1,25(OH)2 D3 inhibits Lewis lung cancer cell migration via NHE1-sensitive metabolic reprograming.

High prevalence and metastasis rates are characteristics of lung cancer. Glycolysis provides energy for the development and metastasis of cancer cells. The 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) has been linked to reducing cancer risk and regulates various physiological functions. We hypothesized that 1,25(OH)2 D3 could be associated with the expression and activity of Na+ /H+ exchanger isoform 1 (NHE1) of Lewis lung cancer cells, thus regulating glycolysis as well as migration by actin reorganization. Followed by online public data analysis, Vitamin D3 receptor, the receptor of 1,25(OH)2 D3 has been proved to be abundant in lung cancers. We demonstrated that 1,25(OH)2 D3 treatment suppressed transcript levels, protein levels, and activity of NHE1 in LLC cells. Furthermore, 1,25(OH)2 D3 treatment resets the metabolic balance between glycolysis and OXPHOS, mainly including reducing glycolytic enzymes expression and lactate production. In vivo experiments showed the inhibition effects on tumor growth as well. Therefore, we concluded that 1,25(OH)2 D3 could amend the NHE1 function, which leads to metabolic reprogramming and cytoskeleton reconstruction, finally inhibits the cell migration.

Spectrum-effect relationship between UPLC fingerprints and anti-lung cancer effect of Panax ginseng.

OBJECTIVES: Lung cancer has the highest mortality rate among the various types of cancer. Panax ginseng (C. A. Mey). is a popular anti-cancer herbal supplement. The quality control of ginseng is crucial to ensure its clinical efficacy. This study aimed to establish new quality control methods for ginseng and to identify its main active components responsible for lung cancer treatment. METHODS: Ultra-high-performance liquid chromatography (UPLC) was used to establish fingerprints of 18 batches of ginseng. CCK-8 test was performed to evaluate the inhibitory activity of ginseng on Lewis lung cancer (LLC) cells. The spectrum-effect relationship analysis of ginseng was assessed by canonical correlation analysis (CCA) and bioactivity validation. KEY FINDINGS: Six common peaks were identified and the variation coefficients were determined. The 18 batches of ginseng inhibited the proliferation of LLC cells to different degrees, showing different half maximal inhibitory concentration (IC50 ) values. Spectrum-effect relationship analysis showed that ginsenoside Ro is the main anti-proliferative constituent of LLC cell. CONCLUSIONS: Spectrum-effect relationship is suitable for quality control of ginseng used for lung cancer. It is also effective in discovering the active ingredients related to the clinical efficacy of traditional Chinese medicine.

Uncovering the anticancer mechanism of petroleum extracts of Farfarae Flos against Lewis lung cancer by metabolomics and network pharmacology analysis.

Lung cancer shows the highest incidence rate in the world. Thus, it has become increasingly important to find therapeutic drugs to treat lung cancer. Farfarae Flos (FF) has been used in traditional Chinese medicine to treat pulmonary diseases such as cough, bronchitis and asthmatic disorders. In this study, the anti-proliferation effects of petroleum extracts of FF (PEFF) on Lewis lung cancer cells and the corresponding mechanisms were studied using cell metabolomics. Fifteen differential metabolites in the cell extracts and the corresponding medium related to the anti-proliferation effect of PEFF were identified, which were probably involved in pyruvate metabolism and glycine, serine and threonine metabolism. For the cellular uptake compounds in PEFF, six metabolites derived from two prototype compounds were also tentatively identified by UHPLC-Q-Orbitrap high-resolution MS. Network pharmacology analysis demonstrated that the anti-proliferation mechanism of PEFF was also probably related to the target genes, including, Aurora-A, glutathione S-transferase Mu 1 (GSTM1), glutathione S-transferase P 1 (GSTP1), progesterone receptor and heme oxygenase-1 (HO-1), and further associated with the proteoglycans and PI3K/Akt signaling pathway. Cell metabolomics and network pharmacology analysis provided a holistic method to investigate the anti-proliferation mechanisms of PEFF. However, further studies were still needed to validate the potential target genes, pathways and active compounds in PEFF.

Evaluation of cytotoxicity of stearoyl gemcitabine-loaded PLGA nanoparticles to Lewis lung cancer cells / 中国免疫学杂志

Objective:To investigate the anti-proliferation effect of 4-(N)-stearoyl gemcitabine-loaded poly(lactic-co-glycolic) acid nanoparticles(GemC18-PLGA-NPs) on Lewis lung cancer cells(LLC) in vitro.Methods: Lewis cells were incubated with GemC18-PLGA-NPs,free GemC18,gemcitabine HCl(GemHCl) or GemC18-free blank nanoparticles(PLGA-NPs) respectively and cell viability was determined using an MTT assay after 24,48 or 72 h of incubation.The apoptosis rate after 48 and 72 h of incubation were measured by flow cytometry.Results:GemC18-PLGA-NPs,GemC18,and GemHCl all significantly inhibited the growth of LLC cells, and the survival rate of GemHCl group was lowest,GemC18-PLGA-NPs group had the highest survival rate.The cell survival rate of GemC18-PLGA-NPs after 72 h was significantly higher than that of GemHCl (P<0.05) at the concentration of 1 μmol/L,indicating that it had a significant drug release effect.PLGA-NPs group produced trifle inhibition on the Lewis cells without correlation to time or concentration.Conclusion:GemC18-PLGA-NPs have significant anti-proliferation effect on mouse Lewis lung cancer cells in vitro.

Effects of Shanxian Granule on antagonistic immunity of Lewis lung cancer mice and level of IFN-γ,TNF-β,IL-10 in peripheral blood / 中国免疫学杂志

Objective:To investigate the effects of Shanxian granule on proliferation of Lewis lung cancer cells and anti-tumor immunity and immune microenvironment of Lewis lung cancer-bearing mice in order to explore the molecular mechanism of anti-tumor of Shanxian Granule and improve the anti-tumor immunity of the body, and provide further theoretical basis for its clinical application. Methods:Lewis lung cancer cells was transplanted to axillary skin to establish mouse tumor model. The mice divided into blank group,model group,chemotherapy group and Shanxian granule group. The tumor tissue of Lewis lung cancer tumor bearing mice was weighed and the tumor inhibition rate was calculated. Immunohistochemical method was used to detect the expression of CD and CD8 in spleen tissue. The effect of lymphocytes on the proliferation of Lewis lung cancer cells was detected by CCK-8 method. The level of IFN-γ,TNF-βand IL-10 in peripheral blood were detected by ELISA. Results:①The tumor inhibition rate of Lewis lung cancer was 45. 99% in Shanxian Granule group,which was significantly higher than that of chemotherapy group (P<0. 05).②The lymphocytes of mouse can inhibit the proliferation of Lewis lung cancer cells and have a positive correlation with lymphocyte concentration and duration of action. Moreover,CD4+ T cells,CD4+/CD8+ratio and lymphocyte inhibition rate of Lewis lung cancer cells in model group and chem-otherapy group were significantly lower than those in blank group (P<0. 05). Shanxian granule group was significantly higher than the model group and chemotherapy group ( P<0. 05 ) . However, there was no significant difference between Shanxian granule group and blank group(P>0. 05).③The levels of IFN-γand TNF-βin peripheral blood of model group and chemotherapy group were significantly lower than those in blank group,while IL-10 was significantly higher than that in blank group (P<0. 05). The levels of IFN-γand TNF-βin peripheral blood of mice in Shanxian granule group were significantly higher than those in model group and chemotherapy group, while IL-10 was significantly lower than that in model group and chemotherapy group (P<0. 05). There was no significant difference in IFN-γ,TNF-β and IL-10 in peripheral blood of mice between Shanxian granule group and blank group. Conclusion:Shanxian granule can significantly inhibit the growth of tumor tissue of Lewis lung cancer tumor bearing mice,increase the spleen index of mice,enhance the activity of T lymphocytes,upregulate IFN-γ and TNF-β in peripheral blood and decrease IL-I. These suggested that the anti-tumor effect of Shanxian granule may be achieved by regulating the content of CD4+ T lymphocyte,the ration of CD4+/CD8+ and Th1/Th2 ratio,in order to restore the immune steady function of tumor patients,improve the immune system and enhance the immune surveillance function.

Effects of Shanxian Granule on antagonistic immunity of Lewis lung cancer mice and level of IFN-γ,TNF-β,IL-10 in peripheral blood / 中国免疫学杂志

Objective:To investigate the effects of Shanxian granule on proliferation of Lewis lung cancer cells and anti-tumor immunity and immune microenvironment of Lewis lung cancer-bearing mice in order to explore the molecular mechanism of anti-tumor of Shanxian Granule and improve the anti-tumor immunity of the body, and provide further theoretical basis for its clinical application. Methods:Lewis lung cancer cells was transplanted to axillary skin to establish mouse tumor model. The mice divided into blank group,model group,chemotherapy group and Shanxian granule group. The tumor tissue of Lewis lung cancer tumor bearing mice was weighed and the tumor inhibition rate was calculated. Immunohistochemical method was used to detect the expression of CD and CD8 in spleen tissue. The effect of lymphocytes on the proliferation of Lewis lung cancer cells was detected by CCK-8 method. The level of IFN-γ,TNF-βand IL-10 in peripheral blood were detected by ELISA. Results:①The tumor inhibition rate of Lewis lung cancer was 45. 99% in Shanxian Granule group,which was significantly higher than that of chemotherapy group (P<0. 05).②The lymphocytes of mouse can inhibit the proliferation of Lewis lung cancer cells and have a positive correlation with lymphocyte concentration and duration of action. Moreover,CD4+ T cells,CD4+/CD8+ratio and lymphocyte inhibition rate of Lewis lung cancer cells in model group and chem-otherapy group were significantly lower than those in blank group (P<0. 05). Shanxian granule group was significantly higher than the model group and chemotherapy group ( P<0. 05 ) . However, there was no significant difference between Shanxian granule group and blank group(P>0. 05).③The levels of IFN-γand TNF-βin peripheral blood of model group and chemotherapy group were significantly lower than those in blank group,while IL-10 was significantly higher than that in blank group (P<0. 05). The levels of IFN-γand TNF-βin peripheral blood of mice in Shanxian granule group were significantly higher than those in model group and chemotherapy group, while IL-10 was significantly lower than that in model group and chemotherapy group (P<0. 05). There was no significant difference in IFN-γ,TNF-β and IL-10 in peripheral blood of mice between Shanxian granule group and blank group. Conclusion:Shanxian granule can significantly inhibit the growth of tumor tissue of Lewis lung cancer tumor bearing mice,increase the spleen index of mice,enhance the activity of T lymphocytes,upregulate IFN-γ and TNF-β in peripheral blood and decrease IL-I. These suggested that the anti-tumor effect of Shanxian granule may be achieved by regulating the content of CD4+ T lymphocyte,the ration of CD4+/CD8+ and Th1/Th2 ratio,in order to restore the immune steady function of tumor patients,improve the immune system and enhance the immune surveillance function.

Ginkgo biloba exocarp extracts inhibits angiogenesis and its effects on Wnt/ß-catenin-VEGF signaling pathway in Lewis lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: A fruit of Ginkgo biloba L. also known as Ginkgo biloba, can be used for the treatment of cancer in Chinese traditional medicine. The scientific name of succulent skin, which is the episperm of Ginkgo nuts, is exocarp. Experiment shows that Ginkgo biloba exocarp extracts (GBEE) has the effects of immune promotion, cancer inhibition and etc. AIM OF STUDY: Study on the activity of GBEE against Lewis lung cancer (LLC) angiogenesis and its partial molecular mechanism. MATERIALS AND METHODS: The effect of GBEE on proliferation of LLC cells was detected by MTT method in vitro. The metastasis model of LLC was set up. The C57BL/6J mice were randomly separated into normal control, model control, positive control and GBEE (50, 100, 200mg/kg) treatment groups, n=10. The mice in normal group and model group were both intragastric gavage (i.g.) normal saline (NS) in a volume of 0.1mL/10g (b.w.), positive group were intraperitoneal (i.p.) injection cyclophosphamide (CPA) at a dose of 20mg/kg (b.w.) , the GBEE treatment groups were respectively i.g. GBEE 50, 100, and 200mg/kg (b.w.), once a day for 20d. After treatment, we calculated the tumor inhibition rate and anti-metastasis rate. The microvessel density (MVD) was measured by immunohistochemistry method in transplanted tumor. The expression levels of vascular en-dothelial growth factor (VEGF) and VEGFR2 mRNA or Wnt3a, ß-catenin, VEGF, VEGFR2 and p-Akt/Akt protein expression were respectively tested by Quantitative Reverse transcription Polymerase chain reaction (qRT-PCR) or western blot in vitro and vivo. RESULTS: GBEE suppressed the growth of LLC cells in a dose-dependent way at the dose of 5, 10, 20, 40, 80 and 160µg/mL in vitro. It can suppressed Wnt3a and ß-catenin protein expression and the content of mRNA of VEGF and VEGFR2 in LLC cells significantly. In vivo, we discovered GBEE can retard the growth of LLC transplanted tumor in a dose-dependent way at the dose of 50, 100, 200mg/kg, suppressing tumor lung metastasis. The expression of CD34 was reduced, which means MVD was inhibited and so do ß-catenin, VEGF, VEGFR2 and p-AKT/AKT protein expression and VEGF and VEGFR2 mRNA expression levels in LLC transplanted tumor of C57BL/6 mice. CONCLUSIONS: GBEE played the effects of anti-tumor and anti-metastatic depending upon the inhibition of tumor angiogenesis, which may be closely relevant to its effect in blockage of Wnt /ß-catenin-VEGF signaling pathway in LLC.

Radiotherapy suppressed tumor-specific recruitment of regulator T cells via up-regulating microR-545 in Lewis lung carcinoma cells.

OBJECTIVE: Radiotherapy is an important treatment for cancer. The main irradiated action is thought to be the irreversible damage to tumor cell DNA, but recent studies showed that high dose radiotherapy related to the tumor immune response. This study was designed to determine the relationship between Lewis lung tumor radiosensitivity and CD4+CD25+ regulatory T cells (Tregs) infiltration and elucidate the underlying mechanisms in vitro. METHODS: With tumor transplantation method to establish mice Lewis lung tumor mice model, to observe the inhibition rate of radiotherapy to tumor growth. Proliferation profiles of CD4+CD25+ Tregs and CD4+ T cells were assessed by flow cytometry. MiR-545 and CCL-22 mRNA were determined by Quantitative Real-Time PCR. CCL-22 protein was determined by western blot assay. RESULTS: Radiotherapy caused a time-dependent inhibition of tumor growth as well as a decrease in the percentage of tumor-infiltrating CD4+CD25+ Tregs of CD4+ T cells compared with no treatment group. And the miR-545 was significantly upregulated and CCL-22 was significantly down-regulated in irradiated tumor and Lewis lung cancer cells. In Lewis lung cancer cell transfection experiments, mimic or inhibitor for miR-545 negatively regulated CCL-22 expression when cells treated or treated without irradiation. Silenced miR-545 promotes CD4+CD25+ Treg proliferation. Additionally, silenced miR-545 reversed radiosensitivity of Lewis lung cancer. CONCLUSION: Radiotherapy suppressed specific recruitment of regulator CD4+CD25+ Treg cells in Lewis lung carcinoma via up-regulating microR-545.

Ganoderic acid Me induces the apoptosis of competent T cells and increases the proportion of Treg cells through enhancing the expression and activation of indoleamine 2,3-dioxygenase in mouse lewis lung cancer cells.

The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression.

Establishment of a mouse model of orthotopic Lewis lung cancer / 中国实验动物学报

Objective To establish a mouse model of orthotopic Lewis lung carcinoma using Matrigel, to evaluate the tumor growth and metastasis, and to provide a more stable mouse model of orthotopic lung cancer, which is more similar to human lung cancer.Methods Logarithmic phase of cultured Lewis lung cancer cells were suspended in Matrigel, vac-cinated into the left lung of inbred C57BL/6 mice.Five mice were killed on the 4th, 7th, 10th, 13th, and 16th days, re-spectively, and to observe the median survival, tumor formation rate, tumor growth, and metastasis.Pathological changes of the mouse lung, liver, kidney and spleen were examined.Results In 5 mice killed on the 7th postoperative day, small tumor nodules were observed on the lung in three mice and no tumor was visible by gross inspection in the other two mice, but small tumor nodules were observed under the microscope.For all the mice killed on the 10th postoperative day, tumors were visible to the naked eye on the lung of all the five mice.On the 13th day, orthotopic tumor was observed on the lung with bloody pleural effusion and pleural metastasis in all the five mice.On the 25th day, in addition to the pleural metasta-sis, one mouse had pericardial metastasis and renal metastasis.The survival periods of the 5 mice were 17 d, 20 d, 22 d, 22 d, and 25 d, respectively, with a median survival period of 21.2 d (17-25 d), and the tumor formation rate was 100%.Conclusions Mouse models of orthotopic Lewis lung carcinoma is successfully established using injection of tumor cells suspended in Matrigel.This model is more similar to the growth of human lung cancer, with good stability, high tumor formation rate and characteristics of distant metastasis, therefore, is worthy of further application.