Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells

OBJECTIVE: The purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. METHODS: Proliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. RESULTS: PENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No cross-resistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. CONCLUSION: Mitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer

Dual targeting of mitochondrial function and mTOR pathway as a therapeutic strategy for diffuse intrinsic pontine glioma

Diffuse Intrinsic Pontine Gliomas (DIPG) are the most devastating of all pediatric brain tumors. They mostly affect young children and, as there are no effective treatments, almost all patients with DIPG will die of their tumor within 12 months of diagnosis. A key feature of this devastating tumor is its intrinsic resistance to all clinically available therapies. It has been shown that glioma development is associated with metabolic reprogramming, redox state disruption and resistance to apoptotic pathways. The mitochondrion is an attractive target as a key organelle that facilitates these critical processes. PENAO is a novel anti-cancer compound that targets mitochondrial function by inhibiting adenine nucleotide translocase (ANT). Here we found that DIPG neurosphere cultures express high levels of ANT2 protein and are sensitive to the mitochondrial inhibitor PENAO through oxidative stress, while its apoptotic effects were found to be further enhanced upon co-treatment with mTOR inhibitor temsirolimus. This combination therapy was found to act through inhibition of PI3K/AKT/mTOR pathway, HSP90 and activation of AMPK. In vivo experiments employing an orthotopic model of DIPG showed a marginal anti-tumour effect likely due to poor penetration of the inhibitors into the brain. Further testing of this anti- DIPG strategy with compounds that penetrate the BBB is warranted

Testing the efficacy and safety of BIO101, for the prevention of respiratory deterioration, in patients with COVID-19 pneumonia (COVA study): a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES As of December, 1, 2020, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, resulted in more than 1 472 917 deaths worldwide and death toll is still increasing exponentially. Many COVID-19 infected people are asymptomatic or experience moderate symptoms and recover without medical intervention. However, older people and those with comorbid hypertension, diabetes, obesity, or heart disease are at higher risk of mortality. Because current therapeutic options for COVID-19 patients are limited specifically for this elderly population at risk, Biophytis is developing BIO101 (20-hydroxyecdysone, a Mas receptor activator) as a new treatment option for managing patients with SARS-CoV-2 infection at the severe stage. The angiotensin converting enzyme 2 (ACE2) serves as a receptor for SARS-CoV-2. Interaction between ACE2 and SARS-CoV2 spike protein seems to alter the function of ACE2, a key player in the renin-angiotensin system (RAS). The clinical picture of COVID-19 includes acute respiratory distress syndrome (ARDS), cardiomyopathy, multiorgan dysfunction and shock, all of which might result from an imbalance of the RAS. We propose that RAS balance could be restored in COVID-19 patients through MasR activation downstream of ACE2 activity, with 20-hydroxyecdysone (BIO101) a non-peptidic Mas receptor (MasR) activator. Indeed, MasR activation by 20-hydroxyecdysone harbours anti-inflammatory, anti-thrombotic, and anti-fibrotic properties. BIO101, a 97% pharmaceutical grade 20-hydroxyecdysone could then offer a new therapeutic option by improving the respiratory function and ultimately promoting survival in COVID-19 patients that develop severe forms of this devastating disease. Therefore, the objective of this COVA study is to evaluate the safety and efficacy of BIO101, whose active principle is 20-hydroxyecdysone, in COVID-19 patients with severe pneumonia. TRIAL DESIGN Randomized, double-blind, placebo-controlled, multi-centre, group sequential and adaptive which will be conducted in 2 parts. Part 1: Ascertain the safety and tolerability of BIO101 and obtain preliminary indication of the activity of BIO101, in preventing respiratory deterioration in the target population Part 2: Re-assessment of the sample size needed for the confirmatory part 2 and confirmation of the effect of BIO101 observed in part 1 in the target population. The study is designed as group sequential to allow an efficient run-through, from obtaining an early indication of activity to a final confirmation. And adaptive - to allow accumulation of early data and adapt sample size in part 2 in order to inform the final design of the confirmatory part of the trial. PARTICIPANTS Inclusion criteria 1. Age: 45 and above 2. A confirmed diagnosis of COVID-19 infection, within the last 14 days, prior to randomization, as determined by PCR or other approved commercial or public health assay, in a specimen as specified by the test used. 3. Hospitalized, in observation or planned to be hospitalized due to COVID-19 infection symptoms with anticipated hospitalization duration ≥3 days 4. With evidence of pneumonia based on all of the following: a. Clinical findings on a physical examination b. Respiratory symptoms developed within the past 7 days 5. With evidence of respiratory decompensation that started not more than 4 days before start of study medication and present at screening, meeting one of the following criteria, as assessed by healthcare staff: a. Tachypnea: ≥25 breaths per minute b. Arterial oxygen saturation ≤92% c. A special note should be made if there is suspicion of COVID-19-related myocarditis or pericarditis, as the presence of these is a stratification criterion 6. Without a significant deterioration in liver function tests: a. ALT and AST ≤ 5x upper limit of normal (ULN) b. Gamma-glutamyl transferase (GGT) ≤ 5x ULN c. Total bilirubin ≤ 5×ULN 7. Willing to participate and able to sign an informed consent form (ICF). Or, when relevant, a legally authorized representative (LAR) might sign the ICF on behalf of the study participant 8. Female participants should be: at least 5 years post-menopausal (i.e., persistent amenorrhea 5 years in the absence of an alternative medical cause) or surgically sterile; OR a. Have a negative urine pregnancy test at screening b. Be willing to use a contraceptive method as outlined in inclusion criterion 9 from screening to 30 days after last dose. 9. Male participants who are sexually active with a female partner must agree to the use of an effective method of birth control throughout the study and until 3 months after the last administration of the investigational product. (Note: medically acceptable methods of contraception that may be used by the participant and/or partner include combined oral contraceptive, contraceptive vaginal ring, contraceptive injection, intrauterine device, etonogestrel implant, each supplemented with a condom, as well as sterilization and vasectomy). 10. Female participants who are lactating must agree not to breastfeed during the study and up to 14 days after the intervention. 11. Male participants must agree not to donate sperm for the purpose of reproduction throughout the study and until 3 months after the last administration of the investigational product. 12. For France only: Being affiliated with a European Social Security. Exclusion criteria 1. Not needing or not willing to remain in a healthcare facility during the study 2. Moribund condition (death likely in days) or not expected to survive for >7 days - due to other and non-COVID-19 related conditions 3. Participant on invasive mechanical ventilation via an endotracheal tube, or extracorporeal membrane oxygenation (ECMO), or high-flow Oxygen (delivery of oxygen at a flow of ≥16 L/min.). 4. Participant is not able to take medications by mouth (as capsules or as a powder, mixed in water). 5. Disallowed concomitant medication: Consumption of any herbal products containing 20-hydroxyecdysone and derived from Leuzea carthamoides; Cyanotis vaga or Cyanotis arachnoidea is not allowed (e.g. performance enhancing agents). 6. Any known hypersensitivity to any of the ingredients, or excipients of the study medication, BIO101. 7. Renal disease requiring dialysis, or known renal insufficiency (eGFR≤30 mL/min/1.73 m2, based on Cockcroft & Gault formula). 8. In France only: a. Non-affiliation to compulsory French social security scheme (beneficiary or right-holder). b. Being under tutelage or legal guardianship. Participants will be recruited from approximately 30 clinical centres in Belgium, France, the UK, USA and Brazil. Maximum patients' participation in the study will last 28 days. Follow-up of participants discharged from hospital will be performed through post-intervention phone calls at 14 (± 2) and 60 (± 4) days. INTERVENTION AND COMPARATOR Two treatment arms will be tested in this study: interventional arm 350 mg b.i.d. of BIO101 (AP 20-hydroxyecdysone) and placebo comparator arm 350 mg b.i.d of placebo. Administration of daily dose is the same throughout the whole treatment period. Participants will receive the study medication while hospitalized for up to 28 days or until a clinical endpoint is reached (i.e., 'negative' or 'positive' event). Participants who are officially discharged from hospital care will no longer receive study medication. MAIN OUTCOMES Primary study endpoint: The proportion of participants with 'negative' events up to 28 days. 'Negative' events are defined as respiratory deterioration and all-cause mortality. For the purpose of this study, respiratory deterioration will be defined as any of the following: Requiring mechanical ventilation (including cases that will not be intubated due to resource restrictions and triage). Requiring extracorporeal membrane oxygenation (ECMO). Requiring high-flow oxygen defined as delivery of oxygen at a flow of ≥16 L/min. Only if the primary endpoint is significant at the primary final analysis the following Key secondary endpoints will be tested in that order: Proportion of participants with events of respiratory failure at Day 28 Proportion of participants with 'positive' events at Day 28. Proportion of participants with events of all-cause mortality at Day 28 A 'positive' event is defined as the official discharge from hospital care by the department due to improvement in participant condition. Secondary and exploratory endpoints: In addition, a variety of functional measures and biomarkers (including the SpO2 / FiO2 ratio, viral load and markers related to inflammation, muscles, tissue and the RAS / MAS pathways) will also be collected. RANDOMIZATION Randomization is performed using an IBM clinical development IWRS system during the baseline visit. Block-permuted randomization will be used to assign eligible participants in a 1:1 ratio. In part 1, randomization will be stratified by RAS pathway modulator use (yes/no) and co-morbidities (none vs. 1 and above). In Part 2, randomization will be stratified by centre, gender, RAS pathway modulator use (yes/no), co-morbidities (none vs. 1 and above), receiving Continuous Positive Airway Pressure/Bi-level Positive Airway Pressure (CPAP/BiPAP) at study entry (Yes/No) and suspicion of COVID-19 related myocarditis or pericarditis (present or not). BLINDING (MASKING) Participants, caregivers, and the study team assessing the outcomes are blinded to group assignment. All therapeutic units (TU), BIO101 b.i.d. or placebo b.i.d., cannot be distinguished in compliance with the double-blind process. An independent data-monitoring committee (DMC) will conduct 2 interim analyses. A first one based on the data from part 1 and a second from the data from parts 1 and 2. The first will inform about BIO101 safety, to allow the start of recruitment into part 2 followed by an analysis of the efficacy data, to obtain an indication of activity. The second interim analysis will inform about the sample size that will be required for part 2, in order to achieve adequate statistical power. Numbers to be randomised (sample size) Number of participants randomized: up to 465, in total Part 1: 50 (to obtain the proof of concept in COVID-19 patients). Part 2: 310, potentially increased by 50% (up to 465, based on interim analysis 2) (to confirm the effects of BIO101 observed in part 1). TRIAL STATUS The current protocol Version is V 10.0, dated on 24.09.2020. The recruitment that started on September 1 2020 is ongoing and is anticipated to finish for the whole study by March2021. TRIAL REGISTRATION The trial was registered before trial start in trial registries: EudraCT , No. 2020-001498-63, registered May 18, 2020; and Clinicaltrials.gov, identifier NCT04472728 , registered July 15, 2020. FULL PROTOCOL The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol

PET molecular imaging of phosphodiesterase 10A: An early biomarker of Huntington's disease progression

Background Changes in phosphodiesterase 10A enzyme levels may be a suitable biomarker of disease progression in Huntington's disease. Objectives To evaluate phosphodiesterase 10A PET imaging as a biomarker of HD progression using the radioligand, [F-18]MNI-659. Methods The cross-sectional study (NCT02061722) included 45 Huntington's disease gene-expansion carriers stratified into four disease stages (early and late premanifest and Huntington's disease stages 1 and 2) and 45 age- and sex-matched healthy controls. The primary analysis compared striatal and pallidal phosphodiesterase 10A availability between Huntington's disease gene-expansion carriers and healthy controls as assessed by [F-18]MNI-659 binding. We assessed changes in phosphodiesterase 10A expression using several PET methodologies and compared with previously proposed measures of Huntington's disease progression (PET imaging of D-2/3 receptors and anatomical volume loss on MRI). The longitudinal follow-up study (NCT02956148) continued evaluation of phosphodiesterase 10A availability in 35 Huntington's disease gene-expansion carriers at a mean of 18 months from baseline of the cross-sectional study. Results Primary analyses revealed that phosphodiesterase 10A availability in caudate, putamen, and globus pallidus was significantly lower in Huntington's disease gene-expansion carriers versus healthy controls across all stages. Striatal and pallidal phosphodiesterase 10A availability progressively declined in the premanifest stages and appeared to plateau between stages 1 and 2. The percentage decline of phosphodiesterase 10A availability measured cross-sectionally between Huntington's disease gene-expansion carriers and healthy controls was greater than that demonstrated by D-2/3 receptor availability or volumetric changes. Annualized rates of phosphodiesterase 10A change showed a statistically significant decline between the cross-sectional study and follow-up. Conclusions [F-18]MNI-659 PET imaging is a biologically plausible biomarker of Huntington's disease progression that is more sensitive than the dopamine-receptor and volumetric methods currently used. (c) 2020 International Parkinson and Movement Disorder SocietyNeurological Motor Disorder