Hypoxic Cytotoxicity Research Articles

Introducing urea into tirapazamine derivatives to enhance anticancer therapy.

Tirapazamine (TPZ) has been approved for multiple clinical trials relying on its excellent anticancer potential. However, as a typical hypoxia-activated prodrug (HAP), TPZ did not exhibit survival advantages in Phase III clinical trials when used in combination therapy due to the insufficient hypoxia levels in patients' tumors. In this study, to improve the therapeutic effects of TPZ, we first introduced urea to synthesize a series of urea-containing derivatives of TPZ. All urea-containing TPZ derivatives showed increased hypoxic cytotoxicity (9.51-30.85-fold) compared with TPZ, while maintaining hypoxic selectivity. TPZP, one of these derivatives, showed 20-fold higher cytotoxicity than TPZ while maintaining a similar hypoxic cytotoxicity ratio. To highly efficiently deliver TPZP to the tumors and reduce its side effects on healthy tissues, we further prepared TPZP into a nanodrug with fibrin-targeting ability: FT11-TPZP-NPs. CA4-NPs, a vascular disrupting agent, was used to increase the fibrin level within tumors and exacerbate tumor hypoxia. By being combined with CA4-NPs, FT11-TPZP-NPs can accumulate in the hypoxia-aggravated tumors and activate sufficiently to kill tumor cells. After a single-dose treatment, FT11-TPZP-NPs+CA4-NPs showed a high inhibition rate of 98.1% against CT26 tumor models with an initial volume of ∼480 mm3 and four out of six tumors were completely eliminated; it thereby exerted a significant antitumor effect. This study provides a new strategy for improving the therapeutic effect of TPZ and other HAPs in anticancer therapy.

WITHDRAWN: Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines

In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O2 levels (normoxic) which are 4-fold higher than O2 levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O2 microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical cancer drug discovery.

Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines.

In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O2 levels (normoxic) which are 4-fold higher than O2 levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O2 microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical cancer drug discovery.

Suppressing DRP1-mediated mitochondrial fission and mitophagy increases mitochondrial apoptosis of hepatocellular carcinoma cells in the setting of hypoxia

Transarterial embolization/transarterial chemoembolization (TAE/TACE) is the acceptable palliative treatment for hepatocellular carcinoma (HCC), mainly through ischemic necrosis induced by arterial embolization. However, how HCC cells survive under such ischemic hypoxic condition remains unclear, which can be exploited to potentiate TAE/TACE treatment. We hypothesized that targeting mitophagy can increase HCC cell apoptosis during hypoxia. HCC cells were subjected to hypoxia and then mitophagy was quantified. The role of dynamin-related protein 1 (DRP1) in hypoxia-induced HCC mitophagy was determined. Moreover, the synergistic effect of hypoxia and DRP1 inhibitor on HCC apoptosis was assessed in vitro and in vivo. Clinical association between DRP1 expression and outcome for HCC patients was validated. HCC cells that survived hypoxia showed significantly increased DRP1-mediated mitochondrial fission and mitophagy compared with cells in normoxia. Hypoxia induced mitophagy in surviving HCC cells by enhancing DRP1 expression and its translocation into the mitochondria and excessive mitochondrial fission into fragments. Blocking the DRP1 heightened the possibility of hypoxic cytotoxicity to HCC cells due to impaired mitophagy and increased the mitochondrial apoptosis, which involved decreased in mitochondrial membrane potential and mitochondrial release of apoptosis-inducing factor and cytochrome c. Additionally, DRP1 inhibitor Mdivi-1 suppressed the in vivo growth of hypoxia-exposed HCC cells. High expression of DRP1 was significantly associated with shorter survival in HCC patients. In conclusion, our results demonstrate that blocking DRP1-mediated mitochondrial fission and mitophagy increases the incidence of mitochondrial apoptosis of HCC cells during hypoxia, suggesting the new approach of targeting mitophagy to potentiate TAE/TACE.

Liposomal formulation of hypoxia activated prodrug for the treatment of ovarian cancer

In this work, a new sphingomyelin-cholesterol liposomal formulation (CPD100Li) for the delivery of a hypoxia activated prodrug of vinblastine, mon-N-oxide (CPD100), is developed. The optimized liposomal formulation uses an ionophore (A23187) mediated pH-gradient method. Optimized CPD100Li is characterized for size, drug loading, and stability. The in vitro toxicity of CPD100Li is assessed on different aspects of cell proliferation and apoptosis of ES2 ovarian cancer under normoxic and hypoxic conditions. The pharmacokinetics of CPD100Li in mice as well as the influence of A23187 on the retention of CPD100 are assessed. The dose limiting toxicity (DLT) and maximum tolerated dose (MTD) for CPD100Li are evaluated in nude mice.CPD100 is loaded in the liposome at 5.5 mg/mL. The sizes of CPD100Li using DLS, qNano and cryo-TEM techniques are 155.4 ± 4.2 nm, 132 nm, and 112.6 ± 19.8 nm, respectively. There is no difference between the in vitro characterization of CPD100Li with and without ionophore. Freshly prepared CPD100Li with ionophore are stable for 48 h at 4 °C, while the freeze-dried formulation is stable for 3 months under argon at 4 °C. The hypoxic cytotoxicity ratios (HCR) of CPD100 and CPD100Li are 0.16 and 0.11, respectively. CPD100Li under hypoxic conditions has a 9.2-fold lower IC50 value as compared to CPD100Li under normoxic conditions, confirming the hypoxia dependent activation of CPD100. CPD100Li treated ES2 cells show a time dependent enhanced cell death, along with caspase production and an increase in the number of cells in G0/G1 and higher cell arrest. The blood concentration profile of CPD100Li in mice without A23187 has a 12.6-fold lower area under the curve (AUC) and 1.6-fold lower circulation time compared to the CPD100Li with A23187. The DLT for both CPD100 and CPD100Li is 45 mg/kg and the MTD is 40 mg/kg in nude mice.Based on the preliminary data obtained, we clearly show that the presence of ionophore affects the in vivo stability of CPD100. CPD100Li presents a unique opportunity to develop a first-in-kind chemotherapy product based on achieving selective drug activation through the hypoxic physiologic microenvironment of solid tumors.

Protective roles of NRF2 signaling pathway in cobalt chloride-induced hypoxic cytotoxicity in human HaCaT keratinocytes

Hypoxia is a key pathological process involved in many cutaneous diseases. Nuclear factor E2-related factor 2 (NRF2) is a central regulator of antioxidant response element (ARE)-dependent transcription and plays a pivotal role in the cellular adaptive response to oxidative stress. Kelch-like ECH-associated protein 1 (KEAP1) is a cullin-3-adapter protein that represses the activity of NRF2 by mediating its ubiquitination and degradation. In the present study, we examined the role of NRF2 signaling pathway in the cytotoxicity induced by cobalt chloride(CoCl2), a hypoxia-mimicking agent, in human keratinocyte HaCaT cells with stable knockdown of NRF2 (NRF2-KD) and KEAP1 (KEAP1-KD). Acute CoCl2 exposure markedly increased the levels of intracellular reactive oxygen species (ROS), and resulted in hypoxic damage and cytotoxicity of HaCaT cells. Stable knockdown of NRF2 dramatically reduced the expression of many antioxidant enzymes and sensitized the cells to acute CoCl2-induced oxidative stress and cytotoxicity. In contrast, KEAP1-KD cells observably enhanced the activity of NRF2 and ARE-regulated genes and led to a significant resistance to CoCl2-induced cellular damage. In addition, pretreatment of HaCaT cells with tert-butylhydroquinone, a well-known NRF2 activator, protected HaCaT cells from CoCl2-induced cellular injury in a NRF2-dependent fashion. Likewise, physical hypoxia-induced cytotoxicity could be significantly ameliorated through NRF2 signaling pathway in HaCaT cells. Together, our results suggest that NRF2 signaling pathway is involved in antioxidant response triggered by CoCl2-induced oxidative stress and could protect human keratinocytes against acute CoCl2 -induced hypoxic cytotoxicity.

Honey Extracted Polyphenolics Reduce Experimental Hypoxia in Human Keratinocytes Culture.

Hypoxic assault affects fundamental cellular processes and generates oxidative stress on healthy cells/molecules. Honey extracted polyphenolics (HEP) as a natural antioxidant reduced hypoxic cytotoxicity in this study. Different honey samples were physicochemically characterized to identify preferred (jamun) honey [pH 3.55 ± 0.04, conductivity (μs/cm) = 6.66 ± 0.14, water content % (w/w) = 14.70 ± 0.35, total solid content % (w/w) = 85.30 ± 0.35, phenol content (mg GAE/100 g) = 403.55 ± 0.35, flavonoid content (mg QE/100 g) = 276.76 ± 4.10, radical scavenging activity (% 500 μL) = 147.75 ± 3.13, catalase activity (absorbance at 620 nm) = 0.226 ± 0.01]. HEP was tested in different doses on hypoxic and normoxic cells (HaCaT) using viability and antioxidant assays. Cardinal molecular expressions such as cadherin-catenin-cytoskeleton complex (namely, E-cadherin, β-catenin, and F-actin), hypoxia marker (Hif 1 α), proliferation marker (Ki67), and epithelial master regulator (p63) were studied by immuno-cytochemisty (ICC) and qRT-PCR. The 0.063 mg/mL HEP demonstrated better vitality and functionality of HaCaT cells as per viability assay (*, P < 0.01) even under hypoxia. ICC and qRT-PCR observations indicated restoration of cellular survival and homeostasis under 0.063 mg/mL HEP after hypoxic assault. Furthermore, major spectral changes for nucleic acid and membrane phospholipid reorganizations by Fourier transform infrared spectroscopy illustrated a positive impact of 0.063 mg/mL HEP on hypoxic cells considering proliferation and cellular integrity. It was concluded that a specific dose of jamun HEP reduces hypoxic cytotoxicity.

Pre-Clinical Activity of Novel Hypoxia-Activated FLT3 Inhibitors in FLT3-Mutated AML

Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are most frequently observed molecular abnormalities in AML, which lead to constitutive activation of the tyrosine kinase. While several mechanisms of resistance to FLT3 tyrosine kinase inhibitors (TKIs) have been identified, commonly failure is associated with lack of complete and sustained inhibition of FLT3 tyrosine kinase activity. FLT3 inhibition is particularly challenging in blasts within the hypoxic bone marrow (BM) microenvironment, which plays a crucial role in AML survival. Widespread hypoxia has been shown by us and others to be prevalent within the confines of the leukemic BM space, and to limit the efficacy of standard chemotherapy (Benito et al., PLoS One 6(8):e23108, 2011). With the goal of exploiting hypoxia as a physiological target and increasing selectivity to FLT3-mutant AML blasts, we have developed a series of hypoxia-activated prodrugs of FLT3 TKIs (HA-TKIs) based on the known clinical compounds sunitinib, AC220, MLN-518 and crenolanib. Bioreductive trigger conjugation was performed at the final step of the synthesis to provide the prodrugs as previously described (Lu et al., Tetrahedron, 69, p9130-9138, 2013). Under hypoxia, these compounds can undergo enzymatic one-electron reduction and then fragment selectively to release the cell permeable TKI, generating higher local concentrations of TKIs and limiting normal tissue exposures.Pulse radiolysis was used to determine the electron affinity (E(1)) and rate of fragmentation of each prodrug. The E(1) values ranged from -430 mV to -497 mV, while the rate of fragmentation of the prodrugs following one-electron reduction under hypoxia ranged from 28 s-1 to 400 s-1. SN37156 and SN37169 in particular demonstrated E(1) and kfrag parameters ideal for hypoxia-selective cellular metabolism (-449 mV, 85 s-1; -449 mV, 62 s-1, respectively).With the purpose of identifying the HA-TKIs with the most selective and dose-potent in vitro efficacy, we first performed an Oxic/Hypoxic screening of the hypoxia-dependent activity of HA-TKIs against FLT3-mutant AML cell lines. From the 10 compounds originally developed, SN37156 and SN37169 showed hypoxia-dependent apoptosis and growth arrest. SN37156 demonstrated the best activity against Molm13, Molm14 and MV4-11 cells under hypoxia with hypoxic cytotoxicity ratios (oxic/hypoxic IC50 ratios) of 4.9, 11 and 17.6, respectively.In our secondary screening we compared the activity of SN37156, SN37169 and their parental inhibitors on target modulation. Both HA-TKIs selectively inhibited FLT3 and ERK auto phosphorylation in MV4-11 cell line under hypoxic but not oxic conditions, with similar potency to the parental inhibitor under normal oxygen culture conditions.We next investigated the effects of both HA-TKIs on inhibition of FLT3 downstream targets, including AKT, ERK, pS6 and STAT5 using time-of-flight mass cytometry (CyTOF). The data demonstrated distinct patterns of signaling inhibition, with a dose-dependent p-ERK and p-S6 inhibition in MV4-11 and Molm-14 cells upon treatment with both HA-TKIs. In turn, both compounds at 100nM inhibited p-AKT and p-STAT5.Single dose tolerability studies were conducted in tumor-free C57BL6 mice. Prodrugs were administered by IP injection in 20% β-hydroxypropylcyclodextrin solution starting at 17.8 µmol/kg and escalating in 1.33-fold increments until lethality or severe mortality (mean body weight loss of >10%) was observed. The maximal tolerated dose for both, SN37156 and SN37169 was established at 56.1 µmol/kg. To test efficacy of HA-TKIs in vivo, we injected C57BL6 mice with genetically engineered Baf3-ITD/luc/GFP cells. Mice were randomized to treatment groups on day 8 when engraftment was documented by bioluminescent imaging (BLI), and treated with either vehicle or 56.1 µmol/kg of SN37156 or SN37169 I.P. every 3 days. Multiple doses (up to 10 so far; study is ongoing) were well tolerated. BLI demonstrated reduced leukemia burden in both treatment groups compared to control.In summary, our data indicate that HA-TKIs SN37156 and SN37169 inhibit mutant FLT3 and its selected downstream signaling targets under conditions resembling marrow hypoxia, and induce hypoxia-dependent growth arrest and apoptosis in FLT3-mutant AML cells. Planned studies in FLT3-mutant AML PDX will evaluate in vivo efficacy to eliminate primary human AML cells from hypoxic BM microenvironment. DisclosuresDaver:Kiromic: Research Funding; Ariad: Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding. Konopleva:Cellectis: Research Funding; Calithera: Research Funding.

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβ phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury.

Abstract 167: Possible New Application of HTK Solution to Protect Cardiac Arrest--Induced Brain Injury After Cardiopulmonary Resuscitation

Background: The cardioplegic solution, histidine-ketoglutarate-tryptophan (HTK), is a preservative solution for organ transplantation. Here we tested whether HTK may protect neurons against poor oxygen-induced cell damage and thus alleviate ischemic injury in brain after CA. Methods: The CA-mediated brain damage was induced by stop of ventilation for 4 min 30 sec (4’30’’) or 6 min 30 sec (6’30’’), and followed by manual CPR and epinephrine. HTK (200 μl) was given into brain via left carotid arterial catheter after CA. The sterile saline was given as controls. Neurological deficit scoring for motor function and mortality were evaluated on day 0 to 3 during recovery. The neuronal-like NG108-15 cells were applied and exposed to 2% oxygen or H2O2 to induce severe hypoxic (SH) injury or cytotoxicity in the presence or absence of HTK. Cell viability was assayed by the release of lactate dehydrogenase (LDH). Changes in caspase-3 activity for cell death were evaluated. Results: Compared with the 6’30’’ CA groups, the 4’30’’ groups had higher survival rates despite either in saline- or HTK-treated rats after 3-day CA (5/8 of 4’30’’ vs. 4/15 for 6’30’’ in saline-treated groups; 6/8 of 4’30’’ vs. 4/9 for 6’30’’ in HTK-treated groups). Neurological scores were significantly improved by HTK on day 1 for the 4’30’’ groups and days 1-3 for the 6’30” group. Most of improvements in the HTK-treated rats were related to increases in level of conscious, corneal reflex, righting reflex, motor coordination, movement activity but not respiration. Compared to the untreated NG108-15 cells, replacement of culture medium with HTK attenuated SH- and H2O2- mediated cytotoxicity in a volume- and time-dependent manner. Interestingly, these effects were associated with reduction of caspase-3 activation. Conclusion: These results suggest HTK is also an effective neuroprotective solution to protect neuron against hypoxia and treat CA-induced brain dysfunction via the mechanism of caspase-3 inhibition.

Synthesis, preferentially hypoxic apoptosis and anti-angiogenic activity of 1,2,4-benzotrazin-3-amine 1,4-dioxide bearing alkyl linkers with 1,2,4-benzotrazin-3-amine 1-oxide derivatives.

3-(Aminoalkylamino)-1,2,4-benzotriazine-1,4-dioxide-extended derivatives were synthesized by the structural modification of 3-amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) that incorporated homologue-alkyl linkers, without or with an extended 3-amino-1,2,4-benzotriazine-1-oxide moiety at the 3-position of the TPZ. According to sequential evaluation of preferentially normoxic and hypoxic cytotoxicities against MCF-7, NCI-H460 and HCT-116, most of the synthesized compounds exhibited hypoxic cytotoxicity greater than or comparable to that of TPZ. Among them, compounds 9a and 9b more powerfully inhibited the proliferation of MCF-7, NCI-H460 and HCT-116 in hypoxia than did TPZ. The representative of 3-(aminoalkylamino)-1,2,4-benzotriazine-1,4-dioxide-extended derivatives, 9a exhibited greater hypoxic cytotoxicity than TPZ, mediated by cell cycle arrest. The induction of DNA damage, the activation of caspase 3/7 and cleaved poly(ADP-ribose) polymerase-related apoptosis, which were detected in HCT-116 cells in both normoxia and hypoxia. In vitro anti-angiogenic assay of co-cultured HUVECs and fibroblasts that were exposed to the selected 7b, 8g, 9a and 9b exhibited 80-90% inhibition of tube formation at 20 μM, whereas TPZ exhibited approximately 50% inhibition of tube formation at 20 μM. At 2 μM, 9a and 9b significantly reduced the areas, lengths, paths and joints of tube formation by 70-80% and 45-50%, respectively. These results reveal that most of synthesized TPZ derivatives in this study exhibited more potent anti-angiogenesis than TPZ.

Synthesis, in vitro aerobic and hypoxic cytotoxicity and radiosensitizing activity of novel metronidazole tethered 5-fluorouracil

Background and the purpose of the studySeveral 2, 4-dinitrophenyl and 2,4-dinitrophenylamine tethered 5-FU (5-fluorouracil) compared to their components have shown minimal or no cytotoxicity to HT-29 cell line under aerobic conditions but high cytotoxicity and radiosensitizing effects under hypoxic conditions. In the present study the cytotoxicity and radiation potentiation of three novel analogues of these compounds by replacing 2,4-dinitrophenyl moiety with 2-methyl-5-nitroimidazole, a known radiosensitizer and cytotoxic agent was investigated.MethodsTethered compounds 7–9 were prepared by the reaction of 1-(t-butoxycarbonyl)-5-fluorouracil 6 with metronidazole esters 2–4 followed by removal of the t-butoxycarbonyl protecting group. Cytotoxicity of compounds in HT-29 cells with or without radiation were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI)-digitonin and clonogenic assays.ResultsTethered compounds 7–9 induced time-and concentration–dependent cytotoxicity under hypoxia but had no significant effect under aerobic conditions. These compounds also showed selective and concentration- dependent radiosensitization effects under hypoxic conditions.ConclusionTethered compounds 7–9 compared to 5-FU 5 showed minimal cytotoxicities under aerobic and selective radiosensitizing activities under hypoxic conditions. Also effects of these compounds were higher than those of metronidazole 1 which is a known cytotoxin and radiosensitizer under hypoxic conditions.

In vitro hypoxic cytotoxicity and hypoxic radiosensitization

Tumor hypoxia is a major problem in radiation therapy of solid tumors because of the radiosensitizing effect of oxygen. Nitroimidazole-containing compounds are oxygen mimetics accumulating in hypoxic tumor areas. However, the broad use of 2-nitroimidazoles as a hypoxic radiosensitizer is limited by their partially low efficacy and/or high neurotoxicity. Here, we characterized the in vitro hypoxic cytotoxicity and hypoxic radiosensitizing efficacy of N,N,N-tris [2-(2-nitro-1H-imidazol-1-yl)ethyl]amine (PRC) in a hypoxia-sensitive lymphoma and a hypoxia-resistant glioblastoma cell line by colony formation assay and flow cytometry. PRC exerted high hypoxic cytotoxic and radiosensitizing action on both cell lines at almost absent toxicity under normoxic conditions. In particular, under hypoxia, but not normoxia, PRC targeted the mitochondria resulting in oxidative stress, G(2)/M cell cycle arrest, and triggering of the intrinsic apoptosis pathway. Our in vitro findings suggest that PRC might be a promising new 2-nitroimidazole for improving radiation therapy of hypoxic tumors in vivo.

Synthesis and Biological Evaluation of 3-Aryl-quinoxaline-2-carbonitrile 1,4-Di-N-oxide Derivatives as Hypoxic Selective Anti-tumor Agents

A series of 3-aryl-2-quinoxaline-carbonitrile 1,4-di-N-oxide derivatives were designed, synthesized and evaluated for hypoxic and normoxic cytotoxic activity against human SMMC-7721, K562, KB, A549 and PC-3 cell lines. Many of these new compounds displayed more potent hypoxic cytotoxic activity compared with TX-402 and TPZ in the tumor cells based evaluation, which confirmed our hypothesis that the replacement of the 3-amine with the substituted aryl ring of TX-402 increases the hypoxic anti-tumor activity. The preliminary SAR revealed that 3-chloro was a favorable substituent in the phenyl ring for hypoxic cytotoxicity and 7-methyl or 7-methoxy substituted derivatives exhibited better hypoxic selectivity against most of the tested cell lines. The most potent compound, 7-methyl-3-(3-chlorophenyl)-quinoxaline-2-carbonitrile 1,4-dioxide (9h) was selected for further anti-tumor evaluation and mechanistic study. It also exhibited significant cytotoxic activity against BEL-7402, HepG2, HL-60, NCI-H460, HCT-116 and CHP126 cell lines in hypoxia with IC50 values ranging from 0.31 to 3.16 μM, and preliminary mechanism study revealed that 9h induced apoptosis in a caspase-dependent pathway.

Abstract 4641: Hypoxia activated pro-drug TH-302 induces hypoxia-dependent anti-leukemia activity in vitro and in vivo

Abstract Hypoxia in tumors is generally associated with chemoresistance and radioresistance. Hypoxia-inducible factor-1α (HIF-1α) was expressed in the bone marrow in 58% of primary acute myelogenous (AML) cases by immunohistochemistry (n=156). We have recently demonstrated a marked expansion of hypoxia in leukemia bone marrow (Benito PLoSOne 2011) and demonstrated hypoxia promotes chemoresistance in acute leukemias. Normoxic stabilization of HIF-1α using inhibitor of prolyl hydroxylases DMOG, or overexpression through infection with lentiviral non-degradable mutant HIF-1α, significantly diminished apoptosis induced by vincristine and etoposide in REH and Nalm-6 cells. These findings suggest that hypoxia mediates chemoresistance at least in part through HIF-1a. TH-302 is a 2-nitroimidazole of the cytotoxin bromo-isophosphoramide mustard that upon hypoxia-dependent activation induces DNA alkylation, a novel approach to target leukemia. We report the in vitro and in vivo anti leukemia activity of TH-302. In vitro, TH-302 exhibited potent anti-leukemia activity in pre-B ALL (REH, NALM6), AML (OCI-AML3, MOLM-13, KG-1) and CML cell lines (KBM5), with IC50s at 1% O2 ranging from 0.04µM to 2.3µM. The hypoxic cytotoxicity ratios (aerobic IC50/hypoxic IC50) were 11 and 92 in ALL (REH, NALM6) and 14, 80 and 116 in AML (OCI-AML3, KG-1, KBM5). The drug at 5-7.5µM also exhibited specific hypoxia dependent anti-leukemia activity in primary ALL and AML samples (normoxic vs hypoxic specific apoptosis defined as %AnnV positive cells +/− SEM normalized to untreated control cells): ALL-1: 5.1+/−0.5% vs 65.3+/−4.2%; ALL-2: 8.3+/−1.9% vs 28.4+/−6.3%; AML 1: 2.6+/−0.2% vs 40.3+/−1.5%). In in vivo leukemia models TH-302 (50 mg/kg IP 3 times a week for three weeks) reduced the number of circulating AML cells (control, 13.2+/−5.7 x106/ml; TH-302, 2.5+/−2.1 x106/ml) and prolonged survival of NSG mice engrafted with primary AML cells compared to the vehicle treated mice (median survival time: TH-302=75 days; Control=56 days; P=0.003). Finally, anti-leukemia efficacy of TH-302 was tested in vitro in combination with chemotherapy commonly used for treatment of AML (AraC, doxorubicin) and with demethylating agents azacitidine (5-AZA) and decitabine (DAC). Combination TH-302 with AraC or with DAC were synergistic in killing OCI-AML3 and MOLM-13 AML cells under hypoxia (combination index values of 0.4 and 0.67 for AraC plus TH-302 in MOLM13 and OCI-AML3, respectively; 0.3 for DAC plus TH-302 in OCI-AML3). In summary, our findings suggest that hypoxia and HIF-1α constitute important factors in the survival of leukemic blasts within the BM microenvironment. The results support targeting hypoxia with a hypoxia-activated drug such as TH-302 to enhance the efficacy of therapeutic regimens in AML. A Phase 1 clinical study of TH-302 in patients with relapsed/refractory hematologic malignancies is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4641. doi:1538-7445.AM2012-4641

Nitro seco Analogues of the Duocarmycins Containing Sulfonate Leaving Groups as Hypoxia-Activated Prodrugs for Cancer Therapy

The synthesis of 19 (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate prodrugs containing sulfonate leaving groups and 7-substituted electron-withdrawing groups is reported. These were designed to undergo hypoxia-selective metabolism to form potent DNA minor groove-alkylating agents. Analogues 17 and 24, containing the benzyl sulfonate leaving group and a neutral DNA minor groove-binding side chain, displayed hypoxic cytotoxicity ratios (HCRs) of >1000 in HT29 human cancer cells in vitro in an antiproliferative assay. Four analogues maintained large HCRs across a panel of eight human cancer cell lines. In a clonogenic assay, 19 showed an HCR of 4090 in HT29 cells. Ten soluble phosphate preprodrugs were also prepared and evaluated in vivo, alone and in combination with radiation in SiHa human tumor xenografts at a nontoxic dose. Compounds 34 and 39 displayed hypoxic log(10) cell kills (LCKs) of 1.78 and 2.71, respectively, equivalent or superior activity to previously reported chloride or bromide analogues, thus showing outstanding promise as hypoxia-activated prodrugs.

The effect of a bromide leaving group on the properties of nitro analogs of the duocarmycins as hypoxia-activated prodrugs and phosphate pre-prodrugs for antitumor therapy

Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkylating agents. A series of four nitroCBI alcohol prodrugs containing a bromide rather than chloride or sulfonate leaving group was synthesized. In assays for in vitro hypoxia-selective cytotoxicity against human tumor cell lines the two bromides with DNA minor groove binding basic side chains displayed hypoxic cytotoxicity ratios (HCRs) of 52–286 in HT29 cells and 41–43 in SiHa cells. These values compare well with a related previously reported chloride analog. The corresponding more water soluble phosphate pre-prodrugs of the bromides were synthesized and evaluated for in vivo antitumor activity against SiHa human tumor xenografts. All four phosphates, with both neutral and basic side chains, demonstrated activity providing statistically significant hypoxic log 10 cell kills of 0.87–2.80 at non-toxic doses, matching or proving superior to those of their chloride analogs.

The effect of sulfonate leaving groups on the hypoxia-selective toxicity of nitro analogs of the duocarmycins

A series of 3-substituted (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate (nitroCBI) prodrugs containing sulfonate leaving groups undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents. They were evaluated (along with chloride leaving group analogs for comparison) for their cytotoxicity against cultures of SKOV3 and HT29 human tumor cell lines under both aerobic and hypoxic conditions. Sulfonates with neutral side chains (e.g., 5,6,7-trimethoxyindole; TMI) show consistently higher hypoxic cytotoxicity ratios (HCRs) (34–246) than the corresponding chloro analogs (2.8–3.1) in SKOV3 cells, but these trends do not hold for compounds with cationic or polar neutral side chains.

Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells

ObjectiveThe efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of hypoxia-inducible-factor 1α (HIF1A). Overexpression of the hypoxia-responsive gene class III β-tubulin (TUBB3) has been associated with docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel. MethodsThe cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with methylation status at the HIF1A binding site. ResultsProstate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression. ConclusionThese data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer patients.

Structural modifications on the phenazine N, N′-dioxide-scaffold looking for new selective hypoxic cytotoxins

We have identified phenazine 5,10-dioxides as prodrugs for antitumour therapy that undergo hypoxic-selective bioreduction to form cytotoxic species. Here, we investigated some structural modifications in order to find new selective hypoxic cytotoxins and to establish the structural requirements for adequate activity. Three different chemical-series were prepared and the clonogenic survival of V79 cells on aerobic and anaerobic conditions was determined. Electrochemical- and DNA-interaction studies were done for the most relevant derivatives. The new fluoro-derivative 7-fluoro-2-aminophenazine 5,10-dioxide displayed selective toxicity towards hypoxic V79 cells having adequate hypoxic cytotoxicity ratio (HCR = 6.8) and being the most potent hypoxic cytotoxins ( P = 2.5 μM) described for this family of bioreductive agents. The reduction potential of the N-oxide moiety in this new fluoro-derivative was in the range for adequate bioreduction property. According to the fluorescence studies, the DNA-interaction mechanism was especially operative in the phenazine drugs more than in the corresponding prodrugs, phenazine dioxides.