A375 Human Melanoma Research Articles

Cloning of the XPD gene and its function in malignant melanoma cells.

The xeroderma pigmentosum group D (XPD) gene is a member of the transcription factor IIH complex and serves an important role in gene repair. Previous studies have suggested that genetic variants of the XPD gene may be associated with an increased risk of cutaneous melanoma. However, the exact mechanism remains unclear. In the present study, the XPD gene was cloned, and its localization and function in malignant melanoma cells were investigated. The human full length XPD gene was cloned via reverse transcription-PCR using the total RNA extracted from human cervical squamous cell carcinoma epithelial HeLa cells. Subsequently, the gene was inserted into a plasmid fused to green fluorescent protein (GFP; pEGFP-N1/XPD), and pEGFP-N1/XPD and pcDNA3.1(+)/XPD were transfected into human malignant melanoma A375 cells using Lipofectamine® 2000. The expression levels of XPD were detected by western blotting. The Golgi marker GM130 and the endoplasmic reticulum membrane protein marker KDEL were used for immunofluorescence staining, and the subcellular localization of XPD was observed under a fluorescence microscope. Cell proliferation was measured using an MTT assay. The recombinant pEGFP-N1/XPD plasmid expressing the human wild-type XPD gene was successfully constructed by restriction enzyme digestion and assessed by gene sequencing. XPD was localized in the endoplasmic reticulum of malignant melanoma A375 cells, as confirmed by immunofluorescence staining. Furthermore, MTT assays indicated that XPD inhibited the proliferation of malignant melanoma A375 cells. The present study provides a basis for further investigation of the biological effects and functions of XPD in malignant melanoma cells.

Characterization of water-soluble esters of nitrobenzoxadiazole-based GSTP1-1 inhibitors for cancer treatment

The 7-nitrobenzo[c][1,2,5]oxadiazole (NBD) derivative NBDHEX (compound 1) and its analogue MC3181 (compound 2) have been found to be potent inhibitors of tumor cell growth in vitro and therapeutically active and safe in mice bearing human melanoma xenografts. To enhance the aqueous solubility of these compounds, we synthesized the hemisuccinate of 1 (compound 3) and the phosphate monoesters of 1 and 2 (compound 4 and 5, respectively). These novel NBD derivatives displayed a solubility in the conventional phosphate-buffered saline up to 150-fold higher than that of 1, and up to 4-fold higher than that of 2. Notably, solubility of phosphates 4 and 5 in a potassium phosphate buffer at pH 7.4, was up to 500-fold higher than that of 1, and ~10-fold higher than that of 2. Compounds 3–5 retained high cytotoxicity towards cultured human melanoma and osteosarcoma cells and were cleaved in vitro by both human and murine hydrolases, thus releasing the corresponding parent compound (i.e., 1 or 2). Interestingly, esters 3–5 displayed high inhibitory activity towards the glutathione transferase (GST) isoform GSTP1-1 and showed a reactivity towards reduced glutathione comparable to that of the respective parent compound. Finally, both 4 and 5 were safe and effective when administered intravenously or orally as an aqueous solution to mice xenografted with A375 human melanoma tumors. Collectively, these results and the previously observed synergistic interaction between 1 and 2 and various approved anticancer drugs, suggest the possible utility of phosphates 4 and 5 as single agents and in combination regimens in cancers with unmet medical need, including melanoma.

Mitochondrial Fatty Acid Oxidation Is Not Vital for Melanoma Cell Proliferation and Migration

Abstract Objectives Melanoma, the 5th most common cancer in the US, is the most aggressive form of skin cancer. Excess adiposity is associated with an increased risk of melanoma in males, and a high-fat diet promotes melanoma tumor growth in mice. Our group found that lipid droplet (LD) content increases with melanoma cell aggressiveness and that fatty acid uptake inhibition reduces cell proliferation and migration; however, the function of these lipids in melanoma is unknown. Since lipids can be used to produce energy by fatty acid oxidation (FAO), we sought to determine whether melanoma cells were using FAO to maintain aggressiveness. Methods Gene expression microarray was used to identify differences in gene expression, which was confirmed by RT-qPCR. WM983A, WM983B, 1205Lu and A375 human melanoma cells were treated with 5 µM Etomoxir and cell proliferation, migration, and respiration were quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit, Boyden Chamber transwell migration, and Seahorse XFe96 Flux Analyzer, respectively. Results The most significantly altered genes expressed between four invasive, lipid-rich and four non-invasive, lipid-poor melanoma cells pertained to fatty acid metabolism. Carnitine palmitoyltransferase 1A (CPT1a), the rate limiting enzyme in mitochondrial FAO (mFAO), mRNA was increased in lipid-rich cells compared to the lipid-poor cells (p < 0.05, n = 6–7), indicating that melanoma cells may use LD lipids for mFAO. To determine the importance of mFAO to melanoma cells, we inhibited mFAO with etomoxir, an irreversible CPT1 inhibitor. Treatment of lipid-rich 1205Lu and A375 cells or lipid-poor WM983A and WM983B cells with etomoxir for 4 days had no effect on cell proliferation. Extracellular flux analysis of cells with or without etomoxir showed no difference in ATP production, indicating that melanoma cells do not use mFAO to generate energy under normal conditions. In motile cells, etomoxir reduced lipid-rich A375 cell migration by 8.8% (p = 0.05, n = 2). Conclusions Lipids play a role in melanoma aggressiveness; however, our results indicate that mFAO of lipids is not vital to melanoma cell proliferation or migration. Further studies are required to understand the implication of excess adiposity and circulating lipids in melanoma development and progression. Funding Sources Institutional Department Fund

RNAi-mediated IARS2 knockdown inhibits proliferation and promotes apoptosis in human melanoma A375 cells.

IARS2, which encodes the mitochondrial form of isoleucyl-tRNA synthetase, has been found to play an important role in a range of diseases, including cancer. However, the relationship between IARS2 and melanoma is still unclear. To evaluate the role of IARS2 in melanoma, we constructed a stable A375 cell line with IARS2 knockdown via lentivirus-mediated small interfering RNAs. The expression of IARS2 was measured by real time-quantitative Polymerase Chain Reaction and western blot analysis. Cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and colony formation assay were conducted to assess the effect of IARS2 on melanoma cell proliferation. Flow cytometry assay was used to determine cell apoptosis and cell cycle distribution in melanoma A375 cells. Finally, immunohistochemistry was employed to validate the expression of IARS2 protein in melanoma tissues. In this study it was found that IARS2 was highly expressed in melanoma cell lines. Furthermore, IARS2 protein also exhibited elevated expression in the tumour tissues obtained from melanoma patients. After suppression of the mRNA expression of IARS2, the proliferation and colony formation ability of the A375 cells were significantly inhibited, while the proportion of apoptotic A375 cells increased significantly, as indicated by an enhanced phosphatidylserine externalization and caspase 3/7 activity after IARS2 knockdown. Further investigations found that knockdown of IARS2 arrested cells in the G1 phase. The results suggested that IARS2 is critical for proliferation and apoptosis of melanoma cells.

Inhibition of the STAT3 Signaling Pathway Contributes to the Anti-Melanoma Activities of Shikonin.

BackgroundMalignant melanoma is an extremely aggressive and metastatic cancer, and highly resistant to conventional therapies. Signal transducer and activator of transcription 3 (STAT3) signaling promotes melanoma development and progression, which has been validated as an effective target in melanoma treatment. Natural naphthoquinone shikonin is reported to exert anti-melanoma effects. However, the underlying mechanisms have not been fully elucidated.PurposeThis study aims to evaluate the anti-melanoma activities of shikonin and explore the involvement of STAT3 signaling in these effects.MethodsZebrafish tumor model was established to evaluate the anti-human melanoma effects of shikonin in vivo. MTT assay and colony formation assay were employed to investigate the anti-proliferative effects of shikonin on human melanoma A375 and A2058 cells. Flow cytometry was used to analyze cell cycle distribution and apoptosis induction. Wound healing assay and Transwell chamber assay were conducted to examine the cell migratory and invasive abilities. Immunofluorescence assay was used to observe F-actin, Tubulin, and STAT3 localization. Western blotting was used to determine the expression levels of proteins associated with apoptosis and key proteins in the STAT3 signaling pathway. Immunoblotting was performed in DSS cross-linked cells to determine the homo-dimerization of STAT3. Gelatin zymography was employed to evaluate the enzymatic activity of MMP-2 and MMP-9. Transient transfection was used to overexpress STAT3 in cell models.ResultsShikonin suppressed melanoma growth in cultured cells and in zebrafish xenograft models. Shikonin induced melanoma cells apoptosis, inhibited cell migration and invasion. Mechanistic study indicated that shikonin inhibited the phosphorylation and homo-dimerization of STAT3, thus reduced its nuclear localization. Further study showed that shikonin decreased the levels of STAT3-targeted genes Mcl-1, Bcl-2, MMP-2, vimentin, and Twist, which are involved in melanoma survival, migration, and invasion. More importantly, overexpression of constitutively active STAT3 partially abolished the anti-proliferative, anti-migratory, and anti-invasive effects of shikonin.ConclusionThe anti-melanoma activity of shikonin is at least partially attributed to the inhibition on STAT3 signaling. These findings provide new insights into the anti-melanoma molecular mechanisms of shikonin, suggesting its potential in melanoma treatment.

Genetic Target Modulation Employing CRISPR/Cas9 Identifies Glyoxalase 1 as a Novel Molecular Determinant of Invasion and Metastasis in A375 Human Malignant Melanoma Cells In Vitro and In Vivo.

Metabolic reprogramming is a molecular hallmark of cancer. Recently, we have reported the overexpression of glyoxalase 1 (encoded by GLO1), a glutathione-dependent enzyme involved in detoxification of the reactive glycolytic byproduct methylglyoxal, in human malignant melanoma cell culture models and clinical samples. However, the specific role of GLO1 in melanomagenesis remains largely unexplored. Here, using genetic target modulation, we report the identification of GLO1 as a novel molecular determinant of invasion and metastasis in malignant melanoma. First, A375 human malignant melanoma cells with GLO1 deletion (A375-GLO1_KO) were engineered using CRISPR/Cas9, and genetic rescue clones were generated by stable transfection of KO clones employing a CMV-driven GLO1 construct (A375-GLO1_R). After confirming GLO1 target modulation at the mRNA and protein levels (RT-qPCR, immunodetection, enzymatic activity), phenotypic characterization indicated that deletion of GLO1 does not impact proliferative capacity while causing significant sensitization to methylglyoxal-, chemotherapy-, and starvation-induced cytotoxic stress. Employing differential gene expression array analysis (A375-GLO1_KO versus A375-GLO1_WT), pronounced modulation of epithelial mesenchymal transition (EMT)-related genes [upregulated: CDH1, OCLN, IL1RN, PDGFRB, SNAI3; (downregulated): BMP1, CDH2, CTNNB1, FN1, FTH1, FZD7, MELTF, MMP2, MMP9, MYC, PTGS2, SNAI2, TFRC, TWIST1, VIM, WNT5A, ZEB1, and ZEB2 (up to tenfold; p < 0.05)] was observed—all of which are consistent with EMT suppression as a result of GLO1 deletion. Importantly, these expression changes were largely reversed upon genetic rescue employing A375-GLO1_R cells. Differential expression of MMP9 as a function of GLO1 status was further substantiated by enzymatic activity and ELISA analysis; phenotypic assessment revealed the pronounced attenuation of morphological potential, transwell migration, and matrigel 3D-invasion capacity displayed by A375-GLO1_KO cells, reversed again in genetic rescue clones. Strikingly, in a SCID mouse metastasis model, lung tumor burden imposed by A375-GLO1_KO cells was strongly attenuated as compared to A375-GLO1_WT cells. Taken together, these prototype data provide evidence in support of a novel function of GLO1 in melanoma cell invasiveness and metastasis, and ongoing investigations explore the function and therapeutic potential of GLO1 as a novel melanoma target.

Phytochemical and Biological Screening of Oenothera Biennis L. Hydroalcoholic Extract.

Oenothera biennis L. (OB), also commonly known as evening primrose, belongs to the Onagraceae family and has the best studied biological activity of all the members in the family. In therapy, the most frequently used type of extracts are from the aerial part, which are the fatty oils obtained from the seeds and have a wide range of medicinal properties. The aim of this study was to evaluate the phytochemical composition and biological activity of OB hydroalcoholic extract and to provide directions for the antimicrobial effect, antiproliferative and pro-apoptotic potential against A375 melanoma cell line, and anti-angiogenic and anti-inflammatory capacity. The main polyphenols and flavonoids identified were gallic acid, caffeic acid, epicatechin, coumaric acid, ferulic acid, rutin and rosmarinic acid. The total phenolic content was 631.496 µgGAE/mL of extract and the antioxidant activity was 7258.67 μmolTrolox/g of extract. The tested extract had a mild bacteriostatic effect on the tested bacterial strains. It was bactericidal only against Candida spp. and S. aureus. In the set of experimental conditions, the OB extract only manifested significant antiproliferative and pro-apoptotic activity against the A375 human melanoma cell line at the highest tested concentration, namely 60 μg/mL. The migration potential of A375 cells was hampered by the OB extract in a concentration-dependent manner. Furthermore, at the highest tested concentration, the OB extract altered the mitochondrial function in vitro, while reducing the angiogenic reaction, hindering compact tumor formation in the chorioallantoic membrane assay. Moreover, the OB extract elicited an anti-inflammatory effect on the experimental animal model of ear inflammation.

Antiproliferative activity of side-chain truncated vitamin D analogs (PRI-1203 and PRI-1204) against human malignant melanoma cell lines

Vitamin D compounds are versatile molecules widely considered as promising agents in cancer prevention and treatment, including melanoma. Previously we investigated series of double point modified vitamin D2 analogs as well as non-calcemic 20S-hydroxyvitamin D3 and 21-hydroxypregnacalciferol as to their anti-melanoma activity. Surprisingly, short side-chain vitamin D analogs were found to be biologically active compounds. Thus, here we tested novel derivatives of pregnacalciferol with an additional hydroxyl at the end of the truncated side chain, PRI-1203 and PRI-1204, as to their potency against human melanoma A375 and RPMI7951 cell lines. Tested compounds are geometric isomers, with 19-methylene positioned in PRI-1203 like in a calcitriol molecule, but reversed in the PRI-1204 analog to the (5E,7E) geometry (5,6-trans). We noticed a decrease in cells viability exerted by PRI-1203. The antiproliferative effect of PRI-1204 was very low, emphasizing the importance of the natural 19-methylene geometry in the PRI-1203. PRI-1203 was also effective in inhibition of A375 melanoma cells migration. PRI-1203, but not PRI-1204, increased the percentage of A375 and RPMI7951 melanoma cells in the G0/G1 phase of cell cycle, possibly in a p21 and p27 independent manner. Both, analogs have very low effect on the level of CYP24A1 mRNA, in comparison to active form of vitamin D – 1.25(OH)2D3. In addition, both tested compounds failed to elicit VDR translocation to the nucleus. Thus, it could be postulated that side chain shortening strongly affects binding of analogs to VDR and activation of genomic responses, however do not impair their antiproliferative activities.

Novel 1,2,4-triazole analogues as mushroom tyrosinase inhibitors: synthesis, kinetic mechanism, cytotoxicity and computational studies.

We have created a novel series of mushroom tyrosinase inhibitors with 1,2,4-triazole as fundamental skeleton. The target compound 1,2,4-triazol-3-ylthio)-N-phenyl acetamide derivatives 9(a-l) were synthesized by the reaction of 4- and 5-substituted 1,2,4-triazole-3-thiol derivatives 6(a-c) with 2-chloro-N-sub/un-substituted phenyl acetamide derivatives 8(a-d) under basic condition. By using the analytical techniques for instance, FTIR, LC-MS, 1H NMR and 13C NMR, the structural verification was evaluated. The novel series of the target compounds 9(a-l) has been scanned for biological activity (mushroom tyrosinase inhibition potential) which demonstrates adequate results. Interestingly, compound 9k (IC50 = 0.0048 ± 0.0016µM) exhibits 3500 times more activity compared with standard drug kojic acid (IC50 = 16.8320 ± 1.1600µM) against mushroom tyrosinase inhibitor. Furthermore, the cytotoxicity experiment was carried out for the highly effective target compounds (9d, 9i, 9j and 9k) by using MTT assay method for A375 human melanoma cells to define the nontoxic performance of the most effective compounds ranging from 1 to 25µM. Furthermore, the molecular docking study delivers the thought concerning the interface of the ligand with an enzyme. Also, the dynamic simulation was accomplished for compound 9k to govern the plausible binding model.

Dual SMO/BRAF Inhibition by Flavonolignans from Silybum marianum

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

Polymethoxyflavones from Gardenia oudiepe (Rubiaceae) induce cytoskeleton disruption-mediated apoptosis and sensitize BRAF-mutated melanoma cells to chemotherapy

A series of 10 natural and semisynthetic flavonoids (1 to 10) were obtained from Gardenia oudiepe (Rubiaceae), an endemic plant from New Caledonia. Most of them were polymethoxylated flavones (PMFs) of rare occurrence. After a cell viability screening test, PMFs 2 and 3 showed significant cytotoxic activity against A2058 human melanoma cells (IC50 = 3.92 and 8.18 μM, respectively) and were selected for in-depth pharmacological assays. Both compounds inhibited cell migration and induced apoptosis and cell cycle arrest after 72h of treatment. Immunofluorescence assays indicated that these outcomes were possibly related to the induction of cytoskeleton disruption associated to actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. As A2058 are considered as chemoresistant to conventional chemotherapy, compounds 2 and 3 (½IC50) were associated to clinically-used antimelanoma drugs (vemurafenib and dacarbazine) and combined therapies efficacy was assessed by the MTT assay. PMFs 2 restored the sensitivity of A2058 cells to dacarbazine treatment (IC50 = 49.38 μM vs. >100 μM). Taken together, these data suggest that PMFs from G. oudiepe could be potential leaders for the design of new antimelanoma drugs.

Pinus maritimaExtract Induces Apoptosis in Human Malignant Melanoma Cells via ROS/Caspase-3 Signaling

Melanoma is the most aggressive type of skin cancer due to its rapid metastasis with a high recurrence rate following conventional therapy. Pine bark extract (PBE) from Pinus maritima contains numerous phenolic compounds and functions as a potent antioxidant. The present study aimed to analyze the potential anticancer properties of PBE on human malignant melanoma A375 cells. The chemical composition of PBE was determined by high-performance liquid chromatography/photodiode array detector. The effects of PBE on cell death, migration, and invasion were determined using xCELLigence Technology real-time cell analysis. Annexin/propidium iodide flow cytometry and Hoechst 33342 staining were conducted to detect cell apoptosis. PBE induced apoptosis and inhibited cell migration and invasion. Cleaved caspase-3 expression and activity were significantly increased ( P &lt; 0.01) in cells treated with PBE compared with control cells. PBE ameliorated hydrogen peroxide (H2O2)-induced reactive oxygen species (ROS) formation. Treatment of the cells with PBE in the presence of H2O2led to significant ( P &lt; 0.001) reduction of matrix metallopeptidase-9, which is a mediator responsible for advanced melanoma. PBE induces A375 programmed cell death and suppresses cellular invasion by attenuating the ROS-dependent pathway associated with MMP-9 reduction.

Combination of TEW-7197 and adoptively transferred NK cells leads to Improved Antitumor Responses in vivo

Abstract Transforming growth factor beta (TGF-β) supports tumor cell proliferation, metastasis, and invasion in later stages of cancer while suppresses the proliferation and survival of lymphocytes including Natural Killer (NK) cells. The current therapeutic approaches of targeting TGF-β include inhibiting its transcription through anti-sense oligonucleotides, blocking TGF-β receptor binding through the use of antibodies, or suppressing the TGF-beta signaling pathways using small molecules, such as TEW-7197. Here, we report the anti-tumorigenic role of TEW-7197, an orally administered TGF-b receptor kinase ALK5 inhibitor, by synergizing with adoptively transferred NK cells in the human xenograft A375 melanoma model. Addition of TEW-7197, at its therapeutic doses, in cultures of A375 melanoma cells did affect neither the growth nor the apoptotic killing of tumor cells in vitro, however, it caused reduction of tumor cell growth in vivo. This was possibly due to the upregulation of TGF-b mRNA upon tumor implantation in the xenograft tissues, which became a target of TEW-7197. Adoptive transfer of ex-vivo expanded human NK cells into these mice also inhibited tumor growth but co-administration of TEW-7197 resulted in significantly higher degree of tumor regression, by both inhibiting TGFb signaling as well as increasing the NK cell’s availability within tumor tissues. These data highlight that the anti-tumor response exhibited by TEW-7197 and NK cells are distinct, and can be synergized by the combination of the two. Therefore, our data provide the scientific rationale for the combination therapies of TGF-b small molecule kinase inhibitors with adoptively transferred NK cells in advanced cancer patients.

3PO as a Selective Inhibitor of 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 in A375 Human Melanoma Cells

The occurrence of BRAFV600E mutation causes an up-regulation of the B-raf kinase activity leading to the stabilization of hypoxia-inducible factor 1-alpha (HIF-1α) - the promoter of the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) enzyme. The aim of the study was to examine the effect of the (2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), as an inhibitor of PFKFB3, on human melanoma cells (A375) with endogenous BRAFV600E mutation. A375 cells were exposed to different concentrations of 3PO and the following tests were performed: docking, cytotoxicity assay, immunocytochemistry staining glucose uptake, clonogenic assay, holotomography imaging, and flow cytometry. Our studies revealed that 3PO presents a dose-dependent and time-independent cytotoxic effect and promotes apoptosis of A375 cells. Furthermore, the obtained data indicate that 3PO induces cell cycle arrest in G1/0 and glucose uptake reduction. Taking all together, our research demonstrated a here should be proapoptotic and antiproliferative effect of 3PO on A375 human melanoma cells.

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

Antiproliferative activity of caged xanthones from the leaves of Garcinia wightii T. Anderson

Two new caged xanthones, wightiic acid (1) 16-O-methyl wightiic acid (2), along with eight known compounds, gaudichaudic acid E (3), isogaudichaudic acid E (4), ursolic acid (5) stigmasterol (6), lupeol (7), glutinol (8), lupenone (9) and stigmasteryl linoleate (10) were isolated from Garcinia wightii T. Anderson. The structures of the compounds were elucidated by spectroscopic means, including 1D and 2D NMR, HR-ESIMS, and the absolute configuration of the new compounds 1 and 2 were determined by analysis of ECD data. Anti-proliferation activities of the four caged xanthones (1–4) were evaluated by MTT assay on MCF-7 and SKBR-3 human breast cancer cells and A-375 human melanoma cells by MTT assay. All the tested compounds exhibited dose dependent antiproliferative activity. Wightiic acid (1) showed remarkable activity with IC50 value of 4.7 μM and 5.2 μM respectively in A-375 and MCF-7 cells. The compound isogaudichaudic acid E (4) induced potent antiproliferation against SKBR-3 cells with an IC50 value of 6.1 μM.

Controlled Synthesis and Characterization of Micrometric Single Crystalline Magnetite With Superparamagnetic Behavior and Cytocompatibility/Cytotoxicity Assessments

A new class of magnetite (Fe3O4) particles, coined as “Single Crystalline Micrometric Iron Oxide Particles” (SCMIOPs), were obtained by hydrothermal synthesis. Both the single Fe3O4 phase content and the particle sizes range, from 1 µm to 30 µm, can be controlled by synthesis. The notable finding states that these particles exhibit vanishing remanent magnetization (σr=0.28 emu/g) and coercive force (Hc=1.5 Oe), which indicate a superparamagnetic-like behavior (unexpected at micrometric particles size), and remarkably high saturation magnetization (σs=95.5 emu/g), what ensures strong magnetic response, and the lack of agglomeration after the magnetic field removal. These qualities make such particles candidates for biomedical applications, to be used instead of magnetic nanoparticles which inevitably involve some drawbacks like aglommeration and insufficient magnetic response. In this sense, cytocompatibility/cytotoxicity tests were performed on human cells, and the results have clearly indicated that SCMIOPs are cytocompatible for healthy cell lines HaCaT (human keratinocytes) and HEMa (primary epidermal melanocytes) and cytotoxic for neoplastic cell lines A375 (human melanoma) and B164A5 (murine melanoma) in a dose-dependent manner.

Abstract B26: Tumor reduction by a small molecule human PD-1/PD-L1 inhibitor in a melanoma/PBMC co-implantation model

Abstract Introduction: FDA-approved antibody-based therapies targeting the Programmed cell Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) immune checkpoint axis have gained considerable attention and success in cancer immunotherapy recently. As a next-generation therapy, small-molecule PD-1/PD-L1 checkpoint inhibitors may provide the potential for increased tumor penetration, shorter half-life (to better manage immune related adverse events), and lower cost of goods. We embarked on an effort to identify and develop small molecules capable of targeting the immune checkpoint molecules PD-1/PD-L1, with an aim to improve anticancer immune responses in vivo. Methods: Co-crystallized human PD-1/PD-L1 provided structural information from which we developed a number of small-molecule checkpoint inhibitors. Active compounds were first profiled by an ELISA assay measuring inhibition of the PD-1/PD-L1 interaction, followed by functional cell-based reporter and mixed lymphocyte reaction (MLR) assays. PD-1/PD-L1 inhibitory compounds thus identified were further selected for in vivo model testing. Since our human-specific PD-1/PD-L1 inhibitors did not cross-react with murine PD-1/PD-L1, we co-implanted A375 human melanoma cells along with human peripheral blood mononuclear cells (PBMCs) into immunodeficient NOD/SCID mice to test their efficacy in vivo. Results: The optimized human PD-1/PD-L1 inhibitors exhibited marked activities in both the cell-based reporter and MLR assays. Moreover, lead compound CCX4503 reduced tumor growth in vivo to a similar extent as the positive control anti-human PD-L1 antibody. Antitumor activity was completely dependent on the presence of human PBMCs. The tumor microenvironment analysis by flow cytometry indicated that the antitumor activity of CCX4503 was accompanied by a significantly higher CD8+ T-Cell/CD4+ T-cell ratio. An X-ray structure of CCX4503 co-crystallized with PD-L1 revealed several vital interactions within the PD-1-binding-region of PD-L1, providing information about the structural basis by which the compound disrupts the PD-1/PD-L1 immune checkpoint interaction. Summary: We have identified and advanced unique small-molecule inhibitors of human PD-1/PD-L1 by rational design. Molecules resulting from these efforts, such as CCX4503, exhibited marked inhibition of the PD-1/PD-L1 interaction and signaling in vitro, and also clear antitumor effects in an animal model system in vivo. Citation Format: Marta Vilalta, Sreenivas Punna, Shijie “Chris” Li, Viengkham Malathong, Christopher Lange, Darren McMurtrie, Ju Yang, Howard Roth, Jeffrey McMahon, James J. Campbell, Linda S. Ertl, Ryan Ong, Yu Wang, Niky Zhao, Vicky Chhina, Alice Kumamoto, Simon Yau, Tong Dang, Penglie Zhang, Thomas J. Schall, Rajinder Singh. Tumor reduction by a small molecule human PD-1/PD-L1 inhibitor in a melanoma/PBMC co-implantation model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B26.

RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells.

The availability of nucleotides has a direct impact on transcription. Inhibition of dihydroorate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. Progesterone additionally bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. Ddx21-deficient zebrafish show resistance to leflunomide-induced stress. On a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.

Drug Delivery and Drug Efficacy from Amorphous Poly(thioether anhydrides).

The correlation between erosion and drug (lidocaine and 6-mercaptopurine, 6-MP) release from amorphous poly(thioether anhydrides), which are synthesized using radical-mediated thiol-ene polymerization, is reported. Cytotoxicity studies of the polymer toward human fibroblast human dermal fibroblasts adult, melanoma A-375, and breast cancer MCF-7 cells are conducted, and drug efficacy of a cancer and autoimmune disease drug (6-MP) when released from the poly(thioether anhydrides) is examined against two cancerous cell types (A-375 and MCF-7). Erosion and drug release studies reveal that lidocaine release is governed by network erosion whereas 6-MP is released by a combination of erosion and diffusion. The cytotoxicity studies show that all three cell types demonstrate high viability, thus cytocompatibility, to poly(thioether anhydrides). Toxicity to the material is dose dependent and comparable to other polyanhydride systems. The 6-MP cancer drug is shown to remain bioactive after encapsulation in the poly(thioether anhydride) matrix and the polymer does not appear to modify the efficacy of the drug.