Pseudomonas aeruginosa Exotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

Effects of Hypoxia on the Ubiquitin-proteasome System in Primary Cortical Neuronal Cell Cultures / 체질인류학회지

The ubiquitin-proteasome system is crucial in maintaining cellular growth and metabolism. Dysfunction of this system may contribute to neurodegenerative diseases, such as Parkinson's disease. But its effects on primary neurons are largely unknown. In the present study, we investigated the effects of proteasome inhibitor and hypoxia on primary neuronal cultures to determine whether proteasomal malfunction induces neuronal death. Neuronal apoptosis increased in primary cultured cortical neurons with treatment of proteasomal inhibitor in normoxic condition and in the presence or absence of proteasomal inhibitor in hypoxic condition. Also expression of PARP and activated caspase 3 increased. NF-kappaB, a key transcription factor in this system expression increased in hypoxic condition and proteasomal inhibition. Interestingly, hypoxic condition induced an expression and accumulation of alpha-synuclein in neuron, one of components of Lewy body in Parkinson's disease. Our findings determine that hypoxic condition may affect the ubiquitin-proteasome system. Furthermore, it suggests that hypoxic condition and proteasomal inhibitors are involved, at least in parts, in neurodegeneration of mouse model for Parkinson's disease.

Induction of Apoptosis by Genistein in Human Melanoma Cells / 대한해부학회지

Genistein is a naturally occurring isoflavone that has been identified predominantly in soybean. It has been found that genistein can inhibit the growth of various cancer cell lines. Melanoma continues to increase in incidence in many parts of the world and remains among the top six cancers as a cause of death and morbidity. Understanding and overcoming resistance mechanism(s) of melanoma to apoptosis would therefore facilitate identification of new therapeutic targets and development of new treatments. This study was undertaken to investigate whether genistein induced apoptosis on human melanoma cells (G361). Genistein had a significant dose- and time-dependent inhibitory effect on the viability of G361 cells. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by hoechst staining, and DNA electrophoresis. p53 levels were not altered by genistein treatment. Genistein treatment induced caspase-3 cleavage and activation. Poly (ADP-ribose)-polymerase (PARP) and DNA fragmentation factor 45 (DFF45), which are caspase-3 substrates, were cleaved during genistein-induced apoptosis. It was found that the caspase-6 substrate lamin A was cleaved, whose cleavage has been reported to be necessary for complete condensation of DNA during apoptosis. The expression level and phosphorylation of focal adhesion kinase (FAK) were reduced by genistein treatment. These results suggest that genistein may constitute a potential antitumor compound against melanoma occurring at oral mucosa and skin.

The mechanism of apoptosis induced by eugenol in human osteosarcoma cells

Eugenol is commonly used in dentistry for the sedation of toothache, pulpitis, and dental hyperalgesia. This study was performed to investigate the apoptotic effect of eugenol to human osteosarcoma (HOS) cells and the potential use of this compound in osteosarcoma cells. Eugenol showed the apoptotic effect in HOS cells in dose- and time-dependent manner. Fragmentation and condensation of DNA were showed by TUNEL assay, Hemacolor stain and Hoechst stain. In the DNA electrophoresis analysis, cells showed DNA degradation characteristic of apoptosis with a ladder pattern of DNA fragments. Apoptosis-related factors were analyzed by western blotting. Cells treated with eugenol showed caspase-3, PARP, lamin A and DFF-45 cleavage. Eugenol treatment induced caspase-3 cleavage and activation. Cleavages of PARP, DFF-45 and lamin A were accompanied with activation of caspase triggered by eugenol in HOS cells. Though this study needs more investigations, these results suggest that eugenol induce apoptosis via caspase dependent pathway in HOS cells and eugenol may constitute a potential antitumor compound against osteosarcoma cells.

Apoptotic Effect of Combination Treatment with a Proteasome Inhibitor, Lactacystin, and a Histone Deacetylase Inhibitor, Trichostatin A, on MCF-7 Cells / 대한해부학회지

Although much information has been accumulated about the synergistic interaction of proteasome inhibitors and HDAC inhibitors to induce apoptosis in a certain type of cells, much less is known currently about the underlying mechanism. This study was undertaken to explore the combination effect of a histone deacetylase inhibitor, TSA, and a proteasome inhibitor, lactacystin, on the induction of apoptosis. Pretreatment of TSA and subsequent treatment of lactacystin showed the strong antitumor activity and nuclear condensation. Western blot assay showed that combination treatment of TSA and lactacystin increased Bax/Bcl-2 ratio and decreased level of XIAP. Activation of caspase-7 and cleavage of PARP were demonstrated after the combination treatment. In combination treatment group, cell cycle arrest was induced at G2/M phase and abolished increase in proteasome activity. This study is elucidating the mechanims whereby targeting apoptotic machineries may help in directing therapeutic strategies.

Effects of Electroacupuncture on the Expression of Spinal Neuronal Nitric Oxide Synthase in Adjuvant-induced Arthritic Rat Model / 체질인류학회지

To examine the effects of electroacupuncture (EA) on central nociceptive modulation, expressional changes of spinal neuronal nitric oxide synthase (nNOS) were investigated in complete Freund's adjuvant (CFA)-injected rats. Inflammation was induced by an intraplantar injection of CFA into the hindpaw of male Sprague-Dawley rats. Bilateral EA stimulation at 2 Hz, 15 Hz and 120 Hz was applied at those acupoints corresponding to Zusanli and Sanyinjiao in man with 3-day intervals for 30 days. At 30 days after CFA-injection, effects of EA on nNOS expression were observed in the dorsal horn of the spinal cord using immunohistochemical methods. The mean integrated optical density of nNOS immunoreaction was significantly increased in the dorsal horn throughout L1 to L5 lumbar segments in CFA-injected rats. The nNOS expression was attenuated in all regions of the dorsal horn by all types of EA. Especially, these reaction was markedly decreased in the superficial laminae and nucleus proprius of L1 and L3 lumbar segments by three types of EA, but a marked decrease in the neck of the dorsal horn was observed only in 2 Hz stimulation. The marked decrease of nNOS also showed in nucleus proprius and the neck of L5 lumbar segments in 2 Hz and 15 Hz EA stimulated rats. It is concluded that EA treatment can attenuate chronic inflammatory process in CFA-injected rats through modulating expression of nNOS in the dorsal horn of the spinal cord.

Inflammatory Response of Secretoneurin -Immunoreactive Nerves in the Rat Uterus after Formalin Exposure / 체질인류학회지

It is known that there are numerous chemotatic secretoneurin -immunoreactive nerve fibers and movable MHC class II -immunoreactive dendritic cells in the normal uterine cervix. And the relationships between them are not fully understood. The aim of this study is to reveal that secretoneurin could give to chemotatic influence to dendritic cells in inflammational state. Virgin female Sprague -Dawley rats (n = 20; approximately 2 months old; 200 ~250 g body weight) were used in this study. Animals (n = 10) were injected with 5% formalin (0.5 ml/day, 5 days) in experiment group. Animals were deeply anesthetized with 3.5% chloral hydrate (100 mg/kg, i.p.) and uterine cervix were removed. Immunostaining was done according to standard methods used routinely. In brief, tissue sections were incubated with primary antibodies generated in mouse anti -rat MHC class II antibody and mouse or rabbit anti -rat secretoneurin antibody for single and double immunostains. FITC for secretoneurin and rhodamine for MHC class II were used as secondary antibodies in double stains. Tissue sections were observed by using light and confocal laser scanning microscopes. The results were as follows; 1. Numerous secretoneurin -immunoreactive nerve fibers were located in the lamina propria and those were not found in the epithelium of normal rat uterine cervix. 2. MHC class II -immunoreactive dendritic cells were mainly located in the epithelium and the lamina propria of normal rat uterine cervix. 3. On the inflammation state, MHC class II -immunoreactive dendritic cells were mainly located in the lamina propria and those were not found in the epithelium of rat uterine cervix. According to above results, it is suggested that secretoneurin can give to chemotatic influence to dendritic cells in inflammational state. Therefore, secretoneurin is considered to be used for dendritic cell immunotheraphy.

Lectin Histochemistry on the Surface Mucous Cells, Mucous Neck Cells and Chief Cells in the Gastric Mucosa of Developing Rat / 체질인류학회지

To localize glycoconjugates of surface mucous cells, mucous neck cells and chief cells in the developing rat, nine of biotinylated lectin(SBA, DBA, PNA, BSL-1, RCA-1, sWGA, UEA-1, Con A and LCA) were applied with ABC method. In the surface and gastric pit epithelium of body of the stomach, DBA affinity was not demonstrated. Although Con A and LCA affinity were slightly increased after birth, these affinities with RCA-1 and sWGA maintained constantly from fetal to adult rat. And UEA-1 affinity gradually increased from the end of suckling period. BSL-1 and PNA affinity showed a tendency to decrease and was not observed in most cells from the suckling and weanling period respectively. In the gastric gland proper, mucous neck cells and chief cells were not distinguished until the early weanling period. All affinities examined except DBA and BSL-1 were observed and increased in the gland of postnatal rat. With the approach of weanling period, more intense affinity for PNA, RCA-1, sWGA and UEA-1 were found on lower portion of the gastric gland proper and more intense affinity for SBA on upper portion. The mucous neck cells showed a similar affinities as gastric gland proper from the weanling period and two affinities for PNA and Con A were detected in the chief cell.

Immunohistochemical Expression of Inducible Nitric Oxide Synthase on the LPS-induced Shock and Wound Healing in Rats / 체질인류학회지

Inducible nitric oxide synthase (iNOS) expression of several organs on the lipopolysaccharides (LPS)-injected rats and on excisional wound was observed by immunohistochemical methods to investigate iNOS-positive cells during inflammation. iNOS expression was induced in response to LPS in the brain and these reactions were observed in the choroidal epithelium, ependymal cells and a few of nerve cells and fiber. A more intensive reaction of nerve cell and fiber was mainly observed in the corpus callosum and hypothalamus. Induction of iNOS of the lung was observed in alveolar macrophage, smooth muscle, pneumocytes and inflammatory cells infilterated in the alveolar septum. iNOS expression of the liver was detected in Kupffer cells, hepatocytes, bile duct and inflammatory cells of spotty necrosis. The cardiac muscle and endothelial cell of the heart showed positive iNOS expression. In the excisional wound, inflammatory cells including macrophages, neutrophil and fibrobast showed iNOS expression and mainly detected necrobiotic layer. Collectively, iNOS expression was induced in the several cell types during inflammatory process. So for better understanding the function of iNOS, more research should be done in relation to each cell type of organ.

The Expression of Transforming Growth Factor -alpha and Epidermal Growth Factor on the Cardiovascularsystem of Developing Mouse Embryo / 체질인류학회지

The distribution of transforming growth factor -alpha (TGF -alpha ) and epidermal growth factor (EGF) on the cardiovascular system of developing mouse embryos of gestational age 7 to 12 days were immunohistochemically (ABC method) studies to investigate the differential expression of these growth factors. Paraffin embedded sections were immunostained with antibodies for TGF -alpha and EGF. In the 8 -day -old mouse embryos, the endocardial tissue, myocardial tissue and cardiac jelly were all TGF -alpha stained. EGF stain was observed in the cardiac jelly and myocardial tissue but was not observed in the endocardial tissue. This suggests that in the initial phase of the cardiovascular system development, TGF -alpha function as earlier growth factor than EGF. The 9, 10 and 11 -day -old embryos showed TGF -alpha stain in the broad spectrum of developing cardiovascular tissues such as, the bulbus cordis, primitive atrium, sinus venosus, aortic sac, dorsal aorta, vitelline artery, endocardial cushion tissue, and myocardium of primitive ventricle. However, EGF stain was observed only in the bulbus cordis, primitive atrium and endocardial tissue. This finding indicates that TGF -alpha function as a more extensive growth factor than EGF. The 12 -day -old embryos showed stronger EGF stain than TGF -alpha in the primitive ventricle, bulbus cordis, and endocardial tissue. This suggests that EGF function as a more growth factor than TGF -alpha at this particular developmental stage and plays important role at the end stage of the primitive heart development.