scholarly journals Hadron Therapy, Magnetic Nanoparticles and Hyperthermia: A Promising Combined Tool for Pancreatic Cancer Treatment

Nanomaterials ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1919 ◽  
Author(s):  
Francesca Brero ◽  
Martin Albino ◽  
Antonio Antoccia ◽  
Paolo Arosio ◽  
Matteo Avolio ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Magnetic Nanoparticles ◽  
Ion Irradiation ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Magnetic Fluid Hyperthermia ◽  
Strand Breaks

A combination of carbon ions/photons irradiation and hyperthermia as a novel therapeutic approach for the in-vitro treatment of pancreatic cancer BxPC3 cells is presented. The radiation doses used are 0–2 Gy for carbon ions and 0–7 Gy for 6 MV photons. Hyperthermia is realized via a standard heating bath, assisted by magnetic fluid hyperthermia (MFH) that utilizes magnetic nanoparticles (MNPs) exposed to an alternating magnetic field of amplitude 19.5 mTesla and frequency 109.8 kHz. Starting from 37 °C, the temperature is gradually increased and the sample is kept at 42 °C for 30 min. For MFH, MNPs with a mean diameter of 19 nm and specific absorption rate of 110 ± 30 W/gFe3o4 coated with a biocompatible ligand to ensure stability in physiological media are used. Irradiation diminishes the clonogenic survival at an extent that depends on the radiation type, and its decrease is amplified both by the MNPs cellular uptake and the hyperthermia protocol. Significant increases in DNA double-strand breaks at 6 h are observed in samples exposed to MNP uptake, treated with 0.75 Gy carbon-ion irradiation and hyperthermia. The proposed experimental protocol, based on the combination of hadron irradiation and hyperthermia, represents a first step towards an innovative clinical option for pancreatic cancer.

scholarly journals DUSP6 regulates radio-sensitivity in glioblastoma by modulating the recruitment of p-DNAPKcs at DNA double-strand breaks

2021 ◽  
Author(s):  
Jyothi Nair ◽  
Safiulla Basha Syed ◽  
Tejashree Mahaddalkar ◽  
Madhura Ketkar ◽  
Rahul Thorat ◽  
...  
Keyword(s):  
Progression Free Survival ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Canonical Function ◽  
Free Survival ◽  
Strand Breaks ◽  
Radio Sensitivity ◽  
Recurrent Gbm

Glioblastoma (GBM) has poor median survival due to its resistance to chemo-radiotherapy regimen, resulting in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is up-regulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies (n=11) compared to primary biopsies (n=11). Importantly, although reported as cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse population of GBM cell lines generated from in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radio-sensitized primary and importantly, relapse GBM cells by inhibiting the recruitment of p-DNAPKcs, subsequently down-regulating the recruitment of γH2AX and 53BP1. This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.

scholarly journals Phytotherapeutics oridonin and ponicidin show additive effects combined with irradiation in pancreatic cancer in vitro

2017 ◽  
Vol 51 (4) ◽  
pp. 407-414 ◽  
Author(s):  
Jakob Liermann ◽  
Patrick Naumann ◽  
Franco Fortunato ◽  
Thomas E. Schmid ◽  
Klaus-Josef Weber ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Additive Effects ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Double Strand Break

Abstract Background Chemoradiation of locally advanced non-metastatic pancreatic cancer can lead to secondary operability by tumor mass reduction. Here, we analyzed radiomodulating effects of oridonin and ponicidin in pancreatic cancer in vitro. Both agents are ent-kaurane diterpenoids, extracted from Isodon rubescens, a plant that is well known in Traditional Chinese Medicine. Cytotoxic effects have recently been shown in different tumor entities for both agents. Materials and methods Pancreatic cancer cell lines AsPC-1, BxPC-3, Panc-1 and MIA PaCa-2 were pretreated with oridonin or ponicidin and irradiated with 2 Gy to 6 Gy. Long-term survival was determined by clonogenic assay. Cell cycle effects and intensity of γH2AX as indicator for DNA double-strand breaks were investigated by flow cytometry. Western blotting was used to study the DNA double-strand break repair proteins Ku70, Ku80 and XRCC4. Results Oridonin and ponicidin lead to a dose-dependent reduction of clonogenic survival and an increase in γH2AX. Combined with irradiation we observed additive effects and a prolonged G2/M-arrest. No relevant changes in the levels of the DNA double-strand break repair proteins were detected. Conclusions Pretreatment with oridonin or ponicidin followed by irradiation lead to an additional reduction in survival of pancreatic cancer cells in vitro, presumably explained by an induced prolonged G2/M-arrest. Both agents seem to induce DNA double-strand breaks but do not interact with the non-homologous end joining (NHEJ) pathway.

scholarly journals RepID-deficient cancer cells are sensitized to a drug targeting p97/VCP segregase

2021 ◽  
Author(s):  
Sang-Min Jang ◽  
Christophe E. Redon ◽  
Haiqing Fu ◽  
Fred E. Indig ◽  
Mirit I. Aladjem
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Cell Sorting ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ubiquitinated Proteins ◽  
Damage Response ◽  
Survival Assay

Abstract Background The p97/valosin-containing protein (VCP) complex is a crucial factor for the segregation of ubiquitinated proteins in the DNA damage response and repair pathway. Objective We investigated whether blocking the p97/VCP function can inhibit the proliferation of RepID-deficient cancer cells using immunofluorescence, clonogenic survival assay, fluorescence-activated cell sorting, and immunoblotting. Result p97/VCP was recruited to chromatin and colocalized with DNA double-strand breaks in RepID-deficient cancer cells that undergo spontaneous DNA damage. Inhibition of p97/VCP induced death of RepID-depleted cancer cells. This study highlights the potential of targeting p97/VCP complex as an anticancer therapeutic approach. Conclusion Our results show that RepID is required to prevent excessive DNA damage at the endogenous levels. Localization of p97/VCP to DSB sites was induced based on spontaneous DNA damage in RepID-depleted cancer cells. Anticancer drugs targeting p97/VCP may be highly potent in RepID-deficient cells. Therefore, we suggest that p97/VCP inhibitors synergize with RepID depletion to kill cancer cells.

scholarly journals Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

2021 ◽  
Vol 22 (14) ◽  
pp. 7638
Author(s):  
Yvonne Lorat ◽  
Judith Reindl ◽  
Anna Isermann ◽  
Christian Rübe ◽  
Anna A. Friedl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dna Damage ◽  
Spatial Clustering ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Stimulated Emission ◽  
Nuclear Chromatin ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

scholarly journals Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

2021 ◽  
Author(s):  
Xinrui Zhang ◽  
Mariana Bobeica ◽  
Michael Unger ◽  
Anastasia Bednarz ◽  
Bjoern Gerold ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Metabolic Activity ◽  
Focused Ultrasound ◽  
Double Strand Breaks ◽  
High Intensity Focused Ultrasound ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

scholarly journals In Vitro Evaluation of No-carrier-added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons

2021 ◽  
Author(s):  
Honoka Obata ◽  
Atsushi B. Tsuji ◽  
Hitomi Sudo ◽  
Aya Sugyo ◽  
Katsuyuki Minegishi ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Short Range ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Primary Target ◽  
Auger Electrons ◽  
Strand Breaks ◽  
Chemical Damage ◽  
No Carrier Added

Due to their short range (2–500 nm), Auger electrons (Auger e-) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Au-ger e-, it remains challenging to maximize the interaction between Auger e- and DNA. To assess the DNA-damaging effect of Auger e- released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by im-munofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incuba-tion), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by release of Auger e- very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e-.

scholarly journals DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Xueqiu Chen ◽  
Yimin Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxoplasma Gondii ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

scholarly journals A global view of meiotic double-strand break end resection

Science ◽  
2017 ◽  
Vol 355 (6320) ◽  
pp. 40-45 ◽  
Author(s):  
Eleni P. Mimitou ◽  
Shintaro Yamada ◽  
Scott Keeney
Keyword(s):  
Meiotic Recombination ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Naked Dna ◽  
Global View ◽  
Genome Wide ◽  
End Resection

DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution inSaccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.

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