Based on the epidermal growth factor receptor(EGFR)/mitogen-activated protein kinase(MAPK) signaling pathway-mediated cell proliferation, this study explores the effect of cisplatin combined with Guiqi Yiyuan Ointment on Lewis lung cancer-bearing mice. A total of 60 male C57BL/6 mice were randomly divided into a blank group with 10 mice and a modeling group with 50 mice. After modeling, they were randomly divided into the model group, cisplatin group, and low-, medium-, and high-dose groups of cisplatin combined with Guiqi Yiyuan Ointment, with 10 mice in each group. After 14 days of medication, the general condition of the mice was observed; body weight was measured, and organ index and tumor inhibition rate were calculated. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology changes in tumor tissue. Immunohistochemistry was used to detect the positive rate of Ki-67 antigen(Ki-67) and proliferating cell nuclear antigen(PCNA) in tumor tissue. Western blot and real time-quantitative polymerase chain reaction(qPCR) were used to detect the expression of related proteins and mRNA in tumor tissue. Flow cytometry was used to detect the cell cycle of tumor cells in tumor tissue. The results showed that compared with that in the blank group, the general condition of mice in the model group deteriorated; the body weight, as well as thymus and spleen index of mice in the model group decreased after 14 days of medication. Compared with that in the model group, the general condition of mice in the cisplatin group deteriorated, while the condition of mice in the combined groups improved; the body weight, as well as thymus and spleen index of mice in the cisplatin group decreased, while the three indicators in the combined groups increased; the tumor weight of each medication group decreased, and the tumor inhibition rate increased; there were varying degrees of necrosis in tumor cells of each medication group, and the tightness of tumor cells, the increase in the number of cell nuclei and chromatin, and mitosis all decreased. The positive rate of Ki-67 and PCNA, as well as the protein expression and ratio of p-EGFR/EGFR, rat sarcoma viral oncogene homolog(Ras), phosphorylated Raf-1 protein kinase(p-Raf-1)/Raf-1, phosphorylated mitogen-activated protein kinase kinase(p-MEK)/MEK, phosphorylated extracellular signal-regulated kinase(p-ERK)/ERK and the mRNA expression of EGFR, Ras, Raf-1, MEK, and ERK all decreased. The proportion of tumor cells in the G_0/G_1 phase of each medication group increased, and that in the S phase decreased. In addition, there was no significant difference in the G_2/M phase. Compared with that of the cisplatin group, the tumor weight of the combined groups decreased, and the tumor inhibition rate increased. The necrosis and mitosis of tumor cells in the combined groups were more pronounced; the positive rate of Ki-67 and PCNA, the protein expression and ratio of p-EGFR/EGFR, Ras, p-Raf-1/Raf-1, p-MEK/MEK, and p-ERK/ERK, as well as the mRNA expression of EGFR, Ras, Raf-1, MEK, and ERK in the combined groups all decreased. The proportion of tumor cells in the G_0/G_1 phase of the combined medium-and high-dose groups increased, and that in the S phase decreased. There was no significant difference in the proportion of tumor cells of the combined groups in the G_2/M phase. This indicates that the combination of cisplatin and Guiqi Yiyuan Ointment can enhance the anti-tumor effect of cisplatin on tumor-bearing mice, and the mechanism may be associated with the inhibition of the EGFR/MAPK pathway, which accelerates the arrest of tumor cells in the G_0/G_1 phase, thereby inhibiting the proliferation of tumor cells. At the same time, the study also indicates that Guiqi Yiyuan Ointment may reduce the damage of tumors to mice and the toxic side effects brought by cisplatin chemotherapy.
Animals ; Male ; Carcinoma, Lewis Lung/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; ErbB Receptors/genetics* ; Mice ; Cisplatin/administration & dosage* ; Mice, Inbred C57BL ; Cell Proliferation/drug effects* ; Ointments/administration & dosage* ; MAP Kinase Signaling System/drug effects* ; Humans ; Antineoplastic Agents/administration & dosage* ; Lung Neoplasms/metabolism*

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

Traditional Chinese medicine believes that the occurrence and development of tumors is related to the body's Qi deficiency. " Invigorating Qi for consolidation of exterior" has became an effective way to treat tumors by traditional Chinese medicine. This study is based on the " invigorating Qi for consolidation of exterior" to explore the effect of flavonoid components in Qi-invigorating herbs Astragali Radix( AR) on the growth and immune function of mouse Lewis lung cancer xenografts,and further explore its mechanism of action. In the present study,high performance liquid chromatography was performed to analyze the flavonoid components in AR.The Lewis lung cancer model of C57 BL/6 mice was constructed,and the tumor volume of mice was determined by Visual Sonics Vevo2100 high frequency color ultrasound. The levels of IL~(-1)7 and RORγt in serum and tumor tissues were detected by ELISA and immunohistochemistry. The expression of IRE~(-1)/XBP~(-1) pathway-related proteins in tumor tissues was detected by Western blot. The results revealed that treatment of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR significantly inhibited tumor growth of C57 BL/6 tumorbearing mice. The inhibition rates at the dose of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR were( 29. 5±4. 4) % and( 43. 4±5. 2) %,respectively. The expression of IL~(-1)7 and RORγt in serum and tumor tissues of Lewis lung cancer mice were decreased,and the spleen index and thymus index were significantly enhanced by the flavonoid components in AR. Flavonoid components in AR could decrease the expression of X-box binding protein 1( XBP1),inositol-requiring enzyme( IRE1) and glucose regulated protein 78 k D( GRP78),and increase the expression of C/EBP homologous protein( CHOP),and the high-dose group is better,suggesting that the anti-lung cancer effect of flavonoid components in AR is related to the regulation of XBP1 mediated ERs. This study provides new evidence that the flavonoid components in AR could inhibit the tumor growth of C57 BL/6 tumor-bearing mice by regulating the body's immune function through " invigorating Qi for consolidation of exterior".
Animals ; Astragalus Plant/chemistry* ; Carcinoma, Lewis Lung/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Endoplasmic Reticulum Chaperone BiP ; Flavonoids/therapeutic use* ; Mice ; Mice, Inbred C57BL ; Qi ; Xenograft Model Antitumor Assays

OBJECTIVEEscape from the body's immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe.

METHODSAn orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography.

RESULTSCompared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4CD25 T-cells and Foxp3 T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells.

CONCLUSIONThe molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

Animals ; Apoptosis ; drug effects ; Carcinoma, Lewis Lung ; drug therapy ; enzymology ; immunology ; physiopathology ; Cell Proliferation ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Growth Inhibitors ; administration & dosage ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; genetics ; immunology ; Lung Neoplasms ; drug therapy ; enzymology ; immunology ; physiopathology ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; drug effects ; immunology

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

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.
Abietanes ; administration & dosage ; Animals ; Antineoplastic Agents ; administration & dosage ; CD8-Positive T-Lymphocytes ; drug effects ; immunology ; Carcinoma, Lewis Lung ; drug therapy ; genetics ; immunology ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; Drug Synergism ; Humans ; Interferon-gamma ; genetics ; immunology ; Lung Neoplasms ; drug therapy ; genetics ; immunology ; Matrix Metalloproteinase 9 ; genetics ; Mice ; Mice, Inbred C57BL ; Myeloid-Derived Suppressor Cells ; drug effects ; immunology ; Plant Extracts ; administration & dosage ; Rosmarinus ; chemistry

The incidence of lung cancer has rapidly increased and cancer patients at a later cancer stage frequently suffer from unbearable cancer-associated pain. However, the pathophysiology of lung cancer pain has not been fully described due to a lack of appropriate animal models. This study was designed to determine the effect of Lewis lung carcinoma (LLC) cell inoculation on formalin-induced pain behavior and spinal Fos expression in C57BL/6 mice. LLC cells (1.5 × 10⁵, 2.5 × 10⁵, 3.0 × 10⁵ or 5.0 × 10⁵) were inoculated into back or peri-sciatic nerve areas. Back area inoculation was adopted to determine the effect of cancer cell circulating factors and the peri-sciatic nerve area was used to evaluate the possible effects of cancer cell contacting and circulating factors on formalin-induced pain. At postinoculation day 7, LLC cell (5.0 × 10⁵) inoculations in both back and peri-sciatic nerve area significantly increased formalin-induced paw-licking time and spinal Fos expression over those in cell-media-inoculated (control) mice. Enhanced pain behavior and spinal Fos expression were significantly suppressed by ibuprofen pretreatment (250 mg/kg). The results of this study suggest that LLC cell circulating factors and inflammatory responses may be critical in enhancing pain sensation in the early stage of lung cancer cell inoculation.
Animals ; Carcinoma, Lewis Lung* ; Formaldehyde ; Humans ; Ibuprofen ; Incidence ; Lung Neoplasms ; Mice* ; Models, Animal ; Sensation

Cancer is one of the leading causes of morbidity and mortality worldwide; therefore there is a need to discover new therapeutic modules with improved efficacy and safety. Immune-(cell) therapy is a promising therapeutic strategy for the treatment of intractable cancers. The effectiveness of certain chemotherapeutics in inducing immunogenic tumor cell death thus promoting cancer eradication has been reported. Ginsenoside Rg3 is a ginseng saponin that has antitumor and immunomodulatory activity. In this study, we treated tumor cells with Rg3 to verify the significance of inducing immunogenic tumor cell death in antitumor therapy, especially in DC-based immunotherapy. Rg3 killed the both immunogenic (B16F10 melanoma cells) and non-immunogenic (LLC: Lewis Lung Carcinoma cells) tumor cells by inducing apoptosis. Surface expression of immunogenic death markers including calreticulin and heat shock proteins and the transcription of relevant genes were increased in the Rg3-dying tumor. Increased calreticulin expression was directly related to the uptake of dying tumor cells by dendritic cells (DCs): the proportion of CRT+ CD11c+ cells was increased in the Rg3-treated group. Interestingly, tumor cells dying by immunogenic cell death secreted IFN-gamma, an effector molecule for antitumor activity in T cells. Along with the Rg3-induced suppression of pro-angiogenic (TNF-alpha) and immunosuppressive cytokine (TGF-beta) secretion, IFN-gamma production from the Rg3-treated tumor cells may also indicate Rg3 as an effective anticancer immunotherapeutic strategy. The data clearly suggests that Rg3-induced immunogenic tumor cell death due its cytotoxic effect and its ability to induce DC function. This indicates that Rg3 may be an effective immunotherapeutic strategy.
Animals ; Apoptosis ; Calreticulin ; Carcinoma, Lewis Lung ; Cell Death* ; Dendritic Cells ; Heat-Shock Proteins ; Immunotherapy* ; Melanoma ; Mortality ; Panax ; Saponins ; T-Lymphocytes

OBJECTIVETo study the effect of Feiji Recipe (FR) intervening indoleamine 2,3-dioxygenase (IDO) induced immune escape on the murine model of Lewis lung carcinoma. Methods Totally 48 C57BL/6 mice inoculated with Lewis lung cancer cells transfected with human (enhanced green fluorescent protein,EGFP)-IDO gene were divided into four groups according to radom digit table, i.e., the model group (administered with normal saline by gastrogavage) , the Chinese medicine group (treated with FR Decoction at the daily dose of 100 mg/g by gastrogavage), the 1-methyl-D-trytaphan (1-MT) group (administered with 1-MT mixed liquor at the daily dose of 100 mg/kg by gastrogavage), and the Paclitaxel group (treated with Paclitaxel at the daily dose of 15 mg/kg by peritoneal injection), 12 in each group. The intervention was started from the 2nd day of modeling. The survival time was observed in 24 of them. Ratios of CD4+ CD25+ FoxP3+ regulatory T cells (Treg) in the spleen were detected in the rest 24 mice by flow cytometry respectively.

RESULTSCompared with the model group, the survival time was significantly prolonged in the Chinese medicine group and the 1-MT group (P < 0.01); ratios of Treg cells remarkably decreased in the Chinese medicine group, the 1-MT group, and the Paclitaxel group (P < 0. 01). Compared with the Paclitaxel group, the survival time was significantly prolonged in the Chinese medicine group and the 1-MT group (P < 0.01); ratios of Treg cells decreased significantly in the 1-MT group (P < 0.05).

CONCLUSIONFR could inhibit the proliferation of lung cancer cells and immune eseape, improve the immune function, and prolong the survival of tumor-bearing mice.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Carcinoma, Lewis Lung ; drug therapy ; immunology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; Lung Neoplasms ; Mice ; Mice, Inbred C57BL ; Paclitaxel ; T-Lymphocytes, Regulatory