The mitogen-stimulated serine/threonine kinase $p70^{S6k}$ plays an important role in the progression of cells from $G_0/G$_1$$ to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts family of mRNA transcripts which contain polypyrimidine tract at their 5 transcriptional start site. Here, we report that $p70^{S6k}$ was constitutively phosphorylated and activated to various degrees in serum-deprived AGS, A2058, HT-1376, MG63, MCF7, MDA-MB-435S, MDA-MB-231 and MB-157. Rapamycin treatment induced a significant dephosphorylation and inactivation of $p70^{S6k}$ in all cancer cell lines, while wortmannin, a specific inhibitor of PI3-K, caused a mild dephosphorylation of $p70^{S6k}$ in AGS, MDA-MB-435S and MB-157. In addition, SQ20006, methylxanthine phosphodiesterase inhibitor, reduced the phosphorylation of $p70^{S6k}$ in all cancer cells tested. Consistent with inhibitory effect of rapamycin on $p70^{S6k}$ activity, rapamycin inhibited [$^3H$]-thymidine incorporation and increased the number of cells at $G_{0}G_{1}$ phase. Furthermore, these inhibitory effects were accompanied by the decrease in growth of cancer cells. Taken together, the results indicate that the antiproliferative activity of rapamycin might be attributed to cell cycle arrest at $G_{0}G_{1}$ phase in human cancer cells through the inhibition of constitutively activated $p70^{S6k}$ of cancer cells and suggest $p70^{S6k}$ as a potential target for therapeutic strategies aimed at preventing or inhibiting tumor growth.
Objectives : The use of natural products with therapeutic properties is as ancient as human civilisation and, for a long time, mineral, plant and animal products were the main sources of drugs. Catalposide, the major iridoid glycoside isolated from the stem bark of Catalpa ovata G. Don (Bignoniceae) has been shown to possess anti-microbial and anti-tumoral properties. Heme oxygenase-1 (HO-1) is a stress response protein and is known to play a protective role against the oxidative injury. In this study, we examined whether catalposide could protect Neuro 2A cells, a kind of neuronal cell lines, from oxidative damage through the induction of HO-1 protein expression and HO activity. We also examined the effects of catalposide on the productions of tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$ and nitric oxide (NO) on RAW 264.7 macrophages activated with the endotoxin lipopolysaccharide. Methods : HO-1 expression in Neuro 2A cells was measured by Western blotting analysis. NO and $TNF--{\alpha}$ produced by RAW 264.7 macrophage were measured by Griess reagent and enzyme-linked immunosorbent assay, respectively. Results : The treatment of the cells with catalposide resulted in dose- and time-dependent up-regulations of both HO-1 protein expression and HO activity. Catalposide protected the cells from hydrogen peroxide-induced cell death. The protective effect of catalposide on hydrogen peroxide-induced cell death was abrogated by zinc protoporphyrin IX, a HO inhibitor. Additional experiments revealed the involvement of CO in the cytoprotective effect of catalposide-induced HO-1. In addition, catalposide inhibited the productions of $TNF--{\alpha}$ and NO with significant decreases in mRNA levels of $TNF--{\alpha}$ and inducible NO synthase. Conclusions : Our results indicate that catalposide is a potent inducer of HO-1 and HO-1 induction is responsible for the catalposide-mediated cytoprotection against oxidative damage and that catalposide may have therapeutic potential in the control of inflammatory disorders.
We investigated a method to improve anticancer activities of Acer mono wood extracts by ultra high pressure extraction process. The A. mono was extracted by water at $40^{\circ}C$ and 300 MPa for 15 min (High Pressure Extraction, HPE). The extraction yield by ultra high pressure extraction process was 5.42%. The cytotoxicity on human normal lung cell (HEL299) of the extracts from HPE showed 21.54% lower than that from conventional water extraction at $100^{\circ}C$ in adding the maximum concentration of 1.0 mg/$m{\ell}$. Ultra high pressure extracts process for 15 minutes extracts (HPE15) showed more potent scavenging effect than the control, BHA. On SOD-like test, the HPE15 showed highest activity as 32.4% at 1.0 mg/$m{\ell}$ concentration. Human stomach adenocarcinoma, liver adenocarcinoma, breast adenocarcinoma and lung adenocarcinoma cell growth were inhibited up to about 67~79%, in adding 1.0 mg/$m{\ell}$ of extracts from HPE. HPE was 20~25% higher than conventional water extraction. It was interesting that, among several cancer cell lines (stomach adenocarcinoma, liver adenocarcinoma), the growth of digestive related cancer cells were most effectively inhibited as about 75~79%. On in vivo experiment using ICR mice, the variation of body weight of mice group treated A. mono wood extracts from HPE of 100 mg/kg/day concentration was very lower than control and other group. The survival times of group treated this extracts was 61.96% longer than that of the control group and this extracts showed the lower tumor weight, which were 10.49 g than positive control as 16.17 g. Based on these results, we could tell that the HPE wood extracts of A. mono had higher anticancer activity than conventional water extraction. The results of HPE showed obvious advantages in higher efficiency, shorter extraction time, at lower energy costs.
The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation [1-3]. The intracellular β-catenin level, which is regulated by a proteasomal degradation pathway, is critical to Wnt/β-catenin pathway control [4]. Normally, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with the scaffolding protein Axin and the tumor suppressor protein adenomatous polyposis coli (APC), phosphorylate β-catenin at Ser45, Thr41, Ser37, and Ser33 [5, 6]. Phosphorylated β-catenin is ubiquitinated by the β-transducin repeat-containing protein (β-TrCP), an F-box E3 ubiquitin ligase complex, and ubiquitinated β-catenin is degraded via a proteasome pathway [7, 8]. Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis [9]. Genetic mutations in the APC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [10]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene were found in patients with colorectal cancer [11]. These mutations cause β-catenin to accumulate in the nucleus, where it forms complexes with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family to stimulate the expression of β-catenin responsive genes, such as c-Myc and cyclin D1, which leads to colorectal tumorigenesis [12-14]. Therefore, downregulating β-catenin response transcription (CRT) is a potential strategy for preventing and treating colorectal cancer. Plant cytokinins are N6-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid (isopentenyladenine, zeatin, and dihydrozeatin), aromatic (benzyladenine, topolin, and methoxytopolin), or furfural (kinetin and kinetin riboside), depending on their structure [15, 16]. Kinetin riboside was identified in coconut water and is a naturally produced cytokinin that induces apoptosis and exhibits antiproliferative activity in several human cancer cell lines [17]. However, little attention has been paid to kinetin riboside's mode of action. In this study, we show that kinetin riboside exerts its cytotoxic activity against colon cancer cells by suppressing the Wnt/β-catenin pathway and promoting intracellular β-catenin degradation.
Journal of the Korean Society of Food Science and Nutrition
/
v.44
no.4
/
pp.516-523
/
2015
This study investigated the antimutagenic and anticancer effects of Platycodon grandiflorum extract (PGE) and its fractions against carcinogenic N-nitrosodimethylamine (NDMA) and genotoxicity. The Ames Salmonella mutagenicity test employing histidine mutants of Salmonella Typhimurium TA98 and TA100 was used to examine the mutagenicity of PGE and its fractions. Bacterial reversion assay with S. Typhimurium TA98 and TA100 did not show a significantly increased number of revertant colonies. The same test was used to examine the ability of PGE and its fractions to prevent acquisition of N-methyl-N'-nitro-N-nitrosoguanidine- and 4-introquino-line-1-oxide-induced mutations. PGE and its fractions inhibited mutagenesis in a dose-dependent manner. Among the fractions, ethyl acetate fraction from PGE (PGEA) exhibited a higher antimutagenic effect than other fractions. PGE and its fractions suppressed the growth of cancer cell lines, including human cervical adenocarcinoma, human hepatocellular carcinoma, human breast adenocarcinoma, human lung carcinoma, and transformed primary human embryonic kidney cells. In addition, we evaluated the antitumor activity of PGEA and its fractions in sacorma-180 solid tumor-bearing mice. In vivo anticancer activity results showed that PGE and its fractions could more effectively suppress tumor growth than the control. PGEA showed higher in vitro and in vivo anticancer effects than PGE and other fractions, and PGEA inhibited NDMA formation. Thus, we showed that PGEA has antimutagenic and anticancer activities, making it a candidate anticancer material under these experimental conditions.
The role of protease-activated receptors (PARs) in lung tumors is controversial. Although PAR4 is preferentially expressed in human lung tissues, its possible significance in lung cancer has not been defined. The studies reported herein used a combination of clinical observations and molecular methods. Surgically resected lung adenocarcinomas and associated adjacent normal lung tissues were collected and BEAS-2B and NCI-H157 cell lines were grown in tissue culture. PAR4 expression was evaluated by RT-PCR, RT-qPCR, Western blotting and immunohistochemistry analysis. The results showed that PAR4 mRNA expression was generally decreased in lung adenocarcinoma tissues as compared with matched noncancerous tissues (67.7%) and was associated with poor differentiation (p=0.017) and metastasis (p=0.04). Western blotting and immunohistochemical analysis also showed that PAR4 protein levels were mostly decreased in lung adenocarcinoma tissues (61.3%), and were also associated with poor differentiation (p=0.035) and clinical stage (p=0.027). Moreover, PAR4 expression was decreased in NCI-H157 cells as compared with BEAS-2B cells. In conclusion, PAR4 expression is significantly decreased in lung adenocarcinoma, and down-regulation of PAR4 is associated with a more clinically aggressive phenotype. PAR4 may acts as a tumor suppressor in lung adenocarcinoma.
For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously upregulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, ${\beta}$-actin, RPII and 18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines. The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.
Hu Key-Soon;Choi Jong-Whan;Choi Soon-Chul;Park Tae-Won;You Dong-Soo
Journal of Korean Academy of Oral and Maxillofacial Radiology
/
v.28
no.1
/
pp.245-259
/
1998
Cellular transforming genes have been identified in a number of different tumor cell lines and tumor types. A significant number of these oncogenes belong to the ras gene family. The ras gene family consists of three closely related genes:H-ras, K-ras and N-ras which code for a related 21 kDa protein. Mutations in codon 12, 13 and 61 of one of the three ras genes convert these genes into acute oncogenes. The presence of H-ras gene mutations has important prognostic implications in various tumors. Each genomic DNA was isolated from tumors induced by implantation with DMBA, or by treatment with DMBA -implantation/irradiation. When genome DNA was transfected into NIH 3T3 cells and investigated by two-step PCR-RFLP, the fOllowing results were concluded: 1. Transformation foci developed in two groups when the genome DNA of two experimental groups were transfected into NIH 3T3 cells. 2. Transformation efficiency was 0.01-0.02 foci/㎍DNA in the experimental group with the DMBA-implantation, 0.01-0.03 foci/㎍lgDNA in the experimental group with the DMBA-implantation/irradiation according to results of transfection assay. 3. When the point mutation of H-ras gene was investigated by a two-step PCR-RFLP, there was 13.9% (5/36) in the experimental group with the DMBA implantation, 15.4 % (6/39) in the experimental group with the DMBA -implantation/irradiation. 4. The point mutation in codon 12 and 61 of H-ras was 5.6%(2/36) and 8.3%(3/36) in the experimental group with the DMBA implantation. 5. The point mutation in codon 12 and 61 of H-ras gene was 7.7%(3/39) in the experimental group with the DMBA -implantation/irradiation.
Park, Eun-Mi;Ye, Eun-Ju;Kim, Soo-Jung;Kim, Sol-Ah;Bae, Man-jong
Journal of the Korean Society of Food Science and Nutrition
/
v.34
no.7
/
pp.953-958
/
2005
This study was conducted to investigate the effect of beverage (beverage HC and beverage PG) using herbs on antimicrobial activity, proliferation of hepatic cancer cell (Hep3B) lines and sarcoma 180 (S-180) and antiallergy, respectively. Beverage PG showed higher antimicrobial activity than beverage HC against Staphylococcus aureus and Pseudomonas aeruginosa. Beverage HC and PG showed the tumor suppressive effect in mice injected with S-180 cells. The growth-inhibitoy ratio against tumor cells were $66\%\;for\;10\%$ beverage HC, $61\%\;for\;10\%$ beverage PG. In an anti-cancer test using Hep3B cells, beverage PG showed higher anti-proliferating effect than beverage HC. Beverage PG showed growth-inhibitory effect of $69.2\%\;at\;100\%$ beverage PG. Beverage PG inhibited histamine release from rat peritoneal mast cells (RPMC) activated by compound 48/80. In conclusion, these results suggest that beverage using herbs have an antimicrobial activity, anti-proliferating effect against Hep3B cell and S-180 tumor and will be beneficial in treatment of allergic reaction.
Choi, Jeong Su;Heo, Ji Hye;Kim, Dae Jin;Namkung, Su Min;Lee, Tae Bok;Lee, Min Woo;Kim, Suhng Wook
Korean Journal of Clinical Laboratory Science
/
v.49
no.2
/
pp.69-78
/
2017
Cordyceps militaris has been used in traditional Chinese medicine owing to its anticancer and immunomodulatory activities. Germanium compounds have also been shown to be associated with many pharmacological functions, such as antimicrobial, antiviral, antitumor, antimutagenic, and immunomodulating effects. In this study, we examined the biological properties of hot water extract from mycelial liquid culture of germanium-enriched C. militaris (CMGe). CMGe displayed a concentration-dependent antiproliferation activity against four human cancer cell lines. The antiproliferative activity of CMGe was 2-4-fold lower than that of hot water extract from mycelial liquid culture in C. militaris (CM). However, CM had a concentration-dependent cytotoxicity to human bone marrow-derived mesenchymal stem cells (MSCs). Contrastingly, CMGe did not cause any cellular damage to MSCs. MSCs cultured with CMGe displayed an increased proliferative activity with no cytotoxic effect. The oral administration of CMGe inhibited increased tumor volume and weight compared with the control group. CMGe has the potential to be used as an industrial product in medicinal foods as well as in pharmaceutical products.
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