Rottlerin Inhibits ROS Formation and Prevents NFκB Activation in MCF-7 and HT-29 Cells

Rottlerin, a polyphenol isolated from Mallotus Philippinensis, has been recently used as a selective inhibitor of PKC δ, although it can inhibit many kinases and has several biological effects. Among them, we recently found that Rottlerin inhibits the Nuclear Factor κB (NFκB), activated by either phorbol esters or H2O2. Because of the redox sensitivity of NFκB and on the basis of Rottlerin antioxidant property, we hypothesized that Rottlerin could prevent NFκB activation acting as a free radicals scavenger, as other natural polyphenols. The current study confirms the antioxidant property of Rottlerin against the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in vitro and against oxidative stress induced by H2O2 and by menadione in culture cells. We also demonstrate that Rottlerin prevents TNFα-dependent NFκB activation in MCF-7 cells and in HT-29 cells transfected with the NFκB-driven plasmid pBIIX-LUC, suggesting that Rottlerin can inhibit NFκB via several pathways and in several cell types

Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers

Abstract Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general.</p

Critical Appraisal of the MTT Assay in the Presence of Rottlerin and Uncouplers

<p>Abstract</p> <p>Rottlerin is a natural product isolated from <it>Mallotus philippinensis</it>. This polyphenolic compound, originally described as a selective inhibitor of PKC&#948;, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor &#954;B in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general.</p

ADMISSION TO PALLIATIVE CARE AND INDICATORS OF END-OF-LIFE INTENSITY OF CARE IN CANCER PATIENTS FROM THE LOMBARDY REGION, ITALY

Objectives: Hospital-based and home palliative care have been associated to a reduction of aggressive treatments in the end-of-life, but data in the Italian context are scanty. We aim therefore investigate the role of palliative care on indicators of end-of-life intensity of care among cancer patients in Lombardy, the largest Italian region. Methods: Within a retrospective study using the healthcare utilization databases of Lombardy, we selected all residents who died in 2019 with a diagnosis of cancer. We considered as exposure variables admission to palliative care and time at palliative care admission, and as indicators of aggressive care hospitalizations, diagnostic/therapeutic procedures, in-hospital death, emergency department visits, and chemotherapy over a time-window of 30 days before death; chemotherapy in the last 14 days was also considered. Results: Our cohort included 26,539 individuals; of these, 14,320 (54%) were admitted to palliative care before death. Individuals who were admitted to palliative care had an odds ratio of 0.27 for one hospitalization, 0.14 for ≥2 hospitalizations, 0.25 for hospital stay ≥12 days, 0.38 for minor diagnostic/therapeutic procedures, 0.18 for major diagnostic/therapeutic procedures, 0.02 for in-hospital death, 0.35 for one emergency department visit, 0.29 for ≥2 emergency department visits, and 0.66 for chemotherapy use in the last 30 days; the odds ratio was 0.56 for chemotherapy use in the last 14 days. Conclusions: This large real-world analysis confirms and further support the importance of palliative care assistance for cancer patients in the end-of-life; this is associated to a significant reduction in unnecessary treatments

Le cure palliative in Lombardia: analisi dell’offerta assistenziale nel 2019

Summary. Objective. The Lombardy Region has one of the most widespread and advanced models of Palliative Care (PC) in the country. However, there is a relative lack of updated data referring to the activity of the Lombardy PC network. Methods. This work aims to describe the activity carried out in the two main PC care settings (home care and hospice) in 2019 through the analysis of the data sent by each Unit to the Lombardy Region. Data were analysed on a regional basis and considering the 8 Health Protection Agencies (ATS) separately. Results. In 2019, PC activity was provided by 114 home care units (1.14/100,000 inhabitants) and 70 hospice for a total of 812 beds (8.1 beds/100,000 inhabitants). Overall, 25,514 patients were assisted for a total of 29,892 care pathway. 77.6% of the patients assisted were oncologic and about 67% of the patients who died of cancer in Lombardy were intercepted by CP. 54.4% of patients were taken care of in home care, although with a significant difference between cancer patients (58%) and non-cancer patients (42%). In home care, average activation time was 2.8 days and in 81% of cases the assistance was activated within 24-48 hours; in hospice, average activation time was 3.5 days, with 60% of admissions within 24 hours and over 30% with waiting time ≥3 days. The median duration of home palliative care was approximately 21 days (average 40.5), that in hospice was 9 days (average 17.2). In the home care pathways, the prevalent outcome was the death of the patient at home (64%) and hospitalization in hospice (17.2%), while 86% of hospitalizations in hospice ended with death. Conclusions. This accurate report of the Lombardy PC activity indicates that PC satisfy various qualitative indicators of structural and care process identified by national regulations. However, PC still remain predominantly intended for cancer patients, with relatively short duration of care, particularly in the hospice setting