PURPOSE: Although the effect of lysosome-associated protein transmembrane 4 beta (LAPTM4B) on the proliferation, migration, and invasion of breast cancer (BC) cells has already been studied, its specific role in BC progression is still elusive. Here, we evaluated the effect of different levels of LAPTM4B expression on the proliferation, invasion, adhesion, and tumor formation abilities of BC cells in vitro, as well as on breast tumor progression in vivo. METHODS: We investigated the influence of LAPTM4B expression on MCF-7 cell proliferation, invasion, adhesion, and tube formation abilities in vitro through its overexpression or knockdown and on breast tumor progression in vivo. RESULTS: Cell growth curves and colony formation assays showed that LAPTM4B promoted the proliferation of breast tumor cells. Cell cycle analysis results revealed that LAPTM4B promoted the entry of cells from the G1 into the S phase. Transwell invasion and cell extracellular matrix adhesion assays showed that LAPTM4B overexpression increased the invasion and adhesion capabilities of MCF-7 cells. More branches were observed in MCF-7 cells overexpressing LAPTM4B under an electron microscope. In comparison with LAPTM4B overexpression, LAPTM4B knockdown decreased the expression of vascular endothelial growth factor-A and significantly inhibited the vasculogenic tube formation ability of tumors. These results were also verified with western blot analysis. CONCLUSION: LAPTM4B promoted the proliferation of MCF-7 cells through the downregulation of p21 (WAF1/CIP1) and caspase-3, and induced cell invasion, adhesion, and angiogenesis through the upregulation of hypoxia-inducible factor 1 alpha, matrix metalloproteinase 2 (MMP2), and MMP9 expression. This specific role deems LAPTM4B as a potential therapeutic target for BC treatment.
Blotting, Western ; Breast Neoplasms ; Breast ; Caspase 3 ; Cell Cycle ; Disease Progression ; Down-Regulation ; Extracellular Matrix ; Hypoxia-Inducible Factor 1 ; In Vitro Techniques ; Matrix Metalloproteinase 2 ; MCF-7 Cells ; S Phase ; Up-Regulation ; Vascular Endothelial Growth Factor A

PURPOSE: This study investigated the role of natriuretic peptide receptor 2 (NPR2) on cell proliferation and testosterone secretion in mouse Leydig cells. MATERIALS AND METHODS: Mouse testis of different postnatal stages was isolated to detect the expression C-type natriuretic peptide (CNP) and its receptor NPR2 by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Leydig cells isolated from mouse testis were cultured and treated with shNPR2 lentiviruses or CNP. And then the cyclic guanosine monophosphate production, testosterone secretion, cell proliferation, cell cycle and cell apoptosis in mouse Leydig cells were analyzed by ELISA, RT-qPCR, Cell Counting Kit-8, and flow cytometry. Moreover, the expression of NPR2, cell cycle, apoptosis proliferation and cell cycle related gene were detected by RT-qPCR and Western blot. RESULTS: Knockdown of NPR2 by RNAi resulted in S phase cell cycle arrest, cell apoptosis, and decreased testosterone secretion in mouse Leydig cells. CONCLUSIONS: Our study provides more evidences to better understand the function of CNP/NPR2 pathway in male reproduction, which may help us to treat male infertility.
Animals ; Apoptosis ; Blotting, Western ; Cell Count ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Germ Cells ; Guanosine Monophosphate ; Humans ; Infertility, Male ; Lentivirus ; Leydig Cells ; Male ; Mice ; Natriuretic Peptide, C-Type ; Polymerase Chain Reaction ; Receptors, Peptide ; Reproduction ; Reverse Transcription ; RNA Interference ; S Phase ; Testicular Diseases ; Testis ; Testosterone

BACKGROUND/OBJECTIVES: Re-epithelialization has an important role in skin wound healing. Astaxanthin (ASX), a carotenoid found in crustaceans including shrimp, crab, and salmon, has been widely used for skin protection. Therefore, we investigated the effects of ASX on proliferation and migration of human skin keratinocyte cells and explored the mechanism associated with that migration. MATERIAL/METHOD: HaCaT keratinocyte cells were exposed to 0.25-1 µg/mL of ASX. Proliferation of keratinocytes was analyzed by using MTT assays and flow cytometry. Keratinocyte migration was determined by using a scratch wound-healing assay. A mechanism for regulation of migration was explored via immunocytochemistry and western blot analysis. RESULTS: Our results suggest that ASX produces no significant toxicity in human keratinocyte cells. Cell-cycle analysis on ASX-treated keratinocytes demonstrated a significant increase in keratinocyte cell proliferation at the S phase. In addition, ASX increased keratinocyte motility across the wound space in a time-dependent manner. The mechanism by which ASX increased keratinocyte migration was associated with induction of filopodia and formation of lamellipodia, as well as with increased Cdc42 and Rac1 activation and decreased RhoA activation. CONCLUSIONS: ASX stimulates the migration of keratinocytes through Cdc42, Rac1 activation and RhoA inhibition. ASX has a positive role in the re-epithelialization of wounds. Our results may encourage further in vivo and clinical study into the development of ASX as a potential agent for wound repair.
Blotting, Western ; Carotenoids ; Cell Movement ; Cell Proliferation ; Clinical Study ; Flow Cytometry ; Humans* ; Immunohistochemistry ; Keratinocytes* ; Pseudopodia ; Re-Epithelialization ; S Phase ; Salmon ; Skin* ; Wound Healing ; Wounds and Injuries

PURPOSE: Prolactinoma (prolactin-secreting pituitary adenoma) is one of the most common estrogen-related functional pituitary tumors. As an agonist of the dopamine D2 receptor, bromocriptine is used widely to inhibit prolactinoma progression. On the other hand, it is not always effective in clinical application. Although a dopamine D2 receptor deficiency contributes to the impaired efficiency of bromocriptine therapy to some extent, it is unknown whether there some other underlying mechanisms leading to bromocriptine resistance in prolactinoma treatment. That is the main point addressed in this project. MATERIALS AND METHODS: Human prolactinoma samples were used to analyze the S-phase kinase associated protein 2 (SKP2) expression level. Nutlin-3/adriamycin/cisplatin-treated GH3 and MMQ cells were used to analyze apoptosis in SKP2 overexpression or knockdown cells. SKP2 expression and the interaction partners of SKP2 were also detected after a bromocriptine treatment in 293T. Apoptosis was analyzed in C25 and bromocriptine-treated GH3 cells. RESULTS: Compared to normal pituitary samples, most prolactinoma samples exhibit higher levels of SKP2 expression, which could inhibit apoptosis in a p53-dependent manner. In addition, the bromocriptine treatment prolonged the half-life of SKP2 and resulted in SKP2 overexpression to a greater extent, which in turn compromised its pro-apoptotic effect. As a result, the bromocriptine treatment combined with C25 (a SKP2 inhibitor) led to the maximal apoptosis of human prolactinoma cells. CONCLUSION: These findings indicated that SKP2 inhibition sensitized the prolactinoma cells to bromocriptine and helped promote apoptosis. Moreover, a combined treatment of bromocriptine and C25 may contribute to the maximal apoptosis of human prolactinoma cells.
Apoptosis* ; Bromocriptine ; Half-Life ; Hand ; Humans* ; Pituitary Neoplasms ; Prolactinoma* ; Receptors, Dopamine D2 ; S-Phase Kinase-Associated Proteins

Chemotherapy-induced cancer cell secretomes promote resistance due, in part, to a predominant glycolytic energy metabolism, which drives aggressive cancer cell proliferation. However, the characterization of these secretomes and the molecular events that associate them with acquired drug resistance remain poorly understood. In this study, we show that secretomes of cancer cells with high-level paclitaxel resistance stimulated cell proliferation and suppressed drug-induced apoptosis of drug-sensitive cells. We also found that drug (docetaxel)-stimulated induction of interferon-α (IFN-α), IFN-λ and tumor necrosis factor-α (TNF-α) release in drug-sensitive cells was lowered by these secretomes. The promotion of cell proliferation by paclitaxel-resistant (PacR) cancer cell secretomes was associated, in part, with an increase in S phase of the cell cycle and downregulation of the cell death pathway that supports escape from apoptosis. In addition, we also found that the regulation of targeted glycolysis in PacR cancer cells alters the effects of the secretomes on cell growth, apoptosis, ATP generation and acquired drug resistance. Further study revealed that the deletion of FOXO3a transcription exacerbates glycolytic shift-induced apoptosis by rescuing TRAIL expression. By generating a docetaxel–cross-resistant PacR cancer cell line (PacR/DCT), we further clarified the role of FOXO3a in glycolysis-associated mediation of P-glycoprotein/ABCB1 hyperactivity that induces docetaxel cross-resistance. These findings suggest that suppression of the cellular energy supply by targeting glycolysis may inhibit the multiplicity of acquired chemotherapy resistance. Therefore, the therapeutic inhibition of FOXO3a might direct glycolysis to induce apoptosis and overcome multidrug resistance in cancer cells.
Adenosine Triphosphate ; Apoptosis* ; Cell Cycle ; Cell Death ; Cell Line ; Cell Proliferation ; Down-Regulation ; Drug Resistance ; Drug Resistance, Multiple ; Drug Therapy ; Energy Metabolism ; Glycolysis ; Necrosis ; Negotiating ; Paclitaxel ; S Phase ; United Nations*

OBJECTIVE: Glycogen synthase kinase 3β (GSK3β) is a pluripotent protein kinase involved in the development of cancers through regulation of numerous oncogenic molecules. Cyclin D1, an important regulator of G1 to S phase transition in various cells, is one of target proteins that GSK3β regulate. Our objective was to assess the expression of GSK3β and cyclin D1 in cervical neoplasm of different histologic grades and to identify their correlation in cervical carcinogenesis. METHODS: Immunohistochemical analysis of GSK3β and cyclin D1 was performed in a total of 137 patients with 12 normal, 62 cervical intraepithelial neoplasia (CIN) (31 CIN1 and 31 CIN3) and 63 invasive cancers including 56 squamous cell carcinomas and 7 adenocarcinomas. RESULTS: The expression of GSK3β increased in parallel with the lesion grade, while that of cyclin D1 decreased with severity of the lesion (P<0.001). There was a significant inverse correlation between GSK3β and cyclin D1 expression in overall cervical neoplasia (Φ=-0.413, P<0.001). GSK3β expression was higher in squamous cell carcinoma than in adenocarcinoma (P=0.049). CONCLUSION: These results suggest that the expressional increase in GSK3β plays a role in cervical carcinogenesis and has inverse correlation with cyclin D1 expression in this process. In addition, GSK3β expression appears to be associated with the histologic type of cervical cancer, especially squamous cell carcinoma.
Adenocarcinoma ; Carcinogenesis* ; Carcinoma, Squamous Cell ; Cervical Intraepithelial Neoplasia ; Cyclin D1* ; Cyclins* ; Glycogen Synthase Kinases* ; Glycogen Synthase* ; Glycogen* ; Humans ; Immunohistochemistry ; Protein Kinases ; S Phase ; Uterine Cervical Neoplasms

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.
Animals ; Cell Count ; Cell Cycle* ; Cell Line ; Cell Proliferation* ; Exosomes* ; Flow Cytometry ; Microarray Analysis ; MicroRNAs ; Microscopy, Confocal ; Myoblasts ; Rats ; S Phase ; Toxoplasma* ; Toxoplasmosis ; Trypan Blue

Orthodenticlehomeobox 1 (OTX1) overexpression had previously been associated with the progression of several tumors. The present study aimed to determine the expression and role of OTX1 in human hepatocellular carcinoma (HCC). The expression level of OTX1 was examined by quantitative real-time PCR (qRT-PCR) in 10 samples of HCC and paired adjacent non-cancerous tissues, and by immunohistochemistry (IHC) analysis in 128 HCC samples and matched controls. The relationship between OTX1 expression and the clinicopathological features werealso analyzed. Furthermore, the effects of OTX1 knockdown on cell proliferation and migration were determined in HCC cell lines. Axenograft mouse model was also established to investigate the role of OTX1 in HCC tumor growth. TheqRT-PCR and IHC analyses revealed that OTX1 was significantly elevated in HCC tissues compared with the paired non-cancerous controls. Expression of OTX1 was positively correlated with nodal metastasis status (P = 0.009) and TNM staging (P = 0.001) in HCC tissues. In addition, knockdown of OTX1 by shRNA significantly inhibited the proliferation and migration, and induced cell cycle arrest in S phase in vitro. Tumor growth was markedly inhibited by OTX1 silencing in the xenograft. Moreover, OTX1 silencing was causable for the decreased phosphorylation level of ERK/MAPK signaling. In conclusion, OTX1 contributes to HCC progression possibly by regulation of ERK/MAPK pathway. OTX1 may be a novel target for molecular therapy towards HCC.
Aged ; Animals ; Blotting, Western ; Carcinoma, Hepatocellular/metabolism/*pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Disease Progression ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Liver/metabolism/pathology ; Liver Neoplasms/metabolism/*pathology ; Lymphatic Metastasis ; MAP Kinase Signaling System ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; Neoplasm Staging ; Otx Transcription Factors/antagonists & inhibitors/genetics/*metabolism ; Phosphorylation ; RNA Interference ; Real-Time Polymerase Chain Reaction ; S Phase Cell Cycle Checkpoints ; Transplantation, Heterologous

BACKGROUND/AIMS: Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. METHODS: The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. RESULTS: The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27(kip-1) increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. CONCLUSIONS: Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27(kip-1).
Adenocarcinoma ; Cell Cycle* ; Cell Line* ; Cell Survival ; Humans* ; Immunoblotting ; Phosphorylation ; Phosphotransferases ; S Phase ; Stomach Neoplasms*

OBJECTIVETo investigate the anti-tumor activity and molecular mechanism of Tonglian Decoction (, TLD) on esophageal carcinoma Eca109 cells.

METHODSEca109 cells were treated with TLD and its separated formulae, including the clearing-heat and detoxification formula (Q), activating-blood and promoting-qi formula (H) and nourishing-yin and blood formula (Z). Cell proliferation was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, cell morphology was observed using a microscope, the cell cycle was measured using flow cytometry and the activity of the nuclear factor-kappa B (NF-κB) signal pathway was detected by Western blot.

RESULTSThe half maximal inhibitory concentrations of TLD, Q and H were 386, 771 and 729 mg/L, respectively. TLD, Q and H significantly inhibited cell proliferation, with 69.43%, 60.84% and 61.90% of treated cells in the G phase of the cell cycle. The percentage of cells in S phase increased significantly after treatment with TLD, Q, and H compared with the control group (P<0.05), and TLD showed the strongest effect. Z had no influence on the cell cycle compared with the control group (P>0.05). Western blot detection indicated slight differences in the inhibition of the NF-κB pathway by the different formulae. TLD formula strongly inhibited IKKβ, NF-κB, interleukin-6 and tumor necrosis factor-α expression compared with the control group.

CONCLUSIONSTLD inhibited Eca109 cell proliferation by arresting cells in S phase. The possible mechanism might be related to inhibiting the NF-κB transduction cascade. The combination of the herbs found in the three separate formulae, H, Q and Z, work synergistically in TLD to produce the inhibitory effects of TLD treatment on Eca109 proliferation.

Blotting, Western ; Cell Count ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Esophageal Neoplasms ; metabolism ; pathology ; Flow Cytometry ; Humans ; Inhibitory Concentration 50 ; NF-kappa B ; metabolism ; S Phase ; drug effects ; Signal Transduction ; drug effects