제 3 회 신약개발 연구발표회 초록집 ( 1994 . 4 . 15 ~ 16 ) : 포스터발표 제 3 분과 ( 임상 , 안정성 평가 , 제제 ) ; 2-Acetylaminofluorene 의 면역독성 기작에 대한 연구

이미가엘,양규환 한국응용약물학회 1994 학술대회 Vol.1994 No.1

Differential Physiological Effects of Raf-1 Kinase Pathways Linked to Protein Kinase C Activation Depending on the Stimulus in v-H-ras-transformed Cells

이미가엘 대한암학회 2008 Cancer Research and Treatment Vol.40 No.2

Purpose: We investigated the molecular mechanism by which the Raf-1 kinase pathways that are linked to protein kinase C induce differential physiological effects, depending on the stimulus, by employing the pharmacological PKC activator PMA. Materials and Methods: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems and these cells were transiently transfected with the pMTH vector that encodes dominant-negative (DN) PKC-ε with using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. The Raf-1 kinase activity was measured by assessing the phosphorylation of recombinant MEK with using the immunoprecipitated Raf-1 proteins. The phosphorylated MEK protein bands were quantified by using Quantity One analysis software. Results: The pharmacological PKC activator phorbol- 12-myristate-13-acetate (PMA) and platelet-derived growth factor (PDGF) were able to induce the activation of Raf-1 kinase in the v-H-ras-transformed NIH3T3 fibroblasts. However, PMA was found to be much less sensitive PI3 kinase inhibitor or the chemical antioxidant than is PDGF. Especially, PMA mediated growth arrest while PDGF induced mitogenic signaling through the PKC-ε activation. Thus, the regulation of the Raf-1 cascade by both PDGF and PMA is likely to be intimately linked and they converge at the PKC level through different upstream pathways, as was shown by the inhibition of PDGF-induced Raf-1 kinase activation by the transient transfection with a dominant-negative mutant of PKC-ε. Conclusions: Taken together, these results imply that, depending on the stimulus, Raf-1 kinase leads to different physiological effects.

The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH 3T3 Cells

이미가엘,안준호,엄기환 대한암학회 2009 Cancer Research and Treatment Vol.41 No.2

Purpose : We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. Materials and Methods : Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. Results : The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells’ proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. Conclusion : Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.

Genotoxicity Assessment of Erythritol by Using Short-term Assay

이미가엘,정영신 한국독성학회 2013 Toxicological Research Vol.29 No.4

Erythritol is a sugar alcohol that is widely used as a natural sugar substitute. Thus, the safety of its usage is very important. In the present study, short-term genotoxicity assays were conducted to evaluate the potential genotoxic effects of erythritol. According to the OECD test guidelines, the maximum test dose was 5,000 μg/plate in bacterial reverse mutation tests, 5,000 μg/ml in cell-based assays, and 5,000 mg/kg for in vivo testing. An Ames test did not reveal any positive results. No clastogenicity was observed in a chromosomal aberration test with CHL cells or an in vitro micronucleus test with L5178Y tk+/− cells. Erythritol induced a marginal increase of DNA damage at two high doses by 24 hr of exposure in a comet assay using L5178Y tk+/− cells. Additionally, in vivo micronucleus tests clearly demonstrated that oral administration of erythritol did not induce micronuclei formation of the bone marrow cells of male ICR mice. Taken together, our results indicate that erythritol is not mutagenic to bacterial cells and does not cause chromosomal damage in mammalian cells either in vitro or in vivo.

신규합성물 KNF1002의 세균을 이용한 복귀돌연변이시험

최기섭,이미가엘,양경형,유용만 한국농약과학회 2002 한국농약과학회 학술발표대회 논문집 Vol.- No.-

Autophagy inhibition in 3T3-L1 adipocytes breaks the crosstalk with tumor cells by suppression of adipokine production

황성희,이미가엘 한국통합생물학회 2020 Animal cells and systems Vol.24 No.1

Several studies have revealed the functional importance of autophagy in both adipogenesis and carcinogenesis. Here, we investigated autophagy as a link between tumorigenesis and adipogenesis using 3T3-L1 cells, which have been shown to closely mimic the in vivo differentiation process. The relative levels of LC3-II/I showed that autophagy was the highest after 4–6 days of initiation of differentiation and it diminished thereafter. Furthermore, chloroquine (CQ), a late autophagy inhibitor, effectively inhibited adipogenic differentiation of 3T3-L1 cells, suggesting that autophagy may have a positive impact on adipogenic differentiation. Notably, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that CQ completely blocked the mRNA expression of three adipokines (adiponectin, leptin, and peroxisome proliferator-activated receptor-γ (PPARγ)), which increased proportionally to adipocyte differentiation. Using adipokine antibody arrays, we also found that among 38 adipokines examined, 6 adipokines were significantly differentially regulated in mature adipocytes compared to those in preadipocytes. A comparative analysis of adipokine production revealed that CQ-treated adipocytes displayed a profile similar to that of preadipocytes. Subsequently, CQ treatment significantly inhibited the migration capacity of v-Haras-transformed cells in both 3T3-L1 adipocyte-conditioned medium and co-culture with 3T3-L1 using a transwell plate. Taken together, our results suggest that autophagy inhibition blocks the production of mediators relevant to the adipogenic process and may significantly contribute to reducing obesity-related cancer risk.

The pro-death role of autophagy and apoptosis in cell death induced by the BH3 mimetic gossypol

김나연,이미가엘 한국통합생물학회 2014 Animal cells and systems Vol.18 No.3

Gossypol has been identified as a natural BH3 mimetic and a small-molecule inhibitor of Bcl-2 that can induce apoptosis invarious cancer cell lines. In this study, we evaluated the effect of gossypol on apoptosis-proficient (wild-type [WT] mouseembryonic fibroblasts, MEFs) and apoptosis-deficient cells (Bax–/–/Bak–/– MEFs) in order to determine whether apoptosisindeed plays an essential death-promoting role in gossypol-mediated cell death. Unexpectedly, we found that Bax–/–/Bak–/–double-knockout (DKO) MEFs were more sensitive than the WT cells to gossypol-induced cell death. Gossypol causedprogressive emergence of both apoptotic and necrotic cells in DKO MEFs; this result suggests that gossypol induces Bax/Bak-independent apoptosis. Conversely, fluorescence-activated cell sorting profiles showed no apoptotic cell death in WTMEFs. Rather, incubation of WT MEFs with gossypol markedly induced autophagy, as evidenced by punctate patterns ofLC3 immunoreactivity, indicative of apoptosis-independent autophagic cell death. Nonetheless, there was only limitedinduction of autophagy in DKO MEFs. No significant differences between WT and DKO MEFs were observed in cell cycleprofiles. Gossypol caused a weak increase of the percentage of cells at the G2/M checkpoint in the two cell lines, with aconcomitant small decrease in the number of cells in the S phase. Taken together, our results suggest that apoptotic celldeath is induced by gossypol in DKO MEFs, whereas autophagy plays a death-promoting role in gossypol-mediated deathof WT MEFs cells.

Assessment of the Dermal and Ocular Irritation Potential of Lomefloxacin by Using In Vitro Methods

안준호,엄기환,이미가엘 한국독성학회 2010 Toxicological Research Vol.26 No.1

The evaluation of eye and skin irritation potential is essential to ensuring the safety of human in contact with a wide variety of substances. Despite this importance of irritation test, little is known with respect to the irritation potency of lomefloxacin, a fluoroquinolone antibiotic, which has been known to cause phototoxicity with an abnormal reaction of the skin. Thus, to investigate the tendency of lomefloxacin to cause eye and skin irritation, we carried out in vitro eye irritation test using Balb/c 3T3, and in vitro skin irritation test using KeraSkinTM human skin model system. 3T3 neutral red uptake assay has been proposed as a potential replacement alternative for the Draize Eye irritation test. In this study, the IC50 value obtained for lomefloxacin was 375 μg. According to the classification model used for determining in vitro categories,lomefloxacin was classified as moderately irritant. For evaluation of skin irritation, engineered epidermal equivalents (KeraSkinTM) were subjected to 10 and 25 mg of lomefloxacin for 15 minutes. Tissue damage was assessed by tissue viability evaluation, and by the release of a pro-inflammatory mediator, interleukin-1α. Lomefloxacin increased the interleukin-1α release after 15 minutes of exposure and 42 hours of post incubation, although no decrease in viability was observed. Therefore, lomefloxacin is considered to be moderately irritant to skin and eye.

Upregulation of S100A9 contributes to the acquired resistance to BRAF inhibitors

황성희,안준호,이미가엘 한국유전학회 2019 Genes & Genomics Vol.41 No.11

Backgrounds Acquired resistance is a significant clinical challenge in targeted therapy of melanomas using BRAF inhibitors. We previously identified that downregulation of miR-92a-1-5p confers acquired resistance to BRAF inhibition using an miRNA array platform. Objective In this study, we investigated the target genes of miR-92a-1-5p and their functional significance in BRAF inhibitor resistance. Methods The miRNA target prediction data were combined with RNA-Seq data to identify possible target genes for miR- 92a-1-5p. Cellular effects of target genes were further examined using siRNA knockdown, WST-1 assay, and immunoblotting analysis. Results We selected S100 calcium-binding protein A9 (S100A9) as a possible target gene for functional validation. S100A9 knockdown abrogated resistance to PLX4720 in A375P/Mdr cells. This result was similar to those described earlier for miR-92a-1-5p, indicating that miR-92a-1-5p inhibits cell viability by targeting S100A9. S100A9 overexpression partially conferred PLX4720 resistance to A375P cells. We also demonstrated that MAPK re-activation does not contribute to the promotion of BRAF inhibitor resistance by S100A9. Conclusion Taken together, our results indicate that S100A9 might be functionally involved in development of resistance to BRAF inhibitors and might be a target for melanoma therapy in the future.

Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells

안준호,한별이,이미가엘 한국응용약물학회 2015 Biomolecules & Therapeutics(구 응용약물학회지) Vol.23 No.4

The clinical benefi ts of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.