Influence of chronic inflammation on the malignant phenotypes and the plasticity of colorectal cancer cells

Sporadic adenoma or adenocarcinoma is often detected during endoscopic surveillance of patients with ulcerative colitis (UC). However, it is occasionally difficult to distinguish these neoplasms from dysplasia or colitis-associated cancers because of the influence of inflammation. However, the influence of inflammation on sporadic neoplasms is not well characterised. To assess this influence, we established a long-term inflammation model of colon cancer cells by inflammatory stimulation with tumour necrosis factor-α, flagellin and interleukin-1β for 60 weeks. Then, the malignant phenotypes were evaluated using the MTS assay, Annexin V fluorescence assay, cell migration assay and sphere formation assay. The influence of P53 function on these phenotypes was assessed with a TP53 mutation model using the CRISPR/Cas9 system. A long-term inflammation model of LS174T cells was established for the first time with continuous inflammatory signalling. Chronic inflammation induced apoptosis and suppressed the proliferation and stemness of these cancer cells via the action of P53. It also enhanced the invasiveness of LS174T cells. Moreover, these phenotypic changes and changes in inflammatory signalling were recoverable after the removal of inflammatory stimuli, suggesting that colon cancer cells have higher plasticity than normal intestinal epithelial cells. In conclusion, our results suggest that sporadic neoplasms in patients with UC are affected by chronic inflammation but are not essentially altered.     

Effects of salts on the nonequivalent stability of the α-helices of isomeric block copolypeptides

The stability of the α-helices of isomeric block copolypeptides is nonequivalent, as reported previously. In order to explore the origin of the nonequivalence, the stability of α-helix of two block copolypeptides, (L -Ala)₂₀-(L -Glu)₂₀-(L -Phe) (designated as AEF) and (L -Glu)₂₀-(L -Ala)₂₀-(L -Phe) (EAF), in aqueous solution was investigated as a function of pH, temperature, and salt concentration by the measurement of the α-helical content using CD at 223 nm. The transition temperature, T_m, as a measure of the stability of the α-helix, decreased with increasing the salt concentration for EAF, while T_m increased for AEF. The results indicate that electrostatic interactions affect the nonequivalence of such helical stability. Thermodynamic quantities, ΔG, ΔH, and ΔS, of the thermal transition from random coil to α-helix were obtained by applying the curve-fitting method to the data. The major contribution to the effects of salts seems to be the entropic term, not the enthalpy term. This is unexpected, since the salt ions would weaken electrostatic interactions between ionized groups and the dipole along the helical axis, which affect the enthalpy term. In addition, the dependence of the electrostatic effect on the salt concentration is different for EAF and AEF. There fore, the nonequivalence cannot be accounted for by only the electrostatic effect, suggesting that it originates from some intrinsic property of the α-helix.     

Studies on co-operative properties of tropomyosin-actin and tropomyosin-troponin-actin complexes by the use of N-ethylmaleimide-treated and untreated species of myosin subfragment 1

When subfragment-1 of rabbit skeletal myosin was extensively modified with N-ethylmaleimide, the protein became strongly associable to actin in the presence of MgATP at low ionic strength, while the ATPase ceased to be activated by actin. Various concentrations of the modified protein were mixed with 10 μmol of pure actin or actin complexed with tropomyosin, and the fraction β of actin saturated with the modified protein in each mixture was determined by an ultracentrifugal method. We then added 0.3 μmol of unmodified subfragment-1 to the same sets of mixtures as used in the above experiments and determined the rate of ATP hydrolysis V by unmodified subfragment-1 as a function of β. A biphasic V-β relation was obtained for the tropomyosin-actin complex: when β was increased continuously from zero, the rate first increased substantially, had a maximum value more than tenfold larger than the initial at β ～- 0.3, and finally decreased to zero. In contrast, the V-β profile for pure actin deviated downwards from a linear relation, showing that there was a weak repulsive interaction between the modified and unmodified subfragment-1 species bound to the actin filament. The occurrence of such a repulsion was interpreted in terms of a steric hinderance model. Assuming that the same kind of repulsion underlay the biphasic V-β relation for the tropomyosin-actin complex, we calculated the relation of V′-β in an ideal case where it was absent. The result was also biphasic. We studied regulated actin in the presence and absence of Ca²⁺ by the same method and obtained biphasic V′-β relations in both cases.The experimental results were analyzed by a two-state model based on the proposal of Bremel & Weber (1972) that, within tropomyosin-actin or the regulated actin complex, n actin monomers undergo “off”/“on” transitions as a unit. Interactions between units were ignored in order to estimate the apparent size n, as well as the equilibrium constant L for the transition in the absence of myosin heads. Within the framework of allosteric theory (Monod et al., 1965), we derived formulae fit for data analysis, found a satisfactory agreement of the experimental and theoretical results, and obtained values of n = 11, and L = 37 for the tropomyosin-actin complex, and n = 16, L = 9 for regulated actin in the presence of Ca²⁺. The parameters in its absence could not be determined separately from the V?β relation which, however, was well-approximated with a combination of n = 16 and L = 10,000. It was also shown that tropomyosin-actin complex in the “on” state activated subfragment-1 ATPase eightfold more strongly than pure actin, and 2.2 to 2.6-fold more strongly than regulated actin in the “on” state. The results are compared with those provided by Greene & Eisenberg (1980), Hill et al. (1980) and Trybus & Taylor (1980) and discussed in conjunction with the double helical structure of tropomyosin-actin and regulated actin filaments.A simple allosteric calculation is presented in the Appendix to explain the well-known biphasic dependence on substrate concentration of the rate of regulated actin-subfragment-1 MgATPase (Bremel et al., 1972; Weber & Murray, 1973), with a reference to Deshcherevsky (1977).     

Inflammation-driven senescence-associated secretory phenotype in cancer-associated fibroblasts enhances peritoneal dissemination

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TGF-β1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition

Epithelial–mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-β1) and whether EMT could be induced through TGF-β1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-β1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-β1. Analysis of EMT markers showed that mRNA levels changed with TGF-β1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-β1 treatment. Invasion assays showed that TGF-β1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-β1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.     

Effects of temperature on life histories of three endangered Japanese diving beetle species

To elucidate population-increasing factors in the diving beetle Cybister tripunctatus lateralis (Fabricius) (Coleoptera: Dytiscidae) in Japan in recent years, life histories and oviposition patterns were compared among three endangered diving beetle species, Cybister brevis Aubé (qualified by the Japanese Red Data List as ‘near threatened’), Cybister chinensis Motschulsky (vulnerable), and C. tripunctatus lateralis (vulnerable). Oviposition in C. brevis, C. chinensis, and C. tripunctatus lateralis was observed from late April to mid-June, from late April to early July, and from late May to mid-August, respectively, under semi-outdoor conditions. There were no interspecies differences in total hatchling production during the reproductive season. In rearing experiments at various temperatures (20, 23, 25, 28, and 30 °C), the mortality of C. tripunctatus lateralis larvae was higher at 20 °C, and gradually lower with increasing temperature up to 30 °C. Adult body size of females in C. tripunctatus lateralis is larger than that of males but there were no significant differences among temperatures (25–30 °C). Cybister brevis had a higher emergence rate at 23–28 °C than at 20 and 30 °C. In C. brevis, the body size of adults reared at 25 or 28 °C was significantly larger than at other temperatures. Cybister chinensis did not differ in emergence rate and adult body size among the five temperature conditions. The developmental zero (i.e., the lower developmental threshold) from the first instar to adult emergence was 11.1 °C for C. brevis, 8.7 °C for C. chinensis, and 16.8 °C for C. tripunctatus lateralis. We speculate how the influence of global warming may have a positive impact on the growth and survival of C. tripunctatus lateralis.     

miR-30a attenuates drug sensitivity to 5-FU by modulating cell proliferation possibly by downregulating cyclin E2 in oral squamous cell carcinoma

We aimed to determine the functional role of the miRNA, which affects drug sensitivity to 5-FU in oral squamous cell carcinoma (OSCC), using two types of 5-FU-resistant and parental OSCC cell lines. MiRNA microarray data showed that miR-30a was significantly upregulated in two resistant cell lines. Therefore, we investigated the effects and molecular mechanism of miR-30a on 5-FU sensitivity. Stable overexpression of miR-30a in parental OSCC cells decreased cell proliferation and attenuated drug sensitivity to 5-FU. Cell cycle analysis indicated that miR-30a overexpression increased the proportion of G1 phase cells and decreased the proportion of S phase cells. MiR-30a knockdown using siRNA reversed the effects of miR-30a overexpression. DNA microarray analysis using miR-30a-overexpressing cell lines and a TargetScan database search showed that cyclin E2 (CCNE2) is a target of miR-30a. A luciferase reporter assay confirmed that a miR-30a mimic interacted with the specific binding site in the 3' UTR of CCNE2. CCNE2 knockdown with siRNA in OSCC cells yielded decreased drug sensitivity to 5-FU, similar to miR-30a overexpressing cells. These findings suggest that miR-30a in OSCC may be a novel biomarker of 5-FU-resistant tumors, as well as a therapeutic target for combating resistance.     

New insights into the role of Arabidopsis RABA1 GTPases in salinity stress tolerance

RAB11 GTPases, widely conserved members of RAB small GTPases, have evolved in a unique way in plants; plant RAB11 has notable diversity compared with animals and yeast. Recently, we have shown that members of RABA1, a subgroup in Arabidopsis RAB11 group, are required for salinity stress tolerance. To obtain a clue to understand its underlying mechanism, here we investigate whether RABA1 regulates sodium transport across the plasma membrane and accumulation in the vacuole. The results indicate that the raba1 quadruple mutant is not defective in the import and intracellular distribution of sodium, implying that RABA1 members are involved in a more indirect way in the responses to salinity stress.