- Research article
- Open Access
- Open Peer Review
Differential cytotoxicity induced by the Titanium(IV)Salan complex Tc52 in G2-phase independent of DNA damage
© The Author(s). 2016
- Received: 14 October 2015
- Accepted: 8 July 2016
- Published: 13 July 2016
Chemotherapy is one of the major treatment modalities for cancer. Metal-based compounds such as derivatives of cisplatin are in the front line of therapy against a subset of cancers, but their use is restricted by severe side-effects and the induction of resistance in treated tumors. Subsequent research focused on development of cytotoxic metal-complexes without cross-resistance to cisplatin and reduced side-effects. This led to the discovery of first-generation titanium(IV)salan complexes, which reached clinical trials but lacked efficacy. New-generation titanium (IV)salan-complexes show promising anti-tumor activity in mice, but their molecular mechanism of cytotoxicity is completely unknown.
Four different human cell lines were analyzed in their responses to a toxic (Tc52) and a structurally highly related but non-toxic (Tc53) titanium(IV)salan complex. Viability assays were used to reveal a suitable treatment range, flow-cytometry analysis was performed to monitor the impact of dosage and treatment time on cell-cycle distribution and cell death. Potential DNA strand break induction and crosslinking was investigated by immunostaining of damage markers as well as automated fluorometric analysis of DNA unwinding. Changes in nuclear morphology were analyzed by DAPI staining. Acidic beta-galactosidase activity together with morphological changes was monitored to detect cellular senescence. Western blotting was used to analyze induction of pro-apoptotic markers such as activated caspase7 and cleavage of PARP1, and general stress kinase p38.
Here we show that the titanium(IV)salan Tc52 is effective in inducing cell death in the lower micromolar range. Surprisingly, Tc52 does not target DNA contrary to expectations deduced from the reported activity of other titanium complexes. Instead, Tc52 application interferes with progression from G2-phase into mitosis and induces apoptotic cell death in tested tumor cells. Contrarily, human fibroblasts undergo senescence in a time and dose-dependent manner. As deduced from fluorescence studies, the potential cellular target seems to be the cytoskeleton.
In summary, we could demonstrate in four different human cell lines that tumor cells were specifically killed without induction of major cytotoxicity in non-tumorigenic cells. Absence of DNA damaging activity and the cell-cycle block in G2 instead of mitosis makes Tc52 an attractive compound for further investigations in cancer treatment.
- Titanium(IV)salan complex
Cancer is the second most frequent cause of death in industrial countries. Treatments to cure the disease range from classical surgery, high-energy irradiation [1–3] or DNA damaging drugs [4–9] and chemicals interfering with DNA repair [10–12] to compounds tackling signaling cascades [13–18] or the cytoskeleton [19–21] and combinations thereof. One success story was the discovery of cisplatin as chemotherapeutic agent . Platinum-compounds still play a role in chemotherapy, but their efficiency is limited to a minor cancer-panel and hampered by severe side-effects and acquired resistance . Current research focuses on other metal-complexes, with little cross-reactivity to cisplatin and reduced side-effects. Several cytotoxic titanium-based complexes have been investigated, with titanocene-dichloride and budotitane reaching clinical trials [24–27]. Unfortunately, both substances display a fast rate of hydrolysis , resulting in low efficacy and cancelling of phase-II trials. Titanium(IV)salans displaying much longer half-life in aqueous environments  and an antitumor-efficacy comparable to cisplatin [29, 30], but with no cross-resistance . Apoptosis induction by two differently substituted complexes in tumor cell lines was described , and efficacy was shown in a mouse tumor-model . Titanium(IV)salans mode of action is unknown, but titanocene-dichloride was enriched in chromatin regions , bound to DNA and inhibited DNA-synthesis and topoisomerase II . In order to elucidate the cytotoxic mechanism of titanium(IV)salans, we treated four different human cell lines with the cytotoxic compound Tc52  or the - despite a high degree in structural identity - non-toxic Tc53 [34, 35] (Additional file 1: Figure S1). We could not detect any signs of DNA damage, but cytotoxicity was selective for the tumorigenic cell lines investigated, accompanied by a G2-phase cell-cycle block. Non-tumor cells were spared from death, and fibroblasts underwent senescence. By analyzing different mitosis-targeting toxins together with Tc52, we could show that the putative target is the cytoskeleton.
DNA damage is not induced by Tc52
Tc52 blocks cells in G2-phase
Tc52 induces apoptosis in cancer cells and senescence in fibroblasts
Efficacy of cancer treatment relies on higher sensitivity of tumor cells towards chemotherapeutic agents compared to surrounding tissue. The search for metal-based chemotherapeutics other than cisplatin-derivatives led to the discovery of titanium as potential replacement, with titanocene-dichloride and budotitane reaching clinical trials. Unfortunately, both compounds lacked anti-tumorigenic potential due to rapid hydrolysis in aqueous solutions . Titanium(IV)salans are much more stable  and have been shown to display promising activity against cancer cells in vitro as well as in mouse models, including some selectivity for tumor cells . But the underlying mechanism of cytotoxicity and the cellular targets have not been defined yet. We investigated this for the toxic titanium(IV)salan Tc52 and the non-toxic Tc53, which display a high degree of structural similarity with only small variations, in different human cell lines. Viability was similarly affected by Tc52 in all lines analyzed, with EC50 values between 2 μM and 6 μM Tc52 (Additional file 2: Figure S2 and Additional file 10: Figure S9), whereas Tc53 had no impact. This discrepancy in toxicity cannot be attributed to differential uptake, as similar timing in cellular accumulation has been reported for related titanium(IV)complexes . Importantly, viability assays do not discriminate between cytotoxic and cytostatic effects, as in both cases cell numbers are reduced compared to controls. Deduced from titanocene-dichloride , we expected genomic DNA to be the target of Tc52. We monitored for DNA strand-break induction with a variety of different assays, using the very sensitive FADU and reverseFADU method able to detect strand-breaks induced by 0.13 Gy of X-ray  as well as DNA crosslinks , respectively, and checked for the appearance of the two different damage markers PAR and γH2AX. Whereas synthesis of PAR is an early marker for DNA strand-breaks, detectable within seconds to minutes , γH2AX foci appear later and correlate finally with the amount of double-strand breaks . We failed to observe any DNA damage induction (Fig. 1) after Tc52 application in contrast to H2O2 treatment (Additional file 3: Figure S3). These data exclude that nuclear DNA is targeted by Tc52. Analyzing the cell-cycle profile of HeLa after Tc52 treatment revealed major cell death induction concomitant with a transient block in G2/M-phase (Fig. 2). A reasonable explanation could be that Tc52 interferes with a step during mitosis or cytokinesis, similar to biological toxins such as cytochalasin B from fungi, colchicine from autumn crocus or taxanes from yew. CytB induces binuclear cells as it interferes with formation of the contractile actin-ring in cytokinesis. In contrast, the colchicine-derivate colcemid or the taxane docetaxel target tubulin, leading to metaphase arrest, but whereas Col prevents tubulin polymerization, Doc blocks disassembly of tubulin. As a consequence of taxane treatment, multipolar spindles are induced, and cells escaping mitotic block show characteristic lobed or fragmented nuclei . Tc52 was applied in combination with all three compounds. Tc52 toxicity was additive to CytB in viability assays, but protected from cytotoxicity of higher doses of Col and Doc (Fig. 3a). Whereas addition of Tc52 mildly changed cell-cycle profile of Col and Doc treated samples, the percent of cells containing >4 N DNA content, i.e., binucleated cells, was drastically reduced in CytB/Tc52-treated cells (Fig. 3b). DAPI-staining of nuclei revealed that addition of Tc52 led to a severe drop in mitotic index (Fig. 3c). In concert with the cell-cycle analysis, these data indicate that Tc52 is not affecting mitosis, but targets an unknown step in G2, preventing progression into M-phase. We hypothesize that steps before or at G2-M transition as centrosome-separation, kinase-signaling or the cytoskeleton might be affected. Interestingly, it has been reported that tubulin targeting colcemid has two different modes of action: At low doses it inhibits proper plus-end dynamics and impacts on cell migration, whereas at high doses it seems to induce cell death by microtubule fragmentation during mitosis . Indeed, visualization of tubulin and F-actin indicated that Tc52 induced small alterations in the tubulin network, displaying increased bundling at the cellular periphery also in combination with other toxins (Fig. 3d). Whether Tc52 also impacts on migration needs to be determined in future research. But probably Tc52 acts independent of this as treated cells are blocked in a step before condensation of chromosomes takes place and mitotic microtubules are formed. Actin was mildly affected by Tc52, which could be due to modified tubulin functionality. F-actin staining was severely affected by CytB as expected, reshuffling actin into bright foci without network-formation. Col and Doc affected only the tubulin network. Of note, combined Doc/Tc52 treatment "rescued" actin and tubulin network compared to Doc-only treated samples, which supports the idea of acting in advance of Doc and thus mitosis. In summary, the protection from Col- and Doc-dependent cytotoxicity, together with the cell-cycle analysis and data from immunofluorescence suggests that Tc52 acts on the same pathway as Col or Doc, but independent of actin-targeting CytB. Monitoring cell-cycle distribution after Tc52 pulse-treatment of HeLa and VH7 revealed massive apoptosis-induction in tumorigenic HeLa, whereas fibroblasts were blocked in G2/M without signs of cell death (Fig. 4a/b). Appearance of apoptotic figures in HeLa confirmed the time- and dose-dependent increase in toxicity by Tc52 (Fig. 4c/e), which was absent in VH7 as expected from cell-cycle data (Fig. 4d/f). Instead, VH7 nuclei displayed changes in chromatin structure, i.e., an increase of DAPI-dense foci resembling SAHF [39, 40] (Fig. 4d/g), together with characteristic morphological changes such as enlargement and flattening (Fig. 5). Testing for SAβGal activity confirmed a time- and concentration-dependent induction of senescence (Fig. 5). To support our findings, we analyzed the rapid release of free Ca2+ in the cytosol in HeLa and VH7 after Tc52 exposure. This method was developed to quickly determine the cytotoxic potential of a substance . Both low and high concentrations induced in HeLa a strong increase in Ca2+-dependent fluorescence, which was absent in fibroblasts (Additional file 7: Figure S7), supporting the cell-specific difference in Tc52 response. Activation of the general stress-kinase p38 by phosphorylation at T180/Y182 is central to many cell fate pathways such as cell death, senescence, differentiation and tumorigenesis (reviewed in ). In order to analyze whether Tc52 and Tc53 application leads to activation of p38, we probed for total and phosphorylated p38 in four cell lines. Indeed, Tc52 led to increased p-p38 in a time and dose-dependent manner in all of them (Figs. 6, 7). In contrast, non-cytotoxic Tc53 did not show enhanced p-p38 levels (Additional file 8: Figure S8). To confirm the observed differential impact of Tc52 on cell death, we probed for apoptosis-dependent cleavage of caspase7 . Indeed, only HeLa and U2OS displayed dose- and time-dependent activation of caspase7 (Figs. 6, 7), and Tc53 was without effect (Additional file 9: Figure S8). Supporting our data, apoptotic PARP1-cleavage was evident only in HeLa and U2OS cultures treated with Tc52, but not in VH7 and HEK293 (Figs. 6, 7).
In summary, the titanium(IV)salan Tc52 is effective with an EC50 value in the low-micromolar range. Tc52 interferes with cell-cycle progression in G2 and deduced from our data, tubulin-related signaling is targeted, as Tc52 treatment induces alterations in the tubulin network and partially rescues from cytotoxicity of tubulin-interacting toxins that induce mitotic arrest. Alternatively, Tc52 may interfere with centrosomal regulation and prevent in this way entry into mitosis, but this would not explain the differential response towards combination with actin- or tubulin-targeting toxins. The actual target of Tc52 has to be elucidated in future research. Nevertheless, as a consequence tumor cells respond with apoptotic death, whereas normal fibroblasts show induction of senescence. We hypothesize that the constant block in G2 imposed by Tc52 cannot be handled properly by tumor cells, leading to induction of apoptosis.
We provided several layers of evidence for the differential cytotoxic potential of the titanium(IV)salan Tc52 by a pathway that is not affecting genomic integrity. As proof-of-principle, we compared two well-established cancer cell lines, HeLa and U2OS, with VH7 fibroblasts and low-passage HEK293. Importantly, Tc52 does not target DNA or block progression through mitosis, which maintains genomic information and stability. In this way, there is no risk of inducing mutations in normal tissue, which may lead to the outgrowth of secondary tumors after cancer therapy . P38 stress kinase is activated in all cells, but only tumor cell lines responded with cell death as evidenced by caspase7 activation and PARP1-cleavage. Supporting this, HeLa cells treated with Tc52 displayed an increase in subG1-fraction, apoptotic figures and cytosolic Ca2+, but not human fibroblasts, which showed induction of senescence. Further exploration of the potential anti-tumorigenic effect of this new titanium(IV)salan could be of great clinical benefit. If chemotherapy would actually discriminate between tumor and normal cells, fighting cancer could be far more effective.
Cell culture and toxins
Cells were cultured in DMEM-GlutaMax (Life-Technologies)/10 % FCS/1 % PenStrep (Life-Technologies). VH7 normal fibroblasts were used from passage 20–30, HEK293 from passage 24–34, HeLa and U2OS from routine culture. Authentication of cell lines was performed by short tandem repeat (STR) DNA-typing. Toxins were applied in medium at the concentrations and for the time periods specified with appropriate solvent controls in parallel. DMSO concentrations in medium were either 0.2 % (titanium(IV)salans alone) or 0.3 % in combination treatments. Titanium(IV)salans (Chemistry-Department University of Konstanz), cytochalasinB (Sigma) and docetaxel (LC-Laboratories) were solubilized in DMSO, colcemid (KaryoMax, Life-Technologies) and H2O2 (Sigma) diluted in medium.
Cells were seeded into 96well-plates (clear bottom/white walls, Corning) and grown for 24 h. Medium was exchanged against medium containing toxins and incubated for 24 h or 48 h. Supernatant was replaced with fresh medium containing 9 μg/ml resazurin (Sigma) and incubated until medium in control wells turned purple. Fluorescence (excitation/emission 560 nm/590 nm) was measured using LS55-spectrometer (Perkin-Elmer). Each experiment was performed in technical duplicates or triplicates.
Cells were seeded 24 h in advance onto glass cover-slips. Medium was supplemented with the respective toxin and incubated for the times specified. Cells were fixed with 3.7 % formaldehyde/PBS for 20 min at room temperature (RT), washed 1x with 0.1 M glycine/PBS for 3 min at RT and permeabilized for 5 min at RT in 0.4 % TritonX100/TBS except for detection of PAR. For PAR, cells were fixed by incubation for 7 min in −20 °C methanol at 4 °C. Subsequent procedures were identical. After three washes with TBS, cells were blocked in TBS/0.3 % Tween 20/1 % BSA for 30 min at 37 °C and incubated for 1 h at 37 °C with 1.antibody diluted in blocking-solution, followed by three washes for 10 min at RT in TBS/0.3 % Tween 20. 2.antibody incubation was performed for 1 h at 37 °C in blocking-solution, followed by three washes as above. Nuclei were stained with 20 ng/ml DAPI-solution, air-dried and mounted with AquaPolymount (PolySciences). Epifluorescence pictures were taken using a Nikon-EclipseTS100 microscope and NIS-ElementsD3.2 software. Subsequent analysis was performed using ImageJv1.47n and CS-Photoshop5.
Antibodies: 10H anti-PAR , anti-γH2AX (Millipore), anti-αTubulin (Sigma), goat-anti-mouse Alexa594 (Molecular Probes, Invitrogen). Phalloidin-Atto488 anti-F-actin (Sigma) was solubilized in DMSO to 40 μM and further diluted to 400 nM.
Automated FADU and reverseFADU
Methods have been described in detail in [36, 37]. Briefly, cells were seeded 24 h before toxin treatment. After incubation with respective concentrations of mitomycinC, 10 μM Tc52 or Tc53 for the indicated times, cells were harvested and analyzed for strand-breaks by FADU or in parallel for crosslinks by reverseFADU. For reverseFADU, cells were additionally irradiated with 25 Gy X-rays before FADU procedure. Liquid handling was performed on a Genesis RSP100 robot (Tecan). Cells were lysed and unwinding of DNA was induced by alkaline conditions. After neutralization, the relative amount of double-stranded DNA in each sample was measured by SYBR-Green fluorescence (excitation/emission 492 nm/520 nm) in FLx800-fluorescence microplate-reader (Bio-TEK Company).
Cells were seeded on 10 cm-dishes and incubated for 24 h. Medium was replaced with medium containing respective toxins and incubated for the time periods indicated. In samples exposed for 6 h, medium was replaced with fresh standard medium and further incubated for 24 h. Floating cells were collected, combined with adherent cells harvested by trypsin, and pelleted by centrifugation. Cells were washed twice with ice-cold PBS, fixed by resuspending in ice-cold 70 % ethanol to a concentration of 1x106 cells/ml and stored at −20 °C. For analysis, fixed cells were pelleted by centrifugation and washed once with PBS. Pellets were suspended in PBS containing 100 μg/ml RNaseA and 25 μg/ml propidium iodide to 1x106 cells/ml. Cell-cycle distribution was monitored using FACSCantoII (BD-Biosciences) and about 10000 events were counted in each single run. Subsequent analysis was performed using Flowing-Software2.1.
SAβGal activity assay
The SAβGal-kit from Cell-Signaling was used following manufacturer's instructions. VH7 were seeded into 6well-plates and incubated for 24 h. Afterwards, medium was exchanged against medium containing Tc52 at concentrations of 2 μM/5 μM/10 μM, 10 μM Tc53 or solvent. Cells were incubated for 2 h/6 h/30 h with Tc52 before exchange of medium against standard medium. After a total incubation of 30 h, cells were fixed and SAβGal-activity assay was performed. To analyze signal intensity, bright-field photomicrographs were taken with fixed exposure time using Nikon EclipseTS100 microscope and gray-values were measured with ImageJv1.47n-software. Nuclei were counted to normalize for cell number, and gray-values/cell were calculated.
Cells were seeded on 10 cm-dishes and incubated for 24 h. Medium was replaced with medium containing respective toxins and incubated for the indicated times. After 2 h and 6 h medium was exchanged in the respective samples against fresh standard medium. After 30 h incubation of all samples, medium was removed and floating cells were harvested by centrifugation. Pelleted cells and cells on the dish were lysed in 95 °C Laemmli-buffer and combined. Equal volumes were run on SDS-gels and blotted onto nitrocellulose- (PARP1 detection) or PVDF-membrane. Membranes were blocked with TBS/0.1 % Tween20/3 % BSA/10 % non-fat dry-milk for 2 h at 37 °C and incubated overnight at 4 °C with 1.antibody diluted in TBS/0.1 % Tween20/3 % BSA. Membranes were washed thrice for 20 min at RT in TBS/0.1 % Tween20/3 % non-fat dry-milk and incubated for 1 h at RT with 2.antibody in TBS/0.1 % Tween20/3 % non-fat dry-milk. After three washes in TBS/0.1 % Tween20, ECL detection was performed (Pierce ECL-SuperSignal-Femto, ThermoFisher Scientific). Antibodies: rabbit-polyclonal anti-GAPDH (Ambion); mouse-monoclonal anti-actin (Millipore); rabbit-polyclonal anti-p38 kinase, rabbit-monoclonal anti-phospho-p38 kinase, rabbit-polyclonal anti-tubulin, mouse-monoclonal anti-cleaved caspase7 (Cell-Signaling); goat-anti-rabbit IgG-HRP, goat-anti-mouse IgG-HRP (Sigma), mouse-monoclonal anti-PARP1 C-2-10 (SantaCruz-Biotechnologies).
All experiments were performed at least three times independently. Statistics were calculated using GraphPad-Prism5 software and tests suggested by the program were used. Presented are means +/− SEM.
Col, colcemid; CytB, cytochalasin B; DAPI, 4',6-diamidino-2-phenylindole; Doc, docetaxel; FADU, Fluorometric Analysis of DNA Unwinding; MI, mitotic index; MMC, mitomycin C; PAR, poly(ADP-ribose); PARP1, poly(ADP-ribose)polymerase1; p-p38, phosphorylated p38 stress-kinase; RT, room temperature; SAHF, senescence-associated heterochromatic foci; SAβGal, senescence-associated beta-galactosidase
We wish to thank Prof. Felix Althaus for stimulating discussions regarding the manuscript and his support.
PW was supported by Lotte and Adolf Hotz-Sprenger Foundation, TI was supported by Konstanz Research School of Chemical Biology.
Availability of data and materials
The datasets supporting the conclusions of this article are available in the Zenodo repository, https://zenodo.org/record/51689.
TP carried out most of the western blotting experiments, HS carried out and analyzed the FADU experiments and revised the manuscript. PW carried out and analyzed the calcium-toxicity assay and revised the manuscript, TI synthesized titanium(IV)salan complexes, WD was responsible for genotyping and revised the manuscript. TH supervised TI, participated in study design and revised the manuscript, AB supervised HS, participated in study design and revised the manuscript. SB designed the study, supervised TP and HS, drafted the manuscript, carried out and analyzed the immunofluorescence experiments, the viability assays, the flow cytometry and senescence analysis and some western blots, including all statistical evaluations. All authors read and approved the final manuscript.
The authors declare that they have no competing interest.
Consent for publication
Ethics approval and consent to participate
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- Lohia S, Rajapurkar M, Nguyen SA, Sharma AK, Gillespie MB, Day TA. A Comparison of Outcomes Using Intensity-Modulated Radiation Therapy and 3-Dimensional Conformal Radiation Therapy in Treatment of Oropharyngeal Cancer. JAMA Otolaryngol Head Neck Surg. 2014.Google Scholar
- Durante M. New challenges in high-energy particle radiobiology. Br J Radiol. 2014.Google Scholar
- Mima M, Demizu Y, Jin D, Hashimoto N, Takagi M, Terashima K, Fujii O, Niwa Y, Akagi T, Daimon T, et al. Particle therapy using carbon ions or protons as a definitive therapy for patients with primary sacral chordoma. Br J Radiol. 2014;87(1033):20130512.View ArticlePubMedGoogle Scholar
- Puisset F, Schmitt A, Chatelut E. Standardization of chemotherapy and individual dosing of platinum compounds. Anticancer Res. 2014;34(1):465–70.PubMedGoogle Scholar
- Griffiths TR. Current perspectives in bladder cancer management. Int J Clin Pract. 2013;67(5):435–48.View ArticlePubMedGoogle Scholar
- Zhu W, Zhou L, Qian JQ, Qiu TZ, Shu YQ, Liu P. Temozolomide for treatment of brain metastases: A review of 21 clinical trials. World J Clin Oncol. 2014;5(1):19–27.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang LJ, Zhou CF, Lin ZX. Temozolomide and radiotherapy for newly diagnosed glioblastoma multiforme: a systematic review. Cancer Invest. 2014;32(2):31–6.View ArticlePubMedGoogle Scholar
- Kelsey CR, Beaven AW, Diehl LF, Prosnitz LR. Combined-modality therapy for early-stage Hodgkin lymphoma: maintaining high cure rates while minimizing risks. Oncology (Williston Park). 2012;26(12):1182–9. 1193.Google Scholar
- Nicholson S, Hall E, Harland SJ, Chester JD, Pickering L, Barber J, Elliott T, Thomson A, Burnett S, Cruickshank C, et al. Phase II trial of docetaxel, cisplatin and 5FU chemotherapy in locally advanced and metastatic penis cancer (CRUK/09/001). British J Cancer. 2013;109(10):2554–9.View ArticleGoogle Scholar
- Curtin NJ, Szabo C. Therapeutic applications of PARP inhibitors: Anticancer therapy and beyond. Mol Aspects Med. 2013;34(6):1217–56.View ArticlePubMedGoogle Scholar
- Papeo G, Casale E, Montagnoli A, Cirla A. PARP inhibitors in cancer therapy: an update. Expert Opin Ther Pat. 2013;23(4):503–14.View ArticlePubMedGoogle Scholar
- Pommier Y. Drugging topoisomerases: lessons and challenges. ACS Chem Biol. 2013;8(1):82–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Singh P, Alex JM, Bast F. Insulin receptor (IR) and insulin-like growth factor receptor 1 (IGF-1R) signaling systems: novel treatment strategies for cancer. Med Oncol. 2014;31(1):805.View ArticlePubMedGoogle Scholar
- Grimaldi AM, Guida T, D'Attino R, Perrotta E, Otero M, Masala A, Carteni G. Sunitinib: bridging present and future cancer treatment. Ann Oncol. 2007;18 Suppl 6:vi31–4.PubMedGoogle Scholar
- Ye F, Gao Q, Cai MJ. Therapeutic targeting of EGFR in malignant gliomas. Expert Opin Ther Targets. 2010;14(3):303–16.View ArticlePubMedGoogle Scholar
- Husseinzadeh N, Husseinzadeh HD. mTOR inhibitors and their clinical application in cervical, endometrial and ovarian cancers: a critical review. Gynecol Oncol. 2014;133(2):375–81.View ArticlePubMedGoogle Scholar
- Li J, Kim SG, Blenis J. Rapamycin: one drug, many effects. Cell Metab. 2014;19(3):373–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Pitts TM, Davis SL, Eckhardt SG, Bradshaw-Pierce EL. Targeting nuclear kinases in cancer: development of cell cycle kinase inhibitors. Pharmacol Ther. 2014;142(2):258–69.View ArticlePubMedGoogle Scholar
- Mikstacka R, Stefanski T, Rozanski J. Tubulin-interactive stilbene derivatives as anticancer agents. Cell Mol Biol Lett. 2013;18(3):368–97.View ArticlePubMedGoogle Scholar
- Fox E, Mosse YP, Meany HM, Gurney JG, Khanna G, Jackson HA, Gordon G, Shusterman S, Park JR, Cohn SL, et al. Time to disease progression in children with relapsed or refractory neuroblastoma treated with ABT-751: A report from the Children's Oncology Group (ANBL0621). Pediatr Blood Cancer. 2013.Google Scholar
- Rohena CC, Mooberry SL. Recent progress with microtubule stabilizers: new compounds, binding modes and cellular activities. Nat Prod Rep. 2014;31(3):335–55.View ArticlePubMedPubMed CentralGoogle Scholar
- Rosenberg B. Platinum coordination complexes in cancer chemotherapy. Naturwissenschaften. 1973;60(9):399–406.View ArticlePubMedGoogle Scholar
- Shen DW, Pouliot LM, Hall MD, Gottesman MM. Cisplatin resistance: a cellular self-defense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol Rev. 2012;64(3):706–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Schilling T, Keppler KB, Heim ME, Niebch G, Dietzfelbinger H, Rastetter J, Hanauske AR. Clinical phase I and pharmacokinetic trial of the new titanium complex budotitane. Invest New Drugs. 1996;13(4):327–32.View ArticlePubMedGoogle Scholar
- Lümmen G, Sperling H, Luboldt H, Otto T, Rübben H. Phase II trial of titanocene dichloride in advanced renal-cell carcinoma. Cancer Chemother Pharmacol. 1998;42(5):415–7.View ArticlePubMedGoogle Scholar
- Korfel A, Scheulen ME, Schmoll HJ, Grundel O, Harstrick A, Knoche M, Fels LM, Skorzec M, Bach F, Baumgart J, et al. Phase I clinical and pharmacokinetic study of titanocene dichloride in adults with advanced solid tumors. Clin Cancer Res. 1998;4(11):2701–8.PubMedGoogle Scholar
- Mross K, Robben-Bathe P, Edler L, Baumgart J, Berdel WE, Fiebig H, Unger C. Phase I Clinical Trial of a Day-1, −3, −5 Every 3 WeeksPhase I Clinical Trial of Day-1, −3, −5 Every 3 Weeks Schedule with Titanocene Dichloride (MKT 5) in Patients with Advanced Cancer. (Phase I Study Group of the AIO of the German Cancer Society). Onkologie. 2000;23(6):576–9.PubMedGoogle Scholar
- Toney JH, Marks TJ. Hydrolysis chemistry of the metallocene dichlorides M (. eta. 5-C5H5) 2Cl2, M = titanium, vanadium, or zirconium. Aqueous kinetics, equilibria, and mechanistic implications for a new class of antitumor agents. J Am Chem Soc. 1985;107(4):947–53.View ArticleGoogle Scholar
- Immel TA, Debiak M, Groth U, Bürkle A, Huhn T. Highly selective apoptotic cell death induced by halo-salane titanium complexes. Chem Med Chem. 2009;4(5):738–41.View ArticlePubMedGoogle Scholar
- Manna CM, Braitbard O, Weiss E, Hochman J, Tshuva EY. Cytotoxic salan-titanium(IV) complexes: high activity toward a range of sensitive and drug-resistant cell lines, and mechanistic insights. Chem Med Chem. 2012;7(4):703–8.View ArticlePubMedGoogle Scholar
- Immel TA, Groth U, Huhn T, Ohlschlager P. Titanium salan complexes displays strong antitumor properties in vitro and in vivo in mice. PLoS One. 2011;6(3), e17869.View ArticlePubMedPubMed CentralGoogle Scholar
- Köpf-Maier P. Intracellular localization of titanium within xenografted sensitive human tumors after treatment with the antitumor agent titanocene dichloride. J Struct Biol. 1990;105(1–3):35–45.View ArticlePubMedGoogle Scholar
- Harding MM, Mokdsi G. Antitumour metallocenes: structure-activity studies and interactions with biomolecules. Curr Med Chem. 2000;7(12):1289–303.View ArticlePubMedGoogle Scholar
- Balsells J, Carroll PJ, Walsh PJ. Achiral tetrahydrosalen ligands for the synthesis of C(2)-symmetric titanium complexes: a structure and diastereoselectivity study. Inorg Chem. 2001;40(22):5568–74.View ArticlePubMedGoogle Scholar
- Immel TA, Groth U, Huhn T. Cytotoxic titanium salan complexes: surprising interaction of salan and alkoxy ligands. Chemistry. 2010;16(9):2775–89.View ArticlePubMedGoogle Scholar
- Moreno-Villanueva M, Pfeiffer R, Sindlinger T, Leake A, Muller M, Kirkwood TB, Burkle A. A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks. BMC Biotechnol. 2009;9:39.View ArticlePubMedPubMed CentralGoogle Scholar
- Debiak M, Panas A, Steinritz D, Kehe K, Burkle A. High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay. Toxicology. 2011;280(1–2):53–60.View ArticlePubMedGoogle Scholar
- Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27–55.View ArticlePubMedGoogle Scholar
- Narita M, Nunez S, Heard E, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell. 2003;113(6):703–16.View ArticlePubMedGoogle Scholar
- Lawless C, Wang C, Jurk D, Merz A, Zglinicki T, Passos JF. Quantitative assessment of markers for cell senescence. Exp Gerontol. 2010;45(10):772–8.View ArticlePubMedGoogle Scholar
- Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92(20):9363–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Wyrsch P, Blenn C, Pesch T, Beneke S, Althaus FR. Cytosolic Ca2+ shifts as early markers of cytotoxicity. Cell Commun Signal. 2013;11(1):11.View ArticlePubMedPubMed CentralGoogle Scholar
- Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994;371(6495):346–7.View ArticlePubMedGoogle Scholar
- Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993;53(17):3976–85.PubMedGoogle Scholar
- Shen C, Gu M, Song C, Miao L, Hu L, Liang D, Zheng C. The tumorigenicity diversification in human embryonic kidney 293 cell line cultured in vitro. Biologicals. 2008;36(4):263–8.View ArticlePubMedGoogle Scholar
- Schur J, Manna CM, Tshuva A, Ott I. Quantification of the titanium content in metallodrugexposed tumor cells using HR-CS AAS. Metallodrugs. 2014;1:1–9.View ArticleGoogle Scholar
- Bürkle A, Virag L. Poly(ADP-ribose): PARadigms and PARadoxes. Mol Asp Med. 2013;34(6):1046–65.View ArticleGoogle Scholar
- Firsanov DV, Solovjeva LV, Svetlova MP. H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues. Clin Epigenetics. 2011;2(2):283–97.View ArticlePubMedPubMed CentralGoogle Scholar
- Paoletti A, Giocanti N, Favaudon V, Bornens M. Pulse treatment of interphasic HeLa cells with nanomolar doses of docetaxel affects centrosome organization and leads to catastrophic exit of mitosis. J Cell Sci. 1997;110(Pt 19):2403–15.PubMedGoogle Scholar
- Yang H, Ganguly A, Cabral F. Inhibition of cell migration and cell division correlates with distinct effects of microtubule inhibiting drugs. J Biol Chem. 2010;285(42):32242–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Coulthard LR, White DE, Jones DL, McDermott MF, Burchill SA. p38(MAPK): stress responses from molecular mechanisms to therapeutics. Trends Mol Med. 2009;15(8):369–79.View ArticlePubMedPubMed CentralGoogle Scholar
- Lamkanfi M, Kanneganti TD. Caspase-7: a protease involved in apoptosis and inflammation. Int J Biochem Cell Biol. 2010;42(1):21–4.View ArticlePubMedGoogle Scholar
- Fiset PO, Wou K, Arseneau J, Gilbert L. Vulvar Carcinosarcoma Secondary to Radiotherapy: A Case Report and Review of the Literature. J Low Genit Tract Dis. 2014.Google Scholar
- Kawamitsu H, Hoshino H, Okada H, Miwa M, Momoi H, Sugimura T. Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures. Biochemistry. 1984;23(16):3771–7.View ArticlePubMedGoogle Scholar