scholarly journals A375 Melanoma Cells are Sensitized to Cisplatin-Induced Toxicity by a Synthetic Nitro-Flavone Derivative 2-(4-Nitrophenyl)-4H-Chromen-4-one Through Inhibition of PARP1

2021 ◽  
Author(s):  
Anindita Mitra ◽  
Rita Ghosh
Keyword(s):  
Combination Therapy ◽  
In Silico ◽  
Nuclear Dna ◽  
Binding Mode ◽  
Melanoma Cells ◽  
Parp1 Inhibitors ◽  
Flavone Derivative ◽  
A375 Melanoma ◽  
Isobologram Analysis ◽  
A375 Cells

Abstract Background: Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for the treatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses. Methods and Results: In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD+) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis. Conclusion: The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

scholarly journals Acid ceramidase controls apoptosis and increases autophagy in human melanoma cells treated with doxorubicin

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michele Lai ◽  
Rachele Amato ◽  
Veronica La Rocca ◽  
Mesut Bilgin ◽  
Giulia Freer ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Melanoma Cells ◽  
Chemotherapeutic Agents ◽  
Human Melanoma ◽  
Acid Ceramidase ◽  
Doxorubicin Treatment ◽  
The Impact ◽  
A375 Melanoma ◽  
Striking Contrast ◽  
A375 Cells

AbstractAcid ceramidase (AC) is a lysosomal hydrolase encoded by the ASAH1 gene, which cleaves ceramides into sphingosine and fatty acid. AC is expressed at high levels in most human melanoma cell lines and may confer resistance against chemotherapeutic agents. One such agent, doxorubicin, was shown to increase ceramide levels in melanoma cells. Ceramides contribute to the regulation of autophagy and apoptosis. Here we investigated the impact of AC ablation via CRISPR-Cas9 gene editing on the response of A375 melanoma cells to doxorubicin. We found that doxorubicin activates the autophagic response in wild-type A375 cells, which effectively resist apoptotic cell death. In striking contrast, doxorubicin fails to stimulate autophagy in A375 AC-null cells, which rapidly undergo apoptosis when exposed to the drug. The present work highlights changes that affect melanoma cells during incubation with doxorubicin, in A375 melanoma cells lacking AC. We found that the remarkable reduction in recovery rate after doxorubicin treatment is strictly associated with the impairment of autophagy, that forces the AC-inhibited cells into apoptotic path.

Effects of Glutathione Transferase-Targeting Nitrobenzoxadiazole Compounds in Relation to PD-L1 Status in Human Melanoma Cells

Chemotherapy ◽  
2019 ◽  
Vol 64 (3) ◽  
pp. 138-145
Author(s):  
Francesca Sciarretta ◽  
Chiara Fulci ◽  
Camilla Palumbo ◽  
Dante Rotili ◽  
Lucio Tentori ◽  
...  
Keyword(s):  
Western Blot ◽  
Cytotoxic Activity ◽  
Glutathione Transferase ◽  
Braf Inhibitor ◽  
Melanoma Cells ◽  
Lower Sensitivity ◽  
Cytotoxic Effects ◽  
Autophagy Inhibitor ◽  
A375 Melanoma ◽  
A375 Cells

Background: PD-L1 is a membrane protein with inhibitory effects on immune responses, whose expression has been correlated with high aggressiveness and the propensity of melanoma to metastasize. The nitrobenzoxadiazole (NBD) NBDHEX and its analog MC3181 are endowed with strong antitumor activity towards melanoma and a significant ability to reduce its adhesion and invasiveness. Therefore, we investigated whether PD-L1 status could affect cell sensitivity to the cytotoxic effects of NBDs. We then evaluated the effects of NBDHEX on PD-L1 expression and autophagy in melanoma cells. We used the BRAF-mutated A375 melanoma cell line and an A375 variant population enriched for PD-L1+ cells as a model. The cytotoxic effects of NBDs were evaluated in comparison to those of the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. Methods: The effect of NBDHEX on autophagy was determined by measuring LC3-II and p62 protein levels by Western blot. The cytotoxic activity of the compounds was evaluated by sulforhodamine B assay. PD-L1 expression and plasma membrane localization were analyzed by FACS and Western blot analysis. Results: NBDHEX behaves as a late-autophagy inhibitor in A375 melanoma cells, as previously found in other tumor cell lines. NBDHEX and MC3181 showed strong and comparable cytotoxic activity in both parental and PD-L1+ A375 cells, with IC50 values in the sub-micromolar range. Conversely, cells sorted for high PD-L1 expression had lower sensitivity to both the BRAF inhibitor vemurafenib and the autophagy inhibitor chloroquine. NBDHEX treatment did not change the total expression and cell surface localization of PD-L1 in both parental and PD-L1+ A375 cells. Conclusions: Our data suggest that NBDs may represent a promising treatment strategy for melanoma with elevated PD-L1 expression.

scholarly journals Exploring aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction: an in silico study

2021 ◽  
Author(s):  
Chiara Luise ◽  
Dina Robaa ◽  
Wolfgang Sippl
Keyword(s):  
Molecular Dynamic ◽  
In Silico ◽  
Binding Pocket ◽  
Binding Mode ◽  
Molecular Dynamic Simulations ◽  
Flexible Docking ◽  
Dynamic Simulations ◽  
Binding Mode Prediction ◽  
Aromatic Cage ◽  
The Right

AbstractSome of the main challenges faced in drug discovery are pocket flexibility and binding mode prediction. In this work, we explored the aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction by means of in silico approaches. We first investigated the Spindlin1 aromatic cage plasticity by analyzing the available crystal structures and through molecular dynamic simulations. Then we assessed the ability of rigid docking and flexible docking to rightly reproduce the binding mode of a known ligand into Spindlin1, as an example of a reader protein displaying flexibility in the binding pocket. The ability of induced fit docking was further probed to test if the right ligand binding mode could be obtained through flexible docking regardless of the initial protein conformation. Finally, the stability of generated docking poses was verified by molecular dynamic simulations. Accurate binding mode prediction was obtained showing that the herein reported approach is a highly promising combination of in silico methods able to rightly predict the binding mode of small molecule ligands in flexible binding pockets, such as those observed in some reader proteins.

Putative binding mode of Escherichia coli exopolyphosphatase and polyphosphates based on a hybrid in silico/biochemical approach

2016 ◽  
Vol 606 ◽  
pp. 64-72 ◽  
Author(s):  
Cristhian Boetsch ◽  
Daniel R. Aguayo-Villegas ◽  
Fernando D. Gonzalez-Nilo ◽  
Á. Teresita Lisa ◽  
Paola R. Beassoni
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Binding Mode ◽  
Biochemical Approach

scholarly journals A Novel Fast Photothermal Therapy Using Hot Spots of Gold Nanorods for Malignant Melanoma Cells

Nanomaterials ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 880 ◽  
Author(s):  
Yanhua Yao ◽  
Nannan Zhang ◽  
Xiao Liu ◽  
Qiaofeng Dai ◽  
Haiying Liu ◽  
...  
Keyword(s):  
Cell Death ◽  
Malignant Melanoma ◽  
Gold Nanorods ◽  
Plasmon Resonance ◽  
Photothermal Therapy ◽  
Treatment Time ◽  
High Energy ◽  
Melanoma Cells ◽  
Photothermal Effect ◽  
A375 Cells

In this paper, the plasmon resonance effects of gold nanorods was used to achieve rapid photothermal therapy for malignant melanoma cells (A375 cells). After incubation with A375 cells for 24 h, gold nanorods were taken up by the cells and gold nanorod clusters were formed naturally in the organelles of A375 cells. After analyzing the angle and space between the nanorods in clusters, a series of numerical simulations were performed and the results show that the plasmon resonance coupling between the gold nanorods can lead to a field enhancement of up to 60 times. Such high energy localization causes the temperature around the nanorods to rise rapidly and induce cell death. In this treatment, a laser as low as 9.3 mW was used to irradiate a single cell for 20 s and the cell died two h later. The cell death time can also be controlled by changing the power of laser which is focused on the cells. The advantage of this therapy is low laser treatment power, short treatment time, and small treatment range. As a result, the damage of the normal tissue by the photothermal effect can be greatly avoided.

scholarly journals The Induction of Apoptosis in A375 Malignant Melanoma Cells bySutherlandia frutescens

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Nicola B. van der Walt ◽  
Zahra Zakeri ◽  
Marianne J. Cronjé
Keyword(s):  
Malignant Melanoma ◽  
Basic Mechanism ◽  
Melanoma Cells ◽  
Caspase Activation ◽  
Nuclear Condensation ◽  
Induced Apoptosis ◽  
Apoptosis Inducing Factor ◽  
A375 Melanoma Cell Line ◽  
A375 Cells

Sutherlandia frutescensis a medicinal plant indigenous to Southern Africa and is commonly known as the “cancer bush.” This plant has traditionally been used for the treatment of various ailments, although it is best known for its claims of activity against “internal” cancers. Here we report on its effect on melanoma cells. The aim of this study was to investigate whether an extract ofS. frutescenscould induce apoptosis in the A375 melanoma cell line and to outline the basic mechanism of action.S. frutescensextract induced apoptosis in A375 cells as evidenced by morphological features of apoptosis, phosphatidylserine exposure, nuclear condensation, caspase activation, and the release of cytochromecfrom the mitochondria. Studies in the presence of a pan-caspase inhibitor allude to caspase-independent cell death, which appeared to be mediated by the apoptosis inducing factor. Taken together, the results of this study show thatS. frutescensextract is effective in inducing apoptosis in malignant melanoma cells and indicates that furtherin vivomechanistic studies may be warranted.

scholarly journals 248 Empiric profiling of peripheral T cell recall responses to tumor mutanomes versus in silico predictions in NSCLC patients undergoing pembrolizumab treatment ± chemotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A268-A268
Author(s):  
Madison Milaszewski ◽  
James Loizeaux ◽  
Emily Tjon ◽  
Crystal Cabral ◽  
Tulin Dadali ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
In Silico ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Patient Specific ◽  
In Silico Prediction ◽  
Cell Responses ◽  
In Silico Predictions

BackgroundEffective immune checkpoint blockade (ICB) treatment is dependent on T-cell recognition of patient-specific mutations (neoantigens). Empirical identification of neoantigens ex vivo has revealed shortcomings of in silico predictions.1 To better understand the impact of ICB treatment on T cell responses and differences between in silico and in vitro methods, neoantigen-specific T cell responses were evaluated in patients with non-small cell lung cancer undergoing first-line therapy with pembrolizumab ± chemotherapy.MethodsTumor and whole blood samples were collected from 14 patients prior to and after immunotherapy; seven each in monotherapy and combination therapy cohorts. The ex vivo ATLAS™ platform was used to profile neoantigen-specific T-cell responses. Patient-specific tumor mutations identified by next-generation sequencing (NGS) were expressed individually as ATLAS clones, processed patient-specific autologous antigen presenting cells, and presented to their T cells in vitro. ATLAS-verified antigens were compared with epitope predictions made using algorithms.ResultsOn average, 150 (range 37–339) non-synonymous mutations were identified. Pre-treatment, ATLAS identified T cell responses to a median of 15% (9–25%) of mutations, with nearly equal proportions of neoantigens (8%, 5–15%) and Inhibigens™, targets of suppressive T cell responses (8%, 3–13%). The combination therapy cohort had more confirmed neoantigens (46, 20–103) than the monotherapy cohort (7, 6–79). After treatment, the median ratio of CD4:CD8 T cells doubled in the monotherapy but not combination cohort (1.2 to 2.4 v. 1.6 to 1.3). Upon non-specific stimulation, T cells from patients on combination therapy expanded poorly relative to monotherapy (24 v. 65-fold, p = 0.014); no significant differences were observed pre-treatment (22 v. 18-fold, p = 0.1578). Post-treatment, the median number of CD8 neoantigens increased in the combination therapy cohort (11 to 15) but in monotherapy were mostly unchanged (6 to 7). Across timepoints, 36% of ATLAS-identified responses overlapped. In silico analysis resulted in 1,895 predicted epitopes among 961 total mutations; among those, 30% were confirmed with ATLAS, although nearly half were Inhibigens, which could not be predicted. Moreover, 50% of confirmed neoantigens were missed by in silico prediction.ConclusionsMonotherapy and combination therapy had differential effects on CD4:CD8 T cell ratios and their non-specific expansion. A greater proportion of neoantigens was identified than previously reported in studies employing in silico predictions prior to empirical verification.2 Overlap between confirmed antigens and in silico prediction was observed, but in silico prediction continued to have a large false negative rate and could not characterize Inhibigens.AcknowledgementsWe would like to acknowledge and thank the patients and their families for participating in this study.ReferencesLam H, McNeil LK, Starobinets H, DeVault VL, Cohen RB, Twardowski P, Johnson ML, Gillison ML, Stein MN, Vaishampayan UN, DeCillis AP, Foti JJ, Vemulapalli V, Tjon E, Ferber K, DeOliveira DB, Broom W, Agnihotri P, Jaffee EM, Wong KK, Drake CG, Carroll PM, Davis TA, Flechtner JB. An empirical antigen selection method identifies neoantigens that either elicit broad antitumor T-cell responses or drive tumor growth. Cancer Discov 2021;11(3):696–713. doi: 10.1158/2159- 8290.CD-20-0377. Epub 2021 January 27. PMID: 33504579. Rosenberg SA. Immersion in the search for effective cancer immunotherapies. Mol Med 27,63(2021). https://doi.org/10.1186/s10020-021-00321-3

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