Cyclin D3 deficiency inhibits skin tumor development, but does not affect normal keratinocyte proliferation

Lee, Sung Hyun,Wang, Xian,Kim, Sun Hye,Kim, Yongbaek,Rodriguez-Puebla, Marcelo L. D.A. Spandidos 2017 Oncology letters Vol.14 No.3

<P>Rearrangement and amplification of the D-type cyclin genes have been reported in human cancer. Previous studies have demonstrated that Ras-mediated skin tumorigenesis depends on pathways that act through cyclin D1 and D2; however, the role of cyclin D3 remains unknown. The present study demonstrates that cyclin D3 ablation does not affect keratinocyte proliferation, but instead increases apoptosis levels in the bulge region of the hair follicle. Consequently, cyclin D3 ablation reduces skin papilloma development in a Ras-dependent carcinogenesis model. Previous results revealed that cyclin D3 preferentially binds to cyclin-dependent kinase 6 (CDK6) in mouse keratinocytes and transgenic expression of CDK6 (K5CDK6 mice) inhibits skin tumor development. Thus, we hypothesized that the inhibitory effect of CDK6 is dependent on cyclin D3 expression. To test this hypothesis, a mouse model that overexpresses CDK6 and does not express cyclin D3 (K5CDK6/cyclin D3-/− compound mouse) was developed. Biochemical analysis of the epidermis of K5CDK6/cyclin D3-/− mice revealed that cyclin D3 ablation resulted in increased expression of cyclin D1 protein, with a consequent elevation in the level of CDK6/cyclin D1 and CDK4/cyclin D1 complexes. These findings were concurrent with the increase skin papilloma malignant progression observed in K5CDK6/cyclin D3-/− mice. In summary the absence of cyclin D3 led to fewer number of papillomas in cyclin D3-ablated mice than in the wild-type owing to increased apoptosis, suggesting that alterations in cell survival are a crucial mechanism for crippling cellular defense against neoplasia. The results of the current study also suggest that although cyclin D3 expression does not alter the tumor suppressive role of CDK6 in skin carcinogenesis, the compensatory increase in cyclin D1 can shift the balance towards malignant progression.</P>

Expression of genes and their responses to enzyme replacement therapy in a Fabry disease mouse model.

Park, Eun-Sook,Choi, Jin-Ok,Park, Joo-Won,Lee, Mi Hee,Park, Hae-Young,Jung, Sung-Chul D.A. Spandidos 2009 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.24 No.3

<P>Fabry disease is a lysosomal storage disease caused by a deficiency of alpha-galactosidase A, which results in aberrant glycosphingolipid metabolism and accumulation of globotriaosylceramide (Gb3). Since a correlation between the level of Gb3 and clinical manifestations of Fabry disease has not been observed, we investigated potential diagnostic biomarkers. Hepatic and renal gene expression of male alpha-galactosidase A-deficient mice (Fabry mice) was compared with that of wild-type mice. Microarray analyses were performed using samples taken before and after intravenous infusion of alpha-galactosidase A. The identified genes were validated using quantitative real-time PCR and Western blot assay. Expression of hepatic Serum Amyloid A1 (Saa1), S100 Calcium-binding protein A8 and A9 (S100a8 and a9), and Lipocalin 2 (Lcn2) and renal Neuropeptide Y (Npy), Thrombospondin 2 and 4 (Tsp-2 and -4) was significantly upregulated in Fabry mice compared with wild-type mice and normalized by enzyme replacement therapy. Plasma concentrations of Lcn2 and Npy were also greater in Fabry mice and reduced to wild-type levels after enzyme replacement therapy, although the plasma concentrations of these proteins show heterogeneity. Upregulation of Saa1, S100a8, S100a9 and Lcn2 may modulate inflammation and Lcn2, Npy and Tsp may be associated with vascular and renal involvement in Fabry disease. Furthermore, these genes are promising targets for developing biomarkers for monitoring disease progression and therapeutic efficacy in patients with Fabry disease.</P>

Anti-apoptotic effects of human placental hydrolysate against hepatocyte toxicity <i>in vivo</i> and <i>in vitro</i>

Bak, Dong-Ho,Na, Jungtae,Choi, Mi Ji,Lee, Byung Chul,Oh, Chang Taek,Kim, Jeom-Yong,Han, Hae Jung,Kim, Moo Joong,Kim, Tae Ho,Kim, Beom Joon D.A. Spandidos 2018 International journal of molecular medicine Vol.42 No.5

<P>Apoptosis and oxidative stress are essential for the pathogenesis of acute liver failure and fulminant hepatic failure. Human placental hydrolysate (hPH) has been reported to possess antioxidant and anti-inflammatory properties. In the present study, the protective effects of hPH against D-galactosamine (D-GalN)- and lipopolysaccharide (LPS)-induced hepatocyte apoptosis were investigated <I>in vivo</I>. In addition, the molecular mechanisms underlying the anti-apoptotic activities of hPH against D-GalN-induced cell death <I>in vitro</I> were examined. Male Sprague-Dawley rats were injected with D-GaIN/LPS with or without the administration of hPH. Rats were sacrificed 24 h after D-GaIN/LPS intraperitoneal injection, and the blood and liver samples were collected for future inflammation and hepatotoxicity analyses. Changes in cell viability, apoptosis protein expression, mitochondrial mass, mitochondrial membrane potential, reactive oxygen species generation, and the levels of proteins and mRNA associated with a protective mechanism were determined in HepG2 cells pretreated with hPH for 2 h prior to D-GalN exposure. The findings suggested that hPH treatment effectively protected against D-GalN/LPS-induced hepatocyte apoptosis by reducing the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, interleukin-6, and tumor necrosis factor-α, and increasing the level of proliferating cell nuclear antigen. It was also found that hPH inhibited the apoptotic cell death induced by D-GalN. hPH activated the expression of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, and catalase, which were further upregulated by the Kelch-like ECH2-associated protein 1-p62-nuclear factor-erythroid 2-related factor 2 pathway, a component of oxidative stress defense mechanisms. Furthermore, hPH markedly reduced cytosolic and mitochondrial reactive oxygen species and rescued mitochondrial loss and dysfunction through the reduction of damage-regulated autophagy modulator, p53, and C/EBP homologous protein. Collectively, hPH exhibited a protective role in hepatocyte apoptosis by inhibiting oxidative stress and maintaining cell homeostasis. The underlying mechanisms may be associated with the inhibition of endoplasmic reticulum stress and minimization of the autophagy progress.</P>

PTEN/pAkt/p53 signaling pathway correlates with the radioresponse of non-small cell lung cancer.

Jung, Il Lae,Kang, Hyo Jin,Kim, Kug Chan,Kim, In Gyu D.A. Spandidos 2010 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.25 No.4

<P>The sensitivity or resistance of cancer cells and normal tissues to ionizing radiation plays an important role in the clinical setting of lung cancer treatment. However, to date the exact molecular mechanisms of intrinsic radiosensitivity have not been well explained. In this study, we compared the radiosensitivity or radioresistance in two non-small cell lung cancers (NSCLCs), H460 and A549, and investigated the signaling pathways that confer radioresistance. H460 cells showed a significant G2/M arrest after 12 h of irradiation (5 Gy), reaching 60% of G2/M phase arrest. A549 cells also showed a significant G2/M arrest after 12 h of exposure; however, this arrest completely disappeared after 24 h of exposure. A549 has higher methylated CpG sites in PTEN, which is correlated with tumor radioresistance in some cancer cells, than H460 cells, and the average of the extent of the methylation was approximately 4.3 times higher in A549 cells than in H460 cells. As a result, PTEN expression was lower in A549 than in H460. Conducting Western blot analysis, we found that PTEN acted as a negative regulator for pAkt, and the pAkt acted as a negative regulator for p53 expression. According to the above results, we concluded that the radiosensitivity shown in H460 cells may be due to the higher expression of PTEN through p53 signaling pathway.</P>

Melanoma antigen-encoding gene family member A1-6 and hTERT in the detection of circulating tumor cells following CD45 ⁻ depletion and RNA extraction

Kim, Dae-Dong,Yang, Chun-Seok,Chae, Hyun-Dong,Kwak, Sang-Gyu,Jeon, Chang-Ho D.A. Spandidos 2017 Oncology letters Vol.14 No.1

<P>A total of 76 blood samples from patients without malignant disease and 107 blood samples from patients with malignant disease were investigated for the presence of circulating tumor cells (CTCs). To detect CTCs, hematopoietic cells were removed from the blood samples and different RNA extraction methods were used to amplify the melanoma antigen-encoding gene family member A1-family member A6 (MAGE A1-6) and the human telomerase reverse transcriptase (hTERT) gene as potential CTC markers. Comparison between four methods for extracting RNA from the blood was performed. The samples were enriched by cluster of differentiation 45 (CD45) antibody capturing, and the reverse transcription-quantitative polymerase chain reaction was used to amplify the MAGE A1-6 and hTERT genes. MAGE A1-6 and hTERT gene expression levels were also evaluated in 14 cancer cell lines, and the MAGE A1-6 and hTERT expression levels were 85.7 and 100%, respectively. The RNeasy method demonstrated the most sensitivity in the SNU1 cells mixed with blood, although the differences between methods was non-significant. The positive expression levels of MAGE A1-6 and hTERT was 11.8% in the control group and 58.9% in those with malignant disease. In the 70 patients with colorectal cancer, positive expression levels of MAGE A1-6 or hTERT were significantly higher in stages T3 and T4 compared with in stages T1 and T2. The CTC detection method involving CD45 antibody capture, RNA extraction and MAGE A1-6 and hTERT reverse transcription resulted in good rates of sensitivity and specificity. Thus, the present study concluded that MAGE A1-6 and hTERT genes may be potential and practical markers for CTCs in a clinical setting.</P>

Elevated HDL2-paraoxonase and reduced CETP activity are associated with a dramatically lower ratio of LDL-cholesterol/total cholesterol in a hypercholesterolemic and hypertriglyceridemic patient.

Lee, Ji-Hye,Park, Jung-Heun,Lee, Sang-Hak,Kim, Jae-Ryong,Cho, Kyung-Hyun D.A. Spandidos 2010 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.25 No.6

<P>A female patient (64 years of age; body mass index, 26) had a markedly and relatively low low-density lipoprotein-cholesterol (LDL-C) level (97 mg/dl) despite high serum total cholesterol (TC) (331 mg/dl) and triacylglyceride levels (307 mg/dl). Since the expected LDL-C was 222 mg/dl, there was a significant difference between the calculation and measurement based on direct enzyme assay. Only 30% of serum cholesterol was associated with LDL-C in this patient. To determine the basis for the markedly low LDL-C/TC ratio, we isolated and analyzed lipoproteins from the patient as well as age- and gender-matched controls. The patient had lowered serum CETP activity and elevated paraoxonase activity with GOT and GPT values in the normal range. The very low-density lipoprotein particles from the patient were larger than those of the controls and enriched with lipid and protein, while the LDL from the patient (LDL-P) had a lower particle number and protein content than the controls. The LDL-P was more resistant to cupric ion-mediated oxidation. HDL2 from the patient (HDL2-P) had highly enhanced paraoxonase activity and antioxidant ability. The patient had a 1.5-fold higher level of apolipoprotein (apo) A-I expression in HDL2. ApoA-I in HDL2 and HDL3 from the patient showed no fragmentation, while the control had fragmented bands (17 and 21 kDa) in the HDL. The HDL2-P also had a larger particle size and greater protein content with less lipid content. HDL3-associated cholesteryl ester transfer protein was reduced in the patient, although the particle size was similar to the controls. In conclusion, a patient who had a markedly lower LDL-C/TC ratio despite hyperlipidemia associated with higher paraoxonase activity, higher apoA-I level and lower CETP activity without fragmentation of apoA-I in the HDL fraction is presented. The enhanced antioxidant and anti-inflammatory activity of HDL might contribute to the low LDL-C/TC ratio in this patient.</P>

Anticancer effects of an extract from the scallop <i>Patinopecten yessoensis</i> on MCF-7 human breast carcinoma cells

Lee, Chu,Chun, Wonjoo,Zhao, Rongjie,Kim, Young Dae,Nam, Myung Mo,Jung, Dae Hwa,Cho, Il Je,Jegal, Kyung Hwan,Lee, Tae Hoon,Kim, Young Woo,Park, Sang Mi,Ju, Seong A.,Lee, Chul Won,Kim, Sang Chan,An, Won D.A. Spandidos 2017 Oncology letters Vol.14 No.2

<P><I>Patinopecten yessoensis</I>, is a species of scallop and a marine bivalve mollusk. In traditional East Asian medicine, scallop meat is used as a drug for the treatment of diabetes, pollakisuria, and indigestion. The present study was conducted in order to examine the potential anticancer effects of scallop flesh extract (SE) on MCF-7 human breast cancer cells. An MTT assay was used to evaluate cell viability and flow cytometry was used for the assessment of cell cycle distribution and apoptosis. The alteration in protein expression level was determined by western blot analysis, and the amounts of docosahexaenoic acid and eicosapentaenoic acid in the SE were measured by gas chromatography. SE inhibited the growth of MCF-7 human breast cancer cells in a dose-dependent manner by inducing G0/G1 phase arrest. The cell cycle arrest was associated with the upregulation of p53 and p21, and downregulation of G1 phase-associated cyclin D1/cyclin-dependent kinase (Cdk) 4 and cyclin E1/Cdk 2. In addition, SE-mediated cell cycle arrest was associated with the promotion of apoptosis, as indicated by the expression of apoptosis-associated proteins and changes in nuclear morphology. SE appeared to induce the mitochondrial apoptotic cascade, as indicated by a decreased expression of Bcl-2, activation of Bcl-2 associated X protein, release of cytochrome c, decrease in procaspase-3, and an increase in cleaved-poly (ADP-ribose) polymerase (PARP). Furthermore, the expression levels of Fas-associated via death domain and cleaved caspase-8 were increased in a SE dose-dependent manner. Taken together, these results suggest that the intrinsic and extrinsic pathways of apoptosis are associated with the anticancer effects of SE on MCF-7 cells. Thus, SE may be a suitable candidate for the treatment and prevention of human breast cancer.</P>

Evaluation of annexin II as a potential serum marker for hepatocellular carcinoma using a developed sandwich ELISA method.

Ji, Na Young,Park, Mi-Young,Kang, Yun Hee,Lee, Chung Il,Kim, Dae Ghon,Yeom, Young Il,Jang, Ye Jin,Myung, Pyung-Keun,Kim, Jong Wan,Lee, Hee Gu,Kim, Jae Wha,Lee, Kyeong,Song, Eun Young D.A. Spandidos 2009 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.24 No.6

<P>Annexin II (Annexin A2, ANXA2) is a 36 kDa calcium-dependent phospholipid-binding protein that is located on the surface of most eukaryotic cells. ANXA2 is involved in several biological processes, including anti-inflammatory effects, Ca27+-dependent exocytosis, immune responses, Ca2+ transport and phospholipase A2 regulation. In our previous study, ANXA2 was identified as an up-regulated gene in hepatocellular carcinoma (HCC) tissue by cDNA microarray. In the present study, we have evaluated ANXA2 as a tumor-associated marker of HCC. We determined the ANXA2 levels in human liver tissues with HCC using real-time RT-PCR and Western blot analysis. For quantitative analysis of the ANXA2 protein in body fluids, we developed a sandwich ELISA system in which a polyclonal antibody and a monoclonal antibody specific to ANXA2 were employed as a capture antibody and a probe antibody, respectively. We detected the ANXA2 concentration in human serum using our newly developed system and evaluated its usefulness as a tumor marker. Overexpression of ANXA2 in human liver tissue was confirmed by real-time RT-PCR and Western blot analysis. The sandwich ELISA system for ANXA2 was developed for the detection of ANXA2 in human samples. The dose-response relationship between ANXA2 and optical density was linear in the range of 0-10 microg/ml and the sensitivity was 0.02 microg/ml. We determined the ANXA2 concentration in serum specimens using the newly developed sandwich ELISA. The serum ANXA2 concentrations of the patients with HCC (53.38+/-36.23 microg/ml) were significantly elevated when compared with those of normal individuals (28.81+/-24.94 microg/ml). These results suggest that expression of ANXA2 may be increased in HCC patients and may play an important role in liver cancer progression. This new ELISA method can be used as a tool for the detection of ANXA2 in human serum, particularly for cancer diagnostics.</P>

Apoptotic and anti-metastatic effects of the whole skin of <i>Venenum bufonis</i> in A549 human lung cancer cells

PARK, JEONG-SEOK,SHIN, DONG YEOK,LEE, YEON-WEOL,CHO, CHONG-KWAN,KIM, GI YOUNG,KIM, WUN-JAE,YOO, HWA-SEUNG,CHOI, YUNG HYUN D.A. Spandidos 2012 International journal of oncology Vol.40 No.4

<P>In the present study, the effects of the whole skin of <I>Venenum bufonis</I> on apoptotic and anti-invasive activity in A549 human lung cancer cells were investigated. Treatment with extract of the whole skin of <I>V. bufonis</I> (SVB) resulted in a significant decrease in cell growth of A549 cells, depending on dosage, which was associated with apoptosis induction, as proved by chromatin condensation and accumulation of apoptotic fraction. SVB treatment induced expression of death receptor-related proteins, such as death receptor 4, which further triggered activation of caspase-8 and cleavage of Bid. In addition, the increase in apoptosis by SVB treatment was correlated with dysfunction of mitochondria, activation of caspase-9 and -3, downregulation of IAP family proteins, such as XIAP, cIAP-1 and cIAP-2, and concomitant degradation of activated caspase-3-specific target proteins, such as poly (ADP-ribose) polymerase and β-catenin proteins. However, z-DEVD-fmk, a caspase-3-specific inhibitor, blocked SVB-induced apoptosis and increased the survival rate of SVB-treated cells, indicating that activation of caspase-3 plays a key role in SVB-induced apoptosis. In addition, within concentrations that were not cytotoxic to A549 cells, SVB induced marked inhibition of cell motility and invasiveness. Activities of matrix metalloproteinase (MMP)-2 and MMP-9 in AGS cells were dose-dependently inhibited by treatment with SVB, and this was also correlated with a decrease in expression of their mRNA and proteins, and upregulation of tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 mRNA expression. Further studies are needed; however, the results indicated that SVB induces apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways. Our data also demonstrated that MMPs are critical targets of SVB-induced anti-invasiveness in A549 cells.</P>

Effects of Asiasari radix on the morphology and viability of mesenchymal stem cells derived from the gingiva.

Jeong, Su-Hyeon,Lee, Ji-Eun,Jin, Seong-Ho,Ko, Youngkyung,Park, Jun-Beom D. A. Spandidos 2014 MOLECULAR MEDICINE REPORTS Vol.10 No.6

<P>Medicinal herbs used in traditional Oriental medicine, which have been in use clinically for thousands of years, are attractive sources of novel therapeutics or preventatives. Asiasari radix (A. radix) has been suggested for use in the treatment of dental diseases, including toothache and aphthous stomatitis. The aim of this study was to evaluate the effects of A. radix extracts on the morphology and viability of human stem cells derived from the gingiva. An Asiasarum heterotropoides extract was centrifuged and freeze-dried in a lyophilizer. Stem cells derived from the gingiva were grown in the presence of A. radix at concentrations ranging between 0.1 ?g/ml and 1 mg/ml (0, 0.1, 1, 10, 100 and 1,000 ?g/ml). Cell morphology was evaluated with an optical microscope and the viability of the cells was quantitatively analyzed with a cell counting kit-8 (CCK-8) assay for up to seven days. The untreated control group exhibited normal fibroblast morphology. The shapes of the cells following 0.1, 1, 10 and 100 ?g/ml A. radix treatments were similar to those of the control group. However, a significant change was noted in the 1,000 ?g/ml group on day 1, when compared with the untreated group. Furthermore, on day 7, the shapes of the cells following 100 and 1,000 ?g/ml A. radix treatments were rounder and fewer cells were present, when compared with those of the control group. The cultures that grew in the presence of A. radix did not exhibit any changes in the CCK?8 assay on day 2; however, significant reductions in cell viability were noticed following 100 and 1,000 ?g/ml A. radix treatment on days 5 and 7. Within the limits of this study, A. radix influenced the viability of the stem cells derived from the gingiva. Thus, the direct application of A. radix to oral tissues may produce adverse effects at high doses. Therefore, the concentration and application time of A. radix requires meticulous control to obtain optimal results. These effects require consideration, if the use of A. radix is planned for the treatment of dental diseases.</P>