Predicting the herbal medicine triggering innate anti-tumor immunity from a system pharmacology perspective.

Although the main focus of immuno-oncology has been manipulating the adaptive immune system, tumor associated macrophages (TAMs) are the main infiltrating component in the tumor microenvironment (TME) and play a critical role in cancer progression. TAMs are mainly divided into two different subtypes: macrophages with antitumor or killing activity are called M1 while tumor-promoting or healing macrophages are named M2. Therefore, controlling the polarization of TAMs is an important strategy for cancer treatment, but there is no particularly effective means to regulate the polarization process. Here, combined systems pharmacology targets and pathways analysis strategy, we uncovered Scutellariae Radix (SR) has the potential to regulate TAMs polarization to inhibit the growth of non-small cell lung cancer (NSCLC). Firstly, systems pharmacology approach was used to reveal the active components of SR targeting macrophages in TME through compound target prediction and target-microenvironment phenotypic association analysis. Secondly, in vitro experiment verified that WBB (wogonin, baicalein and baicalin), major active ingredients of SR are significantly related to macrophages and survival, initiated macrophages programming to M1-like macrophages to promoted the apoptosis of tumor cells. Finally, we evidenced that WBB effectively inhibited tumor growth in LLC (Lewis lung carcinoma) tumor-bearing mice and increased the infiltration of M1-type macrophages in TME. Overall, the systems pharmacology strategy offers a paradigm to understand the mechanism of polypharmacology of natural products targeting TME.

Metabolomics and integrated network pharmacology analysis reveal Tricin as the active anti-cancer component of Weijing decoction by suppression of PRKCA and sphingolipid signaling.

Currently, conventional methods of treating non-small cell lung cancer (NSCLC) have many disadvantages. An alternative effective therapy with minimal adverse reactions is urgently needed. Weijing decoction (WJD), which is a classic ancient Chinese herbal prescription, has been used successfully to treat pulmonary system diseases containing lung cancer in the clinic. However, the key active component and target of Weijing decoction are still unexplored. Therefore, for the first time, our study aims to investigate the pharmacological treatment mechanism of Weijing decoction in treating NSCLC via an integrated model of network pharmacology, metabolomics and biological methods. Network pharmacology results conjectured that Tricin is a main bioactive component in this formula which targets PRKCA to suppress cancer cell growth. Metabolomics analysis demonstrated that sphingosine-1-phosphate, which is regulated by sphingosine kinase 1 and sphingosine kinase 2, is a differential metabolite in plasma between the WJD-treated group and the control group, participating in the sphingolipid signaling. In vitro experiments demonstrated that Tricin had vital effects on the proliferation, pro-apoptosis, migration and colony formation of Lewis lung carcinoma cells. Through a series of validation assays, Tricin inhibited the tumor growth mainly by suppressing PRKCA/SPHK/S1P signaling and antiapoptotic signaling. On the other hand, Weijing formula could inhibit the tumor growth and prolong the survival time. A high dosage of Tricin was much more potent in animal experiments. In conclusion, we confirmed that Weijing formula and its primary active compound Tricin are promising alternative treatments for NSCLC patients.

Zi Shen Decoction Inhibits Growth and Metastasis of Lung Cancer via Regulating the AKT/GSK-3ß/ß-Catenin Pathway.

Lung cancer has become the leading cause of cancer-related death worldwide. Oxidative stress plays important roles in the pathogenesis of lung cancer. Many natural products show antioxidative activities in cancer treatment. Zi Shen decoction (ZSD) is a classic prescription for the treatment of lung disease. However, its effect on lung cancer lacks evidence-based efficacy. In this study, we investigated the anticancer effects of ZSD on lung cancer in vivo and in vitro. Our results showed that oral administration of ZSD suppressed the Lewis lung cancer (LLC) growth in a subcutaneous allograft model and promoted necrosis and inflammatory cell infiltration in the tumor tissues. Furthermore, ZSD not only inhibited tumor cell proliferation and migration but also induced cell apoptosis in lung cancer cells. PI3K/AKT signaling is well characterized in response to oxidative stress. The bioinformatics analysis and western blot assays suggested that ZSD decreased the enzyme activity of PI3K and AKT in vivo and in vitro. We also found that the AKT/GSK-3ß/ß-catenin pathway medicated anticancer effect of ZSD in lung cancer cells. In conclusion, we demonstrate for the first time that ZSD possesses antitumor properties, highlighting its potential use as an alternative strategy or adjuvant treatment for lung cancer therapy.

Immunomodulatory and antitumour bioactive labdane diterpenoids from Leonurus japonicus.

OBJECTIVES: Two labdane diterpenoids, leojapone B and heteronone B, were isolated from Leonurus japonicus Houtt., and their biological activity were evaluated in this study. METHODS: Human and mouse cancer cells, human peripheral blood mononuclear cells (PBMCs) and mouse macrophages (RAW264.7 cells) were used to evaluate the activity of leojapone B and heteronone B, while the in vivo effects of leojapone B were further examined in Lewis Lung Cancer tumour-bearing mice. KEY FINDINGS: In vitro studies showed that leojapone B selectively inhibited the proliferation of lung cancer cells, and both leojapone B and heteronone B inhibited the production of pro-inflammatory cytokines in activated PBMCs. In tumour-bearing mice model, lung tumours were reduced in size in mice treated with intraperitoneal injections of leojapone B at 20 and 30 mg/kg for 14 days. The population ratio of CD4+ /CD8+ T cells in mouse spleens was found to be increased, while regulatory T cells were decreased after leojapone B treatment. CONCLUSIONS: The inhibitory effects of leojapone B in mouse lung tumours were demonstrated for the first time in this study. The immunomodulatory activity of heteronone B were also demonstrated. Our findings indicated that both leojapone B and heteronone B may act as active components in L. japonicus.

Effects of Tussilago farfara L. Polysaccharides on the Expression of PD-1 (CD279) and PD-L1 (CD274) in Peripheral Blood and Tumor Tissue Lymphocytes in Mice with Lewis Lung Carcinoma.

One of prospective methods for immunotherapy of tumors is modulation via immunological checkpoints, specifically, via the PD-1(CD279)/PD-L1(CD274) system. Interactions between tumor cell receptor (CD279) and the ligand on lymphocytes (CD274) leads to lymphocyte inactivation, which allows tumor escape from the immune control. Experiments on C57BL/6 mice with Lewis lung carcinoma demonstrate the possibility of reducing the expression of CD279 and CD274 on the peripheral blood and tumor tissue lymphocytes under the effects of Tussilago farfara L. polysaccharides. This phenomenon can underlie the antitumor and antimetastatic effects of these substances.

HYR-2 plays an anti-lung cancer role by regulating PD-L1 and Akkermansia muciniphila.

Traditional Chinese medicine (TCM) plays a vital part in cancer treatment due to its unique superiority. Huoxue Yiqi Recipe-2 (HYR-2) was supposed to have therapeutic effect on lung cancer, which came from Ze Qi Decoction in one of the four great classics of TCM called "Synopsis of Prescriptions of the Golden Chamber". Network pharmacology demonstrated that the targets of active components from HYR-2 were significantly enriched in the signaling pathways, which were closely associated with non-small cell lung cancer (NSCLC) and programmed death ligand 1 (PD-L1). Then, data about NSCLC was downloaded from Gene Expression Omnibus database (GEO). The Cancer Genome Atlas (TCGA) and DisGeNET was analyzed by bioinformatics, and 214 biomarkers for NSCLC were obtained, containing 14 targets of active components from HYR-2 (which were significantly enriched in the PD-L1 related signaling pathway). In vivo and in vitro experiments showed that HYR and HYR-2 could inhibit the growth of lung cancer and down-regulate the expression of PD-L1, which might be related to the blocking effect of HYR-2 on the PI3K/Akt signaling pathway. Furthermore, HYR-2 promoted the transformation of M2 macrophages into M1 macrophages as well. It is deserved to be mentioned that the level of Akkermansia muciniphila was also significantly elevated by HYR-2, which was believed to enhance the therapeutic effect of PD-L1 antibodies. To sum up, HYR-2 might play an anti-lung cancer effect by down-regulating PD-L1 together with up-regulating Akkermansia muciniphila.

Real-time fluorescence imaging for visualization and drug uptake prediction during drug delivery by thermosensitive liposomes.

Objective: Thermosensitive liposomal doxorubicin (TSL-Dox) is a promising stimuli-responsive nanoparticle drug delivery system that rapidly releases the contained drug in response to hyperthermia (HT) (>40 °C). Combined with localized heating, TSL-Dox allows highly localized delivery. The goals of this study were to demonstrate that real-time fluorescence imaging can visualize drug uptake during delivery, and can predict tumor drug uptake. Methods: Nude mice carrying subcutaneous tumors (Lewis lung carcinoma) were anesthetized and injected with TSL-Dox (5 mg/kg dose). Localized HT was induced by heating tumors for 15, 30 or 60 min via a custom-designed HT probe placed superficially at the tumor location. In vivo fluorescence imaging (excitation 523 nm, emission 610 nm) was performed before, during, and for 5 min following HT. After imaging, tumors were extracted, drug uptake was quantified by high-performance liquid chromatography, and correlated with in vivo fluorescence. Plasma samples were obtained before and after HT to measure TSL-Dox pharmacokinetics. Results: Local drug uptake could be visualized in real-time during HT. Compared to unheated control tumors, fluorescence of heated tumors increased by 4.6-fold (15 min HT), 9.3-fold (30 min HT), and 13.2-fold (60 min HT). HT duration predicted tumor drug uptake (p = .02), with tumor drug concentrations of 4.2 ± 1.3 µg/g (no HT), 7.1 ± 5.9 µg/g (15 min HT), 14.1 ± 6.7 µg/g (30 min HT) and 21.4 ± 12.6 µg/g (60 min HT). There was good correlation (R2 = 0.67) between fluorescence of the tumor region and tumor drug uptake. Conclusions: Real-time in vivo fluorescence imaging can visualize drug uptake during delivery, and can predict tumor drug uptake.

Synthesis and in vivo antitumor evaluation of an orally active potent phosphonamidate derivative targeting IDO1/IDO2/TDO.

Targeting Trp-Kyn pathways has been identified as an attractive approach for the cancer immunotherapies. In this study, a novel phosphonamidate containing compound was designed, synthesized and evaluated for its inhibitory activity against key dioxygenases in Trp-Kyn pathway, including IDO1, IDO2 and TDO. This compound showed potent IDO1 inhibitory activity with an IC50 value of 94 nM in an enzymatic assay and 12.6 nM in HeLa cells. In addition, this compound showed promising IDO2 inhibition and TDO inhibition with IC50 values of 310 nM and 2.6 µM, respectively, in enzyme assay. Based on the promising enzyme inhibitory activity toward IDO/TDO, compound F04 was evaluated of its antitumor effects in two tumor models. Further evaluation of mechanism demonstrated compound F04 with the remarkable capacity of reducing kynurenine level in plasma/TME and restoring anti-tumor immune response. F04 could be further developed as a potential immunotherapeutic agent combined with immune checkpoint inhibitors or chemotherapeutic drugs for cancer treatment.

Immunomodulatory effects exerted by Poria Cocos polysaccharides via TLR4/TRAF6/NF-κB signaling in vitro and in vivo.

OBJECTIVE: Poria cocos polysaccharide (PCP) is the major active ingredients of P. cocos and possesses various pharmacological effects, including anti-oxidative and anti-apoptosis effects and activity against cancer. This study investigated the immunomodulatory mechanism by which PCP acts on RAW 264.7 macrophages and LLC tumors in mice. METHODS: The concentrations of nitric oxide, and Th1, Th2, and Th17 cytokines were examined by Griess reaction and using a bead-based cytokine assessment kit. qRT-PCR and western blotting were used to investigate relevant signaling molecule expression. RESULTS: Levels of nitric oxide, IL-2, IL-6, IL-17 A, TNF, and IFN-γ were increased by PCP while levels of IL-4 and IL-10 were unaffected. The addition of TAK-242 (TLR4 inhibitor) or assessment in C57BL/10ScNJ (TLR4-deficient) mice markedly reduced this effect. In C57BL/10 J (TLR4+/+wild-type) mice, the indices of organ immune activity were all elevated, and oral PCP delivery resulted in a significant reduction in tumor volume over a 25 day period. Relative to controls, TLR4, MyD88, TRAF-6, p-NF-κB and p-c-JUN expression significantly increased, while TRAM expression did not change. Nevertheless, there was no PCP-dependent activation of MyD88, TRAF-6, TRAM, p-NF-κB or p-c-JUN in TLR4-deficient mice. CONCLUSION: These results support the concept that PCP may exhibit immunomodulatory activity through TLR4/TRAF6/NF-κB signaling both in vitro and in vivo.

Antrodia cinnamomea induces anti-tumor activity by inhibiting the STAT3 signaling pathway in lung cancer cells.

We examined the effects of an Antrodia cinnamomea ethanol extract (ACEE) on lung cancer cells in vitro and tumor growth in vivo. ACEE produced dose-dependent cytotoxic effects and induced apoptosis in Lewis lung carcinoma (LLC) cells. ACEE treatment increased expression of p53 and Bax, as well as cleavage of caspase-3 and PARP, while reducing expression of survivin and Bcl-2. ACEE also reduced the levels of JAK2 and phosphorylated STAT3 in LLC cells. In a murine allograft tumor model, oral administration of ACEE significantly inhibited LLC tumor growth and metastasis without affecting serum biological parameters or body weight. ACEE increased cleavage of caspase-3 in murine tumors, while decreasing STAT3 phosphorylation. In addition, ACEE reduced the growth of human tumor xenografts in nude mice. Our findings therefore indicate that ACEE inhibits lung tumor growth and metastasis by inducing apoptosis and by inhibiting the STAT3 signaling pathway in cancer cells.

Effects of low-dose leucine supplementation on gastrocnemius muscle mitochondrial content and protein turnover in tumor-bearing mice.

Many forms of cancer are associated with loss of lean body mass, commonly attributed to decreased protein synthesis and stimulation of proteolytic pathways within the skeletal muscle. Leucine has been shown to improve protein synthesis, insulin signaling, and mitochondrial biogenesis, which are key signaling pathways influenced by tumor signaling. The purpose of this study was to examine the effects of leucine supplementation on mitochondrial biogenesis and protein turnover in tumor-bearing mice. Twenty male C57BL/6 mice were divided into 4 groups (n = 5): Chow, leucine (Leu), Lewis lung carcinoma (LLC) implant, and LLC+Leu. At 9-10 weeks of age, mice were inoculated and supplemented with 5% leucine (w/w) in the diet. C2C12 myotubes were treated with 2.5 mmol/L leucine and 25% LLC conditioned media to further elucidate the direct influence of the tumor and leucine on the muscle. Measures of protein synthesis, mitochondrial biogenesis, and inflammation in the gastrocnemius were assessed via Western blot analysis. Gastrocnemius mass was decreased in LLC+Leu relative to LLC (p = 0.040). Relative protein synthesis rate was decreased in LLC mice (p = 0.001). No change in protein synthesis was observed in myotubes. Phosphorylation of STAT3 was decreased in the Leu group relative to the control in both mice (p = 0.019) and myotubes (p = 0.02), but did not significantly attenuate the inflammatory effect of LLC implantation (p = 0.619). LLC decreased markers of mitochondrial content; however, PGC-1α was increased in LLC+Leu relative to LLC (p = 0.001). While leucine supplementation was unable to preserve protein synthesis or mitochondrial content associated with LLC implantation, it was able to increase mitochondrial biogenesis signaling. Novelty This study provides novel insights on the effect of leucine supplementation on mitochondrial biogenesis and protein turnover in tumor-bearing mice. Leucine increased signaling for mitochondrial biogenesis in the skeletal muscle. Leucine supplementation decreased inflammatory signaling in skeletal muscle.

Extracts from Huangqi (Radix Astragali Mongoliciplus) and Ezhu (Rhizoma Curcumae Phaeocaulis) inhibit Lewis lung carcinoma cell growth in a xenograft mouse model by impairing mitogen-activated protein kinase signaling, vascular endothelial growth factor production, and angiogenesis.

OBJECTIVE: To study the anti-tumor effects of the extracts from Huangqi (Radix Astragali Mongolici) and Ezhu (Rhizoma Curcumae Phaeocaulis) on the growth of Lewis lung carcinoma (LLC) in a xenograft mouse model and to investigate the possible underlying mechanism. METHODS: LLC tumor-bearing C57BL/6 mice were treated with normal saline, cisplatin (2 mg/kg intraperitoneally every other day), or Huangqi (Radix Astragali Mongolici) and Ezhu (Rhizoma Curcumae Phaeocaulis) (1∶1, 2∶1, or 3∶1 ratio; 5 , 8 , or 11 g/kg crude drug intragastrically every day) for 15 d. Body weights and tumor volumes were measured every other day. Tumors were excised on day 15 and analyzed. Tumor microvessel density (MVD) was assessed by immunohistochemical staining of CD34; and expression of vascular endothelial cell growth factor (VEGF), the mitogen-activated protein kinases p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and Jun N-terminal kinase (JNK) and their phosphorylated forms were assessed by Western blotting. RESULTS: Treatment with cisplatin caused a significant loss of body weight compared with controls, whereas Huangqi (Radix Astragali Mongolici) and Ezhu (Rhizoma Curcumae Phaeocaulis) extract combinations had no effect. Extracts from Huangqi (Radix Astragali Mongolici) and Ezhu (Rhizoma Curcumae Phaeocaulis) significantly decreased tumor weight and tumor MVD compared with controls, and at the 3∶1 treatment group had similar efficacy to cisplatin in reducing MVD. Tumors from Huangqi (Radix Astragali Mongolici) and Ezhu (Rhizoma Curcumae Phaeocaulis) treatments also showed decreased p38 MAPK, p-p38 MAPK, ERK1/2, p-ERK1/2, JNK, and p-JNK expression compared with the control group (all P < 0.01). VEGF protein expression was significantly reduced in the 2∶1 and 3∶1 treatment groups compared with the control group (P < 0.01). CONCLUSION: Extracts from Huangqi (Radix Astragali Mongolici) and Ezhu (Rhizoma Curcumae Phaeocaulis) hindered LLC growth in the xenograft mouse model, possibly via inhibition of the MAPK signaling pathway, VEGF production, and tumor angiogenesis.

Intervention effect of Qi-Yu-San-Long Decoction on Lewis lung carcinoma in C57BL/6 mice: Insights from UPLC-QTOF/MS-based metabolic profiling.

Qi-Yu-San-Long Decoction (QYSLD) has been used to treat lung carcinoma for over twenty years in clinical practices, and its curative effect is considered credible. However, the therapeutic mechanism of this effect has not been thoroughly elucidated to date. In this study, a MTT dye reduction assay and DAPI staining were first used to evaluate the cell viability and apoptosis of A549 cells with and without QYSLD-treatment, respectively. The weight/volume of Lewis lung carcinoma (LLC) sarcoma was used to assess the therapeutic effect of QYSLD on LLC mice. Second, an UPLC-QTOF/MS-based untargeted metabolomics method was employed to identify and relatively quantify functional metabolites that were responsible for the intervention effect of QYSLD on LLC. As a result, the MTT dye reduction assay and DAPI staining demonstrated that QYSLD could inhibit the proliferation and induce the apoptosis of A549 cells. The weight/volume test of LLC sarcoma showed that QYSLD could restrain the development of LLC. Next, 21 potential biomarkers that could contribute to the curative mechanism of QYSLD on LLC were screened by the untargeted metabolomics method. The down-regulated metabolites induced by QYSLD included PC(16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(20:2(11Z,14Z)/16:0), PC(22:4(7Z,10Z,13Z,16Z)/14:0), PC(22:5(7Z,10Z,13Z,16Z,19Z)/14:0), arachidonic acid, gamma-glutamylisoleucine, cholesterol sulfate, CL (8:0/10:0/11:0/a-13:0) and CDP-DG (16:0/18:1(11Z)). The up-regulated metabolites were LysoPC(16:0), LysoPC(18:0), LysoPE(18:2(9Z,12Z)/0:0), LysoPE(22:0/0:0), LysoPE(22:1(13Z)/0:0), LysoPE(22:2(13Z,16Z)/0:0), triglylcarnitine, 1­arachidonoylglycerophosphoinositol, 1­palmitoylglycerophosphoinositol, 2­stearoylglycerophosphoinositol, sphingosine 1­phosphate(d19:1-P) and SM(d18:0/16:1(9Z)). The metabolic pathway analysis revealed that the potential biomarkers were primarily involved in glycerophospholipid metabolism, sphingolipid metabolism, steroid hormone biosynthesis, fatty acid degradation and arachidonic acid metabolism. This study demonstrated that QYSLD has a good antitumor effect and that a UPLC-QTOF/MS-based untargeted metabolomics method is a promising means of elucidating the intervention mechanism of traditional Chinese medicine formulas.

Brucea javanica oil emulsion alleviates cachexia induced by Lewis lung cancer cells in mice.

This study was conducted to evaluate the efficacy and possible mechanism of Brucea javanica oil emulsion (BJOE) on cachexia, by observing changes in related indexes in mice with cachexia and identifying the genes responsible based on gene chip analysis. In the BJOE treatment group, body weight loss, tumour growth and metastasis were found obviously inhibited, food and water intake had markedly increased, and survival time was significantly prolonged, as compared to the control group. Moreover, the BJOE witnessed improvement in body weight, prevention of tumour metastasis and overall increase in survival time, as compared to Indometacin (IND, the positive control medicine). It was also found that TNF-α and IL-6 in serum were significantly lower in both groups of BJOE and IND, than in the control group (p < .01). Based on the gene expression data, seven and six hub genes of BJOE and IND groups were found in the potential prognostic impacts networks, and three common genes comprising of Nmd3, Bcl2 and Nhp2l1 were screened. Thus, BJOE could reduce tumour growth and effectively alleviate cancer cachexia, due to inhibition of pro-inflammatory cytokines. Nmd3, Bcl2, Nhp2l1 may be important drug targets, establishing the role of BJOE in the treatment of lung cancer induced cachexia.

Effect of Acorus calamus L. Polysaccharide on CD274 and CD326 Expression by Lewis Lung Carcinoma Cells in Mice.

Tumor cells can maintain their growth via immunosuppression and escape from host antitumor immunity by controlling the PD-1/PD-L1 system. Expression of PD-L1 (CD274) is an inhibitory signal for T cells, while the increase in CD326 expression in the tumor tissue correlates with metastasis development. The experimental preparation on the basis of α(1,2)-L-rhamno-α(1,4)-D-galactopyranosyluronan from Acorus calamus L. produces an antitumor effect: it reduces tumor node size and the number and area of metastases after transplantation of Lewis lung carcinoma. Using flow cytometry, we demonstrated a decrease in the population of tumor cells expressing surface CD274 (PD-L1) and CD326 antigens after 20-day course of α(1,2)-L-rhamno-α(1,4)-D-galactopyranosyluronan.

[Effect of Grain-moxibustion on IL-6 and STAT 3 in Inflammatory Microenvironment of Lewis Lung Cancer Mice].

OBJECTIVE: To observe the effect of grain-moxibustion on serum interleukin-6(IL-6) and signal transducers and activators of transcription 3 (STAT 3) in inflammatory microenvironment of Lewis lung cancer mice, so as to explore its possible mechanism underlying relieving Lewis lung cancer. METHODS: C 57 BL/6 mice were randomly divided into blank control group, model group, and moxibustion group (n=10 in each group). Lewis lung cancer model was established by hypodermic injection of Lewis tumor cell suspension. Mice in the moxibustion group were treated by grain-moxibustion at bilateral "Housanli"(ST 36), 5 zhuang each acupoint, once a day for 10 days. The body weight, survival state score, volume of tumor, and survival rate were observed. Serum IL-6 level was detected by ELISA, and the expression of STAT 3 in tumor tissue was determined by Wes-tern blot. RESULTS: Compared with the blank control group, the body weight was significantly increased in the model group (P<0.05). The volume of tumor increased significantly in the model group, while it markedly decreased in the moxibustion group after 8 and 10 days treatment compared with the model group (P<0.05). The survival state of mice deteriorated companied with decreased survival rate in the model group, however, after 6, 8 or 10 days treatment, the survival state scores were increased in the moxibustion group than in the model group (P<0.05). Compared with the blank control group, both serum IL-6 level and STAT 3 expression in tumor tissue were increased significantly in the model group (P<0.001). Compared with the model group, both serum IL-6 level and STAT 3 expression in tumor tissue were decreased significantly in the moxibustion group (P<0.001). CONCLUSIONS: Grain-moxibustion has remarkable effect on Lewis lung cancer, by improving survival state, reducing death rate, and controlling growth rate of tumor, which might be associated with its function of inhibiting the activation of IL-6 and STAT 3 in inflammatory microenvironment.

Ginkgo biloba exocarp extracts induces autophagy in Lewis lung cancer cells involving AMPK / mTOR / p70S6k signaling pathway.

OBJECTIVES: Ginkgo biloba L. is called a living fossil plant, and could be used for the treatment of cancer thousands of years ago in China. The extracts prepared from the Ginkgo biloba exocarp (Ginkgo biloba exocarp extracts, GBEE) has a significant anti-cancer effect. Autophagy plays an important role in the occurrence and development of cancer as programmed cell death (PCD) type II. Thus it would be interesting to study the effects and mechanisms of GBEE inducing autophagy in Lewis lung cancer (LLC) cells. METHODS: MTT method was used to detect the inhibitory effect of GBEE on LLC cells. Monodansylcadaverine (MDC) staining method was applied to observe the formation of acidic vacuoles in cells. The ultrastructure of LLC cells was observed using transmission electron microscope (TEM) to confirm the formation of autophagosomes. Quantify reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA levels of Beclin1 and Atg5. Western Blot was used to detect the protein levels of Beclin1, Atg5, LC3I/II, p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6k and p70S6K in LLC cells. RESULTS: GBEE (5-160µg/mL) inhibited the proliferation of LLC cells in vitro with the half maximal inhibitory concentration (IC50) value of 161.26µg/mL. The formation and activation of acidic vacuoleswere increased by the action of GBEE (10, 20 and 40µg/mL) on LLC cells. The autophagosomes were also increased. Meanwhile, it up-regulated both the mRNA and protein levels of Beclin1 and Atg5. The ratio of LC3-I/LC3-II protein was down-regulated. In addition, the protein level of p-AMPK was increased, and the p-mTOR and p-p70S6K was decreased. But the AMPK, mTOR and p70S6K proteins were not significantly changed. CONCLUSIONS: The inhibitory effect of GBEE on LLC is associate with inducing autophagy in LLC cells, which may be closely relevant to the regulation of AMPK/mTOR/p70S6k signaling pathways.

Yangfei Kongliu Formula, a compound Chinese herbal medicine, combined with cisplatin, inhibits growth of lung cancer cells through transforming growth factor-ß1 signaling pathway.

OBJECTIVE: To investigate the tumor inhibition effect of Yangfei Kongliu Formula (YKF), a compound Chinese herbal medicine, combined with cisplatin (DDP) and its action mechanisms. METHODS: C57BL/6 mice with Lewis lung carcinoma were divided into six groups: control group (C), DDP group (2 mg/kg, DDP), low-dose YKF group (2.43 g/kg, L), high-dose YKF group (24.3 g/kg, H), low-dose YKF combined with DDP group (L + DDP) and high-dose YKF combined with DDP group (H + DDP). Transforming growth factor-ß1 (TGF-ß1), mothers against decapentaplegic homolog 3 (Smad3) and Smad7 levels were measured with quantitative real-time polymerase chain reaction (qPCR), Western blotting and immunohistochemistry. An enzyme-linked immunosorbent assay was used to analyze the expressions of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). RESULTS: YKF combined with DDP significantly inhibited the growth and metastasis of tumors relative to the control group, and YKF groups (P < 0.05). There was no significant difference between high-dose YKF group and low-dose YKF group (P > 0.05). We also found that the expression levels of TGF-ß1 and Smad3 were both significantly decreased by YKF relative to the control group (P < 0.05). Furthermore, after treatment with YKF combined with DDP, the expression levels of TGF-ß1 and Smad3 were decreased but the expression level of Smad7 was increased relative to the DDP group (P < 0.05). Compared to the DDP group, the combination of YKF and DDP enhanced the effect of tumor inhibition (P < 0.05), showing obvious synergy between YKF and DDP. Treatment with DDP or YKF decreased serum levels of IL-2 and TNF-α relative to the control group (P < 0.05). Furthermore, the expression levels of IL-2 and TNF-α were significantly decreased when treated with YKF in combination with DDP. Co-treatment with YKF and DDP significantly inhibited tumor growth, decreased the expressions of TGF-ß1, Smad3, IL-2 and TNF-α and increased the expression of Smad7; these differences were significant relative to both YKF groups and the control group (P < 0.05). CONCLUSION: YKF can inhibit tumor growth synergistically with DDP, mainly through the TGF-ß1 signaling pathway.

Carboxyamidotriazole Synergizes with Sorafenib to Combat Non-Small Cell Lung Cancer through Inhibition of NANOG and Aggravation of Apoptosis.

Lung cancer is currently the leading cause of cancer-related deaths worldwide. In this study, we investigated the combination of carboxyamidotriazole (CAI) and sorafenib in non-small cell lung cancer (NSCLC) in vitro and in vivo to test whether CAI enhances the antitumor effects of sorafenib and reduces its side effects. The combination index (CI) showed that coadministration of CAI and sorafenib synergistically inhibited the proliferation of NSCLC cells (Lewis lung carcinoma, A549, and NCI-H1975 cells). Cell death as a result of the combination treatment was attributed to apoptosis, which was accompanied by activation of caspase-3 and poly(ADP-ribose) polymerase. In addition, combination therapy induced the accumulation of mitochondrial-associated reactive oxygen species, as well as depolarization of mitochondrial and reduced NANOG (homeobox protein NANOG) mRNA and protein expression. Basic fibroblast growth factor, a stimulator of NANOG, was applied to identify the possible mechanism. The addition of basic fibroblast growth factor followed by combined treatment may stimulate NANOG expression and synchronously rescue the accumulation of reactive oxygen species. C57BL/6J mice bearing Lewis lung carcinoma were randomized to receive vehicle (polyethylene glycol 400), CAI (30 mg/kg), low-dose sorafenib (SFB-L; 10 mg/kg), high-dose sorafenib (SFB-H; 30 mg/kg), or a CAI and SFB-L combination. Tumor growth was significantly suppressed in the combination group, and the efficacy of combination treatment was equivalent to that of the SFB-H monotherapy group. Furthermore, the combination group had reduced side effects compared with the SFB-H group, as indicated by weight preservation in mice. Our study illustrates that CAI enhances the antitumor activity of sorafenib in NSCLC and provides a novel strategy for NSCLC treatment.

[Inhibitory effect and the mechanism of Astragalus polysaccharide combined with cisplatin on growth of inplanted Lewis lung carcinoma in mice].

Objective To observe the effect of Astragalus polysaccharides (APS) combined with cisplatin (DDP) on the expressions of cytochrome C (CytC) and high temperature required serine protease A2 (Omi/HtrA2) in the mice with Lewis lung carcinoma (LLC) transplantated tumors. Methods Ninty C57BL/6J mice were randomly divided into normal control group, model group, and (50, 100, 200) µg/mL APS groups, 6 mg/kg DDP group, 3 mg/kg DDP combined with (50, 100, 200) µg/mL APS groups. Each group included 10 mice. Except the mice in the normal group, the rest mice were inoculated subcutaneously with LLC cells (1×107 mL) at the right fore axillary fossa to establish tumor-bearing mouse models. In the second day of building models, the mice in the treatment group were given intraperitoneal injection of 0.3 mL of the drug. DDP was given once a week, and the other drugs once a day. The mice in the normal group and the model group were administrated the same amount of saline injection for continuous 20 days. All mice were killed at the 21st day. The pathological changes of tumor tissues were observed by HE staining. The expressions and location of CytC and Omi/HtrA2 proteins in the transplanted tumor tissues were detected by immunohistochemical staining and image analysis. Results The mass of tumor decreased in the mice of (100, 200) µg/mL APS group and 3 mg/kg DDP combined with (100, 200) µg/mL APS group. Compared with the model group, the necrosis of tumor tissues in 200 µg/mL APS combined with 3 mg/kg DDP group was the most obvious. The expressions of CytC and Omi/HtrA2 increased in the treatment groups, and the increase was the most remarkable in 200 µg/mL APS combined with 3 mg/kg DDP group. Conclusion APS and APS combined with DDP can restrain the growth of Lewis Lung cancer in C57BL/6J mice, which may be related to the increased expressions of CytC and Omi/HtrA2.