This study aims to investigate the anti-tumor effect and mechanism of Guiqi Yiyuan Ointment combined with cisplatin on Lewis lung carcinoma-bearing mice via the protein kinase RNA-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2α(eIF2α)/activated transcription factor 4(ATF4)/C/EBP homologous protein(CHOP) signaling pathway. Sixty SPF-grade male C57BL/6 mice were selected and assigned into a blank group and a modeling group by the random number table method. After modeling of the Lewis lung carcinoma, the mice in the modeling group were randomized into model, cisplatin(5 mg·kg~(-1), once a week), and low-, medium-, and high-dose(1.7, 3.5, and 7.05 g·kg~(-1), respectively, once a day) Guiqi Yiyuan Ointment+cisplatin(5 mg·kg~(-1)) groups(n=10). After 14 days of continuous intervention, the spleen, thymus, and tumor samples of the mice were collected, weighed, and recorded, and the spleen index, thymus index, and tumor suppression rate were calculated. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes in the tumor tissue. The morphological changes of the endoplasmic reticulum of tumor cells were observed by transmission electron microscopy. The positive expression of phosphorylated eIF2α(p-eIF2α) and ATF4 in the tumor tissue was detected by immunofluorescence. Western blot was employed to determine the protein levels of phosphorylated PERK(p-PERK), p-eIF2α, ATF4, CHOP, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cyclin-dependent kinase inhibitor 1A(p21), and cyclinD1 in the tumor tissue. Real-time fluorescent quantitative PCR was employed to determine the mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, p21, and cyclinD1 in the tumor tissue. Compared with the blank group, the model group showed decreases in spleen index and thymus index(P<0.05). Compared with the model group, the cisplatin group showed decreases in spleen index and thymus index(P<0.05), and the medium-and high-dose Guiqi Yiyuan Ointment+cisplatin groups presented increases in spleen index and thymus index(P<0.05). In addition, the treatment groups all showed decreased tumor mass(P<0.05), increased tumor cell lysis and nuclear rupture, widened gap between rough endoplasmic reticulum, enhanced average fluorescence intensity of p-eIF2α and ATF4(P<0.05), up-regulated protein levels of p-PERK/PERK, p-eIF2α/eIF2α, ATF4, CHOP, Bax, and p21(P<0.05), down-regulated protein and mRNA levels of Bcl-2 and cyclinD1(P<0.05), and up-regulated mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, and p21(P<0.05). Compared with the cisplatin group, the combination groups showed increases in spleen index and thymus index(P<0.05) as well as mean optical density(P<0.05), and the high-dose Guiqi Yiyuan Ointment+cisplatin group showed decreased tumor mass(P<0.05). In addition, the medium-and high-dose Guiqi Yiyuan Ointment+cisplatin groups showcased enhanced average fluorescence intensity of p-eIF2α and ATF4(P<0.05), up-regulated protein levels of p-PERK/PERK, p-eIF2α/eIF2α, ATF4, CHOP, Bax, and p21(P<0.05), down-regulated protein and mRNA levels of Bcl-2 and cyclinD1(P<0.05), and up-regulated mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, and p21(P<0.05). In conclusion, Guiqi Yiyuan Ointment combined with cisplatin can effectively inhibit the growth of Lewis lung carcinoma in mice by regulating the expression of proteins related to the PERK/eIF2α/ATF4/CHOP signaling pathway and promoting cell cycle arrest and apoptosis.
Animals ; Cisplatin/administration & dosage* ; Activating Transcription Factor 4/genetics* ; Eukaryotic Initiation Factor-2/genetics* ; eIF-2 Kinase/genetics* ; Carcinoma, Lewis Lung/pathology* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Mice ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Transcription Factor CHOP/genetics* ; Ointments/administration & dosage* ; Humans ; Cell Proliferation/drug effects* ; Antineoplastic Agents/administration & dosage*

Objective This study aimed to establish a pre-metastatic niche mouse model utilizing luciferase-labeled Lewis (Luc-Lewis) lung cancer cells and to assess the efficacy of this model employing both qualitative and quantitative methods. Methods C57BL/6 mice were categorized into two groups: a normal control group and a model group, each containing 15 individual mice. The pre-metastatic niche model was established via tail vein injection of Luc-Lewis lung cancer cells. Body mass were measured daily for all groups. Tumor fluorescence signals within the mice were detected using a high-throughput enzyme marker instrument. Lung tissue specimens were harvested to evaluate metastatic progression. HE staining was used to assess histopathological changes. Real-time quantitative PCR and Western blot analysis were used to detect the mRNA and protein expression of lysyl oxidase (LOX), matrix metalloproteinase 9 (MMP9), versican (VCAN), and fibronectin (FN), which are the specific markers for the formation of the microenvironment of lung tissues before metastasis. Results Significant declines in body mass and observable lethargy were noted in the model group when compared to the control group. Distinct fluorescence signals were observed in the lung tissue of the model group, demonstrating a positive correlation with the duration of model establishment. By day 14, elevated mRNA and protein expression levels of LOX, MMP9, VCAN, and FN were significantly evident. In addition, histopathological evaluations revealed augmented interstitial thickness, alveolar atrophy and significant inflammatory cell infiltration within the lung tissues of the model group. By the 21st day, metastatic lesions manifested in the lung tissues of the model group, suggesting an approximate pre-metastatic niche maturation timeline of 14 days. Conclusion A pre-metastatic niche mouse model for Lewis lung cancer is successfully established.
Mice ; Animals ; Lung Neoplasms/pathology* ; Matrix Metalloproteinase 9 ; Mice, Inbred C57BL ; Carcinoma, Lewis Lung ; Disease Models, Animal ; RNA, Messenger ; Tumor Microenvironment

The aim of this paper was to observe the effect of Feiliuping Gao and its combination with different types of drugs intervention on the expression of PI3K/AKT/NF-κB in lung metastatic microenvironment, and to reveal the advantage of Chinese medicine intervention time on the key molecule in lung metastatic microenvironment. The mouse model of Lewis lung carcinoma was established, and lung tissues were collected at 14 days, 21 days and 28 days after the intervention of Feiliuping Gao, and the expressions of PI3K, AKT and NF-κB were detected by immunohistochemistry and Western blot. At 14 days, there was no significant difference in PI3K expression between each group and the control group. The expression of AKT protein was significantly inhibited in the celecoxib (CLB) group, the Feiliuping Gao (FLP) combination with cyclophosphamide (FLP+CTX) group, and the Feiliuping Gao combination with celecoxib (FLP+CLB) group (<0.05). The inhibition of AKT protein expression in FLP+CLB group was superior. The FLP+CLB group can inhibit the expression of NF-κB protein (<0.05). At 21 days, compared with the control group, the expression of PI3K was inhibited in FLP group and the FLP+CTX group (<0.05), while the expression of PI3K was best inhibited in the FLP+CLB group (<0.001). Only the FLP+CLB group could significantly inhibit the expression of AKT protein (<0.01). The FLP+CTX group had the best effect in inhibiting the expression of NF-κB protein (<0.001). At 28 days, compared with the control group, the expression of PI3K and AKT was inhibited in the FLP+CLB group (<0.001). Feiliuping ointment combination with celecoxib has an advantage in regulating the expression of PI3K/AKT/NF-κB molecules in lung metastatic microenvironment.
Animals ; Carcinoma, Lewis Lung ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Lung ; Mice ; NF-kappa B ; metabolism ; Neoplasm Metastasis ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

Coptidis Rhizoma and Aconiti Kusnezoffii Radix represent hot Chinese medicine and cold Chinese medicine respectively. The purpose of this study is to observe the differentiation effect of Coptidis Rhizoma and Aconiti Kusnezoffii Radix on lewis lung cancer and compare effect of hot Chinese medicine and cold Chinese medicine on tumor progression. In this study, the rat serum containing Coptidis Rhizoma or Aconiti Kusnezoffii Radix was prepared to treat lewis lung cancer cells in vitro, and effects of the serum containing Coptidis Rhizoma or Aconiti Kusnezoffii Radix on cell differentiation, proliferation, adhesion, succinic dehydrogenase (SDH) activity and gap-junction intercellular communication (GJIC) were investigated. In vivo, the subcutaneous implant model and pulmonary metastasis model of lewis lung cancer were established. Tumor bearing mice were taken water decoction of coptis chinensis or aconite by intragastric administration bid for four weeks, and the influences of coptis chinensis and aconite on tumor progression were evaluated by body temperature, blood oxygen saturation, red cell ATPase, blood rheology, intratumor hypoxia, capillary permeability and GJIC. The results showed that the serum containing aconite could induce cell differentiation, inhibit cell proliferation and migration, promote SDH activity and GJIC in lewis lung cancer cells. The serum containing Coptidis Rhizoma increased cell adhesion and decreased SDH activity and GJIC without cell differentiation although it also suppressed cell proliferation. Aconiti Kusnezoffii Radix water decoction could keep body temperature, blood oxygen saturation, red cell ATPase and blood rheology, and improve intratumor hypoxia, capillary permeability and GJIC in tumor bearing mice, which led to slower tumor growth and less metastasis. Coptidis Rhizoma water decoction decreased body temperature, blood oxygen saturation, red cell ATPase, blood rheology and GJIC, and promoted intratumor hypoxia and capillary permeability, which resulted to more tumor metastasis although it also prevented tumor growth. These results suggested that the hot Chinese medicine could induce tumor cell differentiation and prevent tumor poison invagination, which is better for tumor treatment than cold Chinese medicine.
Aconitum ; chemistry ; Animals ; Antineoplastic Agents ; pharmacology ; Carcinoma, Lewis Lung ; pathology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Curcuma ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Mice ; Neoplasm Metastasis ; Rats ; Xenograft Model Antitumor Assays

OBJECTIVETo observe the effect of alcohol extracts from Pharbitidis Semen on the proliferation and metastasis of Lewis lung cancer, and study its anti-tumor mechanism.

METHODIn 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.

RESULTIn 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.

CONCLUSIONAlcohol 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.

Animals ; Antineoplastic Agents ; administration & dosage ; Aquaporin 1 ; genetics ; metabolism ; Carcinoma, Lewis Lung ; drug therapy ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Connexin 43 ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Ipomoea ; chemistry ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis ; Seeds ; chemistry

Tumors are believed to consist of a heterogeneous population of tumor cells originating from rare cancer stem cells (CSCs). However, emerging evidence suggests that tumor may also originate from non-CSCs. To support this viewpoint, we are here to present definitive evidence indicating that the number of tumorigenic tumor cells is greater than that of CSCs in tumor, and tumor can also derive from non-CSCs. To achieve this, an idealized mathematical model was employed in the present study and theoretical calculation revealed that non-CSCs could initiate the occurrence of tumor if their proliferation potential was adequate. Further, experimental studies demonstrated that 17.7%, 38.6% and 5.2% of tumor cells in murine B16 solid melanoma, H22 hepatoma and Lewis lung carcinoma, respectively, were potentially tumorigenic. Thus, based on the aforementioned findings, we propose that the scarce CSCs, if exist, are not the sole source of a tumor.
Algorithms ; Animals ; Carcinogenesis ; pathology ; Carcinoma, Lewis Lung ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Liver Neoplasms, Experimental ; pathology ; Melanoma, Experimental ; pathology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Biological ; Neoplasms, Experimental ; pathology ; Neoplastic Stem Cells ; pathology ; Time Factors ; Tumor Stem Cell Assay ; methods

OBJECTIVETo explore the radiosensitization effect of black garlic extract (BGE) on lung cancer cell line Lewis cells.

METHODSThe inhibition rate of lung cancer cells after BGE action was detected by MTT. Effect of BGE combined radiotherapy on the colony formation rate was observed by cloning formation assay. Changes of the cell morphology were observed by Hoechst staining. Changes of the cell cycle were detected by flow cytometry. Real time PCR was used to detect mRNA expressions of bcl-2 and bax.

RESULTSBGE could have significant inhibitory action on the growth of lung cancer Lewis cells. The combination of BGE and radiotherapy (by 60Co gamma) significantly induced Lewis cells' apoptosis in G2/M stage, obviously decreased the expression of bcl-2, and up-regulated the expression of bax.

CONCLUSIONSBGE could sensitize the lung cancer Lewis cells to ionizing irradiation. This effect might be probably caused by changing the cell cycles and affecting expressions of bax and bcl-2.

Animals ; Apoptosis ; Carcinoma, Lewis Lung ; metabolism ; pathology ; Cell Line, Tumor ; drug effects ; radiation effects ; Garlic ; Mice ; Plant Extracts ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Radiation-Sensitizing Agents ; pharmacology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the mechanism of polypeptide extract from scorpion venom (PESV) on promoting anti-tumor effects of cyclophosphamide (CTX).

METHODSThe Lewis lung tumor model was established by subcutaneously implanting Lewis lung cells into C57BL/6 mice. The tumor-bearing mice were randomly divided into 4 groups, i. e., the model group, the cyclophosphamide (CTX) group, the polypeptide extract from scorpion venom (PESV) group, and the combination group (CTX + PESV), 10 mice in each group. The tumor growth curve was recorded. Changes of vascular endothelial growth factor-A (VEGF-A) and transforming growth factor-beta1 (TGF-beta1) expressions in the tumor microenvironment were detected using reverse transcription PCR and immunohistochemical assay. Changes of dendritic cells (DCs) phenotype CD80 and CD86 expressions in the tumor tissue were detected using immunofluorescence chemical assay.

RESULTSAfter 21 successive days of treatment, the growth of Lewis lung cancer transplantation tumor in the combination group was obviously inhibited (P<0.05). Compared with the model group,the expressions of CD80 and CD86 in the PESV group was somewhat enhanced, while those in the CTX group was somewhat lowered. Compared with the CTX group, the fluorescent signal strength and expressions in the combination group somewhat increased. Compared with the model group, the expressions of TGF-beta1 and VEGF-A mRNA decreased in the PESV group and the CTX group (both P<0.05). Compared with the PESV group and the CTX group, the expressions of TGF-beta1 and VEGF-A in the combination group both decreased (both P<0.05).

CONCLUSIONPESV could inhibit the expressions of VEGF and TGF-beta1, promote the maturation of DCs, recover its antigen uptake presentation function, and reverse the immune injury to the body by CTX, thus playing a role in inducing the tumor cell apoptosis.

Animals ; B7-1 Antigen ; B7-2 Antigen ; Carcinoma, Lewis Lung ; immunology ; metabolism ; pathology ; Cyclophosphamide ; pharmacology ; Dendritic Cells ; immunology ; Lung Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Peptides ; pharmacology ; Scorpion Venoms ; pharmacology ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo reveal the different molecular mechanisms between Chinese drugs for activating blood (CDAB) and Chinese drugs for nourishing qi and activating blood (CDNQAB) in the metastasis process of Lewis lung carcinoma, thus providing experimental reliance for Chinese drugs to reverse immune escape.

METHODSThe inhibition rate of lung metastasis was observed in each group. The dynamic percentage and ratio changes of Th17 and Treg cells in spleen CD4+ T lymphocytes were detected using flow cytometry. The dynamic levels of IL-17, IL-23, and gamma interferon (IFN-gamma) in the culture supernatant of CD4+ T lymphocytes were detected by ELISA. The dynamic mRNA expressions of Foxp3 and RORgammat in CD4+ T lymphocytes were detected by RT-PCR.

RESULTSCDNQAB (sapanwood +astragalus) showed better lung metastasis inhibiting rate than CDAB (sapanwood alone) (P<0.05), similar to the effects of cyclophosphamide (P>0.05). Except the CDNQAB group, spleen Th17 and Treg cells showed a rising tendency in mice of each tumor-bearing group. The effectors of Th17 and Treg cells (IL-17, IL-23, and IFN-gamma) and key transcription molecules of Th17 and Treg cells (RORgammat and Foxp3) showed dynamic changes corresponding to Th17 and Treg cells.

CONCLUSIONSThe immune inflammatory reactions of CDNQAB (sapanwood +astragalus) were superior to those of CDAB (sapanwood alone) and of cyclophosphamide during the process of inhibiting tumor immunotolerance and of the formation of tumor. All drugs showed certain inhibition on the mechanisms for neoplasm metastasis. But CD-NQAB was superior to CDAB and chemotherapeutics.

Animals ; Carcinoma, Lewis Lung ; drug therapy ; immunology ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Forkhead Transcription Factors ; metabolism ; Interferon-gamma ; immunology ; Interleukin-17 ; immunology ; Interleukin-23 ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; T-Lymphocytes, Helper-Inducer ; immunology ; T-Lymphocytes, Regulatory ; immunology

A variety of novel 2-(1-substituted-piperidine-4-ylamino)quinazoline derivatives were prepared and their antiproliferative activities on five cancer cell lines were evaluated by MTT assay. Quinazolines 4j-4l, 5a, 5b and 5d bearing a small hydrophobic alkyl group on piperidine ring exhibited potent antitumor activities with IC50 values at micromolar level. Compound 41 displayed significant in vivo antitumor activity with 72.9% inhibition on H22 tumor growth and 80% inhibition on Lewis lung cancer growth at a dose of 200 mg x kg(-1).
Animals ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Carcinoma, Lewis Lung ; pathology ; Cell Line, Tumor ; Drug Screening Assays, Antitumor ; Inhibitory Concentration 50 ; Liver Neoplasms ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Quinazolines ; chemical synthesis ; chemistry ; pharmacology