Abstract Background The neuropathological changes of early Alzheimer’s disease (AD) include neurodegenerative loss of noradrenaline neurons in the locus coeruleus with decreasing noradrenaline availability in their projection areas such as the hippocampus. This diminishing noradrenaline availability is thought to play an important role pathophysiologically in the development of cognitive impairment in AD, because noradrenaline is not only essential for maintaining cognitive functions such as memory, learning and attention, but also its anti-inflammatory action, where its lack is known to accelerate the progression of AD in the mouse model. Therefore, the availability of in vivo biomarkers of the integrity of noradrenaline neurons may be beneficial for furthering our understanding of the role played by the noradrenaline system in the progressive cognitive dysfunction seen in AD patients. In this study, we investigated if PET imaging of noradrenaline transporters can predict the level of noradrenaline in the brain. Our hypothesis was PET measured noradrenaline transporter densities could predict the level of noradrenaline concentrations in the rat hippocampus after lesioning of noradrenaline neurons in this region. Results We chemically lesioned the hippocampus of rats (n = 15) by administering a neurotoxin, DSP-4, in order to selectively damage axonal terminals of noradrenergic neurons. These rats then underwent PET imaging of noradrenaline transporters using [¹¹C]MRB ((S,S)-[¹¹C]Methylreboxetine). To validate our hypothesis, postmortem studies of brain homogenates of these rats were performed to measure both noradrenaline transporter and noradrenaline concentrations. [¹¹C]MRB PET showed decreased noradrenaline transporter densities in a DSP-4 dose-dependent manner in the hippocampus of these rats. In turn, these PET measured noradrenaline transporter densities correlated very well with in vitro measured noradrenaline concentrations as well as in vitro transporter densities. Conclusions [¹¹C]MRB PET may be used as an in vivo biomarker of noradrenaline concentrations in the hippocampus of the neurodegenerating brain. Further studies appear warranted to extend its applicability to AD studies.

To develop antitumor drugs capable of targeting energy metabolism in the tumor microenvironment, we produced a series of potent new biguanide derivatives via structural modification of the arylbiguanide scaffold. We then conducted biological screening using hypoxia inducible factor (HIF)-1- and unfolded protein response (UPR)-dependent reporter assays and selective cytotoxicity assay under low glucose conditions. Homologation studies of aryl-(CH2)n-biguanides (n = 0-6) yielded highly potent derivatives with an appropriate alkylene linker length (n = 5, 6). The o-chlorophenyl derivative 7l (n = 5) indicated the most potent inhibitory effects on HIF-1- and UPR-mediated transcriptional activation (IC50; 1.0 ± 0.1 μM, 7.5 ± 0.1 μM, respectively) and exhibited selective cytotoxicity toward HT29 cells under low glucose condition (IC50; 1.9 ± 0.1 μM). Additionally, the protein expression of HIF-1α induced by hypoxia and of GRP78 and GRP94 induced by glucose starvation was markedly suppressed by the biguanides, thereby inhibiting angiogenesis. Metabolic flux and fluorescence-activated cell sorting analyses of tumor cells revealed that the biguanides strongly inhibited oxidative phosphorylation and activated compensative glycolysis in the presence of glucose, whereas both were strongly suppressed in the absence of glucose, resulting in cellular energy depletion and apoptosis. These findings suggest that the pleiotropic effects of these biguanides may contribute to more selective and effective killing of cancer cells due to the suppression of various stress adaptation systems in the tumor microenvironment.

We recently characterized the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition and found that 2-Cl-Phen attenuated ATF4 and GRP78, typical downstream targets of the unfolded protein response (UPR), together with c-Myc protein expression in a transcriptional and posttranscriptional manner. In the current study, we focused on the expression of ER-associated protein degradation (ERAD) components after treatment with 2-Cl-Phen under a serum- and glucose-deprived condition. Among nine ER-localizing factors regulating protein quality control within the ER, the amounts of Herp, GRP78, GRP94, and OS9 proteins were significantly downregulated by treatment with 2-Cl-Phen. In particular, replacement of the culture medium with the serum- and glucose-deprived medium induced the expression of Herp protein at the early phase. This increase in Herp protein was accompanied by an increase in its mRNA, and its induction was significantly dampened by 2-Cl-Phen. However, cotreatment with a proteasome inhibitor, MG132, restored Herp expression only to a limited extent. Taken together, these results show that 2-Cl-Phen changed the expression of several ERAD components, especially by transcriptional inhibition of Herp induction by 2-Cl-Phen when it occurred at an early phase, and this finding provides new insights into understanding the mechanisms of 2-Cl-Phen-mediated cytotoxicity.

We recently demonstrated the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition. In that study, we showed that the ATF6 arm of the ER stress pathway and c-Myc expression were downregulated 12 h after the treatment with 2-Cl-Phen. Through characterization of intracellular events at the early phase of the 2-Cl-Phen treatment before noticeable morphological changes, we found rapid fluctuations in the c-Myc and ATF4 proteins but not in their mRNAs in 2-Cl-Phen-treated HT-29 cells under the serum- and glucose-deprived condition. The 2-Cl-Phen-mediated downregulation of ATF4 protein was not paralleled by the phosphorylation status of PERK and eIF2α. Reduction of c-Myc expression by 2-Cl-Phen was more profound than that of ATF4 expression, and phosphorylated c-Myc was downregulated within 2 h. Pharmacological studies on the expression of c-Myc and ATF4 proteins showed that this decrease was mediated through proteasomal degradation but not by autophagy. Interestingly, treatment with lithium chloride, which is a well-known inhibitor of GSK3β, partially recovered the expression of ATF4 protein, but its effect on the level of total c-Myc protein was negligible. Treatment with 2-Cl-Phen increased the expression of phosphorylated AMPK, but Compound C, an AMPK inhibitor, did not influence the expression of c-Myc protein in HT-29 cells. Finally, we observed that 2-Cl-Phen partially attenuated the gene expression of integrin subunit α1 (ITGA1), a downstream target of c-Myc. Taken together, these results show that 2-Cl-Phen rapidly downregulated the expression of c-Myc in addition to ER stress responses in a post-translational manner. Further elucidation and improvement of this multi-target-directed compound will provide new insights for developing therapeutic strategies against cancer.

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.