Exploring heterogeneity: a dive into preclinical models of cancer cachexia.

Cancer cachexia (CC) is a multifactorial and complex syndrome experienced by up to 80% of patients with cancer and implicated in ∼40% of cancer-related deaths. Given its significant impact on patients' quality of life and prognosis, there has been a growing emphasis on elucidating the underlying mechanisms of CC using preclinical models. However, the mechanisms of cachexia appear to differ across several variables including tumor type and model and biologic variables such as sex. These differences may be exacerbated by variance in experimental approaches and data reporting. This review examines literature spanning from 2011 to March 2024, focusing on common preclinical models of CC, including Lewis Lung Carcinoma, pancreatic KPC, and colorectal colon-26 and Apcmin/+ models. Our analysis reveals considerable heterogeneity in phenotypic outcomes, and investigated mechanisms within each model, with particular attention to sex differences that may be exacerbated through methodological differences. Although searching for unified mechanisms is critical, we posit that effective treatment approaches are likely to leverage the heterogeneity presented by the tumor and pertinent biological variables to direct specific interventions. In exploring this heterogeneity, it becomes critical to consider methodological and data reporting approaches to best inform further research.

Decoupling FcRn and tumor contributions to elevated immune checkpoint inhibitor clearance in cancer cachexia.

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia.

A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus.

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Characterization of neoantigen-specific T cells in cancer resistant to immune checkpoint therapies.

Neoantigen-specific T cells are strongly implicated as being critical for effective immune checkpoint blockade treatment (ICB) (e.g., anti-PD-1 and anti-CTLA-4) and are being targeted for vaccination-based therapies. However, ICB treatments show uneven responses between patients, and neoantigen vaccination efficiency has yet to be established. Here, we characterize neoantigen-specific CD8+ T cells in a tumor that is resistant to ICB and neoantigen vaccination. Leveraging the use of mass cytometry combined with multiplex major histocompatibility complex (MHC) class I tetramer staining, we screened and identified tumor neoantigen-specific CD8+ T cells in the Lewis Lung carcinoma (LLC) tumor model (mRiok1). We observed an expansion of mRiok1-specific CD8+ tumor-infiltrating lymphocytes (TILs) after ICB targeting PD-1 or CTLA-4 with no sign of tumor regression. The expanded neoantigen-specific CD8+ TILs remained phenotypically and functionally exhausted but displayed cytotoxic characteristics. When combining both ICB treatments, mRiok1-specific CD8+ TILs showed a stem-like phenotype and a higher capacity to produce cytokines, but tumors did not show signs of regression. Furthermore, combining both ICB treatments with neoantigen vaccination did not induce tumor regression either despite neoantigen-specific CD8+ TIL expansion. Overall, this work provides a model for studying neoantigens in an immunotherapy nonresponder model. We showed that a robust neoantigen-specific T-cell response in the LLC tumor model could fail in tumor response to ICB, which will have important implications in designing future immunotherapeutic strategies.

Elucidation of protective effects of oxime derivatives against cisplatin-induced cytotoxicity in LLC-PK1 kidney cells.

This study aimed to explore the renoprotective effects of oxime derivatives against cisplatin-mediated cell death in LLC-PK1 porcine kidney epithelial cells. Treatment with compounds 161-A and 161-F improved cisplatin-mediated LLC-PK1 cell damage and increased cell viability by more than 80% of the control value when compared with that of cisplatin-treated cells. In addition, 161-A and 161-F reduced cisplatin-induced apoptosis. Analysis of the molecular mechanisms underlying the effects exerted by these compounds revealed that treatment with 161-A and 161-B inhibited the protein expression of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and cleaved caspase-3 in cisplatin-treated LLC-PK1 cells. Thus, these findings provide in vitro scientific evidence that oxime derivatives may be useful as pharmacological candidates for the prevention of cisplatin-mediated nephrotoxicity.

Challenges and engineering application of landfill leachate concentrate treatment.

Landfill leachate concentrate (LLC) is a concentrated waste stream from landfill leachate treatment systems and has been recognized as a key challenge due to its high concentration of salts, heavy metals, organic matters, etc. Improper management of LLC (e.g. reinjection) would exacerbate the performance of upstream treatment processes and pose risks to the surrounding environments near landfill sites. Addressing the challenge and recovering resources from LLC have thus been attracting considerable attention. Although many LLC treatment technologies have been developed, a comprehensive discussion about the challenges still lacks. This review critically evaluates mainstream LLC treatment technologies, namely incineration, coagulation, advanced oxidation, evaporation and solidification/stabilization. We then introduce a geopolymer-based solidification (GS) process as a promising technology owning to its simple casting process and reusable final product and summarize engineering applications in China. Finally, we suggest investigating hybrid systems to minimize LLC production and achieve the on-site reuse of LLC. Collectively, this review provides useful information to guide the selection of LLC treatment technologies and suggests a sustainable alternative for large-scale application, while also highlighting the need of joint efforts in the industry to achieve efficient, ecofriendly and economical on-site management of landfill waste streams.

Spectroscopic Characterization and Biological Activity of Hesperetin Schiff Bases and Their Cu(II) Complexes.

The three Schiff base ligands, derivatives of hesperetin, HHSB (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]isonicotinohydrazide), HIN (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]benzhydrazide) and HTSC (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]thiosemicarbazide) and their copper complexes, CuHHSB, CuHIN, and CuHTSC were designed, synthesized and analyzed in terms of their spectral characterization and the genotoxic activity. Their structures were established using several methods: elemental analysis, FT-IR, UV-Vis, EPR, and ESI-MS. Spectral data showed that in the acetate complexes the tested Schiff bases act as neutral tridentate ligand coordinating to the copper ion through two oxygen (or oxygen and sulphur) donor atoms and a nitrogen donor atom. EPR measurements indicate that in solution the complexes keep their structures with the ligands remaining bound to copper(II) in a tridentate fashion with (O-, N, Oket) or (O-, N, S) donor set. The genotoxic activity of the compounds was tested against model tumour (HeLa and Caco-2) and normal (LLC-PK1) cell lines. In HeLa cells the genotoxicity for all tested compounds was noticed, for HHSB and CuHHSB was the highest, for HTSC and CuHTSC-the lowest. Generally, Cu complexes displayed lower genotoxicity to HeLa cells than ligands. In the case of Caco-2 cell line HHSB and HTSC induced the strongest breaks to DNA. On the other side, CuHHSB and CuHTSC induced the highest DNA damage against LLC-PK1.

Renoprotective Effect of Liraglutide Is Mediated via the Inhibition of TGF-Beta 1 in an LLC-PK1 Cell Model of Diabetic Nephropathy.

Background: Recently published research demonstrated direct renoprotective effects of the glucagon-like peptide-1 receptor agonist GLP 1 RA, but the relevant molecular mechanisms are still not clear. The aim of this research was to assess the effects of Liraglutide in a cell culture model of diabetic nephropathy on cell viability, antioxidant (GSH) and transforming growth factor beta 1 (TGF- ß1) levels and extracellular matrix (ECM) expression. The metabolic activity in hyperglycemic conditions and the effect of Liraglutide treatment were assessed by measuring Akt, pAkt, GSK3ß, pGSK3ß, pSTAT3, SOCS3, iNOS and NOX4 protein expression with Western blot. F actin distribution was used to assess the structural changes of the cells upon treatment. Materials and methods: The cells were exposed to high glucose (HG30 mM) followed by 0.5 mM H2O2 and a combination of glucose and H2O2 during 24 h. Subsequently, the cells were treated with different combinations of HG30, H2O2 and Liraglutide. Cell viability was determined by an MTT colorimetric test, and the GSH, TGF-ß1 concentration and ECM expression were measured using a spectrophotometric/microplate reader assay and an ELISA kit, respectively. Western blotting was used to detect the protein level of Akt, pAkt, GSK3ß, pGSK3ß, pSTAT3, SOCS3, iNOS and NOX4. The F-actin cytoskeleton was visualized with Phalloidin stain and subsequently quantified. Results: Cell viability was decreased as well as GSH levels in cells treated with a combination of HG30/H2O2, and HG30 alone (p < 0.001). The addition of Liraglutide improved the viability in cells treated with HG30, but it did not affect the cell viability in the cell treated with the addition of H2O2. GSH increased with the addition of Liraglutide in HG30/H2O2 (p < 0.001) treated cells, with no effect in cells treated only with HG30. TGF-ß1 levels (p < 0.001) were significantly increased in HG30 and HG30/H2O2. The addition of Liraglutide significantly decreased the TGF-ß1 levels (p < 0.01; p < 0.05) in all treated cells. The synthesis of collagen was significantly increased in HG30/H2O2 (p < 0.001), while the addition of Liraglutide in HG30/H2O2 significantly decreased collagen (p < 0.001). Akt signaling was not significantly affected by treatment. The GSK3b and NOX4 levels were significantly reduced (p < 0.01) after the peroxide and glucose treatment, with the observable restoration upon the addition of Liraglutide suggesting an important role of Liraglutide in oxidative status regulation and mitochondrial activity. The treatment with Liraglutide significantly upregulated STAT3 (p < 0.01) activity, with no change in SOCS3 indicating a selective regulation of the STAT 3 signaling pathway in glucose and the oxidative overloaded environment. A significant reduction in the distribution of F-actin was observed in cells treated with HG30/H2O2 (p < 0.01). The addition of Liraglutide to HG30-treated cells led to a significant decrease of distribution of F-actin (p < 0.001). Conclusion: The protective effect of Liraglutide is mediated through the inhibition of TGF beta, but this effect is dependent on the extent of cellular damage and the type of toxic environment. Based on the WB analysis we have revealed the signaling pathways involved in cytoprotective and cytotoxic effects of the drug itself, and further molecular studies in vitro and vivo are required to elucidate the complexity of the pathophysiological mechanisms of Liraglutide under conditions of hyperglycemia and oxidative stress.

Signaling Cascade Mediating the Effect of FTY720P on the Na+/K+ ATPase in LLC-PK1.

BACKGROUND/AIMS: In renal ischemia, the Na+/K+ ATPase of the kidney epithelial cells translocates to intracellular compartments, resulting in altered kidney functions. Sphingosine-1-phosphate (S1P) was shown to play a protective role against this ischemic injury. Whether the sphingolipid targets the Na+/K+ ATPase is a possibility that has not been explored before. This work aims at investigating the effect of S1P on renal Na+/K+ ATPase using its analogue FTY720P and LLC-PK1 cells. METHODS: The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme while its protein expression was studied by western blot analysis. RESULTS: FTY720P increased the activity of the ATPase in a dose and time dependent manner, with a highest effect observed at 15 minutes and a dose of 80 nM. The protein expression was also increased. The stimulation of the Na+/K+ ATPase disappeared completely in presence of JTE-013, a specific blocker of S1PR2, as well as in presence of Y-27632, a Rho kinase inhibitor, BAPTA-AM, a Ca2+ chelator, wortmannin, a PI3K inhibitor, carboxy-PTIO, a scavenger for nitric oxide (NO), and KT 5823, a PKG inhibitor. CYM 5520, a S1PR2 agonist mimicked the effect of FTY720P. FTY720P increased the expression of p-Akt, a direct effector of PI3K, however, this increase disappeared when Rho kinase was inhibited, revealing that Rho kinase acts upstream PI3K. Glyco-SNAP-1, a NO donor, activated the pump in both presence and absence of wortmannin, indicating that PI3K is upstream NO. Interestingly, glyco-SNAP-1 and 8-bromo-cGMP, a PKG activator, exerted no effect on the Na+/K+ ATPase in absence of free Ca2+ revealing that the NO mediated effect is calcium-dependent. The involvement of calcium was further confirmed by the translocation of NFAT to the nucleus. The presence of verapamil or extracellular EGTA abolished the stimulatory effect of FTY720P, indicating that the source of calcium is extracellular. CONCLUSION: The results suggest that FTY720P activates sequentially S1PR2, Rho kinase, PI3K, leading to NO release and PKG stimulation. The latter phosphorylates calcium channels in the cell membrane, leading to calcium influx, and translocation of the ATPase units to the membrane.

Highly Modulated In-Fiber Mach-Zehnder Interferometer Based on an Ultracompact Leaky-Guided Liquid Core.

We proposed a novel sensor based on an ultracompact leaky-guided liquid core fiber Mach-Zehnder interferometer (LLCFMZI) for high modulation of an interference spectrum. The sensor structure is based on a micro-sized hollow-core fiber (HCF) splicing a tilt end face single-mode fiber (SMF) to create a miniature oblique gap for the effective access of different liquids. The liquid core with a relatively lower refractive index (RI) than the cladding can achieve a leaky-mode optical waveguide (LMOW) mechanism, and its volume is only approximately 7.85 pL. In addition, the utilized micro-length HCF can reduce the energy loss of core in the LMOW to obtain an acceptable extinction ratio (>30 dB) with high temperature (T) sensitivity in the interference spectra. Experimental results show that the interference spectra can be highly modulated within the wide measurement range of 1250-1650 nm with a steadily linear response for thermal effect. The measured temperature sensitivities (T-sensitivities) of various liquids of DI water, ethanol, and Cargille-liquid (nD = 1.305) are 0.8869, 4.4754, and 4.8229 nm/°C, and the corresponding measured thermal optics coefficient (TOC) are -4.16 × 10-5, -2.11 × 10-4, and -3.6 × 10-4 °C-1, respectively. Measurement results demonstrate that the used liquids with a higher TOC can obtain better T-sensitivity modulation. The highest experimental sensitivity of the liquid-core filled with Cargille-liquid (nD = 1.40) is up to +13.87 nm/°C with a corresponding TOC of -4.07 × 10-4 °C-1. Furthermore, the experimental and theoretical values are in good agreement according to FSR the measuring scheme that investigates the effectiveness of the proposed LLCFMZI.

Improving Water-Absorption and Mechanical Strength: Lyotropic Liquid Crystalline-Based Spray Dressings as a Candidate Wound Management System.

A spray dressing based on lyotropic liquid crystalline (LLC) with adjustable crystalline lattices was investigated in this study. It possesses water-triggering phase transition property and ease of spraying on wound, as well as stable drug encapsulation and controllable drug release. When it comes to wound with exudate, adequate water absorption and sustainable mechanical strength after water absorption was important for a good dressing, while most of the normal LLC dressings were still unable to meet such standards. Herein, a type of hyaluronic acid (HA)-incorporated LLC-based spray dressing (HLCSD) was developed to overcome the above limitations. After comparing HAs with different molecular weights (MWs) and concentrations, 3% HA with MW of 800~1000 kD was chosen as an ideal amount of excipients to add into the HLCSD. The water absorption of HLCSD precursor increased by 150% with the appearance of enlarged water channels. The complex modulus of HLCSD gel also increased from 1 to 100 kPa, which suggested lasting wound coverage and good patient compliance when used clinically. The spraying and phase transition properties of HLCSD was studied and showed acceptable changes. Moreover, good safety comparable with the commercial product Purilon® was also demonstrated in an in vivo acute skin irritation test. Thus, the improved HLCSD was a promising dressing for exudation wound treatment.

The circular RNA circSlc7a11 promotes bone cancer pain pathogenesis in rats by modulating LLC-WRC 256 cell proliferation and apoptosis.

Treatment of bone cancer pain (BCP) caused by bone metastasis in advanced cancers remains a challenge in clinical oncology, and the underlying mechanisms of BCP are poorly understood. This study aimed to investigate the pathogenic roles of circular RNAs (circRNAs) in regulating cancer cell proliferation and BCP development. Eight differentially expressed circRNAs in the rat spinal cord were validated by agarose gel electrophoresis and Sanger sequencing. Expression of circRNAs and mRNAs was detected by quantitative RT-PCR. MTS assay and flow cytometry were performed to analyze cell proliferation and apoptosis, respectively. Differentially expressed mRNA profiles were characterized by deep RNA sequencing, hierarchical clustering, and functional categorization. The interactions among circRNAs, microRNAs (miRNAs), and mRNAs were predicted using TargetScan. Additionally, western blot was performed to determine the protein levels of Pax8, Isg15, and Cxcl10. Multiple circRNAs were differentially expressed in the spinal cords of BCP model rats; of these, circSlc7a11 showed the greatest increase in expression. The overexpression of circSlc7a11 significantly promoted cell proliferation and repressed apoptosis of LLC-WRC 256 and UMR-106 cells, whereas circSlc7a11 silencing produced the opposite effects. Altered expression of circSlc7a11 also induced substantial changes in the mRNA expression profiles of LLC-WRC 256 cells; these changes were linked to multiple apoptotic processes and signaling pathways, such as the chemokine signaling pathway, and formed a complex circRNA/miRNA/mRNA network. Additionally, Pax8, Isg15, and Cxc110 protein level in LLC-WRC 256 cells was consistent with the mRNA results. The circRNA circSlc7a11 regulates rat BCP development by modulating LLC-WRC 256 cell proliferation and apoptosis through multiple-signaling mechanisms.

Cloning, Expression and Inhibitory Effects on Lewis Lung Carcinoma Cells of rAj-Tspin from Sea Cucumber (Apostichopus japonicus).

In recent years, sea cucumber has become a favorite healthcare food due to its characteristic prevention of cardiovascular diseases, suppression of tumors, as well as enhancement of immunity. In order to screen the anti-tumoral proteins or peptides from sea cucumber (Apostichopus japonicus), its cDNA library was analyzed, and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13)-like was found. ADAMTS13-like contains 10 thrombospondin 1 (TSP1) domains. Based on analysis of bioinformatics, the third TSP1 domain of this protein, which is further named Aj-Tspin, contains an arginine-glycine-aspartate (RGD) motif. Since our previous studies showed that the recombinant RGD-containing peptide from lampreys showed anti-tumoral activity, the third TSP1 domain of ADAMTS13-like was chosen to evaluate it's effect on tumor proliferation and metastasis, despite the fact it shares almost no homologue with disintegrins from other species. After artificial synthesis, its cDNA sequence, Aj-Tspin, which is composed of 56 amino acids, was subcloned into a pET23b vector and expressed as a recombinant Aj-Tspin (rAj-Tspin) in a soluble form with a molecular weight of 6.976 kDa. Through affinity chromatography, rAj-Tspin was purified as a single protein. Both anti-proliferation and immunofluorescence assays showed that rAj-Tspin suppressed the proliferation of Lewis lung carcinoma (LLC) cells through apoptosis. Adhesion assay also displayed that rAj-Tspin inhibited the adhesion of LLC cells to ECM proteins, including fibronectin, laminin, vitronectin and collagen. Lastly, rAj-Tspin also suppressed the migration and invasion of LLC cells across the filter in transwells. Thus, the above indicates that rAj-Tspin might act as a potential anti-tumoral drug in the future and could also provide information on the nutritional value of sea cucumber.

Impact of In Vitro Passive Permeability in a P-gp-transfected LLC-PK1 Model on the Prediction of the Rat and Human Unbound Brain-to-Plasma Concentration Ratio.

PURPOSE: More accurate prediction of the extent of drug brain exposure in early drug discovery and understanding potential species differences could help to guide medicinal chemistry and avoid unnecessary animal studies. Hence, the aim of the current study was to validate the use of a P-gp transfected LLC-PK1 model to predict the unbound brain-to-plasma concentration ratio (Kpuu,brain) in rats and humans. METHODS: MOCK-, Mdr1a- and MDR1-transfected LLC-PK1 monolayers were applied in a transwell setup to quantify the bidirectional transport for 12 specific P-gp substrates, 48 UCB drug discovery compounds, 11 compounds with reported rat in situ brain perfusion data and 6 compounds with reported human Kpuu,brain values. The in vitro transport data were introduced in a minimal PBPK model (SIVA®) to determine the transport parameters. These parameters were combined with the differences between in vitro and in vivo passive permeability as well as P-gp expression levels (as determined by LC-MS/MS), to predict the Kpuu,brain. RESULTS: A 10-fold difference between in vitro and in vivo passive permeability was observed. Incorporation of the differences between in vitro and in vivo passive permeability and P-gp expression levels resulted in an improved prediction of rat (AAFE 2.17) and human Kpuu,brain (AAFE 2.10). CONCLUSIONS: We have succesfully validated a methodology to use a P-gp overexpressing LLC-PK1 cell line to predict both rat and human Kpuu,brain by correcting for both passive permeability and P-gp expression levels.

Pharmacokinetics and tissue distribution in rats of a novel anticancer platinum compound LLC-1903.

LLC-1903, a novel anticancer compound, was synthesized by optimizing the structure, which was derived from altering the leaving group of lobaplatin. It has an excellent in vitro anti-cancer activity, high water solubility, high stability in solution and low in vivo toxicity according to our former study.The plasma pharmacokinetics (PK) and tissue distribution of LLC-1903 and lobaplatin in rats were determined after intravenous administration of a single dose (0.06 mmol/kg body weight). Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the concentration of platinum (Pt) in plasma and tissue samples.Most PK parameters of the Pt in LLC-1903 showed a significant difference from those of lobaplatin. The plasma level of LLC-1903 is only half of that of lobaplatin (p < 0.01) which could be the direct result of faster drug clearance. The tissue distribution showed that both LLC-1903 and lobaplatin were mainly found in the liver and kidney, and less in other organs. At four time points (0.083, 0.5, 1 and 4 h) after administration, the tissue concentrations of LLC-1903 were almost always significantly higher than those of lobaplatin (p < 0.05 or p < 0.01).

Effects of 5-Hydroxypyrimidine Derivatives on Tumor Growth and Lifespan of C57BL/6 Mice with Epidermoid Lung Carcinoma.

The effects of 5-hydroxypyrimidine derivatives SNK-411 (2-isobutyl-4,6-dimethyl-5-hydroxypyrimidine) and SNK-578 (2-isobutyl-4,6-dimethyl-5-hydroxypyrimidine chlorohydrate) on the tumor growth and survival of male C57BL/6 mice with transplanted Lewis lung epidermoid carcinoma (LLC) were studied in animals receiving intraperitoneal treatment on days 2-15 of tumor development. Compound SNK-578 in a dose of 10 mg/kg significantly inhibited tumor growth (by 3.6 times; 72.2%) in 7 days after the treatment was discontinued, while compound SNK-411 in a dose of 25 mg/kg only negligibly reduced tumor volume (by 41.7%). A combination of course of SNK-411 (25 mg/kg) and single intraperitoneal dose of doxorubicin (4 mg/kg) significantly inhibited the tumor growth (by 2.2 times; 55.2%), while the combination of SNK-578 (10 mg/kg) with doxorubicin (4 mg/kg) was in fact ineffective. The median survival of animals with untreated LLC was 28 days. Median survival of mice injected with SNK-578 (10 mg/kg) was 43 days, hence, the lifespan of mice with LLC was by 38.6% longer after the treatment. Two of ten mice in this group developed no tumors.

Elevated glucose concentration in culture media decreases membrane trafficking of SGLT2 in LLC-PK1 cells via a cAMP/PKA-dependent pathway.

Na+-dependent glucose reabsorption in the renal proximal tubule is dynamically regulated by changes in blood glucose levels. There is, however, a disparity in reports studying the relationship between hyperglycemia and Na+-glucose-linked transporter (SGLT) function and expression. Similarly, manipulation of the glucose content in growth media of cultured renal cells has been shown to influence SGLT activity. In this investigation, SGLT activity was significantly lower in proximal tubule LLC-PK1 cells cultured in medium containing 17.5 than 5 mM glucose. α-Methyl d-glucopyranoside (AMG) transport kinetics showed reduced apparent Vmax and Km in cells grown in 17.5 mM glucose. SGLT2 was identified as the isoform responsible for glucose transport, and protein expression analyses showed decreased apical membrane localization of SGLT2 in cells grown in 17.5 mM glucose, explaining the reduced activity. Multiple signaling pathways have been implicated in regulation of SGLT activity and trafficking. Elevated media glucose decreased intracellular cAMP and PKA activation, leading to decreased SGLT2 trafficking into the plasma membrane, which was reversed after treatment with 1 µM forskolin. The effects of media glucose on SGLT activity were found to be dependent on p38 MAPK activation due to PKA-mediated signaling. Glucose-modulated AMG uptake is reversible and was associated with altered SGLT2 membrane trafficking and cAMP alterations. In summary, elevated glucose concentrations in culture medium decrease SGLT activity in LLC-PK1 cells by reducing membrane trafficking of SGLT2 via decreasing intracellular cAMP, resulting in a lowered PKA-dependent phosphorylation of p38 MAPK.

Lung tumor cells inhibit bone mineralization and osteoblast activity.

Patients with non-small cell lung cancer (NSLC) often develop skeletal complications and fractures. To understand mechanisms of bone loss, we developed a murine model of non-metastatic NSLC. Decreased bone mineral density, trabecular thickness and mineralization, without an increase in bone resorption, were observed in vivo in mice injected with Lewis lung adenocarcinoma (LLC1) cells in the absence of tumor cell metastases. A decrease in trabecular bone mineral density was observed in mice injected with cell-free LLC1 CM. Plasma osteoblast biomarkers and PTH-related peptide (PTHrP) were reduced, and parathyroid hormone (PTH), 1,25-dihydroxyvitamin D, calcium and phosphate concentrations were normal in tumor-bearing mice. LLC1 cell conditioned medium (CM) inhibited alkaline phosphatase activity, osteoblast mineralization, and expression of Alpl and Ocn/Bglap mRNA in MC3T3 osteoblast cultures, whereas non-CM or CM from NIH/3T3 fibroblasts did not induce similar changes. LLC1 CM reduced Wnt3a-stimulated Tcf/Lef reporter plasmid activity and Wnt5A, Tcf1 and Lef1 mRNA expression in MC3T3 cells. Although concentrations of the Wnt inhibitor, DKK2, were increased in LLC1 CM compared to non-CM, depletion of DKK2 from LLC1 CM did not completely restore Wnt3a activity in MC3T3 cultures, and recombinant DKK2 failed to inhibit osteoblast mineralization. The data indicate that in a model of lung adenocarcinoma without bone metastases, tumor cells elaborate a secreted factor(s) that reduces bone mass, bone formation and osteoblast Wnt signaling without increases in bone resorption or calcium-regulating hormone concentrations. The factor(s) mediating this inhibition of osteoblast mineralization require further characterization.

Hybrid Polyketides from a Hydractinia-Associated Cladosporium sphaerospermum SW67 and Their Putative Biosynthetic Origin.

Five hybrid polyketides (1a, 1b, and 2-4) containing tetramic acid core including a new hybrid polyketide, cladosin L (1), were isolated from the marine fungus Cladosporium sphaerospermum SW67, which was isolated from the marine hydroid polyp of Hydractinia echinata. The hybrid polyketides were isolated as a pair of interconverting geometric isomers. The structure of 1 was determined based on 1D and 2D NMR spectroscopic and HR-ESIMS analyses. Its absolute configuration was established by quantum chemical electronic circular dichroism (ECD) calculations and modified Mosher's method. Tetramic acid-containing compounds are reported to be derived from a hybrid PKS-NRPS, which was also proved by analyzing our 13C-labeling data. We investigated whether compounds 1-4 could prevent cell damage induced by cisplatin, a platinum-based anticancer drug, in LLC-PK1 cells. Co-treatment with 2 and 3 ameliorated the damage of LLC-PK1 cells induced by 25 µM of cisplatin. In particular, the effect of compound 2 at 100 µM (cell viability, 90.68 ± 0.81%) was similar to the recovered cell viability of 88.23 ± 0.25% with 500 µM N-acetylcysteine (NAC), a positive control.

Augmentation of cadmium-induced oxidative cytotoxicity by pioglitazone in renal tubular epithelial cells.

The aim of this study was to examine whether a peroxisome proliferator-activated receptor (PPAR)-γ agonist could affect cadmium (Cd)-induced cytotoxicity via the increased expression of megalin, one of the uptake pathways, using renal epithelial LLC-PK1 cells. The treatment with 1 µM Cd for 24 h was not cytotoxic; however, when the cells were pretreated with 0.1 µM pioglitazone for 12 h and then exposed to 1 µM Cd for 24 h, significant accumulation of Cd and cytotoxicity were detected, with an increase in megalin mRNA expression. In addition, pretreatment with pioglitazone significantly increased the Cd-induced generation of hydrogen peroxide and cell apoptosis. The augmented Cd-induced cytotoxicity and apoptosis on preincubation with pioglitazone were inhibited by prior treatment with GW 9662 (PPAR-γ antagonist). These findings suggest that a PPAR-γ agonist could augment Cd-induced oxidative injury and cell apoptosis, possibly dependent on the expression level of the uptake pathway.