Molecular and Biochemical Basis of Minocycline-Induced Hyperpigmentation-The Study on Normal Human Melanocytes Exposed to UVA and UVB Radiation.

Minocycline is a drug which induces skin hyperpigmentation. Its frequency reaches up to 50% of treated patients. The adverse effect diminishes the great therapeutic potential of minocycline, including antibacterial, neuroprotective, anti-inflammatory and anti-cancer actions. It is supposed that an elevated melanin level and drug accumulation in melanin-containing cells are related to skin hyperpigmentation. This study aimed to evaluate molecular and biochemical mechanism of minocycline-induced hyperpigmentation in human normal melanocytes, as well as the contribution of UV radiation to this side effect. The experiments involved the evaluation of cyto- and phototoxic potential of the drug using cell imaging with light and confocal microscopes as well as biochemical and molecular analysis of melanogenesis. We showed that minocycline induced melanin synthesis in epidermal melanocytes. The action was intensified by UV irradiation, especially with the UVB spectrum. Minocycline stimulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) gene. Higher levels of melanin and increased activity of tyrosinase were also observed in treated cells. Moreover, minocycline triggered the supranuclear accumulation of tyrosinase, similar to UV radiation. The decreased level of premelanosome protein PMEL17 observed in all minocycline-treated cultures suggests disorder of the formation, maturation or distribution of melanosomes. The study revealed that minocycline itself was able to enhance melanin synthesis. The action was intensified by irradiation, especially with the UVB spectrum. Demonstrated results confirmed the potential role of melanin and UV radiation minocycline-induced skin hyperpigmentation.

Adoptive transfer of Tc1 or Tc17 cells elicits antitumor immunity against established melanoma through distinct mechanisms.

Adoptive cell transfer (ACT) of ex vivo-activated autologous tumor-reactive T cells is currently one of the most promising approaches for cancer immunotherapy. Recent studies provided some evidence that IL-17-producing CD8(+) (Tc17) cells may exhibit potent antitumor activity, but the specific mechanisms have not been completely defined. In this study, we used a murine melanoma lung-metastasis model and tested the therapeutic effects of gp100-specific polarized type I CD8(+) cytotoxic T (Tc1) or Tc17 cells combined with autologous bone marrow transplantation after total body irradiation. Bone marrow transplantation combined with ACT of antitumor (gp100-specific) Tc17 cells significantly suppressed the growth of established melanoma, whereas Tc1 cells induced long-term tumor regression. After ACT, Tc1 cells maintained their phenotype to produce IFN-γ, but not IL-17. However, although Tc17 cells largely preserved their ability to produce IL-17, a subset secreted IFN-γ or both IFN-γ and IL-17, indicating the plasticity of Tc17 cells in vivo. Furthermore, after ACT, the Tc17 cells had a long-lived effector T cell phenotype (CD127(hi)/KLRG-1(low)) as compared with Tc1 cells. Mechanistically, Tc1 cells mediated antitumor immunity primarily through the direct effect of IFN-γ on tumor cells. In contrast, despite the fact that some Tc17 cells also secreted IFN-γ, Tc17-mediated antitumor immunity was independent of the direct effects of IFN-γ on the tumor. Nevertheless, IFN-γ played a critical role by creating a microenvironment that promoted Tc17-mediated antitumor activity. Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective antitumor immunity through distinct effector mechanisms, but Tc1 cells are superior to Tc17 cells in mediating tumor regression.

Analysis of the processing of seven human tumor antigens by intermediate proteasomes.

We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (ß5i) or two (ß1i and ß5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3(114-122), MAGE-C2(42-50), MAGE-C2(336-344)) or the standard proteasome (Melan-A(26-35), tyrosinase(369-377), gp100(209-217)). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2(336-344) was only produced by intermediate proteasome ß1i-ß5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2(191-200) is produced only by intermediate proteasome ß1i-ß5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.

Control of amyloid assembly by autoregulation.

The assembly of proteins into amyloid fibrils can be an element of both protein aggregation diseases and a functional unit in healthy biological pathways. In both cases, it must be kept under tight control to prevent undesired aggregation. In normophysiology, proteins can self-chaperone amyloidogenic segments by restricting their conformational flexibility in an overall stabilizing protein fold. However, some aggregation-prone segments cannot be controlled in this manner and require additional regulatory elements to limit fibrillation. The present review summarizes different molecular mechanisms that proteins use to control their own assembly into fibrils, such as the inclusion of a chaperoning domain or a blocking segment in the proform, the controlled release of an amyloidogenic region from the folded protein, or the adjustment of fibrillation propensity according to pH. Autoregulatory elements can control disease-related as well as functional fibrillar protein assemblies and distinguish a group of self-regulating amyloids across a wide range of biological functions and organisms.

Shikonin enhances efficacy of a gene-based cancer vaccine via induction of RANTES.

BACKGROUND: Shikonin, a phytochemical purified from Lithospermum erythrorhizon, has been shown to confer diverse pharmacological activities, including accelerating granuloma formation, wound healing, anti-inflammation and others, and is explored for immune-modifier activities for vaccination in this study. Transdermal gene-based vaccine is an attractive approach for delivery of DNA transgenes encoding specific tumor antigens to host skin tissues. Skin dendritic cells (DCs), a potent antigen-presenting cell type, is known to play a critical role in transmitting and orchestrating tumor antigen-specific immunities against cancers. The present study hence employs these various components for experimentation. METHOD: The mRNA and protein expression of RANTES were detected by RT-PCR and ELISA, respectively. The regional expression of RANTES and tissue damage in test skin were evaluated via immunohistochemistry assay. Fluorescein isothiocyanate sensitization assay was performed to trace the trafficking of DCs from the skin vaccination site to draining lymph nodes. Adjuvantic effect of shikonin on gene gun-delivered human gp100 (hgp100) DNA cancer vaccine was studied in a human gp100-transfected B16 (B16/hgp100) tumor model. RESULTS: Among various phytochemicals tested, shikonin induced the highest level of expression of RANTES in normal skin tissues. In comparison, mouse RANTES cDNA gene transfection induced a higher level of mRANTES expression for a longer period, but caused more extensive skin damage. Topical application of shikonin onto the immunization site before gene gun-mediated vaccination augmented the population of skin DCs migrating into the draining lymph nodes. A hgp100 cDNA gene vaccination regimen with shikonin pretreatment as an adjuvant in a B16/hgp100 tumor model increased cytotoxic T lymphocyte activities in splenocytes and lymph node cells on target tumor cells. CONCLUSION: Together, our findings suggest that shikonin can effectively enhance anti-tumor potency of a gene-based cancer vaccine via the induction of RANTES expression at the skin immunization site.

Premelanosome-negative inflammatory angiomyolipoma of liver with expression of cathepsin K and TFE3.

We report the first case of inflammatory variant of hepatic angiomyolipoma (AML) with expression of transcription factor E3 (TFE3) protein but negativity for HMB45 and melan A in a 62-year-old female. Imaging studies revealed a tumor in the left lobe of liver, sized 5.8 cm in maximum diameter. Microscopically, the lesion was composed of large polygonal or epithelioid cells with copious eosinophilic granular cytoplasm. There was a very prominent stromal lymphoplasmacytic infiltrate. Immunohistochemically, the tumor cells showed very strong and diffuse positivity for smooth muscle actin, and cathepsin K, while S-100 protein, keratin, desmin, HMB45 and Melan-A are negative. However, there was multifocal and very convincing nuclear positivity for TFE3, thus confirms the diagnosis.

Melanosome-autonomous regulation of size and number: the OA1 receptor sustains PMEL expression.

Little is known as to how cells ensure that organelle size and number are coordinated to correctly couple organelle biogenesis to the demands of proliferation or differentiation. OA1 is a melanosome-associated G-protein-coupled receptor involved in melanosome biogenesis during melanocyte differentiation. Cells lacking OA1 contain fewer, but larger, mature melanosomes. Here, we show that OA1 loss of function reduces both the basal expression and the α-melanocyte-stimulating hormone/cAMP-dependent induction of the microphthalmia-associated transcription factor (MITF), the master regulator of melanocyte differentiation. In turn, this leads to a significant reduction in expression of PMEL, a major melanosomal structural protein, but does not affect tyrosinase and melanin levels. In line with its pivotal role in sensing melanosome maturation, OA1 expression rescues melanosome biogenesis, activates MITF expression and thereby coordinates melanosome size and number, providing a quality control mechanism for the organelle in which resides. Thus, resident sensor receptors can activate a transcriptional cascade to specifically promote organelle biogenesis.

Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.

Skin cancer is the most common type of all cancers. However, it comprises several different types of cancers, one of which is malignant melanoma. Even though melanomas only make up about 5% of skin cancers, they are responsible for the majority of skin cancer deaths due to the poor chance of survival once the tumor has metastasized. In the present study, we have developed a new assay for quantitative analysis of B16 melanoma metastasis in the lungs. We have used a triplex Q-PCR to determine the expression of the melanoma genes GP100/Pmel and tyrosinase-related protein 2 (TRP-2), and found that B16.F10gp cells were detectable in the lungs as early as 2 hours after intravenous challenge with ≥ 10(4) tumor cells. When investigating the gene expression as a function of time, we observed a gradual decrease from 2-24 hours post tumor challenge followed by an increase of approximately 2 log10 on day 11. The early decrease was accelerated in the presence of activated NK cells. To further evaluate our assay, we also investigated the level of metastasis in the context of vaccination with replication defective adenoviral vectors, Ad-Ii-GP and Ad-GP, previously found to significantly delay the outgrowth of subcutaneous melanomas. Results obtained using Q-PCR were compared to conventional counting of metastatic foci under a dissection microscope. A marked reduction in gene expression was observed in the lungs after vaccination with both vectors; however, Ad-Ii-GP showed the highest protection, and matching results were obtained by enumeration of visible tumor nodules on the lung surfaces. Finally, we could show that inhibition of tumor metastasis required antigen-specific CD8 T cells and IFNγ, but not perforin. In conclusion, the presented results validate triplex Q-PCR as a fast, objective, and quantitative method for analysis of melanoma metastasis in the lungs.

Skin-test infiltrating lymphocytes early predict clinical outcome of dendritic cell-based vaccination in metastatic melanoma.

The identification of responding patients early during treatment would improve the capability to develop effective new immunotherapies more rapidly. Here, we describe a bioassay that may link early T-cell-mediated immune responses to later clinical benefits. This bioassay rests upon the tenet of immunotherapy that tumor-specific effector T cells capable of invading peripheral tissue can recognize tumor antigens and exert cytotoxic functions there. To show its utility, we conducted a retrospective study of a large cohort of metastatic melanoma patients (n = 91) enrolled in dendritic cell (DC)-based vaccination protocols to examine a hypothesized correlation of posttreatment skin-infiltrating lymphocytes (SKIL) with overall survival (OS). Stringent immunologic criteria were defined to identify long-term survivors. The presence of tumor-associated antigen (TAA)-specific CD8(+) T cell populations within SKILs (criterion I) was highly predictive for long-term survival. Further restriction by selecting for the presence of TAA-specific CD8(+) T cells specifically recognizing tumor peptide (criterion II) was also associated with improved OS. Recognition of naturally processed antigen (criterion III) maximized the accuracy of the test, with a median OS of 24.1 versus 9.9 months (P = 0.001). Our results show that detailed characterization of SKILs can permit an accurate selection of metastatic melanoma patients who benefit most from DC-based vaccination. This simple and robust bioassay integrates multiple aspects of cellular functions that mediate effective immune responses, thereby offering an effective tool to rapidly identify patients who are responding to immunotherapy at an early stage of treatment.