OBJECTIVE: To investigate the anti-tumor effects of cinobufacini (CINO) on hepatocellular carcinoma (HCC) induced by des-gamma-carboxy-prothrombin (DCP) and to uncover the underlying mechanisms. METHODS: The inhibitory effect of CINO on HCC cell proliferation was evaluated using the cell counting kit-8 method, and the apoptosis rate was quantified using flow cytometry. Immunofluorescence and Western blot analyses were used to investigate the differential expression of proteins associated with cell growth, apoptosis, migration, and invasion pathways after CINO treatment. The therapeutic potential of CINO for HCC was confirmed, and the possibility of combining cinobufacini with c-Met inhibitor for the treatment of primary HCC was further validated by in vivo experiments. RESULTS: Under the induction of DCP, CINO inhibited the activity of HCC cells, induced apoptosis, and inhibited migration and invasion. Upon the induction of DCP, CINO regulated c-Met activation and the activation of the phosphatidylinositol-3 kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways. In a mouse model of HCC, CINO exhibited significant antitumor effects by inhibiting the phosphorylation of c-Met and the downstream PI3K/AKT and MEK/ERK pathways in tumor tissues. CONCLUSIONS CINO inhibited HCC cell growth, promoted apoptosis, and suppressed HCC cell invasion and migration by targeting c-Met and PI3K/AKT and MEK/ERK signaling pathways under DCP induction.
Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-met/metabolism* ; Liver Neoplasms/drug therapy* ; Signal Transduction/drug effects* ; Animals ; Humans ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; Cell Proliferation/drug effects* ; Amphibian Venoms/therapeutic use* ; Cell Line, Tumor ; Neoplasm Metastasis ; Cell Survival/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Neoplasm Invasiveness ; Mice, Inbred BALB C ; Mice, Nude ; Mice ; Male ; Bufanolides/therapeutic use* ; Protein Precursors ; Prothrombin ; Biomarkers

Lung cancer is a major cause of cancer-related mortality worldwide. Among patients with non-small cell lung cancer (NSCLC), approximately 3%-7% harbor anaplastic lymphoma kinase (ALK) gene fusions. In recent years, multiple tyrosine kinase inhibitors (TKIs) have significantly improved the survival of patients with metastatic ALK-positive NSCLC. However, disease progression due to resistance remains a challenge. This article retrospectively analyzes a case of advanced lung adenocarcinoma with the echinoderm microtubule associated protein like 4 (EML4)-ALK fusion variant 3 (V3). The patient developed resistance to Lorlatinib treatment accompanied by mesenchymal-epithelial transition factor (MET) amplification. Effective tumor control was achieved with the combined use of Crizotinib and Lorlatinib, providing a valuable reference for further exploration of treatment strategies following resistance to ALK-TKIs in clinical practice.
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Humans ; Adenocarcinoma/genetics* ; Adenocarcinoma of Lung/genetics* ; Aminopyridines/therapeutic use* ; Crizotinib/therapeutic use* ; Drug Resistance, Neoplasm/drug effects* ; Lactams/therapeutic use* ; Lung Neoplasms/genetics* ; Oncogene Proteins, Fusion/metabolism* ; Protein Kinase Inhibitors/therapeutic use* ; Proto-Oncogene Proteins c-met/metabolism* ; Pyrazoles/therapeutic use*

MET gene is a proto-oncogene, which encodes MET protein with tyrosine kinase activity. After binding to its ligand, hepatocyte growth factor, MET protein can induce MET dimerization and activate downstream signaling pathways, which plays a crucial role in tumor formation and metastasis. Savolitinib, as a specific tyrosine kinase inhibitor (TKI) targeting MET, selectively inhibits the phosphorylation of MET kinase with a significant inhibitory effect on tumors with MET abnormalities. Based on its significant efficacy shown in the registration studies, savolitinib was approved for marketing in China on June 22, 2021 for the treatment of advanced non-small cell lung cancer with MET 14 exon skipping mutations. In addition, many studies have shown that MET TKIs are equally effective in patients with advanced solid tumors with MET gene amplification or MET protein overexpression, and relevant registration clinical studies are ongoing. The most common adverse reactions during treatment with savolitinib include nausea, vomiting, peripheral edema, pyrexia, and hepatotoxicity. Based on two rounds of extensive nationwide investigations to guide clinicians, the consensus is compiled to use savolitinib rationally, prevent and treat various adverse reactions scientifically, and improve the clinical benefits and quality of life of patients. This consensus was prepared under the guidance of multidisciplinary experts, especially including the whole-process participation and valuable suggestions of experts in Traditional Chinese Medicine, thus reflecting the clinical treatment concept of integrated Chinese and western medicines.
Humans ; Carcinoma, Non-Small-Cell Lung/genetics* ; Lung Neoplasms/pathology* ; Consensus ; Quality of Life ; Proto-Oncogene Proteins c-met/genetics* ; Protein Kinase Inhibitors/adverse effects* ; Drug-Related Side Effects and Adverse Reactions ; Mutation

OBJECTIVETo study the effect and mechanism of bevacizumab on proliferation and invasion of human lung cancer A549 cells.

METHODSA549 cells were treated with bevacizumab. Proliferation and invasion of the bevacizumab-treated A549 cells were detected using cell counting kit CCK-8 and Transwell assay, respectively. The expression of the mRNA and protein of MMP-2, MMP-9 and c-Met were detected by real-time PCR and Western blotting, respectively.

RESULTSProliferation activity was inhibited at the concentration of 10 µg/ml and promoted at the concentration of 100 µg/ml bevacizumab. Bevacizumab in the concentration of 50 µg/ml had a stronger inhibitory effect on the invasion of A549 cells (16 406.19 ± 5 674.23 penetrated cells) than that of control group (36 108.68 6 263.83, P<0.05). The real-time PCR showed that bevacizumab had a stronger inhibitory effect on the expression of MMP-2 and MMP-9 mRNA at the concentration of 50 µg/ml and on the expression c-Met mRNA at the concentration of 10 µg/ml bevacizumabin the A549 cells. However bevacizumab at the concentration of 100 µg/ml showed a promoting effect on the expression of MMP-2, MMP-9 and c-Met mRNA (1.82 ± 0.31, 1.60 ± 0.25, 2.63 ± 0.48), significantly higher than that of the control group (1.00 ± 0.19, 1.00 ± 0.23, 1.00 ± 0.22, P<0.05). The expression of MMP-2, MMP-9 and c-Met mRNA and protein was inhibited by 10 µg/ml bevacizumab in a time-dependent manner. The Western blot assay showed that bevacizumab had a bi-directional effect on the expression of MMP-2 and c-Met proteins in the A549 cells: a promoting effect at 100 µg/ml and inhibitory effect on the expression of MMP-2 at 50 µg/ml bevacizumab, and inhibitory effect on the expression of c-Met protein at 10 µg/ml bevacizumab.

CONCLUSIONSOur findings indicate that in a certain range of concentrations, bevacizumab has prominent inhibitory effect on the proliferation and invasion of A549 cells. However,over the concentration of 100 µg/ml, bevacizumab shows a weakening anti-invasion effect, even has a promoting effect on cell proliferation. This phenomenon may be related to the inhibiting effect on the expression of MMP-2 and c-Met proteins in a non-concentration-dependent manner by bevacizumab.

Angiogenesis Inhibitors ; pharmacology ; Bevacizumab ; pharmacology ; Blotting, Western ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; drug effects ; Enzyme Inhibitors ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-met ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the ability of invasion and migration of breast cancer MDA-MB-231 cells under serum starvation and hypoxia, and the effect of antiangiogenic drugs, rh-endostatin and bevacizumab, on the ability of invasion and migration of breast cancer cells under serum starvation and/or hypoxia, in order to explore the potential risk of antiangiogenic therapy in clinics.

METHODSThe cells were randomized into 4 groups, i.e., group A: 10% fetal bovine serum (FBS) group; group B: hypoxia + 10% FBS group; group C: serum starvation group; group D: hypoxia + serum starvation group; each group was further divided into three subgroups as blank control, treated with rh-endostatin and bevacizumab, respectively. Cell counting kit-8 (CCK-8) was used to assess the inhibition rate of cell growth induced by endostatin and bevacizumab, in order to determine the proper working concentration and time of the two drugs. Transwell assay was conducted to detect the cell invasion and migration in vitro. The expressions of c-Met and MMP-9 were detected by Western blot. The cells treated with rh-endostatin or bevacizumab under serum starvation were tested by hybridization using Exiqon miBase 18.0 microarray. The miRNAs which exibited significant differences (P < 0.05) in miRNA hybridization were verified by real-time PCR assay.

RESULTSCCK-8 assay showed that the inhibition rates of MDA-MB-231 cells cultured with 800 mg/L rh-endostatin for 48 h and 24 h were (32.2 ± 2.5)% and (27.0 ± 1.3)%, respectively, showing a significant difference (P = 0.023). The inhibition rates of MDA-MB-231 cells cultured with 80 mg/L bevacizumab for 48 h and 24 h were (30.5 ± 1.4) % and (26.1 ± 2.4) %, respectively, showing also a significant difference (P = 0.015). The Transwell assay showed that in the starvation blank group, the number of invaded and penetrated cells were 28.8 ± 2.2 and 31.4 ± 1.5, respectively, significantly different from that in the rh-endostatin and bevacizumab groups (P < 0.05). The relative expressions of c-Met and MMP-9 were 0.213 ± 0.017 and 0.542 ± 0.048, respectively, with a significant difference from those of the groups treated with each drug (P < 0.05 for both). The numbers of penetrated cells in the Transwell assay treated with rh-endostatin in hypoxia were 17.5 ± 2.1 and 16.5 ± 2.8, respectively, and the numbers of penetrated cells in the Transwell assay treated with bevacizumab were 16.3 ± 3.5 and 17.5 ± 2.4, respectively, showing no significant difference among them (P > 0.05 for both). The ability of migration and invasion of MDA-MB-231 cells and the expression of c-Met and MMP-9 were not impacted by hypoxia (P > 0.05). Real-time PCR assay showed that only the levels of miR-2355 and miR375 were significantly and stably decreased in the cells which had increased ability of invasion and migration. The relative expression levels of miR375 and miR-2355 in the serum starvation blank group were 0.550 ± 0.036 and 0.852 ± 0.121, respectively, significantly lower than that in the groups treated with rh-endostatin or bevacizumab (P<0.05). In the serum starvation group, the expression levels of miR375 and miR-2355 of cells treated with rh-endostatin were 0.295 ± 0.012 and 0.253 ± 0.011, and the expression levels of cells treated with bevacizumab were 0.234 ± 0.020 and 0.309 ± 0.022, respectively, (P > 0.05 for all). Compared with the serum starvation blank group, the expression levels of miR2355 and miR375 were significantly decreased when cells were treated with rh-endostatin/bevacizumab under serum starvation, but no significant difference was found between the two drugs (P > 0.05). However, hypoxia did not affect the expressions of miR2355 and miR375 (P > 0.05).

CONCLUSIONSThe results of this study suggest that serum starvation can increase the ability of invasion and migration of breast cancer cells. Furthermore, both rh-endostatin and bevacizumab may enhance their invasion and penetration ability under serum starvation condition.

Angiogenesis Inhibitors ; adverse effects ; Bevacizumab ; adverse effects ; Breast Neoplasms ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Culture Media, Serum-Free ; Endostatins ; adverse effects ; Female ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; MicroRNAs ; analysis ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-met ; metabolism ; Random Allocation ; Real-Time Polymerase Chain Reaction ; Time Factors

BACKGROUNDColon cancer is one of the major malignancies worldwide and it still remains resistant to much of the currently available chemotherapy. Downregulation of HGF/c-Met signaling pathway is an emerging therapy for cancer treatment.

METHODSIn this study, the inhibitory effects of c-Met phosphorylation were observed with SU11274 on different colon cancer cell lines in vitro.

RESULTSThe results revealed the significant inhibitory effects of SU11274 on cell proliferation and cell survival, in a time and dose-dependent manner. Furthermore, the inhibitory effects of SU11274 on different subgroups of colon cancer cells via the HGF/c-Met signaling pathway were implicated in this study.

CONCLUSIONThe results suggested the possible selective therapeutic effects of c-Met inhibitor on colon cancer.

Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; drug therapy ; pathology ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Indoles ; pharmacology ; Piperazines ; pharmacology ; Proto-Oncogene Proteins c-met ; antagonists & inhibitors ; physiology ; Signal Transduction ; Sulfonamides ; pharmacology

OBJECTIVETo observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib.

METHODSThe effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib.

RESULTSThe MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin.

CONCLUSIONSHGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.

Adenocarcinoma ; metabolism ; pathology ; Antineoplastic Agents ; pharmacology ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Drug Resistance, Neoplasm ; Epithelial-Mesenchymal Transition ; Fibroblasts ; cytology ; metabolism ; Hepatocyte Growth Factor ; pharmacology ; secretion ; Humans ; Lung ; cytology ; Lung Neoplasms ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-met ; metabolism ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; drug effects ; Transforming Growth Factor alpha ; pharmacology ; Tumor Microenvironment ; Vimentin ; metabolism

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50 = 15.8 microg/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.
Animals ; Autocrine Communication/*drug effects ; Catechin/*analogs & derivatives/metabolism/pharmacology ; Cell Line, Tumor ; Cell Movement/drug effects ; Female ; *Hepatocyte Growth Factor ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasms, Experimental/*metabolism/pathology ; Paracrine Communication/*drug effects ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-met/antagonists & inhibitors/metabolism ; Receptors, Growth Factor/antagonists & inhibitors/metabolism ; Signal Transduction

BACKGROUNDVascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells.

METHODSHepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA. The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells.

RESULTSSerum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells.

CONCLUSIONThis present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways.

Butadienes ; pharmacology ; Cell Line, Tumor ; Chromones ; pharmacology ; Colorectal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Hepatocyte Growth Factor ; blood ; pharmacology ; Humans ; MAP Kinase Signaling System ; physiology ; Morpholines ; pharmacology ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; physiology ; Phosphorylation ; Proto-Oncogene Proteins c-met ; metabolism ; RNA, Messenger ; analysis ; Signal Transduction ; physiology ; Vascular Endothelial Growth Factor A ; genetics

BACKGROUND: Up-regulation of the hepatocyte growth factor (HGF), its transmembrane tyrosine kinase receptor (c-Met), and urokinase type plasminogen activator (uPA), is associated with the development and metastasis of various types of cancers. However, the mechanisms by which HGF/c-Met signaling mediates cancer progression and metastasis are unclear. METHODS: We investigated the roles of HGF/c-Met in tumor progression and metastasis in NUGC-3 and MKN-28 stomach cancer cell lines. RESULTS: Treatment with HGF increased c-Met phosphorylation in a dose-dependent manner, as well as increasing cell proliferation. HGF treatment also increased the protein level and the activity of uPA in NUGC-3 and MKN-28 cells. A monoclonal antibody against human uPA receptor (uPAR), mAb 3936, inhibited HGF-mediated tumor cell invasion in a dose-dependent manner. Down-regulation of uPA using uPA-shRNA induced a decrease in in vitro cell invasion in NUGC-3 cells. CONCLUSIONS: These results suggest that NUGC-3 and MKN-28 cells express functional c-Met, which may provide a therapeutic target for interfering with metastases of cancer cells by inhibiting uPA and uPAR-mediated proteolysis.
Urinary Plasminogen Activator/antagonists & inhibitors/*metabolism ; Stomach Neoplasms/drug therapy/*enzymology ; Signal Transduction/*drug effects ; Receptors, Growth Factor/*drug effects ; Receptor Protein-Tyrosine Kinases/*drug effects ; Proto-Oncogene Proteins c-met/*drug effects ; Neoplasm Metastasis ; Humans ; Hepatocyte Growth Factor/*metabolism ; Disease Progression ; Adenocarcinoma/drug therapy/enzymology