• Biomolecules & Therapeutics
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• 제21권2호
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• pp.114-120
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• 2013

Most patients with mutant B-Raf melanomas respond to inhibitors of oncogenic B-Raf but resistance eventually emerges. To better understand the mechanisms that determine the long-term responses of mutant B-Raf melanoma cells to B-Raf inhibitor, we used chronic selection to establish B-Raf (V600E) melanoma clones with acquired resistance to the new oncogenic B-Raf inhibitor UI-152. Whereas the parental A375P cells were highly sensitive to UI-152 ($IC_{50}$ < $0.5{\mu}M$), the resistant sub-line (A375P/Mdr) displayed strong resistance to UI-152 ($IC_{50}$ < $20{\mu}M$). Immunofluorescence analysis indicated the absence of an increase in the levels of P-glycoprotein multidrug resistance (MDR) transporter in A375P/Mdr cells, suggesting that resistance was not attributable to P-glycoprotein overexpression. In UI-152-sensitive A375P cells, the anti-proliferative activity of UI-152 appeared to be due to cell-cycle arrest at $G_0/G_1$ with the induction of apoptosis. However, we found that A375P/Mdr cells were resistant to the apoptosis induced by UI-152. Interestingly, UI-152 preferentially induced autophagy in A375P/Mdr cells but not in A375P cells, as determined by GFP-LC3 puncta/cell counts. Further, autophagy inhibition with 3-methyladenine (3-MA) partially augmented growth inhibition of A375P/Mdr cells by UI-152, which implies that a high level of autophagy may protect UI-152-treated cells from undergoing growth inhibition. Together, our data implicate high rates of autophagy as a key mechanism of acquired resistance to the oncogenic B-Raf inhibitor, in support of clinical studies in which combination therapy with autophagy targeted drugs is being designed to overcome resistance.

• 한국식품과학회지
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• 제38권3호
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• pp.419-426
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• 2006

전국 각지에서 채집한 토양에서 분리한 25종의 내열성 균주 중 내열성 단백질 기수분해효소 활성을 갖는 균주 strain JE 375를 선별하였다. 본 균주는 gram 양성 간균의 특징을 나타냈으며 Bergey's Manual of Systematic Bacteriology와 Biochemical Tests for Identification of Medical Bacteria에 준하여 생화학적 특성을 검토한 결과 catalase 양성, 포자형성, motility 양성, glucose 발효, mannitol 발효, xylose 산화, hemolysis ${\beta}$균임을 보아 Bacillus sp.으로 추정 되었다. Strain JE 375의 whole cell fatty acid를 gas chromatography로 분석한 결과 $C_{15:0}$ iso 26.17%, $C_{16:0}$ iso 13.01%, $C_{17:0}$ iso 30.19%로 분석되어 Bacillus 계열로 동정되었다. 16S rDNA sequence분석 결과 strain JE 375는 Bacillus caldoxylolyticus와 sequence가 97.6% 일치하는 유사성을 보였으나 부분적으로 sequence의 차이가 있고 gene bank data base상에서 16S rDNA sequence가 일치하는 균주는 검색되지 않았다. 이 같은 실험 결과에 따라 strain JE 375는 기존에 발표되지 않은 새로운 균주로 판단되어 Bacillus sp. JE 375로 명명하였다. Bacillus sp. JE 375은 tryptone 1%, yeast extract 0.5%, NaCl 1%, maltose 1%의 배지조성분과 배양 온도 $65^{\circ}C$에서 20시간 동안 배양하였을 때 최대의 단백질 분해 효소를 생산하였다. Bacillus sp. JE 375로부터 단백질 분해 효소를 acetone으로 침전시키고 DEAE-sepharose column chromatography를 통하여 효소를 정제하여 SDS-PAGE를 통해 확인한 결과 55 kDa 크기의 band를 확인할 수 있었다. 이 효소의 최적 배양 온도는 $65^{\circ}C$이었으며 배지의 최적 pH는 6.5로 나타났다. pH에 대한 안정성은 중성 부근의 pH에서 효소 활성의 안정성이 높게 나타났다. 본 효소의 반응 조건을 검토한 결과 중성 조건에서 안정하였으며, $60^{\circ}C$의 고온에서 활성을 가졌다. 효소 활성은 1 mM $CaCl_2$ 첨가에 의해 증가하였다.

• 한국식품위생안전성학회지
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• 제34권2호
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• pp.183-190
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• 2019

우르솔릭산은 항암, 항산화, 항염증 작용과 같은 다양한 효과를 지니고 있다. 본 연구에서는 우르솔릭산이 인간 흑색종 암세포인 A375SM과 A375P 세포에 항암효과가 있는지 확인하였다. 두 세포의 생존율은 MTT assay를 통하여 확인하였으며 증식률은 Wound healing assay로 확인하였다. 두 세포의 apoptotic body와 apoptosis 비율의 확인을 위한 DAPI 염색과 유세포 분석을 진행하였다. 그리고 웨스턴 블로팅을 통하여 흑색종 세포의 우르솔릭산의 농도에 따른 apoptosis 단백질의 유도를 조사하였다. 우르솔릭산의 처리 농도에 따라 흑색종 세포의 생존율 감소와 증식률 감소를 확인하였다. DAPI 염색을 통하여 우르솔릭산의 농도가 증가함에 따라 흑색종 세포의 염색체 응축이 농도 의존적으로 증가하였고, 유세포 분석을 통하여 우르솔릭산에 대하여 농도 의존적으로 흑색종 세포의 apoptosis 비율의 증가를 확인하였다. 그리고 웨스턴 블로팅을 통해 흑색종 세포 A375SM과 A375P의 우르솔릭산 $12{\mu}M$ 농도에서 cleaved-PARP와 Bax의 증가와 Bcl-2의 감소를 확인하였다. 본 연구는 우르솔릭산의 농도를 0 에서 $20{\mu}M$ 수준의 저농도에서 진행하였으며, 물질 처리 후 24 시간 뒤 결과를 가지고 분석하였다. 본 연구의 결과로 보아 우르솔릭산은 흑색종 세포 A375SM과 A375P에서 apoptosis 관련 단백질들의 조절을 통해 항암효과를 일으키는 것으로 사료된다.

• Bulletin of the Korean Chemical Society
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• 제33권11호
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• pp.3635-3639
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• 2012

A series of new diarylureas possessing aminoisoquinoline scaffold was synthesized, and their in vitro antiproliferative activity against A375P human melanoma cell line was tested. Compounds 1d, 1l, 1n, 1p, 1q, and 1t showed superior potency against A375P to Sorafenib. The highest potency was shown by compound 1p possessing 3,5-bis(trifluoromethyl)phenyl terminal ring with $IC_{50}$ value of $0.41{\mu}M$.

• Biomolecules & Therapeutics
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• 제23권4호
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• pp.320-326
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• 2015

The clinical benefits 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.

• International Journal of Oral Biology
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• 제42권3호
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• pp.107-115
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• 2017

Human malignant melanoma is an aggressive skin cancer which has been rising at a greater rate than any other cancers. Although various new therapeutic methods have been developed in previous studies, this disease has properties of high proliferation and metastasis rate which remain obstacles that have lead to a poor prognosis in patients. It has been reported that a specific Lactobacillus extract has anti-cancer and -metastasis effect in vitro and in vivo. However, previous research has not specified precisely what effect the Lactobacillus rhamnosus GG (LGG) extract has had on human malignant melanomas. In this study, we showed that the LGG extract has anti-cancer and -metastasis effects on the human malignant melanoma cell lines, A375P and A375SM. At first, it was found that, while the LGG extract affects human neonatal dermal fibroblasts slightly, it induced the dose-dependent anti-cancer effect on A375P and A375SM by a WST-1 proliferation assay. As a result of a real-time PCR analysis, the expression patterns of several genes related to cell cycle, proliferation, and apoptosis were modulating in a manner that inhibited the growth of both malignant melanoma cell lines after the treatment of the LGG extract. Furthermore, genes related to the epithelial-mesenchymal transition were down-regulated, and migration rates were also decreased significantly by the LGG extract. Our study showed that the LGG extract could be used as a potential therapeutic source.

• Bulletin of the Korean Chemical Society
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• 제33권9호
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• pp.2991-2998
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• 2012

A series of new diarylureas and diarylamides possessing 1,3,4-triarylpyrazole scaffold was synthesized and their in vitro antiproliferative activities against A375P human melanoma cell line and NCI-60 cell line panel were tested. Compounds 9, 11, 12, 14, and 17-21 showed superior potency against A375P to Sorafenib. Over the NCI-60 cancer cell line panel, compound 14 possessing a methoxy group, amide linker, and 4-chloro-3-(trifluoromethyl)phenyl terminal ring showed the highest potency and broad-spectrum anticancer activity. Compound 13 showed high selectivity towards leukemia subpanel over other cancer types.

• Asian Pacific Journal of Cancer Prevention
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• 제16권2호
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• pp.699-705
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• 2015

Vemurafenib has recently been used as drug for treatment of melanomas with $BRAF^{V600E}$ mutation. Unfortunately, treatment with only vemurafenib has not been sufficiently effective, with recurrence after a short period. In this study, three vemurafenib-resistant $BRAF^{V600E}$ melanoma cell lines, $A375P^R$, $A375M^R$ and SKMEL-$28^R$, were established from the original A375P, A375M and SKMEL-28 cell lines. Examination of the molecular mechanisms showed that the phosphorylation levels of MEK and ERK, which play key roles in the RAS/RAF/MEK/ERK signaling pathway, were reduced in these three cell lines, with increased phosphorylation levels of pAKTs limited to SKMEL-$28^R$ cells. Treatment of SKMEL-$28^R$ cells with 100 nM paclitaxel resulted in increased apoptosis and decreased cellular proliferation, invasion and colony formation via reduction of expression levels of EGFR and pAKTs. Moreover, vemurafenib-induced pAKTs in SKMEL-$28^R$ were decreased by treatment with an AKT inhibitor, MK-2206. Taken together, our results revealed that resistance mechanisms of $BRAF^{V600E}$-mutation melanoma cells to vemurafenib depended on the cell type. Our results suggested that paclitaxel should be considered as a drug in combination with vemurafenib to treat melanoma cells.

• Archives of Pharmacal Research
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• 제28권1호
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• pp.68-72
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• 2005

Pseudolaric acid B is a major compound found in the bark of Pseudolarix kaempferi Gordon. In our study, pseudolaric acid B inhibited growth of human melanoma cells, A375-S2 in a time and dose-dependent manner. A375-S2 cells treated with pseudolaric acid B showed typical characteristics of apoptosis including morphologic changes, DNA fragmentation, sub-diploid peak in flow cytometry, cleavage of poly-ADP ribose polymerase (PARP) and degradation of inhibitor of caspase-activated DNase (ICAD). P53 protein expression was upregulated while cells were arrested at the $G_2/M$ phase of the cell cycle. There was a decrease in the expression of anti-apoptotic Bcl-2 and Bcl-xL proteins, whereas pro-apoptotic Bax was increased. The two classical caspase substrates, PARP and ICAD, were both decreased in a time-dependent manner, indicating the activation of downstream caspases.

• 대한수학회보
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• 제45권2호
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• pp.375-384
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• 2008

Let $k={\mathbb{F}}_q(T)$ and ${\mathbb{A}}={\mathbb{F}}_q[T]$. Fix a prime divisor ${\ell}$ q-1. In this paper, we consider a ${\ell}$-cyclic real function field $k(\sqrt[{\ell}]P)$ as a subfield of the imaginary bicyclic function field K = $k(\sqrt[{\ell}]P,\;(\sqrt[{\ell}]{-Q})$, which is a composite field of $k(\sqrt[{\ell}]P)$ wit a ${\ell}$-cyclic totally imaginary function field $k(\sqrt[{\ell}]{-Q})$ of class number one. und give various conditions for the class number of $k(\sqrt[{\ell}]{P})$ to be one by using invariants of the relatively cyclic unramified extensions $K/F_i$ over ${\ell}$-cyclic totally imaginary function field $F_i=k(\sqrt[{\ell}]{-P^iQ})$ for $1{\leq}i{\leq}{\ell}-1$.