The major advance of drug discovery for targeted therapy to cancer cells can be achieved by exploiting their unique biological property. The biological property unique to the melanocyte and melanoma cell resides the biosynthesis of melanin pigments within specific compartments, melanosomes. Melanogenesis begins with the conversion of amino acid, tyrosine to dopa and subsequently to dopaquinone in the presence of tyrosinase. This pathway is unique to all of melanocytes and melanoma cells including “amelanotic” melanoma. With the interaction of melanocyte-stimulating hormone (MSH)/ melanocortin 1 receptor (MC1R), the melanogenesis cascade begins from activation of microphthalmia transcription factor (MITF) for induction of either euor pheomelanin biosynthesis. Tyrosinase is the major player in this cascade. Tyrosinase is a glycoprotein and its glycosylation process is regulated by a number of molecular chaperons, including calnexin in the endoplasmic reticulum [22,23]. Vesicular transport then occurs to carry tyrosinase and its related proteins from trans-Golgi network to melanosomal compartments. In this process a significant number of transporters, such as small GTPbinding protein, adaptor proteins and PI3-kinase are involved, and early and late endosomes are closely associated with the production of these compartments. Once melanin biosynthesis is completed to conduct either euor pheomelanogenesis within melanosomal compartments, they will move along dendritic processes and transferred to surrounding keratinocytes in normal skin [24-26]. In metastatic melanoma cells, however, there will be no melanosome transfer inasmuch as there will be no receptor cells such as keratinocytes, and melanosomes synthesized are aggregated in autophagic vacuoles of melanoma cells. Thus a chemically modified melanogenesis substrate can be retained in melanoma cells once they are incorporated into their melanogenesis cascade, hence providing a unique drug delivery system (DDS) to melanoma cells.

In order to utilize melanin biosynthesis pathway for developing cytocidal compounds in controlling melanoma growth, N-acetyl and N-propionyl derivatives of cysteaminylphenols (CAPs) have been synthesized [27,28] (Figures 1 and 2). These compounds are found to possess cytocidal effect on in vivo and in vitro melanocytes through the oxidative stress. For example, both NPrCAP and NAcCAP can selectively disintegrate follicular melanocytes after single or multiple ip administration to new-born or adult C57 black mice [3,29]. In the case of adult mice after repeated ip administration of NPrCAP, white follicles with 100% success can be seen at the site where hair follicles were plucked to stimulate new melanocyte growth and to activate tyrosinase synthesis. A single ip injection of NPrCAP into a new born mouse resulted in the development of silver follicles in the entire body coat. The selective disintegration of melanocytes can be seen as early as in 12 hr after a single ip administration. None of surrounding keratinocytes or fibroblasts showed such membrane degeneration and cell death.

A high, specific uptake of NAcCAP was seen by melanoma cell lines compared to non-melanoma cells. A melanoma-bearing mouse showed, on the whole body autoradiogram, the selective uptake and covalent binding of NAcCAP in melanoma tissues of lung and skin. The specific cytotoxicity of NPrCAP and NAcCAP was examined on various types of culture cells by MTT assay [30], showing that only melanocytic cells except HeLa possessed the low IC50. The cytotoxicity on DNA synthesis inhibition was time-dependent and irreversible on melanoma cells, but was transient on HeLa cells [21].

We also examined to what extent the melanoma growth can be blocked in both in vitro culture and in vivo lung metastasis assays by administration of NAcCAP combined with buthionine sulfoximine (BSO), which blocks the effect of anti-oxidants through reducing glutathione levels. There was a marked growth inhibition of cultured melanoma cells in the presence of BSO, indicating that the selective cytotoxicity by our NPrand NAc-CAP is mediated by the production of cytotoxic free radicals. The in vivo lung metastasis experiment also showed the decreased number of lung melanoma colonies [17]. The problem was, however, that a fairly large number of amelanotic melanoma lesions were seen to grow in the lung. NPrCAP has been developed and con-

jugated with magnetite nanoparticles in the hope of increasing the cytotoxicity and overcoming the problem.

Magnetite nanoparticles have been employed for thermotherapy in a number of cancer treatments including human gliomas and prostate cancers [31-33]. They consist of 10 - 100 nm-sized iron oxide (Fe₃O₄) with a surrounding polymer coating and become magnetized when placed in AMF [3]. We expected the combination of NPrCAP and magnetite nanoparticles to be a potential source for developing not only anti-melanoma pharmacologic but also immunogenic agent. It was expected that NPrCAP/magnetite nanoparticles complex could be selectively incorporated into melanoma cells because NPrCAP possesses a specific effect on DDS. The degradation of melanoma tissues occurs from oxidative and heat stresses by exposure of NPrCAP to tyrosinase and by exposure of magnetite nanoparticles to AMF. These two stress processes produce the synergistic or additive effect for generating tumor-infiltrating lymphocytes (TIL) that will kill melanoma cells in distant metastases (Figure 3).

We synthesized, in our initial study, the conjugates of NPrCAP with magnetite nanoparticles, NPrCAP/ML and NPrCAP/CML, in which NPrCAP was embedded in neutral and cationic liposomes respectively (Figure 4). There were, however, non-specific electrostatic interacttion between cationic magneto-liposomes (NPrCAP/ CML) and various non-target cells [33] as well as nonspecific aggregations in neutral magneto-liposomes (NPrCAP/ML). A promising technique is the use of tumor-targeted magnetite nanoparticles, and this approach was extended by synthesizing another type of magnetite nanoparticles, NPrCAP/M and NPrCAP/PEG/M, on which NPrCAP is superficially and directly bound on the surface of magnetite nanoparticles without using liposomes. They are chemically stable, can be produced in

large quantities and may develop effective mela-nomaargeted chemotherapy (by NPrCAP) and thermo-immunotherapy (by magnetite nanoparticles with HSP), hence providing a basis for a novel CTI therapy. Most of the experiments described below were carried out by employing NPrCAP/M. A preliminary clinical trial, however, used NPrCAP/PEG/M to which polyethylene glycol (PEG) was employed to conjugate NPrCAP and magnetite nanoparticles.

In our studies, we could see that NPrCAP/M nanoparticle conjugates were selectively aggregated in melanoma cells compared to non-melanoma cells (Figure 5(a)). Specifically NPrCAP/M nanoparticles were found to be incorporated into and aggregated in melanosomal compartments after ip administration by electron microscopy (Figure 5(b)). After AMF exposure, there will be selective disintegration of melanoma tissues as can be seen by Berlin Blue staining [34,35] (Figure 5(c)). The conjugates of NPrCAP and magnetite nanoparticles will be selectively aggregated on the cell surface of melanoma cells through still unknown surface receptor and then incorporated into melanoma cells by early and late endosomes. The conjugates will then be incorporated into melanosomal compartment as the stage I melanosomes and late endosomes share common compartments, to which tyrosinase will be transported from trans-Golgi network by vesicular transport (Figure 6).

In this study, we employed three cell lines of B16 melanoma, i.e., B16F1, B16F10 and B16-ovalbmine (OVA)

cells and compared the thermotherapeutic protocols in detail by evaluating the growth of the re-challenge melanoma as well as the duration and rates of survival of melanoma bearing mice (Figure 7).

By employing B16F1 and F10 cells, we first evaluated the chemotherapeutic effect of NPrCAP/M with or without AMF exposure which generates heat. NPrCAP/ M without heat inhibited growth of primary transplants to the same degree as did NPrCAP/M with heat, indicating that NPrCAP/M alone has a chemotherapeutic effect. However, there was a significant difference in the melanoma growth inhibition of re-challenge transplants between the groups of NPrCAP/M with and without heat. NPrCAP/M with AMF exposure showed the most significant growth inhibition in re-challenge melanoma and increased life span of the host animals, i.e., almost complete rejection of re-challenge melanoma growth whereas NPrCAP/M without heat was much less, i.e., 30% - 50%, indicating that NPrCAP/M with heat possesses a thermoimmunotherapeutic effect (Figures 8(a)-(c)).

Specifically our study indicated that the most effective thermo-immunotherapy for re-challenge B16 F1 and F10 melanoma cells can be obtained at a temperature of 43˚C for 30 min with the treatment repeated three times on every other day intervals without complete degradation of the primary melanoma (Figure 8(b)). Our therapeutic conditions and their effects differ from those of magnetically mediated hyperthermia on the transplanted melanomas reported previously by Suzuki et al. [36]. Cationic magneto-liposomes-mediated hyperthermia for B16 melanoma showed that hyperthermia at 46˚C once or twice led to regression of 40% - 90% of primary tumors and to 30% - 60% survival of mice, whereas their hyperthermia at 43˚C failed to induce regression of the secondary tumors with 0% survival of mice [36].

It has been reported that the intracellular hyperthermia using magnetic nanoparticles is effective for treating certain types of cancer in not only primary but also metastatic lesions [37-43]. Incorporated magnetic nanoparticles generate heat within the cells after exposure to the AMF due to hysteresis loss or relaxational loss [44,45]. Hyperthermic treatment using CMLs, which are cationic liposomes containing 10 nm magnetite nanoparticles, induced antitumor immunity by enhancement of HSP expression [38,46-48].

In our animal study, those animals bearing B16F1 and B16F10 melanoma cells showed, to certain degree, rejecttion of second re-challenge melanoma transplantation by administration of both NPrCAP alone and NPrCAP/M minus AMF exposure [49]. Our working hypothesis for this finding is that there is a difference in the cytotoxic mechanism and immunogenic property of NPrCAP/M between experimental groups with and without AMF exposure. The animals with NPrCAP/M without AMF exposure resulted in non-necrotic, apoptotic cell death. The animals with NPrCAP/M plus AMF exposure, on the other hand, resulted in non-apoptotic, necrotic cell death with immune complex production of melanoma peptide as well as Hsp70 and a small amount of Hsp 90. The latter group of NPrCAP/M plus AMF exposure showed the most significant growth inhibition of the re-challenged melanoma growth which resulted in the almost complete survival of the host animals as long as for 3 months that we have conducted our experimental protocol [34]. In the latter study [34], we also found that repeated hyperthermia (3 cycles of NPrCAP/M administration and AMF irradiation) was required to induce the maximal antitumor immune response.

In the study using B16-OVA cells, the hyperthermia of melanoma cells using NPrCAP/M with AMF exposure further showed antitumor immune responses via crosspresentation of HSP-chaperoned antigen (Figures 9(a), 9(b), 10(a)-(d)). Moreover, the HSPs-antigen peptide complex released from melanoma cells treated with this intracellular hyperthermia was taken-up by dendritic cells (DCs) and cross-presented HSP-chaperoned peptide in the context of MHC class I molecules [49]. As stated above, our CTI therapy with AMF exposure induced NPrCAPas well as heat-mediated melanoma cell necrosis to NPrCAP/M incorporated cells. If melanoma cells escaped from necrotic cell death, repeated hyperthermia should produce necrotic cell death of previously heat shockedmelanoma cells in which HSPs were induced. In addition, our data of CTI therapy with AMF exposure using B16- OVA cells suggested that Hsp72/Hsc73, Hsp90, and ERresident HSPs participated in the induction of CD8⁺ T

cell response. In particular, among HSPs, Hsp72 was largely responsible for the augmented antigen presentation to CD8⁺ T cells. As Hsp72 is known to up-regulate in response to hyperthermia or heat shock treatment [46], newly synthesized Hsp72 has a chance to bind to the heat-denatured melanoma-associated antigen.

Monobenzyl ether of hydroquinone has long been known to produce the skin depigmentation at both drug-applied area by direct chemical reaction with tyrosinase and non-applied distant area by immune reaction with still unknown mechanism [50-53]. The melanogenesis-related cytotoxicity primarily derives from tyrosinase-mediated formation of dopaquinone and other quinone intermediates, which produce reactive oxygen species such as superoxide and H₂O₂ [19,54-56]. This unique biological property of melanin intermediates not only causes cell death, but also may produce immunogenic properties. We postulated that the cytotoxic action of NAcCAP and NPrCAP appears to involve two major biological processes. One is cytostatic process which derives from the DNA synthesis inhibition through the interaction of quinone and free radicals with SH-enzymes and thymidine synthase. Another is the cytocidal process by damage of DNA and mitochondrial ATP through oxidative stress and interaction with SH-enzyme [21]. They bind protein disulphide isomerise [57]. Although we have not yet studied which one of these two processes is responsible for the immune reaction, it is likely that the cyotocidal process of NPrCAP is involved in the induction of immune reaction.

Monobenzy ether of hydroquinone was shown to produce a reactive ortho-quinone generated by tyrosinasecatalyzed oxidation and self-coupling and thiol conjugation reactions [58]. It was also shown to induce cell death without activating the caspase cascade or DNA fragmentation, indicating that the death pathway is nonapoptotic [58,59]. It was further reported that monobenzyl ether of hydroquinone induced the immunogenicity to melanocytes and melanoma cells by forming quinonehaptens to tyrosinase protein and by inducing the release of tyrosinaseand melanoma antigen recognized by T cells-1 (MART-1) containing CD63⁺ exosomes following melanosome oxidative stress induction. The drug further augmented the processing and shedding of melanocyte differentiation antigens by inducing melanosome autophagy and enhanced tyrosinase ubiquitination, ultimately activating DCs, which induced cytotoxic human melanoma-reactive cells. These T cells eradicated melanoma in vivo [59]. It is necessary to examine if NPrCAP will also take an immune-biological process similar to that reported in the case of monobenzyl ether of hydroquinone.