Antitumor activity and expression profiles of genes induced by sulforaphane in human melanoma cells

Purpose Human melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistance to apoptosis, reprogramming, proliferation and survival during tumour progression. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, plays a role in carcinogenesis in many cancer types. However, the cytotoxic molecular mechanisms and gene expression profiles promoted by SFN in human melanoma remain unknown. Methods Three different cell lines were used: two human melanoma A375 and 501MEL and human epidermal melanocytes (HEMa). Cell viability and proliferation, cell cycle analysis, cell migration and invasion and protein expression and phosphorylation status of Akt and p53 upon SFN treatment were determined. RNA-seq of A375 was performed at different time points after SFN treatment. Results We demonstrated that SFN strongly decreased cell viability and proliferation, induced G2/M cell cycle arrest, promoted apoptosis through the activation of caspases 3, 8, 9 and hampered migration and invasion abilities in the melanoma cell lines. Remarkably, HEMa cells were not affected by SFN treatment. Transcriptomic analysis revealed regulation of genes involved in response to stress, apoptosis/cell death and metabolic processes. SFN upregulated the expression of pro-apoptotic genes, such as p53, BAX, PUMA, FAS and MDM2; promoted cell cycle inhibition and growth arrest by upregulating EGR1, GADD45B, ATF3 and CDKN1A; and simultaneously acted as a potent inhibitor of genotoxicity by launching the stress-inducible protein network (HMOX1, HSPA1A, HSPA6, SOD1). Conclusion Overall, the data show that SFN cytotoxicity in melanoma derives from complex and concurrent mechanisms during carcinogenesis, which makes it a promising cancer prevention agent

Comparative analysis of lactaptin activity when produced in bacterial or eukaryotic expression systems

Despite the multitude of anticancer cytostatic drugs available to oncologists today, most of such drugs have serious side effects that may preclude their use in some groups of patients. Hence, selective induction of apoptosis in cancer but not normal cells remains an attractive goal of molecular medicine. Lactaptin, a proteolytic fragment of the human milk kappa-casein, has been previously identified as a protein displaying potent killing of cancer cells in vitro. Its recombinant analog (RL2) produced in E. coli has been shown to delay solid tumor growth in vivo. Given that lactaptin is of human origin and is not immunogenic, it can be administered to patients multiple times without running the risk of immune response that could dampen the therapy efficacy. In the present study, we demonstrate that the combination of RL2 and cyclophosphamide treatments has an additive therapeutic effect against hepatoma tumor in immunocompetent mice. We asked whether production of lactaptin in human rather than bacterial cells would result in a protein with increased cytotoxic activity. Using lentiviral vector pCDH as a backbone, two constructs, pEL1 and pEL2, encoding secreted forms of lactaptin that differ in their signal sequences were created. Lactaptin expression in human cell lines was confirmed using Western-blot analysis, whereas ELISA was used for quantification of secreted lactaptin. Next, we measured the cytotoxic effects of the media conditioned by pEL1-transfected HEK293T cells, as assayed against the panel of three human cancer cell lines: MDA-MB-231 (adenocarcinoma), PC3 (prostate cancer), and T98G (glioblastoma). We show that EL1-derived lactaptin is at least 100-fold more cytotoxic than RL2. Taken together, our results provide an opportunity for developing armored immune cells as an “off-the-shelf” platform for targeted delivery of lactaptin to cancer cells

Cytotoxic effect of the VVGMCSF-Lact oncolytic virus against 3D cultures of human glioblastoma cells U-87 MG

Background. One of the promising methods of treating tumors is virotherapy, which is based on direct lysis of cancer cells by a virus and a virus-mediated antitumor immune response of the body. For the recombinant vaccinia virus strain VVGMCSF-Lact, producing human GMCSF and the oncotoxic protein lactaptin, cytotoxic and antitumor effects were shown in experiments in vitro and in vivo, respectively, when using adhesive cultures of U-87 MG human glioblastoma cells. 3D cultures are a more relevant tumor model than adhesive models, as they more fully reflect the realistic scenario of cancer development, as well as the response of the tumor to anticancer therapy.The aim. To evaluate the cytotoxic effect of the oncolytic virus VV-GMCSF-Lact against 3D cultures of human glioblastoma U-87 MG.Materials and methods. The following methods were used in the work: cultivation of 3D cell cultures, cytofluorometry, microscopic analysis, virus titration, statistical analysis.Results. U-87 MG cells were transduced with a lentiviral vector carrying the GFP reporter gene. The cytotoxicity of the VV-GMCSF-Lact virus (IC50) against the studied cells was 0.024 PFU/cell. U-87 MG cells were cultured under conditions for the formation of 3D structures. Microscopic analysis showed the oncolytic effect of the virus on the cells of 3D cultures as early as 24 hours after the start of incubation. Flow cytometry showed an increase in the granularity of glioblastoma cells under the action of the virus, which indicates active replication of the virus in the cells. The virus titer was 0.44 PFU/cell.Conclusions. The recombinant VV-GMCSF-Lact virus has a cytotoxic effect on 3D human glioblastoma U-87 MG cell cultures and actively replicates in them. In the future, to test the oncolytic effect of VV-GMCSF-Lact, it is planned to use not only 3D human glioblastoma cultures, but also cerebral organelles obtained in the process of cocultivation of glioblastoma cells and induced human pluripotent cells

Multiple Pathway-Based Genetic Variations Associated with Tobacco Related Multiple Primary Neoplasms

BACKGROUND: In order to elucidate a combination of genetic alterations that drive tobacco carcinogenesis we have explored a unique model system and analytical method for an unbiased qualitative and quantitative assessment of gene-gene and gene-environment interactions. The objective of this case control study was to assess genetic predisposition in a biologically enriched clinical model system of tobacco related cancers (TRC), occurring as Multiple Primary Neoplasms (MPN). METHODS: Genotyping of 21 candidate Single Nucleotide Polymorphisms (SNP) from major metabolic pathways was performed in a cohort of 151 MPN cases and 210 cancer-free controls. Statistical analysis using logistic regression and Multifactor Dimensionality Reduction (MDR) analysis was performed for studying higher order interactions among various SNPs and tobacco habit. RESULTS: Increased risk association was observed for patients with at least one TRC in the upper aero digestive tract (UADT) for variations in SULT1A1 Arg²¹³His, mEH Tyr¹¹³His, hOGG1 Ser³²⁶Cys, XRCC1 Arg²⁸⁰His and BRCA2 Asn³⁷²His. Gene-environment interactions were assessed using MDR analysis. The overall best model by MDR was tobacco habit/p53(Arg/Arg)/XRCC1(Arg³⁹⁹His)/mEH(Tyr¹¹³His) that had highest Cross Validation Consistency (8.3) and test accuracy (0.69). This model also showed significant association using logistic regression analysis. CONCLUSION: This is the first Indian study on a multipathway based approach to study genetic susceptibility to cancer in tobacco associated MPN. This approach could assist in planning additional studies for comprehensive understanding of tobacco carcinogenesis

CYP17 genetic polymorphism, breast cancer, and breast cancer risk factors: Australian Breast Cancer Family Study

INTRODUCTION: Because CYP17 can influence the degree of exposure of breast tissues to oestrogen, the interaction between polymorphisms in this gene and hormonal risk factors is of particular interest. We attempted to replicate the findings of studies assessing such interactions with the -34T→C polymorphism. METHODS: Risk factor and CYP17 genotyping data were derived from a large Australian population-based case-control-family study of 1,284 breast cancer cases and 679 controls. Crude and adjusted odds ratio (OR) estimates and 95% confidence intervals (CIs) were calculated by unconditional logistic regression analyses. RESULTS: We found no associations between the CYP17 genotype and breast cancer overall. Premenopausal controls with A(2)/A(2 )genotype had a later age at menarche (P < 0.01). The only associations near statistical significance were that postmenopausal women with A(1)/A(1 )(wild-type) genotype had an increased risk of breast cancer if they had ever used hormone replacement therapy (OR 2.40, 95% CI 1.0 to 5.7; P = 0.05) and if they had menopause after age 47 years (OR 2.59, 95% CI 1.0 to 7.0; P = 0.06). We found no associations in common with any other studies, and no evidence for interactions. CONCLUSION: We observed no evidence of effect modification of reproductive risk factors by CYP17 genotype, although the experiment did not have sufficient statistical power to detect small main effects and modest effects in subgroups. Associations found only in subgroup analyses based on relatively small numbers require cautious interpretation without confirmation by other studies. This emphasizes the need for replication in multiple and large population-based studies to provide convincing evidence for gene–environment interactions

Аптамеры для диагностики и лечения глиальных опухолей человека

Purpose of the study: to evaluate the feasibility of using functional analogues of protein antibodies – dNa/ RNa aptamers in diagnostics, treatment and prognosis of human brain glial tumors.Material and Methods. The relevant literature sources were searched in scopus, Web of science, pubmed, elibrary with inclusion of publications from 2000 to 2023. sixty articles are presented in the review.Results. The analysis of the literature devoted to classification, diagnostics and therapy of brain glioblastomas was carried out and the feasibility of using for in vivo diagnostics and therapy of this disease aptamers, which are molecular recognition elements based on DNA/RNA oligonucleotides, capable of binding to the given molecular targets and distinguishing even separate functional groups in them, was studied. A list of aptamers to human glial brain tumors and their molecular targets that can be used for diagnostics and therapy of glioblastoma, including tumor imaging by pet/ct, mRi, plasmon resonance, fluorescence and confocal microscopy, etc., is presented. literature data suggest that DNA/RNA aptamers can be used to search for circulating tumor cells in the blood of glioblastoma patients, to target therapeutic drugs to the tumor and to inhibit tumor growth.Conclusion. Brain glioblastoma is a heterogeneous tumor consisting of cells at different stages of malignancy and, accordingly, with a different set of oncogenes. For this reason, a multitarget strategy that includes combined suppression of angiogenesis, invasion, metastasis, proliferation and survival of tumor cells should be proposed for the therapy of this disease. DNA/RNA aptamers tailored to key proteins involved in oncogenic transformation may be suitable candidates for the implementation of multitarget therapy for brain glioblastoma.Цель исследования – оценить возможность использования функциональных аналогов белковых антител – ДНК/РНК-аптамеров в диагностике, лечении и прогнозировании развития глиальных опухолей головного мозга человека.Материал и методы. Поиск соответствующих источников литературы проводили в системах scopus, Web of science, pubmed, elibrary с включением публикаций с 2000 по 2023 г. В обзоре представлены данные из 60 статей.Результаты. Проведен анализ литературы, посвященной классификации, диагностике и терапии глиобластом головного мозга и изучению возможности использования для in vivo диагностики и терапии этого заболевания аптамеров, которые представляют собой молекулярные распознающие элементы на основе ДНК/РНК-олигонуклеотидов, способных связываться с заданными молекулярными мишенями и различать в них даже отдельные функциональные группы. Приведен список из аптамеров к глиальным опухолям головного мозга человека и их молекулярных мишеней, которые могут быть использованы для диагностики и терапии глиобластомы, в том числе для визуализации опухоли методами ПЭТ/КТ, МРТ, плазмонного резонанса, флуоресцентной и конфокальной микроскопии и др. Данные литературы свидетельствуют о том, что с помощью ДНК/РНК-аптамеров можно осуществлять поиск циркулирующих опухолевых клеток в крови больных глиобластомой, адресную доставку к опухоли терапевтических препаратов и подавлять опухолевый рост.Заключение. Глиобластома головного мозга – гетерогенная опухоль, состоящая из клеток, находящихся на разной стадии злокачественности и, соответственно, с различным набором онкогенов. Именно поэтому для терапии этого заболевания должна быть предложена мультитаргетная стратегия, которая включает в себя комбинированное подавление ангиогенеза, инвазии, метастазирования, пролиферации и выживаемости опухолевых клеток. Подходящими кандидатами для реализации мультитаргетной терапии глиобластомы головного мозга могут стать ДНК/РНК-аптамеры, подобранные к ключевым белкам, участвующим в онкогенной трансформации