38 research outputs found
Modeling of the molecular and cellular response to ionizing radiations : impact of the nucleo-shuttling of the ATM protein
No full textDepuis plus d'un siècle que les rayons X ont été découverts, les effets biologiques des radiations ionisantes ne sont pas encore entièrement expliqués. Pourtant, une description précise et la modélisation mathématique des événements physico-chimiques, moléculaires et cellulaires contribueraient significativement à l'effet des risques liés à une irradiation. Le groupe de Radiobiologie de l'UMR1052 Inserm (Lyon) a accumulé un nombre considérable de données sur la radiosensibilité individuelle et la réparation des dommages radioinduits de l'ADN qui nous permettent aujourd'hui de valider des modèles nouveaux qui sont souvent en contradiction avec les paradigmes actuels. En particulier, alors que les cassures double-brin de l'ADN semblent être les dommages clés de la létalité cellulaire, aucun protocole ni biomarqueur n'est considéré comme prédictif de la radiosensibilité. Le but de la thèse a donc été de déterminer les paramètres précis qui peuvent prédire la réponse aux radiations. Un grand nombre de protéines se relocalisent sous forme de foci nucléaire autour des sites de CDB. Dans une première étape, nous avons pu proposer une formule générale qui lie induction, reconnaissance et réparation des CDB, valable pour toutes les protéines relocalisantes après irradiation. Cette formule a été appelée « Formule de Bodgi ». La validité de cette formule a pu être vérifiée sur différents biomarqueurs et sur différents types de cellules de patients montrant des radiosensibilités différentes. Dans une deuxième étape, nous avons pu modéliser le processus de transit cytonucléaire de la protéine ATM. Nous avons pu alors apporter une interprétation nouvelle et cohérente des paramètres α et β du modèle linéaire-quadratique qui décrit la relation entre la dose de radiation et la survie cellulaire. Notre théorie s'est avérée également utile pour expliquer certaines autres énigmes de la radiobiologie, notamment le phénomène d'hypersensibilité aux faibles dosesMore than a century after the discovery of X-rays, the biological effects of ionizing radiation are still not entirely explained. Nevertheless, a relevant description and a mathematical model of the physico-chemical, molecular and cellular events would significantly contribute to the evaluation of the related risks. The Radiobiology Group of the UMR1052 Inserm Unit (Lyon) has collected a considerable number of data concerning individual radiosensitivity and DNA damage repair, which allows us today to validate actual modeling approaches that are often contradicting actual paradigms. Particularly, while the DNA double-strand breaks (DSB) appear to be the key-damages of cell lethality, there is still no experimental protocol or biomarker that is considered to be predictive for radiosensitivity. The purpose of this thesis was to determine the precise parameters that can predict the response to radiation. An important number of proteins relocalize as nuclear foci in the DSB sites. As a first step, we proposed a general formula that links the DSB induction, recognition and repair, valid for all the relocalized proteins after irradiation. We called this formula the ‘’Bodgi’s formula’’. The validity of this model was verified with different biomarkers, but also on different cell types from patients showing different radiosensitivity. In a second step, we proposed a model for the whole process of the nucleo-shuttling of the ATM protein that occurs after irradiation. We provided a novel and coherent interpretation of the α and β parameters of the linear-quadratic model that describes the relation between radiation dose and cell survival. Our theory was also shown to be useful in explaining some other enigmas of radiobiology, including the hypersensitivity to lowdose phenomen
Radio-sensitizing prostate cancer cells: A possible role for zoledronic acid and pravastatin (ZoPra)
No full textProstate cancer is highly prevalent in men worldwide and in Lebanon specifically. Radiotherapy is one of the first-line treatments aimed at reducing tumor size and further disease progression. Its efficacy relies on the radio sensitivity of tumor cells and the patient. However, it’s significantly reduced by the radioresistance of tumor cells and deleterious effects on surrounding normal cells. Radioresistance of tumor cells is brought about by the hyperactivation of non-homologous end-joining DNA repair proteins, pATM and H2AX, that increase repair signaling and recognition respectively. Therefore, targeting these proteins in tumor cells to impede resistance is of high importance.
Bisphosphonates such as Zoledronic acid are widely used in the treatment of bone loss-related diseases. Statins, such as Pravastatin, are used in the management of lipid levels. Recently, a combination of both ZoPra was shown to radioprotect normal tissues and radiosensitize cancer cells.
Therefore, the aim of this study is to assess the effect of Zoledronic acid and Pravastatin, alone and in combination on the radio-response of human prostate cancer cell lines, DU-145 and PC-3, in vitro, on a cellular and molecular level.
The cytotoxic effect of different concentrations of Zoledronic acid and Pravastatin were tested using MTT assay. The optimum concentration of both was determined to be 1 μM and was therefore used in further experiments. Cells were treated with 1 μM Zoledronic acid and Pravastatin, alone and in combination, prior to a 2 Gy irradiation. Clonogenic assay was performed to assess cell survival and colony forming ability in both cell lines with treatment. Immunofluorescence analysis of pATM and γH2AX was performed to study DNA DSB repair kinetics.
Pre-treatment with 1 μM ZoPra prior to a 2 Gy irradiation was shown to radiosensitize DU-145 and PC3 cell lines. The treatment was shown to increase the residual number of γH2AX foci. A significant decrease in cell survival in both cell lines was observed. This study presents novel findings on the potential use of ZoPra as a radio-sensitizing agent for radio-resistant prostate cancer cells
Assessing the Radioprotecting Effect of ZoPra on Human Prostate Epithelial Cells and Human Fibroblast Cells.
No full textProstate cancer is the most prevalent cancer in men and the second leading cause of cancer-related deaths in men around the world. One of the first-line treatments for lowering tumor size and preventing further cancer development is radiotherapy. Its effectiveness is based on the patient's and tumor cells' radiosensitivity. The radioresistance of tumor cells and harmful effects on the surrounding normal cells, however, greatly limit it.
Radiation exposure to normal tissue can cause both acute and chronic toxicities. Radiotherapy induces DNA double strand breaks and the cell’s response translates into the activation of DSB repair through ATM nucleoshuttling and phosphorylation of H2AX Radioprotectors are chemicals that are meant to decrease the effects of radiation on normal tissues. Numerous disorders linked to bone loss are treated using bisphosphonates, such as zoledronic acid (Zo). Pravastatin (Pra) is one of many statins that are used to control lipid levels.
The ZoPra combination has been found to improve the speed of ATM nucleo-shuttling by blocking nucleus membrane farnesylation. This results in faster and better DSB signaling, which improves the cell's ability to repair DNA. As a result, the combination of Zoledronate and Pravastatin may have a radio protecting effect on normal prostate and fibroblast cells.
Therefore, the aim of this study is to assess the radioprotecting effect of Zoledronic acid and Pravastatin, alone and in combination on normal epithelial human prostate cell line, RWPE-1, in vitro, on a cellular and molecular level.
The cytotoxic effect of different concentrations of Zo and Pra was tested using MTT assay. The optimum concentration of both was determined to be 1 μM and was therefore used in further experiments. Cells were treated with 1 μM Zoledronic acid and Pravastatin, alone and in combination, prior to a 2 Gy irradiation. Clonogenic assay was performed to assess cell survival and colony forming ability with treatment. Immunofluorescence analysis of pATM and γH2AX was performed to study DNA DSB repair kinetics.
Pre-treatment with 1 μM ZoPra prior to a 2 Gy irradiation was shown to decrease the residual number of γH2AX foci. However no significant change was observed in cell survival of RWPE-1 cells. This study presents novel findings on the potential use of ZoPra as a radioprotecting agent for normal human prostate epithelial cells
Assessing the Anticancer Potential of ONC201 and ONC206 Imipridones on Human Prostate Cancer Using 2D and 3D Cell Models
No full textBackground: Prostate cancer (PC) is the second most commonly diagnosed cancer and the fifth leading cause of cancer-related deaths among men worldwide. Although many therapeutic approaches have been used to manage PC, the disease often develops resistance and progresses into an aggressive and lethal state, known as metastatic castration-resistant prostate cancer (mCRPC). Therefore, defining new targets and elucidating novel therapeutics for treating and managing PC are of utmost priority. Imipridones represent a novel class of anti-cancer compounds that showed promising results in several cancer types. ONC201, the first-in-class clinical imipridone, showed to have anticancer effects in PC. ONC206, an analog derivative of ONC201, possesses enhanced nanomolar potency against several cancers. However, there are no published studies assessing the anti-cancer activity of ONC206 in PC. With ONC206 being more potent than ONC201, it might be able to target the tumors that have acquired therapy resistance mechanisms.
Objective: The aim of this study is to investigate the anti-cancer potential of ONC206, in comparison to ONC201, on human PC using two-dimensional (2D) and three-dimensional (3D) in vitro cell models.
Methods: ONC201 and ONC206 drugs were tested on two PC cell lines (DU145 and PC3) using several in vitro assays. MTT assay was performed to evaluate the cytotoxic effect of a wide range of concentrations of ONC201 and ONC206 on PC cells. Trypan blue exclusion assay was then used to assess the effect of both drugs on cellular viability. In addition, cell migration ability was investigated using the “wound-healing” scratch assay. Furthermore, the 3D sphere-forming assay was applied to examine the effect of both drugs in targeting the enriched population of PC stem/progenitor cells.
Results: Our MTT data showed that ONC206 exerts a more potent cytotoxic effect on the DU145 and PC3 cell lines compared to ONC201, in a time and dose-dependent manner. These results were confirmed through the trypan-blue viability assay. Similarly, ONC206 displayed a more significant attenuation in the migration ability of PC cells in comparison to ONC201. Importantly, these results were validated in a 3D culture system with the sphere-forming assay, where both imipridones decreased the size and the sphere forming ability of prostatospheres. ONC206 was also more potent than ONC201 in targeting the subpopulation of prostate cancer stem cells.
Conclusion: Imipridones represent a novel therapeutic approach for the management of cancer. Our data shows that ONC206, the analog derivative of ONC201, shows more potent anti-cancer effects on PC cells at nanomolar concentrations, paving the way for new effective therapeutics and better clinical management of PC
In Vitro Assessment of the Radiosensitivity of Friedreich’s Ataxia Fibroblasts
No full textIntroduction: Friedreich’s Ataxia (FRDA) is a neurodegenerative disorder that stands as the most common form of inherited ataxia. FRDA affects 15,000 individuals worldwide, with approximately 250 documented cases in Lebanon. FRDA is an autosomal recessive disorder caused by a GAA triplet expansion on the FXN gene, that encodes for the protein Frataxin. The onset of symptoms typically manifests in childhood or early adolescence, Rare studies have shown that individuals with FRDA have an increased radiosensitivity compared to the normal population. In fact, radiation or exposure to any genotoxic stress induces different types of DNA damage, with double-strand breaks (DSBs) being the most lethal. Recent advancements in radiobiology have highlighted the importance of assessing the kinetics of the DSBs damage and repair pathway proteins in assessing radiosensitivity.
Aim: To assess the in vitro radiosensitivity of FRDA fibroblasts.
Methods: Five human skin fibroblasts (three FRDA and two normal fibroblasts) were used in this study. Cells were exposed to a 2 Gy irradiation, and the DNA DSB signaling and repair pathways were analyzed by anti-pATM and anti-γH2AX immunofluorescence. The clonogenic assay was performed to characterize the cellular radiosensitivity.
Results: FRDA cell lines exhibited an impaired DSB repair mechanism when compared with the normal cell lines. Additionally, the post-irradiation pATM activity was significantly lower in FRDA cell lines than in normal fibroblasts.
Conclusion: Our results show that FRDA cells have a significant radiosensitivity caused by the impairment of their DNA DSB repair mechanism. These findings, if confirmed by other studies, will lead to the modification and development of FRDA treatment strategies that will take into account their increased radiosensitivity
Novel Role for Sphingomyelin Phosphodiesterase Acid-like 3b in Radiation Nephropathy
No full textBackground: Radiation nephropathy remains a challenging complication for cancer patients who receive abdominal or genitourinary radiation therapy (RT). The kidneys are highly radiosensitive organs that impose an ablative dose limitation. Sphingolipids are key bioactive lipids that cross-talk with DNA damage response (DDR) effectors to determine cell fate after genotoxic injuries. The specific expression of sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in podocytes modulates stress signaling and ceramide-1-phosphate (C1P) levels. Prior work suggested that radiation-induced loss of SMPDL3b mediates podocyte injury through cytoskeletal remodeling, filopodia effacement, and altering sphingolipids homeostasis. However, the molecular mechanisms involved in radiation podocytopathy remain to be further explored.
Aim: Our study investigates the role of SMPDL3b in regulating the DDR of renal podocytes after radiation injury.
Methods: Wild-type (WT), SMPDL3b overexpressors (OE), and SMPDL3b knock-down (KD) human podocytes were used as in vitro models for this study and were exposed to a single radiation dose of 2 Gy. We assessed the kinetics of DNA double-strand breaks (DSBs) recognition and repair along with ATM pathway activation post-irradiation. We also assessed the extent of DNA damage repair and apoptosis in an in vivo model using C57BL6 WT and podocyte-specific SMPDL3b-knock out (KO) mice at 24 hours after a single 14 Gy dose of focal renal irradiation. Additionally, we examined the effect of SMPDL3b expression on nuclear sphingolipids and nuclear membrane fluidity along with their impact on the DDR.
Results: SMPDL3b overexpression enhanced DSBs recognition and repair through the modulation of ATM nuclear shuttling and pathway activation. On the other hand, a significant delay in DSBs repair was observed when SMPDL3b was knocked down in vitro and in vivo. Ionizing Radiation (IR) altered the expression and subcellular localization of SMPDL3b from the lipid rafts of plasma membranes to the perinuclear and nuclear regions. Moreover, the expression of SMPDL3b regulated the nuclear membrane fluidity by altering its sphingomyelin content. Furthermore, SMPDL3b overexpression prevented radiation-induced alterations in the nuclear levels of C1P and ceramide. Exogenous administration of C1P radiosensitized OE podocytes by delaying ATM foci formation and activity, and subsequent DSBs repair. Conversely, pretreatment with ceramide kinase inhibitor (CERKI) radioprotected WT podocytes by enhancing ATM foci formation and activity, DSBs repair, and cell survival. These results suggest potential roles for the SMPDL3b and CERK/C1P axes in modulating radiation-induced podocyte injury.
Conclusion: We suggest that SMPDL3b regulates nuclear membrane fluidity, nuclear sphingolipids, ATM nuclear shuttling and pathway activation, DSBs repair, and consequently podocytes survival. The current work unmasks novel roles for SMPDL3b and C1P in radiation-induced DDR and paves the way towards further investigations on promising, novel therapeutic targets that may prevent radiation nephropathy
Repurposing Piroxicam Enhances the Antineoplastic Effects of Docetaxel and Enzalutamide in Prostate Cancer Cells In Vitro
No full textIntroduction:
Ranked second most diagnosed cancer and the fifth leading cause of cancer-related deaths
among men, prostate cancer (PCa) constitutes a major medical health issue. Despite the fact that many
therapeutic approaches exist against PCa, most cases often develop into a more advanced and lethal
stage known as metastatic castration-resistant prostate cancer (mCRPC). Hence, unfolding new
therapeutics to treat and manage PCa is of crucial importance. Drug repurposing is a growing research
field based on using previously approved drugs with known pharmacokinetic and pharmacodynamic
characteristics, for indications other than their traditional ones, like cancer. Non-steroidal anti-
inflammatory drugs (NSAIDs) have been studied as anti-neoplastic agents in several malignancies.
Objective:
In this study, we are interested in assessing the anti-tumorigenic effects of the NSAID
Piroxicam (PXM), alone and in combination with conventional therapies, on two PCa cell lines in vitro.
Material and Methods:
PXM, alone and in combination with conventional therapies (docetaxel and enzalutamide), will be tested on two cell lines
established in our laboratory that have the same defined Pten‑/‑TP53‑/‑ background representing two
different stages of PCa disease: PLum-AD (androgen-dependent) and PLum-AI (androgen-
independent). MTT assay will be performed to screen the cytotoxic effect of a wide battery of PXM
concentrations on those two cell lines. Then, trypan blue exclusion assay will be done to study PXM’s
effect on cell viability. Afterwards, wound healing will be used to study the ability of PXM in inhibiting
cell migration. Furthermore, 3D-sphere formation assay will be performed to assess the effect of PXM
in targeting PCa progenitor/stem cells.
Results/anticipated results:
MTT preliminary results showed that PXM has a dose-dependent cytotoxic effect and a synergistic effect with both conventional drugs on PCa cells.
Similar effects are obtained using trypan blue exclusion method. In addition, PXM inhibits and synergistically increases the effect of docetaxel and enzalutamide on migratory index of PCa cells and the sphere-forming abilities of cells.
Conclusion:
NSAIDs represent a potential candidate to be repurposed against PCa. We expect PXM, an
FDA-approved drug, to show anti-cancer effects against PCa cells including progenitor cells, and to
enhance the effect of already existing PCa treatments, overcoming resistance obstacles limiting
conventional treatments leading to a more efficient management of the disease
Assessing the Effects of Thymoquinone (TQ) alone and in Combination with Cisplatin (CDDP) on Ovarian Cancer Cells
No full textBackground: Ovarian cancer, the most lethal gynecologic malignancy, ranks among the top five causes of cancer-related deaths in women. The high mortality rate is attributed to late-stage diagnosis, the absence of effective public screening methods, and resistance to platinum-based treatments. Consequently, combination drug therapy emerges as a promising avenue in ovarian cancer management, aiming to augment therapeutic efficacy and circumvent resistance to conventional therapies. Thymoquinone (TQ), a phytochemical compound derived from the Nigella sativa herb, has demonstrated anti-tumorigenic and anti-proliferative properties across various cancer types on ovarian cancer specifically when combined with platinum agents such as cisplatin (CDDP). Therefore, this study seeks to explore the impact of TQ on ovarian cancer cells, both alone and in combination with CDDP. Methods: Two ovarian cancer cell lines OVCAR-420 and SKOV-3 were used in this study. A range of two-dimensional (2D) in vitro assays, including MTT, trypan blue assay, and wound healing, were used to assess cell proliferation, viability, and migration respectively. Additionally, a three-dimensional (3D) MatrigelTM-based cell culture assay was employed to evaluate stemness. Both TQ and CDDP were administered as monotherapies and in combination to ascertain their effectiveness. Results: Our results revealed that TQ, both alone and in combination with cisplatin, significantly and synergistically reduced the proliferation, viability, migration, and sphere growth of ovarian cancer cells. TQ specifically at 25μM for OVCAR-420 and 10μM for SKOV-3, significantly lowered the concentration of CDDP when used in combination enhancing the susceptibility of cancer cells to treatment. Conclusion: Our findings show a strong synergistic effect between TQ and CDDP on ovarian cancer cells. Further validation of these findings could pave the way for a more potent therapeutic strategy for women diagnosed with ovarian cancer
Radiation-Induced Fibrosis in Patients with Head and Neck Cancer: A Review of Pathogenesis and Clinical Outcomes
No full textRadiotherapy-related fibrosis remains one of the most challenging treatment related side effects encountered by patients with head and neck cancer. Several established and ongoing novel therapies have been studied with paucity of data in how to best treat these patients. This review aims to provide researchers and health care providers with a comprehensive review on the presentation, etiology, and therapeutic options for this serious condition. © The Author(s) 2021
Seventy Years of Dose-response Models: From the Target Theory to the Use of Big Databases Involving Cell Survival and DNA Repair
No full textInternational audienceRadiobiological data, whether obtained at the clinical, biological or molecular level has significantly contributed to a better description and prediction of the individual dose-response to ionizing radiation and a better estimation of the radiation-induced risks. Particularly, over the last seventy years, the amount of radiobiological data has considerably increased, and permitted the mathematical formulas describing dose-response to become less empirical. A better understanding of the basic radiobiological mechanisms has also contributed to establish quantitative inter-correlations between clinical, biological and molecular biomarkers, refining again the mathematical models of description. Today, big data approaches and, more recently, artificial intelligence may finally complete and secure this long process of thinking from the multi-scale description of radiation-induced events to their prediction. Here, we reviewed the major dose-response models applied in radiobiology for quantifying molecular and cellular radiosensitivity and aimed to explain their evolution: Specifically, we highlighted the advances concerning the target theory with the cell survival models and the progressive introduction of the DNA repair process in the mathematical models. Furthermore, we described how the technological advances have changed the description of DNA double-strand break (DSB) repair kinetics by introducing the important notion of DSB recognition, independent of that of DSB repair. Initially developed separately, target theory on one hand and, DSB recognition and repair, on the other hand may be now fused into a unified model involving the cascade of phosphorylations mediated by the ATM kinase in response to any genotoxic stress
