scholarly journals Role of P53 protein in activation of atm- and parp-mediated dna damage repair (DDR) pathways induced by topoisomerase type II inhibitors

2016 ◽  
Vol 97 (2) ◽  
pp. 245-249
Author(s):  
B R Ramazanov ◽  
R R Khusnutdinov ◽  
A R Galembikova ◽  
P D Dunaev ◽  
S V Boichuk

Aim. To study the mechanisms of doxorubicin genotoxic effects in terms of poly-(ADP)-ribose-polymerase (PARP) and the ATM-kinase (Ataxia Telangiectasia Mutated) inhibition in cell lines with different p53 status.Methods. The study was conducted on BJ and BJp53DD human fibroblasts cell lines, cultured in DMEM medium supplemented with fetal bovine serum, L-glutamine and antibiotics. Inhibition of PARP and ATM-kinase activity was attained by adding synthetic inhibitors Nu1025 and Ku55933 respectively. Chemotherapy drug doxorubicin was used to induce deoxyribonucleic acid (DNA) damages. Cell viability analysis was performed using MTS-test. Repair system proteins and apoptotic markers expression was assessed by western blotting. Cells distribution by cell cycle phases was performed by flow cytometry.Results. Adding PARP and ATM-kinase inhibitors to the BJ p53DD cell line culture resulted in a significant reduction in the viable cells number amid DNA damage induction caused by doxorubicin. Cell death in these samples occurs according to the apoptosis mechanism, what was confirmed by the increase in hypodiploid cells number and increased expression of cleaved forms of PARP-1 and caspase-3. The above-described effects of the type II topoisomerase inhibitor doxorubicin were significantly higher in BJ fibroblasts line with non-functional p53 protein (p53DD) compared with conventional BJ human fibroblasts line.Conclusion. In the context of the failure of p53-dependent mechanisms of cell cycle regulation in BJ p53DD human fibroblasts, PARP and ATM-kinase activity inhibition leads to increased cell death by apoptosis mechanism induced by the doxorubicin action.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5157-5157
Author(s):  
Choon Kee Lee ◽  
Shuiliang Wang ◽  
Xiaoping Huang ◽  
John Ryder ◽  
Peter Ordentlich ◽  
...  

Abstract One of the main mechanisms of action of HDAC inhibitors is the transcriptional reactivation of dormant tumor-suppressor genes through acetylation of histones, thereby inducing apoptosis. Treatment with HDACI has also been shown to induce chromatin destabilization in a transcription independent way. In the current study, we sought to determine whether HDAC inhibition induces DNA damage and amplifies alkylator-induced mitotic cell death in both melphalan sensitive- and resistant-MM cell lines (RPMI8226, 8226/LR5). The IC50 values of SNDX275, a class I HDACI agent, and melphalan on the 72-hour MTT assay were 268.05 nM and 245.94 nM in the RPMI8226, and 309.91 nM and 8657.46 nM in the 8226/LR5, respectively. When combined together at clinically attainable concentrations, the combination index by the Chou-Talalay method ranged from 0.27 to 0.75 for the RPMI8226 and from 0.33 to 0.7 for the 8226/LR5, indicating a powerful synergism. For elucidation of molecular mechanisms, MM1S and RPMI8226 cell lines were investigated for apoptosis, histone acetylation, cell cycle analysis, DNA double strand break and DNA damage response serially in 48-hour culture with SNDX-275 at 500 nM and melphalan at 10 μM, alone and in combination. Cleavage of PARP was seen following treatment with each SNDX275 and melphalan, but was highest at 48 hours with the combination of both. Apoptosis was associated with cleavage of caspases of 8, 3 and 9, which was most intense on combination. Melphalan amplified SNDX275-induced acetylation of H3. In cell cycle analysis by flow cytometry, SNDX275 caused an increase in G0-G1 and a decrease in S and G2-M. Cyclin D1, E2F-1 and p53 on western blot were not affected but expression of p21 increased. Melphalan arrested the cell cycle at G2, increased expression of p53 in the RPMI8226 and of p21 in the MM1S. The combination intensified the increase in p21 in both cell lines and in p53 only in the RPMI8226. Phosphorylation of H2AX, a marker of DNA double strand break, increased in a time dependent manner following each drug, along with an increase in phosphorylation of CHK1 and CHK2, indicative of initiation of DNA damage response. The increase in γH2AX and pCHK1 & 2, however, was considerably higher on combination than each drug alone. Furthermore, morphologic assessment of dead cells by the 48 hours of culture revealed a significant increase in mitotic catastrophe on combination in the MM1S: 0% on SNDX275 alone; 10% on melphalan alone; 43.4% on combination. The current study suggests that HDAC inhibition synergizes with melphalan in MM cells and that intensification of DNA damage is one of the mechanisms. Further studies are necessary to understand the role of HDAC inhibition for induction of mitotic catastrophe.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4505-4505 ◽  
Author(s):  
Jeffrey D. Altenburg ◽  
Shuhong Zhang ◽  
Michelle Grimard ◽  
Xingkui Xue ◽  
Sherif S Farag

Abstract The PIM kinases are a family of proteins recently identified as promising therapeutic targets in several cancers, including pancreatic, B-cell malignancies, acute leukemia, and prostate cancer among others. The family of PIM kinases is composed of three different members (PIM-1, -2, and -3) that are short-lived serine/threonine kinases involved in the regulation of a number of cellular pathways that are important for cancer cell growth and survival. The PIM kinases show high homology with each other, and exhibit functional redundancy in vitro and in vivo. Overexpression of PIM kinases promotes tumor growth through activation of several key cell-cycle progression and anti-apoptotic proteins, including BAD, p21, p27KIP, c-Myc, and AKT-1. Recently, overexpression of PIM-2 has been shown to have a protective effect against ultraviolet light induced DNA damage (Zirkin et al. J Biol Chem288:21770-83, 2013). We investigated the protective role of PIM kinases in chemotherapy-induced DNA damage, and whether inhibition of PIM kinases enhances anthracycline-induced DNA damage by inhibiting DNA repair, thus enhancing cell death in lymphoma cells lines. Using immunobloting and RT-PCR, we found similarly low levels of PIM-1 and PIM-3, but a wide range of PIM-2 expression, in a panel of non-Hodgkin lymphoma (NHL) cell lines, including Raji, HS Sultan, Daudi, Farage, Granta519, and Toledo. Treatment of cells with doxorubicin (200-400 nM) resulted in up to a five fold increase gene transcription and expression of PIM-1 and PIM-2, which was maximal at 6 hours, and was associated with an increase in DNA damage as detected using acridine orange flow cytometry assay. We also tested the single agent effect of the pan-PIM kinase inhibitor, CX6258 on the cell lines. CX6258 alone inhibited cell growth in all NHL cell lines with varying degrees of potency with IC50ranging from 0.2 – 12.9 µM. The anti-cancer was associated most with PIM-2 expression, with the most sensitive cell lines, Daudi and Toledo, expressing the most PIM-2. Suppression of PIM-2 expression by shRNA significantly decreased proliferation, indicating that PIM-2 is a significant factor in cell growth. Treatment of NHL cells with CX6258 resulted in increased caspase-3 activation and PARP cleavage, decreased BAD phosphorylation, and apoptosis. Treatment with CX6258 also increased expression of p21, decreased expression of cyclins A1 and B1, and induced G2-M cell cycle arrest. The effect of combinations of CX6258 (5-50 µM) and doxorubicin (50-500 nM) on DNA damage and cell death was tested on HS sultan and Daudi cells. While doxorubicin alone resulted in a two-fold increase in DNA damage, this was significantly increased in the presence of CX6258 (12 fold). The addition of CX6258 inhibited the phosphorylation of the DNA repair proteins H2.AX, ATM, and Chk2 that occurred when the cells were treated with doxorubicin alone. The combination of CX6258 and doxorubicin was synergistic in inducing lymphoma cell death, with combination indexes ranging from 0.32-0.85. Our findings suggest a mechanism for synergy where doxorubicin damages cellular DNA and initiates the DNA damage response, while CX6258 inhibits the upregulated PIM kinases from activating the proteins involved in the response. This synergistic anti-tumor activity is further strengthened by the CX6258 inhibition of cell cycle progression and anti-apoptotic proteins activated by the PIM kinases. Taken together, our results provide pre-clinical rationale for clinical testing of PIM kinase inhibitors in combination with doxorubicin in patients with NHL. It also suggests that CX6258 may similarly enhance the anti-cancer effects of other DNA damaging agents. Disclosures No relevant conflicts of interest to declare.


Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1768
Author(s):  
Cerquetti ◽  
Bucci ◽  
Carpinelli ◽  
Lardo ◽  
Proietti ◽  
...  

Mitotane (MTT) is an adrenolytic drug used in adjuvant and advanced treatments of adrenocortical carcinoma (ACC). Ionizing radiation (IR) is also used in adrenal cancer treatment, even though its biological action remains unknown. To provide a reliable in vivo preclinical model of ACC, we used mouse xenografts bearing human ACC to test the effects of MTT and IR alone and in combination. We evaluated tumor growth inhibition by the RECIST criteria and analyzed the cell cycle by flow cytometry (FCM). In the xenograft ACC model treated with MTT/IR in combination, we observed a marked inhibition of tumor growth, with strong tumor regression (p < 0.0001) compared to MTT and IR given alone (p < 0.05). The MTT results confirm its antisteroidogenic activity (p < 0.05) in the xenograft ACC model, revealing its ability to render cancer cells more prone to radiotherapy treatment. In addition, to explain the biological effect of these treatments on the Mismatch Repair System (MMR), we interfered with the MSH2 gene expression in untreated and MTT/IR-treated H295R and SW13 cell lines. Moreover, we observed that upon treatment with MTT/IR to induce DNA damage, MSH2 gene inhibition in both the H295R and SW13 cell lines did not allow DNA damage repair, thus inducing cell death. In conclusion, MTT seems to have a radiosensitizing property and, when given in combination with IR, is able to promote neoplastic growth inhibition, leading to a significant reduction in tumor size due to cell death.


2018 ◽  
Vol 18 (5) ◽  
pp. 739-746 ◽  
Author(s):  
Raj Kaushal ◽  
Nitesh Kumar ◽  
Archana Thakur ◽  
Kiran Nehra ◽  
Pamita Awasthi ◽  
...  

Abstract: Background: After the discovery of cisplatin, first non platinum anticancer drugs having excellent efficacy were budotitane and TiCl2(cp)2 but action mechanism is not clear. Therefore, we hereby reporting synthesis and biological activities novel titanium complexes to explore their mode of action. Objectives: Synthesis, spectral characterization, antibacterial and anticancer activity of some titanium complexes. Antibacterial studies on various bacterial strains and anticancer studies on HeLa, C6, CHO cancerous cell lines have been performed. Further, the cell death mechanistic study was done on CHO cell lines. Method: Titanium complexes with and without labile groups have been synthesized by reacting of TiCl4 with nitrogen containing ligands viz. 1,2-diaminocyclohexane, 1,10-Phenanthroline, adamantylamine, 2,2'-bipyridine, 4,4'-dimethyl-2,2'-bipyridine in predetermined molar ratios. Antibacterial and anticancer studies were performed by agar well diffusion method and MTT assay respectively. Cell cycle analysis is done by using flow cytometry. Results: Complex 2 i.e TiCl2(Phen)2 showed better activity than other complexes as an antibacterial as well as anticancer agent. Phase contrast imaging indicates that observed morphological changes of cells was dose dependent. Cell death mechanistic study have shown the increase in sub G0 phase population as well as formation of blebbing and fragmentation of chromatin material which is an indicative measure of apoptosis. Conclusion: Complex 2 proved to be more effective bactericide and cytotoxic agent. Cell cycle analysis showed cell arrest in G0 phase. Apoptosis percentage was found to increase in a dose dependent manner. So, prepared titanium complexes can be put to use as an important chemotherapeutic agents.


2019 ◽  
Vol 18 (10) ◽  
pp. 1457-1468
Author(s):  
Michelle X.G. Pereira ◽  
Amanda S.O. Hammes ◽  
Flavia C. Vasconcelos ◽  
Aline R. Pozzo ◽  
Thaís H. Pereira ◽  
...  

Background: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. Objectives: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. Methods: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. Results: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. Conclusion: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kalyan Mahapatra ◽  
Sujit Roy

AbstractAs like in mammalian system, the DNA damage responsive cell cycle checkpoint functions play crucial role for maintenance of genome stability in plants through repairing of damages in DNA and induction of programmed cell death or endoreduplication by extensive regulation of progression of cell cycle. ATM and ATR (ATAXIA-TELANGIECTASIA-MUTATED and -RAD3-RELATED) function as sensor kinases and play key role in the transmission of DNA damage signals to the downstream components of cell cycle regulatory network. The plant-specific NAC domain family transcription factor SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) plays crucial role in transducing signals from both ATM and ATR in presence of double strand breaks (DSBs) in the genome and found to play crucial role in the regulation of key genes involved in cell cycle progression, DNA damage repair, endoreduplication and programmed cell death. Here we report that Arabidopsis exposed to high salinity shows generation of oxidative stress induced DSBs along with the concomitant induction of endoreduplication, displaying increased cell size and DNA ploidy level without any change in chromosome number. These responses were significantly prominent in SOG1 overexpression line than wild-type Arabidopsis, while sog1 mutant lines showed much compromised induction of endoreduplication under salinity stress. We have found that both ATM-SOG1 and ATR-SOG1 pathways are involved in the salinity mediated induction of endoreduplication. SOG1was found to promote G2-M phase arrest in Arabidopsis under salinity stress by downregulating the expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1, and CYCB1;1, while upregulating the expression of WEE1 kinase, CCS52A and E2Fa, which act as important regulators for induction of endoreduplication. Our results suggest that Arabidopsis undergoes endoreduplicative cycle in response to salinity induced DSBs, showcasing an adaptive response in plants under salinity stress.


Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 925
Author(s):  
Eva-Maria Faulhaber ◽  
Tina Jost ◽  
Julia Symank ◽  
Julian Scheper ◽  
Felix Bürkel ◽  
...  

(1) Kinase inhibitors (KI) targeting components of the DNA damage repair pathway are a promising new type of drug. Combining them with ionizing radiation therapy (IR), which is commonly used for treatment of head and neck tumors, could improve tumor control, but could also increase negative side effects on surrounding normal tissue. (2) The effect of KI of the DDR (ATMi: AZD0156; ATRi: VE-822, dual DNA-PKi/mTORi: CC-115) in combination with IR on HPV-positive and HPV-negative HNSCC and healthy skin cells was analyzed. Cell death and cell cycle arrest were determined using flow cytometry. Additionally, clonogenic survival and migration were analyzed. (3) Studied HNSCC cell lines reacted differently to DDRi. An increase in cell death for all of the malignant cells could be observed when combining IR and KI. Healthy fibroblasts were not affected by simultaneous treatment. Migration was partially impaired. Influence on the cell cycle varied between the cell lines and inhibitors; (4) In conclusion, a combination of DDRi with IR could be feasible for patients with HNSCC. Side effects on healthy cells are expected to be limited to normal radiation-induced response. Formation of metastases could be decreased because cell migration is impaired partially. The treatment outcome for HPV-negative tumors tends to be improved by combined treatment.


2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.


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