An update on central nervous system tumors in germline replication-repair deficiency syndromes.

DNA replication-repair deficiency (RRD) arises from pathogenic variants in the mismatch repair and/or polymerase-proofreading genes. Multiple germline cancer predisposition syndromes in children and young adults, including constitutional mismatch repair deficiency (CMMRD), Lynch, polymerase-proofreading deficiency, and rare digenic syndromes can lead to RRD cancers. The most frequent brain tumors in these children are high-grade gliomas. Embryonal tumors like medulloblastoma have also been described. Lower-grade tumors are reported from cancer surveillance initiatives. The latter has an extremely high rate of malignant transformation. Novel functional assays quantifying the genomic microsatellite indel load have been demonstrated to be highly sensitive and specific for the diagnosis of RRD cancers and children with germline CMMRD. Importantly, RRD brain tumors uniformly harbor high mutation and microsatellite burden. High T-cell infiltration makes these aggressive cancers amenable to immune checkpoint inhibition, irrespective of their germline genetic background. Synergistic combinations are reported to be successful in patients failing checkpoint inhibitor monotherapy. Future directions include the development of innovative approaches to improve immune surveillance for RRD brain cancers. Additionally, the use of novel tools including circulating tumor DNA and quantifying microsatellite indel load over time can be useful to monitor disease burden and treatment responses in patients.

4-{3-[(Pyridin-4-ylmethyl)amino]-[1,2,4]triazolo[4,3-b][1,2,4]triazin-6-yl}phenol: An improved anticancer agent in hepatocellular carcinoma and a selective MDR1/MRP modulator.

Hepatocellular carcinoma is the most common type of primary liver cancer. However, multidrug resistance (MDR) is a major obstacle to the effective chemotherapy of cancer cells. This report documents the rational design, synthesis, and biological evaluation of a novel series of triazolotriazines substituted with CH2NH-linked pyridine for use as dual c-Met/MDR inhibitors. Compound 12g with IC50 of 3.06 µM on HepG2 cells showed more potency than crizotinib (IC50 = 5.15 µM) in the MTT assay. In addition, 12g inhibited c-Met kinase at a low micromolar level (IC50 = 0.052 µM). 12g significantly inhibited P-gp and MRP1/2 efflux pumps in both cancerous HepG2 and BxPC3 cells starting from the lower concentrations of 3 and 0.3 µM, respectively. 12g did not inhibit MDR1 and MRP1/2 in noncancerous H69 cholangiocytes up to the concentration of 30 and 60 µM, respectively. Current results highlighted that cancerous cells were more susceptible to the effect of 12g than normal cells, in which the inhibition occurred only at the highest concentrations, suggesting a further interest in 12g as a selective anticancer agent. Overall, 12g, as a dual c-Met and P-gp/MRP inhibitor, is a promising lead compound for developing a new generation of anticancer agents.

Clinical and biological landscape of constitutional mismatch-repair deficiency syndrome: an International Replication Repair Deficiency Consortium cohort study.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) syndrome is a rare and aggressive cancer predisposition syndrome. Because a scarcity of data on this condition contributes to management challenges and poor outcomes, we aimed to describe the clinical spectrum, cancer biology, and impact of genetics on patient survival in CMMRD. METHODS: In this cohort study, we collected cross-sectional and longitudinal data on all patients with CMMRD, with no age limits, registered with the International Replication Repair Deficiency Consortium (IRRDC) across more than 50 countries. Clinical data were extracted from the IRRDC database, medical records, and physician-completed case record forms. The primary objective was to describe the clinical features, cancer spectrum, and biology of the condition. Secondary objectives included estimations of cancer incidence and of the impact of the specific mismatch-repair gene and genotype on cancer onset and survival, including after cancer surveillance and immunotherapy interventions. FINDINGS: We analysed data from 201 patients (103 males, 98 females) enrolled between June 5, 2007 and Sept 9, 2022. Median age at diagnosis of CMMRD or a related cancer was 8·9 years (IQR 5·9-12·6), and median follow-up from diagnosis was 7·2 years (3·6-14·8). Endogamy among minorities and closed communities contributed to high homozygosity within countries with low consanguinity. Frequent dermatological manifestations (117 [93%] of 126 patients with complete data) led to a clinical overlap with neurofibromatosis type 1 (35 [28%] of 126). 339 cancers were reported in 194 (97%) of 201 patients. The cumulative cancer incidence by age 18 years was 90% (95% CI 80-99). Median time between cancer diagnoses for patients with more than one cancer was 1·9 years (IQR 0·8-3·9). Neoplasms developed in 15 organs and included early-onset adult cancers. CNS tumours were the most frequent (173 [51%] cancers), followed by gastrointestinal (75 [22%]), haematological (61 [18%]), and other cancer types (30 [9%]). Patients with CNS tumours had the poorest overall survival rates (39% [95% CI 30-52] at 10 years from diagnosis; log-rank p<0·0001 across four cancer types), followed by those with haematological cancers (67% [55-82]), gastrointestinal cancers (89% [81-97]), and other solid tumours (96% [88-100]). All cancers showed high mutation and microsatellite indel burdens, and pathognomonic mutational signatures. MLH1 or MSH2 variants caused earlier cancer onset than PMS2 or MSH6 variants, and inferior survival (overall survival at age 15 years 63% [95% CI 55-73] for PMS2, 49% [35-68] for MSH6, 19% [6-66] for MLH1, and 0% for MSH2; p<0·0001). Frameshift or truncating variants within the same gene caused earlier cancers and inferior outcomes compared with missense variants (p<0·0001). The greater deleterious effects of MLH1 and MSH2 variants as compared with PMS2 and MSH6 variants persisted despite overall improvements in survival after surveillance or immune checkpoint inhibitor interventions. INTERPRETATION: The very high cancer burden and unique genomic landscape of CMMRD highlight the benefit of comprehensive assays in timely diagnosis and precision approaches toward surveillance and immunotherapy. These data will guide the clinical management of children and patients who survive into adulthood with CMMRD. FUNDING: The Canadian Institutes for Health Research, Stand Up to Cancer, Children's Oncology Group National Cancer Institute Community Oncology Research Program, Canadian Cancer Society, Brain Canada, The V Foundation for Cancer Research, BioCanRx, Harry and Agnieszka Hall, Meagan's Walk, BRAINchild Canada, The LivWise Foundation, St Baldrick Foundation, Hold'em for Life, and Garron Family Cancer Center.

Combined Immunotherapy Improves Outcome for Replication-Repair-Deficient (RRD) High-Grade Glioma Failing Anti-PD-1 Monotherapy: A Report from the International RRD Consortium.

Immune checkpoint inhibition (ICI) is effective for replication-repair-deficient, high-grade gliomas (RRD-HGG). The clinical/biological impact of immune-directed approaches after failing ICI monotherapy is unknown. We performed an international study on 75 patients treated with anti-PD-1; 20 are progression free (median follow-up, 3.7 years). After second progression/recurrence (n = 55), continuing ICI-based salvage prolonged survival to 11.6 months (n = 38; P < 0.001), particularly for those with extreme mutation burden (P = 0.03). Delayed, sustained responses were observed, associated with changes in mutational spectra and the immune microenvironment. Response to reirradiation was explained by an absence of deleterious postradiation indel signatures (ID8). CTLA4 expression increased over time, and subsequent CTLA4 inhibition resulted in response/stable disease in 75%. RAS-MAPK-pathway inhibition led to the reinvigoration of peripheral immune and radiologic responses. Local (flare) and systemic immune adverse events were frequent (biallelic mismatch-repair deficiency > Lynch syndrome). We provide a mechanistic rationale for the sustained benefit in RRD-HGG from immune-directed/synergistic salvage therapies. Future approaches need to be tailored to patient and tumor biology. SIGNIFICANCE: Hypermutant RRD-HGG are susceptible to checkpoint inhibitors beyond initial progression, leading to improved survival when reirradiation and synergistic immune/targeted agents are added. This is driven by their unique biological and immune properties, which evolve over time. Future research should focus on combinatorial regimens that increase patient survival while limiting immune toxicity. This article is featured in Selected Articles from This Issue, p. 201.

Phenelzine protects against acetaminophen induced apoptosis in HepG2 cells.

Acetaminophen (APAP) overdosing is the most common cause of drug-induced liver failure. Despite extensive study, N-acetylcysteine is currently the only antidote utilized for treatment. The purpose of this study was to evaluate the effect and mechanisms of phenelzine, an FDA-approved antidepressant, on APAP-induced toxicity in HepG2 cells. The human liver hepatocellular cell line HepG2 was used to investigate APAP-induced cytotoxicity. The protective effects of phenelzine were determined by examining the cell viability, combination index calculation, Caspase 3/7 activation, Cytochrome c release, H2O2 levels, NO levels, GSH activity, PERK protein levels, and pathway enrichment analysis. Elevated H2O2 production and decreased glutathione (GSH) levels were indicators of APAP-induced oxidative stress. The combination index of 2.04 indicated that phenelzine had an antagonistic effect on APAP-induced toxicity. When compared to APAP alone, phenelzine treatment considerably reduced caspase 3/7 activation, cytochrome c release, and H2O2 generation. However, phenelzine had minimal effect on NO and GSH levels and did not alleviate ER stress. Pathway enrichment analysis revealed a potential connection between APAP toxicity and phenelzine metabolism. These findings suggested that phenelzine's protective effect against APAP-induced cytotoxicity could be attributed to the drug's capacity to reduce APAP-mediated apoptotic signaling.

Efficacy of Nivolumab in Pediatric Cancers with High Mutation Burden and Mismatch Repair Deficiency.

PURPOSE: Checkpoint inhibitors have limited efficacy for children with unselected solid and brain tumors. We report the first prospective pediatric trial (NCT02992964) using nivolumab exclusively for refractory nonhematologic cancers harboring tumor mutation burden (TMB) ≥5 mutations/megabase (mut/Mb) and/or mismatch repair deficiency (MMRD). PATIENTS AND METHODS: Twenty patients were screened, and 10 were ultimately included in the response cohort of whom nine had TMB >10 mut/Mb (three initially eligible based on MMRD) and one patient had TMB between 5 and 10 mut/Mb. RESULTS: Delayed immune responses contributed to best overall response of 50%, improving on initial objective responses (20%) and leading to 2-year overall survival (OS) of 50% [95% confidence interval (CI), 27-93]. Four children, including three with refractory malignant gliomas are in complete remission at a median follow-up of 37 months (range, 32.4-60), culminating in 2-year OS of 43% (95% CI, 18.2-100). Biomarker analyses confirmed benefit in children with germline MMRD, microsatellite instability, higher activated and lower regulatory circulating T cells. Stochastic mutation accumulation driven by underlying germline MMRD impacted the tumor microenvironment, contributing to delayed responses. No benefit was observed in the single patient with an MMR-proficient tumor and TMB 7.4 mut/Mb. CONCLUSIONS: Nivolumab resulted in durable responses and prolonged survival for the first time in a pediatric trial of refractory hypermutated cancers including malignant gliomas. Novel biomarkers identified here need to be translated rapidly to clinical care to identify children who can benefit from checkpoint inhibitors, including upfront management of cancer. See related commentary by Mardis, p. 4701.

Effects of single or combined exposure to bisphenol A and mono(2-ethylhexyl)phthalate on oxidant/antioxidant status, endoplasmic reticulum stress, and apoptosis in HepG2 cell line.

Endocrine disrupting chemicals (EDCs) may affect many biological processes like growth and stress response. Bisphenol A (BPA) is a plasticizer that is used to harden plastics and polycarbonates. Phthalates are used to add flexibility to polyvinyl chloride containing plastics. The main metabolite of di(2-ethylhexyl) phthalate (DEHP) is mono(2-ethylhexyl) phthalate (MEHP) and it is even more toxic than the parent compound. Humans are usually exposed to these chemicals in mixtures by different routes starting from fetal period. However, there are not many studies in literature that investigate the combined effects of these chemicals. The aim of this study is to investigate toxic effects of BPA and/or MEHP on HepG2 cell line. We have evaluated cytotoxicity, cytomorphological, apoptotic changes, oxidative stress, oxidant/antioxidant status alterations, and endoplasmic reticulum (ER) stress. Combined exposure to BPA and MEHP caused alterations in oxidant/antioxidant status and ER stress marker proteins in both cytoplasmic and nuclear cellular fractions. We can suggest that combined exposure to EDCs may cause serious toxicological outcomes and more mechanistic studies are needed to determine the combined toxic effects.

A different approach to immunochemotherapy for colon Cancer: Development of nanoplexes of cyclodextrins and Interleukin-2 loaded with 5-FU.

Immune system deficiencies are crucial in the progression of cancer, predominantly because immune cells are not stimulated by cytokines to eradicate cancer cells. Immunochemotherapy is currently considered an innovative approach that creates pathways in cancer treatment, sometimes also aiding in the efficacy of chemotherapeutics. The aim of this study was to prepare a cyclodextrin (CD) nanoplex based on charge interaction to deliver the anticancer drug 5-fluorouracil (5-FU) and Interleukin-2 (IL-2), thereby forming a nanoscale drug delivery system aimed at chemo-immunotherapy for colorectal cancers. The CD:IL-2 nanoplexes were obtained with a particle size below 100 nm and a cationic surface charge based on the extent of charge interaction of the cationic CD polymer with negatively charged IL-2. The loading capacity of CD nanoplexes was 40% for 5-FU and 99.8% for IL-2. Nanoplexes maintained physical stability in terms of particle size and zeta potential in aqueous solution for 1 week at + 4 °C. Moreover, the structural integrity of IL-2 loaded into CD nanoplexes was confirmed by SDS-PAGE analysis. The cumulative release rates of both 5-FU and IL-2 were found to be more than 80% in simulated biological fluids in 12 h. Cell culture studies demonstrate that CD polymers are safe on healthy L929 mouse fibroblast cells. Drug-loaded CD nanoplexes were determined to have a higher anticancer effect than free drug solution against CT26 mouse colon carcinoma cells. In addition, intestinal permeability studies supported the conclusion that CD nanoplexes could be promising candidates for oral chemotherapy as well. In conclusion, effective cancer therapy utilizing the absorptive/cellular uptake effect of CDs, the synergic effect and co-transport of chemotherapeutic drugs and immunotherapeutic molecules is a promising approach. Furthermore, the transport of IL-2 with this nano-sized system can reduce or avoid its toxicity problem in the clinic.

Genomic predictors of response to PD-1 inhibition in children with germline DNA replication repair deficiency.

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion-deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10-100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in 'immunologically cold' tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy.

Survival Benefit for Individuals With Constitutional Mismatch Repair Deficiency Undergoing Surveillance.

PURPOSE: Constitutional mismatch repair deficiency syndrome (CMMRD) is a lethal cancer predisposition syndrome characterized by early-onset synchronous and metachronous multiorgan tumors. We designed a surveillance protocol for early tumor detection in these individuals. PATIENTS AND METHODS: Data were collected from patients with confirmed CMMRD who were registered in the International Replication Repair Deficiency Consortium. Tumor spectrum, efficacy of the surveillance protocol, and malignant transformation of low-grade lesions were examined for the entire cohort. Survival outcomes were analyzed for patients followed prospectively from the time of surveillance implementation. RESULTS: A total of 193 malignant tumors in 110 patients were identified. Median age of first cancer diagnosis was 9.2 years (range: 1.7-39.5 years). For patients undergoing surveillance, all GI and other solid tumors, and 75% of brain cancers were detected asymptomatically. By contrast, only 16% of hematologic malignancies were detected asymptomatically (P < .001). Eighty-nine patients were followed prospectively and used for survival analysis. Five-year overall survival (OS) was 90% (95% CI, 78.6 to 100) and 50% (95% CI, 39.2 to 63.7) when cancer was detected asymptomatically and symptomatically, respectively (P = .001). Patient outcome measured by adherence to the surveillance protocol revealed 4-year OS of 79% (95% CI, 54.8 to 90.9) for patients undergoing full surveillance, 55% (95% CI, 28.5 to 74.5) for partial surveillance, and 15% (95% CI, 5.2 to 28.8) for those not under surveillance (P < .0001). Of the 64 low-grade tumors detected, the cumulative likelihood of transformation from low-to high-grade was 81% for GI cancers within 8 years and 100% for gliomas in 6 years. CONCLUSION: Surveillance and early cancer detection are associated with improved OS for individuals with CMMRD.

Polycationic cyclodextrin nanoparticles induce apoptosis and affect antitumoral activity in HepG2 cell line: An evaluation at the molecular level.

Hepatocellular carcinoma (HCC) is a highly metastatic primary liver cancer generating molecular alterations that end up escaping the apoptotic machinery and conferring multidrug resistance. Targeted medicines with increased and selective cytotoxicity and minimal drug resistance are essential for the treatment of HCC. In this study, a self-assembled polycationic (PC) amphiphilic ß-cyclodextrin (ßCDC6) nanoparticle formulation was characterized and its efficacy over HCC cell line HepG2 was evaluated in terms of cytotoxicity, apoptotic potential, chemosensitivity and mitochondrial balance utilizing biochemical, gene expression and proteomic approaches without encapsulating an anti-neoplastic agent. Blank PC ßCDC6 exerted an anti-proliferative effect on 3D multicellular HepG2 spheroid tumors. These nanoparticles were able to trigger apoptosis proved by caspase 3/7 activity, gene expression and flow cytometry studies. The subjection of PC restored the chemosensitivity of HepG2 cells by suppressing the function of p-glycoprotein. The proteomic studies with Q-TOF LC/MS revealed 73 proteins that are aberrantly encoded after cells were treated with the blank PC. Metabolomic analysis further confirmed the shift in certain biological pathways. Thus, we confirmed that the hepatocellular carcinoma-targeting ßCDC6 PC nanoparticles induce apoptosis, lower the rate of cell proliferation, hinder multidrug resistance and they are convenient carriers for eventual therapeutic administrations in HCC patients.

Cancers from Novel Pole-Mutant Mouse Models Provide Insights into Polymerase-Mediated Hypermutagenesis and Immune Checkpoint Blockade.

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype-phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients. SIGNIFICANCE: Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy.See related commentary by Wisdom and Kirsch p. 5459.

A kojic acid derivative promotes intrinsic apoptotic pathway of hepatocellular carcinoma cells without incurring drug resistance.

Hepatocellular carcinoma is one of the most pervasive cancers with low prognosis, high frequency of recurrence, and metastasis. Studies conducted have focused on extricating novel strategies for successful treatment. Kojic acid and its derivatives are already proven to have depigmenting, anti-inflammatory, and anti-neoplastic properties. In the present study, kojic acid and its 10 distinct derivatives were tested on HEPG2 cell line for their possible anti-cancer effect and seven of them were observed to be cytotoxic. Compound 6 was chosen to proceed as the IC50 dosage for HEPG2 cells was lower in comparison with the other derivatives and kojic acid itself. Further experiments pointed out that intrinsic apoptotic pathway was triggered with the exposure of the cells to IC50 concentration of the derivative as the treatment led to escalation of intracellular ROS, induction of TP53 gene, and activation of caspase 3/7. Pro-apoptotic Jnk and Bax genes were not triggered suggesting that the apoptotic pathway advance through an alternative route. Complementary experiments are in need; howbeit, the current findings suggest that the derivative offers a novel promising approach against hepatocellular carcinoma as it is not detrimental to healthy cells within the concentrations applied, and it does not induce drug resistance.

Synthesis and Anticancer Activity of Benzimidazole/Benzoxazole Substituted Triazolotriazines in Hepatocellular Carcinoma.

BACKGROUND: Receptor Tyrosine Kinases (RTK) are the main family of cell surface receptors for growth factors, hormones and cytokines which are responsible for cell growth and differentiation and are considered as an important therapeutic target in cancer. OBJECTIVE: The aim of this study was to design, synthesise and conduct the biological evaluation of benzimidazole/ benzoxazole substituted triazolotriazines as new anticancer agents. METHODS: A series of benzimidazolyl and benzoxazolyl-linked triazolotriazines 8a-e and 9a-e were synthesized as receptor tyrosine kinase inhibitors. Target compounds were evaluated in HGF-induced cell proliferation assay in A549, MCF-7, HepG2 and MDA-MB-231 cancer cells. RESULTS: Hepatocellular carcinoma was the most sensitive cell line towards the tested compounds and 8e was the most potent one on HepG2 cells with an IC50 value of 5.13µM which was close to crizotinib (HepG2 IC50 = 4.35µM) as a standard c-Met kinase inhibitor. c-Met kinase assay of 8e showed that this compound is not capable of inhibiting this enzyme and subsequently molecular docking confirmed the low affinity of 8e towards c- Met active site and its possible anticancer mechanism through VEGFR-2 inhibition. CONCLUSION: Further in silico predictions revealed that 8e can be a drug candidate with favorable pharmacokinetic properties.

Synthesis, computational molecular docking analysis and effectiveness on tyrosinase inhibition of kojic acid derivatives.

Tyrosinase inhibitors have become increasingly important as whitening agents and for the treatment of pigmentary disorders. In this study, the synthesis of kojic acid derivatives having 2-substituted-3-hydroxy-6-hyroxymethyl/chloromethyl/methyl/morpholinomethylpiperidinyl- methyl/pyrrolidinylmethyl-4H-pyran-4-one structure (compounds 1-30) with inhibitory effects on tyrosinase enzyme were described. One-pot Mannich reaction was carried out by using kojic acid/chlorokojic acid/allomaltol and substituted benzylpiperazine derivatives in presence of formaline. Subsequently, cyclic amine (morpholine, piperidine and pyrrolidine) derivatives of the 6th-position of chlorokojic acid were obtained with nucleophilic substitutions in basic medium. The structures of new compounds were identified by FT-IR, 1H- and 13C NMR, ESI-MS and elemental analysis data. The potential mushroom tyrosinase inhibitory activity of the compounds were evaluated by the spectrophotometric method using l-DOPA as a substrate and kojic acid as the control agent. The potential inhibitory activity was also investigated in silico using molecular docking simulation method. Tyrosinase inhibitory action was significantly more efficacious for several compounds (IC50: 86.2-362.1 µM) than kojic acid (IC50: 418.2). Compound 3 bearing 3,4-dichlorobenzyl piperazine moiety was proven to have the highest inhibitory activity. The results of docking studies showed that according to the predicted conformation of compound 3 in the enzyme binding site, hydroxymethyl group provides a metal complex with copper ions and enzyme. Thus, this interaction explain the high inhibitory activities of the compounds 1, 3 and 4 possessing hydroxymethyl substituent supporting the mushroom assay results with docking studies. In accordance with the results, it is suggested that Mannich bases of kojic acid bearing substituted benzyl piperazine groups (compounds 1, 3, 4, 11, 13, 14, 23, 24, 28, and 29) could be promising antityrosinase agents. Additionally, considering the relationship between tyrosinase inhibitory activity results and molecular docking, a new tyrosinase inhibition mechanism can be proposed.

Global omics strategies to investigate the effect of cyclodextrin nanoparticles on MCF-7 breast cancer cells.

Cyclodextrins (CD) are natural macrocyclic oligosaccharides linked by α(1,4) glycosidic bonds. Hydrophobic cavity of CDs are able to incorporate small molecules, ions, macromolecules which makes them excellent delegates for forming nanoparticulate carriers upon chemical modification to render amphiphilicity to CDs. In this study, blank 6OCaproßCD nanoparticle was prepared and administered to MCF-7 breast cancer cells. The effects of these nanoparticles on the cells were investigated in depth through biochemical and proteomic tests following 48 h of incubation. Proteomics studies revealed that apoptosis-related protein levels of hnRNP and CBX1 were increased while HDGF was not affected supporting the idea that 6OCaproßCD nanoparticles prevent cell proliferation. Gene expression studies were generally in correlation with protein levels since gene expression was significantly stimulated while protein levels were lower compared to the control group suggesting that a post-transcriptional modification must have occurred. Furthermore, 6OCaproßCD was observed to not trigger multidrug resistance as proved with RT-PCR that effectuates another exquisite characteristic of 6OCaproßCD nanoparticle as carrier of chemotherapeutic drugs. Metabolomic pathways of CD effect on MCF7 cells were elucidated with HMDB as serine biosynthesis, transmembrane transport of small molecules, metabolism of steroid hormones, estrogen biosynthesis and phospholipid biosynthesis. In conclusion, 6OCaproßCD is a promising nanoparticulate carrier for chemotherapeutic drugs with intrinsic apoptotic effect to be employed in treatment of breast cancer and further studies should be conducted in order to comprehend the exact mechanism of action.

Synthesis and Cytotoxic Evaluation of Kojic Acid Derivatives with Inhibitory Activity on Melanogenesis in Human Melanoma Cells.

BACKGROUND: Malignant melanoma is an agressive tumour related to the overproduction of melanin, which provides colors of skin, eyes and hair. In addition contributing to the risk of malignant melanoma, abnormal production of melanin has many drawbacks, including hyperpigmentation, post-inflammatory pigmentation, melasma and skin aging. Kojic acid is currently employed in order to lighten skin pigmentation and provide depigmentation. OBJECTIVE: Mannich bases of kojic acid with the structure of 2-substituted-3-hydroxy-6-hyroxymethyl/chloromethyl/ methyl/morpholinomethyl/piperidinylmethyl/pyrrolidinylmethyl-4H-pyran-4-one (compounds 1-23) were synthesized by the reaction of kojic acid/chlorokojic acid/allomaltol and substituted benzylpiperazine derivatives in the presence of formaline. To obtain the cyclic amine (morpholine, piperidine and pyrrolidine) derivatives, nucleophilic substitutions were carried out. METHOD: Cytotoxic effects on A375 human malignant melanoma, HGF-1 human gingival fibroblasts, and MRC-5 human lung cell lines were investigated by sulphorhodamine B assay. Control agents were vemurafenib, dacarbazine, temozolomide, and lenalidomide, which are the commercially available drugs for the treatment of malignant melanoma. RESULTS: Cytotoxic action against melanoma cells was significantly more efficacious (IC50: 11.26-68.58 µM) than the FDA-approved drugs except for vemurafenib. Fourteen of the compounds were proven to have higher IC50 values for the non-cancerous cell lines, HGF-1, and MRC-5 cells. Melanogenesis inhibition assay was performed to observe the ability of the drugs to inhibit melanin production and certain compounds were shown to be capable of actively inhibiting melanin production in melanoma cells. CONCLUSION: Mannich bases of kojic acid derivatives may be promising therapeutic agents, since some have more potent effects on melanoma cells than previously FDA-approved drugs for the treatment of malignant melanoma.

Cholesterol-Targeted Anticancer and Apoptotic Effects of Anionic and Polycationic Amphiphilic Cyclodextrin Nanoparticles.

Amphiphilic cyclodextrins (CDs) are biocompatible derivatives of natural CDs and are able to form nanoparticles or polyplexes spontaneously. In this study, nanoparticles prepared from nonionic (6OCaproßCD) or cationic amphiphilic CD (PC ßCDC6) were used comparatively to develop nanoparticles intended for breast cancer therapy. The characterization of these nanoparticles was performed both by in vitro and cell culture studies. Furthermore, the apoptotic and cytotoxic effects of blank amphiphilic CDs were demonstrated by various mechanistic methods including Caspase-8 activity, lipid peroxidation assay, TUNEL assay, Tali(®)-based image analysis, cholesterol assay, and gene expression studies. Blank nanoparticles exerted cytotoxicity against a variety of cancer cells (MCF-7, HeLa, HepG2, and MB49) but none to healthy cells (L929, G/G). Interestingly, blank 6OCaproßCD and blank PC ßCDC6 derivatives were found to be intrinsically effective on cell number and membrane integrity of MCF-7 cells in apoptosis studies. Further in-depth studies were performed to elucidate the selective mechanism of anticancer action in MCF-7 cells caused by these amphiphilic CDs. In conclusion, blank amphiphilic CD nanoparticles induced apoptosis through mitochondrial pathway targeted to cholesterol microdomains in cancer cell membrane.

Reproducibility and optimization of in vivo human diffusion-weighted MRS of the corpus callosum at 3 T and 7 T.

Diffusion-weighted MRS (DWS) of brain metabolites enables the study of cell-specific alterations in tissue microstructure by probing the diffusion of intracellular metabolites. In particular, the diffusion properties of neuronal N-acetylaspartate (NAA), typically co-measured with N-acetylaspartyl glutamate (NAAG) (NAA + NAAG = tNAA), have been shown to be sensitive to intraneuronal/axonal damage in pathologies such as stroke and multiple sclerosis. Lacking, so far, are empirical assessments of the reproducibility of DWS measures across time and subjects, as well as a systematic investigation of the optimal acquisition parameters for DWS experiments, both of which are sorely needed for clinical applications of the method. In this study, we acquired comprehensive single-volume DWS datasets of the human corpus callosum at 3 T and 7 T. We investigated the inter- and intra-subject variability of empirical and modeled diffusion properties of tNAA [D(avg) (tNAA) and D(model) (tNAA), respectively]. Subsequently, we used a jackknife-like resampling approach to explore the variance of these properties in partial data subsets reflecting different total scan durations. The coefficients of variation (C(V)) and repeatability coefficients (C(R)) for D(avg) (tNAA) and D(model) (tNAA) were calculated for both 3 T and 7 T, with overall lower variability in the 7 T results. Although this work is limited to the estimation of the diffusion properties in the corpus callosum, we show that a careful choice of diffusion-weighting conditions at both field strengths allows the accurate measurement of tNAA diffusion properties in clinically relevant experimental time. Based on the resampling results, we suggest optimized acquisition schemes of 13-min duration at 3T and 10-min duration at 7 T, whilst retaining low variability (C(V) ≈ 8%) for the tNAA diffusion measures. Power calculations for the estimation of D(model )(tNAA) and D(avg) (tNAA) based on the suggested schemes show that less than 21 subjects per group are sufficient for the detection of a 10% effect between two groups in case-control studies.