Cytotoxicity Of Etoposide Research Articles

Methylselenocysteine Promotes Etoposide Cytotoxicity by Enhancing Homotypic Gap Junctions Composed of Connexin 26

To investigate the effect of methylselenocysteine (MSC) on the function of homotypic gap junction (GJ) composed of connexin (Cx) 26 and its regulation of chemotherapeutic drug cytotoxicity. The Tet-on HeLa cells transfected with and stably expressing Cx26 were used as the tool cells. Effects of MSC on cell growth, GJ function, and Cx26 protein expression were examined by MTT method, parachute assay, and Western blot analysis, respectively. The cytotoxicity of chemotherapeutic drugs was determined by standard colony-forming assay, and the relationship between MSC's effect on cytotoxicity of these chemotherapeutic drugs and its regulation of GJ was further analyzed. In Tet-on HeLa cells, doxycycline (Dox) can induce the expression of Cx26, which could then form functional GJs. Within a concentration range of 50 μmol/L, MSC had no significant effect on HeLa cell growth. Non-toxic concentrations of MSC can enhance GJs in a concentration-dependent manner and exert its effect at the nanomolar level. This effect was associated with an induction of Cx26 protein expression by MSC. Among the three common chemotherapeutic agents with different mechanisms of action, etoposide (Eto) presented cytotoxicity differences between HeLa cells cultured at low density (nonconfluent, no GJ formed) and high density (confluent, GJ formed). What's more, the inhibitory effect of Eto combined with MSC on HeLa cell colony formation was stronger than that of Eto alone, and this effect occurred only in HeLa cells with GJ formation. MSC can potentiate the cytotoxicity of Eto by enhancing the GJs composed of Cx26, indicating that combined strategy of selenide and chemotherapy shows potential value in the treatment of malignant tumors.

Effects of thymoquinone and etoposide combination on cell viability and genotoxicity in human cervical cancer hela cells

Background and Aims: It is thought that thymoquinone might have a crucial role in preventing DNA damage, regulating DNA repair mechanisms, and inhibiting the formation of a cancer. Studies on the cytotoxic and genotoxic effects of thymoquinone together with etoposide in cervical carcinoma cells (HeLa) are not adequate. The objective of this study is to evaluate the effect of combinations with thymoquinone on etoposide cytotoxicity and genotoxicity in HeLa cells. Methods: Cytotoxicity was evaluated by MTT assay and genotoxicity was determined by Comet assay. Results: The IC50 values of thymoquinone were 233.6 μM and 145.5 μM, and the IC50 values of etoposide were 167.3 μM and 52.7 μM for 24 and 48 h, respectively. Thymoquinone significantly decreased the approximate IC50 value of etoposide in doses of 15.63 μM and above for 24 h and 31.5 μM and above for 48 h in a dose-dependent manner. 0.1-5 μM thymoquinone and 1 μM etoposide alone did not cause DNA damage, but at higher doses increased DNA damage significantly in a dose-dependent manner. Thymoquinone significantly reduced DNA damage induced by 10 μM etoposide at the doses of 0.1-10 μM. Conclusion: Our results show that thymoquinone might increase the cytotoxic and genotoxic effects of etoposide in HeLa cells at high doses and reduce DNA damage at low doses that are not cytotoxic, which suggests that etoposide may increase its anticancer effect at high doses, but comprehensive studies are needed on this subject. This study is a preliminary study and will contribute to the development of new treatment strategies.

Etoposide in combination with erastin synergistically altered iron homeostasis and induced ferroptotic cell death through regulating IREB2/FPN1 expression in estrogen receptor positive-breast cancer cells

AimFerroptosis is an iron-dependent cell death mechanism that substantially differs from apoptosis. Since its mechanism involves increased oxidative stress and rich iron content, cancer cells are particularly vulnerable to ferroptotic death compared to healthy tissues. In the present study, the effect of etoposide in combination with a ferroptotic agent, erastin, was investigated in breast cancer. Main methodsCell viability was assessed by the MTT assay. Oxidative stress, lipid peroxidation and glutathione peroxidase activity were detected using the relevant kits. Intracellular iron levels were measured by HPLC. Ferroptosis markers were explored by western blotting. Key findingsResults demonstrated that although etoposide didn't induce a significant cell death up to 50 μM in MCF-7 cells, with the addition of erastin, a significant synergistic activity was achieved at a dose as low as 1 μM (p < 0.05), contrary to normal breast epithelial cells. This cytotoxic effect was blocked by ferrostatin-1, which is a specific inhibitor of ferroptosis. The combined treatment of etoposide and erastin synergistically induced oxidative stress and lipid peroxidation, while suppressing glutathione peroxidase activity. More importantly, the combination treatment synergistically increased iron accumulation, which was associated with altered expression of IREB2/FPN1. Additionally, ferroptosis-regulating proteins ACSF2 and GPX4 were altered more potently by the combination treatment, compared to untreated cells and erastin treatment alone (p < 0.05). SignificanceIn conclusion, this is the first study that reports enhanced cytotoxicity of etoposide, in combination with erastin, in ER-positive breast cancer cells via activation of ferroptotic pathways, and offers a new perspective for future regimens.

Discovery, enantioselective synthesis of myrtucommulone E analogues as tyrosyl-DNA phosphodiesterase 2 inhibitors and their biological activities

As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 2 (TDP2) can specifically repair topoisomerase 2 (TOP2)-mediated DNA damage, resulting in cancer cell resistance to TOP2 inhibitors. Its inhibitors can enhance the anticancer efficacy of TOP2 inhibitors. In this work, we report the discovery of natural product myrtucommulone E as selective TDP2 inhibitor and its first enantioselective total synthesis through a pivotal CPA-catalyzed Michael addition reaction. A series of myrtucommulone E analogues were asymmetrically synthesized and evaluated for TDP2 and TDP1 inhibitions, and cytotoxicity. Analogue (+)-29 shows good TDP2 inhibition potency (5.4 ± 0.25 μM), but no TDP1 inhibition at 100 μM concentration, and can significantly enhance the cytotoxicity of TOP2 inhibitor etoposide in both DU145 (CI = 0.26) and DT40 hTDP2 cells (CI = 0.48).

Cytotoxic triterpene and steroidal glycosides from the seeds of Digitalis purpurea and the synergistic cytotoxicity of steroidal glycosides and etoposide in SBC-3 cells

The phytochemical investigations of the seeds of Digitalis purpurea have revealed their richness in cardenolide and pregnane glycosides exhibiting potent cytotoxicity; further chemical examinations of the D. purpurea seeds have achieved the isolation of six triterpene glycosides (1–6), six spirostanol glycosides (7–12), and three furostanol glycosides (13–15), including seven previously unidentified compounds (1–3, 10–12, and 14). Here, the structures of 1–3, 10–12, and 14 were determined via extensive spectroscopic analyses, including two-dimensional (2D) NMR; hydrolysis, followed by chromatographic and spectroscopic analyses; and X-ray crystallographic analysis. The cytotoxic activities of the isolated compounds (1–15) against SBC-3 small cell lung carcinoma and TIG-3 normal human diploid fibroblast cells were evaluated. Triterpene glycoside 3 and spirostanol glycoside 9 exhibited considerable cytotoxicity with IC50 values of 1.0 and 1.7 µM, respectively; they induced apoptotic cell death, which was accompanied by the activation of caspase-3 in SBC-3 cells. Spirostanol glycoside 7 exhibited cytotoxicity toward the SBC-3 cells (IC50 1.3 μM). Additionally, 7 at 0.1 and 1.0 µM synergistically enhanced the cytotoxicity of etoposide against SBC-3 cells; compound 7 induced the release of DAMPs; the release of HMGB1, the secretion of ATP, and the exposure of CALR in the SBC-3 cells. Furthermore, the combination of 7 and etoposide resulted in increasing the extracellular release of DAMPs. These data indicated that 7, as well as its combination with etoposide, might potentially cause immunogenic cell death.

PLAC8 promotes the autophagic activity and improves the growth priority of human trophoblast cells.

Autophagy plays an important role in the normal development and function of trophoblast cells and is precisely regulated during pregnancy. Dysregulated autophagy contributes to the abnormal proliferation of trophoblasts, which is closely related to the occurrence of pregnancy-related diseases. Placenta specific 8 (PLAC8, Onzin) is a multifaceted protein proven to promote autophagy and potentiate various tumor progression. Its role in trophoblasts remains elusive. In our present study, PLAC8 expression was detected in tissues of first-trimester placentas (n=5), term placentas (n=5), choriocarcinoma (n=5), and placental site trophoblastic tumor (n=5). PLAC8 expression was increased in gestational neoplasms compared with normal pregnancies. mCherry-EGFP-LC3B reporter and transmission electron microscopy confirmed PLAC8 promoted the autophagic flux of human trophoblast cells. Both gain-of-function and loss-of-function experiments demonstrated PLAC8-regulated autophagy-related genes, including ATG5, ATG12, and Beclin-1. In addition, our data showed that PLAC8 co-localized with p53 and promoted its degradation, and p53 re-expression partially abrogated the PLAC8-induced autophagy activity. Furthermore, the overexpression of PLAC8 promoted cell viability and proliferation, acting as a protective mechanism of trophoblasts against the cytotoxicity of etoposide (VP-16). Such a phenomenon was effectively abrogated by autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ). In conclusion, PLAC8-induced autophagy to promote the proliferation of trophoblasts. This study provided insights into the mechanism of PLAC8-induced autophagy in trophoblasts, which is significant for a wide range of gestational diseases and may contribute to developing novel treatment strategies for trophoblastic diseases.

Drivers of topoisomerase II poisoning mimic and complement cytotoxicity in AML cells.

Recently approved cancer drugs remain out-of-reach to most patients due to prohibitive costs and only few produce clinically meaningful benefits. An untapped alternative is to enhance the efficacy and safety of existing cancer drugs. We hypothesized that the response to topoisomerase II poisons, a very successful group of cancer drugs, can be improved by considering treatment-associated transcript levels. To this end, we analyzed transcriptomes from Acute Myeloid Leukemia (AML) cell lines treated with the topoisomerase II poison etoposide. Using complementary criteria of co-regulation within networks and of essentiality for cell survival, we identified and functionally confirmed 11 druggable drivers of etoposide cytotoxicity. Drivers with pre-treatment expression predicting etoposide response (e.g., PARP9) generally synergized with etoposide. Drivers repressed by etoposide (e.g., PLK1) displayed standalone cytotoxicity. Drivers, whose modulation evoked etoposide-like gene expression changes (e.g., mTOR), were cytotoxic both alone and in combination with etoposide. In summary, both pre-treatment gene expression and treatment-driven changes contribute to the cell killing effect of etoposide. Such targets can be tweaked to enhance the efficacy of etoposide. This strategy can be used to identify combination partners or even replacements for other classical anticancer drugs, especially those interfering with DNA integrity and transcription.

Gypsophila saponins enhance the cytotoxicity of etoposide in HD-MY-Z lymphoma cells

Glucuronide Oleanane-type Triterpenoid Carboxylic Acid 3,28-Bidesmosides (GOTCAB) are accumulated in Gypsophila L. roots. In the study we aimed at investigating the possible synergistic effects of Gypsophila trichotoma GOTCABs and cytostatic etoposide towards the Hodgkin lymphoma cell line HD-MY-Z. The combination effects with etoposide were evaluated using the symbolic mathematical software MAPLE. Liquid chromatography-mass spectrometry allowed the identification or tentative assignment of 28 core GOTCAB structures together with 6 monodesmosides in the root extract. Tested gypsogenin-based saponins possessed C-28 ester-bonded chain substituted with acetyl, cis/trans methoxycinnamoyl and both acetyl and sulfate groups. No cytotoxic effect was observed up to 20 μg/mL on normal mice fibroblasts (CCL-1 cell line) and lymphoma cells. Etoposide alone exerted IC50 93 μg/mL. In the presence of acetylated saponins (20 μg/mL), a strong synergism (Fa = 0.8, CI = 0.1) was observed with IC50 11 μg/mL. The combination induced apoptosis witnessed by caspase activation, elevated levels of cytosolic mono- and oligonucleosomes, and nuclear fragmentation together with discernible increase in ROS generation. The results emphasize the arabinose in the C-3 chain and acetylation pattern of the C-28 chain of the saponins as important structural features for cytotoxicity enhancing activity. Triterpenoid saponins are a valuable tool to improve the efficacy of cytostatics.

Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense.

Background/AimsCytotoxicity induced by reactive oxygen species (ROS) is critical for the effectiveness of chemotherapeutic drugs used in the treatment of acute myeloid leukemia (AML). This study aimed to investigate whether ROS contributes to cytotoxicity in AML cells when treated with homoharringtonine (HHT) and etoposide (ETP) in combination.MethodsAML cell lines THP1 and HL60 and primary AML cells from patients were treated with HHT and ETP alone or in combination, and cell viability was determined by trypan blue exclusion test, and apoptosis was analyzed by annexin-V/propidium iodide double staining as well as Western blot for measuring expression of cleaved caspase-9 and cleaved caspase-3. Intracellular ROS level was detected by DCFH-DA fluorescence assay, and N-Acetyl-L-cysteine (NAC) was used to scavenge intracellular ROS. Retroviral infection was applied to mediate stable overexpression in AML cells.ResultsWe show that HHT and ETP exhibit synergistic cytotoxicity in AML cell lines and primary AML cells in vitro, and meanwhile, HHT causes elevated ROS generation in ETP-treated AML cells. We next reveal that the elevated ROS is a critical factor for the synergistic cytotoxicity, since ROS scavenge by NAC remarkably diminishes this effect. Mechanistically, we demonstrate that HHT causes elevated ROS generation by disabling thioredoxin-mediated antioxidant defense. Finally, similar to HHT treatment, depletion of thioredoxin sensitizes AML to ETP treatment.ConclusionThese results provide the foundation for augmenting the efficacy of ETP in treating AML with HHT, and also highlight the importance of targeting ROS in improving treatment outcome in AML.

Promoting Effect of Adriamycin, Cisplatinum and Etoposide on Trk Genes Expression in Human Neuroblastoma Cells

Purpose: Tropomyosin receptor kinases (Trk) gene family had been strongly correlated with tumor progression and found to be highly predictive of clinical behavior. The aim of the study is to evaluated the cytotoxicity and cell cycle effect of adriamycin (ADM), cisplatinum (DDP) and etoposide (VP16) on neuroblastoma(NB) cells, and then assessed effect of three anti-cancer agents on Trk gene family expression in NB in vitro conditions. Methods: SK-N-SH NB cells were exposed in vitro to ADM, DDP and VP16. Cell counting kit (CCK-8) assay was performed to determine the effect of anti-cancer agents on cell viability, and the cell cycle change was measured by flow cytometry. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis was utilized to detect the expression levels of Trk gene family (TrkA, TrkB and TrkC) in a wide range of concentration of ADM, DDP and VP16 at different time. Results: Our data illustrated that ADM, DDP and VP16 had cytotoxic activity as a single agent in both a time- and dose-dependent manner in vitro. However, NB cells resistance to high concentrations of ADM. It was confirmed that NB cells showed in vitro sensitivity to ADM, DDP and Vp16, with concentration that observed with the half-maximal inhibitory concentration (IC50) values on SK-N-SH cells being 5μg/ml, 8μg/ml and 100μg/ml, respectively. Flow cytometry analysis showed that these three drugs also leaded to enhanced accumulation of cell populations in S phase. Additionally, our results showed that in NB cells, these three drugs treatment dramatically increased expression of TrkA, TrkB and TrkC in RT-PCR analysis. Conclusions: Collectively, our results may help to develop tailored treatment approaches and enhance the efficacy of cytotoxic agents treatment for high-risk NB with minimal or no additional toxicity by adjusting the concentration and action time of ADM, DDP and VP16 depending on different NB stages.

In vitro and in vivo evaluation of etoposide - silk wafers for neuroblastoma treatment

High-risk neuroblastoma requires surgical resection and multi-drug chemotherapy. This study aimed to develop an extended release, implantable and degradable delivery system for etoposide, commonly used for neuroblastoma treatment. Different concentrations of silk, a biodegradable, non-toxic, non-immunogenic material were employed to prepare etoposide-loaded wafer formulations. Secondary structure of silk in the formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy and optimized based on the crystalline structure. Accelerated in vitro degradation studies under different conditions such as acidic, alkaline, oxidizing mediums and high temperature, were performed. The integrity of the silk wafer structure was maintained unless exposed to 0.1 N NaOH for 24 h. In vitro release of etoposide was performed in PBS (phosphate buffered saline) at 37 °C. Silk coated 6% wafers released the drug up to 45 days, while uncoated wafers released the drug for 30 days. Cytotoxicity study was performed on KELLY cells to evaluate the etoposide cytotoxicity (LC50) and the long-term efficacy of the etoposide wafer formulations. The results showed that etoposide killed 50% of the cells at 1 μg/mL concentration and the wafer formulations demonstrated significant cytotoxicity up to 22 days when compared to untreated cells. Using an orthotopic neuroblastoma mouse model, intra-tumoral implantation of the coated 6%, uncoated 6%, or uncoated 3% silk wafers were all effective at decreasing tumor growth. Histological examination revealed tumor cell necrosis adjacent to the drug-loaded silk wafer.

Abstract 904: The novel C-terminal truncated 90-kDa isoform of topoisomerase IIα, TOP2α/90, is a determinant of etoposide resistance in K562 leukemia cells via heterodimerization with the TOP2α/170 isoform

Abstract DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells since it resolves DNA topologic entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated TOP2α/90 isoform, detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide (J Pharmacol Exp Ther 2017;360:152-63). TOP2α/90 (786 amino acids) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 (Tyr805) required to generate double-strand DNA breaks as well as the nuclear localization signals present in the TOP2α/170 isoform (1531 amino acids). The function of TOP2α/90 is unknown. Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Importantly, co-immunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, while siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated K562 or K/VP.5 cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by soft agar colony formation assays. qPCR and immunoassays demonstrated expression of TOP2α/90 mRNA and protein in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents. Citation Format: Ragu Kanagasabai, Soumendra Karmahapatra, Yang Yu, Victor A. Hernandez, Corey A. Kientz, Evan E. Kania, Terry S. Elton, Jack C. Yalowich. The novel C-terminal truncated 90-kDa isoform of topoisomerase IIα, TOP2α/90, is a determinant of etoposide resistance in K562 leukemia cells via heterodimerization with the TOP2α/170 isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 904.

17-(Allylamino)-17-Demethoxygeldanamycin Enhances Etoposide-Induced Cytotoxicity via the Downregulation of Xeroderma Pigmentosum Complementation Group C Expression in Human Lung Squamous Cell Carcinoma Cells

Etoposide (VP16) is a topoisomerase II inhibitor and has been used for the treatment of non-small cell lung cancer (NSCLC). Xeroderma pigmentosum complementation group C (XPC) protein is a DNA damage recognition factor in nucleotide excision repair and involved in regulating NSCLC cell proliferation and viability. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) enhanced etoposide-induced cytotoxicity in NSCLC cells through modulating the XPC expression. We found that etoposide increased XPC expression in an AKT activation manner in 2 squamous cell carcinoma H1703 and H520 cells. Knockdown of XPC using siRNA or inactivation of AKT by pharmacological inhibitor PI3K inhibitor (LY294002) enhanced the cytotoxic effects of etoposide. In contrast, enforced expression of XPC cDNA or AKT-CA (a constitutively active form of AKT) reduced the cytotoxicity and cell growth inhibition of etoposide. Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of etoposide in NSCLC cells, which were associated with the downregulation of XPC expression and inactivation of AKT. Our findings suggested that the Hsp90 inhibition induced XPC downregulation involved in enhancing the etoposide-induced cytotoxicity in H1703 and H520 cells.

Cytotoxic prenylated acylphloroglucinols from Hypericum annulatum

A phytochemical investigation of the aerial parts of Hypericum annulatum Moris led to the isolation of five new prenylated acylphloroglucinol derivatives hyperannulatins A-E (1–3, 5 and 7) in addition to the known hypercalyxone A (4) and 3-geranyl-1-(2′-methylpropanoyl)phloroglucinol (6). The structures were determined by 1D and 2D NMR and MS spectroscopic techniques. Compounds 1 and 2 have in their structure evgenyl group, a rare hydrocarbon side chain. The cytotoxicity of isolated compounds was established on a panel of tumor cell lines (HL-60, HL-60/DOX, MDA-MB, SKW-3 and K-562) and was determined using MTT based assays. The compounds 1 and 2 showed to be the most potent cytotoxic agents, whose IC50 values against the chemosensitive cell lines ranged 3.42–5.87 μM and 1.48–8.21 μM, respectively. Noteworthy, albeit all tested compounds were less potent than podophyllotoxin their IC50 values were comparable to that of the other reference drug etoposide. In some of the cell lines compounds 1 and 2 even outclassed the cytotoxicity of etoposide.

The Novel C-terminal Truncated 90-kDa Isoform of Topoisomerase IIα (TOP2α/90) Is a Determinant of Etoposide Resistance in K562 Leukemia Cells via Heterodimerization with the TOP2α/170 Isoform.

DNA topoisomerase IIα (170 kDa, TOP2α/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2α/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2α/90 (786 aa) is the translation product of a TOP2α mRNA that retains a processed intron 19. TOP2α/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2α/170 isoform (1531 aa). Here, we found that TOP2α/90, like TOP2α/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2α/90 and TOP2α/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2α/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2α/90 in K/VP.5 cells enhanced, etoposide-mediated DNA strand breaks compared with similarly treated cells transfected with empty vector or control siRNAs, respectively. In addition, forced expression of TOP2α/90 in K562 cells inhibited etoposide cytotoxicity assessed by clonogenic assays. qPCR and immunoassays demonstrated TOP2α/90 mRNA and protein expression in normal human tissues/cells and in leukemia cells from patients. Together, results strongly suggest that TOP2α/90 expression decreases drug-induced TOP2α-DNA covalent complexes and is a determinant of chemoresistance through a dominant-negative effect related to heterodimerization with TOP2α/170. Alternative processing of TOP2α pre-mRNA, and subsequent synthesis of TOP2α/90, may be an important mechanism regulating the formation and/or stability of cytotoxic TOP2α/170-DNA covalent complexes in response to TOP2α-targeting agents.

Helicobacter pylori CagA protein activates Akt and attenuates chemotherapeutics-induced apoptosis in gastric cancer cells.

Infection with cagA-positive Helicobacter pylori is associated with a higher risk of gastric cancer. The cagA gene product, CagA, is translocated into gastric epithelial cells and perturbs host cellular biological functions. Etoposide, a topoisomerase II inhibitor widely used to couple DNA damage to apoptosis, is a common cytotoxic agent used for advanced gastric cancer. We investigate the effect of CagA on etoposide-induced apoptosis in gastric cancer cells to elucidate whether CagA play a role in gastric carcinogenesis via impairing DNA damage-dependent apoptosis. AGS cell lines stably expressing CagA isolated from H. pylori 26695 strain were established. In the presence of etoposide, viability of parental AGS cells was decreased in a time-and dose-dependent manner, whereas CagA-expressing AGS cells were less susceptible to etoposide induced cell-killing effect. Suppression of etoposide-induced apoptosis was shown in CagA-expressing but not in parental AGS cells by DNA fragmentation, cell cycle, and annexin-V assays. This inhibitory effect of etoposide-induced apoptosis conferred by CagA was also demonstrated in SCM1 and MKN45 gastric cancer cell lines, with two additional chemotherapeutics, 5-FU and cisplatin. The effect of Akt activation on inhibition of etoposide-induced cytotoxicity by CagA was also evaluated. CagA expression and etoposide administration activate Akt in a dose-dependent manner. Enhancement of etoposide cytotoxicity by a PI-3-kinase inhibitor, LY294002, was evident in parental but was attenuated in CagA-expressing AGS cells. CagA may activate Akt, either in the absence or presence of etoposide, potentially contributing to gastric carcinogenesis associated with H. pylori infection and therapeutic resistance by impairing DNA damage-dependent apoptosis.

A Src/Abl kinase inhibitor, bosutinib, downregulates and inhibits PARP enzyme and sensitizes cells to the DNA damaging agents

Abstract Background Poly(ADP-ribosyl)ation (PARylation) catalyzed mainly by PARP1 is a highly regulated posttranslational modification associated with several pathways in cellular physiology and genotoxic deoxyribonucleic acid (DNA) damage response. PAR polymers and PARP enzyme function in DNA integrity maintenance and several PARP inhibitors have entered clinical phase studies for cancer therapies. Material and methods The effect of bosutinib, a dual Src/Abl kinase inhibitor, on PARylation was fluorometrically measured. The cytotoxic and chemosensitizing effects were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of DNA repair proteins and PARP enzyme were examined by immunoblotting. Results In this study, bosutinib is characterized as a novel PARP inhibitor. Bosutinib inhibited oxidative stress-induced cellular PARylation and nuclear foci formation by downregulating PARP1 levels. Bosutinib was found to be more cytotoxic on Capan1 cells with BRCA2 mutation. Furthermore by acting as a chemosensitizer, bosutinib enhanced the cytotoxicity of doxorubicin (DOXO) and etoposide (ETP) by decreasing phosphorylation of DNA repair enzymes checkpoint kinase 1 (Chk1) and ataxia-telangiectasia mutated (ATM). Conclusion By inhibition of both PARP and DNA damage checkpoint kinases, bosutinib increased the phospho-H2AX levels, an early indicator of DNA double strand breaks.

Protective effect of metoclopramide against organophosphate‐induced apoptosis in the murine skin fibroblast L929

This study was performed to evaluate the protective efficacy of metoclopramide (MCP) against the organophosphates paraoxon (POX)- and malathion (MLT)-induced apoptosis in the murine L929 skin fibroblasts. L929 cells were exposed to either POX (10nm) or 1.0μm MLT in the absence and presence of increased concentrations of MCP. The protective effect of MCP on these organophosphate-stimulated apoptotic events was evaluated by flow cytometry analysis after staining with annexin-V/propidium iodide, processing and activation of the executioner caspase-3, cleavage of the poly-ADP ribose polymerase, fragmentation of the nucleosomal DNA and disruption of the mitochondrial membrane potential (Δψ). Our results showed that increased doses of MCP alone (≥10μm) did not induce apoptosis or activation of caspase-3. Pretreatment of the cells with MCP attenuated all the apoptotic events triggered by the organophosphate compounds in a dose-dependent manner reaching ~70-80% protection when they were preincubated at 1 and 5μm of the drug before the addition of POX and MLT, respectively. Interestingly, MCP did not offer a significant protective effect against the cytotoxicity of tumor necrosis factor-α, cisplatinum, etoposide or paclitaxel, which stimulate apoptosis by various mechanisms, suggesting that the anti-apoptotic effect of the drug is specific to organophosphates. The strong and specific anti-apoptotic activity of subclinical doses of MCP against the cytotoxicity of organophosphate compounds suggests its potential clinical application in treating their poisoning.

Sensitizing Ewing sarcoma to chemo- and radiotherapy by inhibition of the DNA-repair enzymes DNA protein kinase (DNA-PK) and poly-ADP-ribose polymerase (PARP) 1/2.

BackgroundDNA-PK and PARP inhibitors sensitize cancer cells to chemo- and radiotherapy. ETS transcription factors (EWS-FLI1) have been described as biomarkers for PARP-inhibitor sensitivity. Sensitivity to single agent PARP inhibitors has so far been limited to homologous recombination repair (HRR) deficient tumors, exploiting synthetic lethality.ResultsIn clonogenic assays, single agent rucaparib LD50 values for continuously exposed cells were similar to those observed in HRR-defective cells (CAPAN-1 cell line, BRCA2 defective); however, both ES cell lines (TC-71, CADO-ES1) had functional HRR. In vivo rucaparib administration (10 mg/kg daily) showed no responses. In clonogenic assays, rucaparib enhanced temozolomide, camptothecin and radiation cytotoxicity, which was most profound for temozolomide (15–29 fold enhancement). NU7441 increased the cytotoxicity of etoposide, doxorubicin and radiation.Materials and MethodsWe assessed PARP1/2 (rucaparib) and DNA-PK (NU7441) inhibitors in Ewing sarcoma (ES) cell lines by performing growth inhibition and clonogenic assays. HRR was measured by RAD51 focus formation. Single agent rucaparib was assessed in an in vivo orthotopic model.ConclusionsSingle agent rucaparib ES sensitivity in vitro was not replicated in vivo. DNA-PK and PARP inhibitors are good chemo-/radiosensitizers in ES. The future of these inhibitors lies in their combination with chemo-/radiotherapy, which needs to be evaluated in clinical trials.

A novel histone deacetylase inhibitor TMU-35435 enhances etoposide cytotoxicity through the proteasomal degradation of DNA-PKcs in triple-negative breast cancer

Triple-negative breast cancer (TNBC) treatment offers only limited benefits, and it is very relevant given the significant number of deaths that it causes. DNA repair pathways can enable tumor cells to survive DNA damage that is induced by chemotherapeutic or radiation treatments. Histone deacetylase inhibitors (HDACi) inhibited DNA repair proteins. However, the detailed mechanisms for this inhibition remain unclear. In the present study, we investigated whether a newly developed HDACi, TMU-35435, could enhance etoposide cytotoxicity by inhibiting DNA repair proteins in triple-negative breast cancer. We found synergistic cytotoxicity following treatment of 4T1 cells with etoposide and TMU-35435. Furthermore, TMU-35435 enhances etoposide-induced DNA damage by inhibiting the DNA repair pathway (non-homologous end joining, NHEJ). TMU-35435 suppresses the NHEJ pathway through the ubiquitination of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In addition, TMU-35435 ubiquitinated DNA-PKcs by inducing the interaction between RNF144A (an E3 ligase) and DNA-PKcs. The combined treatment induced apoptosis and autophagic cell death in 4T1 cells. In an orthotopic breast cancer model, combined treatment with TMU-35435 and etoposide showed anti-tumor growth through the increase of DNA damage and cell death. Taken together, our data suggest that TMU-35435 enhances etoposide cytotoxicity by regulating ubiquitin–proteasome system and inhibiting the DNA repair pathway in TNBC.