Constructing a Prognostic Gene Signature for Lung Adenocarcinoma Based on Weighted Gene Co-Expression Network Analysis and Single-Cell Analysis.

Purpose: Lung adenocarcinoma (LUAD) has a high degree of intratumor heterogeneity. Advanced single-cell RNA sequencing (scRNA-seq) technologies have offered tools to analyze intratumor heterogeneity, which improves the accuracy of identifying biomarkers based on single-cell expression data, and thus helps in predicting prognosis of cancer patients and assisting decision-makings for cancer treatment. Patients and Methods: ScRNA-seq data containing two LUAD and two para-cancerous tissue samples were included to identify different cell clusters in tumor tissues. To identify the most relevant modules and important cell subpopulations (clusters) in LUAD tissues, weighted gene co-expression network analysis (WGCNA) was performed. Subsequently, LUAD molecular subtypes were constructed by unsupervised consensus clustering based on genes in key modules. Using differential analysis, univariate Cox regression analysis, and least absolute shrinkage and selection operator (LASSO) regression analysis, a prognostic model of LUAD was established. Results: A total of 14 cell clusters belonging to 10 cell types in LUAD were identified. The turquoise module was the most relevant to LUAD among all the modules; cluster 10 (C10, lung epithelial cells) was found to be the most strongly associated with the turquoise module. LUAD samples were divided into two groups of distinct molecular subtypes. Based on the 165 shared genes between the turquoise module and C10, 511 DEGs between the two molecular subtypes were obtained, and five of them were selected to construct the gene signature, which was validated to be an independent prognostic marker of LUAD. Conclusion: Fourteen cell clusters co-existed in LUAD, which contributed to its intratumor heterogeneity. Two molecular subtypes of LUAD were identified and a five-gene signature was developed and validated to be significantly associated with prognostic and clinical characteristics of LUAD patients.

He-Chan Pian inhibits the metastasis of non-small cell lung cancer via the miR-205-5p-mediated regulation of the GREM1/Rap1 signaling pathway.

BACKGROUND: He-Chan Pian (HCP), a traditional Chinese medicinal formula, shows promising efficacy for the treatment of lung cancer. PURPOSE: Gremlin (GREM1) plays an important role in gastrointestinal tumor metastasis; however, little is known about its role in lung cancer. We determined the mechanism underlying the protective effect of HCP against metastasis in a mouse model of non-small cell lung cancer (NSCLC) and demonstrated the role of GREM1. METHODS: Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to analyze the herbal components and metabolites from the serum of HCP-treated mice. The tumor, liver, and kidney were examined histologically, and the antitumor effects and toxicity of HCP were evaluated. Levels of epithelial-mesenchymal transition (EMT)-associated transcription factors were measured using western blotting in tumors from five groups (i.e., model, HCP [L], HCP [M], HCP [H], and positive control [cisplatin, DDP]). Differentially expressed proteins and genes were identified using protein chip and sequencing analyzes, respectively. Short hairpin RNAs and overexpression plasmids were introduced into cells to evaluate the effects of GREM1. To evaluate proliferation, migration, and invasion, the expression levels of proteins involved in the Rap1 pathway and EMT were measured in vitro. Xenograft tumors with overexpression-GREM1 (OE-GREM1) in A549 cells were examined for cell proliferation. A dual-luciferase assay was performed to verify the direct interaction of GREM1 with miR-205-5p in lung cancer. RESULTS: Thirty-six ingredients and bioactive constituents detected in the serum of HCP-treated mice were identified as the key compounds involved in the inhibition of tumor growth. Animal experiments revealed that HCP significantly decreased tumor volumes and had no adverse effects on the liver or kidney or side effects. GREM1 upregulation was closely related to tumor metastasis and was regulated by miR-205-5p, as confirmed using a dual-luciferase reporter assay. OE-GREM1 promoted A549 cell migration and invasion, promoted EMT, and increased the expression of Rap1 pathway intermediaries, whereas shGREM1 had the opposite effects. Furthermore, the effects of OE-GREM1 on proliferation in the A549 xenograft mouse model were attenuated, although HCP has an inhibitory effect on tumors. CONCLUSION: Our results suggest that HCP contributes to the inhibition of NSCLC metastasis via the Gremlin/Rap1 signaling pathway regulated by miR-205-5p.

The Ciji-Hua'ai-Baosheng II Formula Attenuates Chemotherapy-Induced Anorexia in Mice With H22 Hepatocellular Carcinoma.

Background: Ciji-Hua'ai-Baosheng II Formula (CHB-II-F) is a traditional Chinese medicine formula, which specifically targets different aspects of chemotherapy-induced adverse effects in patients with cancer. In our clinical application, CHB-II-F significantly alleviated chemotherapy-induced anorexia (loss of appetite) and improved the quality of life for patients with tumor during and after chemotherapy. However, the mechanism of CHB-II-F in alleviation of chemotherapy-induced anorexia remains to be further investigated. Aim of Study: To explore the therapeutic effect and mechanism of CHB-II-F on chemotherapy-induced anorexia in the mice model of H22 hepatoma. Materials and Methods: A total of 72 Kunming mice of SPF grade were inoculated subcutaneously with H22 hepatoma cells into the right anterior armpit of the mice. After 1 week of seeding, mice were injected intraperitoneally with a high dose of 5-fluorouracil (200 mg/kg 5-FU) to establish the model of chemotherapy. The mice were randomly divided into six groups: untreated group, 5-FU group, 5-FU plus Yangzheng Xiaoji capsule (YZXJC) group, and three groups of 5-FU plus different concentrations of CHB-II-F. All the mice in each group were treated for 14 days. The body weight, food intake, tumor volume, and tumor weight of mice were measured, and pathological examinations of tumor tissue, stomach, and duodenum were carried out. Expressions of serum Leptin, Neuropeptide Y (NPY), epidermal cell growth factor (EGF), Motilin (MTL), Orexin A (OXA), Gastrin (GAS), Ghrelin, Prostaglandin E2 (PGE2), and jejunum superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were examined. The protein and mRNA levels of proopiomelanocortin (POMC), Orexin receptor 1 (OX1R), neuropeptide Y (NPY), cocaine and amphetamine regulated transcript peptide (CART), Agouti gene-related protein (AgRP), Leptin receptor (Ob-R), and Ghrelin receptor (GHSR) were examined in hypothalamus, and the protein levels of substance P (SP) and 5-hydroxytryptamine (5-HT) in duodenum were measured. Results: The combination of CHB-II-F and 5-FU could enhance the inhibitory effect of 5-FU on tumor. The tumor inhibition rates of 5-FU group, YZXJC group, CHB-II-F(H) group, CHB-II-F(M) group, and CHB-II-F(L) group were 58.88, 28.08, 54.96, 37.69, and 28.61%, respectively. Compared with untreated group and 5-FU group, CHB-II-F significantly increased the body weight and food intake of tumor-bearing mice; increased the content of NPY, Orexin A, Ghrelin, GAS, MTL, EGF, and PGE2 in serum and the activity of SOD in jejunum; and decreased the content of Leptin in serum and the content of MDA in jejunum. Compared with untreated group and 5-FU group, CHB-II-F also enhanced the expression of OX1R, GHSR, NPY, and AgRP protein and gene and decreased the expression of Ob-R, POMC, and CART protein and gene in hypothalamus of mice, and the gene expression was consistent with the protein expression. In addition, CHB-II-F decreased the expression of 5-HT and SP protein in duodenum. Conclusion: In the murine model of H22 hepatocellular carcinoma (HCC) receiving chemotherapy, CHB-II-F enhances the inhibitory effect of 5-FU on tumor, significantly improves the pathological injury of gastrointestinal tract caused by chemotherapy, and regulates the secretion of gastrointestinal hormones. It may alleviate chemotherapy-induced anorexia by affecting appetite regulatory factors in the feeding area of hypothalamus central nervous system and peripheral appetite regulatory factors.

Safety Evaluation of a New Traditional Chinese Medical Formula, Ciji-Hua'ai-Baosheng II Formula, in Adult Rodent Models.

BACKGROUND: Ciji-Hua'ai-Baosheng II Formula (CHB-II-F) is a new traditional Chinese medical formula that has been shown to reduce toxicity and side effects of chemotherapy and increase the probability of cancer patient survival. Whether CHB-II-F is safe as an adjunctive therapy for cancer patients receiving chemotherapy has yet to be determined. PURPOSE: To evaluate the acute and subchronic toxic effects of CHB-II-F in rodent models. METHODS: In acute toxicity test, 24 Kunming mice were divided into 2 groups: untreated control and CHB-II-F 1.05 g/mL (31.44 g/kg) treated group. Treatment was administered to the treated group 3 times a day for 14 days. The overall health, adverse reactions, and mortality rate were documented. In subchronic toxicity test, 96 Sprague-Dawley rats were divided into 4 groups: untreated control, high dose CHB-II-F (H) (26.20 g/kg), medium dose CHB-II-F (M) (13. 10 g/kg), and low dose CHB-II-F (L) (6.55 g/kg) [equal to 24.375 g (dried medicinal herb)/kg] treated groups. Treated groups were given the treatments once a day for 4 weeks. The overall health and mortality rate were recorded every day. Body weight and food consumption were measured once a week. Hematologic and biochemical parameters, organ weights, and histopathologic markers were analyzed after 4 weeks. An additional 2 weeks were given as the treatment recovery period before end-point euthanization, and biochemical analyses were performed. RESULTS: The maximum tolerated dose (MTD) of CHB-II-F on mice was found to be 94.31 g/kg [equal to 351 g (dried medicinal herb)/kg], which is 108 times the human adult dose. In the acute toxicity test, administration of CHB-II-F 31.44 g/kg showed no adverse effect and did not cause mortality. In the subchronic toxicity test, after 4 weeks of treatment, compared to the controls, total cholesterol (TCHO) level, cardiac and splenic indexes, body weights of female rats, and mean corpuscular hemoglobin concentration (MCHC) in the CHB-II-F (H) group were significantly increased; triglyceride (TG) in the CHB-II-F (M) group and liver and splenic indexes in the CHB-II-F (L) group were increased. After the two-week recovery period, biofluid analyses, food consumption, and histopathologic examinations showed no abnormalities. CONCLUSION: Administration of CHB-II-F had no obvious adverse effect on the overall health of rodent models. A daily maximum dose of less than 94.31 g/kg or 6.55 g/kg CHB-II-F for 4 continuous weeks was considered safe.

The effects of Ciji-Hua'ai-Baosheng on immune function of mice with H22 hepatocellular carcinoma receiving chemotherapy.

BACKGROUND: Ciji-Hua'ai-Baosheng Decoction (CHBD) is a traditional Chinese formula that may attenuate the toxicity and side-effects of chemotherapy. The formula may also prolong the life of cancer patients. Whether CHBD should be employed as adjunctive therapy for cancer patients receiving chemotherapy has yet to be determined as does the mechanism whereby CHBD exerts its beneficial effects. AIM OF THE STUDY: To document the potential effects of CHBD on tumor growth and immune function in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy. MATERIALS AND METHODS: Sixty Kunming mice were injected subcutaneously with H22 hepatoma cells in the right anterior armpit. After seven days, the mice with formed tumors were injected with Cytoxan (CTX) (200 mg/kg) to establish the chemotherapy model. These mice were randomly divided into 5 groups: model (untreated controls), control (CTX,33.33 mg/kg), and high CHBD (H) (117 g/kg), moderate CHBD (M) (58.5 g/kg) and low CHBD (L) (29.25 g/kg) treated groups. Tumor weights and inhibitory ratio (decrease in tumor dimensions), histology of tumor, colon, spleen and liver, and biochemical tests of liver and kidney function were documented after 10 days. Serum and tumor IL-2, IFN-γ, IL-6, and TNF-α levels were determined by enzyme-linked immunosorbent assay (ELISA) and Western blot respectively. The potential bioactive compounds in CHBD were characterized by UHPLC-MS. RESULTS: Although tumor weights were decreased in CTX alone and CHBD (H) and CHBD (M) groups (-66%, -41% and -25% respectively), tumor cell density was reduced to the greatest extent in the CHBD (H) group. CHBD had no evident effects on liver and kidney function. CTX-induced colon inflammation and decrease in spleen lymphocytes were attenuated with CHBD treatment. CHBD increased serum IL-2, IFN-γ and TNF-α, but decreased IL-6 levels in serum and tumor tissue. UHPLC-MS analysis of CHBD revealed the presence of 11 bioactive compounds. CONCLUSIONS: In this murine model of HCC receiving chemotherapy, CHBD inhibited tumor growth, improved immune function and pro-inflammatory cytokine responses while attenuating CTX-associated side effects.

The Protective Effects of Ciji-Hua'ai-Baosheng II Formula on Chemotherapy-Treated H22 Hepatocellular Carcinoma Mouse Model by Promoting Tumor Apoptosis.

Ciji-Hua'ai-Baosheng II Formula (CHB-II-F) is a traditional Chinese medical formula that has been shown in clinical practice to relieve side effects of chemotherapy and improve quality of life for cancer patients. In order to understand the mechanism of its protective effects on chemotherapy, mice with transplanted H22 hepatocellular carcinoma were employed in this study. Ninety-two mice were injected subcutaneously with H22 HCC cell suspension into the right anterior armpit. After mice were treated with 5-fluorine pyrimidine (5-FU), they were divided into six groups as untreated group, 5-FU group, 5-FU plus Yangzheng Xiaoji Capsule group and three groups of 5-FU plus different concentrations of CHB-II-F. Twenty mice were euthanized after 7 days of treatment in untreated and medium concentration of CHB-II-F groups and all other mice were euthanized after 14 days of treatment. Herbal components/metabolites were analyzed by UPLC-MS. Tumors were evaluated by weight and volume, morphology of light and electron microscope, and cell cycle. Apoptosis were examined by apoptotic proteins expression by western blot. Four major components/metabolites were identified from serum of mice treated with CHB-II-F and they are ß-Sitosterol, Salvianolic acid, isobavachalcone, and bakuchiol. Treatment of CHB-II-F significantly increased body weights of mice and decreased tumor volume compared to untreated group. Moreover, CHB-II-F treatment increased tumor cells in G0-G1 transition instead of in S phase. Furthermore, CHB-II-F treatment increased the expression of pro-apoptotic proteins and decreased the expression anti-apoptotic protein. Therefore, CHB-II-F could improve mice general condition and reduce tumor cell malignancy. Moreover, CHB-II-F regulates apoptosis of tumor cells, which could contribute its protective effect on chemotherapy.

The combination effects of Shen-Ling-Bai-Zhu on promoting apoptosis of transplanted H22 hepatocellular carcinoma in mice receiving chemotherapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Shen-Ling-Bai-Zhu Powder (SLBZP) is a classic traditional Chinese medical formula that has been used for several decades in the treatment of patients with gastrointestinal malignancies. Whether SLBZP is best employed as single agent or adjunctive therapy has yet to be determined as does the mechanism whereby SLBZP exerts its anti-tumor effects. AIM OF THE STUDY: To investigate the effects of SLBZP alone and in combination with Cytoxan (CTX) on tumor growth, malignant cell apoptosis and Akt/Nuclear Factor kappa B (NF-КB) signaling in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy. MATERIALS AND METHODS: Sixty-four adult mice developed HCC following subcutaneous inoculation with H22 hepatocellular carcinoma cells. Seven days later, all received chemotherapy with CTX (200mg/kg) once. Mice were then randomized into eight study groups (N=8/group). Three groups were treated with different concentrations of SLBZP alone (6.00, 3.00, 1.5g/kg), three with SLBZP (6.00, 3.00, 1.5g/kg) plus CTX (20mg/kg), one with CTX (20mg/kg) alone (positive control), and one with physiologic saline (untreated, negative control). All groups were treated for 14 days. Tumor size, histology and serum or tissue levels and/or mRNA expression of PDGF-BB, VEGF, Ang-1, Ang-2, NF-КB, B-cell lymphoma-2 (Bcl-2); B-cell lymphoma-extra large (Bcl-xL); X-linked inhibitor of apoptosis (XIAP), Survivin, Caspase-3, Caspase-9, Caspase-7, Akt and phosphorylated Akt expression were documented at the end of treatment. RESULTS: Compared to untreated negative controls, tumor sizes were decreased in the CTX alone, SLBZP (M)+CTX and SLBZP (H)+CTX groups (-52%,-53% and -58% respectively). Tumor cell density was decreased in all treated groups but most apparent in the SLBZP (H)+CTX group. Electron microscopic evidence of apoptosis was also most apparent in this group. Serum and/or tissue levels and expression of PDGF-BB, VEGF, Ang-1, Ang-2, their downstream signaling proteins and anti-apoptotic markers were lowest and pro-apoptotic markers highest in SLBZP (H)+CTX treated mice. CONCLUSIONS: In this chemotherapy-induced animal model of HCC, SLBZP was most efficacious as adjunctive therapy and appears to act by inhibiting tumor growth promoters and anti-apoptotic proteins while enhancing pro-apoptotic proteins.