OBJECTIVE: To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. METHODS: We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). RESULTS: In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. CONCLUSIONS ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.
Acetates ; pharmacology ; Apoptosis ; drug effects ; genetics ; Autophagy ; drug effects ; Autophagy-Related Proteins ; metabolism ; Benzopyrans ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation ; Humans ; MCF-7 Cells ; Morpholines ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; metabolism ; Pyrans ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Triazines ; pharmacology ; Ubiquitin-Conjugating Enzymes ; metabolism

BACKGROUND: Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored. METHODS: Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests. RESULTS: Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues. CONCLUSIONS Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.
Animals ; Antineoplastic Agents ; therapeutic use ; Apoptosis ; drug effects ; Caco-2 Cells ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HT29 Cells ; Humans ; In Situ Nick-End Labeling ; Matrix Metalloproteinase 3 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Sesquiterpenes ; therapeutic use ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; genetics ; metabolism

OBJECTIVE: To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. METHODS: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. RESULTS: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL (P<0.05). CONCLUSION The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Hepatic Stellate Cells ; drug effects ; physiology ; Humans ; Injections ; Liver Cirrhosis ; drug therapy ; Tumor Suppressor Proteins ; genetics

Colorectal cancer (CRC) is a common malignant tumor in the digestive tract, and 30%-85% of CRCs express epidermal growth factor receptors (EGFRs). Recently, treatments using cetuximab, also named C225, an anti-EGFR monoclonal antibody, for CRC have been demonstrated to cause an S492R mutation in EGFR. However, little is known about the biological function of S492R EGFR. Therefore, we attempted to elucidate its biological function in CRC cells and explore new treatment strategies for this mutant form. Our study indicated that EGFR and S492R EGFR accelerate the growth of CRC cells in vitro and in vivo and monoclonal antibody CH12, which specifically recognizes an EGFR tumor-specific epitope, can bind efficiently to S492R EGFR. Furthermore, mAb CH12 showed significantly stronger growth suppression activities and induced a more potent antibody-dependent cellular cytotoxicity effect on CRC cells bearing S492R EGFR than mAb C225. mAb CH12 obviously suppressed the growth of CRC xenografts with S492R EGFR mutations in vivo. Thus, mAb CH12 may be a promising therapeutic agent in treating patients with CRC bearing an S492R EGFR mutation.
Animals ; Antibodies, Monoclonal ; pharmacology ; Antineoplastic Agents, Immunological ; pharmacology ; Caco-2 Cells ; Cell Proliferation ; drug effects ; Colorectal Neoplasms ; therapy ; ErbB Receptors ; genetics ; immunology ; Female ; HT29 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mutation ; Xenograft Model Antitumor Assays

To explore the mechanism of β-carboline alkaloids inhibiting the migration and invasion of SGC-7901 cells and its correlation with FAK gene expression,CCK-8 method was used to determine the inhibitory rate of β-carboline alkaloids on the proliferation of gastric cancer SGC-7901 cells under different concentrations.The effect of β-carboline alkaloids on the migration and invasion of SGC-7901 cells was used by Transwell compartment.Detection of mRNA and protein expression of FAK genes were used by qRT-PCR and Western blot.Then si-FAK-1051 recombinant plasmid was transfected into SGC-7901 cells.FAK gene silencing effect was identified by qRT-PCR and Western blot technique again.Finally,the effects of FAK gene silencing on proliferation and migration of gastric cancer SGC-7901 cells were detected by CCK-8 kit and Transwell chamber assay respectively.With the increase of the concentration ofβ-carboline alkaloids,the inhibitory rate of SGC-7901 cells in human gastric cancer cells increased gradually,with IC5013.364 mg·L-1.The number of SGC-7901 cells of Transwell compartment in the positive experimental group(5-FU,5 mg·L-1) and the β-carboline alkaloids group decreased significantly(P<0.01) and the number of SGC-7901 cells in the β-carboline alkaloids group was significantly lower than that in the positive experimental group(P<0.01).Compared with the blank control group,the mRNA and protein expression level of FAK genes in the positive experimental group was significantly lower than that in the experimental group of β-carboline alkaloids(P<0.05).After transfection of si-FAK-1051 into gastric cancer SGC-7901 cells,the expression of mRNA and protein of FAK gene was significantly down regulated(P<0.05).SGC-7901 cell proliferation and cell migration ability also decreased significantly(P<0.05).β-carboline alkaloids are more effective than 5-FU in inhibiting migration and invasion of gastric cancer SGC-7901 cells,and the mechanism may be related to the inhibition of mRNA and protein expression of FAK gene by β-carboline alkaloids.
Alkaloids ; pharmacology ; Carbolines ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; Focal Adhesion Kinase 1 ; genetics ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Neoplasm Invasiveness ; Stomach Neoplasms ; drug therapy ; pathology

Integrin is a large family of cell adhesion molecules (CAMs) which involves in the interaction of cells/cells and cells/ extracellular matrix (ECM) to mediate cell proliferation, differentiation, adhesion, migration, etc. In recent years, aberrant expression of integrin has been clearly found in many tumor studies, indicating that integrin is closely related to tumor formation and development. Meanwhile, it has effects on tumor cell differentiation, cell migration, proliferation and tumor neovascularization. The study of drugs targeting integrins is of great significance for the clinical treatment of tumors. Because of its important role in tumorigenesis and development, integrin has become a promising target for the treatment of cancer. This review summarizes the role of integrin in tumor development and the current state of integrin inhibitors to provide a valuable reference for subsequent research.
Antineoplastic Agents ; pharmacology ; therapeutic use ; Biological Products ; pharmacology ; therapeutic use ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Extracellular Matrix ; metabolism ; Humans ; Integrins ; antagonists & inhibitors ; classification ; genetics ; metabolism ; Neoplasms ; drug therapy ; pathology ; Neovascularization, Pathologic ; drug therapy ; pathology ; Signal Transduction ; drug effects

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

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo observe the effect of Shugan Liangxue Decoction (, SGLXD) on estrogen receptor α (ERα) in human breast cancer cells.

METHODSThe effect of SGLXD (0.85-5.10 mg/mL) on the proliferation of breast cancer cells were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The nuclear ERα protein levels in MCF-7, T47D and ZR-75-1 cells which treated by SGLXD for 24 h were examined by western blot and immunofluorescence assay. MCF-7 and MDA-MB-231 cells were treated by 17β-estradiol (E2) with or without SGLXD, for 24 h, and the E2 targeted genes c-myc and bcl-2 protein product was evaluated by western blot.

RESULTSSGLXD showed dose-dependent inhibition on the proliferation of MCF-7, T47D and ZR-75-1 cells, but did not inhibit the proliferation of MDA-MB-231 cells. Furthermore, the promotive effect on cell growth induced by E2 was also significantly inhibited by SGLXD treatment. With the treatment of 1.70, 3.40, 5.10 mg/mL SGLXD, the nuclear ERα protein level was reduced to 88.1%, 70.4% and 60.9% in MCF-7 cells, and was decreased to 43.0%, 38.4% and 5.9% in ZR-75-1 cells as compared with the control group. In T47D cells, the nuclear ERα protein was down-regulated to 51.3% and 4.3% by 3.40 and 5.10 mg/mL SGLXD treatment. The down-regulative effect of SGLXD on nuclear ERα was confirmed by immunofluorescence assay. SGLXD decreased the protein product of c-myc and bcl-2.

CONCLUSIONSSGLXD may exhibit selective inhibition effect on the proliferation of ER positive breast cancer cells. SGLXD reduced the nuclear ERα expression and the protein product of E2 target gene c-myc and bcl-2.

Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; genetics ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; MCF-7 Cells

BACKGROUND: Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells. METHODS: EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991. RESULTS: PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells. CONCLUSIONS PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase 6 ; genetics ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; Mice ; Piperazines ; pharmacology ; Pyridines ; pharmacology