• Asian Pacific Journal of Cancer Prevention
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• 제15권9호
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• pp.4019-4023
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• 2014

Metformin has been shown to be useful in reducing insulin resistance by restoring sensitivity. Recent evidence suggests that metformin might also possess anti-tumour activity. This study aimed to investigate the effects of cisplatin combined with metformin on the proliferation, invasion and migration of HNE1/DDP human nasopharyngeal carcinoma (NPC) cells, and to provide a new target for treating metastasis. The MTT assay was used to assess viability of HNE1/DDP cells after exposure to different concentrations of 2, 5-diaminopyrimidine-4, 6-diol (DDP; 2, 4, 8, 16, and $32{\mu}mol{\cdot}L^{-1}$), metformin (5, 10, 15, 20, and $25{\mu}mol{\cdot}L^{-1}$), and $4{\mu}mol{\cdot}L^{-1}$ of DDP combined with metformin. Wound healing and transwell migration assays were performed to assess cell migration and invasion, and expression of E-cadherin and MMP-9 was detected using Western blotting. MTT assay results showed that DDP could inhibit the proliferation of HNE1/DDP cells in a time- and concentration-dependent manner, with an IC50 of $32.0{\mu}mol{\cdot}L^{-1}$ at 24 h (P < 0.05), whereas low concentrations of DDP had almost no inhibitory effects on cell invasion and migration. DDP combined with metformin significantly inhibited cell invasion and migration. In addition, genes related to migration and invasion, such as those of E-cadherin and MMP-9, showed differential expression in the NPC cell line HNE1/DDP. In the present study, with an increasing concentration of metformin, the expression of MMP-9 was downregulated whereas that of E-cadherin was significantly upregulated. Taken together, our results show that cisplatin combined with metformin has effects on proliferation, invasion, and migration of human NPC cells.

• Asian Pacific Journal of Cancer Prevention
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• 제16권2호
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• pp.571-578
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• 2015

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-${\gamma}$, TNF-${\beta}$ and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-${\gamma}$, TNF-${\beta}$ and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

• Molecules and Cells
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• 제41권3호
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• pp.188-197
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• 2018

Benzidine, a known carcinogen, is closely associated with the development of bladder cancer (BC). Epithelial-mesenchymal transition (EMT) is a critical pathophysiological process in BC progression. The underlying molecular mechanisms of mitogen-activated protein kinase (MAPK) pathway, especially extracellular regulated protein kinases 5 (ERK5), in regulating benzidine-induced EMT remains unclarified. Hence, two human bladder cell lines, T24 and EJ, were utilized in our study. Briefly, cell migration was assessed by wound healing assay, and cell invasion was determined by Transwell assay. Quantitative PCR and western blot were utilized to determine both gene expressions as well as protein levels of EMT and MAPK, respectively. Small interfering RNA (siRNA) was transfected to further determine ERK5 function. As a result, the migration and invasion abilities were enhanced, epithelial marker expression was decreased while mesenchymal marker expression was increased in human BC cell lines. Meanwhile, benzidine administration led to activation of ERK5 and activator protein 1 (AP-1) proteins, without effective stimulation of the Jun N-terminal kinase (JNK) or p38 pathways. Moreover, Benzidine-induced EMT and ERK5 activation were completely suppressed by XMD8-92 and siRNAs specific to ERK5. Of note, ERK1/2 was activated in benzidine-treated T24 cells, while benzidine-induced EMT could not be reversed by U0126, an ERK1/2 inhibitor, as indicated by further study. Collectively, our findings revealed that ERK5-mediated EMT was critically involved in benzidine-correlated BC progression, indicating the therapeutic significance of ERK5 in benzidine-related BC.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10911-10916
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• 2015

Background: Tumor metastases are the main reasons for oncotherapy failure. Paris polyphylla (Chinese name: Chonglou) has traditionally been used for its anti-cancer actions. In this article, we focus on the regulation of human lung cancer A549 cell metastases and invasion by Paris polyphylla steroidal saponins (PPSS). Materials and Methods: Cell viability was evaluated in A549 cells by MTT assay. Effects of PPSS on invasion and migration were investigated by wound-healing and matrigel invasion chamber assays. Adhesion to type IV collagen and laminin was evaluated by MTT assay. Expression and protease activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blotting and gelatin zymography, respectively. Results: PPSS exerted growth inhibitory effects on A549 cells, and effectively inhibited A549 cell adhesion, migration and invasion in a concentration-dependent manner. Western blotting and gelatin zymography analysis revealed that PPSS inhibited the expression and secretion of MMP-2 and MMP-9 in A549 cells. Conclusions: PPSS has the potential to suppress the migration, adhesion and invasion of A549 cells. PPSS could be a potential candidate for interventions against lung cancer metastases.

• Asian Pacific Journal of Cancer Prevention
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• 제15권14호
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• pp.5577-5582
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• 2014

Objective: To observe the effects of metastasis-associated tumor gene family 2 (MTA2) depletion on human breast cancer cell proliferation and metastasis. Methods: A short-hairpin RNA targeting MTA2 was chemically synthesized and transfected into a lentivirus to construct Lv-shMTA2 for infection into the MDA-MB231 human breast cancer cell line. At 48 hours after infection cells were harvested and mRNA and protein levels of MTA2 were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Cell viability and metastasis were assessed by CCK-8, wound-healing assay and Transwell assay, respectively. In addition, a xenograft model of human breast cancer was constructed to investigate cancerous cell growth and capacity for metastasis. Results: After infection with Lv-shMTA2, mRNA and protein levels of MTA2 was significantly reduced (p<0.05) and MDA-MB231 cell proliferation and metastasis were inhibited (p<0.05). In addition, mean tumor size was smaller than that in control group nude mice (p<0.05) and numbers of metastatic deposits in lung were lower than in control group mice (p<0.05). Depletion of MTA2 affected MMP-2 and apoptosis-related protein expression. Conclusions: For the first time to our knowledge we showed that MTA2 depletion could significantly inhibit human breast cancer cell growth and metastasis, implying that MTA2 might be involved in the progression of breast cancer. The role of MTA2 in breast cancer growth and metastasis might be linked with regulation of matrix metalloproteinase and apoptosis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제28권3호
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• pp.216-225
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• 2002

Hyaluronic acid (HA) is an almost essential component of extracellular matrices. Early in embryogenesis mesenchymal cells migrate, proliferate and differentiate, in part, because of the influence of HA. Since the features of embryogenesis are revisited during wound repair, including bone fracture repair, this study was initiated to evaluate whether HA has an effect on calcification and bone formation in an in vitro system of osteogenesis. Mouse calvaria Pre-osteoblast (MC3T3-E1) cells were cultured in ${\alpha}-MEM$ medium with microorganism-derivative hyaluronic acid that was produced by Strep. zooepidemicus which of molecular weight was 3 million units. The dosages were categorized in each 0.5, 1.0 and 2.0 mg/ml concentration experimental groups. After 2 and 4 days cultures in expeirmental and control groups, the tendency of cell proliferation, MTT assay, protein synthesis ability, collagen synthesis and alkaline phosphatase activity were analysed and bone nodule formation capacity were measured with Alizarin Red S stain after 29 days cultures. The cell proliferation was increased in time, especially the group of 0.5 and 1.0 mg/ml concentration of HA were showed prominent cell proliferation. After 2 and 4 days culture, experimental groups in general were greater cell activity in MTT assay. The protein synthesis was increased in all experimental groups compared to control group, especially most prominent in 1.0 mg/ml concentration group. The collagen synthesis capacity were increased in HA experimental groups, especially prominent in 1.0 mg/ml group and the activity of alkaline phosphatase were increased, especially also prominent in 1.0 mg/ml group, compared to control group. Above these, the activity of mouse carvarial pre-osteoblast cells was showed greater bone osteogenesis activity in all applied HA experimental group, especially group of 1.0 mg/ml concentration of HA.

• Clinical and Experimental Pediatrics
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• 제59권9호
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• pp.374-380
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• 2016

Purpose: Patients with unresectable, relapsed, or refractory osteosarcoma need a novel therapeutic agent. Metformin is a biguanide derivative used in the treatment of type II diabetes, and is recently gaining attention in cancer research. Methods: We evaluated the effect of metformin against human osteosarcoma. Four osteosarcoma cell lines (KHOS/NP, HOS, MG-63, U-2 OS) were treated with metformin and cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were evaluated using flow cytometric analysis, and migration and wound healing assay were performed. Fourteen female Balb/c-nude mice received KHOS/NP cell grafts in their thigh, and were allowed access to metformin containing water (2 mg/mL) ad libitum. Tumor volume was measured every 3-4 days for a period of 4 weeks. Results: Metformin had a significant antiproliferative effect on human osteosarcoma cells. In particular, metformin inhibited the proliferation and migration of KHOS/NP cells by activation of AMP-activated protein kinase and consequent inhibition of the mammalian target of rapamycin pathway. It also inhibited the proliferation of cisplatin-resistant KHOS/NP clone cells. Analysis of KHOS/NP xenograft Balb/c-nude models indicated that metformin displayed potent in vivo antitumor effects. Conclusion: Further studies are necessary to explore metformin's therapeutic potential and the possibilities for its use as an adjuvant agent for osteosarcoma.

• Journal of Microbiology and Biotechnology
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• 제18권11호
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• pp.1841-1847
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• 2008

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

• Genomics & Informatics
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• 제11권4호
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• pp.245-253
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• 2013

A radioresistant cell line was established by fractionated ionizing radiation (IR) and assessed by a clonogenic assay, flow cytometry, and Western blot analysis, as well as zymography and a wound healing assay. Microarray was performed to profile global expression and to search for differentially expressed genes (DEGs) in response to IR. H460R cells demonstrated increased cell scattering and acidic vesicular organelles compared with parental cells. Concomitantly, H460R cells showed characteristics of increased migration and matrix metalloproteinase activity. In addition, H460R cells were resistant to IR, exhibiting reduced expression levels of ionizing responsive proteins (p-p53 and ${\gamma}$-H2AX); apoptosis-related molecules, such as cleaved poly(ADP ribose) polymerase; and endoplasmic reticulum stress-related molecules, such as glucose-regulated protein (GRP78) and C/EBP-homologous protein compared with parental cells, whereas the expression of anti-apoptotic X-linked inhibitor of apoptosis protein was increased. Among DEGs, syntrophin beta 2 (SNTB2) significantly increased in H460R cells in response to IR. Knockdown of SNTB2 by siRNA was more sensitive than the control after IR exposure in H460, H460R, and H1299 cells. Our study suggests that H460R cells have differential properties, including cell morphology, potential for metastasis, and resistance to IR, compared with parental cells. In addition, SNTB2 may play an important role in radioresistance. H460R cells could be helpful in in vitro systems for elucidating the molecular mechanisms of and discovering drugs to overcome radioresistance in lung cancer therapy.

• Archives of Plastic Surgery
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• 제38권5호
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• pp.590-593
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• 2011

Purpose: Surgical site infections (SSIs) are the third most frequently reported nosocomial infection. Of these SSIs, mostly were confined to the incision associated with underlying disease as diabetes, cigarette smoking, systemic steroid use, obesity, operating room environment, suture and surgical technique. This study has been planned to reduce the SSIs by using Vicryl $plus^{(R)}$ (Ethicon, USA) which contains triclosan, a broad-spectrum antibacterial agent, into the infected wound to evaluate whether or not Vicryl $plus^{(R)}$ (Ethicon, USA) is effective to nosocomial bacteria using a zone of inhibition assay. Methods: We did a comparison of Vicryl $plus^{(R)}$ suture (with triclosan) size 2-0, 5-0 with $Vicryl^{(R)}$ suture (without triclosan) size 4-0 each as treatment and control group, applied in Mueller-Hinton agar infected by following mircroorganisms: Methicillin-sensitive $Staphylococcus$ $aureus$ (MSSA), Methicillin-resistant $Staphylococcus$ $aureus$ (MRSA), Acinetobacter baumanii, $Escherichia$ $coli$, Enterobacter faecalis, Pseudomonas aeruginosa, Candida albicans. Cultures were made of the selected mircroorganisms, seeding the study strain in agar plates for 24 and 48-hour period in an oven at $37^{\circ}C$ followed by zone of inhibition assay. Results: Vicryl $plus^{(R)}$ group has demonstrated to create a zone of inhibition against MRSA, MSSA and $A.$ $baumanii$, but no effect on $E.$ $faecalis$, $P.$ $aeruginosa$, $C.$ $albicans$. Vicryl $plus^{(R)}$ suture size 2-0 also had antibactericidal effect while Vicryl $plus^{(R)}$ suture size 5-0 did not. $Vicryl^{(R)}$ group had no zones of inhibition showing colonization at all mircroorganisms. Conclusion: Our results seem to warrant the use of Vicryl $plus^{(R)}$ as absorbable buried suture when concerning SSIs as a prophylaxis against surgical nosocomial infection.