Melanoma Cell Lines Research Articles

Abstract B009: The RNA binding activity of the BRAF Kinase

Abstract Multiprotein complexes are key molecular switches of oncogenic signal transduction pathways that relay the flux of information coming from transmembrane receptors and distribute signals to a myriad of effectors. As such the regulation of protein-protein interactions is crucial in signal transduction pathways. For instance, activation of the mitogen-activated protein kinases pathway (MAPK) occurs through a cascade of phosphorylation and protein-protein interactions (PPIs) and is often deregulated in cancer. RNA-protein interactions play key roles in biological processes. More than a thousand RNA binding proteins (RBPs) influence the fate of mRNAs and non-coding RNAs in different ways. It is however less described how RNAs can in turn influence the functions of proteins to which they bind. Here we propose that RNAs scaffold PPIs in the MAPK pathway, thereby fine-tuning oncogenic signaling. We focused our study on cutaneous melanoma for which the MAPK is activated in more than 70% of the cases due to activating mutations in BRAF and NRAS. Using UV-CrossLinking ImmunoPrecipitation (CLIP) experiments, we found that several proteins of the MAPK pathway, including BRAF (but not MEK and ERK) interact with RNAs in RAS-activated melanoma cells. In addition, using a PLA- based imaging technique that allows the visualisation of the proximity of two given proteins, we showed that RNAs promote the stabilisation of BRAF-PPIs, suggesting a scaffolding role of RNAs in the MAPK pathway. Loss of BRAF RNA binding activity (achieved with a single point mutation) is associated with decreased BRAF dimerization and signaling. The oncogenic mutant protein BRAF (V600E) also interacts with RNA in a variety of melanoma cell lines including a BRAF inhibitor-resistant melanoma cell line in which the dimerization of BRAF and CRAF is partly dependent on RNA. Identifying BRAF-bound RNAs will open new therapeutic strategies targeting RNA-protein interactions. We also envision that the level of BRAF-RNA interactions could serve as a predictive marker of response to BRAF (V600E) inhibitors. Citation Format: Alexia Le Barch, Sabrina Mennour, Patricia Uguen, Stephan Vagner. The RNA binding activity of the BRAF Kinase [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B009.

Abstract B027: An Innovative cell-penetrating peptide-based RNAi Therapy to Overcome B-Raf/MEK Inhibitor Resistance in Melanoma

Abstract Small-molecule-targeted therapies have revolutionized cancer treatment, including for B-RafV600E-mutated melanoma, where B-Raf and combined B-Raf/MEK inhibitors (BRAFi/MEKi) prolong progression-free survival and overall survival. However, resistance to these therapies quickly develops, driven by mechanisms such as the expression of alternative spliced isoforms of B-RafV600E and mutated MEK1 variants. The need for novel strategies to overcome such resistance is critical. In this study, we developed and evaluated a novel RNAi-based therapeutic using our designed cell-penetrating peptide HE25 to specifically target and silence resistance-associated spliced isoforms of B-RafV600E, restoring the efficacy of existing treatments. In a recently published work (Tuttolomondo et al., 2024), we demonstrated that HE25 effectively reduced SARS-CoV-2 replication when complexed with siRNA targeting the virus's RNA-dependent RNA polymerase. HE25 facilitated siRNA internalization via multiple receptors involved in viral entry, including ACE2, integrins, and NRP1, through endocytosis, highlighting its versatility as a delivery platform with potential applications beyond cancer research. Initially, we established a vemurafenib-resistant A375 melanoma cell line (A375-VR), which revealed an alternatively spliced B-RafV600E isoform driving resistance to vemurafenib. Through cDNA Sanger sequencing, we confirmed that this alternatively spliced isoform lacks the regulatory domain, leading to constitutive B-Raf activation and rendering vemurafenib ineffective. Given these findings, we hypothesized that specifically silencing this isoform with our siRNA-HE25 complex could synergize with B-Raf inhibition, thereby overcoming vemurafenib resistance. The B-RafV600E-targeting siRNA encapsulated within HE25 significantly inhibited the proliferation of both A375 and A375-VR cells in vitro, with no toxic effects on non-cancerous melanocytes. In vivo, B-RafV600E siRNA-HE25 treatment significantly reduced tumor growth in A375 melanoma xenografts compared to a non-targeting siRNA control. We also developed A375-VTR cells resistant to combined vemurafenib and MEK inhibitor trametinib. This cell line also expressed the shortened B-RafV600E isoform and its silencing by the B-RafV600E siRNA/HE25 system was confirmed by Western blotting. Indeed, in A375-VTR cells, the combination of vemurafenib and B-RafV600E siRNA/HE25 was more effective than the combination of vemurafenib and trametinib. Our strategy holds promise for personalized cancer treatment by targeting resistance-related mutations or alternatively spliced isoforms. Future work will optimize this approach for broader clinical use and explore its efficacy in combination with existing therapies. These findings suggest that siRNA-based therapeutics via HE25 offer a promising solution for overcoming resistance in B-RafV600E-mutated melanoma and resistance mechanisms in other cancer types. This strategy should enable tailored treatments with broad applicability across cancer types. Citation Format: Martina Tuttolomondo, Mikkel Green Terp, Chiara Brienza, Morten Frier Gjerstorff, Henrik Jørn Ditzel. An Innovative cell-penetrating peptide-based RNAi Therapy to Overcome B-Raf/MEK Inhibitor Resistance in Melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B027.

Abstract B063: DiMMER: A robust computational pipeline for differential methylation marker evaluation in R of cell-free DNA fragments

Abstract Background: Decoding the origins of cell-free DNA (cfDNA) released from dying cells in a liquid biopsy sample offers the potential to provide insight into the dynamic, organism-wide changes reflective of health and disease, making cfDNA an ideal target for serial, minimally invasive monitoring of disease-related changes. To this end, cell-type specific DNA methylation patterns offer a promising target to facilitate tissue of origin analysis, yet limited methods exist to identify differentially methylated regions that distinguish cell-types. Methods: We develop a robust differentially methylated marker region identification pipeline, DiMMER, that leverages cfDNA fragment level information of neighboring, co-regulated CpG sites on methylome-wide sequencing reads. Compared to prior methods that utilize simple heuristics in one-vs-all average methylation rate comparisons, we implement an individual pairwise statistical test procedure across all cell-types or groups under consideration. We assess the cell-specific nature of identified differentially methylated marker regions by generating in-silico mixtures from known cell-type of origin at each region, and calculating the area under the receiver operating characteristic curve (AUROC). We utilize our differentially methylated marker finding pipeline to identify melanocyte specific methylation marker regions and assess their functional role through annotations. We show these cell-specific regions are conserved in melanoma cell-lines and are distinct from the aberrant changes in a cancer context. Results: We identified the most cell-type specific differentially methylated marker regions across 24 distinct cell-type groups using our pipeline. Compared to a simpler one-vs-all heuristic, we demonstrate improved cell-type specificity evidenced by a higher AUROC on average. Using our pipeline we identified melanocyte-specific marker regions, which were commonly found in intronic and promoter regions of genes related to melanocyte development and differentiation. When comparing methylation patterns between melanocytes and melanoma cell-lines, we demonstrate melanocyte specific methylation marks are conserved through the transformation to malignant melanoma. Conclusion: As methylome-wide sequencing methods continue to rapidly develop and more cell-specific methylation data is generated, there is unmet need for more statistically robust differentially methylated marker finding tools. Here we present one such tool, DiMMER, and demonstrate the potential utility of identifying such regions to be used to assess organ-specific load as a measure of residual disease, as opposed to traditional circulating tumor DNA quanitification. Citation Format: Arthur P McDeed, Sidharth S Jain, Megan E McNamara, Amber Alley, Anton Wellstein, Jaeil Ahn. DiMMER: A robust computational pipeline for differential methylation marker evaluation in R of cell-free DNA fragments [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B063.

Melanoma Cells from Different Patients Differ in Their Sensitivity to Alpha Radiation-Mediated Killing, Sensitivity Which Correlates with Cell Nuclei Area and Double Strand Breaks

Background/Objective: In this study, for the first time, we examined and compared the sensitivity of four patient-derived cutaneous melanoma cell lines to alpha radiation in vitro and analyzed it in view of cell nucleus area and the formation of double-strand breaks (DSB). Melanoma cells sensitivity to alpha radiation was compared to photon radiation effects. Furthermore, we compared the sensitivity of the melanoma cells to squamous cell carcinoma. Methods: Human melanoma cell lines YDFR.C, DP.C, M12.C, and M16.C, and the squamous cell carcinoma cell line, CAL 27, were irradiated in vitro using Americium-241 as alpha-particle source. Cells were irradiated with doses of 0 to 2.8 gray (Gy). Cell viability, DNA DSB, and nuclear size were measured. Results: 1. Alpha radiation caused death and proliferation arrest of all four melanoma cell lines, but inter-tumor heterogeneity was observed. 2. The most sensitive cell line (DP.C) had a significantly larger nucleus area (408 µm2) and the highest mean number of DSB per cell (9.61) compared to more resistant cells. 3. The most resistant cell, M16.C, had a much lower nucleus area (236.99 µm2) and DSB per cell (6.9). 4. Alpha radiation was more lethal than photon radiation for all melanoma cells. 5. The SCC cell, CAL 27, was more sensitive to alpha radiation than all melanoma cells but had a similar number of DSB (6.67) and nucleus size (175.49 µm2) as the more resistant cells. 6. The cytotoxic effect of alpha radiation was not affected by proliferation arrest after serum starvation. 7. Killing of cells by alpha radiation was marginally elevated by ATR or topoisomerase 1 inhibition. Conclusions: This study demonstrates that various human melanoma cells can be killed by alpha radiation but exhibit variance in sensitivity to alpha radiation. Alpha radiation applied using the Intra-tumoral Diffusing alpha-emitters Radiation Therapy (Alpha DaRT) methodology may serve as an efficient treatment for human melanoma.

DDDR-04. PRECLINICAL INVESTIGATION OF A NOVEL BRAIN PENETRANT COMBINATION THERAPY TARGETING RAF AND MEK FOR MELANOMA BRAIN METASTASIS

Abstract INTRODUCTION Up to 75% of patients with advanced melanoma develop brain metastases. BRAF mutations are found in 50-55% of melanoma brain metastases. Current BRAF/MEK inhibitors, such as dabrafenib and vemurafenib, have shown responses on the order of 50-60% in patients with BRAF-mutant melanoma; however, responses are not durable. Better therapies are needed with improved penetration of the blood brain barrier. This study aims to evaluate the efficacy of two novel brain-penetrant inhibitors, KIN-7136 (MEK inhibitor) and KIN-8391 (RAF inhibitor), in preclinical mouse models of melanoma brain metastases driven by aberrant BRAF-MEK signaling. METHODS CellTiter-Glo® 2.0 Cell Viability Assay (Promega) was used for in vitro cell viability assays. Intracranial A375 melanoma models were generated in athymic nude mice to assess the pharmacodynamics, safety, and efficacy of combination therapy of KIN-7136 and KIN-8391. RESULTS In mice, the brain-to-plasma unbound partition coefficients (Kpuu) of KIN-7136 and KIN-8391 were 0.67 and 0.7 respectively, higher than the comparators binimetinib and belvarafenib, demonstrating improved brain exposure. The IC50s of KIN-7136/ KIN-8391 in melanoma cell lines were 27.0/36.5 nM in A375 (BRAF V600E), 78.6/35.4 nM in HMV2 (BRAF G469V), 183.0/699.0 nM in SK-MEL-2 (NRAS Q61R), and 53.9/110.0 nM in SK-MEL30 (BRAF D287H and E275K), respectively. KIN-7136 and KIN-8391 synergistically inhibited HMV2, SK-MEL-2, and SK-MEL-30 cells. Pharmacodynamic studies showed that both KIN-7136 and KIN-8391 suppressed phospho-ERK, a downstream component of BRAF-MEK signaling, in A375 xenografts in mice. Daily oral treatment with a combination of KIN-7136 and KIN-8391 was well-tolerated and extended overall survival to 64 days (median survival), compared to the vehicle control and monotherapies with KIN-7136 or KIN-8391 (28, 58, and 46.5, respectively) in the A375 intracranial tumor model. CONCLUSION These preclinical data confirm the synergistic effect of KIN-7136 and KIN-8391 in BRAF-mutant melanoma brain metastases models, supporting further research to advance its clinical translation.

EXTH-66. ONCOLYTIC ADENOVIRAL INFECTION SYNERGIZES WITH IMMUNE CHECKPOINT INHIBITION TO MEDIATE INTRACRANIAL TUMOR CONTROL IN DISPARATE MODELS OF MELANOMA BRAIN METASTASES

Abstract Amongst all solid cancers, melanoma brain metastases (MBM) have the highest likelihood of metastasizing to the brain. The emergence of immune checkpoint inhibitors (ICI) for metastatic melanoma have demonstrated increasingly promising results; however, intracranial progression remains a challenging obstacle. Oncolytic viral (OV) therapy leverages tumor cell replication machinery to selectively replicate and kill tumor cells and promote anti-tumor immunity. We propose this OV strategy can be applied to MBM through direct tumor infection that simultaneously sustains a distant antitumor immune response. To counteract immunosuppressive features of the tumor microenvironment (TME), a third-generation oncolytic adenovirus (Delta-24-RGDOX) has been developed which expresses the immune stimulatory OX40 ligand (OX40L). We quantified the cytotoxic capacity of Delta-24-RGDOX and its ability to achieve immunogenic cell death (ICD) in vitro along with cerebral organoid co-culture models. Additionally, we used a series of syngeneic murine melanoma models to explore local and systemic antitumor immunity following Delta-24-RGDOX infection. Delta-24-RGDOX inoculation achieved viral infection, OX40L cell surface expression, tumor cell cytotoxicity, and ICD in vitro. Viral oncolysis was recapitulated in a cerebral organoid melanoma co-culture model with concomitant alterations in microglia activation and detection of viral transcripts within microglia using single cell RNA sequencing, suggesting a viral-clearing role of microglia within the TME. Local tumor clearance was achieved in orthotopic intracranial models following direct intracranial viral inoculation alone, and in combination with ICI. Lastly, we demonstrated improvement in overall survival in a synchronous subcutaneous/intracranial B16F10 model where the subcutaneous tumor was treated with Delta-24-RGDOX in combination with ICI. In summary, Delta-24-RGDOX achieves viral infection, oncolysis, and ICD following infection of melanoma cell lines. These findings translated in multiple in vivo models including immune cell activation and systemic antitumor immunity against uninfected MBM. Future studies are needed to explore the role of immune cell trafficking, myeloid cell modulation, T cell priming, and clonal expansion withing locally infected and the distant melanoma TME.

DDDR-07. FUNCTIONAL GENOMICSIN VIVO USING CRISPR/CAS9 IDENTIFIES POTENTIAL METABOLIC VULNERABILITIES WITHIN MELANOMA BRAIN METASTASIS

Abstract BACKGROUND Inhibition of the oxidative phosphorylation (OxPhos) pathway in mice suppressed the growth of melanoma brain metastasis but not extracranial or primary skin melanoma. This indicates that melanoma brain metastasis may have targetable metabolic vulnerabilities. Human clinical trials, however showed toxicities following OxPhos pathway inhibition. This suggests that more selective metabolic targets are needed to minimize off target effects. METHODS We designed a focused CRISPR guide RNA (gRNA) lentiviral knockout library targeting 106 metabolism genes significantly enriched in human melanoma brain metastasis (n=88) relative to extracranial (n=50) or primary skin melanoma (n=52). We infected mouse B16F10 melanoma cell lines with the CRISPR library and implanted 1X106 cells subcutaneously or intracerebrally into male (n=3) and female (n=3) C57/Bl6 mice. DNA from cell lines and tumor samples were sent for next generation sequencing. Candidate metabolism genes that may drive growth of melanoma lesion within the brain were identified based on loss of gRNA copies in brain relative to skin. RESULTS The CRISPR/Cas9 in vivo screen identified metabolic genes whose knockout resulted in loss of gRNA clones within established B16F10 melanoma brain lesions relative to skin lesions. These putative metabolism targets included specific components in the oxidative phosphorylation pathway, choline metabolism, and ubiquitin cascade activation. CONCLUSION Using a focused CRISPR/Cas9 in vivo screen, we uncovered potential metabolic targets within established mouse melanoma brain lesions. vWe are currently performing the in vivo screen in melanoma mouse models with spontaneous brain metastasis. Top 3 hits across all models will be validated individually prior to a compound screen for inhibitors.

IMMU-55. TUMOR-WIDE RNA SPLICING ABERRATIONS GENERATE IMMUNOGENIC PUBLIC NEOANTIGENS IN GLIOBLASTOMAS AND OTHER CANCER TYPES

Abstract BACKGROUND Immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. Aberrant RNA-splicing (neojunctions) offers a new source for targets, and our neoantigen discovery platform (SNIPP) characterizes a novel class of clonally-expressed splicing-derived neoantigens that elicit a CD8+ T-cell-mediated tumor killing response. METHODS SNIPP identified public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across 12 cancer types. To characterize intratumorally-conserved neojunctions, we performed maximally-distanced multi-site biopsies (n=535) within glioma patients (n=56) and obtained RNA-seq data from each intratumoral site. Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. Neoantigen-specific TCR sequences characterized from subsequent PBMC in vitro sensitization and 10x V(D)J scRNA-seq were transduced into CD8+ T-cells for immunogenicity and cytotoxicity assays against glioma cell lines. RESULTS Our pipeline identified 789 public neojunctions, with 32 neojunctions concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in multiple GBM PDX cell lines, and co-culturing these T-cells against both glioblastoma and melanoma cell lines demonstrated tumor-specific cytotoxicity. Furthermore, IDH1-mutant oligodendroglioma samples demonstrated significantly elevated expression of neojunctions over IDH1-mutant astrocytoma and IDH1wt subtypes. Differential gene expression (DESeq2) identified decreased expression of splicing factors due to oligodendroglioma-specific co-deletion of Chromosomes 1p/19q. siRNA knockdown of these splicing factors (e.g. SF3A3, SNRPD2) in IDH1wt glioma cells resulted in significantly increased expression of corresponding neojunctions. CONCLUSION Our unique SNIPP neoantigen discovery platform identified novel public tumor-wide splice-derived neoantigens and reactive TCRs, and its pan-cancer application characterized candidates that are targetable across multiple diseases.

Non-nucleophilic base promoted synthesis of azo-linked oxazolone-pyrazole hybrids: Antimicrobial, antitubercular, anticancer evaluations and in-silico modeling insights

Ten new non-nucleophilic base (DBU) catalyzed oxazolone-bearing pyrazole derivatives were created to treat infectious diseases caused by bacterial, mycobacterial strains, and cancerous cell lines. Compounds 7b and 7c showed excellent antibacterial and antifungal activity. Compound 7b was highly effective against M. tuberculosis H37Rv, with a MIC of 0.06 μg/mL and significant binding affinities to AcrB of E. coli (−12 Kcal/mol), TriABC of P. aeruginosa (−12.5 Kcal/mol), and MepR of S. aureus (−10.6 Kcal/mol). Based on the findings, compounds 7b, 7c, and 7e exhibit proficient binding affinity with two essential targets, namely Enoyl-[acyl-carrier-protein] reductase and Topoisomerase I, of M. tuberculosis. Compound 7b showed superior cytotoxic activity against all cancer types except leukemia with GI50 values of 1.26–1.83 µM and LC50 values of 5.36–7.88 µM. Compound 7a demonstrated remarkable anticancer activity against colon, melanoma, ovarian, renal, and breast cancer cell lines with GI50 values of 1.60–2.55 µM.

Molecular Imaging of Melanoma VEGF-expressing Tumors through [99mTc]Tc-HYNIC-Fab(Bevacizumab).

Angiogenesis is a process that many tumors depend on for growth, development, and metastasis. Vascular endothelial growth factor (VEGF) is one of the major players in tumor angiogenesis in several tumor types, including melanoma. VEGF inhibition is achieved by bevacizumab, a humanized monoclonal antibody that binds with high affinity to VEGF and prevents its function. In order to successfully enable in vivo VEGF expression imaging in a murine melanoma model, we previously labeled bevacizumab with [99mTc]Tc. We observed that this was feasible, but it had prolonged blood circulation and delayed tumor uptake. The aim of this study was to develop a radiolabeled Fab bevacizumab fragment, [99mTc]Tc-HYNICFab( bevacizumab), for non-invasive in vivo VEGF expression molecular imaging. Flow cytometry was used to examine VEGF presence in the murine melanoma cell line (B16-F10). Bevacizumab was digested with papain for six hours at 37°C to produce Fab(bevacizumab), which was then conjugated to NHS-HYNIC-Tfa for radiolabeling with [99mTc]Tc. Stability and binding affinity assays were also evaluated. Biodistribution and single photon emission computed tomography/computed tomography (SPECT/CT) were performed at 1, 3, and 6 h (n = 4) after injection of [99mTc]Tc-HYNIC-Fab(Bevacizumab) in normal and B16-F10 tumor-bearing C57Bl/6J mice. Using flow cytometry, it was shown that the B16-F10 murine melanoma cell line has intracellular VEGF expression. Papain incubation resulted in the complete digestion of bevacizumab with good purity and homogeneity. The radiolabeling yield of [99mTc]Tc-HYNIC-Fab(bevacizumab) was 85.00 ± 6.06%, with a specific activity of 291.87 ± 18.84 MBq/mg (n=3), showing in vitro stability. Binding assays demonstrated significant intracellular in vitro VEGF expression. Fast blood clearance and high kidney and tumor uptake were observed in biodistribution and SPECT/CT studies. We present the development and evaluation of [99mTc]Tc-HYNIC-Fab(bevacizumab), a novel molecular VEGF expression imaging agent that may be used for precision medicine in melanoma and potentially in other VEGF-expressing tumors.

Synthesis of bis-Chalcones Based on Green Chemistry Strategies and Their Cytotoxicity toward Human MeWo and A375 Melanoma Cell Lines

Synthesis of bis-Chalcones Based on Green Chemistry Strategies and Their Cytotoxicity toward Human MeWo and A375 Melanoma Cell Lines

Electrospun nanofibrous scaffolds reinforced with therapeutic lithium/manganese-doped calcium phosphates: Advancing skin cancer therapy through apoptosis induction

In the current study we fabricated potent materials by incorporating therapeutic elements into calcium phosphates (CPs) to combat cancer. This involved synthesizing manganese (Mn)- and lithium (Li)-doped CPs and loading them into electrospun nanofibers (NFs) composed of chitosan (CS) and polyethylene oxide (PEO). The characterized CPs exhibited excellent properties, including a particle size of 47–75 nm, surface charge of –(30−56) mV, and specific surface area of 75–266 m2/g. The electrochemical analysis revealed that Mn and Mn/Li-doped CPs are promising for generating oxygen free radicals and H2O2, crucial for cancer therapy. Biological evaluation showcased the outstanding performance of the developed materials. MTT assay revealed a cytotoxic effect of nano-constructs on melanoma A375 cell line without adverse effects on normal L929 cells over 72 h. Annexin V/PI apoptosis assay indicated substantial apoptosis rates in A375 cells treated with PC-20 % (62.55 ± 4.59 %). The obtained data of qPCR analysis of pro-apoptotic and anti-apoptotic genes (P53, Bax, Bcl-2) in A375 cells treated with different CP nanoparticles (NPs) showed a significant increase in P53 and Bax gene expression, indicating high levels of A375 cell apoptosis. Additionally, the samples containing Mn ion exhibited high reactive oxygen species (ROS) generation. In conclusion, the fabricated NFs scaffolds hold promising potential for cancer therapy.

Antiproliferative and biochemical evaluation of rose extracts: impact on tumor and normal skin cells

Rose petals (Rosa L.) are rich sources of phenolic compounds, including anthocyanins. Anthocyanins and anthocyanidins are associated with multiple health benefits due to their antioxidant properties. In this study, eighteen rose cultivars were comparatively analyzed to determine their total polyphenol and flavonoid content, as well as their antioxidant activity using the 2,2-diphenylpicrylhydrazyl (DPPH) radical scavenging method. The extracts were purified using Amberlite XAD-7 and Sephadex LH-20 columns to obtain anthocyanin-rich fractions. Individual anthocyanins were separated and identified using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). The three cultivars with the highest anthocyanin content were further examined for cytotoxic effects on cell cultures at various extract concentrations (200-1000 µg/mL) using two skin cell lines: a melanoma cell line (A375) and a normal skin cell line (Hs27). The HPLC-MS analysis identified nine different anthocyanin compounds, with the total anthocyanin content in the rose cultivars varying from 12.42 to 331.95 mg of cyanidin-3-glucoside equivalent/100g of fresh weight. The total polyphenol and total flavonoid contents ranged from 289 to 2703 mg gallic acid equivalent/100g fresh weight and 102 to 603 mg catechin equivalent/100g fresh weight, respectively. Antioxidant activity ranged from 450 to 1304 µmol trolox equivalent/g fresh weight. A significant correlation was observed between antioxidant activity and the content of anthocyanins (R = 0.875, p < 0.001), flavonoids (R = 0.982, p < 0.001), and polyphenols (R = 0.991, p < 0.001). Furthermore, principal component analysis, along with dendrograms and heatmaps, illustrated the relationships among these key compounds and their association with antioxidant activity. The MTT assay showed a substantial suppression of A375 cancer skin cells, while simultaneously exhibiting cell proliferation in Hs27 normal skin cells in a concentration-dependent manner. Altogether, results suggest that the anthocyanins from these rose cultivars could be considered as a promising agent for adjuvant treatment of skin melanoma.

Effect of Amygdalin on Murine Melanoma Cancer Cell Line and Normal Human Fibroblast Cells

Background and Objectives: Melanoma is the most aggressive and fatal type of skin cancer caused by the uncontrolled proliferation of melanocytes. The ineffectiveness of existing therapies has encouraged researchers to use herbal medicines. Amygdalin is a natural cyanogenic glycoside that is found in fruits and seeds of different plants and has antiproliferative, antioxidant, and immune-regulating activities. In this study, we aim to evaluate the effect of amygdalin against murine melanoma cancer cell line and normal human fibroblast cells. Methods: The murine melanoma cancer cell line (B16F10) and human foreskin fibroblasts (HFF) cells were exposed to different concentrations of amygdalin and dacarbazine (a standard anticancer drug) for 24 and 48 hours. The half-maximal inhibitory concentration (IC50) of amygdalin was obtained using the MTT assay and analyzed in GraphPad Prism and Excel applications. Results: Amygdalin had a high cytotoxic effect on the melanoma cell line within 48 hours of exposure, which is comparable to the cytotoxicity of dacarbazine at the same time. Amygdalin had no cytotoxic effect on HFF cells, while dacarbazine caused a high toxicity on HFF cells. Conclusion: Amygdalin, by inhibiting the proliferation of cancer cells in a dose-dependent manner, may prevent the adverse effects of chemical drugs on healthy cells and can be an appropriate treatment option for melanoma.

Hypoxia-Active Iridium(III) Bis-terpyridine Complexes Bearing Oligothienyl Substituents: Synthesis, Photophysics, and Phototoxicity toward Cancer Cells.

In an effort to develop hypoxia-active iridium(III) complexes with long visible-light absorption, we synthesized and characterized five bis(terpyridine) Ir(III) complexes bearing oligothienyl substituents on one of the terpyridine ligands, i.e., nT-Ir (n = 0-4). The UV-vis absorption, emission, and transient absorption spectroscopy were employed to characterize the singlet and triplet excited states of these complexes and to explore the effects of varied number of thienyl units on the photophysical parameters of the complexes. In vitro photodynamic therapeutic activities of these complexes were assessed with respect to three melanoma cell lines (SKMEL28, A375, and B16F10) and two breast cancer cell lines (MDA-MB-231 and MCF-7) under normoxia (∼18.5% oxygen tension) and hypoxia (1% oxygen tension) upon broadband visible (400-700 nm), blue (453 nm), green (523 nm), and red (633 nm) light activation. It was revealed that the increased number of thienyl units bathochromically shifted the low-energy absorption bands to the green/orange spectral regions and the emission bands to the near-infrared (NIR) regions. The lowest triplet excited-state lifetimes and the singlet oxygen generation efficiency also increased from 0T to 2T substitution but decreased in 3T and 4T substitution. All complexes exhibited low dark cytotoxicity toward all cell lines, but 2T-Ir-4T-Ir manifested high photocytotoxicity for all cell lines upon visible, blue, and green light activation under normoxia, with 2T-Ir showing the strongest photocytotoxicity toward SKMEL28, MDA-MB-231, and MCF-7 cells, and 4T-Ir being the most photocytotoxic one for B16F10 and A375 cells. Singlet oxygen, superoxide anion radicals, and peroxynitrite anions were found to likely be involved in the photocytotoxicity exhibited by the complexes. 4T-Ir also showed strong photocytotoxicity upon red-light excitation toward all cell lines under normoxia and retained its photocytotoxicity under hypoxia toward all cell lines upon visible, blue, and green light excitation. The hypoxic activity of 4T-Ir along with its green to orange light absorption, NIR emission, and low dark cytotoxicity suggest its potential as a photosensitizer for photodynamic therapy applications.

Tubulin-Targeted Therapy in Melanoma Increases the Cell Migration Potential by Activation of the Actomyosin Cytoskeleton─An In Vitro Study.

One of the most dangerous aspects of cancers is their ability to metastasize, which is the leading cause of death. Hence, it holds significance to develop therapies targeting the eradication of cancer cells in parallel, inhibiting metastases in cells surviving the applied therapy. Here, we focused on two melanoma cell lines─WM35 and WM266-4─representing the less and more invasive melanomas. We investigated the mechanisms of cellular processes regulating the activation of actomyosin as an effect of colchicine treatment. Additionally, we investigated the biophysical aspects of supplement therapy using Rho-associated protein kinase (ROCK) inhibitor (Y-27632) and myosin II inhibitor ((-)-blebbistatin), focusing on the microtubules and actin filaments. We analyzed their effect on the proliferation, migration, and invasiveness of melanoma cells, supported by studies on cytoskeletal architecture using confocal fluorescence microscopy and nanomechanics using atomic force microscopy (AFM) and microconstriction channels. Our results showed that colchicine inhibits the migration of most melanoma cells, while for a small cell population, it paradoxically increases their migration and invasiveness. These changes are also accompanied by the formation of stress fibers, compensating for the loss of microtubules. Simultaneous administration of selected agents led to the inhibition of this compensatory effect. Collectively, our results highlighted that colchicine led to actomyosin activation and increased the level of cancer cell invasiveness. We emphasized that a cellular pathway of Rho-ROCK-dependent actomyosin contraction is responsible for the increased invasive potential of melanoma cells in tubulin-targeted therapy.

DNA-PK inhibition enhances neoantigen diversity and increases T cell responses to immunoresistant tumors.

Effective antitumor T cell activity relies on the expression and MHC presentation of tumor neoantigens. Tumor cells can evade T cell detection by silencing the transcription of antigens or by altering MHC machinery resulting in inadequate neoantigen-specific T cell activation. We identified DNA-PK inhibitor (DNA-PKi) NU7441 as a promising immunomodulator that reduced immunosuppressive proteins while increasing MHC-I expression in a panel of human melanoma cell lines. In tumor-bearing mice, combination therapy using NU7441 and immune adjuvants STING ligand and CD40 agonist (NU-SL40) substantially increased and diversified the neoantigen landscape, antigen presenting machinery, and consequently substantially increased both the number and repertoire of neoantigen-reactive tumor infiltrating lymphocytes (TILs). DNA-PK-inhibition or knockout promoted transcription and protein expression of various neoantigens in human and mouse melanomas and induced sensitivity to ICB in resistant tumors. In patients, PRKDC levels inversely correlated with MHC I expression and CD8 TILs but positively correlated with increased neoantigen loads and improved responses to ICB. These studies suggest that inhibiting DNA-PK activity can restore tumor immunogenicity by increasing neoantigen expression and presentation and broadening the neoantigen-reactive T cell population.

A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens.

Cancer cells effectively evade immune surveillance, not only through the well-known PD-1/PD-L1 pathway but also via alternative mechanisms that impair patient response to immune checkpoint inhibitors. We present a novel co-culture model that pairs a reporter T-cell line with different melanoma cell lines that have varying immune evasion characteristics. We developed a scalable high-throughput lentiviral arrayed CRISPR interference (CRISPRi) screening protocol to conduct gene perturbations in both T-cells and melanoma cells, enabling the identification of genes that modulate tumor immune evasion. Our study functionally validates the co-culture model system and demonstrates the performance of the CRISPRi-screening protocol by modulating the expression of known regulators of tumor immunity. Together, our work provides a robust framework for future research aimed at systematically exploring mechanisms of tumor immune evasion.

Azacytidine treatment affects the methylation pattern of genomic and cell-free DNA in uveal melanoma cell lines

BackgroundUveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines.MethodsFour primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared.ResultsIn all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA.ConclusionsThis data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool.

Different Doxorubicin Sensitivity Across Various Human Cancer Cell Lines

Doxorubicin (Dox) is a highly potent chemotherapeutic agent, approved by FDA since 1974, for treating a broad spectrum of cancers. However, development of severe side effects and drug resistance are main issues that limit the clinical use of Dox. Herein, we aimed to investigate the sensitivity of Dox in a variety of human cancer cell lines. Eleven cell lines were tested in this study including 2 hepatocellular carcinoma (HCC) cell lines (HepG2 and Huh7), 4 bladder cancer (BlCa) cell lines (UMUC-3, VMCUB-1, TCCSUP and BFTC-905), a lung cancer cell line (A549), a cervical carcinoma cell line (HeLa), a breast cancer cell line (MCF-7), a skin melanoma cell line (M21) and a noncancer human kidney cell line (HK-2). The MTT assay was employed for the determination of cytotoxicity. Cells were treated with various concentrations of Dox for 24 h. The half-maximal inhibitory concentration (IC50) of Dox was calculated to compare the Dox sensitivity among tested cell lines. The result showed that IC50 of Dox in HepG2, Huh7, UMUC-3, VMCUB-1, TCCSUP, BFTC-905, A549, HeLa, MCF-7, M21 and HK-2 cells were 12.2, > 20, 5.1, > 20, 12.6, 2.3, > 20, 2.9, 2.5, 2.8 and > 20 mM, respectively. BFTC-905 had the lowest IC50 value, therefore, it was the most sensitive to Dox. In contrast, Huh7, VMCUB-1 and A549 cells were resistant to Dox with IC50 values > 20 mM. Conclusions, we demonstrated that different human malignant cell lines had different sensitivity to Dox. BFTC-905 BlCa cell line had the highest sensitivity to Dox. Huh7, VMCUB-1 and A549 cell lines were resistant to Dox. Perhaps, the different Dox sensitivity in different cell lines was due to the different acquisition of Dox resistance mechanisms. Huh7, VMCUB-1 and A549 cell lines could be suitable cell models to investigate the molecular mechanism of Dox resistance in different cancers. HIGHLIGHTS Different types of cancer cell lines exhibited different sensitivity to doxorubicin. BFTC-905, MCF-7 and M21 cell lines were sensitive to doxorubicin. HepG2, UMUC-3, TCCSUP and HeLa cells were moderately sensitive to doxorubicin. Huh7, VMCUB-1, A549 and HK-2 cells were resistant to doxorubicin. Huh7, VMCUB-1 and A549 cell lines could be suitable cell models for investigating the molecular pathways of Dox resistance in different types of cancers. GRAPHICAL ABSTRACT