Inhibitory Effect of Chlorogenic Acid Analogues Comprising Pyridine and Pyrimidine on α-MSH-Stimulated Melanogenesis and Stability of Acyl Analogues in Methanol

In continuation of studies for α-MSH stimulated melanogenesis inhibitors, we have evaluated the design, synthesis, and activity of a new series of chlorogenic acid (CGA) analogues comprising pyridine, pyrimidine, and diacyl derivatives. Among nineteen synthesized compounds, most of them (fifteen) exhibited better inhibitions of melanin formation in B16 melanoma cells. The results illustrated that a pyridine analogue 6f and a diacyl derivative 13a of CGA showed superior inhibition profiles (IC50: 2.5 ± 0.7 μM and 1.1 ± 0.1 μM, respectively) of α-MSH activities than positive controls, kojic acid and arbutin (IC50: 54 ± 1.5 μM and 380 ± 9.5 μM, respectively). The SAR studies showed that both –CF3 and –Cl groups exhibited better inhibition at the meta position on benzylamine than their ortho and para positions. In addition, the stability of diacyl analogues of CGA in methanol monitored by HPLC for 28 days indicated the steric bulkiness of acyl substituents as a key factor in their stability.

Human Recombinant DNase I (Pulmozyme®) Inhibits Lung Metastases in Murine Metastatic B16 Melanoma Model That Correlates with Restoration of the DNase Activity and the Decrease SINE/LINE and c-Myc Fragments in Blood Cell-Free DNA

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.

669 Lactate uptake through MCT11, a novel monocarboxylate transporter, enforces dysfunction in terminally exhausted T cells

BackgroundUpon infiltration into tumors, T cells experiencing persistent antigen stimulation progressively differentiate into a state of dysfunction, known as exhaustion. Exhausted T cells are characterized by the sustained upregulation of co-inhibitory molecules and reduced effector cytokine production. Additionally, exhausted T cells exist in a state of metabolic dysfunction in the tumor microenvironment (TME), due to disrupted mitochondrial biogenesis, hypoxia and lack of metabolites. Highly glycolytic tumor and stromal cells outcompete T cells for glucose, and secrete lactate into the TME, acidifying the extracellular space. Recent studies have shown lactate can be metabolized by tumor infiltrating Tregs and macrophages. We hypothesized that CD8+ tumor-infiltrating lymphocytes (TIL) may also take up lactate as an alternative carbon source to meet their metabolic demands.MethodsFor lactate uptake experiments, B16 melanoma single cell suspensions from B6 mice were loaded with the pH sensitive dye pHrodo, then pulsed with 5µM lactic acid. MCT11 KO OT-I T cells were generated via transfection of Slc16a11 sgRNA-Cas9 ribonucleoprotein complexes, and adoptively transferred into B16-OVA bearing mice.ResultsRNA sequencing and flow cytometry data from CD8+ T cell subsets in the TME revealed MCT11 (encoded by Slc16a11), a monocarboxylate transporter (MCT) only recently discovered, to be highly and uniquely expressed in terminally exhausted T cells (Tex). As lactate is an abundant monocarboxylate in tumors, we asked whether MCT11 supports lactate uptake into Tex cells. Antibody blockade of MCT11 resulted in reduced lactic acid uptake, but whether lactic acid promoted or inhibited effector function. Intriguingly, overexpression of MCT11 in OT-I T cells adoptively transferred into B16-OVA bearing mice resulted in accelerated exhaustion: increased co-inhibitory marker expression and decreased TNFa and IFN production. Conversely, knockdown of MCT11 in the same model resulted in decreased co-inhibitory marker expression and increased TNFa and IFN production. Further, MCT11 KO OT-I T cells used therapeutically had decreased tumor burden over mice treated with control OT-I T cells. As MCT11's uptake function was blocked with an antibody, we also used the antibody therapeutically, revealing that single-agent MCT11 antibody treatment reduced tumor burden and increased survival in B16 melanoma bearing mice.ConclusionsOur data support a model where exhausted CD8+ T cells upregulate MCT11, which renders them sensitive to toxic lactic acid in the TME. Our data suggest MCT11 could be deleted on therapeutic T cells or blocked using an antibody on endogenous T cells to render exhausted T cells impervious to lactic acid such and promote tumor eradication.

Tuliposides H–J and Bioactive Components from the Bulb of Amana edulis

Three new tuliposides H–J (1–3) and 11 known compounds were obtained from the methanolic extracts of the bulbs of Amana edulis for the first time. Their structures were elucidated by NMR, MS, and IR spectroscopic data, optical rotation, and Mosher’s method. The melanogenesis properties of all the isolates were evaluated in B16 melanoma cells. Consequently, tributyl citrate (9) had anti-melanogenesis activity but was cytotoxic toward B16. (+)-Pyroglutamic acid (4), (+)-butyl 5-oxopyrrolidine-2-carboxylate (6), (–)-3-hydroxy-2-methylbutyrolactone (10), and 5-(hydroxymethyl)furfural (12) had increased melanin productions and tyrosinase activities. Those active components could be further studied as the candidates against melanoma and vitiligo for skin diseases or whitening/hypopigmentation for hair.

Evaluation of the antitumor activity of 2-[3-(2-chloroethyl)-3-nitrosoureido]-1,3-propanediol (chlonisol) in C57BL/6 mice with intracranially transplanted B16 melanoma

Background. The arsenal of antitumor drug therapy for melanoma brain metastases is limited. The search and study of new agents capable to penetrate the blood-brain barrier and provide a therapeutic effect against intracranial tumors remains an unmet clinical need. The aim is to evaluate the antitumor activity of the domestic derivative of nitrosoalkylureas, chlonisol, in mice with intracranially transplanted syngeneic B16 melanoma. Methods. The experiment was carried out in 18 female inbred C57BL/6 mice. After intracranial tumor transplantation, performed according to modified technique, the animals were randomized into two groups: I. Control (n = 10) – the animals were injected with normal saline 10 ml/kg intraperitoneally; II. Chlonisol (n = 8) – the animals were treated with the test compound at a dose of 15 mg/kg in normal saline intraperitoneally. The single administration of normal saline and chlonisol was performed 24 hours after tumor transplantation. The end point of the study was overall survival (OS) of the animals. Results. Compared with the control group, administration of chlonisol significantly increased the median OS of mice from 13 to 18 days (log rank test, p = 0.0005). Chlonisol significantly decreased the risk of death by 71 % compared with the control group (HR = 0.29; 95 %CI 0.10–0.82). By the 15th day after intracranial transplantation of B16 melanoma, all 10 mice in the control group died from intracerebral tumors (100 %), whereas in the chlonisol group only 2 out of 8 (25 %) mice died (Fisher's exact test, p = 0.0015). Conclusion. Despite the exploratory nature of the present study, it provides a good starting point for further research of chlonisol in brain tumors.

Deciphering Repertoire of B16 Melanoma Reactive TCRs by Immunization, In Vitro Restimulation and Sequencing of IFNγ-Secreting T Cells

Recent advances in cancer immunotherapy have great promise for the treatment of solid tumors. One of the key limiting factors that hamper the decoding of physiological responses to these therapies is the inability to distinguish between specific and nonspecific responses. The identification of tumor-specific lymphocytes is also the most challenging step in cancer cell therapies such as adoptive cell transfer and T cell receptor (TCR) cloning. Here, we have elaborated a protocol for the identification of tumor-specific T lymphocytes and the deciphering of their repertoires. B16 melanoma engraftment following anti-PD1 checkpoint therapy provides better antitumor immunity compared to repetitive immunization with heat-shocked tumor cells. We have also revealed that the most error-prone part of dendritic cell (DC) generation, i.e., their maturation step, can be omitted if DCs are cultured at a sufficiently high density. Using this optimized protocol, we have achieved a robust IFNγ response to B16F0 antigens, but only within CD4+ T helper cells. A comparison of the repertoires of IFNγ-positive and -negative cells shows a prominent enrichment of certain clones with putative tumor specificity among the IFNγ+ fraction. In summary, our optimized protocol and the data provided here will aid in the acquisition of broad statistical data and the creation of a meaningful database of B16-specific TCRs.

Elucidation of Melanogenesis-Associated Signaling Pathways Regulated by Argan Press Cake in B16 Melanoma Cells

The beneficial effect on health of argan oil is recognized worldwide. We have previously reported that the cake that remains after argan oil extraction (argan press-cake or APC) inhibits melanogenesis in B16 melanoma cells in a time-dependent manner without cytotoxicity. In this study, the global gene expression profile of B16 melanoma cells treated with APC extract was determined in order to gain an understanding of the possible mechanisms of action of APC. The results suggest that APC extract inhibits melanin biosynthesis by down-regulating microphthalmia-associated transcription factor (Mitf) and its downstream signaling pathway through JNK signaling activation, and the inhibition of Wnt/β-catenin and cAMP/PKA signaling pathways. APC extract also prevented the transport of melanosomes by down-regulating Rab27a expression. These results suggest that APC may be an important natural skin whitening product and pharmacological agent used for clinical treatment of pigmentary disorders.

Loss of fragile site-associated tumor suppressor promotes antitumor immunity via macrophage polarization

Common fragile sites (CFSs) are specific breakage-prone genomic regions and are present frequently in cancer cells. The (E2-independent) E3 ubiquitin-conjugating enzyme FATS (fragile site-associated tumor suppressor) has antitumor activity in cancer cells, but the function of FATS in immune cells is unknown. Here, we report a function of FATS in tumor development via regulation of tumor immunity. Fats−/− mice show reduced subcutaneous B16 melanoma and H7 pancreatic tumor growth compared with WT controls. The reduced tumor growth in Fats−/− mice is macrophage dependent and is associated with a phenotypic shift of macrophages within the tumor from tumor-promoting M2-like to antitumor M1-like macrophages. In addition, FATS deficiency promotes M1 polarization by stimulating and prolonging NF-κB activation by disrupting NF-κB/IκBα negative feedback loops and indirectly enhances both CD4+ T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) adaptive immune responses to promote tumor regression. Notably, transfer of Fats−/− macrophages protects mice against B16 melanoma. Together, these data suggest that FATS functions as an immune regulator and is a potential target in cancer immunotherapy.