- Email: [email protected]
Hair shaft miRNA-221 levels as a new tumor marker of melanoma
e174
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
observed in most patients with TSC. However, the pathogenesis is still unknown. Recently, autophagy-lysosome pathway has been known as a novel pathway involved in skin pigmentation. Objective: To investigate whether the autophagy-lysosome pathway was involved in TSC-associated hypopigmentation. Method: Using skin tissues from hypopigmented macules of TSC, the activity of autophagy-lysosome pathway was assessed by immunohistochemical staining. Furthermore, we investigated both the pigmentation and autophagy-lysosome pathway in TSC2 knockdown (KD) human primary melanocytes and keratinocytes, by real-time PCR, western blotting analyses and intracellular immunofluorescence staining. Results: Compared to healthy donors, the number of melanocytes was not changed, but autophagy-lysosome activity was dramatically decreased in skin tissue of hypopigmentation macules from TSC. Furthermore, in TSC2 KD melanocytes, melanin contents decreased strikingly, and decreased expression levels of melanogenesis-related proteins were observed. Besides, we found that expression levels of LC3, LAMP1 and the number of autophagosomes and lysosomes were significantly decreased in TSC2 KD melanocytes. Furthermore, to clarify whether the reduction of autophagy was related to hypopigmentation, ATG7 siRNA was transfected into melanocytes. Very interestingly, we found that melanogenesis was significantly inhibited in ATG7 KD melanocytes. Conclusions: These results suggest that dysfunction of autophagy-lysosome pathway might be involved in the pathogenesis of hypopigmentation in patients with TSC. http://dx.doi.org/10.1016/j.jdermsci.2016.08.512 P13-03[C11-08] TLR3 agonist Poly (I:C) enhance Rab27A and melanosome transportation through TLR3 and MAVS pathway in human epidermal melanocytes Saaya Koike ∗ , Kenshi Yamasaki, Takeshi Yamauchi, Mai Inoue, Kenichiro Tsuchiyama, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Japan Innate immune stimuli restlessly influence epidermis where human melanocytes reside. We have examined how innate immunity affects pigmentation and observed that TLR3 agonist poly (I:C) increased melanin release from melanocytes. Poly (I:C) increased Rab27A expression and cell peripheral accumulation, which facilitate melanosome transportation to cell membrane. Knockdown of Rab27A abrogated melanosome transfer to keratinocytes by poly (I:C). Since MITF is known to increase Rab27A transcription, we examined if MITF is involved in Rab27A induction by poly (I:C). Poly (I:C) increased MITF mRNA, but siRNA for MITF did not suppress Rab27A increase, perimembranous localization of Gp100 and Rab27A, and extracellular melanin release by poly (I:C). These suggested that poly (I:C) induces Rab27A and melanosome transportation independent of MITF function. We next knocked down TRIF and MAVS, the molecules involved in TLR3 pathway. We observed that siTRIF and siMAVS suppressed Rab27A induction and melanosome transport to neighboring keratinocytes by poly (I:C) stimulation. We also observed that poly (I:C) increased expression of MAVS mRNA about 7-fold, but poly (I:C) did not altered the mRNA level of TRIF and its downstream molecules IRF3 and TBK1. These indicated that TLR3 agonist poly (I:C) activates melanosome trans-
port by increase Rab27A expression through the TLR3 and MAVS pathway. http://dx.doi.org/10.1016/j.jdermsci.2016.08.513 P13-04[C11-07] IFN- modulates chemokines from tumor-associated macrophages to enhance the therapeutic effect of anti-PD-1 antibodies in B16F10 melanoma Aya Kakizaki 1,∗ , Taku Fujimura 1 , Sadanori Furudate 1 , Yumi Kambayashi 1 , Takeshi Yamauchi 1 , Hideo Yagita 2 , Setsuya Aiba 1 1 Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan 2 Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
Tumor-associated macrophages (TAMs) play roles in maintaining the immunosuppressive microenvironment by recruiting regulatory T cells (Tregs). Since IFN- and anti-PD-1 Ab have been clinically used for the treatment of malignant melanoma, we investigated their immunomodulatory effects on melanoma using the B16F10 mouse melanoma model. B16F10 melanoma cells were subcutaneously injected into C57BL/6 mice, and when the tumor had established, we administered IFN-. The peritumoral administration of IFN- significantly decreased the mRNA expression of Tregs-related chemokines (CCL17, CCL22), which led to the suppressed recruitment of Tregs in B16F10 melanoma. Moreover, to validate the production of chemokines, we isolated CD11b+ TAMs by MACS, analyzed the chemokine production, and confirmed the decrease of CCL22 in the IFN- treated group. Furthermore, the culture supernatant from CD11b+ TAMs significantly increased the chemotactic activity for TRP-2-specific CD4+ T cells, which might suggest that chemotactic factors from CD11b+ TAMs recruit B16F10 melanoma-specific T cells into the tumor microenvironment. Since the administration of IFN- augments the expression of PD-1 on TILs, the co-administration of anti-PD-1 Ab augmented the therapeutic effect of IFN- on the B16F10 melanoma. As in the mouse model, in human, IFN- decreased the production of Th2related chemokines and augmented the production of Th1-related chemokines from monocyte- derived M2 macrophages. Since these immunomodulatory effects of IFN- on macrophages were also observed in the lesional skin of human in- transit melanoma, our present data suggest one of the possible immunomodulatory effects of IFN- and support the use of IFN- in combination with anti-PD1 Ab for the treatment of melanoma patients. http://dx.doi.org/10.1016/j.jdermsci.2016.08.514 P13-05[C11-06] Hair shaft miRNA-221 levels as a new tumor marker of melanoma Satoshi Fukushima ∗ , Taisuke Inada, Masayuki Murai, Masatoshi Jinnin, Azusa Miyashita, Satoshi Nakahara, Aki Tokuzumi, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan Micro RNA-221 (miR-221) is known to be abnormally expressed in many human cancers. We have reported that the serum levels
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
of miR-221is a tumor marker for malignant melanoma (MM). Circulating miR-221 was detectable and could be quantified in serum samples. MM patients had significantly higher miR-221 levels than healthy controls. Among the MM patients, the miR-221 levels were significantly increased in patients with stage I–IV MM compared to those with MM in situ, and their levels were correlated with tumor thickness. Moreover, a longitudinal study revealed a tendency for the miR-221 levels to decrease after surgical removal of the primary tumor, and to increase again at recurrence. Therefore, we hypothesized that the hair shaft miR-221 levels may be increased in patients with MM. In this study, we assessed the possibility that hair shaft miR-221 levels could be a marker for MM. The hair shaft miR-221 levels were significantly higher in patients with MM than those in controls. The rates of increased hair shaft miR-221 levels above the cut-off value were comparable to those of serum 5-S-CD, which is a tumor marker commonly used for MM. Measurements of the hair shaft miR-221 levels could have potential clinical value in the detection of MM. This is the first report investigating the hair shaft levels of a miRNA in patients with MM. Our investigations offer new insight into the relationship between miR-221 and MM, and may provide a new, non-invasive way to screen for melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.515 P13-06[C11-05] Aspirin induces cell death, TRAIL sensitization, and mitochondrial network abnormalities in human melanoma cells Itsuho Fujikawa 1,∗ , Miki Suzuki-Karasaki 1 , Toyoko Ochiai 1 , Yoshihiro Suzuki-Karasaki 2,3 1 Department of Dermatology, Nihon University Hospital, Tokyo, Japan 2 Innovative Therapy Research Group, Nihon University Research Institute of Medical Science, Tokyo, Japan 3 Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, Tokyo, Japan
Aspirin is emerging as a promising antineoplastic drug, because accumulating evidence suggest that the use of aspirin decreases the risks of multiple cancers in a cyclooxygenase-independent manner. Moreover, aspirin alone displays significant cytotoxicity toward multiple cancer cell types and potentiates the efficacy of various antineoplastic drugs. However, the precise mechanisms that underlie these effects remain poorly understood. Here we report that aspirin exerts these effects by modulating Ca2+ homeostasis and network morphology of the mitochondria in human malignant melanoma cells. Application of aspirin (mM) for 24–7 h resulted in non-apoptotic cell death. The cell death occurred relatively slowly and associated with annexin V-negative cells, and independent of caspase activation. In addition, nontoxic concentrations of aspirin sensitizes the cells to apoptosis induced by tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) in a caspase-dependent manner. In parallel, aspirin induced mitochondrial network abnormalities characterized by excessive mitochondrial fragmentation and clustering. The mitochondrial network abnormalities arise from reactive oxygen species (ROS)-mediated mitochondrial Ca2+ loading and were accelerated by the impairment of dynamin related protein 1-dependent mitochondrial fission. Indeed, aspirin at the toxic concentrations markedly increased mitochondrial Ca2+ and ROS levels. The effects of aspirin were independent of cyclooxygenase, since salicylates, which are unable of inhibiting the enzyme, induced all these events more potently than aspirin. Our results
e175
highlight the potential use of aspirin in the treatment of malignant melanoma cells in particular concomitant with TRAIL. http://dx.doi.org/10.1016/j.jdermsci.2016.08.516 P13-07[C11-03] Cell division cycle associated gene 1 as a new therapeutic target for melanoma Aki Tokuzumi ∗ , Satoshi Fukushima, Azusa Miyashita, Junji Yamashita, Satoshi Nakahara, Yosuke Kubo, Takamitsu Makino, Masatoshi Jinnin, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Science, Kumamoto University, Kumamoto, Japan Recently, it has been confirmed that immunotherapy and molecular target therapy for advanced melanoma increase the median survival time. To improve their effects and to reduce adverse reactions, a more ideal melanoma associated antigen is needed. Cell division cycle associated gene 1 (CDCA1), a specific function at the kinetochores to stabilize microtubule attachment, is overexpressed in various cancers, such as, non-small cell lung cancer, cholangiocellular carcinoma, renal cell cancer. On the other hand, CDCA1 does not show detectable levels of expression in normal tissues except for the testis, so it is a member of a cancer-testis antigen. To investigate the expressions of CDCA1 in melanoma, we conducted reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines. We also performed immunohistochemical analysis using tissue samples of melanoma and nevus tissue. In addition, we analyzed the role of CDCA1 in proliferation, migration and invasion of melanoma with melanoma cell lines SK-MEL-2 and WM115, using specific small interfering siRNAs. We found that CDCA1 was expressed in all of the melanoma cell lines, 74% of primary melanomas, 64% of metastatic melanomas and 25% of nevi. The patients whose melanoma was positive for CDCA1 had a significantly poorer overall survival (p = 0.026). CDCA1-specific siRNA inhibited the cell proliferation of SK-MEL-2 and WM115 but not reduced the migration or invasion. Our study suggested that CDCA1 could be a new therapeutic target for melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.517 P13-08[C11-04] Rhododendrol-induced cytotoxicity to melanocytes is enhanced by UVB exposure Noriko Goto 1,∗ , Taro Masaki 1 , Hiroshi Nagai 1 , Shosuke Ito 2 , Chikako Nishigori 1 1 Department of Dermatology, Graduate School of Medicine, Kobe University, Kobe, Japan 2 Department of Chemistry, Fujita Health University School of Health Sciences, Toake, Aichi, Japan
4-(4-Hydroxyphenyl)-2-butanol (rhododendrol, RD) developed for skin-whitening cosmetics in Japan was reported to cause leukoderma in some users of the products. Some patients developed RD-induced leukoderma after long sun-exposures in summer seasons. In this study, we aimed at investigating the effect of UVB on the RD-induced leukoderma. Human melanocytes were incubated with or without 24 h treatment with RD (0.36–0.72 mM) and followed by the irradiation with 20 mJ/cm2 of UVB. The cytotoxicity at 48 h after UVB-irradiation was examined using trypan blue staining, MTT assay and LDH assay. The samples treated
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
observed in most patients with TSC. However, the pathogenesis is still unknown. Recently, autophagy-lysosome pathway has been known as a novel pathway involved in skin pigmentation. Objective: To investigate whether the autophagy-lysosome pathway was involved in TSC-associated hypopigmentation. Method: Using skin tissues from hypopigmented macules of TSC, the activity of autophagy-lysosome pathway was assessed by immunohistochemical staining. Furthermore, we investigated both the pigmentation and autophagy-lysosome pathway in TSC2 knockdown (KD) human primary melanocytes and keratinocytes, by real-time PCR, western blotting analyses and intracellular immunofluorescence staining. Results: Compared to healthy donors, the number of melanocytes was not changed, but autophagy-lysosome activity was dramatically decreased in skin tissue of hypopigmentation macules from TSC. Furthermore, in TSC2 KD melanocytes, melanin contents decreased strikingly, and decreased expression levels of melanogenesis-related proteins were observed. Besides, we found that expression levels of LC3, LAMP1 and the number of autophagosomes and lysosomes were significantly decreased in TSC2 KD melanocytes. Furthermore, to clarify whether the reduction of autophagy was related to hypopigmentation, ATG7 siRNA was transfected into melanocytes. Very interestingly, we found that melanogenesis was significantly inhibited in ATG7 KD melanocytes. Conclusions: These results suggest that dysfunction of autophagy-lysosome pathway might be involved in the pathogenesis of hypopigmentation in patients with TSC. http://dx.doi.org/10.1016/j.jdermsci.2016.08.512 P13-03[C11-08] TLR3 agonist Poly (I:C) enhance Rab27A and melanosome transportation through TLR3 and MAVS pathway in human epidermal melanocytes Saaya Koike ∗ , Kenshi Yamasaki, Takeshi Yamauchi, Mai Inoue, Kenichiro Tsuchiyama, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Japan Innate immune stimuli restlessly influence epidermis where human melanocytes reside. We have examined how innate immunity affects pigmentation and observed that TLR3 agonist poly (I:C) increased melanin release from melanocytes. Poly (I:C) increased Rab27A expression and cell peripheral accumulation, which facilitate melanosome transportation to cell membrane. Knockdown of Rab27A abrogated melanosome transfer to keratinocytes by poly (I:C). Since MITF is known to increase Rab27A transcription, we examined if MITF is involved in Rab27A induction by poly (I:C). Poly (I:C) increased MITF mRNA, but siRNA for MITF did not suppress Rab27A increase, perimembranous localization of Gp100 and Rab27A, and extracellular melanin release by poly (I:C). These suggested that poly (I:C) induces Rab27A and melanosome transportation independent of MITF function. We next knocked down TRIF and MAVS, the molecules involved in TLR3 pathway. We observed that siTRIF and siMAVS suppressed Rab27A induction and melanosome transport to neighboring keratinocytes by poly (I:C) stimulation. We also observed that poly (I:C) increased expression of MAVS mRNA about 7-fold, but poly (I:C) did not altered the mRNA level of TRIF and its downstream molecules IRF3 and TBK1. These indicated that TLR3 agonist poly (I:C) activates melanosome trans-
port by increase Rab27A expression through the TLR3 and MAVS pathway. http://dx.doi.org/10.1016/j.jdermsci.2016.08.513 P13-04[C11-07] IFN- modulates chemokines from tumor-associated macrophages to enhance the therapeutic effect of anti-PD-1 antibodies in B16F10 melanoma Aya Kakizaki 1,∗ , Taku Fujimura 1 , Sadanori Furudate 1 , Yumi Kambayashi 1 , Takeshi Yamauchi 1 , Hideo Yagita 2 , Setsuya Aiba 1 1 Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan 2 Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
Tumor-associated macrophages (TAMs) play roles in maintaining the immunosuppressive microenvironment by recruiting regulatory T cells (Tregs). Since IFN- and anti-PD-1 Ab have been clinically used for the treatment of malignant melanoma, we investigated their immunomodulatory effects on melanoma using the B16F10 mouse melanoma model. B16F10 melanoma cells were subcutaneously injected into C57BL/6 mice, and when the tumor had established, we administered IFN-. The peritumoral administration of IFN- significantly decreased the mRNA expression of Tregs-related chemokines (CCL17, CCL22), which led to the suppressed recruitment of Tregs in B16F10 melanoma. Moreover, to validate the production of chemokines, we isolated CD11b+ TAMs by MACS, analyzed the chemokine production, and confirmed the decrease of CCL22 in the IFN- treated group. Furthermore, the culture supernatant from CD11b+ TAMs significantly increased the chemotactic activity for TRP-2-specific CD4+ T cells, which might suggest that chemotactic factors from CD11b+ TAMs recruit B16F10 melanoma-specific T cells into the tumor microenvironment. Since the administration of IFN- augments the expression of PD-1 on TILs, the co-administration of anti-PD-1 Ab augmented the therapeutic effect of IFN- on the B16F10 melanoma. As in the mouse model, in human, IFN- decreased the production of Th2related chemokines and augmented the production of Th1-related chemokines from monocyte- derived M2 macrophages. Since these immunomodulatory effects of IFN- on macrophages were also observed in the lesional skin of human in- transit melanoma, our present data suggest one of the possible immunomodulatory effects of IFN- and support the use of IFN- in combination with anti-PD1 Ab for the treatment of melanoma patients. http://dx.doi.org/10.1016/j.jdermsci.2016.08.514 P13-05[C11-06] Hair shaft miRNA-221 levels as a new tumor marker of melanoma Satoshi Fukushima ∗ , Taisuke Inada, Masayuki Murai, Masatoshi Jinnin, Azusa Miyashita, Satoshi Nakahara, Aki Tokuzumi, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan Micro RNA-221 (miR-221) is known to be abnormally expressed in many human cancers. We have reported that the serum levels
Abstracts / Journal of Dermatological Science 84 (2016) e89–e180
of miR-221is a tumor marker for malignant melanoma (MM). Circulating miR-221 was detectable and could be quantified in serum samples. MM patients had significantly higher miR-221 levels than healthy controls. Among the MM patients, the miR-221 levels were significantly increased in patients with stage I–IV MM compared to those with MM in situ, and their levels were correlated with tumor thickness. Moreover, a longitudinal study revealed a tendency for the miR-221 levels to decrease after surgical removal of the primary tumor, and to increase again at recurrence. Therefore, we hypothesized that the hair shaft miR-221 levels may be increased in patients with MM. In this study, we assessed the possibility that hair shaft miR-221 levels could be a marker for MM. The hair shaft miR-221 levels were significantly higher in patients with MM than those in controls. The rates of increased hair shaft miR-221 levels above the cut-off value were comparable to those of serum 5-S-CD, which is a tumor marker commonly used for MM. Measurements of the hair shaft miR-221 levels could have potential clinical value in the detection of MM. This is the first report investigating the hair shaft levels of a miRNA in patients with MM. Our investigations offer new insight into the relationship between miR-221 and MM, and may provide a new, non-invasive way to screen for melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.515 P13-06[C11-05] Aspirin induces cell death, TRAIL sensitization, and mitochondrial network abnormalities in human melanoma cells Itsuho Fujikawa 1,∗ , Miki Suzuki-Karasaki 1 , Toyoko Ochiai 1 , Yoshihiro Suzuki-Karasaki 2,3 1 Department of Dermatology, Nihon University Hospital, Tokyo, Japan 2 Innovative Therapy Research Group, Nihon University Research Institute of Medical Science, Tokyo, Japan 3 Division of Physiology, Department of Biomedical Sciences, Nihon University School of Medicine, Tokyo, Japan
Aspirin is emerging as a promising antineoplastic drug, because accumulating evidence suggest that the use of aspirin decreases the risks of multiple cancers in a cyclooxygenase-independent manner. Moreover, aspirin alone displays significant cytotoxicity toward multiple cancer cell types and potentiates the efficacy of various antineoplastic drugs. However, the precise mechanisms that underlie these effects remain poorly understood. Here we report that aspirin exerts these effects by modulating Ca2+ homeostasis and network morphology of the mitochondria in human malignant melanoma cells. Application of aspirin (mM) for 24–7 h resulted in non-apoptotic cell death. The cell death occurred relatively slowly and associated with annexin V-negative cells, and independent of caspase activation. In addition, nontoxic concentrations of aspirin sensitizes the cells to apoptosis induced by tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) in a caspase-dependent manner. In parallel, aspirin induced mitochondrial network abnormalities characterized by excessive mitochondrial fragmentation and clustering. The mitochondrial network abnormalities arise from reactive oxygen species (ROS)-mediated mitochondrial Ca2+ loading and were accelerated by the impairment of dynamin related protein 1-dependent mitochondrial fission. Indeed, aspirin at the toxic concentrations markedly increased mitochondrial Ca2+ and ROS levels. The effects of aspirin were independent of cyclooxygenase, since salicylates, which are unable of inhibiting the enzyme, induced all these events more potently than aspirin. Our results
e175
highlight the potential use of aspirin in the treatment of malignant melanoma cells in particular concomitant with TRAIL. http://dx.doi.org/10.1016/j.jdermsci.2016.08.516 P13-07[C11-03] Cell division cycle associated gene 1 as a new therapeutic target for melanoma Aki Tokuzumi ∗ , Satoshi Fukushima, Azusa Miyashita, Junji Yamashita, Satoshi Nakahara, Yosuke Kubo, Takamitsu Makino, Masatoshi Jinnin, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Science, Kumamoto University, Kumamoto, Japan Recently, it has been confirmed that immunotherapy and molecular target therapy for advanced melanoma increase the median survival time. To improve their effects and to reduce adverse reactions, a more ideal melanoma associated antigen is needed. Cell division cycle associated gene 1 (CDCA1), a specific function at the kinetochores to stabilize microtubule attachment, is overexpressed in various cancers, such as, non-small cell lung cancer, cholangiocellular carcinoma, renal cell cancer. On the other hand, CDCA1 does not show detectable levels of expression in normal tissues except for the testis, so it is a member of a cancer-testis antigen. To investigate the expressions of CDCA1 in melanoma, we conducted reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines. We also performed immunohistochemical analysis using tissue samples of melanoma and nevus tissue. In addition, we analyzed the role of CDCA1 in proliferation, migration and invasion of melanoma with melanoma cell lines SK-MEL-2 and WM115, using specific small interfering siRNAs. We found that CDCA1 was expressed in all of the melanoma cell lines, 74% of primary melanomas, 64% of metastatic melanomas and 25% of nevi. The patients whose melanoma was positive for CDCA1 had a significantly poorer overall survival (p = 0.026). CDCA1-specific siRNA inhibited the cell proliferation of SK-MEL-2 and WM115 but not reduced the migration or invasion. Our study suggested that CDCA1 could be a new therapeutic target for melanoma. http://dx.doi.org/10.1016/j.jdermsci.2016.08.517 P13-08[C11-04] Rhododendrol-induced cytotoxicity to melanocytes is enhanced by UVB exposure Noriko Goto 1,∗ , Taro Masaki 1 , Hiroshi Nagai 1 , Shosuke Ito 2 , Chikako Nishigori 1 1 Department of Dermatology, Graduate School of Medicine, Kobe University, Kobe, Japan 2 Department of Chemistry, Fujita Health University School of Health Sciences, Toake, Aichi, Japan
4-(4-Hydroxyphenyl)-2-butanol (rhododendrol, RD) developed for skin-whitening cosmetics in Japan was reported to cause leukoderma in some users of the products. Some patients developed RD-induced leukoderma after long sun-exposures in summer seasons. In this study, we aimed at investigating the effect of UVB on the RD-induced leukoderma. Human melanocytes were incubated with or without 24 h treatment with RD (0.36–0.72 mM) and followed by the irradiation with 20 mJ/cm2 of UVB. The cytotoxicity at 48 h after UVB-irradiation was examined using trypan blue staining, MTT assay and LDH assay. The samples treated