Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation

Chung, Heesung,Jung, Hyejung,Jho, Eek-hoon,Multhaupt, Hinke A.B.,Couchman, John R.,Oh, Eok-Soo Elsevier 2018 Biochemical and biophysical research communication Vol.503 No.2

<P><B>Abstract</B></P> <P>In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, −3 and −6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Human keratinocyte cells reduce the N-cadherin levels of co-cultured melanoma cells. </LI> <LI> Cell-to-cell contact is necessary for this keratinocyte-mediated N-cadherin reduction. </LI> <LI> Keratinocytes reduce intracellular calcium in co-cultured melanoma cells. </LI> <LI> Keratinocytes reduce the expression of TRPCs in contacting melanoma cells. </LI> </UL> </P>

A New Endothelial Molecule Involved in Melanoma Cell Binding to Human Dermal Microvascular Endothelial Cells

Lee, Kwang Hoon,Chung, Kee-Yang,Thomas J.Lawley,Robert A.Swerlick Korean Dermatological Association 1994 Annals of Dermatology Vol.6 No.1

Characterization of morphological changes of B16 melanoma cells under natural killer cell attack

Kim, Ji Sung,Kim, Boyeong,Lee, Hong Kyung,Kim, Hyung Sook,Park, Eun Jae,Choi, Yeo Jin,Ahn, Gi Beom,Yun, Jieun,Hong, Jin Tae,Kim, Youngsoo,Han, Sang-Bae Elsevier 2019 INTERNATIONAL IMMUNOPHARMACOLOGY Vol.67 No.-

<P><B>Abstract</B></P> <P>Natural killer (NK) cell killing of melanoma cells involves perforin-mediated delivery of granzymes from NK cells to cancer cells; however, how melanoma cells die remains poorly characterized. Here, we examined the dying process of melanoma cells by using time-lapse imaging. Upon contact with NK cells, B16-F10 cells rounded and most of them showed membrane rupture (98 min); however, B16 parent cells showed writhing and delayed membrane rupture (235 min). This morphological difference depended on the expression levels of myosin regulatory light chain 9 (MYL9) but not activating ligands (CD112, CD155, Rae-1, and MULT-1), SPI, FasL, or PD-L1. Taken together, our data show that melanoma cells show two distinct types of morphological changes upon contact with NK cells and suggest that a strategy to decrease MYL9 expression by melanoma cells may improve the efficacy of NK cell–based immunotherapy.</P>

Antitumor Effect of Metformin in Combination with Binimetinib on Melanoma Cells

Lee, Eunsung,Kwon, Yongjae,Kim, Jiwon,Park, Deokbae,Lee, Youngki The Korean Society of Developmental Biology 2021 발생과 생식 Vol.25 No.2

Cutaneous melanoma is a fatal disease for patients with distant metastasis. Metformin is the most widely used anti-diabetic drug, and proved to suppress cell proliferation and metastasis in diverse cancers including melanoma. We previously reported that MEK inhibitor trametinib increases the expression of epithelial-mesenchymal transition (EMT) regulators and melanoma cell motility, which are suppressed by addition of metformin in A375 melanoma cells. To confirm our findings further, we first evaluated the effect of metformin in combination with another MEK inhibitor binimetinib on cell viability in G361 melanoma cells. We then investigated whether binimetinib affects the expression of EMT regulators and cell motility. We finally monitored the effect of metformin on binimetinib-induced cell migration. Cell viability assay showed that combination index (CI) value at ED₅₀ is 0.80, suggesting synergy for the combination of metformin with binimetinib. Our results also revealed that binimetinib increased the expression of EMT regulators such as integrin αV, fibronectin and slug, which correlate well with the enhanced cell migration in wound healing assay. Metformin, on the contrary, suppressed the expression of sparc, integrin αV, fibronectin and N-cadherin with the reduced cell motility. The combination treatment showed that metformin counteracts the binimetinib-induced increase of cell motility. Overall, these results suggest that metformin with binimetinib might be useful as a potential therapeutic adjuvant against cell survival and metastatic activity in melanoma patients.

악성흑색종세포에서의 향신경요소 수용체 발현 양상

이정열,박영립,황규왕 ( Jung Youl Lee,Young Lip Park,Kyu Uang Whang ) 대한피부과학회 1997 대한피부과학회지 Vol.35 No.6

Background: Nerve growth factor(NGF), brain derived neurotrophic factor(BDNF), neurotropin 3(NT-3) and neurotropir-4/5 are neurotrophic factors necessary for the development and maintenance of specific neurors. The tyrosine protein kinase(trk) receptors exhibit specificity for differ ent neurotrophins. NGF is the cognate ligand for the trk A receptor, BDNF binds to trk B receptor and NT-3 binds to irk A, trk B and trk C receptors, Since melanoma cells are devived from neural ectoderm, growth factors which affect. neuronal tissue may have a role in melanoma biology. Objective : The purpose of this study is to demonstrate the presence of trk receptors in rnelanoma cells and observe th effect of K-252a on these melanoma cells growth and differentiation. Methods : After K252a over a range of 0-200nM was added into their cell lines, we exam ined cell viability of SK 28 and SK 30 cells. We performed this to examine the expression of the trk by flow cytometry and immunoblotting. Results : 1. The incubation of . K 28 cells and SK 30 cells with K 252a resulted in a dose dependent inhibition of cell proliferation. 2. In the flowcytometry, SK 28 cells and SK 30 cells showed a high expression of trk A and trk B, not trk C. 3. Using immunoblottiiig, trk in SK 28 cells and SK 30 cells was not expressed. Cpnclusions : These results indicate that. the identification of tyrosine protein kinase reeeptors and their inhibitor which affect differentiation and growth of a melanoma may provide an additional therapeutic option for treatment of melanoma. (Korean J Dermatol 1997;35(6): 1151-1158)

OTUB1 knockdown promotes apoptosis in melanoma cells by upregulating TRAIL expression

이복순,Sung Un Kang,Mei Huang,Yeon Soo Kim,Young-Sun Lee,Jae-Yong Park,김철호 생화학분자생물학회 2021 BMB Reports Vol.54 No.12

Melanoma, the most serious type of skin cancer, exhibits ahigh risk of metastasis. Although chemotherapeutic treatmentfor metastatic melanoma improves disease outcome and patientsurvival, some patients exhibit resistance or toxicity to the drugtreatment regime. OTUB1 is a deubiquitinating enzyme overexpressedin several cancers. In this study, we investigated theeffects of inhibiting OTUB1 expression on melanoma-cell proliferationand viability and identified the underlying molecularmechanism of action of OTUB1. We did endogenous OTUB1knockdown in melanoma cells using short interfering RNA,and assessed the resulting phenotypes via MTT assays, Westernblotting, and cell-cycle analysis. We identified differentiallyexpressed genes between OTUB1-knockdown cells and controlcells using RNA sequencing and confirmed them via Westernblotting and reverse transcription polymerase chain reaction. Furthermore, we investigated the involvement of apoptotic andcell survival signaling pathways upon OTUB1 depletion. OTUB1depletion in melanoma cells decreased cell viability and causedsimultaneous accumulation of cells in the sub-G1 phase, indicatingan increase in the apoptotic-cell population. RNA sequencingof OTUB1-knockdown cells revealed an increase in thelevels of the apoptosis-inducing protein TRAIL. Additionally,OTUB1-knockdown cells exhibited increased sensitivity toPLX4032, a BRAF inhibitor, implying that OTUB1 and BRAFact collectively in regulating apoptosis. Taken together, ourfindings show that OTUB1 induces apoptosis of melanomacells in vitro, likely by upregulating TRAIL, and suggest thatapproaches targeting OTUB1 can be developed to provide noveltherapeutic strategies for treating melanoma.

Anti-Tumor Activity of Acinetobacter baumannii Outer Membrane Protein A on Dendritic Cell-Based Immunotherapy against Murine Melanoma

이준식,Jung Wook Kim,최철희,Won Kee Lee,Hae Young Chung,이제철 한국미생물학회 2008 The journal of microbiology Vol.46 No.2

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a major surface protein that is an important pathogen-associated molecular pattern. Based on our previous findings that AbOmpA induced the phenotypic maturation of dendritic cells (DCs) and drove the Th1 immune response in vitro, we investigated the therapeutic efficacy of AbOmpA-pulsed DC vaccines in a murine melanoma model. The surface expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class I and II molecules was higher in DCs pulsed with AbOmpA alone or with a combination of B16F10 cell lysates than that of DCs pulsed with B16F10 cell lysates. AbOmpA stimulated the maturation of murine splenic DCs in vivo. In a therapeutic model of murine melanoma, AbOmpA-pulsed DCs significantly retarded tumor growth and improved the survival of tumor-bearing mice. AbOmpA-pulsed DCs significantly enhanced CD8+, interleukin-2+ T cells and CD4+, interferon-γ+ T cells in tumor-bearing mice. These results provide evidence that AbOmpA may be therapeutically useful in adjuvant DC immunotherapy against poorly immunogenic melanoma without tumor-specific antigens. Acinetobacter baumannii outer membrane protein A (AbOmpA) is a major surface protein that is an important pathogen-associated molecular pattern. Based on our previous findings that AbOmpA induced the phenotypic maturation of dendritic cells (DCs) and drove the Th1 immune response in vitro, we investigated the therapeutic efficacy of AbOmpA-pulsed DC vaccines in a murine melanoma model. The surface expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class I and II molecules was higher in DCs pulsed with AbOmpA alone or with a combination of B16F10 cell lysates than that of DCs pulsed with B16F10 cell lysates. AbOmpA stimulated the maturation of murine splenic DCs in vivo. In a therapeutic model of murine melanoma, AbOmpA-pulsed DCs significantly retarded tumor growth and improved the survival of tumor-bearing mice. AbOmpA-pulsed DCs significantly enhanced CD8+, interleukin-2+ T cells and CD4+, interferon-γ+ T cells in tumor-bearing mice. These results provide evidence that AbOmpA may be therapeutically useful in adjuvant DC immunotherapy against poorly immunogenic melanoma without tumor-specific antigens.

B16F10 melanoma cell을 이용한 캐모마일(Matricaria chamomilla L.) 추출물의 미백 효과

조재범 ( Jae-bum Jo ),김명욱 ( Myung-uk Kim ),이은호 ( Eun-ho Lee ),김예진 ( Ye-jin Kim ),조은비 ( Eun-bi Cho ),강인규 ( In-kyu Kang ),조영제 ( Young-je Cho ) 한국응용생명화학회(구 한국농화학회) 2018 Journal of Applied Biological Chemistry (J. Appl. Vol.61 No.3

기능성 천연물 소재로서의 가능성을 검토하고자 캐모마일 추출물의 미백 효과를 조사하고 melanin 생성 반응에 관여하는 물질 억제에 대한 기전을 규명하고자 하였다. 캐모마일을 water와 60% ethanol을 이용하여 추출하였고, 얻어진 추출물을 phenolic농도별로 설정하여 tyrosinase 저해 활성을 알아보았을 때, water추출물의 경우 효과가 미비하였고, 60% ethanol 추출물에서는 농도 의존적으로 저해 활성이 나타내어 melanin 생성 저해 효과가 있을 것으로 판단되었다. 캐모마일 60% ethanol 추출물을 동결건조하여 얻어진 분말을 이용하여 B16F10 melanoma cell에 대한 세포 독성을 측정 결과, 75 μg/mL의 농도에서부터 독성이 관찰되어 농도 구간을 10, 25, 50 μg/mL으로 선정하였다. α-MSH로 자극한 B16F10 melanoma cell에서 melanin 생성량을 측정하여 캐모마일의 melanin 성성 억제 효능과 melanin 생성에 영향을 미치는 단백질인 tyrosinase, MITF, TRP-1, TRP-2의 단백질 발현 억제 효과를 알아본 결과, 캐모마일의 농도가 높아짐에 따라 농도 의존적으로 melanin 생성 함량이 감소하였고, tyrosinase, TRP-1, TRP-2, 등의 단백질 발현량 또한 감소하는 것을 확인할 수 있었다. 따라서 캐모마일 추출물은 B16F10 melanoma cell에서 melanin의 생성을 억제하고, melanin생성 관련 단백질발현을 억제하는 효과가 있음을 확인하였다. 위의 결과들로 인하여 캐모마일은 미백 기능성 식품 산업화를 위한 유용한 자원으로 활용 될 것으로 예상되며, 추후 산업적 응용을 위한 지속적인 연구가 진행되어야 할 것으로 판단되었다. Matricaria chamomilla L. has been used as a bath agent in Europe because of its sterilization effect on the skin. Flowers contain terpenes, flavonoids are effective in relieving inflammation. Matricaria chamomilla L. has been reported to have various drug efficacies such as sedation, anti-diabetic effect and anti-arthritic effect, but there is little research on the scientific efficacy of whitening effect. The purpose of this study was to examine the whitening effect of Matricaria chamomilla L. extract and to investigate the mechanism of inhibition of melanogenesis. The extracts were used to determine tyrosinase inhibitory activity. The tyrosinase inhibitory activity of extracts was ineffective for water extract but in 60% ethanol extract was shown in a concentration-dependent manner. B16F10 melanoma cell was measured using a powder obtained by lyophilization of 60% ethanol extract. The toxicity was observed at a concentration of 75 μg/mL. And concentration range was selected to be at most 50 μg/ mL. The effect of tyrosinase, MITF, TRP-1 and TRP-2 on the expression of melanin protein was investigated in melanoma cells of B16F10 melanoma cells. As a result, it was confirmed that as the concentration of the extract increased, the melanogenesis level decreased and the protein expression level also decreased in a concentration dependent manner. Therefore, it was concluded that Matricaria chamomilla L. extract inhibited melanogenesis in cells. Based on the above results, it is expected that it will be used as a useful basic data for industrialization of whitening functional food of Matricaria chamomilla L.

Autophagy-Dependenr Survival of Mutant B-Raff Melanoma Cells Selected for Resistance to Apoptosis Inducef by Inhibitors against Oncogenic B-Raf

( Jun Ho Ahn ),( Mi Chael Lee ) 한국응용약물학회 2013 Biomolecules & Therapeutics(구 응용약물학회지) Vol.21 No.2

Most patuents with mutant B-Raf melanomas respond to inhibitors of oncogenic B-Raf buf 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 vhronic selection to establish B-Raf (V^))E) melanoma clones with acpuired resistance to the new oncogenic B-Raf Inhibitor UI-152. Whereas the parental A375P cells were highly sensitive to UI-152 (IC50<0. 5 M). the resistant sub-line (A375P/Mdr) displayed strong resistance to UI-152 (IC50<0. 5 M). lmmunofluorescence analysis indicated the absence of an increase in the levels of P-giycoprotein mutidrug resistance (MAR) transporter in A375P/Mdr cells, suggesting that resistance was not at-tributabie to P-giycoprotein overexpression. Ln UI-152-sensitive A375P cells, the anti-poliferative activity of UI-152 appeared tobe die to cell-cycle arrest at G₁/G₁ 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, sa 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-tread cells from undergoing growth inhibition. Together. our data implicate high level of autophagy may protect UI-152-treated cells from the oncogenic B-Raf inhibitor, in support of clinical studies in which combiation therapy with autophagy targered drugs is being designed to overcome resistance.

1,25‐Dihydroxyvitamin D₃ enhances NK susceptibility of human melanoma cells via Hsp60‐mediated FAS expression

Lee, Ji H.,Park, Sunyoung,Cheon, Soyoung,Lee, Joo H.,Kim, Sunghan,Hur, Dae Y.,Kim, Tae S.,Yoon, Suk R.,Yang, Yoolhee,Bang, Sa I.,Park, Hyunjeong,Lee, Hoon T.,Cho, Daeho WILEY‐VCH Verlag 2011 European journal of immunology Vol.41 No.10

<P><B>Abstract</B></P><P>The active metabolite of vitamin D<SUB>3</SUB>, 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB>, displays anticancer effects by regulating cell cycle and apoptosis in many cancer cells. However, it has not been determined whether 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB> increases the susceptibility of cancer cells to NK cells. Here, we investigated the anticancer effect of 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB> in human melanoma cell lines by investigating enhancement of NK susceptibility and elucidating the mediator of NK cytotoxicity. 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB>‐resistant melanoma cells (G‐361 and SK‐MEL‐5) treated with 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB> showed higher susceptibility to NK cells with up‐regulation of Fas expression. Furthermore, G‐361 cells treated with 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB> showed significantly increased caspase activity. In addition to Fas up‐regulation, expression of heat shock protein 60 (Hsp60) was elevated by 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB>. Increased expression of Hsp60 by 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB> was related to not only up‐regulation of Fas expression but also to NK susceptibility of G‐361 cells. Taken together, our data suggest that 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB> acts as an anticancer agent by increasing expression of Fas on the surface of melanoma cells through Hsp60 induction and strengthens caspase sensitivity to Fas‐mediated apoptotic pathway by NK cells. 1α,25(OH)<SUB>2</SUB>D<SUB>3</SUB> treatment may therefore have a preventive role in melanoma occurrence or potentiate the anticancer effects of NK‐cell immune therapy.</P>