A375 Melanoma Cells Research Articles

Effect of Arylazo Sulfones on DNA: Binding, Cleavage, Photocleavage, Molecular Docking Studies and Interaction with A375 Melanoma and Non-Cancer Cells.

A set of arylazo sulfones, known to undergo N-S bond cleavage upon light exposure, has been synthesized, and their activity in the dark and upon irradiation towards DNA has been investigated. Their interaction with calf-thymus DNA has been examined, and the significant affinity observed (most probably due to DNA intercalation) was analyzed by means of molecular docking "in silico" calculations that pointed out polar contacts, mainly via the sulfonyl moiety. Incubation with plasmid pBluescript KS II revealed DNA cleavage that has been studied over time and concentration. UV-A irradiation considerably improved DNA damage for most of the compounds, whereas under visible light the effect was slightly lower. Moving to in vitro experiments, irradiation was found to slightly enhance the death of the cells in the majority of the compounds. Naphthylazosulfone 1 showed photo-disruptive effect under UV-A irradiation (IC50 ~13 μΜ) followed by derivatives 14 and 17 (IC50 ~100 μΜ). Those compounds were irradiated in the presence of two non-cancer cell lines and were found equally toxic only upon irradiation and not in the dark. The temporal and spatial control of light, therefore, might provide a chance for these novel scaffolds to be useful for the development of phototoxic pharmaceuticals.

Nano-scaled emulsion and nanogel containing Mentha pulegium essential oil: cytotoxicity on human melanoma cells and effects on apoptosis regulator genes

BackgroundTopical drug delivery using nanoemulsions and nanogels is a promising approach to treating skin disorders such as melanoma.MethodsIn this study, the chemical composition of Mentha pulegium essential oil with five major compounds, including pulegone (68.11%), l-menthone (8.83%), limonene (2.90%), iso-pulegone (2.69%), and iso-menthone (1.48%) was first identified using GC-MS (Gas chromatography–Mass Spectrometry) analysis. Afterward, a nano-scaled emulsion containing the essential oil with a droplet size of 7.70 ± 1 nm was prepared. Nanogel containing the essential oil was then prepared by adding (2% w/v) carboxymethyl cellulose to the nano-scaled emulsion. Moreover, the successful loading of M. pulegium essential oil in the nano-scaled emulsion and nanogel was confirmed using ATR-FTIR (Attenuated total reflectance-Fourier Transform InfraRed) analysis. Then, human A375 melanoma cells were treated with different concentrations of samples, the MTT assay evaluated cell viability, and cell apoptosis was confirmed by flow cytometry. In addition, the expression of apoptotic and anti-apoptotic genes, including Bax and Bcl-2, was evaluated using the qPCR (quantitative Polymerase Chain Reaction) technique.ResultsThe results showed that cell viability was reduced by 90 and 45% after treatment with 300 μg/mL of the nanogel and nano-scaled emulsion. As confirmed by flow cytometry, this effect was mediated by apoptosis. Furthermore, gene expression analysis showed up-regulation of Bax and down-regulation of Bcl-2 genes. Therefore, the prepared nanogel, with high efficacy, could be considered a potent anticancer agent for supplementary medicine and in vivo research.

The Alternative TrkAIII Splice Variant, a Targetable Oncogenic Participant in Human Cutaneous Malignant Melanoma.

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM.

The Antimelanoma Biological Assessment of Triterpenic Acid Functionalized Gold Nanoparticles.

One of several promising strategies for increasing the bioavailability and therapeutic potential of high-lipophilic biologically active compounds is gold nanoparticle formulation. The current study describes the synthesis and biological antimelanoma evaluation of three triterpen-functionalized gold nanoparticles, obtained using our previously reported antimelanoma benzotriazole-triterpenic acid esters. Functionalized gold nanoparticle (GNP) formation was validated through UV-VIS and FTIR spectroscopy. The conjugate's cytotoxic effects were investigated using HaCaT healthy keratinocytes and A375 human melanoma cells. On A375 cells, all three conjugates demonstrated dose-dependent cytotoxic activity, but no significant cytotoxic effects were observed on normal HaCaT keratinocytes. GNP-conjugates were found to be more cytotoxic than their parent compounds. After treatment with all three GNP-conjugates, 4,6'-diamidino-2-phenylindole (DAPI) staining revealed morphological changes consistent with apoptosis in A375 melanoma cells. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis revealed that the triterpene-GNP conjugate treated A375 melanoma cells had a fold change increase in Bcl-2-associated X protein (BAX) expression and a fold change decrease in B-cell lymphoma 2 (Bcl-2) expression. In A735 melanoma cells, high-resolution respirometry studies revealed that all three GNP-conjugates act as selective inhibitors of mitochondrial function. Furthermore, by examining the effect on each mitochondrial respiratory rate, the results indicate that all three conjugates are capable of increasing the production of reactive oxygen species (ROS), an apoptosis trigger in cancer cells.

In Vitro Cytostatic Effect on Tumor Cells by Carborane‐Based Dual Cyclooxygenase‐2 and 5‐Lipoxygenase Inhibitors

AbstractThe selective inhibition of enzymes that catalyze the conversion of arachidonic acid to inflammatory eicosanoids represents a promising approach for cancer therapy. This study, therefore, focuses on the incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes into existing dual cycloxygenase‐2 (COX‐2)/5‐lipoxygenase (5‐LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids. Here, the first carborane‐containing dual COX‐2/5‐LO inhibitors derived from RWJ‐63556 are presented. The replacement of the fluorophenyl moiety by meta‐ or para‐carborane resulted in five carborane‐containing derivatives 3, 6, 9, 13, and 17 that show high inhibitory activities toward COX‐2 and 5‐LO in vitro. Cell viability studies on the A375 melanoma cell line revealed that meta‐carborane derivative 3 shows higher anticancer activity compared to RWJ‐63556 based on accumulation of lipid droplets in the cells due to blockage of the COX‐2 and 5‐LO pathways, indicating a promising approach for the design of potent dual COX‐2/5‐LO inhibitors.

Synthesis and cytotoxicity of diastereomeric benzylamides derived from maslinic acid, augustic acid and bredemolic acid

36 substituted benzylamides were prepared starting from maslinic acid, bredemolic acid, and augustic acid and evaluated for their cytotoxicity in SRB assays employing several human tumor cell lines as well as non-malignant fibroblasts. Thereby, the benzylamides of maslinic acid, however, were found to be more cytotoxic than those obtained from augustic acid or bredemolic acid. The best compound (18, derived from maslinic acid) showed an EC50 value of 1.3 μM against A375 melanoma cells. Additional staining experiments revealed that this compound acted rather by apoptosis than by necrosis.

Cytotoxic Effects of Ethanolic Extract of Polypodium Vulgare on Human Malignant Melanoma Cell Line.

Melanoma is a malignant tumor that originates from the skin's melanocytes and has the highest death rate from skin cancer. Developing more efficacious anticancer medications with fewer adverse effects is the key to effective cancer management. Natural products are considered relevant and cost-effective sources of treatment. The plant (Polypodium vulgare) is a small and evergreen fern. One of the most important chemical compounds in the extract of this herb is flavonoids, which are thought to have beneficial effects in the treatment of melanoma through antioxidant properties. Due to the limitations of current cancer management and cytotoxic drugs available in the country, the need to study drugs of natural origin has become more prominent. In this regard, the present study aims to investigate the cytotoxic effects of the ethanolic extract of Polypodium vulgare on A375 melanoma cells. Polypodium vulgare was extracted in 80% ethanol by the maceration. Then, its effects on the cell death of the melanoma cell line A375 compared to the AGO-1522 cell line as control were measured using the MTT-assay technique. The amount of cellular lipid peroxidation was estimated by TBARS assay. The amount of cellular ROS was calculated by fluorescent reagent 2,7-dichlorofluorescein diacetate. Cytochrome c concentration was measured by a cytochrome c immunoassay kit. In this experiment, the anticancer effects of Polypodium vulgare ethanolic extract on human melanoma cell lines were investigated for the first time. Herb extract with a concentration of 0.123 mg/ml significantly increased the death of A375 melanoma cells (p < 0.001), lipid peroxidation (p < 0.01), and reactive oxygen species (ROS) (p < 0.01) and cytochrome c concentration (p < 0.001). Meanwhile, the same amount was ineffective and safe on AGO-1522 normal fibroblast cells. A 0.123 mg/ml concentration of Polypodium vulgare increases apoptosis in melanoma cells. Meanwhile, the same amount was safe on healthy cells. So, it could be considered an effective treatment without side effects in human melanoma.

Identification of TUBB4A as a Prognostic Biomarker of Melanoma by Transcriptomic Data and In Vitro Experiments.

Background: Melanoma is one of the most malignant skin carcinomas with high metastatic potential. Increasing evidence has demonstrated that β-tubulin 4A (TUBB4A) plays a key role in the development and progression of several types of human cancer. However, the potential function of TUBB4A in cutaneous melanoma remains to be determined. Methods: We first performed a differential expression analysis based on skin melanoma tissues and normal tissues from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets and then a survival analysis to identify prognostic-related key genes. We further conducted in vitro biochemical experiments to verify the functional roles of the key gene TUBB4A. Two small-molecule inhibitors of TUBB4A, Dihydroartemisinin (DHA) and Nocodazole, were used to validate the effect of TUBB4A on the apoptosis and cell cycle of melanoma cells. Results: We found that TUBB4A expression was positively correlated to the overall survival (OS) of cutaneous melanoma patients. The coexpressed genes with TUBB4A were enriched in melanoma-related pathways and functions. The experimental results showed that knockdown of TUBB4A inhibited the proliferation and migration of A375 and B16-F10 melanoma cells. Moreover, DHA and Nocodazole promoted the apoptosis of melanoma cells and blocked the melanoma tumor cell cycle in the G2/M stage. Conclusion: TUBB4A may be a prognostic biomarker and therapeutic target for melanoma.

3D, PEG-Based Hydrogels Induce Spheroid Formation and Higher Viability of A2058 Melanoma Cells

3D, PEG-Based Hydrogels Induce Spheroid Formation and Higher Viability of A2058 Melanoma Cells

Transcriptome analysis reveals tumor antigen and immune subtypes of melanoma.

To screen potential tumor antigens for melanoma vaccine development and identify different immune subtypes. Transcriptional data (HTSEQ-FPKM) and clinical information of a 472 Melanoma cohort GDC TCGA Melanoma (SKCM) were downloaded from the UCSC XENA website (http://xena.ucsc.edu/). Subsequently, transcriptome data and clinical information of 210 melanoma cohort GSE65904 were downloaded from Gene Expression Omnibus (GEO), a large global public database. All the transcriptome expression data matrices were log2 transformed for subsequent analysis. GEPIA, TIMER, and IMMPORT databases are also used for analysis. Cell function experiments were performed to validate the role of the IDO1 gene in melanoma cell line A375. Our study provides potential tumor antigens for vaccine development in melanoma patients: GZMB, GBP4, CD79A, APOBEC3F, IDO1, JCHAIN, LAG3, PLA2G2D, XCL2. In addition, we divide melanoma patients into two immune subtypes that have significant differences in tumor immunity and may have different responses to vaccination. In view of the unclear role of IDO1 in melanoma, we selected IDO1 for cell assay validation. Cell function assay showed that IDO1 was significantly overexpressed in the melanoma A375 cell line. After IDO1 knockdown, the activity, invasion, migration and healing ability of A375 cell lines were significantly decreased. Our study could provide a reference for the development of vaccines for melanoma patients.

A new amide alkaloid induces the apoptosis of human melanoma A375 cellsviainhibition of the STAT3 signaling pathway

A new amide alkaloid (ZYL-01) can inhibit the activity of A375 cells by inducing cell apoptosisviainhibiting STAT3 signaling.

Humic and Fulvic Acids Induced Thermodynamic and Structural Instability of Tyrosinase With Antiproliferative Effect on A375 Melanoma Cancer Cell Line

Background: The tyrosinase enzyme catalysis monophenols to melanin pigments through the melanogenesis process. For this reason, various inhibitors have been studied for enzyme regulations in melanogenesis abnormalities in both the food and cosmetics industries. In this study, the effect of humic acid (Hu) and fulvic acid (Fu) on the structure, activity, and stability of mushroom tyrosinase (MT) was investigated. Methods: These two organic acids are the main components of soil humus. Assessment of the thermodynamic and structural stability of enzymes was obtained through thechemical and thermal denaturations and (8-anilino-naphthalene sulfonic acid) ANS fluorescence analysis. The Hu and Fu impact on A375 melanoma cancer cell viability was achieved by MTT assay. Findings: The results of enzyme half denaturation concentration (Cm), melting points (Tm), ΔG0 values and external fluorescence emissions in the presence of Hu and FA proved the reduction of the thermodynamic and structural stability of MT by these compounds. The anti-proliferation effects of the compounds were confirmed by the inhibitory concentrations of 50% (IC50) of 31.5 and 42.7 µM and 12.5 and µM at time points of 24 and 48 hours treatments of the A375 melanoma cell line by Hu and Fu, respectively. Conclusion: Humic and fulvic acids can be expected to contribute to advancing skin disorder science play a crucial role in tyrosinase related disorders and anti-cancer effects, and good candidates for medical applications.

Extracellular transportation of α-synuclein by HLA class II molecules

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive accumulation of α-synuclein aggregates in form of Lewy bodies. Genome-wide association studies have revealed that human leukocyte antigen (HLA) class II is a PD-associated gene, although the mechanisms linking HLA class II and PD remain elusive. Here, we identified a novel function of HLA class II in the transport of intracellular α-synuclein to the outside of cells. HLA class II molecules and α-synuclein formed complexes and moved to the cell surface at various degrees among HLA-DR alleles. HLA-DR with a DRB5∗01:01 allele, a putative PD-risk allele, substantially translocated normal and conformationally abnormal α-synuclein to the cell surface and extracellular vesicles. α-Synuclein/HLA class II complexes were found in A2058 melanoma cells, which express intrinsic α-synuclein and HLA-DR with DRB5∗01:01. Our findings will expand our knowledge of unconventional HLA class II function from autoimmune diseases to neurodegenerative disorders, shedding light on the association between the GWAS-prioritized PD-risk gene HLA-DR and α-synuclein.

Antiproliferative and Proapoptotic Effects of Erucin, a Diet-Derived H2S Donor, on Human Melanoma Cells.

Melanoma is the most dangerous form of skin cancer and is characterized by chemotherapy resistance and recurrence despite the new promising therapeutic approaches. In the last years, erucin (ERU), the major isothiocyanate present in Eruca sativa, commonly known as rocket salads, has demonstrated great efficacy as an anticancer agent in different in vitro and in vivo models. More recently, the chemopreventive effects of ERU have been associated with its property of being a H2S donor in human pancreatic adenocarcinoma. Here, we investigated the effects of ERU in modulating proliferation and inducing human melanoma cell death by using multiple in vitro approaches. ERU significantly reduced the proliferation of different human melanoma cell lines. A flow cytometry analysis with annexin V/PI demonstrated that ERU was able to induce apoptosis and cell cycle arrest in A375 melanoma cells. The proapoptotic effect of ERU was associated with the modulation of the epithelial-to-mesenchymal transition (EMT)-related cadherins and transcription factors. Moreover, ERU thwarted the migration, invasiveness and clonogenic abilities of A375 melanoma cells. These effects were associated with melanogenesis impairment and mitochondrial fitness modulation. Therefore, we demonstrated that ERU plays an important role in inhibiting the progression of melanoma and could represent a novel add-on therapy for the treatment of human melanoma.

Multidirectional Characterization of Phytochemical Profile and Health-Promoting Effects of Ziziphora bungeana Juz. Extracts.

Ziziphora species (Lamiaceae) have been used in traditional medicine as sedatives, antiseptics, carminatives, or expectorants. Despite their common applications in phytotherapy, there is still lack of evidence about the composition of their extracts and its impact on biological properties of the plants. The aim of this study was to evaluate the content of Ziziphora bungeana, a less studied species growing in Kazakhstan, using HPLC-ESI-QTOF-MS/MS instrumentation and to determine its antimicrobial, antioxidant, and cytotoxic activity together with inhibitory properties against tyrosinase and toxicity in erythrocyte lysis assay. Extracts from Z. bungeana were found to be sources of flavonoids, phenolic acids, organic acids, and terpenes that determined their antiradical activity. The minimum inhibitory concentrations of extracts were lower for Gram-positive bacteria (1.25-10 mg/mL) than for Gram-negative bacteria and fungi (5-20 mg/mL). The EC50 value calculated for antiradical activity ranged between 15.00 ± 1.06 µg/mL and 13.21 ± 3.24 µg/mL for ABTS and DPPH assays, respectively. Z. bungeana extracts were found to decrease the activity of tyrosinase by 50% (at 200 µg/mL) similarly to kojic acid and were slightly cytotoxic for human melanoma A375 cell line (at 200 µg/mL) with no effect on HaCaT keratinocytes. In the end, Z. bungeana did not reveal toxic effects in hemolytic assay as compared to the positive control Triton X-100. The performed tests show potential application of the plant in the treatment of infectious diseases, disorders caused by free radicals, and skin problems.

Increased MARCKS Activity in BRAF Inhibitor-Resistant Melanoma Cells Is Essential for Their Enhanced Metastatic Behavior Independent of Elevated WNT5A and IL-6 Signaling.

Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells.

3-(1,2,3-Triazol-4-yl)-β-Carbolines and 3-(1H-Tetrazol-5-yl)-β-Carbolines: Synthesis and Evaluation as Anticancer Agents.

Herein, the synthesis and anticancer activity evaluation of a series of novel β-carbolines is reported. The reactivity of nitrosoalkenes towards indole was explored for the synthesis of novel tryptophan analogs where the carboxylic acid was replaced by a triazole moiety. This tryptamine was used in the synthesis of 3-(1,2,3-triazol-4-yl)-β-carbolines via Pictet-Spengler condensation followed by an oxidative step. A library of compounds, including the novel 3-(1,2,3-triazol-4-yl)-β-carbolines as well as methyl β-carboline-3-carboxylate and 3-tetrazolyl-β-carboline derivatives, was evaluated for their antiproliferative activity against colorectal cancer cell lines. The 3-(1H-tetrazol-5-yl)-β-carbolines stood out as the most active compounds, with values of half-maximal inhibitory concentration (IC50) ranging from 3.3 µM to 9.6 µM against colorectal adenocarcinoma HCT116 and HT29 cell lines. The results also revealed a mechanism of action independent of the p53 pathway. Further studies with the 3-tetrazolyl-β-carboline derivative, which showed high selectivity for cancer cells, revealed IC50 values below 8 μM against pancreatic adenocarcinoma PANC-1, melanoma A375, hepatocarcinoma HEPG2, and breast adenocarcinoma MCF-7 cell lines. Collectively, this work discloses the 3-tetrazolyl-β-carboline derivative as a promising anticancer agent worthy of being further explored in future works.

In Vitro Photodynamic Treatment Modality for A375 Melanoma Cell Line Using a Sulphonated Aluminum Phthalocyanine Chloride-Photosensitizer-Gold Nanoparticle Conjugate.

Metastatic melanoma cancer stem cells are subpopulations that have been identified and linked to tumor progression, immunoevasive behavior, drug resistance, and metastasis, leading to a poor prognosis. Photodynamic therapy (PDT) is an approach to eradicate cancer through a photochemical process which directly generates reactive oxygen species (ROS). This study investigated the impact of PDT using an aluminum phthalocyanine gold nanoparticle (AlPcS4Cl-AuNP) conjugate for targeting melanoma stem cells. The isolated stem cells were irradiated at 673.2 nm with a radiant exposure of 5 J/cm2. Post-irradiation signs of cell death were determined using microscopy and biochemical assays. A possible enhanced effect of ROS in inducing cell death could be seen when AlPcS4Cl was conjugated to AuNPs. Nanoparticles as carriers promote the efficient cellular uptake of photosensitizers, enhancing organelle accumulation and the targeted therapy of cancerous cells. A biochemical assay revealed significant post-irradiation signs of cell death. The measurement of adenosine triphosphate (ATP) content revealed a decrease in cell proliferation. The study suggested an approach directed at expanding the knowledge on PDT to improve cancer treatment. Understanding the cell death mechanism through which ROS influence cancer stem cells (CSCs) is, therefore, useful for improving PDT efficiency and preventing tumor recurrence and metastasis.

Nanofibrillar peptide hydrogels for self-delivery of lonidamine and synergistic photodynamic therapy

The use of lonidamine (LND) in photodynamic therapy (PDT) provides a viable approach to develop low-dose PDT modules with high efficacy, for LND potentiates cytotoxicity of photosensitizers through dysregulation of mitochondrial function. Yet, the efficacy of LND is restricted by its low accumulation in cancer cells, especially in the mitochondrial compartments. To address the problem, we design an LND-derived self-assembling peptide molecule (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. The targeted cellular delivery of LND-K gives higher efficacy in ablation of mitochondrial function in melanoma cells A375, as compared to free LND or the control molecule that lacks mitochondria-targeting moieties. To integrate LND-K in a typical PDT module, we develop a nanofibrillar hydrogel system through co-assembly of LND-K and TPPS4, an anionic photosensitizer that forms tight electrostatic interactions with cationic residues of LND-K. Notably, hydrogel formulation of LND-K/TPPS4 facilitates slow release of TPPS4 over 14 days in vitro, and displays a longer retention time than aqueous solution of TPPS4in vivo. By integrating a mitochondria-targeted molecule (LND-K) in a typical PDT module, we achieve synergistic killing of A375 cells with dual drugs, where LND-K not only serves as a chemotherapeutic drug, but also potentiates the cytotoxicities of TPPS4 toward A375 cells in vitro and in vivo. The peptide-based drug self-delivery system promises the development of efficacious combination treatments against cancer, that integrate cell sensitization with existing anticancer modules (e.g., chemotherapy and PDT) for enhanced therapeutic efficacy. Statement of significanceThis study reports the design and synthesis of a lonidamine (LND)-derived self-assembling peptide (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. Under the precision guidance of a mitochondria-targeting sequence, LND-K-containing nanofibers target mitochondria and ablate mitochondrial functions. On one hand, the targeted delivery of LND-K reduces cell viabilities through a chemotherapy route; on the other hand, LND-K sensitizes cancer cells for subsequent PDT treatment with enhanced efficacy, which is mediated by induction of ROS, loss of mitochondrial membrane potential, and decrease of cellular ATP level. We believe that the design of mitochondria-targeted drug delivery systems with a self-assembling molecule provides a new approach to potentiate cytotoxicity of photosensitizers in a low-dose PDT module.

CRISPR/Cas9 Edited RAS &amp; MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy

BRAF V600E mutation drives uncontrolled cell growth in most melanomas. While BRAF V600E tumors are initially responsive to BRAF inhibitors, prolonged treatment results in inhibitor resistance and tumor regrowth. Clinical data have linked the NRAS Q61K, KRAS G13D and MEK1 Q56P mutations to the BRAF inhibitor resistance. However, development of novel therapeutics is hindered by the lack of relevant isogeneic cell models. We employed CRISPR/Cas9 genome engineering to introduce NRAS Q61K, KRAS G13D and MEK1 Q56P mutations into the A375 melanoma cell line with endogenously high expression of BRAF V600E. The resulting isogenic cell lines are resistant to BRAF inhibitors. The A375 MEK1 Q56P isogenic cells are additionally resistant to MEK inhibitors as single agent, but interestingly, these cells become sensitive to MEK/BRAF inhibitor combo. Our results suggest that resistance in the NRAS and MEK isogenic lines is driven by constitutive MEK/ERK signaling, while the resistance in the KRAS isogenic line is driven by EGFR overexpression. Interestingly, the KRAS G13D isogenic line displays elevated PD-L1 expression suggesting the KRAS G13D mutation could be a potential indication for immunotherapy. Overall, these three novel isogenic cell models with endogenous level RAS and MEK1 point mutations provide direct bio-functional evidence demonstrating that acquiring a drug-resistant gene drives tumor cell survival and may simultaneously introduce new indications for combo therapy or immunotherapy in the clinic.