ABSTRACT
Crack cocaine users are simultaneously exposed to volatilized cocaine and to its main pyrolysis product, anhydroecgonine methyl ester (AEME). Although the neurotoxic effects of cocaine have been extensively studied, little is known about AEME or its combination. We investigated cell death processes using rat primary hippocampal cells exposed to cocaine (2 mM), AEME (1 mM) and their combination (C + A), after 1, 3, 6 and 12 h. Cocaine increased LC3 I after 6 h and LC3 II after 12 h, but reduced the percentage of cells with acid vesicles, suggesting failure in the autophagic flux, which activated the extrinsic apoptotic pathway after 12 h. AEME neurotoxicity did not involve the autophagic process; rather, it activated caspase-9 after 6 h and caspase-8 after 12 h leading to a high percentage of cells in early apoptosis. C + A progressively reduced the percentage of undamaged cells, starting after 3 h; it activated both apoptotic pathways after 6 h, and was more neurotoxic than cocaine and AEME alone. Also, C + A increased the phosphorylation of p62 after 12 h, but there was little difference in LC3 I or II, and a small percentage of cells with acid vesicles at all time points investigated. In summary, the present study provides new evidence for the neurotoxic mechanism and timing response of each substance alone and in combination, indicating that AEME is more than just a biological marker for crack cocaine consumption, as it may intensify and hasten cocaine neurotoxicity.
Subject(s)
Cocaine/analogs & derivatives , Animals , Cocaine/toxicity , Gas Chromatography-Mass Spectrometry , Hippocampus , Neurons , Neurotoxicity Syndromes , Pyrolysis , RatsABSTRACT
Indoleamine 2,3-dioxygenase (IDO) is associated with the progression of many types of tumors, including melanoma. However, there is limited information about IDO modulation on tumor cell itself and the effect of BRAF inhibitor (BRAFi) treatment and resistance. Herein, IDO expression was analyzed in different stages of melanoma development and progression linked to BRAFi resistance. IDO expression was increased in primary and metastatic melanomas from patients' biopsies, especially in the immune cells infiltrate. Using a bioinformatics approach, we also identified an increase in the IDO mRNA in the vertical growth and metastatic phases of melanoma. Using in silico analyses, we found that IDO mRNA was increased in BRAFi resistance. In an in vitro model, IDO expression and activity induced by interferon-gamma (IFNγ) in sensitive melanoma cells was decreased by BRAFi treatment. However, cells that became resistant to BRAFi presented random IDO expression levels. Also, we identified that treatment with the IDO inhibitor, 1-methyltryptophan (1-MT), was able to reduce clonogenicity for parental and BRAFi-resistant cells. In conclusion, our results support the hypothesis that the decreased IDO expression in tumor cells is one of the many additional outcomes contributing to the therapeutic effects of BRAFi. Still, the IDO production changeability by the BRAFi-resistant cells reiterates the complexity of the response arising from resistance, making it not possible, at this stage, to associate IDO expression in tumor cells with resistance. On the other hand, the maintenance of 1-MT off-target effect endorses its use as an adjuvant treatment of melanoma that has become BRAFi-resistant.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Melanoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Skin Neoplasms/drug therapy , Vemurafenib/pharmacology , Cell Line, Tumor , Databases, Genetic , Drug Resistance, Neoplasm/genetics , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Melanoma/enzymology , Melanoma/genetics , Molecular Targeted Therapy , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Skin Neoplasms/enzymology , Skin Neoplasms/genetics , Tryptophan/analogs & derivatives , Tryptophan/pharmacologyABSTRACT
Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Mutations in the MAPK (Ras-Raf-MEK-ERK) pathway is closely linked to the lack of control of cell proliferation. Especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent inhibitors of v-Raf murine sarcoma viral oncogene homolog B (BRAFi) and mitogen-activated protein kinase kinase (MEKi). Very high rates of response have been achieved, but most patients are relapsed due to the development of resistance, justifying the constant search for new therapeutic compounds. Early results from our group indicated that 4-nerolidylcatechol (4-NC), a catechol compound extracted from Pothomorphe umbellata, induces DNA damage, ROS production, increased p53 expression culminating in apoptosis in melanoma but with no data regarding the 4-NC effects in cells resistant to BRAFi or MEKi. Therefore, here we evaluated the role of 4-NC alone or in combination with BRAFi/MEKi in resistant melanoma cells. Double-resistant cells were generated and characterized by MAPK pathway reactivation. 4-NC alone or in combination (30 µM) with MAPK inhibitors was cytotoxic, inhibited colony formation and decreased invasiveness in two and three-dimensional cell culture models of treatment-naïve, BRAFi-resistant and BRAF/MEKi double-resistant melanoma cells. Apoptosis induction was demonstrated in resistant and double-resistant melanoma cell lines after 4-NC treatments. 4-NC showed important ability to induce apoptosis via Endoplasmatic Reticulum (ER) stress and specifically BiP and CHOP that had increased protein expression in all melanoma cell lines proving to be part of the ER stress pathway activation. CHOP knockdown slightly but enough increases cellular viability following 4-NC treatment indicating that apoptosis observed is partially dependent on CHOP. In summary, we show that 4-NC is a compound with activity against cutaneous melanoma, including resistant cells to clinically approved therapies.
Subject(s)
Antineoplastic Agents/pharmacology , Catechols/pharmacology , Drug Resistance, Neoplasm/drug effects , Endoplasmic Reticulum Stress/drug effects , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Apoptosis/drug effects , Cell Line, Tumor , Humans , Melanoma/drug therapy , Skin Neoplasms/drug therapyABSTRACT
Melanoma is a highly invasive and metastatic cancer with high mortality rates and chemoresistance. Around 50% of melanomas are driven by activating mutations in BRAF that has led to the development of potent anti-BRAF inhibitors. However resistance to anti-BRAF therapy usually develops within a few months and consequently there is a need to identify alternative therapies that will bypass BRAF inhibitor resistance. The curcumin analogue DM-1 (sodium 4-[5-(4-hydroxy-3-methoxy-phenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate) has substantial anti-tumor activity in melanoma, but its mechanism of action remains unclear. Here we use a synthetic lethal genetic screen in Saccharomyces cerevisiae to identify 211 genes implicated in sensitivity to DM-1 toxicity. From these 211 genes, 74 had close human orthologues implicated in oxidative phosphorylation, insulin signaling and iron and RNA metabolism. Further analysis identified 7 target genes (ADK, ATP6V0B, PEMT, TOP1, ZFP36, ZFP36L1, ZFP36L2) with differential expression during melanoma progression implicated in regulation of tumor progression, cell differentiation, and epithelial-mesenchymal transition. Of these TOP1 and ADK were regulated by DM-1 in treatment-naïve and vemurafenib-resistant melanoma cells respectively. These data reveal that the anticancer effect of curcumin analogues is likely to be mediated via multiple targets and identify several genes that represent candidates for combinatorial targeting in melanoma.
Subject(s)
Curcumin/analogs & derivatives , Curcumin/pharmacology , Gene Expression Regulation, Fungal/drug effects , Melanoma/genetics , Saccharomyces cerevisiae/genetics , Cell Line, Tumor , Computational Biology , Humans , Mutation , ToxicogeneticsABSTRACT
The BRAF(V600E) mutation confers constitutive kinase activity and accounts for >90% of BRAF mutations in melanoma. This genetic alteration is a current therapeutic target; however, the antitumorigenic effects of the BRAF(V600E) inhibitor vemurafenib are short-lived and the majority of patients present tumor relapse in a short period after treatment. Characterization of vemurafenib resistance has been essential to the efficacy of next generation therapeutic strategies. Herein, we found that acute BRAF inhibition induced a decrease in active MMP-2, MT1-MMP and MMP-9, but did not modulate the metalloproteinase inhibitors TIMP-2 or RECK in naïve melanoma cells. In vemurafenib-resistant melanoma cells, we observed a lower growth rate and an increase in EGFR phosphorylation followed by the recovery of active MMP-2 expression, a mediator of cancer metastasis. Furthermore, we found a different profile of MMP inhibitor expression, characterized by TIMP-2 downregulation and RECK upregulation. In a 3D spheroid model, the invasion index of vemurafenib-resistant melanoma cells was more evident than in its non-resistant counterpart. We confirmed this pattern in a matrigel invasion assay and demonstrated that use of a matrix metalloproteinase inhibitor reduced the invasion of vemurafenib resistant melanoma cells but not drug naïve cells. Moreover, we did not observe a delimited group of cells invading the dermis in vemurafenib-resistant melanoma cells present in a reconstructed skin model. The same MMP-2 and RECK upregulation profile was found in this 3D skin model containing vemurafenib-resistant melanoma cells. Acute vemurafenib treatment induces the disorganization of collagen fibers and consequently, extracellular matrix remodeling, with this pattern observed even after the acquisition of resistance. Altogether, our data suggest that resistance to vemurafenib induces significant changes in the tumor microenvironment mainly by MMP-2 upregulation, with a corresponding increase in cell invasiveness.
Subject(s)
Antineoplastic Agents/pharmacology , Indoles/pharmacology , Matrix Metalloproteinase 2/metabolism , Melanoma/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Sulfonamides/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm/physiology , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Interleukin-8/metabolism , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Melanoma/genetics , Melanoma/metabolism , Neoplasm Invasiveness , Proto-Oncogene Proteins B-raf/genetics , Tissue Inhibitor of Metalloproteinase-2/genetics , Tissue Inhibitor of Metalloproteinase-2/metabolism , Tumor Microenvironment/drug effects , Up-Regulation , VemurafenibABSTRACT
This work describes the anti-inflammatory effect of the curcumin-analog compound, sodium 4-[5-(4-hydroxy-3-methoxyphenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate (DM1), and shows that DM1 modulates iNOS and COX-2 gene expression in cultured RAW 264.7 cells and induces autophagy on human melanoma cell line A375.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Autophagy/drug effects , Curcumin/analogs & derivatives , Curcumin/pharmacology , Cyclooxygenase 2/genetics , Edema/drug therapy , Gene Expression Regulation, Enzymologic/drug effects , Nitric Oxide Synthase Type II/genetics , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Carrageenan , Cells, Cultured , Curcumin/chemistry , Dose-Response Relationship, Drug , Edema/chemically induced , Gene Expression Profiling , Humans , Male , Mice , Molecular Structure , Nitric Oxide/biosynthesis , Structure-Activity RelationshipABSTRACT
Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.
Subject(s)
Apoptosis/drug effects , Cytoskeleton/metabolism , Maytansine/analogs & derivatives , Melanoma/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Skin Neoplasms/pathology , Blotting, Western , Cell Proliferation/drug effects , Cytoskeleton/drug effects , Humans , Maytansine/pharmacology , Melanoma/drug therapy , Melanoma/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Poly(ADP-ribose) Polymerases/metabolism , Skin Neoplasms/drug therapy , Skin Neoplasms/metabolism , Tumor Cells, Cultured , bcl-2-Associated X Protein/metabolism , bcl-X Protein/metabolismABSTRACT
The main difficulty in the successful treatment of metastatic melanoma is that this type of cancer is known to be resistant to chemotherapy. Chemotherapy remains the treatment of choice, and dacarbazine (DTIC) is the best standard treatment. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and antimetastatic properties. The objective of this study was to evaluate the signaling pathways involved in melanoma cell death after treatment with DM-1 compared to the standard agent for melanoma treatment, DTIC. Cell death was evaluated by flow cytometry for annexin V and iodide propide, cleaved caspase 8, and TNF-R1 expression. Hoechst 33342 staining was evaluated by fluorescent microscopy; lipid peroxidation and cell viability (MTT) were evaluated by colorimetric assays. The antiproliferative effects of the drugs were evaluated by flow cytometry for cyclin D1 and Ki67 expression. Mice bearing B16F10 melanoma were treated with DTIC, DM-1, or both therapies. DM-1 induced significant apoptosis as indicated by the presence of cleaved caspase 8 and an increase in TNF-R1 expression in melanoma cells. Furthermore, DM-1 had antiproliferative effects in this the same cell line. DTIC caused cell death primarily by necrosis, and a smaller melanoma cell population underwent apoptosis. DTIC induced oxidative stress and several physiological changes in normal melanocytes, whereas DM-1 did not significantly affect the normal cells. DM-1 antitumor therapy in vivo showed tumor burden decrease with DM-1 monotherapy or in combination with DTIC, besides survival rate increase. Altogether, these data confirm DM-1 as a chemotherapeutic agent with effective tumor control properties and a lower incidence of side effects in normal cells compared to DTIC.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Curcumin/pharmacology , Lipid Peroxidation/drug effects , Melanoma/pathology , Membrane Potential, Mitochondrial/drug effects , Animals , Antineoplastic Agents, Alkylating/pharmacology , Blotting, Western , Dacarbazine/pharmacology , Flow Cytometry , Free Radicals/metabolism , Humans , Melanoma/drug therapy , Melanoma/metabolism , Mice , Tumor Cells, CulturedABSTRACT
This paper describes a new method for the preparation of sodium 4-[5-(4-hydroxy-3-methoxyphenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate, DM-1, and 3-oxo-penta-1,4-dienyl-bis (2-methoxy-phenolate), DM-2. The aim of this work was to evaluate the antitumor effects of DM-1 in adjuvant chemotherapy for breast cancer treatment. Mice bearing mammary adenocarcinomas (Ehrlich ascites tumors) were treated with paclitaxel alone, DM-1 alone, and paclitaxel + DM-1. Tumor samples were used to perform cytological analysis by the Papanicolaou method and apoptosis analysis by annexin V and phosphorylated caspase 3. The paclitaxel + DM-1 group had decreased tumor areas and tumor volumes, and the frequency of metastasis was significantly reduced. This caused a decrease in cachexia, which is usually caused by the tumor. Furthermore, treatment with paclitaxel + DM-1 and DM-1 alone increased the occurrence of apoptosis up to 40% in tumor cells, which is 35% more than in the group treated with paclitaxel alone. This cell death was mainly caused through phosphorylated caspase 3 (11% increase in paclitaxel + DM-1 compared to the paclitaxel group), as confirmed by reduced malignancy criteria in the ascitic fluid. DM-1 emerges as a potential treatment for breast cancer and may act as an adjuvant in chemotherapy, enhancing antitumor drug activity with reduced side effects.
Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Guaiacol/analogs & derivatives , Ketones/therapeutic use , Mammary Neoplasms, Experimental/drug therapy , Adenocarcinoma/mortality , Animals , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Caspase 3/metabolism , Cell Proliferation/drug effects , Female , Guaiacol/administration & dosage , Guaiacol/pharmacology , Guaiacol/therapeutic use , Ketones/administration & dosage , Ketones/pharmacology , Mammary Neoplasms, Experimental/mortality , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Paclitaxel/administration & dosage , Survival Analysis , Tumor Burden/drug effectsABSTRACT
Information on (10)B distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The (10)B concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the (10)B concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of (10)B uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.
Subject(s)
Boron Neutron Capture Therapy , Boron/pharmacokinetics , Boron/therapeutic use , Melanocytes/metabolism , Melanocytes/radiation effects , Melanoma, Experimental/metabolism , Melanoma, Experimental/radiotherapy , Animals , Cell Line, Tumor , Cell Survival/radiation effects , Female , Humans , Melanins/biosynthesis , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Reproducibility of Results , Tissue DistributionABSTRACT
We previously demonstrated that pan-HDAC inhibitors could limit escape from MEK inhibitor (MEKi) therapy in uveal melanoma (UM) through suppression of AKT and YAP/TAZ signaling. Here, we focused on the role of specific HDACs in therapy adaptation. Class 2 UM displayed higher expression of HDACs 1, 2, and 3 than Class 1, whereas HDACs 6, 8, and 11 were uniformly expressed. Treatment of UM cells with MEKi led to modulation of multiple HDACs, with the strongest increases observed in HDAC11. RNA-seq analysis showed MEKi to decrease the expression of multiple HDAC11 target genes. Silencing of HDAC11 significantly reduced protein deacetylation, enhanced the apoptotic response to MEKi and reduced growth in long-term colony formation assays across multiple UM cell lines. Knockdown of HDAC11 led to decreased expression of TAZ in some UM cell lines, accompanied by decreased YAP/TAZ transcriptional activity and reduced expression of multiple YAP/TAZ target genes. Further studies showed this decrease in TAZ expression to be associated with increased LKB1 activation and modulation of glycolysis. In an in vivo model of uveal melanoma, silencing of HDAC11 limited the escape to MEKi therapy, an effect associated with reduced levels of Ki67 staining and increased cleaved caspase-3. We have demonstrated a novel role for adaptive HDAC11 activity in UM cells, that in some cases modulates YAP/TAZ signaling leading to MEKi escape.
Subject(s)
Melanoma , Uveal Neoplasms , Humans , Cell Line, Tumor , Uveal Neoplasms/drug therapy , Uveal Neoplasms/genetics , Uveal Neoplasms/metabolism , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Mitogen-Activated Protein Kinase Kinases , Histone Deacetylases/geneticsABSTRACT
This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one 1 and its derivatives 2-5. This set of synthetic compounds exhibited high antitumoral activities regarding in vitro screening against several human tumor cell lines as lung carcinoma NCI-460, melanoma UACC-62, breast MCF-7, colon HT-29, renal 786-O, ovarian OVCAR-03 and ovarian expressing the resistance phenotype for adriamycin NCI-ADR/RES, prostate PC-3, and leukemia K-562. Compounds were also tested against murine tumor cell line B16F10 melanoma and lymphocytic leukemia L1210 as well as to their effect toward normal macrophages. Specific activity against colon cancer cells HT-29 was observed for all tested compounds and suggests further studies with models of colon cancer. Compounds 1, 2, and 4 showed significant cytotoxic activity with IC(50) values 2.3 microM for all human cancer cell lines. Intraperitoneal acute administration of compound 1 and 2 showed very low toxicity rate.
Subject(s)
Anisoles/pharmacology , Antineoplastic Agents/pharmacology , Ketones/pharmacology , Animals , Anisoles/chemical synthesis , Anisoles/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Ketones/chemical synthesis , Ketones/chemistry , Mice , Structure-Activity RelationshipABSTRACT
NRAS-mutations arise in 15-20% of all melanomas and are associated with aggressive disease and poor prognosis. Besides, the treatment for NRAS-mutant melanoma are not very efficient and is currently limited to immune checkpoints inhibitors or aggressive chemotherapy. 4-nerolidylcathecol (4-NC), a natural product extracted from Pothomorphe umbellata, induces apoptosis in melanoma cells by ROS production, DNA damage and increased p53 expression, in addition to inhibiting invasion in reconstructed skin. Moreover, 4-NC showed cytotoxicity in BRAF/MEKi-resistant and naive melanoma cells by Endoplasmic Reticulum (ER) stress induction in vitro. We evaluated the in vivo efficacy and the systemic toxicity of 4-NC in a NRAS-mutant melanoma model. 4-NC was able to significantly suppress tumor growth 4-fold compared to controls. Cleaved PARP and p53 expression were increased indicating cell death. As a proof of concept, MMP-2 and MMP-14 gene expression were decreased, demonstrating a possible role of 4-NC in melanoma invasion inhibition. Toxicological analysis indicated minor changes in the liver and bone marrow, but this toxicity was very mild when compared to other proteasome inhibitors and ER stress inductors already described. Our data indicate that 4-NC can counteract melanoma growth in vivo with minor adverse effects, suggesting further investigation as a potential NRAS-mutant melanoma treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Catechols/pharmacology , GTP Phosphohydrolases/genetics , Melanoma/pathology , Membrane Proteins/genetics , Mutation , Skin Neoplasms/pathology , Animals , Antineoplastic Agents/toxicity , Catechols/toxicity , Cell Line, Tumor , Cell Proliferation/drug effects , Endoplasmic Reticulum Stress/drug effects , Female , Humans , Melanoma/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Skin Neoplasms/genetics , Toxicity Tests, Subacute , Xenograft Model Antitumor AssaysABSTRACT
MEK inhibitors (MEKi) demonstrate anti-proliferative activity in patients with metastatic uveal melanoma, but responses are short-lived. In the present study, we evaluated the MEKi trametinib alone and in combination with drugs targeting epigenetic regulators, including DOT1L, EZH2, LSD1, DNA methyltransferases, and histone acetyltransferases. The DNA methyltransferase inhibitor (DNMTi) decitabine effectively enhanced the anti-proliferative activity of trametinib in cell viability, colony formation, and 3D organoid assays. RNA-Seq analysis showed the MEKi-DNMTi combination primarily affected the expression of genes involved in G1 and G2/2M checkpoints, cell survival, chromosome segregation and mitotic spindle. The DNMTi-MEKi combination did not appear to induce a DNA damage response (as measured by γH2AX foci) or senescence (as measured by ß-galactosidase staining) compared to either MEKi or DNMTi alone. Instead, the combination increased expression of the CDK inhibitor p21 and the pro-apoptotic protein BIM. In vivo, the DNMTi-MEKi combination was more effective at suppressing growth of MP41 uveal melanoma xenografts than either drug alone. Our studies indicate that DNMTi may enhance the activity of MEKi in uveal melanoma.
Subject(s)
Decitabine/therapeutic use , Melanoma/drug therapy , Melanoma/enzymology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Uveal Neoplasms/drug therapy , Uveal Neoplasms/enzymology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Decitabine/pharmacology , Female , Humans , Melanoma/pathology , Mice , Pyridones/pharmacology , Pyridones/therapeutic use , Pyrimidinones/pharmacology , Pyrimidinones/therapeutic use , Uveal Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Melanoma is the most aggressive skin cancer, and BRAF (V600E) is the most frequent mutation that led to the development of BRAF inhibitors (BRAFi). However, patients treated with BRAFi usually present recidivism after 6-9 months. Curcumin is a turmeric substance, and it has been deeply investigated due to its anti-inflammatory and antitumoral effects. Still, the low bioavailability and biodisponibility encouraged the investigation of different analogs. DM-1 is a curcumin analog and has shown an antitumoral impact in previous studies. METHODS: Evaluated DM-1 stability and cytotoxic effects for BRAFi-sensitive and resistant melanomas, as well as the role in the metalloproteinases modulation. RESULTS: DM-1 showed growth inhibitory potential for melanoma cells, demonstrated by reduction of colony formation, migration and endothelial tube formation, and cell cycle arrest. Subtoxic doses were able to downregulate important Metalloproteinases (MMPs) related to invasiveness, such as MMP-1, -2 and -9. Negative modulations of TIMP-2 and MMP-14 reduced MMP-2 and -9 activity; however, the reverse effect is seen when increased TIMP-2 and MMP-14 resulted in raised MMP-2. CONCLUSION: These findings provide essential details into the functional role of DM-1 in melanomas, encouraging further studies in the development of combinatorial treatments for melanomas.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Melanoma/drug therapy , Metalloproteases/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Cycle Checkpoints/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Melanoma/metabolism , Melanoma/pathology , Metalloproteases/metabolism , Molecular Structure , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Melanoma cells have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanomas to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia, and UV irradiation leads to an increase in histone deacetylase 8 (HDAC8) activity and the adoption of a drug-resistant phenotype. Mass spectrometry-based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in vivo. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase activation, leading to MAPK signaling. Although HDACs function at the histone level, they also regulate nonhistone substrates, and introduction of HDAC8 decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine 273 increased transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both nonselective and HDAC8-specific inhibitors enhancing the durability of BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors limit the adaptation of melanoma cells to multiple stresses including BRAF-MEK inhibition. SIGNIFICANCE: This study provides evidence that HDAC8 drives transcriptional plasticity in melanoma cells in response to a range of stresses through direct deacetylation of c-Jun.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2947/F1.large.jpg.
Subject(s)
Drug Resistance, Neoplasm/genetics , Histone Deacetylases/metabolism , Melanoma/drug therapy , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Repressor Proteins/metabolism , Skin Neoplasms/drug therapy , Acetylation , Animals , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/genetics , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System/drug effects , Melanoma/metabolism , Mice, Inbred NOD , Panobinostat/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Skin Neoplasms/metabolism , Stress, Physiological/drug effects , Stress, Physiological/physiologyABSTRACT
PURPOSE: The clinical use of MEK inhibitors in uveal melanoma is limited by the rapid acquisition of resistance. This study has used multiomics approaches and drug screens to identify the pan-HDAC inhibitor panobinostat as an effective strategy to limit MEK inhibitor resistance.Experimental Design: Mass spectrometry-based proteomics and RNA-Seq were used to identify the signaling pathways involved in the escape of uveal melanoma cells from MEK inhibitor therapy. Mechanistic studies were performed to evaluate the escape pathways identified, and the efficacy of the MEK-HDAC inhibitor combination was demonstrated in multiple in vivo models of uveal melanoma. RESULTS: We identified a number of putative escape pathways that were upregulated following MEK inhibition, including the PI3K/AKT pathway, ROR1/2, and IGF-1R signaling. MEK inhibition was also associated with increased GPCR expression, particularly the endothelin B receptor, and this contributed to therapeutic escape through ET-3-mediated YAP signaling. A screen of 289 clinical grade compounds identified HDAC inhibitors as potential candidates that suppressed the adaptive YAP and AKT signaling that followed MEK inhibition. In vivo, the MEK-HDAC inhibitor combination outperformed either agent alone, leading to a long-term decrease of tumor growth in both subcutaneous and liver metastasis models and the suppression of adaptive PI3K/AKT and YAP signaling. CONCLUSIONS: Together, our studies have identified GPCR-mediated YAP activation and RTK-driven AKT signaling as key pathways involved in the escape of uveal melanoma cells from MEK inhibition. We further demonstrate that HDAC inhibition is a promising combination partner for MEK inhibitors in advanced uveal melanoma.
Subject(s)
Histone Deacetylase Inhibitors/pharmacology , Melanoma/metabolism , Protein Kinase Inhibitors/pharmacology , Uveal Neoplasms/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Disease Progression , Drug Resistance, Neoplasm , Drug Synergism , Humans , MAP Kinase Signaling System/drug effects , Melanoma/drug therapy , Melanoma/pathology , Mice , Panobinostat/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Proteome , Proteomics/methods , Proto-Oncogene Proteins c-akt/metabolism , Pyridones/pharmacology , Pyrimidinones/pharmacology , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Receptor, IGF Type 1/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Uveal Neoplasms/drug therapy , Uveal Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Cancer cells are highly plastic and adopt multiple phenotypic states that contribute to tumor progression. Heppt et al. demonstrate that the homeodomain transcription factor Msh homeobox 1 reprograms melanoma cells to a precursor state associated with melanoma progression and increased liver metastasis. Identification of this new role for Msh homeobox 1 may facilitate the development of new therapies that limit melanoma dissemination.
Subject(s)
Gene Expression Regulation, Neoplastic , Melanoma/genetics , Disease Progression , Humans , MSX1 Transcription Factor , Neural Crest , Transcription FactorsABSTRACT
Targeted therapy has proven to be beneficial at producing significant responses in patients with a wide variety of cancers. Despite initially impressive responses, most individuals ultimately fail these therapies and show signs of drug resistance. Very few patients are ever cured. Emerging evidence suggests that treatment of cancer cells with kinase inhibitors leads a minor population of cells to undergo a phenotypic switch to a more embryonic-like state. The adoption of this state, which is analogous to an epithelial-to-mesenchymal transition, is associated with drug resistance and increased tumor aggressiveness. In this commentary we will provide a comprehensive analysis of the mechanisms that underlie the embryonic reversion that occurs on targeted cancer therapy and will review potential novel therapeutic strategies designed to eradicate the escaping cells.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Delivery Systems , Neoplasms/drug therapy , Animals , Humans , Treatment OutcomeABSTRACT
Malignant melanoma is a highly aggressive form of skin cancer with a high mortality rate if not discovered in early stages. Although a limited number of treatment options for melanoma currently exist, patients with a more aggressive form of this cancer frequently decline treatment. DM-1 is a sodium phenolate and curcumin analog with proven anticancer, anti-proliferative and anti-metastatic properties. In this paper, the DM-1 compound showed in vivo antitumor activity alone or in combination with chemotherapeutic DTIC in B16F10 melanoma-bearing mice. Beneficial effects such as melanoma tumor burden reduction with pyknotic nuclei, decreased nuclei/cytoplasmic ratio and nuclear degradation occurred after DM-1 treatment. No toxicological changes were observed in the liver, kidneys, spleen and lungs after DM-1 monotherapy or DTIC combined therapy. DTIC+DM-1 treatment induced the recovery of anemia arising from melanoma and immunomodulation. Both DM-1 treatment alone and in combination with DTIC induced apoptosis with the cleavage of caspase-3, -8 and -9. Furthermore, melanoma tumors treated with DM-1 showed a preferential apoptotic intrinsic pathway by decreasing Bcl-2/Bax ratio. Considering the chemoresistance exhibited by melanoma towards conventional chemotherapy drugs, DM-1 compound in monotherapy or in combination therapy provides a promising improvement in melanoma treatment with a reduction of side effects.