The anti-inflammatory agent curcumin can selectively eliminate malignant rather than normal cells. The present study examined the effects of curcumin on the Lewis lung carcinoma (LLC) cell line and characterized a subpopulation surviving curcumin treatments. Cell density was measured after curcumin was applied at concentrations between 10 and 60 μM for 30 hours. Because of the high cell loss at 60 μM, this dose was chosen to select for surviving cells that were then used to establish a new cell line. The resulting line had approximately 20% slower growth than the original LLC cell line and based on ELISA contained less of two markers, NF-κB and ALDH1A, used to identify more aggressive cancer cells. We also injected cells from the original and surviving lines subcutaneously into syngeneic C57BL/6 mice and monitored tumor development over three weeks and found that the curcumin surviving-line remained tumorigenic. Because curcumin has been reported to kill cancer cells more effectively when administered with light, we examined this as a possible way of enhancing the efficacy of curcumin against LLC cells. When LLC cells were exposed to curcumin and light from a fluorescent lamp source, cell loss caused by 20 μM curcumin was enhanced by about 50%, supporting a therapeutic use of curcumin in combination with white light. This study is the first to characterize a curcumin-surviving subpopulation among lung cancer cells. It shows that curcumin at a high concentration either selects for an intrinsically less aggressive cell subpopulation or generates these cells. The findings further support a role for curcumin as an adjunct to traditional chemical or radiation therapy of lung and other cancers.

Asymmetric division (ASD), the unique characteristic of normal stem cells, is regarded as a stemness marker when applied to the study of cancer stem cells (CSCs). However, the role of ASD in the self-renewal of CSCs and its regulation remain largely unknown. Here, we first established a mouse Lewis lung carcinoma CSC cell line that could undergo asymmetric division (LLC-ASD cells) derived from the parental mouse Lewis lung carcinoma cancer cells (LLC-Parental cells). In vitro assessment of stemness by RT-qPCR and western blot analysis of stem cell markers, clonogenic assay (p < 0.001), single cell spheroid formation assay (p < 0.05) and 96-well-plate single-cell cloning assay (p < 0.01) indicated that the LLC-ASD cells exhibited stronger stemness features in comparison to the LLC-Parental cells. In vivo, tumorigenicity of LLC-ASD cells, transplanted subcutaneously to the nude mice, was increased compared to that of LLC-parental cells (p < 0.05). Further, Neuralized1a, a regulator of ASD in normal stem cells, was highly expressed in the LLC-ASD cells. Silencing Neuralized1a expression in LLC-ASD cells by siRNA weakened the stemness features measured by the in vitro assays listed above (p < 0.05). The tumorigenic ability was also decreased in the nude mice upon Neuralized1a silencing (p < 0.05). Collectively, the present study suggests that Neuralized1a regulates the stemness of LLC-ASD cells which could be the new marker and therapeutic target of CSCs.

It has been suggested that particle size plays an important role in determining the genotoxicity of gold nanoparticles (GNPs). The purpose of this study was to compare the potential radio-sensitization effects of two different sized GNPs (3.9 and 37.4 nm) fabricated and examined in vitro in Lewis lung carcinoma (LLC) as a model of non-small cell lung cancer through use of comet and clonogenic assays. After treatment with 2Gy X-ray irradiation, both particle sizes demonstrated increased DNA damage when compared to treatment with particles only and radiation alone. This radio-sensitization was further translated into a reduction in cell survival demonstrated by clonogenicity. This work indicates that GNPs of both sizes induce DNA damage in LLC cells at the tested concentrations, whereas the 37.4 nm particle size treatment group demonstrated greater significance in vitro. The presented data aids in the evaluation of the radiobiological response of Lewis lung carcinoma cells treated with gold nanoparticles.

Epithelial mesenchymal transition (EMT) is thought to be an essential feature of malignant tumor cells when they spread into the stroma. Despite the extracellular acidity of tumor tissues, the effect of acidic extracellular pH (pH e ) on EMT in carcinoma models, including the Lewis lung carcinoma (LLC) model, remains unclear.

The mammalian target of rapamycin (mTOR) regulates the motility and invasion of cancer cells. Cardamonin is a chalcone that exhibits anti-tumor activity. The previous study had proved that the anti-tumor effect of cardamonin was associated with mTOR inhibition. In the present study, the anti-metastatic effect of cardamonin and its underlying molecule mechanisms were investigated on the highly metastatic Lewis lung carcinoma (LLC) cells. The proliferation, invasion and migration of LLC cells were measured by MTT, transwell and wound healing assays, respectively. The expression and activation of mTOR- and adhesion-related proteins were assessed by Western blotting. The in vivo effect of cardamonin on the metastasis of the LLC cells was investigated by a mouse model. Treated with cardamonin, the proliferation, invasion and migration of LLC cells were significantly inhibited. The expression of Snail was decreased by cardamonin, while that of E-cadherin was increased. In addition, cardamonin inhibited the activation of mTOR and its downstream target ribosomal S6 kinase 1 (S6K1). Furthermore, the tumor growth and its lung metastasis were inhibited by cardamonin in C57BL/6 mice. It indicated that cardamonin inhibited the invasion and metastasis of LLC cells through inhibiting mTOR. The metastasis inhibitory effect of cardamonin was correlated with down-regulation of Snail and up-regulation of E-cadherin.

Metastasis is a devastating aspect of cancer. This study tested the hypothesis that metabolome of metastases differs from that of host organs by using the spontaneous metastasis model of Lewis lung carcinoma (LLC). In a 2 × 2 design, male C57BL/6 mice with or without a subcutaneous LLC inoculation were fed the standard AIN93G diet or a high-fat diet (HFD) for 12 weeks. Lung metastases from injected mice and the lungs from non-injected mice were harvested at the end of study for untargeted metabolomics of primary metabolism by using gas chromatography time-of-flight mass spectrometry. We identified 91 metabolites for metabolomic analysis. The analysis demonstrated that amino acid and energy metabolism were altered the most in LLC metastases compared to the lungs. A 60% decrease in glutamine and a 25-fold elevation in sorbitol were observed in metastases. Cholesterol and its metabolite dihydrocholesterol were 50% lower in metastases than in the lungs. The HFD elevated arachidonic acid and its precursor linoleic acid in the lungs from noncancer-bearing mice, reflecting the dietary fatty acid composition of the HFD. This elevation did not occur in metastases from HFD-fed LLC-bearing mice, suggesting alterations in lipid metabolism during LLC metastatic progression. Understanding the differences in metabolome between pulmonary LLC metastases and the normal healthy lungs can be useful in designing targeted studies for prevention and treatment of cancer spread using this LLC spontaneous metastasis model.

Metabolic reprogramming of lipid metabolism is a hallmark of cancer. Consumption of a high-fat obesogenic diet enhances spontaneous metastasis using a Lewis lung carcinoma (LLC) model. In order to gain further insights into the mechanisms by which dietary fats impact cancer progression, we conducted a lipidomic analysis of primary tumors originated from LLC from mice fed with a standard AIN93G diet or a soybean oil-based high-fat diet (HFD). Hierarchical clustering heatmap analysis of phosphatidylcholine (PC) lipids and phosphatidylethanolamine (PE) lipids demonstrated an increase in polyunsaturated fatty acids (PUFA)-containing phospholipids and a decrease in monounsaturated fatty acids (MUFA)-containing lipids in tumors from mice fed the HFD. The quantities of 51 PC and 24 PE lipids differed in primary tumors of LLC from mice fed the control diet and the HFD. Analysis of triacylglycerol (TAG) lipids identified differences in 32 TAG (by brutto structure) between the two groups; TAG analysis by neutral loss identified 46 PUFA-containing TAG species that were higher in mice fed with the HFD than in the controls. Intake of the HFD did not alter the expression of the de novo lipogenesis enzymes (fatty acid synthase, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase-1). Our results demonstrate that the dietary fatty acid composition of the HFD is reflected in the higher order lipidomic composition of primary tumors. Subsequent studies are needed to investigate how these lipidomic changes may be used for targeted dietary intervention to reduce tumor growth and malignant progression.

Spontaneous neoplasm metastasis, a fatalist pathological feature of cancer, is a long-evolving, multi-steps process that can now only be treated or controlled by drugs or immuno-modulators. Probimane (Pro), as a representative of the well-known class of antimetastatic agents âBisdioxopiperazine compounds (Biz)â, is systematically studied for its absorption, distribution and excretion in mice bearing Lewis lung carcinoma by a radioactivity-detective method in this investigation. It is found that the (14)C-Pro concentrations in different normal organs of mice at 2 hrs are very high and dramatically declined at 24 and 48 hrs. However, Pro concentrations in metastatic foci are slightly changed at the same time. Almost no change of Pro concentrations is observed in pulmonary metastatic nodules within 48 hrs. This evidence can be used to explain the characteristics of good metastatic inhibition by Biz compounds. The radioactivity in brain is relatively low because Pro can hardly penetrate into the blood-brain-barrier to eliminate brain tumors. The excretion of (14)C-Pro is observed at the same ratios from both urine and feces and also at constant rates. These data are much useful for better understanding of the general pharmacological characters and possible antimetastatic mechanisms of actions of probimane and other Biz compounds from a new perspective and research angles.

Radiotherapy is an important therapeutic method for lung cancer. However, in clinical situations, cellular resistance to radiotherapy is a significant component of tumor treatment failure. Thus, clarification in cellular mechanism underlying radiosensitivity of cancer cell is urgently needed. In this study, we established a radiation model of Lewis lung carcinoma in C57BL/6 mice and investigated the possible signaling molecule involved in this process.

The present study showed that oral mushroom beta-glucan treatment significantly increased IFN-γ mRNA expression but significantly reduced COX-2 mRNA expression within the lung. For LLC tumor model, oral Ganoderma lucidum or Antrodia camphorata polysaccharides treatments significantly reduced TGF-β production in serum. In addition, IL-12 and IFN-γ mRNA expression were significantly increased, but IL-6, IL-10, COX-2, and TGF-β mRNA expression were substantially following oral mushroom polysaccharides treatments. The study highlights the efficacious effect of mushroom polysaccharides for ameliorating the immune suppression in the tumor microenvironment. Increased M1 phenotype of tumor-associated macrophages and attenuated M2 phenotype of tumor-associated macrophages could be achieved by ingesting mushroom polysaccharides.

Basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor 1 (FGFR1) are associated with drug resistance in lung cancer. In the present study, mouse monoclonal antibodies (mAb) against human bFGF, targeting the binding site of bFGF with FGFR1 were produced, and the antitumor activity and inhibition of metastasis was studied in Lewis lung carcinoma (LLC). A total of four hybridoma cell strains that stably secreted bFGF mAb were obtained. mAbE12 was selected as the most effective for use in the following studies, with a relative affinity constant of 5.66x108 l/mol. mAbE12 was demonstrated to inhibit cell proliferation and tumor growth in vitro and in vivo. Furthermore, mAbE12 blocked migration and metastasis of LLC cells in vitro and in vivo. This occurred due to a mAbE12‑induced upregulation of E‑cadherin expression through the protein kinase B‑glycogen synthase kinase 3 β‑Snail pathway. These results suggested that mAbE12 may be a potential antibody for the treatment of lung cancer.

Background and objectives: There are several studies that sevoflurane could enhance proliferation of cancer cells, while others suggest no effect on clinical outcome. We conducted in vivo and in vitro experiments to investigate the effects of sevoflurane, a volatile anesthetic, on proliferation and outcomes of Lewis lung carcinoma (LLC) cells. Materials and Methods: A total of 37 mice were injected with LLC cells to compare the tumor size and survival of the sevoflurane exposed group (sevo group) and control group. The sevo group was exposed to 2% sevoflurane and 4 L/min of oxygen for 1 h per day 3 times per week, and the control group was exposed only to 4 L/min of oxygen. In vitro study, 12 plates incubated with LCC cells. 6 plates were exposed to 2% sevoflurane for 1 hr/day for 3 days and 6 plates were not exposed, and cell proliferation was compared after 3 days. Results: There were no significant differences in survival or tumor size between mice exposed to sevoflurane and control mice (survival: 29.06 ± 4.45 vs. 28.76 ± 3.75, p = 0.836; tumor size: 0.75 (0.41-1.02) vs. 0.49 (0.11-0.79), p = 0.153). However, in vitro study, the proliferation of LLC cells exposed to sevoflurane increased by 9.2% compared to the control group (p = 0.018). Conclusions: Sevoflurane (2 vol%) exposure could promote proliferation of LLC cells in vitro environment, but may not affect proliferation of LLC cells in vivo environment. These results suggest that in vitro studies on the effects of anesthetics on cancer may differ from those of in vivo or clinical studies.

Phyllanthus urinaria (P. urinaria), a widely used herb medicine, was tested for the anticancer effect in its water extract for the first time. The water extract of P. urinaria significantly decreased the number of Lewis lung carcinoma cells in a dose-and time-dependent manner as determined by MTT assay. However, the water extract of P. urinaria did not exert any cytotoxic effect on normal cells such as endothelial cells and liver cells. Result from flow cytometry revealed a dose-dependent increase of dead cells 24 hours after treating Lewis lung carcinoma cells with P. urinaria extract. The anticancer activity of P. urinaria extract was due to the apoptosis induced in Lewis lung carcinoma cells, which was demonstrated by DNA fragmentation analysis and increased caspase-3 activity. The apoptosis triggered by P. urinaria extract in Lewis lung carcinoma cells was associated with the down-regulation of Bcl-2 gene expression, but not with p53, p21 and Bax. Furthermore, the partial inhibition of P. urinaria-induced apoptosis in Lewis lung carcinoma cells by pretreatment with cyclosporin A, a mitochondria permeability transition pore inhibitor, suggesting that P. urinaria extract induced the apoptosis of Lewis lung carcinoma cells, at least in part, through a mitochondria-associated intrinsic pathway.

Drug combination is considered to be the cornerstone of cancer treatment. Simultaneous administration of two or more drugs but at lower doses not only increases cytotoxic effects on tumor cells, but also reduces side effects and possibly overcomes drug resistance. Salinomycin is a well-known cancer stem cell killer, and dichloroacetate is a pyruvate dehydrogenase kinase inhibitor that exclusively targets cells with altered mitochondrial activity, a characteristic being common to most of the cancer cells. In our recent study, we have demonstrated that salinomycin exerted a cytotoxic effect on colorectal carcinoma cells in the 2D and 3D cultures and provided evidence that the mechanism of their synergy was mediated by dichloroacetate-dependent inhibition of the activity of multidrug resistance proteins. In the current work, we confirmed the synergistic cytotoxic properties of salinomycin and dichloroacetate in the 2D and 3D cultures of Lewis lung carcinoma (LLC1) cells. To verify if a synergistic effect of these compounds persisted in vivo, we performed series of experiments using a syngeneic LLC1-C57BL/6 mouse model and demonstrated that combination therapy with salinomycin and DCA increased the survival rate of allografted mice, inhibited metastatic site formation and reduced the populations of cancer stem cells as well as cells that underwent the epithelial-to-mesenchymal transition. Our results demonstrate that a synergistic effect of salinomycin and dichloroacetate exists not only in vitro but also in vivo and suggest their benefits in the treatment of metastatic cancers.

Angiogenesis plays an essential role in the growth and metastasis of a number of tumors. Anti-angiogenic drugs are able to normalize tumor vasculature and inhibit tumor growth. Therefore, it has been hypothesized that the combination of cytotoxic chemotherapy drugs and angiogenesis inhibitors may exert complementary therapeutic benefits in the treatment of cancer. In the present study, the effect of the angiogenesis inhibitor, recombinant human endostatin (Endostar), in combination with cisplatin, was evaluated in C57/BL/6 mouse xenografts under different administration sequences. The drug combinations and sequences of administration were analyzed within the cancer xenografts for any inhibitory effects. Changes in the cell cycle distribution of the cells were monitored using flow cytometry. The effects of Endostar, particularly a reduction in the density of microvessels, were assessed using a method that employed anti-cluster of differentiation 31 antibodies. The concentration of cisplatin in the blood and tumor tissue at various time-points following administration was detected by high-performance liquid chromatography. The tumor tissues that received simultaneous Endostar and cisplatin exhibited increased inhibition of tumor growth and improved cell cycle distribution compared with those that received cisplatin alone, or those in which Endostar was administered prior to cisplatin. The simultaneous administration of the drugs resulted in the lowest microvessel density in the xenografts. Under these conditions, the concentration of cisplatin was revealed to be the highest in the grafted tumor tissue. The results of the present study suggest that the co-administration of Endostar and cisplatin may aid in the optimization of the antitumor activity of cisplatin.

The tumor stromal microenvironment is an integral part of the occurrence and development of tumor. Cancer-associated fibroblasts (CAFs) are a key component of most tumor stromal microenvironments. The present study aimed to investigate the use of CAFs-targeted immunotherapy to fibroblast activation protein-α (FAP-α) expressed in CAFs. Recombinant adenoviral vectors containing the mouse FAP-α cDNA (rAd-FAP-α) were constructed. C57BL/6 mice were immunized with rAd-FAP-α infected dendritic cells (DCs) against FAP-α, which is overexpress in CAFs. The results demonstrated that mice vaccinated with rAd-FAP-α DCs gave rise to potent FAP-α-specific cytotoxic T lymphocytes capable of lysing Lewis lung cancer (LLC) CAFs. Furthermore, mice vaccinated with rAd-FAP-α-transduced DCs induced an effective therapeutic or protective antitumor immunity to LLC in a subcutaneous model, and prolonged overall survival time compared with mice vaccinated with the control recombinant adenovirus-transduced DCs (rAd-c DCs) or DCs alone. The results of the present study suggested that FAP-α, which is preferentially expressed in CAFs, may be considered as a potential target for killing or destroying CAFs within the tumor stromal microenvironment, and may be exploited to develop immunogenic tumor vaccines.

Natural polysaccharides have shown promising effects on the regulation of immunity in animals. In this study, we examined the immune stimulatory effect of intranasally administered Codium fragile polysaccharides (CFPs) in mice. Intranasal administration of CFPs in C57BL/6 mice induced the upregulation of surface activation marker expression in macrophages and dendritic cells (DCs) in the mediastinal lymph node (mLN) and the production of interleukin-6 (IL-6), IL-12p70, and tumor necrosis factor-α in bronchoalveolar lavage fluid. Moreover, the number of conventional DCs (cDCs) was increased in the mLNs by the upregulation of C-C motif chemokine receptor 7 expression, and subsets of cDCs were also activated following the intranasal administration of CFP. In addition, the intranasal administration of CFPs promoted the activation of natural killer (NK) and T cells in the mLNs, which produce pro-inflammatory cytokines and cytotoxic mediators. Finally, daily administration of CFPs inhibited the infiltration of Lewis lung carcinoma cells into the lungs, and the preventive effect of CFPs on tumor growth required NK and CD8 T cells. Furthermore, CFPs combined with anti-programmed cell death-ligand 1 (PD-L1) antibody (Ab) improved the therapeutic effect of anti-PD-L1 Ab against lung cancer. Therefore, these data demonstrated that the intranasal administration of CFP induced mucosal immunity against lung cancer.

Ghrelin is a potential therapy for cachexia due to its orexigenic properties and anabolic effects on muscle and fat. However, its clinical use is limited by the short half-life of active (acylated) ghrelin (~11 min in humans). EXT418 is a novel long-acting, constitutively active ghrelin analog created by covalently linking it to a vitamin D derivative. Here, we evaluated the effects and mechanisms of action of EXT418 on Lewis lung carcinoma (LLC)-induced cachexia in mice.

Lung cancer is currently the leading cause of cancer-related mortality with very limited effective therapy. Screening of a variety of traditional Chinese medicines (TCMs) for their capacity to inhibit the proliferation of human lung cancer A549 cells and to induce the in vitro maturation of human DCs led to the identification of cryptotanshinone (CT), a compound purified from the TCM Salvia miltiorrhiza Bunge. Here, CT was shown to inhibit the proliferation of mouse Lewis lung carcinoma (LLC) cells by upregulating p53, downregulating cyclin B1 and Cdc2, and, consequently, inducing G2/M cell-cycle arrest of LLC cells. In addition, CT promoted maturation of mouse and human DCs with upregulation of costimulatory and MHC molecules and stimulated DCs to produce TNFα, IL-1β, and IL-12p70, but not IL-10 in vitro. CT-induced maturation of DCs depended on MyD88 and also involved the activation of NF-κB, p38, and JNK. CT was effective in the treatment of LLC tumors and, when used in combination with low doses of anti-PD-L1, cured LLC-bearing mice with the induction of subsequent anti-LLC long-term specific immunity. CT treatment promoted T-cell infiltration and elevated the expression of genes typical of Th1 polarization in LLC tumor tissue. The therapeutic effect of CT and low doses of anti-PD-L1 was reduced by depletion of CD4 and CD8 T cells. This paper provides the first report that CT induces immunological antitumor activities and may provide a new promising antitumor immunotherapeutic.

Angiogenesis modelling is an important tool to understand the underlying mechanisms yielding tumour growth. Nevertheless, there is usually a gap between models and experimental data. We propose a model based on the intrinsic microscopic reactions defining the angiogenesis process to link experimental data with previous macroscopic models. The microscopic characterisation can describe the macroscopic behaviour of the tumour, which stability analysis reveals a set of predicted tumour states involving different morphologies. Additionally, the microscopic description also gives a framework to study the intrinsic stochasticity of the reactive system through the resulting Langevin equation. To follow the goal of the paper, we use available experimental information on the Lewis lung carcinoma to infer meaningful parameters for the model that are able to describe the different stages of the tumour growth. Finally we explore the predictive capabilities of the fitted model by showing that fluctuations are determinant for the survival of the tumour during the first week and that available treatments can give raise to new stable tumour dormant states with a reduced vascular network.