Masae Okura

Melanoma-targeted chemo-thermo-immuno (CTI)-therapy using N-propionyl-4-S-cysteaminylphenol-magnetite nanoparticles elicits CTL response via heat shock protein-peptide complex release

Melanogenesis substrate, N‐propionyl‐4‐S‐cysteaminylphenol (NPrCAP) is specifically taken up by melanoma cells and inhibits their growth by producing cytotxic free radicals. By taking advantage of this unique chemical agent, we have established melanoma‐targeting intracellular hyperthermia by conjugating NPrCAP with magnetite nanoparticles (NPrCAP/M) upon exposure to an alternating magnetic field (AMF). This treatment causes cytotoxic reaction as well as heat shock responses, leading to elicitation of antitumor immune response, which was proved by tumor rechallenge test and CTL induction. We found the level of heat shock protein 72 (Hsp72) to be increased in the cell lysate and culture supernatant after intracellular hyperthermia. Melanoma‐specific CD8+ T‐cell response to dendritic cells loaded with hyperthermia‐treated tumor lysate was enhanced when compared with non‐treated tumor lysate. When heat shock protein, particularly Hsp72, was immuno‐depleted from hyperthermia‐treated tumor cell lysate, specific CD8+ T‐cell response was abolished. Thus, it is suggested that antitumor immune response induced by hyperthermia using NPrCAP/M is derived from the release of HSP‐peptide complex from degraded tumor cells. Therefore, this chemo‐thermo‐immuno (CTI)‐therapy might be effective not only for primary melanoma but also for distant metastasis because of induction of systemic antimelanoma immune responses. (Cancer Sci 2010)

Tyrosinase-catalyzed metabolism of rhododendrol (RD) in B16 melanoma cells: production of RD-pheomelanin and covalent binding with thiol proteins

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was reported to induce leukoderma of the skin. To explore the mechanism underlying that effect, we previously showed that oxidation of RD with mushroom tyrosinase produces RD‐quinone, which is converted to secondary quinone products, and we suggested that those quinones are cytotoxic because they bind to cellular proteins and produce reactive oxygen species. We then confirmed that human tyrosinase can oxidize both enantiomers of RD. In this study, we examined the metabolism of RD in B16F1 melanoma cells in vitro. Using 4‐amino‐3‐hydroxy‐n‐butylbenzene as a specific indicator, we detected moderate levels of RD‐pheomelanin in B16F1 cells exposed to 0.3 to 0.5 mM RD for 72 h. We also confirmed the covalent binding of RD‐quinone to non‐protein thiols and proteins through cysteinyl residues. The covalent binding of RD‐quinone to proteins was 20‐ to 30‐fold greater than dopaquinone. These results suggest that the tyrosinase‐induced metabolism of RD causes melanocyte toxicity.

T-cell receptor repertoires of tumor-infiltrating lymphocytes after hyperthermia using functionalized magnetite nanoparticles

AIM Accumulating evidence has indicated that hyperthermia using magnetite nanoparticles induces antitumor immunity. This study investigated the diversity of T-cell receptors (TCRs) in tumor-infiltrating lymphocytes after hyperthermia using magnetite nanoparticles. MATERIALS & METHODS Functionalized magnetite nanoparticles, N-propionyl-4-S-cysteaminylphenol (NPrCAP)/magnetite, were synthesized by conjugating the melanogenesis substrate NPrCAP with magnetite nanoparticles. NPrCAP/magnetite nanoparticles were injected into B16 melanomas in C57BL/6 mice, which were subjected to an alternating magnetic field for hyperthermia treatment. RESULTS Enlargement of the tumor-draining lymph nodes was observed after hyperthermia. The TCR repertoire was restricted in tumor-infiltrating lymphocytes, and expansion of Vβ11(+) T cells was preferentially found. DNA sequences of the third complementaritydetermining regions revealed the presence of clonally expanded T cells. CONCLUSION These results indicate that the T-cell response in B16 melanomas after hyperthermia is dominated by T cells directed toward a limited number of epitopes and that epitope-specific T cells frequently use a restricted TCR repertoire.

N-propionyl-4-S-cysteaminylphenol induces apoptosis in B16F1 cells and mediates tumor-specific T-cell immune responses in a mouse melanoma model

BACKGROUND N-propionyl-4-S-cysteaminylphenol (NPr-4-S-CAP) is selectively incorporated into melanoma cells and degrades them. However, it remains unclear whether NPr-4-S-CAP can induce cell death associated with the induction of host immune responses and tumor suppression in vivo. OBJECTIVE To examine the molecular mechanism of NPr-4-S-CAP-mediated cytotoxicity toward melanoma cells and to test whether NPr-4-S-CAP can suppress transplanted primary and secondary B16F1 melanomas. METHODS Cytotoxicity and apoptosis of melanoma cells were assessed by cell counting, flow cytometry, and detection of reactive oxygen species (ROS) and apoptotic molecules. NPr-4-S-CAP-associated host immunity was studied using a B16F1 mouse melanoma model through the application of CD4- and CD8-specific antibodies and tetramer assay. RESULTS NPr-4-S-CAP suppressed growth of pigmented melanoma cells associated with an increase of intracellular ROS, activation of caspase 3 and DNA fragmentation, suggesting that NPr-4-S-CAP mediated ROS production, eliciting apoptosis of melanoma cells. Growth of transplanted B16F1 melanomas was inhibited after the consecutive intratumoral injections of NPr-4-S-CAP, and the tumor growth after rechallenge of B16F1 was significantly suppressed in the treated mice. This suppression occurred when the treated mice were given the anti-CD4 antibody, but not the anti-CD8 antibody. Tetramer assay demonstrated increased TYRP-2-specific CD8(+) T cells in the lymph node and spleen cells prepared from NPr-4-S-CAP-treated B16F1-bearing mice. CONCLUSIONS These suggest that NPr-4-S-CAP induces apoptosis in melanoma cells through ROS production and generates CD8(+) cell immunity resulting in the suppression of rechallenged B16F1 melanoma.

The potent pro‐oxidant activity of rhododendrol–eumelanin induces cysteine depletion in B16 melanoma cells

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD‐pheomelanin and RD‐quinone bound to non‐protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5‐ to 3‐h exposure, due to oxidation to cystine. This pro‐oxidant activity was then examined using synthetic melanins. Indeed, we find that RD‐eumelanin exerts a pro‐oxidant activity as potent as Dopa‐pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD‐eumelanin with a concomitant production of H2O2. We propose that RD‐eumelanin induces cytotoxicity through its potent pro‐oxidant activity.

Rab7 is a critical mediator in vesicular transport of tyrosinase-related protein 1 in melanocytes

How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase‐related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild‐type (Rab7WT), constitutively active (Rab7Q67L) or dominant‐negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio‐distribution of melanosomal proteins between Rab7WT‐expressing cells and mutant Rab7‐expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.

Biochemical effects of the flavanol-rich lychee fruit extract on the melanin biosynthesis and reactive oxygen species

An ingredient of fruit polyphenol, resveratrol, is known to have an inhibitory effect on melanogenesis. In order to examine the functional differences between resveratrol and other fruit polyphenols, we compared biochemical effects of a resveratrol‐free polyphenol, flavanol‐rich lychee fruit extract (FRLFE), with other phenolic compounds including resveratrol. FRLFE as well as hydroquinone and resveratrol suppressed growth of B16F1 melanoma cells more significantly than rhododendrol or arbutin. Resveratrol suppressed mushroom tyrosinase at the lowest concentration (23.0 μmol/L) among the compounds tested. FRLFE and hydroquinone suppressed tyrosinase at almost the same concentration (half maximal inhibitory concentration [IC50], 83.5 and 94.6 μmol/L, respectively), which was higher than rhododendrol, ascorbic acid and arbutin (IC50, 245, 345 and 421 μmol/L, respectively). Western blot analysis revealed that although resveratrol decreased expressions of tyrosinase and tyrosinase‐related protein 1, FRLFE did not affect their expressions. Both FRLFE and resveratrol suppressed antimycin A‐mediated reactive oxygen species (ROS) production in melanocytic cells. Resveratrol‐mediated ROS suppression was inhibited by nicotinamide, a SIRT1 inhibitor. However, FRLFE‐mediated suppression was not affected by nicotinamide. Moreover, FRLFE directly decreased superoxide in vitro, as detected by superoxide dismutase‐like scavenging activity assay. These results suggest that FRLFE can protect melanocytes from cytotoxicity caused by an excess amount of melanin and ROS in a different manner from resveratrol.

Nagashima-type palmoplantar keratosis caused by compound heterozygous mutations in SERPINB7

Nagashima-type palmoplantar keratosis (NPPK, MIM 615598) is an autosomal recessive disorder, first described by Nagashima [1-4]. It is characterized by diffuse mild palmoplantar hyperkeratosis that extends onto the dorsum of hands and feet and the Achilles tendon areas. Elbows and knees are also involved and palmoplantar hyperhidrosis is common. Recently, causative loss-of-function mutations in the SERPINB7 gene were identified [4-6]. Here we report a case of a patient with compound heterozygous [...]

Diagnosis of eight groups of xeroderma pigmentosum by genetic complementation using recombinant adenovirus vectors

Because patients with xeroderma pigmentosum (XP) must avoid ultraviolet (UV) light from an early age, an early diagnosis of this disorder is essential. XP is composed of seven genetic complementation groups, XP‐A to ‐G, and a variant type (XP‐V). To establish an easy and accurate diagnosis of the eight disease groups, we constructed recombinant adenoviruses that expressed one of the XP cDNA. When fibroblasts derived from patients with XP‐A, ‐B, ‐C, ‐D, ‐F or ‐G were infected with the adenovirus expressing XPA, XPB, XPC, XPD, XPF or XPG, respectively, and UV‐C at 5–20 J/m2 was irradiated, cell viability was clearly recovered by the corresponding recombinant adenoviruses. In contrast, XP‐E and XP‐V cells were not significantly sensitive to UV irradiation and were barely complemented by the matched recombinant adenoviruses. However, co‐infection of Ad‐XPA with Ad‐XPE increased survival rate of XP‐E cells after UV‐C exposure. When XP‐V cell strains, including one derived from a Japanese patient, were infected with Ad‐XPV, exposed to UV‐B and cultured with 1 mmol/L of caffeine, flow cytometry detected a characteristic decrease in the S phase in all the XP‐V cell strains. From these results, the eight groups of XP could be differentiated by utilizing a set of recombinant adenoviruses, indicating that our procedure provides a convenient and correct diagnostic method for all the XP groups including XP‐E and XP‐V.

Verruciform xanthoma developing in eroded skin of recessive dystrophic epidermolysis bullosa.

Verruciform xanthoma (VX), first described by Shafer in 1971 [1], is a benign solitary lesion that commonly involves anogenital sites. We describe a rare case of multiple VXs arising in the skin of the back in a patient with generalized intermediate-type recessive dystrophic epidermolysis bullosa (RDEB).A 23-year-old Japanese woman developed severe generalized blistering immediately after birth; subsequently, she showed digital fusion of the fourth and fifth toes on both feet. Her sister was diagnosed [...]