Mind the Curve: Dose-Response Fitting Biases the Synergy Scores across Software Used for Chemotherapy Combination Studies.

Drug combinations are increasingly studied in the field of anticancer agents. Mathematical models, such as Loewe, Bliss, and HSA, are used to interpret drug combinations, while informatics tools help cancer researchers identify the most effective combinations. However, the different algorithms each software uses lead to results that do not always correlate. This study compared the performance of Combenefit (Ver. 2.021) and SynergyFinder (Ver. 3.6) in analyzing drug synergy by studying combinations involving non-steroidal analgesics (celecoxib and indomethacin) and antitumor drugs (carboplatin, gemcitabine, and vinorelbine) on two canine mammary tumor cell lines. The drugs were characterized, their optimal concentration-response ranges were determined, and nine concentrations of each drug were used to make combination matrices. Viability data were analyzed under the HSA, Loewe, and Bliss models. Celecoxib-based combinations showed the most consistent synergistic effect among software and reference models. Combination heatmaps revealed that Combenefit gave stronger synergy signals, while SynergyFinder produced better concentration-response fitting. When the average values of the combination matrices were compared, some combinations shifted from synergistic to antagonistic due to differences in the curve fitting. We also used a simulated dataset to normalize each software's synergy scores, finding that Combenefit tends to increase the distance between synergistic and antagonistic combinations. We conclude that concentration-response data fitting biases the direction of the combination (synergistic or antagonistic). In contrast, the scoring from each software increases the differences among synergistic or antagonistic combinations in Combenefit when compared to SynergyFinder. We strongly recommend using multiple reference models and reporting complete data analysis for synergy claiming in combination studies.

Indomethacin Induces Spermidine/Spermine-N1-Acetyltransferase-1 via the Nucleolin-CDK1 Axis and Synergizes with the Polyamine Oxidase Inhibitor Methoctramine in Lung Cancer Cells.

Indomethacin is a non-selective NSAID used against pain and inflammation. Although cyclooxygenase (COX) inhibition is considered indomethacin's primary action mechanism, COX-independent ways are associated with beneficial effects in cancer. In colon cancer cells, the activation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) is related to the increase in spermidine/spermine-N1-acetyltransferase-1 (SSAT-1), a key enzyme for polyamine degradation, and related to cell cycle arrest. Indomethacin increases the SSAT-1 levels in lung cancer cells; however, the mechanism relying on the SSAT-1 increase is unclear. Thus, we asked for the influence of the PPAR-γ on the SSAT-1 expression in two lung cancer cell lines: H1299 and A549. We found that the inhibition of PPAR-γ with GW9662 did not revert the increase in SSAT-1 induced by indomethacin. Because the mRNA of SSAT-1 suffers a pre-translation retention step by nucleolin, a nucleolar protein, we explored the relationship between indomethacin and the upstream translation regulators of SSAT-1. We found that indomethacin decreases the nucleolin levels and the cyclin-dependent kinase 1 (CDK1) levels, which phosphorylates nucleolin in mitosis. Overexpression of nucleolin partially reverts the effect of indomethacin over cell viability and SSAT-1 levels. On the other hand, Casein Kinase, known for phosphorylating nucleolin during interphase, is not modified by indomethacin. SSAT-1 exerts its antiproliferative effect by acetylating polyamines, a process reverted by the polyamine oxidase (PAOX). Recently, methoctramine was described as the most specific inhibitor of PAOX. Thus, we asked if methoctramine could increase the effect of indomethacin. We found that, when combined, indomethacin and methoctramine have a synergistic effect against NSCLC cells in vitro. These results suggest that indomethacin increases the SSAT-1 levels by reducing the CDK1-nucleolin regulatory axis, and the PAOX inhibition with methoctramine could improve the antiproliferative effect of indomethacin.

In vitro and in vivo activity of voriconazole and benznidazole combination on trypanosoma cruzi infection models.

Combination therapy has been proposed as an ideal strategy to reduce drug toxicity and improve treatment efficacy in Chagas disease. Previously, we demonstrated potent in vivo anti-Trypanosoma cruzi activity of voriconazole. In this work, we aimed to study the synergistic effect of voriconazole (VCZ) and benznidazole (BZ) both in vitro and in vivo models of T. cruzi infection using the Tulahuen strain. Combining VCZ and BZ at fixed concentrations, the inhibitory concentration 50% (IC50) on amastigotes was lower than the obtained IC50 for BZ alone and the Fractional Inhibitory Concentration Index (∑FIC) suggested an in vitro additive effect on T. cruzi amastigotes inhibition at concentrations devoid of cytotoxic effects. Treatment response in the in vivo model was evaluated by comparing behavior and physical aspects, parasitemia and mortality of mice infected with Tulahuen strain. VCZ and BZ treatments alone or in combination were well tolerated. All treated animals displayed significantly lower mean peak parasitemia and mortality compared to infected non-treated controls (p< 0.05). However, VCZ + BZ combination elicited no additional benefits over BZ monotherapy. VCZ efficacy was not enhanced by combination therapy with BZ at the doses studied, requiring further and astringent non-clinical studies to establish the VCZ efficacy and eventually moving forward to clinical trials.

Inflammatory and Pro-resolving Lipids in Trypanosomatid Infections: A Key to Understanding Parasite Control.

Pathogenic trypanosomatids (Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp.) are protozoan parasites that cause neglected diseases affecting millions of people in Africa, Asia, and the Americas. In the process of infection, trypanosomatids evade and survive the immune system attack, which can lead to a chronic inflammatory state that induces cumulative damage, often killing the host in the long term. The immune mediators involved in this process are not entirely understood. Most of the research on the immunologic control of protozoan infections has been focused on acute inflammation. Nevertheless, when this process is not terminated adequately, permanent damage to the inflamed tissue may ensue. Recently, a second process, called resolution of inflammation, has been proposed to be a pivotal process in the control of parasite burden and establishment of chronic infection. Resolution of inflammation is an active process that promotes the normal function of injured or infected tissues. Several mediators are involved in this process, including eicosanoid-derived lipids, cytokines such as transforming growth factor (TGF)-ß and interleukin (IL)-10, and other proteins such as Annexin-V. For example, during T. cruzi infection, pro-resolving lipids such as 15-epi-lipoxin-A4 and Resolvin D1 have been associated with a decrease in the inflammatory changes observed in experimental chronic heart disease, reducing inflammation and fibrosis, and increasing host survival. Furthermore, Resolvin D1 modulates the immune response in cells of patients with Chagas disease. In Leishmania spp. infections, pro-resolving mediators such as Annexin-V, lipoxins, and Resolvin D1 are related to the modulation of cutaneous manifestation of the disease. However, these mediators seem to have different roles in visceral or cutaneous leishmaniasis. Finally, although T. brucei infections are less well studied in terms of their relationship with inflammation, it has been found that arachidonic acid-derived lipids act as key regulators of the host immune response and parasite burden. Also, cytokines such as IL-10 and TGF-ß may be related to increased infection. Knowledge about the inflammation resolution process is necessary to understand the host-parasite interplay, but it also offers an interesting opportunity to improve the current therapies, aiming to reduce the detrimental state induced by chronic protozoan infections.

Protective role of acetylsalicylic acid in experimental Trypanosoma cruzi infection: evidence of a 15-epi-lipoxin A4-mediated effect.

Chagas' disease, produced by Trypanosoma cruzi, affects more than 8 million people, producing approximately 10,000 deaths each year in Latin America. Migration of people from endemic regions to developed countries has expanded the risk of infection, transforming this disease into a globally emerging problem. PGE2 and other eicosanoids contribute to cardiac functional deficits after infection with T. cruzi. Thus, the inhibition of host cyclooxygenase (COX) enzyme emerges as a potential therapeutic target. In vivo studies about the effect of acetylsalicylic acid (ASA) upon T. cruzi infection are controversial, and always report the effect of ASA at a single dose. Therefore, we aimed to analyze the effect of ASA at different doses in an in vivo model of infection and correlate it with the production of arachidonic acid metabolites. ASA decreased mortality, parasitemia, and heart damage in T. cruzi (Dm28c) infected mice, at the low doses of 25 and 50 mg/Kg. However, this effect disappeared when the high ASA doses of 75 and 100 mg/Kg were used. We explored whether this observation was related to the metabolic shift toward the production of 5-lipoxygenase derivatives, and although we did not observe an increase in LTB4 production in infected RAW cells and mice infected, we did find an increase in 15-epi-LXA4 (an ASA-triggered lipoxin). We also found high levels of 15-epi-LXA4 in T. cruzi infected mice treated with the low doses of ASA, while the high ASA doses decreased 15-epi-LXA4 levels. Importantly, 15-epi-LXA4 prevented parasitemia, mortality, and cardiac changes in vivo and restored the protective role in the treatment with a high dose of ASA. This is the first report showing the production of ASA-triggered lipoxins in T. cruzi infected mice, which demonstrates the role of this lipid as an anti-inflammatory molecule in the acute phase of the disease.

Biological and chemical study of fused tri- and tetracyclic indazoles and analogues with important antiparasitic activity.

A series of fused tri- and tetracyclic indazoles and analogues compounds (NID) with potential antiparasitic effects were studied using voltamperometric and spectroscopic techniques. Nitroanion radicals generated by cyclic voltammetry were characterized by electron spin resonance spectroscopy (ESR) and their spectral lines were explained and analyzed using simulated spectra. In addition, we examined the interaction between radical species generated from nitroindazole derivatives and glutathione (GSH). Biological assays such as activity against Trypanosoma cruzi and cytotoxicity against macrophages were carried out. Finally, spin trapping and molecular modeling studies were also done in order to elucidate the potentials action mechanisms involved in the trypanocidal activity.