• Title/Summary/Keyword: Korean-Western combination treatment

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Hepatitis C Virus Nonstructural 5A Protein (HCV-NS5A) Inhibits Hepatocyte Apoptosis through the NF-κb/miR-503/bcl-2 Pathway

  • Xie, Zhengyuan;Xiao, Zhihua;Wang, Fenfen
    • Molecules and Cells
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    • v.40 no.3
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    • pp.202-210
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    • 2017
  • The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor $(TNF)-{\alpha}$. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse $TNF-{\alpha}$-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

APOPTOTIC EFFECT IN COMBINATION OF CYCLOSPORIN A AND TAXOL ON ORAL SQUAMOUS CELL CARCINOMA CELL LINE THROUGH THE PI-3 KINASE/AKT1 PATHWAY (구강 편평세포암종 세포주에서 Cyclosporin A와 Taxol 투여시 PI-3 kinase/Akt1 Pathway에 의한 세포사멸 병용효과)

  • Kim, Kyu-Young;Lee, Jae-Hoon
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.33 no.5
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    • pp.426-436
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    • 2007
  • Oral cancer take up 2-6% of all carcinomas and squamous cell carcinoma, which is the most common type in oral cancer, has a poor prognosis due to its high metastasis and recurrence rates. In treating oral cancer, chemotherapy to the primary, metastasized and recurrent lesion is a very important and useful treatment, even though its widespread usage is limited due to high general toxicity and local toxicity to other organs. Taxol, a microtubule stabilizing agent, is an anticancer drug that induces cell apoptosis by inhibiting depolymerization of microtubules in between the metaphase and anaphase of the cell mitosis. Recently, its effectiveness and mechanism on various tumor has been reported. However, not much research has been done on the application of Taxol to oral squamous cell carcinoma. Cyclosporin A, which is an immunosuppressant, is being used on cancers and when co-administered with Taxol, effectiveness of Taxol is enhanced by inhibition of Taxol induced multidrug resistance. In this study, Cyclosporin A with different concentration of Taxol was co-administered to HN22, the oral squamous cell carcinomacell line. To observe the cell apoptosis and the mechanisms that take part in this process, mortality evaluation of tumor cell using wortmannin, c-DNA microarray, RT-PCR analysis, cytometry analysis and western blotting were used, and based upon the observation on the effect and mechanism of the agent, the following results were obtained: 1. The HN22 cell line viability was lowest when $100{\mu}M$ of Wortmannin and $5{\mu}g/ml$ of Taxol were co-administered, showing that Taxol participates in P13K-AKT1 pathway. 2. In c-DNA microarray, where $1{\mu}g/ml$ of cyclosporine A and 3mg/ml of Taxol were co-administered, no up regulation of AKT1, PTEN and BAD c-DNA that participate in cell apoptosis was observed. 3. When $1{\mu}g/ml$ of Cyclosporin A was applied alone to HN22 cell line, no difference was found in AKT1, PTEN and BAD mRNA expression. 4. Increased AKT1, mRNA expression was observed when $3{\mu}g/ml$ of Taxol was applied alone to HN22 cell line. 5. When $1{\mu}g/ml$ of Cyclosporin A and Taxol($3{\mu}g/ml\;and\;5{\mu}g/ml$) were co-administered to HN22 cell line, PTEN mRNA expression increased, whereas AKT1 and BAD mRNA decreased. 6. As a result of cytometry analysis, in the group of Cyclosporin A($1{\mu}g/ml$) and Taxol($3{\mu}g/ml$) co-administration, increased Annxin V was observed, which shows that apoptosis occurred by deformation of plasma membrane. However, no significant difference was observed with vary ing concentration. 7. In western blot analysis, no caspase 3 was observed in the group of Cyclosporin A($1{\mu}g/ml$) and Taxol($3{\mu}g/ml$) co-administration. From the results of this study, it can be concluded that synergistic effect can be observed in combination therapy of Taxol and Cyclosporin A on oral squamous cell carcinoma cell line, where decreased activity of the cell line was observed. This resulted in decreased AKT1 and BAD mRNA and increased PTEN mRNA expression and when wortmannin and Taxol were co-administered, the viability decreased which confirms that Taxol decreases the viability of tumor cell line. Hence, when Taxol and cyclosporine A are co-administered, it can be assumed that cell apoptosis occurs through AKt1 pathway.

Apoptotic Effect of co-treatment with HS-1200 and Cisplatin on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line (HS-1200과 cisplatin의 병용처리가 사람구강암세포에 미치는 세포자멸사 효과에 대한 연구)

  • Kim, Duk-Han;Kim, In-Ryoung;Park, Bong-Soo;Ahn, Yong-Woo;Jeong, Sung-Hee
    • Journal of Oral Medicine and Pain
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    • v.38 no.3
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    • pp.221-233
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    • 2013
  • Bile acids are polar derivatives of cholesterol essential for the absorption of dietary lipids and regulate the transcription of genes that control cholesterol homeostasis. Recently it have been identified the synthetic chenodeoxycholic acid (CDCA) derivatives HS-1200 and cisplatin showed apoptisis-inducing activity on various cancer cells in vivo and in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with HS-1200 and cisplatin on human tongue squamous cell carcinoma cells (SCC25 cells). To investigate whether the co-treatment with HS-1200 and cisplatin compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining and an analysis DNA hypoploidy. Westen blot analysis and immunofluorescent staining were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following this co-treatment. Furthermore, proteasome activity and mitochondrial membrane potential (MMP) change were also assayed. In this study, co-treatment with HS-1200 and cisplatin on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, reduction of MMP and proteasome activity, the increase of Bax and the decrease of Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD) whereas each single treated SCC25 cells did not show these patterns. Although the single treatment of $25{\mu}M$ HS-1200 and $4{\mu}g/ml$ cisplatin for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that the combination therapy with HS-1200 and cisplatin could be considered as a novel therapeutic strategy for human squamous cell carcinoma.

Apoptotic Effect of Co-Treatment with Chios Gum Mastic and Eugenol on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line (사람혀편평세포암종세포에서 Chios gum mastic과 eugenol의 병용처리가 미치는 세포자멸사 효과에 관한 연구)

  • Sohn, Hyeon-Jin;Yea, Byeong-Ho;Kim, In-Ryoung;Park, Bong-Soo;Jeong, Sung-Hee;Ahn, Yong-Woo;Ko, Myung-Yun
    • Journal of Oral Medicine and Pain
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    • v.36 no.3
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    • pp.147-160
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    • 2011
  • Eugenol (4-allyl-2-methoxyphenol) is a natural phenolic constituent extensively used in dentistry as a component of zinc oxide eugenol cement and is applied to the mouth environment. Chios gum mastic (CGM) is a resinous exudate obtained from the stem and the main leaves of Pistacia lenticulus tree native to Mediterranean areas. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with a natural product, CGM and natural phenolic compound, eugenol on SCC25 human tongue squamous cell carcinoma cell line. To investigate whether the co-treatment with eugenol and CGM compared to each single treatment efficiently reduces the viability of SCC25 cells, MTT assay was conducted. Induction and augmentation of apoptosis were confirmed by Hoechst staining, TUNEL staining and DNA hypoploidy. Westen blot analysis and immunofluorescent staining were performed to study the alterations of the expression level and the translocation of apoptosis-related proteins in co-treatment. In this study, co-treatment of with eugenol and CGM on SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensations, DNA fragmentation, the increase and decrease of Bax and Bcl-2, decrease of DNA content, the release of cytochrome c into cytosol, translocation of AIF and DFF40 (CAD) onto nuclei, and activation of caspase-3, caspase-6 caspase-7, caspase-9, PARP, Lamin A/C and DFF45 (ICAD) whereas each single treated SCC25 cells did not show or very slightly these patterns. Although the single treatment of 40 ${\mu}g$/ml CGM and 0.5 mM eugenol for 24 h did not induce apoptosis, the co-treatment of these reagents prominently induced apoptosis. Therefore our data provide the possibility that combination therapy with CGM and eugenol could be considered as a novel therapeutic strategy for human oral squamous cell carcinoma.

Liver Protective Effect of the Co-treatment of Rhei Radix et Rhizoma and Silymarin on TAA-induced Liver Injury (대황과 실리마린의 병용투여의 간섬유화 보호 효과)

  • Il-ha Jeong;Sang-woo Ji;Seong-soo Roh
    • The Journal of Internal Korean Medicine
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    • v.44 no.3
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    • pp.402-417
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    • 2023
  • Objective: Liver fibrosis is a highly conserved wound-healing response and the final common pathway of chronic inflammatory injury. This study aimed to evaluate the potential anti-fibrotic effect of the combination of Rhei Radix et Rhizoma water extract (RW) and silymarin in a thioacetamide (TAA)-induced liver fibrosis model. Methods: The liver fibrosis mouse model was established through the intraperitoneal injection of TAA (1 week 100 mg/kg, 2-3 weeks 200 mg/kg, 4-8 weeks 400 mg/kg) three times per week for eight weeks. Animal experiments were conducted in five groups; Normal, Control (TAA-induced liver fibrosis mice), Sily (silymarin 50 mg/kg), RSL (RW 50 mg/kg+silymarin 50 mg/kg), and RSH (RW 100 mg/kg+silymarin 50 mg/kg). Biochemical analyses were measured in serum, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), and ammonia levels. Liver inflammatory cytokines and fibrous biomarkers were measured by Western blot analysis, and liver histopathology was evaluated through tissue staining. Results: A significant decrease in the liver function markers AST and ALT and a reduction in ammonia and total bilirubin were observed in the group treated with RSL and RSH. Measurement of reactive oxygen species and MDA revealed a significant decrease in the RSL and RSH administration group compared to the TAA induction group. The expression of extracellular matrix-related proteins, such as transforming growth factor β1, α-smooth muscle actin, and collagen type I alpha 1, was likewise significantly decreased. All drug-administered groups had increased matrix metalloproteinase-9 but a decreasing tissue inhibitor of matrix metalloproteinase-1. RSL and RSH exerted a significant upregulation of NADPH oxidase 2, p22phox, and p47phox, which are oxidative stress-related factors. Furthermore, pro-inflammatory proteins such as cyclooxygenase 2 and interleukin-1β were markedly suppressed through the inhibition of nuclear factor kappa B activation. Conclusions: The administration of RW and silymarin suppressed the NADPH oxidase factor protein level and showed a tendency to reduce inflammation-related enzymes. These results suggest that the combined administration of RW and silymarin improves acute liver injury induced by TAA.

Apoptotic Effect of Co-Treatment with Valproic Acid and HS-1200 on Human Osteosarcoma Cells (Valproic acid와 HS-1200의 병용처리가 사람골육종세포에 미치는 세포자멸사 효과에 대한 연구)

  • Kim, Duck-Han;Lee, Kee-Hyun;Kim, In-Ryoung;Kwak, Hyun-Ho;Park, Bong-Soo;Jeong, Sung-Hee;Ko, Myung-Yun;Ahn, Yong-Woo
    • Journal of Oral Medicine and Pain
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    • v.35 no.3
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    • pp.165-175
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    • 2010
  • Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent as well, when findings indicated the substance inhibited proliferation and induced differentiation of primitive neuroectocdermal tumor cells in vivo (Cinatl et al., 1997). Antitmor activity of VPA is associated with its targeting histone deacetylases. Bile acids and their synthetic derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. It has been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis-inducing activity on various cancer cells in vitro. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and a CDCA derivative, HS-1200 on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and HS-1200 showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-7, caspase-3 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or $25\;{\mu}M$ HS-1200 for 48 h did not induce apoptosis, the co-treatment of them induced prominently apoptosis. Therefore our data provide the possibility that combination therapy of VPA and HS-1200 could be considered as a novel therapeutic strategy for human osteosarcoma.

Apoptotic Effect of Co-Treatment with Valproic Acid and 17AAG on Human Osteosarcoma Cells (Valproic acid와 17AAG의 병용처리가 사람골육종세포에 미치는 세포자멸사 효과에 대한 연구)

  • Park, Jun-Young;Park, Se-Jin;Kim, In-Ryoung;Park, Bong-Soo;Jeong, Sung-Hee;Ko, Myung-Yun;Ahn, Yong-Woo
    • Journal of Oral Medicine and Pain
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    • v.36 no.1
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    • pp.11-20
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    • 2011
  • Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent. And it is known that antitmor activity of VPA is associated with its targeted at histone deacetylases. 17AAG, Inhibition of HSP90 leads to the proteasome degradation of the HSP90 client proteins, such as Akt, Raf/Ras, Erk, VEGF, cyclin D and p53, and causes potent antitumor activity. It is reported that 17AAG-induced HSP90 inhibition results in prevention of cell proliferation and induction of apoptosis in several types of cancer. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and the HSP90 inhibitor, 17AAG on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and 17AAG showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-3, caspase-7 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or 0.5 ${\mu}M$ 17AAG for 48 h did not induce apoptosis, the co-treatment with them induced prominently apoptosis. Therefore our data in this study provide the possibility that combination therapy with VPA and 17AAG could be considered as a novel therapeutic strategy for human osteosarcoma.

Effects of Mutagenesis for Glycosylation Sites of Recombinant Human EPO During Production from Cultured CHO Cell

  • Lee, Hyun-Gi;Seong, Hwan-Hoo;Im, Seok-Ki;Chung, Hee-Kyoung;Lee, Poongyeon;Lee, Yeun-Kun;Min, Kwan-Sik;Chang, Won-Kyoung;Lee, Hoon-Taek
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.97-97
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    • 2002
  • Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2${\times}$10$\^$6//${\mu}\ell$. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6${\times}$10$\^$6//${\mu}\ell$. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.

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Long Noncoding RNA HOXA11-AS Modulates the Resistance of Nasopharyngeal Carcinoma Cells to Cisplatin via miR-454-3p/c-Met

  • Lin, Feng-Jie;Lin, Xian-Dong;Xu, Lu-Ying;Zhu, Shi-Quan
    • Molecules and Cells
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    • v.43 no.10
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    • pp.856-869
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    • 2020
  • To elucidate the mechanism of action of HOXA11-AS in modulating the cisplatin resistance of nasopharyngeal carcinoma (NPC) cells. HOXA11-AS and miR-454-3p expression in NPC tissue and cisplatin-resistant NPC cells were measured via quantitative reverse transcriptase polymerase chain reaction. NPC parental cells (C666-1 and HNE1) and cisplatin-resistant cells (C666-1/DDP and HNE1/DDP) were transfected and divided into different groups, after which the MTT method was used to determine the inhibitory concentration 50 (IC50) of cells treated with different concentrations of cisplatin. Additionally, a clone formation assay, flow cytometry and Western blotting were used to detect DDP-induced changes. Thereafter, xenograft mouse models were constructed to verify the in vitro results. Obviously elevated HOXA11-AS and reduced miR-454-3p were found in NPC tissue and cisplatin-resistant NPC cells. Compared to the control cells, cells in the si-HOXA11-AS group showed sharp decreases in cell viability and IC50, and these results were reversed in the miR-454-3p inhibitor group. Furthermore, HOXA11-AS targeted miR-454-3p, which further targeted c-Met. In comparison with cells in the control group, HNE1/DDP and C666-1/DDP cells in the si-HOXA11-AS group demonstrated fewer colonies, with an increase in the apoptotic rate, while the expression levels of c-Met, p-Akt/Akt and p-mTOR/mTOR decreased. Moreover, the si-HOXA11-AS-induced enhancement in sensitivity to cisplatin was abolished by miR-454-3p inhibitor transfection. The in vivo experiment showed that DDP in combination with si-HOXA11-AS treatment could inhibit the growth of xenograft tumors. Silencing HOXA11-AS can inhibit the c-Met/AKT/mTOR pathway by specifically upregulating miR-454-3p, thus promoting cell apoptosis and enhancing the sensitivity of cisplatin-resistant NPC cells to cisplatin.

Ozone Inhalation with 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)- 1-butanone and/or Dibutyl Phthalate Induced Cell Cycle Alterations via Wild-type p53 Instability in B6C3F1 Mice

  • Kim, Min-Young;Song, Kyung-Suk;Park, Gun-Ho;Kim, Hyun-Woo;Park, Jin-Hong;Kim, Jun-Sung;Jin, Hwa;Kook-Jong, Eu;Cho, Hyun-Sun
    • Toxicological Research
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    • v.20 no.1
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    • pp.71-82
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    • 2004
  • Changes in cell cycle control in the lungs and liver of the B6C3F1 mice (20 males per each group) exposed to ozone (0.5 ppm), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1.0 mg/kg), and dibutyl phthalate (DBP, 5,000 ppm) after 52 weeks were examined through Western, Northern blot, and immunohistochemistry based on alterations in protein expression levels of G1/S checkpoints (cyclin D1, cyclin E, and PCNA), G2/M checkpoints (cyclin B1, cyclin G, and cyclin A), negative regulators (p53, p21, GADD45, and p27), and positive regulator (mdm2). Expression levels of cyclins D1, E, G, PCNA, mutant p53, and mdm2 proteins were higher in the lungs and livers treated with combination of toxicants than in those treated with ozone only. Expression levels of the wild-type and mutant p53, p21, GADD45, p27, and mdm2 proteins and mRNAs were higher in toxicant-treated groups than those of the control. Immunohistochemical analysis revealed staining intensities of the PCNA, cyclin D1, c-myc and mdm2 protein- treated lungs and livers were stronger than those of the control group. Our results showed that combined treatment of ozone with NNK/DBP altered the cell cycle control through instability of the wild-type p53 gene. Such pivotal p53-mediated cell cycle alterations may be responsible for the toxicity observed under our experimental condition. These results may be applied to risk assessment of mixture-induced toxicity.