Ki-Hwan Eum

Crosstalk between autophagy and apoptosis in the regulation of paclitaxel-induced cell death in v-Ha-ras-transformed fibroblasts

The previous studies by this author group has shown that paclitaxel, a mitotic inhibitor used in breast cancer chemotherapy, inhibits cell growth via induction of Raf-1-dependent apoptosis. In this article, the role of autophagy in paclitaxel anticancer action was investigated using v-Ha-ras-transformed NIH 3T3 cells. Paclitaxel induced a notable increase in the number of fluorescent particles labeled with monodansylcadaverine (MDC), a specific marker for autophagic vacuoles. MDC-labeled vacuoles clearly exhibited the fluorescent-tagged LC3 in cells transiently overexpressing GFP-LC3 (a protein that associates with autophagosome membranes). However, autophagy inhibition with 3-methyladenine (3-MA) failed to rescue v-Ha-ras-transformed NIH 3T3 cells from paclitaxel-induced cell death. More interestingly, the apoptosis inhibition by overexpression of the X-linked inhibitor of apoptosis (XIAP) did not fully block the cell death by paclitaxel, implying that apoptosis inhibition might accelerate the autophagic components of the paclitaxel response. Conversely, Raf-1 shRNA expression protected against paclitaxel-induced cell death through the simultaneous inhibition of both autophagy and apoptosis. These results suggest that both autophagy and apoptosis act as cooperative partners to induce cell death in v-Ha-ras-transformed NIH 3T3 cells treated with paclitaxel.

Targeting the autophagy pathway using ectopic expression of Beclin 1 in combination with rapamycin in drug-resistant v-Ha-ras-transformed NIH 3T3 cells.

The effectiveness of an apoptosis-targeting therapy may be limited in tumor cells with defects in apoptosis. Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target (mTOR), inhibition of which results in autophagic cell death. In our study using multidrug-resistant v-Ha-rastransformed NIH3T3 (Ras-NIH 3T3/Mdr) cells, we demonstrated that rapamycin-induced cell death may result from 2 different mechanisms. At high rapamycin concentrations (≥ 100 nM), cell death may occur via an autophagy-dependent pathway, whereas at lower concentrations (≤ 10 nM), cell death may occur after G1-phase cell cycle arrest. This effect was accompanied by upregulation of p21Cip1 and p27Kip1 expression via an autophagy-independent pathway. We also tested whether inhibition of mTOR with low concentrations of rapamycin and ectopic Beclin-1 expression would further sensitize multidrug resistance (MDR)-positive cancer cells by upregulating autophagy. Rapamycin at low concentrations might be insufficient to initiate autophagosome formation in autophagy but Beclin-1 overexpression triggered additional processes downstream of mTOR during G1 cell cycle arrest by rapamycin. Our findings suggest that these combination strategies targeting autophagic cell death may yield significant benefits for cancer patients, because lowering rapamycin concentration for cancer treatment minimizes its side effects in patients undergoing chemotherapy.

Differential inhibitory effects of two Raf-targeting drugs, sorafenib and PLX4720, on the growth of multidrug-resistant cells

B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade in human cancers, making it an important therapeutic target. Here, we describe the differential effects of two Raf-targeting drugs, sorafenib and PLX4720, on multidrug-resistant v-Ha-ras-transformed cells (Ras-NIH 3T3/Mdr). We demonstrate that the growth of the NIH 3T3/Mdr cell line was affected in a dose-dependent manner more significantly by the pan-Raf inhibitor sorafenib than by the selective mutant B-Raf inhibitor PLX4720. Despite their differential effects on LKB1/AMPK phosphorylation, both sorafenib and PLX4720 inhibited downstream mTOR signaling with concomitant induction of autophagy, implying that the differential effects of sorafenib and PLX4720 on multidrug-resistant cells might not be due to different levels of autophagy and apoptosis. Interestingly, sorafenib caused a dose-dependent increase in rhodamine 123 uptake and retention. More importantly, sorafenib reversed the resistance to paclitaxel in Ras-NIH 3T3/Mdr cells. Moreover, MEK/ERK signaling was hyperactivated by the selective mutant B-Raf inhibitor PLX4720 and inhibited by the pan-Raf inhibitor sorafenib. Our data suggest that sorafenib sensitivity in MDR cells is mediated through the inhibition of P-glycoprotein activity following strong inhibition of Raf/MEK/ERK signaling. Thus, Raf inhibition with sorafenib might be a promising approach to abrogate the multidrug resistance of cancer cells.

Genotoxicity Studies on Carrageenan : Short-term In Vitro Assays

Carrageenan is a naturally-occurring sulfated polygalactan which has been widely used in the dairy industry and a gelling agent in non-dairy products. In this study, four short-term in vitro genotoxicity assays were investigated to evaluate the potential genotoxic effects of carrageenan. The mutagenic-ity of carrageenan was evaluated up to a maximum dose of 5 mg/plate in Ames test. There was no increase in the number of revertant colonies compared to its negative control at any dose in all of strains tested. To assess clastogenic effect, the in vitro chromosomal aberration assay was performed using Chinese hamster lung cells. Carrageenan was not considered to be clastogenic in this assay at up to the highest feasible concentration which could be evaluated. The in vitro comet assay and micronucleus test results obtained on L5178Y cells also revealed that carrageenan has no genotoxicity potential, although there was a marginal increase in micronuclei frequencies and DNA damage in the respective micronucleus and comet assays. Taken together, our results indicate that carrageenan was not genotoxic based on four in vitro genotoxicity results.

The enhancement of Raf-1 kinase activity by knockdown of Spry2 is associated with high sensitivity to paclitaxel in v-Ha -ras -transformed NIH 3T3 fibroblasts

We previously demonstrated that the downregulation of Raf-1 kinase may contribute to the development of acquired resistance in paclitaxel-resistant cells. In this study, we determine whether the sensitivities of parental and its v-Ha-ras-transformed NIH 3T3 cells to paclitaxel were dependent on Raf-1 kinase activity. Paclitaxel sensitivity of v-Ha-ras-transformed cells was found to be significantly higher than that of its parental cells. Paclitaxel transiently increased Raf-1 kinase activity in v-Ha-ras-transformed cells while showing no effect on its parental cells, suggesting that the Raf-1-MAP kinase pathway is proapoptotic. Furthermore, using siRNA-mediated Raf-1 knockdown analysis, we showed that Raf-1 knockdown cells were more resistant than control cells to paclitaxel treatment. In particular, the expression of the gene SPRY2, which has been known to act as an inhibitor on Ras/Raf/MAPK signaling, was downregulated after the treatment with paclitaxel. Methylation-specific PCR also revealed that downregulation of Spry2 was associated with altered methylation of the CpG-rich region of the SPRY2 exon 1. In addition, the Spry2 protein knockdown cells were more susceptible to paclitaxel treatment than control cells. Taken together, our results suggest that the enhancement of Raf-1 kinase activity by knockdown of Spry2 is associated with high sensitivity to paclitaxel.

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

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.

Assessment of the dermal and ocular irritation potential of alcohol hand sanitizers containing aloe vera with in vitro and in vivo methods

Alcohol-based hand sanitizers have been proven effective in reducing the spread of microbial infections. However, frequent use of alcohol-based formulations of hand sanitizers can cause adverse skin effects, such as allergic and irritant contact dermatitis. Thus, we evaluated the irritant potency of alcoholbased hand sanitizers containing aloe vera and clove extract with four irritation studies and compared in vivo and in vitro results. The Draize rabbit eye and skin irritancy tests were conducted to evaluate the in vivo ocular and dermal irritation potential. For in vitro evaluation of skin irritation, tissue damage was assessed with the MTT assay and by examining the release of interleukin-1α (IL-1α) using engineered epidermal equivalents (KeraSkin™). A 3T3-neutral red uptake (NRU) assay was performed as a potential alternative to the Draize eye irritation test. Sanitizers containing aloe vera and clove extract were non-irritating to skin, and the results were comparable with both the in vivo and in vitro MTT viability assay. However, an increase in IL-1α release was observed. The results of the in vivo eye irritation toxicity studies in rabbits demonstrated reversible conjunctival redness and edema, whereas the test article was non-irritating in the in vitro 3T3-NRU study. These data indicate that the eye is more sensitive to alcohol-based hand sanitizerinduced damage than the skin. Furthermore, in vitro tests are useful for screening purposes, but not for the elimination of in vivo testing.

Abstract 941: Discovery of novel VEGFR2 and Raf-1 kinase inhibitorsuppressing MAPK signaling pathway in K-Ras mutant cancer cell-lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Signal transduction in the Ras-Raf-MEK-ERK (MAPK) pathway plays a key role in cell survival, growth and proliferation. The pathway is controlled by extracellular signals through receptor tyrosine kinases (RTK) and is activated by oncogenic mutations in many types of cancer. The B-Raf mutants are found in various cancers, such as melanoma, colorectal, ovarian, and prostate cancer. The Ras mutants are also found in various carcinomas including colon cancer, pancreatic cancer, non-small-cell lung cancer, and liver cancer. The mutated K-Ras gene sends continuous signals to the colorectal cancer cells, telling them to grow independent of the way ERBITUX blocks the EGFR growth signals. Especially, over 90% of detected mutations in B-Raf are a V600E which leads to constitutive kinase activity 500-fold greater than B-Raf wild type and correlates with increased malignancy. Vascular endothelial growth factor (VEGF) and its receptors have been implicated in the angiogenesis that is essential for growth and metastasis of solid tumors. Since formation of solid tumors are angiogenesis dependent, several strategies have been developed to inhibit VEGF signal transduction as part of anticancer therapy. The first VEGFR2 and Raf-1 inhibitor to enter clinical development was the multikinase inhibitor SORAFENIB, which is now approved for the treatment of patients with advanced renal cell carcinoma. More potent dual inhibitors against VEGFR2 and Raf-1 may be beneficial for patients suffering from various tumors. In this study, some of rational-designed compounds were synthesized and carried out the in vitro and in vivo studies to find more efficient VEGFR2 and Raf-1 inhibitors. Methods: About two hundreds of compounds were designed and synthesized. in vitro assays and in vivo studies were performed to characterize the potential of VEGFR2 and Raf-1 inhibitors for solid tumors. Results: Compound UI-162 showed strong kinase inhibition against VEGFR2 (12 nM), B-Raf V600E mutant (0.2 nM), B-Raf wildtype (2.0 nM) and Raf-1(0.6 nM). The compound showed good inhibitory effect in against malignant cells expressing K-Ras or B-Raf V600E mutant type of cell-line (SK-MEL-2 GI50: 133 nM / HCT-116 GI50: 229 nM / MDA-MB231 GI50: 278 nM / A375P GI50: 51 nM / HT29 GI50: 40 nM / COLO 205 GI50: 12 nM, respectively). In mechanism studies, the phosphorylation of MEK and ERK were suppressed by the UI-162 compound in HCT-116 cell-line. In addition, paradoxical effects were not detected compared with VEMURAFENIB. The compound had also a good anti-proliferative activity against HUVEC and showed a good oral bioavailability. Conclusions: The results of in vitro and in vivo studies suggests that dual inhibition of VEGFR2 and Raf-1 may provide strong antitumor efficacy and that UI-162 is a promising candidate for the treatment of various human cancers harboring the K-Ras and B-Raf V600E mutation. Citation Format: Seung Yong Kim, Younho Lee, Hyungtae Bang, Kihwan Eum, Sungpyo Hong, Ky-Youb Nam, Nam Song Choi, Ju Hee Kang, Hara Kang, Kil Won Kim, Michael Lee, Soon Kil Ahn. Discovery of novel VEGFR2 and Raf-1 kinase inhibitorsuppressing MAPK signaling pathway in K-Ras mutant cancer cell-lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 941. doi:10.1158/1538-7445.AM2013-941

In Vitro Studies on the Genotoxic Effects of Wood Smoke Flavors

Smoke flavors based on the thermal decomposition of wood have been applied to a variety of food products as an alternative for traditional smoking. Despite its increasing use, the available genotoxicity data on wood smoke flavors (WSF) are still controversial. Thus, potential genotoxic effects of WSF in four short-term in vitro genotoxicity assays were investigated, which included the Ames assay, chromosomal aberration assay, micronucleus test and the alkaline comet assay. WSF did not cause any mutation in the Ames assay using five tester strains at six concentrations of 0.16, 0.31, 0.63, 1.25, 2.5 and 5 µl/plate. To assess clastogenic effect, the in vitro chromosomal aberration assay was performed using Chinese hamster lung cells. No statistically significant increase in the number of metaphases with structural aberrations was observed at the concentrations of 1.25, 2.5, and 5 µl/ml. The in vitro comet assay and micronucleus test results obtained on L5178Y cells also revealed that WSF has no genotoxicity potential, although there was a marginal increase in micronuclei frequencies and DNA damage in the respective micronucleus and comet assays. Taken together, based on the results obtained from these four in vitro studies, it is concluded that WSF is not a mutagenic agent in bacterial cells and causes no chromosomal and DNA damage in mammalian cells in vitro.