Predicting the mechanism of action of YQYYJD prescription in the treatment of non-small cell lung cancer using transcriptomics analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The efficacy of the herbal formula Yiqi Yangyin Jiedu (YQYYJD) in the treatment of advanced lung cancer has been reported in clinical trials. However, the key anti-lung cancer herbs and molecular mechanisms underlying its inhibition of lung cancer are not well-understood. AIM OF THE STUDY: To identify the key anti-lung cancer herbs in the YQYYJD formula and investigate their therapeutic effect and potential mechanism of action in non-small cell lung cancer (NSCLC) using transcriptomics and bioinformatics techniques. MATERIALS AND METHODS: A mouse Lewis lung carcinoma (LLC) subcutaneous inhibitory tumor model was established with 6 mice in each group. Mice were treated with the YQYYJD split formula: Yiqi Formula (YQ), Yangyin Formula (YY), and Ruanjian Jiedu Formula (RJJD) for 14 days. The tumor volume and mouse weight were recorded, and the status of tumor occurrence was further observed by taking photos. The tumor was stained with hematoxylin-eosin to observe its histopathological changes. Immunohistochemistry was used to detect the expression of the proliferation marker Ki67 and the apoptotic marker Caspase-3 in tumor tissues. Flow cytometry was used to detect the number of CD4+ and CD8+ T cells and cytokines interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in the spleen and tumor tissues. The differential genes of key drugs against tumors were obtained by transcriptome sequencing of tumors. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed on differential genes to obtain pathways and biological processes where targets were aggregated. TIMER2.0 and TISIDB databases were used to evaluate the impact of drugs on immune cell infiltration and immune-related genes. The binding activity of the key targets and compounds was verified by molecular docking. RESULTS: YQ, YY, and RJJD inhibited the growth of subcutaneous transplanted tumors in LLC mice to varying degrees and achieved antitumor effects by inhibiting the expression of tumor cell proliferation, apoptosis, and metastasis-related proteins. Among the three disassembled prescriptions, YQ better inhibited the growth of subcutaneous transplanted tumors in LLC mice, significantly promoted tumor necrosis, significantly increased the expression of Caspase-3 protein in tumor tissue, and significantly decreased the expression of Ki-67 (P < 0.05), thereby increasing the infiltration of CD8+ T cells. YQ significantly increased the expression of CD4+ and CD8+ T cells in tumor and splenic tissues of tumor-bearing mice and up-regulated the expression of IL-2 and IFN-γ. Transcriptome sequencing and bioinformatics results showed that after YQ intervention, differentially expressed genes were enriched in more than one tumor-related pathway and multiple immune regulation-related biological functions. There were 12 key immune-related target genes. CONCLUSION: YQ was the key disassembled prescription of YQYYJD, exerting significant antitumor effects and immune regulation effects on NSCLC. It may have relieved T cell exhaustion and regulated the immune microenvironment to exert antitumor effects by changing lung cancer-related targets, pathways, and biological processes.

Integrating network pharmacology and experimental models to investigate the mechanisms of dihydroartemisinin in preventing NSCLC progression via mTOR/HIF-1α signaling.

Advanced Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a poor prognosis. The anti-malaria compounds dihydroartemisinin (DHA) have shown to regulate multiple targets and signaling pathways in cancers, but a global view of its mechanism of action remains elusive. In present study, we integrated network pharmacology and in vitro and in vivo experimental models to investigate the mechanisms of DHA in preventing NSCLC proliferation. We first proved that DHA inhibits the growth of lung cancer via inducing cell apoptosis and cell cycle arrest, then we integrated information from publicly available databases to predict interactions between DHA and its potential targets in NSCLC, as well as the signaling pathways involved. In this way we identified 118 common targets of DHA and NSCLC, and further analyzed with the correlation between these targets by KEGG and GO analysis. Our data indicate that mTOR/HIF-1α signaling is one of potential critical pathways involved in DHA-induced tumor inhibition in NSCLC. Finally, the data from human and mouse lung cancer cell lines and in mouse Lewis lung cancer models showed that DHA does decrease the expression level of mTOR and HIF-1α which supported the potential roles of mTOR/HIF-1α Signaling in NSCLC and deserves further investigation.

Traditional Chinese Medicine Xihuang Wan Inhibited Lewis Lung Carcinoma in a Syngeneic Model, Equivalent to Cytotoxic Chemotherapy, by Altering Multiple Signaling Pathways.

BACKGROUND/AIM: Xihuang Wan (XHW), a traditional Chinese medicine (TCM), has been used in China for a variety of cancers including lung cancer. The present study evaluated the efficacy of XHW on a Lewis lung mouse model and explored the potential mechanism via transcriptomics. MATERIALS AND METHODS: The mice were randomized into 6 groups: 1) untreated control (n=10); 2) low-dose XHW; 3) medium-dose XHW; 4) high-dose XHW; 5) cisplatin; and 6) untreated blank (n=4). Lewis lung carcinoma (LLC) cells were injected subcutaneously except for the 4 mice in the blank group. The body weight and tumor length and width were measured every 3 days. RNA-sequencing was performed on tumors in the high-dose XHW group and the control group. RESULTS: XHW inhibited the growth of LLC in a syngeneic mouse model, without toxicity, with equivalent efficacy to cisplatin. RNA-sequencing demonstrated that many signaling pathways were involved in XHW-mediated inhibition of LLC, including tumor necrosis factor, estrogen, cyclic guanosine 3', 5'-monophosphate-protein kinase G, apelin and the peroxisome proliferator-activated receptor signaling pathways. CONCLUSION: XHW inhibited LLC carcinoma through different pathways and shows clinical promise for patients who cannot tolerate platinum-based drugs.

α-Cyperone inhibitory effects on tumor-derived DNA trigger microglia by STING pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (Cyperaceae) is a widespread herbal in China and widely used in Traditional Chinese Medicine for multiple effects such as anti-arthritic, anti-genotoxic, anti-mutagenic, anti-bacterial effects, and analgesic. α-Cyperone is an active compound in Cyperus rotundus and has analgesic effects, but the exact molecular mechanisms require further investigations. MATERIALS AND METHODS: Tumor-derived DNA isolated from Lewis cell lines was transfected into microglia, and analyzed for stimulator of interferon genes (STING) effects. The downstream protein, such as interferon regulatory factor 3 (IRF3) and p65 nuclear factor-κB (NF-κB) were treated with STING siRNA and 5,6-dimethyllxanthenone-4-acetic acid (DMXAA) in microglia. The α-Cyperone effect on microglia was also investigated. RESULTS: Tumor-derived DNA activate microglia by upregulation of STING and downstream proteins. STING siRNA was reduced to its downstream expression and neuroinflammation inhibition was caused by tumor-derived DNA. However, DMXAA reversed the STING siRNA effect and increased neuroinflammation. α-Cyperone takes inhibitory effects on tumor-derived DNA that trigger microglia by STING pathway. CONCLUSIONS: α-Cyperone inhibition by tumor-derived DNA activated microglial to neuroinflammation in STING signaling pathway.

Mechanisms of Fritillariae Thunbergii Flos in lung cancer treatment from a systems pharmacology perspective.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae Thunbergii Flos (FTF) included in the Chinese Pharmacopoeia (1977 Edition) is a Chinese medicinal herb traditionally used to treat bronchitis. In recent years, it has been applied in the treatment of lung cancer. However, the molecular mechanism remains largely unknown. METHODS: The screening of bioactive compounds, acquisition of drug targets, network construction, and experimental validation in vivo were combined to explored the mechanism of FTF in the treatment of lung carcinoma with regards to systems pharmacology. RESULTS: The network Lung Cancer Pathway consisted of 114 nodes (44 compounds and 70 potential targets) and 361 edges, as well as modules that included inflammatory response, angiogenesis, negative regulation of the apoptotic process, and positive regulation of cell proliferation and migration. It was examined by conducting experiments that involved the administration of ethanol-based extracts of FTF in Lewis lung carcinoma mice. The extracts exerted excellent anti-lung cancer effects in vivo by significantly inhibiting tumor proliferation, thereby extending the survival period of tumor-bearing mice. Moreover, FTF induced the downregulation of PIK3CG, Bcl-2, eNOS, VEGF, p-STAT3, and STAT3 genes in tumor-bearing mice. CONCLUSIONS: The findings of the present study verify the therapeutic effects and mechanism of FTF on lung cancer and provide a theoretical basis to support the comprehensive utilization of FTF resources.

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.

Systems pharmacology uncover the mechanism of anti-non-small cell lung cancer for Hedyotis diffusa Willd.

Non-small cell lung cancer (NSCLC) has become one of the most general malignancies in the world and has been shown to be the leading cause of cancer-related deaths. Traditional Chinese medicine (TCM) is considered to be a useful medicine for survival, and has been used in Asia for thousands of years. Hedyotis diffusa Willd (HDW) is an important folk herb that is used in clinical treatment of various cancers in various Chinese medicine prescriptions. However, its underlying mechanism of action remains unclear. Presently, we used an innovative system-pharmacology platform to systematically uncover the pharmacological mechanisms of HDW in the treatment of NSCLC from molecules, targets, and pathway levels. The results show that HDW treatment of NSCLC may activate immunity, achieve anti-inflammatory, anti-proliferative and anti-migration therapeutic effects by regulating multiple pathways. This research provides a new idea for understanding the mechanism of TCM and promotes to develop potential drugs from HDW in modern medicine.

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.

Viola Yedoensis Suppresses Cell Invasion by Targeting the Protease and NF-κB Activities in A549 and Lewis Lung Carcinoma Cells.

Cancer metastasis is a vital trait in malignancies with complicated early diagnosis and therapeutic management. Therefore, the development of new remedies and the utilization of natural medicines that target metastasis are of great interest and have been studied extensively. Chinese medicinal herbs have various anti-carcinogenesis properties; however, the in vitro effect and mechanism of Viola yedoensis on cancer cell metastasis remains poorly understood. V. yedoensis extracts (VYE) can suppress the invasion of a highly metastatic human lung cancer cell line, A549 cells. According to gelatin zymography and casein zymography assays, VYE inhibited the activities of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (u-PA). The results of reverse transcription-polymerase chain reaction and Western blotting revealed that VYE can alter the expression of proteinase inhibitor. VYE also suppressed the DNA binding activity of nuclear factor-kappa B. We concluded that VYE may inhibit tumor invasion by suppressing the activities of MMP and u-PA in lung cancer cells.

Carnosic acid enhances the anti-lung cancer effect of cisplatin by inhibiting myeloid-derived suppressor cells.

Cisplatin and other platinum-based drugs are used frequently for treatment of lung cancer. However, their clinical performance are usually limited by drug resistance or toxic effects. Carnosic acid, a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), has been reported to have several pharmacological and biological activities. In the present study, the combination effect of cisplatin plus carnosic acid on mouse LLC (Lewis lung cancer) xenografts and possible underlying mechanism of action were examined. LLC-bearing mice were treated with intraperitoneal injection with cisplatin, oral gavage with carnosic acid, or combination with cisplatin and carnosic acid, respectively. Combination of carnosic acid and cisplatin yielded significantly better anti-growth and pro-apoptotic effects on LLC xenografts than drugs alone. Mechanistic study showed that carnosic acid treatment boosted the function of CD8+ T cells as evidenced by higher IFN-γ secretion and higher expression of FasL, perforin as well as granzyme B. In the meantime, the proportion of MDSC (myeloid-derived suppressor cells) in tumor tissues were reduced by carnosic acid treatment and the mRNA levels of iNOS2, Arg-1, and MMP9, which are the functional markers for MDSC, were reduced. In conclusion, our study proved that the functional suppression of MDSC by carnosic acid promoted the lethality of CD8+ T cells, which contributed to the enhancement of anti-lung cancer effect of cisplatin.

The Chinese medicine JC-001 enhances the chemosensitivity of Lewis lung tumors to cisplatin by modulating the immune response.

BACKGROUND: JC-001 is a Chinese medicine that can modulate the immunity in Hepa 1-6 tumor-bearing mice, and we questioned whether JC-001 can serve as efficient adjuvant chemotherapy. We aimed to identify a novel approach for enhancing cis-diamminedichloroplatinum (II) (CDDP)-based chemotherapy by immunomodulation. METHODS: The anti-tumor activity in vitro was determined based on foci formation and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A LLC1 tumor xenograft model was used to analyze the activity of tumor rejection in vivo. The tumors were analyzed through hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) staining and cytokine arrays. RESULTS: JC-001 suppressed foci formation and reduced the viability of Lewis lung carcinoma (LLC1) cells in vitro. JC-001 suppressed LLC1 tumor growth in immunodeficient BALB/c nude mice and in immunocompetent C57BL/6 mice to an even greater extent. Furthermore, JC-001 up-regulated interferon-γ expression in the tumor microenvironment, enhanced the Th1 response in tumor-bearing mice, and increased the chemosensitivity of LLC1 tumors to CDDP chemotherapy. The results of our study suggest that JC-001 is associated with low cytotoxicity and can significantly suppress tumor growth by enhancing the Th1 response. CONCLUSION: JC-001 is a Chinese medicine with potential clinical applications in CDDP-based chemotherapeutic regimens.

[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.

Pharmacokinetic and anti-cancer properties of high dose ascorbate in solid tumours of ascorbate-dependent mice.

Despite recent evidence for an anti-tumour role for high-dose ascorbate, potential mechanisms of action are still unclear. At mM concentrations that are achieved with high-dose intravenous administration, autoxidation of ascorbate can generate cytotoxic levels of H2O2. Ascorbate is also a required co-factor for the hydroxylases that suppress the transcription factor hypoxia-inducible factor (HIF-1). HIF-1 supports an aggressive tumour phenotype and is associated with poor prognosis, and previous studies have shown that optimizing intracellular ascorbate levels down-regulates HIF-1 activation. In this study we have simultaneously measured ascorbate concentrations and the HIF-1 pathway activity in tumour tissue following high dose ascorbate administration, and have studied tumour growth and physiology. Gulo-/- mice, a model of the human ascorbate dependency condition, were implanted with syngeneic Lewis lung tumours, 1g/kg ascorbate was administered into the peritoneum, and ascorbate concentrations were monitored in plasma, liver and tumours. Ascorbate levels peaked within 30min, and although plasma and liver ascorbate returned to baseline within 16h, tumour levels remained elevated for 48h, possibly reflecting increased stability in the hypoxic tumour environment. The expression of HIF-1 and its target proteins was down-regulated with tumour ascorbate uptake. Elevated tumour ascorbate levels could be maintained with daily administration, and HIF-1 and vascular endothelial growth factor protein levels were reduced in these conditions. Increased tumour ascorbate was associated with slowed tumour growth, reduced tumour microvessel density and decreased hypoxia. Alternate day administration of ascorbate resulted in lower tumour levels and did not consistently decrease HIF-1 pathway activity. Levels of sodium-dependent vitamin C transporters 1 and 2 were not clearly associated with ascorbate accumulation by murine tumour cells in vitro or in vivo. Our results support the suppression of the hypoxic response by ascorbate as a plausible mechanism of action of its anti-tumour activity, and this may be useful in a clinical setting.

[Qiyusanlong decoction inhibits the level of PD-1/PD-L1 in mice bearing Lewis lung carcinoma].

Objective To investigate the anti-tumor effect of Qiyusanlong decoction (QYSL) in the mice with Lewis lung cancer (LLC) and its effect on the expression of programmed death 1 and programmed death ligand 1 (PD-1/PD-L1). Methods The model of lung cancer subcutaneous allograft was established using LLC cells. The model mice were randomly divided into six groups: a model group, a chemotherapy group, three QYSL groups of high, middle and low doses, and a combined group, each group containing 8 mice. On the 11th day, the low-, middle- and high-dose QYSL groups were respectively given intragastric administration of 20.12, 40.24, 80.48 g/(kg.d) QYSL; the chemotherapy group were intraperitoneally injected with 0.4 mL cisplatin; the combined group were administrated with cisplatin and high-dose QYSL; the model group were administrated with the same amount of normal saline, once a day for 10 days. Tumor volume was examined and tumor growth curve was drawn. Tumors were weighed and tumor inhibition rates were calculated. The expressions of PD-1, PD-L1 mRNA and protein in spleens and tumor tissues of mice were detected by real-time quantitative PCR and Western blotting, respectively. Results Compared with the model group, the high-dose QYSL group could inhibit tumor growth with tumor inhibition rate being 23.86%; the tumor inhibition rate between the combined group and the chemotherapy group was equal. The high-dose QYSL could significantly decrease the expression of PD-1 mRNA and protein in spleen and inhibited the expression of PD-L1 mRNA and protein in tumor. The levels of PD-1 and PD-L1 protein in the combined group were obviously lower than those in the chemotherapy group, but the interaction effect of the other indicators had no statistical significance. Conclusion QYSL can moderately inhibit the growth of the transplanted tumor by decreasing PD-1/PD-L1 level.

[Effect of Epimedii Herba alcohol extract on inhibition of lung tumor growth and immunomodulatory].

To observe the effect of Epimedii Herba alcohol extract (HE) on tumor growth of lung cancer by establishing the model of Lewis tumor-bearing mice, ELISA method was used to detect the levels of TNF-α, IL-10, IL-17, IL-2 in serum. Ki67 and P53 protein expression was detected in lung cancer tissues by using Western blot assay method and immunohistochemical assay method. The experimental results showed that HE has certain inhibitory effect on Lewis lung cancer tumor growth, and it can reduce the levels of TNF-α, IL-10 and IL-17 in serum, improve the level of IL-2,significantly decrease the expression of Ki67, and significantly increase P53 expression. HE has obvious inhibitory effect against lung cancer, and has the ability to improve immune regulating effect. This study reveals the anti-lung cancer effect of HE may be related to its ability of improving immunity, thus provides the basis for further research on anti-lung cancer effect of HE.

The effect of perioperative analgesic drugs omnopon and dexketoprofen on the functional activity of immune cells in murine model of tumor surgery.

We aimed to investigate the effect of perioperative analgesia with nonselective cyclooxygenase-2 inhibitor dexketoprofen and opioid drug omnopon on the functional activity of immune cells in tumor excision murine model. Lewis lung carcinoma cells were transplanted into hind paw of C57/black mice. On the 23th day tumor was removed. Analgesic drugs were injected 30 min before and once a day for 3 days after the surgery. Biological material was obtained a day before, 1 day and 3 days after the tumor removal. IFN-γ, IL-4, IL-10 and TGF-ß mRNA levels in splenic cells were assessed by quantitative real-time RT-PCR. Cytotoxic activity of splenocytes was estimated by flow cytometry. We found that in splenocytes of mice received opioid analgesia IL-10 mRNA level was increased 2.3 times on day one after the surgery compared to preoperative level (P < 0.05), while in dexketoprofen group this parameter did not change. IFN-γ gene expression level on day 3 after tumor removal was 40% higher in splenocytes of dexketoprofen treated mice as compared with omnopon treated animals (P < 0.05). Cytotoxic activity of splenocytes on day 3 postsurgery was (62.2 ± 2.4)% in dexketoprofen against (50.2 ± 3.3)% in omnopon group. In conclusion, perioperative analgesia with cyclooxygenase inhibitor dexketoprofen in contrast to opioid analgesia with omnopon preserves higher functional activity of murine immune cells in the experimental model of tumor surgery.

[Tanreqing injection promotes the immune function of mice with Lewis lung carcinoma after chemotherapy].

OBJECTIVE: To investigate the effect of Tanreqing injection on immune function of mice with Lewis lung carcinoma treated by chemotherapy and discuss the immunoregulatory function of this herb. METHODS: All mouse models with Lewis lung carcinoma were randomly divided to four groups: model control group, Tanreqing injection group, chemotherapy group and chemotherapy combined with Tanreqing injection group (8 rats in each group). Other 8 normal mice served as a normal control group. After treatment, peripheral blood was collected from the mice of all groups before they were sacrificed. Tumor tissue, femur, thymus and spleen were obtained to perform the following experiments. Tumor mass and volume were first observed. The apoptosis levels of tumor cells and the ratios of CD3⁺T lymphocyte and CD3⁻NK1.1⁺ cells in the infiltrating lymphocytes in the tumor tissues were analyzed by flow cytometry. Thymus indexes (TI) were counted, and the structure of thymus was observed using HE staining. Cytotoxicity of spleen cytotoxic T cells (CTL) were investigated by MTT assay. The number of lymphocytes in the periphery blood and the nucleated cells in femur were also detected, and the expression levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) mRNA in the tumor tissues were studied by reverse transcription PCR. RESULTS: After chemotherapy, TI, the number of the nucleated cells in femur and lymphocytes in peripheral blood of chemotherapy combined with Tanreqing injection group were obviously higher than those in the chemotherapy group. The ratios of CD3⁺T lymphocytes and CD3⁻NK1.1⁺ cells in the infiltrating lymphocytes in the tumor tissues of chemotherapy combined with Tanreqing injection group were also significantly higher than those in the chemotherapy group. Besides, compared with chemotherapy group, cytotoxicity of CTL in chemotherapy combined with Tanreqing injection group was improved notably. Meanwhile, the expression levels of IFN-γ and TNF-α mRNA in tumor tissues of chemotherapy combined with Tanreqing injection group were dramatically higher than those in chemotherapy group. CONCLUSION: Tanreqing injection has certain protective effects on damaged immune function of body with lung carcinoma induced by chemotherapy, and also improves the anti-tumor immune function of the body.

[Study on anti-tumor and anti-metastasis mechanism of alcohol extracts from pharbitidis semen against Lewis lung cancer].

OBJECTIVE: To observe the effect of alcohol extracts from Pharbitidis Semen on the proliferation and metastasis of Lewis lung cancer, and study its anti-tumor mechanism. METHOD: In vitro, MTT assay and scratch assay were adopted to detect the effect of alcohol extracts from Pharbitidis Semen on the proliferation and metastasis of Lewis lung cancer cells. The cell autophagy was detected by the acridine orange staining. The gap-junction intercellular communication (GJIC) was investigated by the fluorescent yellow transfer. The expression of aquaporin 1 (AQP1) was analyzed by the Western blotting. In vivo, the subcutaneous implant model and the experimental pulmonary metastasis model of Lewis lung cancer in mice were established to evaluate the anti-tumor and anti-metastasis effects of alcohol extract from Pharbitidis Semen. The serum carcinoembryonic antigen (CEA) and beta2 microglobulin (beta2-MG) of mice bearing Lewis lung cancer were detected by the electrochemiluminesence immunoassay. The expressions of lung AQP1 and Connexin 43 (Cx43) were examined by the immunohistochemical method. RESULT: In vitro, alcohol extracts from Pharbitidis Semen inhibited the cell proliferation in a dose-dependent matter, significantly prevented the cell migration, down-regulated AQP1 proteins of cells, promoted GJIC, and decreased the serum-free autophagy of tumor cells. In vivo, compared with untreated model mice, alcohol extracts from Pharbitidis Semen inhibited the tumor growth in a dose-dependent matter, prevented the tumor metastasis and prolonged the life span of mice bearing Lewis lung cancer, while decreasing serum CEA and beta2-MG of mice bearing Lewis lung cancer, enhancing the immumohistochemical staining intensity of Cx43 and weakening aquaporins AQP1 positive intensity. CONCLUSION: Alcohol extracts from Pharbitidis Semen could prevent the proliferation and metastasis in Lewis lung cancer cells. Its mechanism may be related to the promotion of GJIC and the down-regulation of AQP1.

Asian ginseng extract inhibits in vitro and in vivo growth of mouse lewis lung carcinoma via modulation of ERK-p53 and NF-κB signaling.

Asian ginseng (AG) is the most commonly used medicinal herb in Asian countries. It is often prescribed for cancer patients as a complementary remedy. However, whether AG in fact benefits cancer patients remains unknown because some studies reported that AG facilitates tumor growth, which contradicts its usage as a dietary remedy to cancer patients. In addition, most of research works on ginseng for anti-cancer were using single ginsenoside rather than whole root extracts used in clinics. Thus, intensive studies using the type of ginseng as its clinical form are necessary to validate its benefits to cancer patients. In this study, anti-tumor potency and underlying molecular mechanisms of the ethanol extract of AG (EAG) were examined in mice with Lewis lung carcinoma (LLC-1). We showed that EAG significantly suppressed tumor growth in LLC-1-bearing mice with concomitant down-regulation of PCNA proliferative marker, and it exhibited specific cytotoxicity to cancer cells. EAG also induced MAPK and p53 signaling in LLC-1 cells, which suppressed cyclin B-cdc2 complex and in turn induced G2-M arrest and apoptosis. Although EAG could activate NF-κB signaling, the proteasome inhibitor of MG-132 could effectively prevent NF-κB targeted gene expression induced by EAG and then sensitize LLC-1 cells to induce EAG-mediated apoptosis. Collectively, EAG in a relatively high dose significantly suppressed tumor growth in LLC-1-bearing mice, indicating that AG may benefit lung cancer patients as a dietary supplement. This is the first report demonstrating possible combination of EAG with proteasome inhibitors could be a novel strategy in anti-cancer treatment.

Efficiency of lipofection combined with hyperthermia in Lewis lung carcinoma cells and a rodent pleural dissemination model of lung carcinoma.

We have previously reported that hyperthermia at 41 degrees C enhanced lipofection-mediated gene transduction into cultured cells. In this study, we adapted hyperthermia technique to novel cationic liposome (Lipofectamine 2000) mediated gene transfection into Lewis lung carcinoma cells in vitro and in vivo. In vitro, transfection efficiencies were 38.9+/-3.3% by lipofection alone and 52.1+/-2.6% by lipofection with hyperthermia for 30 min, and 62.5+/-5.5% and 81.4+/-3.2% for 1 h, respectively. Hyperthermia significantly enhanced gene transfection efficiency 1.2-1.4 times more than that with lipofection only. We also evaluated the effect of hyperthermia with a pleural dissemination model of lung carcinoma of mice. We developed a model which was well-tolerated with hyperthermia with lipofection by the mice. In spite of repeated treatments, transfection efficiencies were very low and we could not show the augmentation of gene transfection by hyperthermia. Though Lipofectamine 2000 showed strong gene transduction effect and hyperthermia augmented its effect in vitro, further evaluation is needed to adapt both techniques in vivo.