Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species

Curcumin may exert a more selective cytotoxic effect in tumor cells with elevated levels of free radicals. Here, we investigated whether curcumin can modulate etoposide action in myeloid leukemia cells and in normal cells of hematopoietic origin. HL-60 cell line, normal myeloid progenitor cluster of differentiation (CD)-34+ cells, and granulocytes were incubated for 4 or 24 hours at different concentrations of curcumin and/or etoposide. Brown Norway rats with acute myeloid leukemia (BNML) were used to prove the influence of curcumin on etoposide action in vivo. Rats were treated with curcumin for 23 days and etoposide was administered for the final 3 days of the experiment. Curcumin synergistically potentiated the cytotoxic effect of etoposide, and it intensified apoptosis and phosphorylation of the histone H2AX induced by this cytostatic drug in leukemic HL-60 cells. In contrast, curcumin did not significantly modify etoposide-induced cytotoxicity and H2AX phosphorylation in normal CD34+ cells and granulocytes. Curcumin modified the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-l-cysteine (NAC) significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In contrast, NAC did not decrease the cytotoxic effect of etoposide. Thus, oxidative stress plays a greater role in the cytotoxic effect of curcumin than that of etoposide in HL-60 cells. In vitro results were confirmed in a BNML model. Pretreatment with curcumin enhanced the antileukemic activity of etoposide in BNML rats (1.57-fold tumor reduction versus etoposide alone; P<0.05) and induced apoptosis of BNML cells more efficiently than etoposide alone (1.54-fold change versus etoposide alone; P<0.05), but this treatment protected nonleukemic B-cells from apoptosis. Thus, curcumin can increase the antileukemic effect of etoposide through reactive oxygen species in sensitive myeloid leukemia cells, and it is harmless to normal human cells

Simvastatin attenuates abdominal aortic aneurysm formation favoured by lack of Nrf2 transcriptional activity

Surgical intervention is currently the only option for an abdominal aortic aneurysm (AAA), preventing its rupture and sudden death of a patient. Therefore, it is crucial to determine the pathogenic mechanisms of this disease for the development of effective pharmacological therapies. Oxidative stress is said to be one of the pivotal factors in the pathogenesis of AAAs. Thus, we aimed to evaluate the significance of nuclear factor erythroid 2-related factor 2 (Nrf2) transcriptional activity in the development of AAA and to verify if simvastatin, administered as pre- and cotreatment, may counteract this structural malformation. Experiments were performed on mice with inhibited transcriptional activity of Nrf2 (tKO) and wild-type (WT) counterparts. We used a model of angiotensin II- (AngII-) induced AAA, combined with a fat-enriched diet. Mice were administered with AngII or saline for up to 28 days via osmotic minipumps. Simvastatin administration was started 7 days before the osmotic pump placement and then continued until the end of the experiment. We found that Nrf2 inactivation increased the risk of development and rupture of AAA. Importantly, these effects were reversed by simvastatin in tKO mice, but not in WT. The abrupt blood pressure rise induced by AngII was mitigated in simvastatin-treated animals regardless of the genotype. Simvastatin-affected parameters that differed between the healthy structure of the aorta and aneurysmal tissue included immune cell infiltration of the aortic wall, VCAM1 mRNA and protein level, extracellular matrix degradation, TGF-β1 mRNA level, and ERK phosphorylation, but neither oxidative stress nor the level of Angiotensin II Type 1 Receptor (AT1R). Taken together, the inhibition of Nrf2 transcriptional activity facilitates AAA formation in mice, which can be prevented by simvastatin. It suggests that statin treatment of patients with hypercholesterolemia might have not only a beneficial effect in terms of controlling atherosclerosis but also potential AAA prevention

Analysis of tumor growth in a model of xenotransplantation: comparison of the caliper-, ultrasound-, and luminescence-based methods

Substancje o potencjalnym działaniu przeciwnowotworowym, przed wprowadzeniem w fazę testów klinicznych, są poddawane szeregowi badań in vivo o charakterze przesiewowym. W tym celu wykorzystuje się zwierzęce modele chorób nowotworowych, takie jak ksenoprzeszczepy. Polegają one na transplantacji ludzkich komórek nowotworowych do organizmu myszy z defektami układu immunologicznego. Podskórne zaszczepienie komórek umożliwia szybkie i łatwe monitorowanie wpływu badanej substancji na rozwój nowotworu, poprzez śledzenie zmian wielkości guza. Pomiary tego typu najczęściej przeprowadzane są przy pomocy suwmiarki, która pomimo wielu zalet (niski koszt, ograniczenie stresu wywieranego na zwierzęta), nie zawsze w sposób rzetelny i obiektywny ukazuje zmiany zachodzące w guzie po zastosowaniu terapii.Celem przeprowadzonych doświadczeń było zbadanie kinetyki zmian wielkości guza za pomocą alternatywnych metod obrazowania, takich jak ultrasonografia i pomiar luminescencji. Badania in vivo zostały poprzedzone doświadczeniami in vitro – polegającymi na wygenerowaniu linii komórkowej posiadającej stabilną ekspresję lucyferazy (komórki HCT116-GFP-luc), a następnie porównaniu żywotności i przebiegu cyklu komórkowego linii wyjściowej (HCT116) i linii zmodyfikowanej (HCT116-GFP-luc).Wyniki doświadczeń in vitro nie wykazały istotnych statystycznie różnic w przeżywalności i cyklu komórkowym pomiędzy porównywanymi liniami komórek – zatem możemy przypuszczać, że dane uzyskane dla komórek HCT116-GFP-luc mogą znaleźć odniesienie do komórek HCT116. Pomiar wielkości guza za pomocą ultrasonografu wysokiej rozdzielczości Vevo-2100 pozwala stwierdzić, że – pomijając przypadki, w których guz ma wyjątkowo nieregularny kształt – pomiary objętości guza przeprowadzone z wykorzystaniem suwmiarki są wiarygodne. Metody te nie pozwalają jednak na ocenę żywotności komórek guza i mogą prowadzić do nieprawdziwych wniosków. Pomiar luminescencji pochodzącej od żywych komórek nowotworowych pozwala uniknąć błędów w interpretacji wyników spowodowanych powstawaniem obszarów nekrotycznych wewnątrz guza oraz umożliwia śledzenie mikroprzerzutów.Podsumowując, zastosowanie linii komórkowej ze stabilną nadekspresją lucyferazy w doświadczeniach przesiewowych dotyczących testowania potencjalnych leków przeciwnowotworowych w modelu ksenoprzeszczepu pozwala na znacznie dokładniejszą analizę wpływu badanych substancji na komórki nowotworowe, uwzględniającą również powstawanie przerzutów.All substances which are potential anticancer drugs have to undergo series of in vivo experiments before clinical trials. Xenograft is one of the animal cancer model widely used in preclinical experiments for a drug screening. In this model, tumors are generally established by an injection of human cancer cells into immunodeficient mice. Subcutaneous localization of a tumor enables relatively fast and easy monitoring of the tumor growth. The measurement made by a caliper, regardless of many advantages (low cost, minimal amount of stress exerted on animals), sometimes does not provide full, objective and solid information about the effects of anticancer treatment.The main purpose of the study was to monitor tumor growth kinetics using alternative, noninvasive imagining techniques such as high-resolution ultrasound imagining and luminescence imagining. In vivo studies were preceded by in vitro experiments – HCT116 cell line was modified to stably express luciferase reporter gene (HCT116-GFP-luc). Then the viability and cell cycle of those cells were analyzed and compared with that of unmodified counterparts.There were no statistically significant differences in cell viability (as measured using MTT reduction assay) and in cell cycle (as analyzed using flow cytometer) between HCT116 and HCT116-GFP-luc cell lines. Thus, results of the in vivo experiments obtained for cells expressing luciferase may also be referred to the HCT116 cells. Two methods of tumor volume measurement were compared. We found that, as long as the tumor does not have irregular shape, the results obtained from ultrasound imaging and from caliper measurements are comparable. However, those methods cannot provide information about viability of the cells within the tumor. The measurement of luminescence allows not only to specify the amount of viable cells in tumor (which is a very important information in case of tumor necrosis) but also makes it possible to monitor metastasis.In conclusion, the use of the cell line modified to overexpress luciferase in xenografts allows more accurate study of effects of the tested drug on tumor cells, including monitoring of metastases formation

Complex effects of helper relatedness on female extra-pair reproduction in a cooperative breeder

In cooperatively-breeding species, the presence of male helpers in a group often reduces the breeding female’s fidelity to her social partner, possibly because there is more than one potential sire in the group. Using a long-term study of cooperatively-breeding superb fairy-wrens (Malurus cyaneus) and records of paternity in 1936 broods, we show that the effect of helpers on rates of extra-pair paternity varied according to the helpers’ relatedness to the breeding female. The presence of unrelated male helpers in a group increased average rates of extra-pair paternity, from 57% for groups with no unrelated helpers, to 74% with one unrelated helper, to 86% with 2+ unrelated helpers. However, this increase was due in equal part to helpers within the group and males in other groups achieving increased paternity. In contrast, helpers who were sons of the breeding female did not gain paternity, nor did they affect the level of extra-group paternity (which occurred at rates of 60%, 58%, 61% in the presence of 0, 1, 2+ helper-sons respectively). There was no evidence of effects of helpers’ relatedness to the female on nest productivity or nestling performance. Because the presence of helpers per se did not elevate extra-pair reproduction rates, our results undermine the ‘constrained female hypothesis’ explanation for an increase in extra-pair paternity with helper number in cooperative breeders. However, they indicate that dominant males are disadvantaged by breeding in ‘cooperative’ groups. The reasons why the presence of unrelated helpers, but not of helper-sons, results in higher rates of extra-group reproduction are not clear.Hajduk, G.K. (2020), Complex effects of helper relatedness on female extra-pair reproduction in a cooperative breeder, Dryad, Dataset, 10.5061/dryad.prr4xgxk