A Sulfur Containing Melanogenesis Substrate, N-Pr-4-S-CAP as a Potential Source for Selective Chemoimmunotherapy of Malignant Melanoma.

N-propionyl-4-S-cysteaminylphenol (N-Pr-4-S-CAP) is a substrate for tyrosinase, which is a melanin biosynthesis enzyme and has been shown to be selectively incorporated into melanoma cells. It was found to cause selective cytotoxicity against melanocytes and melanoma cells after selective incorporation, resulting in the induction of anti-melanoma immunity. However, the underlying mechanisms for the induction of anti-melanoma immunity remain unclear. This study aimed to elucidate the cellular mechanism for the induction of anti-melanoma immunity and clarify whether N-Pr-4-S-CAP administration could be a new immunotherapeutic approach against melanoma, including local recurrence and distant metastasis. A T cell depletion assay was used for the identification of the effector cells responsible for N-Pr-4-S-CAP-mediated anti-melanoma immunity. A cross-presentation assay was carried out by using N-Pr-4-S-CAP-treated B16-OVA melanoma-loaded bone marrow-derived dendritic cells (BMDCs) and OVA-specific T cells. Administration of N-Pr-4-S-CAP induced CD8+ T cell-dependent anti-melanoma immunity and inhibited the growth of challenged B16F1 melanoma cells, indicating that the administration of N-Pr-4-S-CAP can be a prophylactic therapy against recurrence and metastasis of melanoma. Moreover, intratumoral injection of N-Pr-4-S-CAP in combination with BMDCs augmented the tumor growth inhibition when compared with administration of N-Pr-4-S-CAP alone. BMDCs cross-presented a melanoma-specific antigen to CD8+ T cells through N-Pr-4-S-CAP-mediated melanoma cell death. Combination therapy using N-Pr-4-S-CAP and BMDCs elicited a superior anti-melanoma effect. These results suggest that the administration of N-Pr-4-S-CAP could be a new strategy for the prevention of local recurrence and distant metastasis of melanoma.

Immunomodulation of Melanoma by Chemo-Thermo-Immunotherapy Using Conjugates of Melanogenesis Substrate NPrCAP and Magnetite Nanoparticles: A Review.

A major advance in drug discovery and targeted therapy directed at cancer cells may be achieved by the exploitation and immunomodulation of their unique biological properties. This review summarizes our efforts to develop novel chemo-thermo-immunotherapy (CTI therapy) by conjugating a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP: amine analog of tyrosine), with magnetite nanoparticles (MNP). In our approach, NPrCAP provides a unique drug delivery system (DDS) because of its selective incorporation into melanoma cells. It also functions as a melanoma-targeted therapeutic drug because of its production of highly reactive free radicals (melanoma-targeted chemotherapy). Moreover, the utilization of MNP is a platform to develop thermo-immunotherapy because of heat shock protein (HSP) expression upon heat generation in MNP by exposure to an alternating magnetic field (AMF). This comprehensive review covers experimental in vivo and in vitro mouse melanoma models and preliminary clinical trials with a limited number of advanced melanoma patients. We also discuss the future directions of CTI therapy.

Elucidation of Melanogenesis Cascade for Identifying Pathophysiology and Therapeutic Approach of Pigmentary Disorders and Melanoma.

Melanogenesis is the biological and biochemical process of melanin and melanosome biosynthesis. Melanin is formed by enzymic reactions of tyrosinase family proteins that convert tyrosine to form brown-black eumelanin and yellow-red pheomelanin within melanosomal compartments in melanocytes, following the cascades of events interacting with a series of autocrine and paracrine signals. Fully melanized melanosomes are delivered to keratinocytes of the skin and hair. The symbiotic relation of a melanocyte and an associated pool of keratinocytes is called epidermal melanin unit (EMU). Microphthalmia-associated transcription factor (MITF) plays a vital role in melanocyte development and differentiation. MITF regulates expression of numerous pigmentation genes for promoting melanocyte differentiation, as well as fundamental genes for maintaining cell homeostasis. Diseases involving alterations of EMU show various forms of pigmentation phenotypes. This review introduces four major topics of melanogenesis cascade that include (1) melanocyte development and differentiation, (2) melanogenesis and intracellular trafficking for melanosome biosynthesis, (3) melanin pigmentation and pigment-type switching, and (4) development of a novel therapeutic approach for malignant melanoma by elucidation of melanogenesis cascade.

Molecular Events in the Melanogenesis Cascade as Novel Melanoma-Targeted Small Molecules: Principle and Development.

Malignant melanoma is one of the most malignant of all cancers. Melanoma occurs at the epidermo-dermal interface of the skin and mucosa, where small vessels and lymphatics are abundant. Consequently, from the onset of the disease, melanoma easily metastasizes to other organs throughout the body via lymphatic and blood circulation. At present, the most effective treatment method is surgical resection, and other attempted methods, such as chemotherapy, radiotherapy, immunotherapy, targeted therapy, and gene therapy, have not yet produced sufficient results. Since melanogenesis is a unique biochemical pathway that functions only in melanocytes and their neoplastic counterparts, melanoma cells, the development of drugs that target melanogenesis is a promising area of research. Melanin consists of small-molecule derivatives that are always synthesized by melanoma cells. Amelanosis reflects the macroscopic visibility of color changes (hypomelanosis). Under microscopy, melanin pigments and their precursors are present in amelanotic melanoma cells. Tumors can be easily targeted by small molecules that chemically mimic melanogenic substrates. In addition, small-molecule melanin metabolites are toxic to melanocytes and melanoma cells and can kill them. This review describes our development of chemo-thermo-immunotherapy based on the synthesis of melanogenesis-based small-molecule derivatives and conjugation to magnetite nanoparticles. We also introduce the other melanogenesis-related chemotherapy and thermal medicine approaches and discuss currently introduced targeted therapies with immune checkpoint inhibitors for unresectable/metastatic melanoma.

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.

Growth inhibition of re-challenge B16 melanoma transplant by conjugates of melanogenesis substrate and magnetite nanoparticles as the basis for developing melanoma-targeted chemo-thermo-immunotherapy.

Melanogenesis substrate, N-propionyl-cysteaminylphenol (NPrCAP), is selectively incorporated into melanoma cells and inhibits their growth by producing cytotoxic free radicals. Magnetite nanoparticles also disintegrate cancer cells and generate heat shock protein (HSP) upon exposure to an alternating magnetic field (AMF). This study tested if a chemo-thermo-immunotherapy (CTI therapy) strategy can be developed for better management of melanoma by conjugating NPrCAP on the surface of magnetite nanoparticles (NPrCAP/M). We examined the feasibility of this approach in B16 mouse melanoma and evaluated the impact of exposure temperature, frequency, and interval on the inhibition of re-challenged melanoma growth. The therapeutic protocol against the primary transplanted tumor with or without AMF exposure once a day every other day for a total of three treatments not only inhibited the growth of the primary transplant but also prevented the growth of the secondary, re-challenge transplant. The heat-generated therapeutic effect was more significant at a temperature of 43 degrees C than either 41 degrees C or 46 degrees C. NPrCAP/M with AMF exposure, instead of control magnetite alone or without AMF exposure, resulted in the most significant growth inhibition of the re-challenge tumor and increased the life span of the mice. HSP70 production was greatest at 43 degrees C compared to that with 41 degrees C or 46 degrees C. CD8(+)T cells were infiltrated at the site of the re-challenge melanoma transplant.

Diacylglycerol kinase alpha suppresses tumor necrosis factor-alpha-induced apoptosis of human melanoma cells through NF-kappaB activation.

We investigated the implication of diacylglycerol kinase (DGK) alpha (type I isoform) in melanoma cells because we found that this DGK isoform was expressed in several human melanoma cell lines but not in noncancerous melanocytes. Intriguingly, the overexpression of wild-type (WT) DGKalpha, but not of its kinase-dead (KD) mutant, markedly suppressed tumor necrosis factor (TNF)-alpha-induced apoptosis of AKI human melanoma cells. In the reverse experiment, siRNA-mediated knockdown of DGKalpha significantly enhanced the apoptosis. The overexpression of other type I isoforms (DGKbeta and DGKgamma) had, on the other hand, no detectable effects on the apoptosis. These results indicate that DGKalpha specifically suppresses the TNF-alpha-induced apoptosis through its catalytic action. We found that the overexpression of DGKalpha-WT, but not of DGKalpha-KD, further enhanced the TNF-alpha-stimulated transcriptional activity of an anti-apoptotic factor, NF-kappaB. Conversely, DGKalpha-knockdown considerably inhibited the NF-kappaB activity. Moreover, an NF-kappaB inhibitor blunted the anti-apoptotic effect of DGKalpha overexpression. Together, these results strongly suggest that DGKalpha is a novel positive regulator of NF-kappaB, which suppresses TNF-alpha-induced melanoma cell apoptosis.

Approach for the Derivation of Melanocytes from Induced Pluripotent Stem Cells.

Induced pluripotent stem (iPS) cells have the ability to differentiate into multiple cell types in the body and have an unlimited growth potential. However, iPS cell-derived melanocytes produced by existing protocols have significant limitations in developing novel strategies for regenerative medicine and cell therapies of pigmentation disorders in humans because they involve culture in media containing fetal bovine serum and nonphysiological agents. In this study, we established an in vitro approach to generate iPS cell-derived human melanocytes that have higher proliferation rates and increased melanin production compared with melanocytes prepared by previously reported approaches. Importantly, our iPS cell-derived human melanocytes are prepared in fetal bovine serum-free culture conditions that do not contain any nonphysiological agents. We designed two original methods, transferring black colonies by pipette and recovering black cell pellets from centrifuged medium, and numerous human iPS cell-derived melanocytes proliferated in gelatinous dishes coated with Matrigel after 12 days. We also succeeded in inducing melanin pigmentation in the nude mouse skin in vivo using those human iPS cell-derived melanocytes. We propose that this method using iPS cells established from T cells in the blood of normal human volunteers could be applied clinically to develop regenerative medicine and cell therapies for various forms of human pigmentation disorders.

Papulonecrotic tuberculid-like eruptions after Bacille Calmette-Guerin vaccination.

One of the specific skin lesions occurring after Bacille Calmette-Guerin (BCG) vaccination is generalized tuberculid-like eruptions, which occur rarely, but have a tendency to heal spontaneously. Their pathogenesis and relationship to "true" tuberculids are poorly understood. This report presents a case of a 6-month-old girl who developed generalized papulonecrotic tuberculid-like eruptions after BCG vaccination. The skin lesions healed spontaneously in 3 months. Culture of blood, gastric juice and reverse transcription polymerase chain reaction (RT-PCR) of papulonecrotic skin biopsies were all negative for Mycobacterium tuberculosis. Histopathology of papulonecrotic eruptions revealed marked epidermal necrosis, perivascular lymphocytic infiltrates and epidermotropic infiltration of lymphocytes showing markers of CD3(+) lymphocytes (90-95% of all infiltrating cells), CD4(+) (40-50%), CD8(+) (40-50%), and CD45RO(+) (70%). In contrast, the BCG vaccination site revealed intradermal granuloma with epithelioid cells, occasional giant cells and infiltration of lymphocytes consisting of CD3(+) (60-70%), CD4(+) (40-50%), CD8(+) (30-40%), CD45RO(+) (40%), CD79a(+) (30-40%), and CD20(+) (20-30%). Our patient did not reveal any signs indicative of tuberculosis. Papulonecrotic lesions were therefore called papulonecrotic tuberculid-like eruptions, rather than tuberculids, that occurred after BCG vaccination and appeared to derive from a hypersensitive reaction mediated by immune lymphocytic infiltration.

The real-time, three-dimensional analyses of benign and malignant skin tumors by confocal laser scanning microscopy.

BACKGROUND: In obtain images of skin tumors non-invasively with real-time, confocal laser scanning microscope (CLSM) is introduced. OBJECTIVE: Reconstructed images of given horizontal sections were converted into three-dimensions using the data set of a large number of tomograms in the horizontal directions. METHODS: To develop the multiplaner reconstruction images of skin tumors in vertical directions and three-dimensionally reconstructed images of tumors will be obtained from the continuously collected horizontal image data sets. RESULTS: Three-dimensional analyses of the skin tumors from reconstructed images of the CLSM scanning have provided the information as to their physiological characteristics as well as the extent of deep invasion in real-time with non-invasive manner. High performance three-dimensional conversion software was effective in displaying three-dimensional construction of skin tumors. CONCLUSION: The CLSM scanning images followed by three-dimensional reconstruction using them can provide the real-time and non-invasive diagnoses of skin tumors and analyze the radial growth phase of tumors and the three-dimensional growth characteristics.

Biological monitoring of solar UV radiation at 17 sites in Asia, Europe and South America from 1999 to 2004.

A small and robust dosimeter for determining the biologically effective dose of ambient UV radiation has been developed using UV-sensitive mutant spores of Bacillus subtilis strain TKJ6312. A membrane filter with four spots of the spores was snapped to a slide mount. The slide was wrapped and covered with two or more layers of polyethylene sheet to protect the sample from rain and snow and to reduce monthly-cumulative doses within the measurable range. From 1999, monthly data were collected at 17 sites for more than 1 year, and data for 4 to 6 consecutive years were obtained from 12 sites. Yearly total values of the spore inactivation dose (SID) ranged from 3200 at subarctic Oulu to 96 000 at tropical Denpasar, and the mean yearly values of SID exhibited an exponential dependence on latitude in both hemispheres with a doubling for about every 14 degrees of change. During the observation period, increasing trends of UV doses have been observed at all sites with more than 5 years of data available. Year-to-year variations at high and middle latitude sites are considered due mostly to climatic variation. At three tropical sites, negative correlations between the yearly doses and the column ozone amounts were observed. The results verified the applicability of spore dosimetry for global and long-time monitoring of solar UV radiation, in particular at tropical sites where no monitoring is taking place.

Reconstruction method with a newly-designed bilobed flap after excision of tumours of the skin.

We have devised a bilobed skin flap for reconstruction after excision of small skin tumours. The sutured part serves as a zig-zag that leads to only slight postoperative contracture of the scar. The rotation centre of the flap is nearer to the affected area than other conventional bilobed flaps, resulting in less dog-ear deformity and distortion of tissue.

Tyrosinase and tyrosinase-related protein 1 require Rab7 for their intracellular transport.

We have recently identified the association of Rab7 in melanosome biogenesis and proposed that Rab7 is involved in the transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes, possibly passing through late-endosome-delineated compartments. In order to further investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and tyrosinase-related protein by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection of the recombinant adenoviruses carrying tyrosinase (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected SK-mel-24 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal lgp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization of tyrosinase and lgp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected SK-mel-24 cells, TRP-1, which is reported to be present exclusively in melanosomes, was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules. Our findings indicate that the dominant-negative mutant of Rab7 impairs vesicular transport of tyrosinase and TRP-1, suggesting that the transport of these melanogenic proteins from the trans-Golgi network to maturing melanosomes requires passage through endosome-delineated compartments.

Combination of porous hydroxyapatite and cationic liposomes as a vector for BMP-2 gene therapy.

The clinical significance of hydroxyapatite (HAP) as a bone substitute has become apparent in recent years and bone morphogenetic protein (BMP) a substance which induces bone has attracted much attention. In this study, a 1.2 cm diameter bone defects created on rabbit cranium were treated with the BMP-2 gene (cDNA plasmid) introduced with porous HAP after completion of hemostasis and the resultant bone formation was analyzed histopathologically. The amounts of bone formation was compared BMP-2 cDNA plasmids were not combined with cationic liposomes as a vector. Four groups of rabbits were compared. In the HAP group the cranial bone defect was treated with HAP containing 40 microg of liposomes and a dummy gene (PU). The BMP gene HAP group was treated with HAP soaked in liposomes and 10 microg of the BMP-2 gene. In addition, a group was treated with the gene without implanting HAP. Bone formation on the cranial defects was evaluated 3, 6 and 9 weeks after the operation, by X-ray and histopathological examinations. Three weeks after the operation there was vigorous bone formation in the cranial defect in the group which received the BMP-2 gene without HAP, and complete ossification was observed at 9 weeks. In the group which received HAP containing the BMP-2 gene, although new bone formation was evident surrounding the scaffold 3 weeks post-operation, the induced bone tissue did not fill all the pores of the scaffold even at 9 weeks post-operation. These results confirm the clinical usefulness of gene therapy for bone formation, using the BMP-2 gene combined with cationic liposomes as a vector. It is possible that the effects of administering the BMP-2 gene will be improved by specializing the microstructure of scaffold for gene therapy.

Detection of tyrosinase and tyrosinase-related protein 1 sequences from peripheral blood of melanoma patients using reverse transcription-polymerase chain reaction.

BACKGROUND: malignant melanoma has one of the highest rates of metastasis. Unlike other solid cancers, no sensitive tumor markers or laboratory tests that can provide information of the risk of metastasis and predict the prognosis have yet been established. OBJECTIVE: the study was done to establish a RT-PCR sensitive and specific enough to detect melanocyte-specific transcripts from peripheral blood cells. METHODS: peripheral white blood cells were collected from 30 healthy donors and 43 melanoma patients. Melanocyte-specific tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1) were selected as targets of RT-PCR. The sensitivity of detection using SK-mel-23 melanoma cells and rates of false-positiveness using non-melanoma blood were compared between single-step PCR and nested PCR. Analysis of melanoma blood samples was carried out by the single-step PCR. RESULTS: the nested RT-PCR amplified the TYR and TYRP1 sequences from 14 and 2 of the 30 healthy bloods, respectively. However, the single-step RT-PCR did not amplify TYR or TYRP1 sequences from any of the healthy controls. Our single-step RT-PCR detected 1.7 and 0.8 SK-mel-23 melanoma cells per ml blood for TYR and TYRP1, respectively. Overall, TYR mRNA was detected in 15 of the 43 melanoma patients (34.9%), and TYRP1 mRNA in 16 of the 43 (37.2%). The specificities of detection of TYR and TYRP1 were 83.3 and 88.9%, respectively. CONCLUSION: our single-step RT-PCR for TYR and TYRP1 mRNAs is more specific than the previous nested RT-PCR for TYR and applicable to detect circulating melanoma cells.

Ectopic expression of tyrosinase increases melanin synthesis and cell death following UVB irradiation in fibroblasts from familial atypical multiple mole and melanoma (FAMMM) patients.

Patients with familial atypical multiple mole and melanoma (FAMMM) [so-called familial dysplastic naevus syndrome (FDNS)] have a high risk for the development of malignant melanoma. The underlying gene defect has an autosomal dominant inheritance with variable expression and incomplete penetrance. Fibroblasts derived from FAMMM patients have high sensitivity to UVC and mutagens, e.g. 4-nitroquinoline-1-oxide. We were interested in identifying how the combination of inherent sensitivity to UV light and abnormal melanin synthesis interacts in the development of melanoma in FAMMM patients. Intermediates of melanin synthesis produce free radicals that are toxic to cells. Atypical moles (dysplastic naevi) are engaged in the biosynthesis of abnormal melanin pigments. This study examined whether there was any abnormal melanin pigmentation or cell damage after the ectopic expression of tyrosinase in fibroblasts from FAMMM patients when compared with fibroblasts from normal subjects. Fibroblasts from FAMMM patients (3012T and 3072T) were associated with a higher sensitivity than normal human fibroblasts to the toxicity of UVB. When cells were infected with tyrosinase-expressing adenovirus (Ad-HT) and irradiated with UVB, FAMMM fibroblasts showed higher tyrosinase activity, produced more melanin pigments and were degraded more significantly than normal human fibroblasts. Western blot analysis revealed that Ad-HT-infected 3072T produced a larger amount of tyrosinase protein than did Ad-HT-infected normal fibroblasts after UVB irradiation. Our findings suggest: (1) that FAMMM fibroblasts have an unknown machinery which enhances tyrosinase expression by UVB irradiation; and (2) that the resulting increase in melanin synthesis affects the cytotoxicity of UVB to FAMMM fibroblasts. All of these processes may be involved in the genomic instability and development of melanoma in FAMMM patients.

A case of systemic osteomyelitides due to Mycobacterium avium.

A case of multiple osteomyelitides due to Mycobacterium avium (M. avium) infection with osteosclerotic bone lesions is reported. A 67-year-old male had been suffering from persistent fever and back pain since October 1999, and 20.0-2.5 mg prednisolone per day was prescribed for continuous inflammatory symptoms in January 2000. Six months later, computed tomography revealed osteosclerotic lesions in the left femur and thoracic vertebrae, but no skin lesion associated with mastocytosis or internal malignancy was identified. In September of 2002, a dome-shaped, soft subcutaneous tumor developed on the upper sternum. Histopathological findings revealed subcutaneous adipose tissue with several foci of tiny abscesses. Two weeks later, creamy pus was discharged through a draining sinus at the center of the wound. M. avium was demonstrated in the pus by Zeel-Nielsen staining and microplate hybridization.

Long-term continuous versus intermittent cyclosporin: therapy for psoriasis.

A multicenter randomized controlled study was conducted to assess the long-term efficacy and safety of cyclosporin A therapy for psoriasis using either a continuous or an intermittent regimen. Initially, both regimens consisted of 3-5 mg/kg/day administration of CyA. Once remission was obtained, CyA dose was maintained between 0.5 and 3 mg/kg/day under the continuous regimen, while under the intermittent regimen, CyA dose was tapered off and, when necessary, topical corticosteroids were used until relapse occurred. Thirty-one patients were followed for at least 48 months (mean follow-up period: 55.9+/-4.6 months): 15 received continuous therapy, and 16 received intermittent therapy. With both regimens, the PASI (Psoriasis Area and Severity Index) score was maintained at 5-12 points throughout the follow-up period. The score was decreased by more than 70% from baseline with both regimens: the responses between them were not significantly different. However, overall control of psoriasis, as assessed from the averaged PASI score, was better in the patients receiving continuous therapy. Although the overall frequency of adverse reactions was similar for the two regimens, cancer occurred in two patients on continuous therapy (gastric cancer and hepatocellular carcinoma in one patient each). We could not, however, definitely attribute the cancers in the two patients to continuous therapy itself. There was a significantly higher incidence of renal impairment in elderly patients receiving either regimen when compared with younger patients. In conclusion, CyA administered to psoriasis patients under both regimens exhibited long-term efficacy and tolerability. Despite a lower overall efficacy, it seems proper to conclude that intermittent therapy is more useful than continuous therapy due to the occurrence of malignancies with continuous therapy. Further investigation is required to determine whether intermittent therapy is really safer than continuous therapy, and, if so, how it should be designed to minimize long-term adverse reactions and achieve overall control comparable to that of continuous CyA therapy.

Antitumor immunity by magnetic nanoparticle-mediated hyperthermia.

Magnetic nanoparticle-mediated hyperthermia (MNHT) generates heat to a local tumor tissue of above 43°C without damaging surrounding normal tissues. By applying MNHT, a significant amount of heat-shock proteins is expressed within and around the tumor tissues, inducing tumor-specific immune responses. In vivo experiments have indicated that MNHT can induce the regression of not only a local tumor tissue exposed to heat, but also distant metastatic tumors unexposed to heat. In this article, we introduce recent progress in the application of MNHT for antitumor treatments and summarize the mechanisms and processes of its biological effects during antitumor induction by MNHT. Several clinical trials have been conducted indicating that the MNHT system may add a promising and novel approach to antitumor therapy.

SIRT1 regulates lamellipodium extension and migration of melanoma cells.

Melanoma is highly metastatic, but the mechanism of melanoma cell migration is still unclear. We found that melanoma cells expressed the nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 in the cytoplasm. Cell membrane extension and migration of melanoma cells were inhibited by SIRT1 inhibitors or SIRT1 knockdown, whereas SIRT1 activators enhanced elongation of protrusion and cellular motility. In B16F1 cells, growth factor stimulation induced lamellipodium extension, a characteristic feature at the leading edge of migrating cells, and SIRT1 was found in the lamellipodium. SIRT1 inhibitor nicotinamide (NAM) or SIRT1 small interfering RNAs suppressed the lamellipodium extension by serum or platelet-derived growth factor (PDGF). The lamellipodium formation by dominant-active Rac1 was also inhibited by NAM, a SIRT1 inhibitor. NAM inhibited the accumulation of phosphorylated Akt at the submembrane by serum or PDGF. Using fluorescence resonance energy transfer, we found that NAM impaired PDGF-dependent increase in the phosphatidylinositol-3,4,5-trisphosphate level at the leading edge. NAM inhibited the abdominal metastasis of transplanted B16F1 melanoma cells in C57BL6/J mice and improved survival. Finally, SIRT1-knockdown B16F1 cells showed significantly reduced metastasis in transplanted mice compared with that in control B16F1 cells. These results indicate that SIRT1 inhibition is a strategy to suppress metastasis of melanoma cells.