Any tumor could be controlled by radiation therapy if sufficient dose were delivered to all tumor cells. Although technological advances in physical treatment delivery have been developed to allow more radiation dose conformity, normal tissues are invariably included in any radiation field within the tumor volume and also as part of the exit and entrance doses relevant for particle therapy. Mechanisms of normal tissue injury and related biomarkers are now being investigated, facilitating the discovery and development of a next generation of radiation protectors and mitigators. Bringing recent research advances stimulated by development of radiation countermeasures for mass casualties, to clinical cancer care requires understanding the impact of protectors and mitigators on tumor response. These may include treatments that modify cellular damage and death processes, inflammation, alteration of normal flora, wound healing, tissue regeneration and others, specifically to counter cancer site-specific adverse effects to improve outcome of radiation therapy. Such advances in knowledge of tissue and organ biology, mechanisms of injury, development of predictive biomarkers and mechanisms of radioprotection have re-energized the field of normal tissue protection and mitigation. Since various factors, including organ sensitivity to radiation, cellular turnover rate, and differences in mechanisms of injury manifestation and damage response vary among tissues, successful development of radioprotectors/mitigators/treatments may require multiple approaches to address cancer site specific needs. In this review, we discuss examples of important adverse effects of radiotherapy (acute and intermediate to late occurring, when it is delivered either alone or in conjunction with chemotherapy, and important limitations in the current approaches of using radioprotectors and/or mitigators for improving radiation therapy. Also, we are providing general concepts for drug development for improving radiation therapy.
Acute radiation effects; radiotherapy; radiation mitigator; radioprotector; oral mucositis; lung fibrosis; radiation-induced brain damage
This study tests whether the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA), combines favorably with ionizing radiation (IR) in controlling squamous carcinoma tumor growth. Animals bearing FaDu and A431 xenografts were treated with L-NNA in the drinking water. IR exposure was 10 Gy for tumor growth and survival studies and 4 Gy for ex vivo clonogenic assays. Cryosections were examined immunohistochemically for markers of apoptosis and hypoxia. Blood flow was assayed by fluorescent microscopy of tissue cryosections after i.v. injection of fluorospheres. Orally administered L-NNA for 24 hrs reduces tumor blood flow by 80% (p<0.01). Within 24 hrs L-NNA treatment stopped tumor growth for at least 10 days before tumor growth again ensued. The growth arrest was in part due to increased cell killing since a combination of L-NNA and a single 4 Gy IR caused 82% tumor cell killing measured by an ex vivo clonogenic assay compared to 49% by L-NNA or 29% by IR alone. A Kaplan-Meyer analysis of animal survival revealed a distinct survival advantage for the combined treatment. Combining L-NNA and IR was also found to be at least as effective as a single i.p. dose of cisplatin plus IR. In contrast to the in vivo studies, exposure of cells to L-NNA in vitro was without effect on clonogenicity with or without IR. Western and immunochemical analysis of expression of a number of proteins involved in NO signaling indicated that L-NNA treatment enhanced arginase-2 expression and that this may represent vasculature remodeling and escape from NOS inhibition. For tumors such as head and neck squamous carcinomas that show only modest responses to inhibitors of specific angiogenic pathways, targeting NO-dependent pro-survival and angiogenic mechanisms in both tumor and supporting stromal cells may present a potential new strategy for tumor control.
Kinesin-13 proteins have a critical role in animal cell mitosis, during which they regulate spindle microtubule dynamics through their depolymerisation activity. Much of what is known about Kinesin-13 function emanates from a relatively small sub-family of proteins containing MCAK and Kif2A/B. However, recent work on kinesins from the much more widely distributed, ancestral Kinesin-13 family, which includes human Kif24, have identified a second function in flagellum length regulation that may exist either alongside or instead of the mitotic role.
The African trypanosome Trypanosoma brucei encodes 7 distinct Kinesin-13 proteins, allowing scope for extensive specialisation of roles. Here, we show that of all the trypanosomal Kinesin-13 proteins, only one is nuclear. This protein, TbKIN13-1, is present in the nucleoplasm throughout the cell cycle, but associates with the spindle during mitosis, which in trypanosomes is closed. TbKIN13-1 is necessary for the segregation of both large and mini-chromosomes in this organism and reduction in TbKIN13-1 levels mediated by RNA interference causes deflects in spindle disassembly with spindle-like structures persisting in non-mitotic cells. A second Kinesin-13 is localised to the flagellum tip, but the majority of the Kinesin-13 family members are in neither of these cellular locations.
These data show that the expanded Kinesin-13 repertoire of trypanosomes is not associated with diversification of spindle-associated roles. TbKIN13-1 is required for correct spindle function, but the extra-nuclear localisation of the remaining paralogues suggests that the biological roles of the Kinesin-13 family is wider than previously thought.
Checkpoint kinase 1 and 2 (Chk1/Chk2), and the Aurora kinases play a critical role in the activation of the DNA damage response and mitotic spindle checkpoints. We have identified a novel inhibitor of these kinases and utilized this molecule to probe the functional interplay between these two checkpoints.
Fragment screening, structure guided design, and kinase cross screening resulted in the identification of a novel, potent small molecule kinase inhibitor (VER-150548) of Chk1 and Chk2 kinases with IC50s of 35 and 34 nM as well as the Aurora A and Aurora B kinases with IC50s of 101 and 38 nM. The structural rationale for this kinase specificity could be clearly elucidated through the X-ray crystal structure. In human carcinoma cells, VER-150548 induced reduplication and the accumulation of cells with >4N DNA content, inhibited histone H3 phosphorylation and ultimately gave way to cell death after 120 hour exposure; a phenotype consistent with cellular Aurora inhibition. In the presence of DNA damage induced by cytotoxic chemotherapeutic drugs, VER-150548 abrogated DNA damage induced cell cycle checkpoints. Abrogation of these checkpoints correlated with increased DNA damage and rapid cell death in p53 defective HT29 cells. In the presence of DNA damage, reduplication could not be observed. These observations are consistent with the Chk1 and Chk2 inhibitory activity of this molecule.
In the presence of DNA damage, we suggest that VER-150548 abrogates the DNA damage induced checkpoints forcing cells to undergo a lethal mitosis. The timing of this premature cell death induced by Chk1 inhibition negates Aurora inhibition thereby preventing re-entry into the cell cycle and subsequent DNA reduplication. This novel kinase inhibitor therefore serves as a useful chemical probe to further understand the temporal relationship between cell cycle checkpoint pathways, chemotherapeutic agent induced DNA damage and cell death.
The effectiveness of radiotherapy treatment could be significantly improved if tumour cells could be rendered more sensitive to ionizing radiation without altering the sensitivity of normal tissues. However many of the key, therapeutically exploitable mechanisms that determine intrinsic tumour radiosensitivity are largely unknown. We have conducted a siRNA screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumour radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumour cells (SQ20B) and irradiated normal tissue cells (MRC5). Using γH2AX foci at 24 hours after ionising radiation we identified several genes such as BRCA2, Lig IV and XRCC5, whose knockdown is known to cause increased cell radiosensitivity thereby validating the primary screening endpoint. In addition we identified POLQ (DNA polymerase theta) as a potential tumour-specific target. Subsequent investigations demonstrated that POLQ knockdown resulted in radiosensitisation of a panel of tumour cell lines from different primary sites, whilst having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumour specific radiosensitisation.
High-throughput Screen; siRNA; DNA-repair; POLQ; Tumour Radiosensitivity
The tumor microenvironment is a key factor in cancer treatment response. Recent work has shown that changes in the tumor vasculature can be achieved by inhibiting tumor cell signaling resulting in enhanced tumor oxygenation. These changes could promote responses to both chemo- and radiation therapy.
Hedgehog (Hh) signaling, a vital signaling pathway for the development and homeostasis of vertebrate tissues, is mediated by members of the Gli family of zinc finger transcription factors. Hh signaling increases the transcriptional activity of Gli proteins, at least in part, by inhibiting their proteolytic processing. Conversely, phosphorylation by cAMP-dependent protein kinase (PKA) inhibits Gli transcriptional activity by promoting their ubiquitination and proteolysis. Whether other post-translational modifications contribute to the regulation of Gli protein activity has been unclear.
Here we provide evidence that all three Gli proteins are targets of small ubiquitin-related modifier (SUMO)-1 conjugation. Expression of SUMO-1 or the SUMO E3 ligase, Pias1, increased Gli transcriptional activity in cultured cells. Moreover, PKA activity reduced Gli protein SUMOylation. Strikingly, in the embryonic neural tube, the forced expression of Pias1 increased Gli activity and induced the ectopic expression of the Gli dependent gene Nkx2.2. Conversely, a point mutant of Pias1, that lacks ligase activity, blocked the endogenous expression of Nkx2.2.
Together, these findings provide evidence that Pias1-dependent SUMOylation influences Gli protein activity and thereby identifies SUMOylation as a post-translational mechanism that regulates the hedgehog signaling pathway.
Many inhibitors of the EGFR-RAS-PI3 kinase-AKT signaling pathway are in clinical use or under development for cancer therapy. Here we show that treatment of mice bearing human tumor xenografts with inhibitors that block EGFR, RAS, PI3 kinase or AKT resulted in prolonged and durable enhancement of tumor vascular flow, perfusion and decreased tumor hypoxia. The vessels in the treated tumors had decreased tortuosity and increased internodal length accounting for the functional alterations. Inhibition of tumor growth cannot account for these results as the drugs were given at doses that did not alter tumor growth. The tumor cell itself was an essential target as HT1080 tumors that lack EGFR did not respond to an EGFR inhibitor, but did respond with vascular alterations to RAS or PI3 Kinase inhibition. We extended these observations to spontaneously arising tumors in MMTV-neu mice. These tumors also responded to PI3 kinase inhibition with decreased tumor hypoxia, increased vascular flow and morphological alterations of their vessels including increased vascular maturity and acquisition of pericyte markers. These changes are similar to the vascular normalization that has been described after anti-angiogenic treatment of xenografts. One difficulty in the use of vascular normalization as a therapeutic strategy has been its limited duration. In contrast, blocking tumor cell RAS-PI3K-AKT signaling led to persistent vascular changes that might be incorporated into clinical strategies based on improvement of vascular flow or decreased hypoxia. These results indicate that vascular alterations must be considered as a consequence of signaling inhibition in cancer therapy.
Signaling inhibition; tumor vasculature; hypoxia; EGFR; RAS; PI3 Kinase; AKT
Telomere length is considered as a biomarker of aging, stress, cancer. It has been associated with many chronic diseases such as hypertension and diabetes. Although, telomere shortening due to ionizing radiation has been reported in vitro, no in vivo data is available on natural background radiation and its effect on telomere length.
The present investigation is an attempt to determine the telomere length among human adults residing in high level natural radiation areas (HLNRA) and the adjacent normal level radiation areas (NLNRA) of Kerala coast in Southwest India. Genomic DNA was isolated from the peripheral blood mononuclear cells of 310 individuals (HLNRA: N = 233 and NLNRA: N = 77). Telomere length was determined using real time q-PCR. Both telomere (T) and single copy gene (S) specific primers were used to calculate the relative T/S and expressed as the relative telomere length. The telomere length was determined to be 1.22±0.15, 1.12±0.15, 1.08±0.08, 1.12±0.11, respectively, among the four dose groups (≤1.50, 1.51–3.00, 3.01–5.00 and >5.00 mGy per year), which did not show any dose response. The results suggested that the high level natural chronic radiation did not have significant effect on telomere length among young adult population living in HLNRA, which is indicative of better repair of telomeric ends. No significant difference in telomere length was observed between male and female individuals. In the present investigation, although the determination of telomere length was studied among the adults with an age group between 18 to 40 years (mean maternal age: 26.10±4.49), a negative correlation was observed with respect to age. However, inter-individual variation was (0.81–1.68) was clearly observed.
In this preliminary investigation, we conclude that elevated level of natural background radiation has no significant effect on telomere length among the adult population residing in HLNRAs of Kerala coast. To our knowledge, this is the first report from HLNRAs of the world where telomere length was determined on human adults. However, more samples from each background dose group and samples from older population need to be studied to derive firm conclusions.
Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor α was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.
Ras; EGFR; radiosensitivity; signal transduction; cancer
Despite advances in radiation delivery protocols, exposure of normal tissues during the course of radiation therapy remains a limiting factor of cancer treatment. If the canonical TGF-β/Smad pathway has been extensively studied and implicated in the development of radiation damage in various organs, the precise modalities of its activation following radiation exposure remain elusive. In the present study, we hypothesized that TGF-β1 signaling and target genes expression may depend on radiation-induced modifications in Smad transcriptional co-repressors/inhibitors expressions (TGIF1, SnoN, Ski and Smad7). In endothelial cells (HUVECs) and in a model of experimental radiation enteropathy in mice, radiation exposure increases expression of TGF-β/Smad pathway and of its target gene PAI-1, together with the overexpression of Smad co-repressor TGIF1. In mice, TGIF1 deficiency is not associated with changes in the expression of radiation-induced TGF-β pathway-related transcripts following localized small intestinal irradiation. In HUVECs, TGIF1 overexpression or silencing has no influence either on the radiation-induced Smad activation or the Smad3-dependent PAI-1 overexpression. However, TGIF1 genetic deficiency sensitizes mice to radiation-induced intestinal damage after total body or localized small intestinal radiation exposure, demonstrating that TGIF1 plays a role in radiation-induced intestinal injury. In conclusion, the TGF-β/Smad co-repressor TGIF1 plays a role in radiation-induced normal tissue damage by a Smad-independent mechanism.
Recent studies underline the important role of microRNAs (miRNA) in the development of lung cancer. The main regulators of miRNA biogenesis are the ribonucleases Drosha, Dicer and Ago2. Here the role of core proteins of miRNA biogenesis machinery in the response of human non-small and small cell lung carcinoma cell lines to treatment with ionizing radiation was assessed. We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines. However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation. Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment. Thus, modulation of miRNA biogenesis machinery is not sufficient to increase the radiosensitivity of lung tumors and other strategies are required to combat lung cancer.
The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors.
The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition.
Materials and methods
We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual γH2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well.
Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of γH2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor.
The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.
PI3K; mTOR; Radiosensitization; Endothelial cells; VEGF
Prostate cancer is the most common malignancy in men. In the present study, LNCaP (androgen-sensitive human prostate cancer cells) and PC-3 cells (androgen-independent human prostate cancer cells) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with arsenic trioxide (ATO) and to determine the underlying mechanisms in vitro and in vivo. We found that IR combined with ATO increases the therapeutic efficacy compared to individual treatments in LNCaP and PC-3 human prostate cancer cells. In addition, combined treatment showed enhanced reactive oxygen species (ROS) generation compared to treatment with ATO or IR alone in PC-3 cells. Combined treatment induced autophagy and apoptosis in LNCaP cells, and mainly induced autophagy in PC-3 cells. The cell death that was induced by the combined treatment was primarily the result of inhibition of the Akt/mTOR signaling pathways. Furthermore, we found that the combined treatment of cells pre-treated with 3-MA resulted in a significant change in AO-positive cells and cytotoxicity. In an in vivo study, the combination treatment had anti-tumor growth effects. These novel findings suggest that combined treatment is a potential therapeutic strategy not only for androgen-dependent prostate cancer but also for androgen-independent prostate cancer.
Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units – fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursor's proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions.
Ionizing irradiation is a commonly accepted treatment modality for lung cancer patients. However, the clinical outcome is hampered by normal tissue toxicity and tumor hypoxia. Since tumors often have higher levels of active heat shock protein 90 (Hsp90) than normal tissues, targeting of Hsp90 might provide a promising strategy to sensitize tumors towards irradiation. Hsp90 client proteins include oncogenic signaling proteins, cell cycle activators, growth factor receptors and hypoxia inducible factor-1α (HIF-1α). Overexpression of HIF-1α is assumed to promote malignant transformation and tumor progression and thus might reduce the accessibility to radiotherapy.
Herein, we describe the effects of the novel Hsp90 inhibitor NVP-AUY922 and 17-allylamino-17-demethoxygeldanamycin (17-AAG), as a control, on HIF-1α levels and radiosensitivity of lung carcinoma cells under normoxic and hypoxic conditions. NVP-AUY922 exhibited a similar biological activity to that of 17-AAG, but at only 1/10 of the dose. As expected, both inhibitors reduced basal and hypoxia-induced HIF-1α levels in EPLC-272H lung carcinoma cells. However, despite a down-regulation of HIF-1α upon Hsp90 inhibition, sensitivity towards irradiation remained unaltered in EPLC-272H cells under normoxic and hypoxic conditions. In contrast, treatment of H1339 lung carcinoma cells with NVP-AUY922 and 17-AAG resulted in a significant up-regulation of their initially high HIF-1α levels and a concomitant increase in radiosensitivity.
In summary, our data show a HIF-1α-independent radiosensitization of normoxic and hypoxic H1339 lung cancer cells by Hsp90 inhibition.
Exposure to uncontrolled irradiation in a radiologic terrorism scenario, a natural disaster or a nuclear battlefield, will likely be concomitantly superimposed on other types of injury, such as trauma. In the central nervous system, radiation combined injury (RCI) involving irradiation and traumatic brain injury may have a multifaceted character. This may entail cellular and molecular changes that are associated with cognitive performance, including changes in neurogenesis and the expression of the plasticity-related immediate early gene Arc. Because traumatic stimuli initiate a characteristic early increase in polyamine metabolism, we hypothesized that treatment with the polyamine inhibitor alpha-difluoromethylornithine (DFMO) would reduce the adverse effects of single or combined injury on hippocampus structure and function. Hippocampal dependent cognitive impairments were quantified with the Morris water maze and showed that DFMO effectively reversed cognitive impairments after all injuries, particularly traumatic brain injury. Similar results were seen with respect to the expression of Arc protein, but not neurogenesis. Given that polyamines have been found to modulate inflammatory responses in the brain we also assessed the numbers of total and newly born activated microglia, and found reduced numbers of newly born cells. While the mechanisms responsible for the improvement in cognition after DFMO treatment are not yet clear, the present study provides new and compelling data regarding the potential use of DFMO as a potential countermeasure against the adverse effects of single or combined injury.
Hypoxia-inducible factor (HIF) is an attractive therapeutic target for renal cell carcinoma (RCC) as its high expression due to the loss of von Hippel-Lindau (VHL) promotes RCC progression. Considering this, we hypothesized that ELR510444, a novel orally available small molecule inhibitor of HIF activity, would reduce angiogenesis and possess significant activity in RCC. The mechanism of action and therapeutic efficacy of ELR510444 were investigated in in vitro and in vivo models of RCC.
ELR510444 decreased HIF-1α and HIF-2α levels, reduced RCC cell viability and clonogenic survival, and induced apoptosis. VHL-deficient RCC cells were more sensitive to ELR510444-mediated apoptosis and restoration of VHL promoted drug resistance. Higher concentrations of ELR51044 promoted apoptosis independently of VHL status, possibly due to the microtubule destabilizing properties of this agent. ELR510444 significantly reduced tumor burden in the 786-O and A498 RCC xenograft models. These effects were associated with increased necrosis and apoptosis and inhibition of angiogenesis.
ELR510444 is a promising new HIF inhibitor that reduced RCC cell viability, induced apoptosis, and diminished tumor burden in RCC xenograft models. ELR510444 also destabilized microtubules suggesting that it possesses vascular disrupting and anti-angiogenic properties. Further investigation of ELR510444 for the therapy of RCC is warranted.
Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.
Different types of ionizing radiation produce different dependences of cancer risk on radiation dose/dose rate. Sparsely ionizing radiation (e.g. γ-rays) generally produces linear or upwardly curving dose responses at low doses, and the risk decreases when the dose rate is reduced (direct dose rate effect). Densely ionizing radiation (e.g. neutrons) often produces downwardly curving dose responses, where the risk initially grows with dose, but eventually stabilizes or decreases. When the dose rate is reduced, the risk increases (inverse dose rate effect). These qualitative differences suggest qualitative differences in carcinogenesis mechanisms. We hypothesize that the dominant mechanism for induction of many solid cancers by sparsely ionizing radiation is initiation of stem cells to a pre-malignant state, but for densely ionizing radiation the dominant mechanism is radiation-bystander-effect mediated promotion of already pre-malignant cell clone growth. Here we present a mathematical model based on these assumptions and test it using data on the incidence of dysplastic growths and tumors in the mammary glands of mice exposed to high or low dose rates of γ-rays and neutrons, either with or without pre-treatment with the chemical carcinogen 7,12-dimethylbenz-alpha-anthracene (DMBA). The model provides a mechanistic and quantitative explanation which is consistent with the data and may provide useful insight into human carcinogenesis.
The link between inflammation and colorectal carcinoma has been acknowledged. However, the impact of bacterial lipopolysaccharide (LPS) binding to Toll-like receptor 4 (TLR4) on chemokine receptors in human colorectal carcinoma cells still remains to be elucidated. The present study shows that exposure to LPS elevated CXC chemokine receptor 7 (CXCR7) expression in colorectal carcinoma SW480 and Colo 205 cell lines expressing TLR4/myeloid differential protein (MD-2). CXCR7 is associated with SW480 cell proliferation and migration. However, exposure of SW480 and Colo 205 cells to LPS had no effect on CXCR4 expression. To further support the above results, the expression of TLR4, MD-2, and CXCR7 was analyzed in human colorectal carcinoma tissues. Higher rates of TLR4 (53%), MD-2 (70%), and CXCR7 (29%) expression were found in colorectal carcinoma tissues than in normal tissues. We demonstrated that the recombination of TLR4, MD-2 and CXCR7 strongly correlated with tumor size, lymph node metastasis and distant metastasis in colorectal carcinoma tissue samples (p = 0.037, p = 0.002, p = 0.042, resp.). Accordingly, simultaneous examination of the expression of TLR4, MD-2 and CXCR7 in cancer tissues of colorectal carcinoma may provide valuable prognostic diagnosis of carcinoma growth and metastasis. Interplay of TLR4, MD-2 and CXCR7 may be of interest in the context of novel immunomodulatory therapies for colorectal carcinoma.
Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.
Humans are exposed to the DNA damaging agent, ionizing radiation (IR), from background radiation, medical treatments, occupational and accidental exposures. IR causes changes in transcription, but little is known about alternative transcription in response to IR on a genome-wide basis. These investigations examine the response to IR at the exon level in human cells, using exon arrays to comprehensively characterize radiation-induced transcriptional expression products. Previously uncharacterized alternative transcripts that preferentially occur following IR exposure have been discovered. A large number of genes showed alternative transcription initiation as a response to IR. Dose-response and time course kinetics have also been characterized. Interestingly, most genes showing alternative transcript induction maintained these isoforms over the dose range and times tested. Finally, clusters of co-ordinately up- and down-regulated radiation response genes were identified at specific chromosomal loci. These data provide the first genome-wide view of the transcriptional response to ionizing radiation at the exon level. This study provides novel insights into alternative transcripts as a mechanism for response to DNA damage and cell stress responses in general.
Dynamic contrast enhanced (DCE-) MRI is commonly applied for the monitoring of antiangiogenic therapy in oncology. Established pharmacokinetic (PK) analysis methods of DCE-MRI data do not sufficiently reflect the complex anatomical and physiological constituents of the analyzed tissue. Hence, accepted endpoints such as Ktrans reflect an unknown multitude of local and global physiological effects often rendering an understanding of specific local drug effects impossible. In this work a novel multi-compartment PK model is presented, which for the first time allows the separation of local and systemic physiological effects. DCE-MRI data sets from multiple, simultaneously acquired tissues, i.e. spinal muscle, liver and tumor tissue, of hepatocellular carcinoma (HCC) bearing rats were applied for model development. The full Markov chain Monte Carlo (MCMC) Bayesian analysis method was applied for model parameter estimation and model selection was based on histological and anatomical considerations and numerical criteria. A population PK model (MTL3 model) consisting of 3 measured and 6 latent (unobserved) compartments was selected based on Bayesian chain plots, conditional weighted residuals, objective function values, standard errors of model parameters and the deviance information criterion. Covariate model building, which was based on the histology of tumor tissue, demonstrated that the MTL3 model was able to identify and separate tumor specific, i.e. local, and systemic, i.e. global, effects in the DCE-MRI data. The findings confirm the feasibility to develop physiology driven multi-compartment PK models from DCE-MRI data. The presented MTL3 model allowed the separation of a local, tumor specific therapy effect and thus has the potential for identification and specification of effectors of vascular and tissue physiology in antiangiogenic therapy monitoring.