A375 Melanoma Cells Research Articles

In Vitro/In Vivo Translation of Synergistic Combination of MDM2 and MEK Inhibitors in Melanoma Using PBPK/PD Modelling: Part I.

Translation of the synergy between the Siremadlin (MDM2 inhibitor) and Trametinib (MEK inhibitor) combination observed in vitro into in vivo synergistic efficacy in melanoma requires estimation of the interaction between these molecules at the pharmacokinetic (PK) and pharmacodynamic (PD) levels. The cytotoxicity of the Siremadlin and Trametinib combination was evaluated in vitro in melanoma A375 cells with MTS and RealTime-Glo assays. Analysis of the drug combination matrix was performed using Synergy and Synergyfinder packages. Calculated drug interaction metrics showed high synergy between Siremadlin and Trametinib: 23.12%, or a 7.48% increase of combined drug efficacy (concentration-independent parameter β from Synergy package analysis and concentration-dependent δ parameter from Synergyfinder analysis, respectively). In order to select the optimal PD interaction parameter which may translate observed in vitro synergy metrics into the in vivo setting, further PK/PD studies on cancer xenograft animal models coupled with PBPK/PD modelling are needed.

Patulin and LL-Z1640-2 induce apoptosis of cancer cells by decreasing endogenous protein levels of Zic family member 5.

Zic family member 5 (ZIC5) is a transcription factor that promotes the survival of several cancer cell types. As ZIC5 is expressed at minimal levels in normal human adult tissues, it is a potential therapeutic target. In this study, we screened a chemical library containing 3398 compounds that includes pre-existing drugs and compounds with known effects to identify ZIC5 inhibitors. In the first screening, 18 hit compounds decreased GFP intensity in melanoma A375 cells overexpressing GFP-tagged ZIC5. In the second screening, five compounds that attenuated ZIC5 protein levels in A375 cells were identified. Among them, LL-Z1640-2 and patulin selectively induced apoptosis in melanoma cells expressing ZIC5, while only inducing very low levels of apoptosis in normal human melanocytes, which have no detectable ZIC5 expression. LL-Z1640-2 and patulin also induced apoptosis in BRAF inhibitor-resistant melanoma, pancreatic cancer, cholangiocarcinoma and colorectal cancer cells. LL-Z1640-2- and patulin-mediated suppression of melanoma proliferation were rescued by ZIC5 overexpression. These results suggest that LL-Z1640-2 and patulin are promising compounds that decrease ZIC5 expression to induce apoptosis in cancer cells.

Structure-activity relationship study of 4,5-didehydroguadiscine, an aporphine alkaloid showing potent melanogenesis-inhibitory activity in B16 melanoma cells

Although 4,5-didehydroguadiscine (12a), an alkaloid with potent melanogenesis-inhibitory activity isolated from Hornschuchia obliqua (Annonaceae), consists of an aporphine nucleus with an aromatized B-ring, to date, it has not been utilized as a template for structure–activity relationship (SAR) studies of pharmacological activities because of its exceptional structure. Accordingly, herein, five analogs (12b–12f) of 12a and five benzylisoquinoline analogs (13b–13f) lacking the C11a–C11b bond of 12b–12f were prepared. The inhibitory effects of 12b–12f and 13b–13f on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells were examined and compared with those of 12a. Melanogenesis-inhibitory activities of 12b–12f were the same as that of 12a, whereas the melanogenesis-inhibitory activities of 13b–13f were significantly inferior to those of 12a and 12b–12f. These results suggest that the C11a–C11b bond plays an essential role in the melanogenesis-inhibitory activities of 12a–12e.

Identification of an ergosterol derivative with anti-melanoma effect from the sponge-derived fungus Pestalotiopsis sp. XWS03F09.

It is difficult to treat malignant melanoma because of its high malignancy. New and effective therapies for treating malignant melanoma are urgently needed. Ergosterols are known for specific biological activities and have received widespread attention in cancer therapy. Here, LH-1, a kind of ergosterol from the secondary metabolites of the marine fungus Pestalotiopsis sp., was extracted, isolated, purified, and further investigated the biological activities against melanoma. In vitro experiments, the anti-proliferation effect on tumor cells was detected by MTT and colony formation assay, and the anti-metastatic effect on tumor cells was investigated by wound healing assay and transwell assay. Subcutaneous xenograft models, histopathology, and immunohistochemistry have been used to verify the anti-tumor, toxic, and side effect in vivo. Besides, the anti-tumor mechanism of LH-1 was studied by mRNA sequencing. In vitro, LH-1 could inhibit the proliferation and migration of melanoma cells A375 and B16-F10 in a dose-dependent manner and promote tumor cell apoptosis through the mitochondrial apoptosis pathway. In vivo assays confirmed that LH-1 could suppress melanoma growth by inducing cell apoptosis and reducing cell proliferation, and it did not have any notable toxic effects on normal tissues. LH-1 may play an anti-melanoma role by upregulating OBSCN gene expression. These findings suggest that LH-1 may be a potential for the treatment of melanoma.

TRIP13/FLNA Complex Promotes Tumor Progression and Is Associated with Unfavorable Outcomes in Melanoma.

Cutaneous melanoma is a high-grade malignant tumor originating from skin melanocytes with high risk of recurrence and metastasis. Further study on the mechanism of melanoma development is urgently needed. Here, we performed a bioinformatic analysis to identify critical genes in melanoma using public datasets in the Gene Expression Omnibus database. Among these differentially expressed genes, thyroid hormone receptor interactor 13 (TRIP13) has been reported to exert an important role in the development of various tumors, while its role in melanoma remains unclear. We selected TRIP13 as a candidate gene for further study. TRIP13 expression in clinical specimens was evaluated by immunohistochemistry, and its association with patient prognosis was analyzed by the Kaplan-Meier method and log-rank test. MV3 and A2058 melanoma cells were transfected with lentiviral vector to overexpress or knockdown TRIP13 expression level, and then, its biological function was studied using a series of in vitro and in vivo assays. RNA sequencing, co-immunoprecipitation, and mass spectrometry were used to identify the underlying mechanism of TRIP13. The results of this study exhibited that TRIP13 expression was upregulated in melanoma tissue compared with normal tissues, and high levels of TRIP13 were closely correlated with poor prognoses of melanoma patients. Elevated TRIP13 promoted the invasion and migration of melanoma cells in vitro and enhanced lung metastasis in vivo, without an influence on tumor growth. Importantly, elevated TRIP13 promoted the epithelial-mesenchymal transition (EMT) of melanoma cells, indicating a higher metastatic potential of these cells. Mechanically, TRIP13 physically interacted with filamin A (FLNA) and then activated the PI3K/AKT pathway to transcriptional activation of EMT-related genes. The present study revealed that TRIP13 is a novel prognostic biomarker and potential therapeutic target for melanoma treatment.

Human Melanoma Cells Differentially Express RNASEL/RNase-L and miR-146a-5p under Sex Hormonal Stimulation.

Polymorphisms in the ribonuclease L (RNASEL) coding gene and hsa-miR-146a-5p (miR-146a) have been associated with melanoma in a sex-specific manner. We hypothesized that RNASEL and miR-146a expression could be influenced by sex hormones playing a role in the female advantages observed in melanoma incidence and survival. Thus, we explored the effects of testosterone and 17β-estradiol on RNASEL and miR-146a expression in LM-20 and A375 melanoma cell lines. Direct targeting of miR-146a to the 3′ untranslated region (3′UTR) of RNASEL was examined using a luciferase reporter system. Our results indicate that RNASEL is a direct target of miR-146a in both melanoma cell lines. Trough qPCR and western blot analyses, we explored the effect of miR-146a mimic transfection in the presence of each hormone either on RNASEL mRNA level or on protein expression of RNase-L, the enzyme codified by RNASEL gene. In the presence of testosterone or 17β-estradiol, miR-146a overexpression did not influence RNASEL transcript level in LM-20 cell line, but it slightly induced RNASEL mRNA level in A375 cells. Remarkably, miR-146a overexpression was able to repress the protein level of RNase-L in both LM-20 and A375 cells in the presence of each hormone, as well as to elicit high expression levels of the activated form of the extracellular signal-regulated kinases (ERK)1/2, hence confirming the pro-tumorigenic role of miR-146a overexpression in melanoma. Thereafter, we assessed if the administration of each hormone could affect the endogenous expression of RNASEL and miR-146a genes in LM-20 and A375 cell lines. Testosterone exerted no significant effect on RNASEL gene expression in both cell lines, while 17β-estradiol enhanced RNASEL transcript level at least in LM-20 melanoma cells. Conversely, miR-146a transcript augmented only in the presence of testosterone in either melanoma cell line. Importantly, each hormone acted quite the opposite regarding the RNase-L protein expression, i.e., testosterone significantly decreased RNase-L expression, whereas 17β-estradiol increased it. Overall, the data show that, in melanoma cells treated with 17β-estradiol, RNase-L expression increased likely by transcriptional induction of its gene. Testosterone, instead, decreased RNase-L expression in melanoma cell lines with a post-transcriptional mechanism in which miR-146a could play a role. In conclusion, the pro-tumor activity of androgen hormone in melanoma cells could be exacerbated by both miR-146a increase and RNase-L downregulation. These events may contribute to the worse outcome in male melanoma patients.

Metabolomic profile and in vitro evaluation of the cytotoxic activity of Asphodelus microcarpus against human malignant melanoma cells A375

Melanoma is a huge worldwide health problem that must be handled more effectively with better therapeutic options. As a result, new treatment drugs are required to treat this condition. The goal of this study was to investigate the cytotoxic activity of the anthraquinone-rich fractions obtained from Asphodelus microcarpus against human melanoma cell A375. On these melanoma cell lines; the cytotoxicity of these fractions had never been studied before. Liquid chromatography linked to mass spectrometry (LC-MS-MS) and Nuclear Magnetic Resonance was used to determine the chemical profiles of these fractions. The cytotoxicity of the fractions studied was determined by measuring cell viability and calculating IC 50 values. Both ethyl acetate (EtOAC) and the precipitate fractions (PPT) exhibited selective cytotoxicity on human melanoma A 375 cell line with IC 50 values of 83 and 65 µg/mL, respectively. The antiproliferative properties of EtOAc fraction and PPT were supported by a noticeable decrease in cell numbers during the G2/M cell cycle arrest. Our findings suggest that the anthraquinone content of A. microcarpus tubers is responsible for its anti-proliferative and apoptotic properties and that further in vivo investigations should be conducted to establish the viability of using them to treat human melanomas.

Homozygous CRISPR/Cas9 Knockout Generated a Novel Functionally Active Exon 1 Skipping XPA Variant in Melanoma Cells.

Defects in DNA repair pathways have been associated with an improved response to immune checkpoint inhibition (ICI). In particular, patients with the nucleotide excision repair (NER) defect disease Xeroderma pigmentosum (XP) responded impressively well to ICI treatment. Recently, in melanoma patients, pretherapeutic XP gene expression was predictive for anti-programmed cell death-1 (PD-1) ICI response. The underlying mechanisms of this finding are still to be revealed. Therefore, we used CRISPR/Cas9 to disrupt XPA in A375 melanoma cells. The resulting subclonal cell lines were investigated by Sanger sequencing. Based on their genetic sequence, candidates from XPA exon 1 and 2 were selected and further analyzed by immunoblotting, immunofluorescence, HCR and MTT assays. In XPA exon 1, we established a homozygous (c.19delG; p.A7Lfs*8) and a compound heterozygous (c.19delG/c.19_20insG; p.A7Lfs*8/p.A7Gfs*55) cell line. In XPA exon 2, we generated a compound heterozygous mutated cell line (c.206_208delTTG/c.208_209delGA; p.I69_D70delinsN/p.D70Hfs*31). The better performance of the homozygous than the heterozygous mutated exon 1 cells in DNA damage repair (HCR) and post-UV-C cell survival (MTT), was associated with the expression of a novel XPA protein variant. The results of our study serve as the fundamental basis for the investigation of the immunological consequences of XPA disruption in melanoma.

108 (PB098) - MYC inhibition by Omomyc unveils a prognostic gene signature in Melanoma

Background: Melanoma is one of the most frequent causes of cancer death and despite significant treatment improvements, non-responder patients and those who develop resistance constitute a significant proportion of patients still in dire need of alternative and improved therapeutic options. Omomyc is a MYC inhibitor designed by Dr. Soucek that is extensively preclinically validated and currently being tested in a Phase I/IIa Clinical Trial. We have seen potent antitumour and antimetastatic effects when Omomyc is transgenically expressed in melanoma cells. Material and methods: We performed microarray analysis on BRAF-mutated A375 and BRAF wild-type SkMel147 human melanoma cells in vivo after one week of Omomyc expression and analysed them in R with Bioconductor. We assessed the overlap on transcriptional reprogramming exerted by Omomyc and investigated how these genes are associated with patient prognosis, by retrieving clinical and transcriptional data from the Skin Cutaneous Melanoma TCGA, PanCancer Atlas study in cBioPortal. Results: We found 620 differentially expressed genes in common, 436 downregulated by Omomyc (DN) and 184 upregulated (UP). DN genes cluster into Cell Cycle, RNA processing, Metabolism and Chromatin modification, organisation, and remodelling, while UP genes are related to Immune Response and several subcellular processes. Expression lower than the median of 45% of the DN genes or higher than the median of 54% of the UP genes are associated with significantly increased survival. We then generated a 6-gene signature combining DN and UP genes, that clearly separates patients in good and bad prognosis cohorts. Those with simultaneous low expression of these DN genes and high expression of these UP genes have significantly increased OS (205 (162-NA) vs. 42 (28– 63) months, p = 7 × 10−7) Additionally, 63% of UP genes are enriched in patients with Good Prognosis whereas 56% of DN genes are enriched in Poor Prognosis patients. Thus, Omomyc in vivo-regulated genes overlap considerably with those genes enriched in patients pointing to a reasonable similarity among in vivo results with Omomyc and clinical and transcriptional data from patients. Finally, pre-ranked GSEA in these patients showed that those with Good Prognosis have lower expression of MYC target genes and genes involved in melanoma relapse and metastases, and increased expression of Adaptive Immune Response. Conclusion: We show that specific gene sets reflecting decreased MYC transcriptional activity, restricted metastatic capacity and increased immune response are identified in Omomyc-expressing melanoma xenografts and indicate patients with Good Prognosis. We can therefore define an Omomyc-derived gene signature that clearly discriminates patients with different prognoses, whose transcriptional signatures resemble those of vehicle or Omomyc-expressing tumours in vivo. Conflict of interest: Ownership: L. Soucek and M. Beaulieu are cofounders and shareholders of Peptomyc, a company focused on developing Myc inhibitors for cancer treatment. M.F. Zacarias Fluck, S. Casacuberta-Serra, and J.R. Whitfield are shareholders of Peptomyc S.L.

Tyrosinase inhibition of major secondary metabolites isolated from endangered Meconopsis bhutanica Tosh.Yoshida & Grey-Wilson

Meconopsis bhutanica Tosh.Yoshida & Grey-Wilson ( Meconopsis Vig., fam: Papaveraceae), is a Critically Endangered species utilized in Tibetan medicine. Due to the very limited numbers of wild individuals, the chemical and biological basis of its medicinal value has not been studied. The objective of this study was to explore the chemical constituents of Meconopsis bhutanica and to determine the antityrosinase activity and influence on human melanoma cell lines A375. The chemical constituents were mainly isolated through column chromatography and semipreparative high performance liquid chromatography , and the structures were elucidated by nuclear magnetic resonance spectra. The inhibition on tyrosinase was examined by microplate reader. High performance liquid chromatography, spectrofluorophotometer, and molecular docking were used to analyze the effects on the function and structure of tyrosinase. The influence on A375 cells were measured by the Cell Counting Kit. Finally, a total of eleven phenolic compounds were isolated and identified from Meconopsis bhutanica . All of these compounds were found in this plant for the first time. The results of antityrosinase activity assay showed that compound 9 exhibited tyrosinase inhibition obviously. Furthermore, compound 9 could decrease the oxidation products and fluorescence emission intensity of tyrosinase. However, compound 9 had no effect on the proliferation of human melanoma A375 cells at high concentrations. The present paper uncovered the chemical composition of Meconopsis bhutanica and assessed the tyrosinase inhibition activity, which will provide data for comparing bioactive chemical constituents between the wild and cultivated ones. This study paved the way for the conservation, cultivation, and application of Meconopsis bhutanica . • The tyrosinase inhibition constituents of Meconopsis bhutanica were first studied. • (E)-2-(β- D -glucopyranosyloxy)-4-methoxycinnamic acid exhibited tyrosinase inhibition. • Meconopsis bhutanica had application value as regulators of tyrosinase activity. • The distribution of endangered Meconopsis bhutanica in China was first discovered. • Meconopsis bhutanica demonstrated potential to be cultivated.

Terminalia catappa leaf extracts inhibited metastasis of A2058 and A375 melanoma cells via downregulating p-Src and β-catenin pathway in vitro.

Background: Melanoma is a highly aggressive, lethal, and malignant cancer. Once diagnosed early, it can be easily removed and cured with satisfaction. Although many methods such as surgery, chemotherapy, radiotherapy, and immunotherapy have been used to treat this disease at an advanced stage, the outcomes are poor. Terminalia catappa leaves have been shown to have various biological benefits, including antitumor activity. The specific effects and molecular mechanisms of Terminalia catappa leaf in treating A2058 and A375 melanoma cells in vitro need to be clarified. Methods: The A2058 and A375 melanoma cancer cells were treated with Terminalia catappa leaf extracts, and then the effect of Terminalia catappa leaf extracts on migration and invasion was examined. The cell migration/invasion capacities of A2058 and A375 cells were investigated by a modified Boyden chamber assay. Zymography was used to clarify the activities of matrix metalloproteinases-2 and urinary type plasminogen activator. We performed a Western blot to verify the related expression of phospho-Src (Tyr416), phospho-Focal adhesion kinase (Tyr397), Vimentin, and β-catenin. Results: Modified Boyden chamber assays demonstrated that treatment of Terminalia catappa leaf extracts significantly inhibited A2058 and A375 cell migration/invasion capacities. In the zymography results, we showed that Terminalia catappa leaf extracts negatively modulated the activities of matrix metalloproteinases-2 and urinary type plasminogen activator. Western blot indicated that Terminalia catappa leaf extracts reduced the expression of phospho-Src (Tyr416), phospho-Focal adhesion kinase (Tyr397), Vimentin, and β-catenin. Conclusion: Terminalia catappa leaf extracts affected the antimetastasis of the A2058 and A375 melanoma cell lines by inhibiting the Focal adhesion kinase/Src interaction and Wingless-int1/β-catenin pathways in vitro. Terminalia catappa leaf extracts may serve as an effective chemopreventive agent against metastasis of melanoma cancer.

Cytotoxic activity of several ent-kaurane derivatives of atractyligenin. Synthesis of unreported diterpenic skeleton by chemical rearrangement

Atractyloside, carboxyatractyloside, their aglycon atractyligenin, and several synthetic derivatives were tested and found to be active against a panel of human tumor cell lines. Atractyligenin was subjected to oxidation, bromination, and elimination reactions, obtaining several compounds. A singular skeleton was synthesized by chemical rearrangement starting from 3β-bromo-2,15-diketoatractyligenin methyl ester. The synthesized compounds resulted active against all cell lines tested. In particular, 15-ketoatractyligenin methyl ester and 3β-bromo-2,15-diketoatractyligenin methyl ester resulted the most active with IC50 values of 0.427 and 0.723 μM against A375 melanoma cell line. Excellent results were also obtained against the colon cancer cell line CaCo2, with slightly lower antiproliferative activity. An interesting extension of the study should be to analyze the atractyligenin derivatives also as target for human melanoma and human colon cancer cells.

Understanding of black salve toxicity by multi-compound cytotoxicity assays

BackgroundBlack salve is a controversial complementary and alternative medicine (CAM) associated with skin toxicity and skin cancer treatment failures. Black salve formulations vary between manufacturers and contain a number of botanical and synthetic constituents. The skin cancer cytotoxicity of a number of these constituents has not been assessed to date. The alkaloids from the rhizomes of Sanguinaria canadensis, a key black salve ingredient, have had their single compound cytotoxicity assessed; however, whether they possess synergistic cytotoxicity with other compounds has not been studied and is of direct clinical relevance. This research aimed to improve our understanding of the skin cancer cytotoxicity of black salve constituents.MethodsThe cytotoxicity of individual and combination black salve constituents were assessed against the A375 melanoma and A431 squamous cell carcinoma cell lines. Cytotoxicity was determined using the Resazurin assay with fluorescence measured using a Tecan Infinite 200 Pro Microplate reader, compound cytotoxicity being compared to that of the topical cancer therapeutic agent, 5- fluouracil. Docetaxal was used as a positive control. Dunnetts p value was used to determine whether significant synergistic cytotoxicity was present.ResultsSanguinarine was the most cytotoxic compound tested with a 24-hour IC50 of 2.1 μM against the A375 Melanoma cell line and 3.14 μM against the A431 SCC cell line. All black salve constituents showed greater cytotoxicity against the two skin cancer cell lines tested than the skin cancer therapeutic 5-Fluouracil with 24 hours of compound exposure. Chelerythrine and minor Quaternary Benzophenanthridine Alkaloids (QBAs) present in black salve, at concentrations not having a cytotoxic effect by themselves, boosted the cytotoxic effects of sanguinarine. This could be a synergistic rather than additive cytotoxic effect although the synergistic effect was cell line and concentration dependent.ConclusionsBlack salve contains several cytotoxic compounds, a number of which have been found to possess synergistic cytotoxicity for the first time against skin cancer cell lines. In addition, these compounds together increase the overall cytotoxic effect. Assessing multi-compound cytotoxicity in herbal medicine can provide additional information about both their therapeutic and toxicity potential. As black salve is currently being used by patients, further cytotoxicity work should be undertaken to assess whether synergistic cytotoxicity exists when tested in normal skin cells.

Green Biosynthesis of Silver Nanoparticles from Moringa oleifera Leaves and Its Antimicrobial and Cytotoxicity Activities.

The plant occupied the largest area in the biosynthesis of silver nanoparticles, especially the medicinal plants, and it has shown great potential in biotechnology applications. In this study, green synthesis of silver nanoparticles from Moringa oleifera leaves extract and its antifungal and antitumor activities were investigated. The formation of silver nanoparticles was observed after 1 hour of preparation color changing. The ultraviolet and visible spectrum, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy techniques were used to characterize synthesis particles. Ultraviolet and visible spectroscopy showed a silver surface plasmon resonance band at 434 nm. Fourier transform infrared analysis shows the possible interactions between silver and bioactive molecules in Moringa oleifera leaves extracts, which may be responsible for the synthesis and stabilization of silver nanoparticles. X-ray diffraction showed that the particles were a semicubic crystal structure and with a size of 38.495 nm. Scanning electron microscopy imaging shows that the atoms are spherical in shape and the average size is 17 nm. The transmission electron microscopy image demonstrated that AgNPs were spherical and semispherical particles with an average of (50–60) nm. The nanoparticles also showed potent antimicrobial activity against pathogenic bacteria and fungi using the well diffusion method. Candida glabrata found that the concentration of 1000 μg/mL exhibited the highest inhibition. As for bacteria, the concentration of 1000 μg/mL appeared to be the inhibition against Staphylococcus aureus. Moringa oleifera AgNPs inhibited human melanoma cells A375 line significant concentration-dependent cytotoxic effects. The powerful bioactivity of the green synthesized silver nanoparticles from medical plants recommends their biomedical use as antimicrobial as well as cytotoxic agents.

STING mediates nuclear PD-L1 targeting-induced senescence in cancer cells

Immune checkpoint molecule programmed death-ligand 1 (PD-L1) is overexpressed in cancer cells and imparts resistance to cancer therapy. Although membrane PD-L1 has been targeted for cancer immune therapy, nuclear PD-L1 was reported to confer cancer resistance. Therefore, it is important to regulate the nuclear PD-L1. The mechanisms underlying the therapeutic efficacy of PD-L1 targeting have not been well-established. Cellular senescence has been considered a pivotal mechanism to prevent cancer progression, and recently, PD-L1 inhibition was shown to be involved in cancer cell senescence. However, the relevance of PD-L1 targeting-induced senescence and the role of stimulator of interferon genes (STING) has not been reported. Therefore, we aimed to identify the role of PD-L1 in cancer progression and how it regulates cancer prevention. In this study, we found that PD-L1 depletion-induced senescence via strong induction of STING expression in mouse melanoma B16-F10 and colon cancer CT26 cells, and in human melanoma A375 and lung cancer A549 cells. Interestingly, nuclear PD-L1 silencing increased STING promoter activity, implying that PD-L1 negatively regulates STING expression via transcriptional modulation. Furthermore, we showed that PD-L1 binds to the STING promoter region, indicating that PD-L1 directly controls STING expression to promote cancer growth. In addition, when we combined PD-L1 silencing with the senescence-inducing chemotherapeutic agent doxorubicin, the effect of PD-L1-targeting was even more powerful. Overall, our findings can contribute to the understanding of the role of PD-L1 in cancer therapy by elucidating a novel mechanism for PD-L1 targeting in cancer cells.

Chemical inhibition of ER aminopeptidase 1 as a tool for regulating the immunopeptidome of cancer cells

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, is often limited by ineffective presentation of antigenic peptides that can elicit T-cell mediated anti-tumor cytotoxic responses. Therefore, manipulating antigen presentation is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides that can bind onto MHC class I molecules (MHC-I). We hypothesized that pharmacologically inhibiting ERAP1 in cells can regulate the global cellular immunopeptidome. we treated the A375 melanoma cell line with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting the bound peptides and identifying them using capillary chromatography and tandem mass spectrometry. Although the inhibitor did not negatively affect overall MHC-I presence on the cell surface, it induced significant changes on the presented peptidomes, both at the qualitative and quantitative levels. Specifically, inhibitor treatment altered about half of the total 3204 identified peptides and about one third of the peptides predicted to be good ligands for MHC-I, affected length and sequence without however interfering with basic binding motifs. Surprisingly, the inhibitor enhanced predicted overall MHC-I binding affinity by reducing presentation of sub-optimal long peptides and generating many high-affinity 9-12mers suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 is a valid approach for manipulating the immunopeptidome of cancer with potential applications in standalone or combination immunotherapy.

Precision Killing of Sinoporphyrin Sodium-Mediated Photodynamic Therapy against Malignant Tumor Cells.

Photodynamic therapy (PDT) has significant advantages in the treatment of malignant tumors, such as high efficiency, minimal invasion and less side effects, and it can preserve the integrity and quality of the organs. The power density, irradiation time and photosensitizer (PS) concentration are three main parameters that play important roles in killing tumor cells. However, until now, the underlying relationships among them for PDT outcomes have been unclear. In this study, human malignant glioblastoma U-118MG and melanoma A375 cells were selected, and the product of the power density, irradiation time and PS concentration was defined as the total photodynamic parameter (TPP), in order to investigate the mechanisms of PS sinoporphyrin sodium (DVDMS)-mediated PDT (DVDMS-PDT). The results showed that the survival rates of the U-118MG and A375 cells were negatively correlated with the TPP value in the curve, and the correlation exactly filed an e-exponential function. Moreover, according to the formula, we realized controllable killing effects of the tumor cells by randomly adjusting the three parameters, and we finally verified the accuracy and repeatability of the formula. In conclusion, the establishment and implementation of a newly functional relationship among the PDT parameters are essential for predicting PDT outcomes and providing personalized precise treatment, and they are contributive to the development of PDT dosimetry.

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells.

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

The Inhibitory Effect of Polyphenon 60 from Green Tea on Melanin and Tyrosinase in Zebrafish and A375 Human Melanoma Cells.

Polyphenon 60 (PP60) from green tea has long been used as an antioxidant, anticancer, antimicrobial, and antimutagenic. Aim of the Study. To investigate tyrosinase inhibition-related kinetic mechanism and antimelanogenesis potential of PP60. Materials and Methods. The effect of PP60 on melanin and tyrosinase was evaluated in A375 melanoma cells and zebrafish embryos. The melanoma cells were treated with 20, 40, and 60 µg/mL of PP60, and tyrosinase expression was induced by using L-DOPA. The western blot method was used for the evaluation of tyrosinase expression. Cell lysates were prepared from treated and untreated cells for cellular tyrosinase and melanin quantification. Furthermore, zebrafish embryos were treated with 20, 40, and 60 µg/mL of PP60 and reference drug kojic acid for determination of depigmentation and melanin quantification. In vitro assays were also performed to examine the impact of PP60 on mushroom tyrosinase activity. To determine cytotoxicity, MTT was used against melanoma cell line A375. Results. PP60 showed good tyrosinase inhibitory activity with an IC50 value of 0.697 ± 0.021 µg/mL as compared to kojic acid a reference drug with an IC50 value of 2.486 ± 0.085 µg/mL. Kinetic analysis revealed its mixed type of inhibition against mushroom tyrosinase. In addition, western blot analysis showed that at 60 µg/mL dose of PP60 significantly reduced L-DOPA-induced tyrosinase expression in melanoma cells. PP60 significantly inhibits the cellular tyrosinase (p < 0.05) and reduces the melanin (p < 0.05) contents of melanoma cells. Furthermore, PP60 was found to be very potent in significantly reducing the zebrafish embryos' pigmentation (p < 0.05) and melanin (p < 0.05) content at the dose of 60 µg/mL. Conclusions. Our results demonstrate that PP60 has a strong potency to reduce pigmentation. It may be useful for the cosmetic industries to develop skin whitening agents with minimal toxic effects.

Novel Triterpenic Acid-Benzotriazole Esters Act as Pro-Apoptotic Antimelanoma Agents.

Pentacyclic triterpenes, such as betulinic, ursolic, and oleanolic acids are efficient and selective anticancer agents whose underlying mechanisms of action have been widely investigated. The introduction of N-bearing heterocycles (e.g., triazoles) into the structures of natural compounds (particularly pentacyclic triterpenes) has yielded semisynthetic derivatives with increased antiproliferative potential as opposed to unmodified starting compounds. In this work, we report the synthesis and biological assessment of benzotriazole esters of betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA) (compounds 1–3). The esters were obtained in moderate yields (28–42%). All three compounds showed dose-dependent reductions in cell viability against A375 melanoma cells and no cytotoxic effects against healthy human keratinocytes. The morphology analysis of treated cells showed characteristic apoptotic changes consisting of nuclear shrinkage, condensation, fragmentation, and cellular membrane disruption. rtPCR analysis reinforced the proapoptotic evidence, showing a reduction in anti-apoptotic Bcl-2 expression and upregulation of the pro-apoptotic Bax. High-resolution respirometry studies showed that all three compounds were able to significantly inhibit mitochondrial function. Molecular docking showed that compounds 1–3 showed an increase in binding affinity against Bcl-2 as opposed to BA, OA, and UA and similar binding patterns compared to known Bcl-2 inhibitors.