin. The number of actin filaments was increased by 3.5-fold after THz irradiation for 20 min. When the THz irradiation was applied to a steady-state actin solution, in which elongation and depolymerization of actin filaments were equilibrated, increased actin polymerization was observed, suggesting that the THz irradiation activates actin polymerization, at least in the elongation process. These results suggest that THz waves could be applied for manipulating biomolecules and cells.

Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway.

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt-independent pathway, which results in EGFR-TKI resistance. Combination therapy using EGFR-TKI and mTOR inhibitor could be a possible strategy to combat this resistance.

Nuclear F-actin enhances the transcriptional activity of ß-catenin by increasing its nuclear localization and binding to chromatin.

Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor ß-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of ß-catenin. Our results also show that the nuclear F-actin colocalizes with ß-catenin and enhances the binding of ß-catenin to the downstream target genes of the Wnt/ß-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/ß-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/ß-catenin signaling.

Contribution of nuclear actin to transcription regulation.

Actin, an integral component of the cytoskeleton, plays crucial roles in a variety of cell functions, including cell migration, adhesion, polarity and shape change. Studies performed during the last couple of decades have revealed that the actin also exists in the nucleus. However, the function and properties of nuclear actin remained elusive so far. Recently, we showed that an actin tagged with EYFP and fused with a nuclear localization signal (EYFP-NLS-actin) formed visible filamentous (F)-actin bundles in cells. To obtain further details about the individual genes that are affected by the nuclear actin, we have used the microarray analysis to determine the changes in the expression levels of RNAs in HeLa cells as a result of EYFP-NLS-actin expression. Our results suggest that the nuclear actin plays a role in the activation of genes rather than their repression. The data has been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE59799.

Nuclear actin activates human transcription factor genes including the OCT4 gene.

RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

Nuclear actin filaments recruit cofilin and actin-related protein 3, and their formation is connected with a mitotic block.

Although actin monomers polymerize into filaments in the cytoplasm, the form of actin in the nucleus remains elusive. We searched for the form and function of ß-actin fused to nuclear localization signal and to enhanced yellow fluorescent protein (EN-actin). Our results reveal that EN-actin is either dispersed in the nucleoplasm (homogenous EN-actin) or forms bundled filaments in the nucleus (EN-actin filaments). Formation of such filaments was not connected with increased EN-actin levels. Among numerous actin-binding proteins tested, only cofilin is recruited to the EN-actin filaments. Overexpression of EN-actin causes increase in the nuclear levels of actin-related protein 3 (Arp3). Although Arp3, a member of actin nucleation complex Arp2/3, is responsible for EN-actin filament nucleation and bundling, the way cofilin affects nuclear EN-actin filaments dynamics is not clear. While cells with homogenous EN-actin maintained unaffected mitosis during which EN-actin re-localizes to the plasma membrane, generation of nuclear EN-actin filaments severely decreases cell proliferation and interferes with mitotic progress. The introduction of EN-actin manifests in two mitotic-inborn defects-formation of binucleic cells and generation of micronuclei-suggesting that cells suffer aberrant cytokinesis and/or impaired chromosomal segregation. In interphase, nuclear EN-actin filaments passed through chromatin region, but do not co-localize with either chromatin remodeling complexes or RNA polymerases I and II. Surprisingly presence of EN-actin filaments was connected with increase in the overall transcription levels in the S-phase by yet unknown mechanism. Taken together, EN-actin can form filaments in the nucleus which affect important cellular processes such as transcription and mitosis.

Anti-aging effects of high molecular weight proteoglycan from salmon nasal cartilage in hairless mice.

Proteoglycans comprise a family of complex macromolecules consisting of a core protein with covalently attached glycosaminoglycan (GAG) chains. The skin anti-aging effects of oral administration of proteoglycan fractions with different molecular weights from salmon nasal cartilage were investigated in a hairless mouse model of skin aging; aging was caused by repeated ultraviolet B (UVB) irradiation. Three proteoglycan fractions of different molecular weights were prepared from salmon nasal cartilage water extract by ion-exchange column chromatography and gel filtration column chromatography. Physiological and histological analysis of the skin indicated that oral administration of high molecular weight proteoglycan inhibited UVB-induced skin aging, defined as increased erythema, increased transepidermal water loss (TEWL), decreased hydration, and epidermal and dermal hypertrophies. The serum and dorsal skin inflammatory cytokine levels indicated that high molecular weight proteoglycan acts on gut immunity and improves skin by inhibiting surplus inflammatory cytokines produced by UVB irradiation. These results suggest that high molecular weight proteoglycan from salmon nasal cartilage is effective in preventing skin aging.

The Hsp90 inhibitor 17-AAG represses calcium-induced cytokeratin 1 and 10 expression in HaCaT keratinocytes.

Hsp90 is essential for maintaining the activity of numerous signaling factors, and plays a key role in cellular signal transduction networks. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an ansamycin antibiotic that binds to Hsp90 and inhibits its function. HaCaT human keratinocytes were used to investigate the cellular and molecular functions of Hsp90 in keratinocyte differentiation. Inhibition of Hsp90 by 17-AAG leads to downregulation of the differentiation markers cytokeratin 1 and cytokeratin 10 at the protein and mRNA levels.