Beyond Traditional Use of Alchemilla vulgaris: Genoprotective and Antitumor Activity In Vitro.

Alchemilla vulgaris L. (lady's mantle) was used for centuries in Europe and Balkan countries for treatments of numerous conditions and diseases of the reproductive system, yet some of the biological activities of lady's mantle have been poorly studied and neglected. The present study aimed to estimate the potential of A. vulgaris ethanolic extract from Southeast Serbia to prevent and suppress tumor development in vitro, validated by antioxidant, genoprotective, and cytotoxic properties. A total of 45 compounds were detected by UHPLC-HRMS analysis in A. vulgaris ethanolic extract. Measurement of antioxidant activity revealed the significant potential of the tested extract to scavenge free radicals. In addition, the analysis of micronuclei showed an in vitro protective effect on chromosome aberrations in peripheral human lymphocytes. A. vulgaris extract strongly suppressed the growth of human cell lines derived from different types of tumors (MCF-7, A375, A549, and HCT116). The observed antitumor effect is realized through the blockade of cell division, caspase-dependent apoptosis, and autophagic cell death. Our study has shown that Alchemilla vulgaris L. is a valuable source of bioactive compounds able to protect the subcellular structure from damage, thus preventing tumorigenesis as well as suppressing tumor cell growth.

In-vitro activation of natural killer cells from regional lymph nodes of melanoma patients with interleukin-2 and interleukin-15.

Regional lymph nodes (LNs) represent the first barrier in lymphogenic tumor dissemination in melanoma. Natural killer (NK) cells, the effector cell subpopulation of the innate immune system, are in the first line of antitumor immune defense. Therefore, the aim of this study was to investigate the effect of interleukin (IL)-2 and IL-15, two cytokines with similar immune-enhancing effects, on antitumor cytotoxic function and immunophenotype of NK cells from regional LNs of melanoma patients. Mononuclear cells purified from regional LNs of 50 melanoma patients in clinical stage II-IV were treated in vitro for 72 h and 7 days with 200 IU/ml rhIL-2 and 25 ng/ml IL-15 at 37°C in 5% CO2. Both cytokines significantly augmented NK cell cytotoxic activity, transcription of the cytotoxic molecule perforin, and the level of functionally mature perforin in both nonmetastatic and metastatic regional LNs. IL-2 treatment increased the percentage of CD3CD56 NK cells by increasing the CD56 NK cell subset in both nonmetastatic and metastatic LNs, whereas IL-15 treatment did not affect the percentage of NK cells and their subsets. Both cytokines increased on NK cells from nonmetastatic and metastatic LNs the expression of CD69 early activation antigen, the NKG2D activating receptor, as well as CD16 and inhibitory killer-cell immunoglobulin-like receptor CD158b, both inherent to the mature and the cytotoxic NK cell phenotype. In conclusion, our data may indicate the therapeutic potential of the NK cell population from regional LNs either as immunotherapeutic targets or as adoptively transferred after activation with IL-2 or IL-15.

The impact of TP53 and RAS mutations on cerebellar glioblastomas.

Cerebellar glioblastoma (cGBM) is a rare, inadequately characterized disease, without detailed information on its molecular basis. This is the first report analyzing both TP53 and RAS alterations in cGBM. TP53 mutations were detected in more than half of the samples from our cohort, mainly in hotspot codons. There were no activating mutations in hotspot codons 12/13 and 61 of KRAS and HRAS genes in cGBM samples but we detected alterations in other parts of exons 2 and 3 of these genes, including premature induction of STOP codon. This mutation was present in 3 out of 5 patients. High incidence of RAS mutations, as well as significantly longer survival of cGBM patients compared to those with supratentorial GBM suggest that cGBM may have different mechanisms of occurrence. Our results suggest that inactivation of TP53 and RAS may play an important role in the progression of cerebellar GBM.

Decreased expression of NKG2D, NKp46, DNAM-1 receptors, and intracellular perforin and STAT-1 effector molecules in NK cells and their dim and bright subsets in metastatic melanoma patients.

Although natural killer (NK) cells play an important antitumor role, melanoma cells may affect their effector functions. In this study, we analyzed the expression of various receptors and effector molecules in NK cells and their subsets in metastatic melanoma (MM) patients compared with healthy controls (HCs). In HC and MM patients, we analyzed NK cell activity using a chromium release assay and the expression of CD107a degranulation marker, activating NKG2D, NKp46, DNAM-1, and inhibitory CD158a and CD158b receptors, IL-12R beta 1, IL-12R beta 2, intracellular interferon (IFN)-γ, perforin, and STAT-1 in CD3-CD56+ NK cells, and cytotoxic CD3-CD56 and immunoregulatory CD3-CD56 subsets by flow cytometry. MM patients compared with HC not only had significantly decreased NK cell activity, lower expression of CD107a, and impaired IFN-γ production but also had decreased expression of activating NKG2D, NKp46, and DNAM-1 receptors, which was followed by lower expression of perforin, STAT-1, and both IL-12R subunits in NK cells. In MM patients only, there was a positive correlation between NKG2D expression and degranulation capacity, as well as IFN-γ production in NK cells. Analysis of the expression of various parameters of NK cell effector functions between MM patients with different localization of distant metastases showed that patients in the unfavorable M1c subclass had decreased expression of NKG2D and NKp46 on NK cells compared with patients in the M1a+b group. Downregulated NKG2D, NKp46, and DNAM-1 receptors associated with impaired NK cell effector function are important biomarkers of advanced disease with a poor prognosis in melanoma patients.