New approach of delivering cytotoxic drugs towards CAIX expressing cells : A concept of dual-target drugs

Abstract Carbonic anhydrase IX (CAIX) is a hypoxia-regulated and tumor-specific protein that maintains the pH balance of cells. Targeting CAIX might be a valuable approach for specific delivery of cytotoxic drugs, thereby reducing normal tissue side-effects. A series of dual-target compounds were designed and synthesized incorporating a sulfonamide, sulfamide, or sulfamate moiety combined with several different anti-cancer drugs, including the chemotherapeutic agents chlorambucil, tirapazamine, and temozolomide, two Ataxia Telangiectasia and Rad3-related protein inhibitors (ATRi), and the anti-diabetic biguanide agent phenformin. An ATRi derivative (12) was the only compound to show a preferred efficacy in CAIX overexpressing cells versus cells without CAIX expression when combined with radiation. Its efficacy might however not solely depend on binding to CAIX, since all described compounds generally display low activity as carbonic anhydrase inhibitors. The hypothesis that dual-target compounds specifically target CAIX expressing tumor cells was therefore not confirmed. Even though dual-target compounds remain an interesting approach, alternative options should also be investigated as novel treatment strategies

Mitochondrial DNA D-loop variants correlate with a primary open-angle glaucoma subgroup

IntroductionPrimary open-angle glaucoma (POAG) is a characteristic optic neuropathy, caused by degeneration of the optic nerve-forming neurons, the retinal ganglion cells (RGCs). High intraocular pressure (IOP) and aging have been identified as major risk factors; yet the POAG pathophysiology is not fully understood. Since RGCs have high energy requirements, mitochondrial dysfunction may put the survivability of RGCs at risk. We explored in buffy coat DNA whether mtDNA variants and their distribution throughout the mtDNA could be risk factors for POAG.MethodsThe mtDNA was sequenced from age- and sex-matched study groups, being high tension glaucoma (HTG, n=71), normal tension glaucoma patients (NTG, n=33), ocular hypertensive subjects (OH, n=7), and cataract controls (without glaucoma; n=30), all without remarkable comorbidities.ResultsNo association was found between the number of mtDNA variants in genes encoding proteins, tRNAs, rRNAs, and in non-coding regions in the different study groups. Next, variants that controls shared with the other groups were discarded. A significantly higher number of exclusive variants was observed in the D-loop region for the HTG group (~1.23 variants/subject), in contrast to controls (~0.35 variants/subject). In the D-loop, specifically in the 7S DNA sub-region within the Hypervariable region 1 (HV1), we found that 42% of the HTG and 27% of the NTG subjects presented variants, while this was only 14% for the controls and OH subjects. As we have previously reported a reduction in mtDNA copy number in HTG, we analysed if specific D-loop variants could explain this. While the majority of glaucoma patients with the exclusive D-loop variants m.72T>C, m.16163 A>G, m.16186C>T, m.16298T>C, and m.16390G>A presented a mtDNA copy number below controls median, no significant association between these variants and low copy number was found and their possible negative role in mtDNA replication remains uncertain. Approximately 38% of the HTG patients with reduced copy number did not carry any exclusive D-loop or other mtDNA variants, which indicates that variants in nuclear-encoded mitochondrial genes, environmental factors, or aging might be involved in those cases.ConclusionIn conclusion, we found that variants in the D-loop region may be a risk factor in a subgroup of POAG, possibly by affecting mtDNA replication

Mitochondrial Dysfunction Inhibits Hypoxia-Induced HIF-1 alpha Stabilization and Expression of Its Downstream Targets

mtDNA variations often result in bioenergetic dysfunction inducing a metabolic switch toward glycolysis resulting in an unbalanced pH homeostasis. In hypoxic cells, expression of the tumor-associated carbonic anhydrase IX (CAIX) is enhanced to maintain cellular pH homeostasis. We hypothesized that cells with a dysfunctional oxidative phosphorylation machinery display elevated CAIX expression levels. Increased glycolysis was observed for cytoplasmic 143B mutant hybrid (m.3243A>G, >94.5%) cells (p 500 mm(3)) took longer for mutant hybrid xenografts, but growth rates were comparable with control tumors upon establishment. Previously, it has been shown that HIF-1 alpha is responsible for tumor establishment. In agreement, we found that HIF-1 alpha expression levels and the pimonidazole-positive hypoxic fraction were reduced for the mutant hybrid xenografts. Our results demonstrate that OXPHOS dysfunction leads to a decreased HIF-1 alpha stabilization and subsequently to a reduced expression of its downstream targets and hypoxic fraction in vivo. In contrast, hypoxia-inducible factor 2-alpha (HIF-2 alpha) expression levels in these xenografts were enhanced. Inhibition of mitochondrial function is therefore an interesting approach to increase therapeutic efficacy in hypoxic tumors

HIF-1α and HIF-2α Differently Regulate the Radiation Sensitivity of NSCLC Cells

The hypoxia-inducible transcription factors (HIF)-1/2α are the main oxygen sensors which regulate the adaptation to intratumoral hypoxia. The aim of this study was to assess the role of the HIF proteins in regulating the radiation response of a non-small cell lung cancer (NSCLC) in vitro model. To directly assess the unique and overlapping functions of HIF-1α and HIF-2α, we use CRISPR gene-editing to generate isogenic H1299 non-small cell lung carcinoma cells lacking HIF-1α, HIF-2α or both. We found that in HIF1 knockout cells, HIF-2α was strongly induced by hypoxia compared to wild type but the reverse was not seen in HIF2 knockout cells. Cells lacking HIF-1α were more radiation resistant than HIF2 knockout and wildtype cells upon hypoxia, which was associated with a reduced recruitment of γH2AX foci directly after irradiation and not due to differences in proliferation. Conversely, double-HIF1/2 knockout cells were most radiation sensitive and had increased γH2AX recruitment and cell cycle delay. Compensatory HIF-2α activity in HIF1 knockout cells is the main cause of this radioprotective effect. Under hypoxia, HIF1 knockout cells uniquely had a strong increase in lactate production and decrease in extracellular pH. Using genetically identical HIF-α isoform-deficient cells we identified a strong radiosensitizing of HIF1, but not of HIF2, which was associated with a reduced extracellular pH and reduced glycolysis

Fitter mitochondria are associated with radioresistance in human head and neck SQD9 cancer cells

The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more and fitter mitochondria could help to preserve mitochondrial functions upon irradiation

Fitter Mitochondria Are Associated With Radioresistance in Human Head and Neck SQD9 Cancer Cells

The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more abundant and fitter mitochondria could help to preserve mitochondrial functions upon irradiation

Distinct radiation responses after <i>in vitro</i> mtDNA depletion are potentially related to oxidative stress

<div><p>Several clinically used drugs are mitotoxic causing mitochondrial DNA (mtDNA) variations, and thereby influence cancer treatment response. We hypothesized that radiation responsiveness will be enhanced in cellular models with decreased mtDNA content, attributed to altered reactive oxygen species (ROS) production and antioxidant capacity. For this purpose BEAS-2B, A549, and 143B cell lines were depleted from their mtDNA (ρ⁰). Overall survival after irradiation was increased (p<0.001) for BEAS-2B ρ⁰ cells, while decreased for both tumor ρ⁰ lines (p<0.05). In agreement, increased residual DNA damage was observed after mtDNA depletion for A549 and 143B cells. Intrinsic radiosensitivity (surviving fraction at 2Gy) was not influenced. We investigated whether ROS levels, oxidative stress and/or antioxidant responses were responsible for altered radiation responses. Baseline ROS formation was similar between BEAS-2B parental and ρ⁰ cells, while reduced in A549 and 143B ρ⁰ cells, compared to their parental counterparts. After irradiation, ROS levels significantly increased for all parental cell lines, while levels for ρ⁰ cells remained unchanged. In order to investigate the presence of oxidative stress upon irradiation reduced glutathione: oxidized glutathione (GSH:GSSG) ratios were determined. Irradiation reduced GSH:GSSG ratios for BEAS-2B parental and 143B ρ⁰, while for A549 this ratio remained equal. Additionally, changes in antioxidant responses were observed. Our results indicate that mtDNA depletion results in varying radiation responses potentially involving variations in cellular ROS and antioxidant defence mechanisms. We therefore suggest when mitotoxic drugs are combined with radiation, in particular at high dose per fraction, the effect of these drugs on mtDNA copy number should be explored.</p></div