Oridonin inhibits SASP by blocking p38 and NF-κB pathways in senescent cells.

Cellular senescence is a state of irreversible cell growth arrest that functions as a biological defense mechanism against severe DNA damage. Senescent cells with DNA damage produce pro-inflammatory cytokines, such as IL-6 and IL-8, and this phenomenon is called the senescence-associated secretory phenotype (SASP). SASP factors have been implicated in various disorders, including cancer. We performed a screening assay and identified oridonin as a candidate SASP inhibitor. Oridonin is an active diterpenoid that is isolated from Isodon plants and has been reported to exhibit anti-inflammatory, antibacterial, antioxidant, and antitumor activities. It reduced the secretion of IL-6 and IL-8 in senescent cells at the protein and mRNA levels. Oridonin also inhibited p65 subunit of NF-κB activity. However, oridonin did not affect SA ß-gal activity and enhanced the expression of p21. The expression and phosphorylation of p38 were down-regulated by oridonin. The p38 inhibitor SB203580 inhibited the secretion of IL-8, slightly inhibited the secretion of IL-6, and did not affect NF-κB activity. Therefore, the NF-κB and p38 pathways may contribute to the inhibition of SASP by oridonin. Oridonin has potential as a therapeutic agent for SASP-related diseases.

A chemoproteoinformatics approach demonstrates that aspirin increases sensitivity to MEK inhibition by directly binding to RPS5.

MEK inhibitors are among the most successful molecularly targeted agents used as cancer therapeutics. However, to treat cancer more efficiently, resistance to MEK inhibitor-induced cell death must be overcome. Although previous genetic approaches based on comprehensive gene expression analysis or RNAi libraries led to the discovery of factors involved in intrinsic resistance to MEK inhibitors, a feasible combined treatment with the MEK inhibitor has not yet been developed. Here, we show that a chemoproteoinformatics approach identifies ligands overcoming the resistance to cell death induced by MEK inhibition as well as the target molecule conferring this resistance. First, we used natural products, perillyl alcohol and sesaminol, which induced cell death in combination with the MEK inhibitor trametinib, as chemical probes, and identified ribosomal protein S5 (RPS5) as their common target protein. Consistently, trametinib induced cell death in RPS5-depleted cancer cells via upregulation of the apoptotic proteins BIM and PUMA. Using molecular docking and molecular dynamics (MD) simulations, we then screened FDA- and EMA-approved drugs for RPS5-binding ligands and found that acetylsalicylic acid (ASA, also known as aspirin) directly bound to RPS5, resulting in upregulation of BIM and PUMA and induction of cell death in combination with trametinib. Our chemoproteoinformatics approach demonstrates that RPS5 confers resistance to MEK inhibitor-induced cell death, and that aspirin could be repurposed to sensitize cells to MEK inhibition by binding to RPS5.

Novel RAF/MEK inhibitor CH5126766/VS-6766 has efficacy in combination with eribulin for the treatment of triple-negative breast cancer.

Various molecular-targeting drugs have markedly improved the treatment of patients with breast cancer. As yet, therapies for triple-negative breast cancer are mainly cytotoxic agents. To investigate the novel therapy for triple-negative breast cancer, we herein examined the effects of a new combination therapy comprising a RAF/MEK inhibitor CH5126766, also known as VS-6766, which we originally discovered, and eribulin. The combination of CH5126766 and eribulin potently inhibited cell growth in the triple-negative breast cancer cell lines tested. The underlying mechanism in the efficacy of this combination treatment in vitro and in vivo was due to enhanced apoptosis through the suppression of survivin and Bcl-2 family proteins. We also showed the suppressed expression of programmed cell death ligand 1 (PD-L1) in combination therapy in vivo. We found that combination therapy with eribulin and CH5126766 for triple-negative breast cancer inhibited cell growth by apoptosis and raised a possibility that immune responses through suppression of PD-L1 might partially contribute to inhibition of tumor growth, indicating the potential of this combination as a novel strategy for triple-negative breast cancer.

Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2.

Natural products have numerous bioactivities and are expected to be a resource for potent drugs. However, their direct targets in cells often remain unclear. We found that rabdosianone I, which is a bitter diterpene from an oriental herb for longevity, Isodon japonicus Hara, markedly inhibited the growth of human colorectal cancer cells by downregulating the expression of thymidylate synthase (TS). Next, using rabdosianone I-immobilized nano-magnetic beads, we identified two mitochondrial inner membrane proteins, adenine nucleotide translocase 2 (ANT2) and prohibitin 2 (PHB2), as direct targets of rabdosianone I. Consistent with the action of rabdosianone I, the depletion of ANT2 or PHB2 reduced TS expression in a different manner. The knockdown of ANT2 or PHB2 promoted proteasomal degradation of TS protein, whereas that of not ANT2 but PHB2 reduced TS mRNA levels. Thus, our study reveals the ANT2- and PHB2-mediated pleiotropic regulation of TS expression and demonstrates the possibility of rabdosianone I as a lead compound of TS suppressor.

Deactivation of Glutaminolysis Sensitizes PIK3CA-Mutated Colorectal Cancer Cells to Aspirin-Induced Growth Inhibition.

Aspirin is one of the most promising over-the-counter drugs to repurpose for cancer treatment. In particular, aspirin has been reported to be effective against PIK3CA-mutated colorectal cancer (CRC); however, little information is available on how the PIK3CA gene status affects its efficacy. We found that the growth inhibitory effects of aspirin were impaired upon glutamine deprivation in PIK3CA-mutated CRC cells. Notably, glutamine dependency of aspirin-mediated growth inhibition was observed in PIK3CA-mutated cells but not PIK3CA wild type cells. Mechanistically, aspirin induced G1 arrest in PIK3CA-mutated CRC cells and inhibited the mTOR pathway, inducing the same phenotypes as glutamine deprivation. Moreover, our study including bioinformatic approaches revealed that aspirin increased the expression levels of glutaminolysis-related genes with upregulation of activating transcription factor 4 (ATF4) in PIK3CA-mutated CRC cells. Lastly, the agents targeting glutaminolysis demonstrated significant combined effects with aspirin on PIK3CA-mutated CRC cells. Thus, these findings not only suggest the correlation among aspirin efficacy, PIK3CA mutation and glutamine metabolism, but also the rational combinatorial treatments of aspirin with glutaminolysis-targeting agents against PIK3CA-mutated CRC.

xCT Inhibition Increases Sensitivity to Vorinostat in a ROS-Dependent Manner.

As histone deacetylase inhibitors (HDACIs) have limited efficacy against solid tumors, we investigated whether and how oxidative stress is involved in sensitivity to HDACIs to develop a novel therapeutic option of HDACIs treatment. We first tested whether a reduction of the antioxidant glutathione (GSH) by glutamine deprivation affects sensitivity to a commercially available HDACI vorinostat and reactive oxygen species (ROS) accumulation. Next we investigated the relationship between a glutamate-cystine transporter xCT and the efficacy of vorinostat using siRNA of xCT and bioinformatic analyses. Finally, we verified the combinatory effects of vorinostat and the xCT inhibitor salazosulfapyridine (SASP) on ROS accumulation, cell death induction, and colony formation. Glutamine deprivation increased vorinostat-mediated cell death with ROS accumulation. Genetic ablation of xCT improved the efficacy of vorinostat, consistent with the results of public data analyses demonstrating that xCT expressions positively correlate with insensitivity to HDACIs in many types of cancer cell lines. Vorinostat caused ROS accumulation when combined with SASP, possibly resulting in synergistic ferroptosis. Our study provides a novel mechanistic insight into the mechanism underlying sensitivity to HDACIs involving xCT, suggesting xCT to be a promising predictive marker of HDACIs and rationalizing combinatory therapy of HDACIs with xCT inhibitors to induce ferroptosis.

Mevalonate pathway blockage enhances the efficacy of mTOR inhibitors with the activation of retinoblastoma protein in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common malignancy of kidney and remains largely intractable once it recurs after resection. mTOR inhibitors have been one of the mainstays used against recurrent RCC; however, there has been a major problem of the resistance to mTOR inhibitors, and thus new combination treatments with mTOR inhibitors are required. We here retrospectively showed that regular use of antilipidemic drug statins could provide a longer progression free survival (PFS) in RCC patients prescribed with an mTOR inhibitor everolimus than without statins (median PFS, 7.5 months vs. 3.2 months, respectively; hazard ratio, 0.52; 95% CI, 0.22-1.11). In order to give a rationale for this finding, we used RCC cell lines and showed the combinatorial effects of an mTOR inhibitor with statins induced a robust activation of retinoblastoma protein, whose mechanisms were involved in statins-mediated hindrance of KRAS or Rac1 protein prenylation. Finally, statins treatment also enhanced the efficacy of an mTOR inhibitor in RCC xenograft models. Thus, we provide molecular and (pre)clinical data showing that statins use could be a drug repositioning for RCC patients to enhance the efficacy of mTOR inhibitors.

Blockage of the mevalonate pathway overcomes the apoptotic resistance to MEK inhibitors with suppressing the activation of Akt in cancer cells.

With increasing clinical demands for MEK inhibitors in cancer treatment, overcoming the resistance to MEK inhibitors is an urgent problem to be solved. Numerous reports have shown that MEK inhibition results in the activation of PI3K-Akt signaling, which may confer apoptotic resistance to MEK inhibitors. We here demonstrate that the blockade of the mevalonate pathway using the antilipidemic drug statins represses Akt activation following MEK inhibition and induces significant apoptosis when co-treated with CH5126766 or trametinib. These events were clearly negated by the addition of mevalonate or geranylgeranyl pyrophosphate, indicating that the protein geranylgeranylation is implicated in the apoptotic resistance to MEK inhibitors. Furthermore, mechanistically, the combined treatment of CH5126766 with statins upregulated TNF-related apoptosis-inducing ligand (TRAIL), which was dependent on inhibition of the mevalonate pathway and is involved in apoptosis induction in human breast cancer MDA-MB-231 cells. The present study not only revealed that the mevalonate pathway could be targetable to enhance the efficacy of MEK inhibitors, but also proposes that combinatorial treatment of MEK inhibitors with statins may be a promising therapeutic strategy to sensitize cancer cells to apoptosis.

Role of the 5-HT4 receptor in chronic fluoxetine treatment-induced neurogenic activity and granule cell dematuration in the dentate gyrus.

BACKGROUND: Chronic treatment with selective serotonin (5-HT) reuptake inhibitors (SSRIs) facilitates adult neurogenesis and reverses the state of maturation in mature granule cells (GCs) in the dentate gyrus (DG) of the hippocampus. Recent studies have suggested that the 5-HT4 receptor is involved in both effects. However, it is largely unknown how the 5-HT4 receptor mediates neurogenic effects in the DG and, how the neurogenic and dematuration effects of SSRIs interact with each other. RESULTS: We addressed these issues using 5-HT4 receptor knockout (5-HT4R KO) mice. Expression of the 5-HT4 receptor was detected in mature GCs but not in neuronal progenitors of the DG. We found that chronic treatment with the SSRI fluoxetine significantly increased cell proliferation and the number of doublecortin-positive cells in the DG of wild-type mice, but not in 5-HT4R KO mice. We then examined the correlation between the increased neurogenesis and the dematuration of GCs. As reported previously, reduced expression of calbindin in the DG, as an index of dematuration, by chronic fluoxetine treatment was observed in wild-type mice but not in 5-HT4R KO mice. The proliferative effect of fluoxetine was inversely correlated with the expression level of calbindin in the DG. The expression of neurogenic factors in the DG, such as brain derived neurotrophic factor (Bdnf), was also associated with the progression of dematuration. These results indicate that the neurogenic effects of fluoxetine in the DG are closely associated with the progression of dematuration of GCs. In contrast, the DG in which neurogenesis was impaired by irradiation still showed significant reduction of calbindin expression by chronic fluoxetine treatment, suggesting that dematuration of GCs by fluoxetine does not require adult neurogenesis in the DG. CONCLUSIONS: We demonstrated that the 5-HT4 receptor plays an important role in fluoxetine-induced adult neurogenesis in the DG in addition to GC dematuration, and that these phenomena are closely associated. Our results suggest that 5-HT4 receptor-mediated phenotypic changes, including dematuration in mature GCs, underlie the neurogenic effect of SSRIs in the DG, providing new insight into the cellular mechanisms of the neurogenic actions of SSRIs in the hippocampus.

Electroconvulsive seizure-induced changes in gene expression in the mouse hypothalamic paraventricular nucleus.

Electroconvulsive therapy is an effective and rapid treatment for depression. In patients with depression, the function of the paraventricular nucleus of the hypothalamus (PVN) is frequently altered. Electroconvulsive seizure (ECS), which is a model of electroconvulsive therapy, upregulates the expression of c-fos in the PVN of animal models. Therefore, we hypothesized that ECS alters gene expression and function in the PVN. The PVN was microdissected from mouse brain sections following ECS treatment, and total RNA was analyzed by microarray. Two hours after ECS, the levels of expression of 2.6% (589 genes) of the genes showed a greater than 2-fold decrease and 0.9% (205 genes) showed a greater than 2-fold increase. Among these genes, 72 of the downregulated genes and 12 of the upregulated genes have been proposed to be associated with psychiatric disorders, such as depression, by knowledge database analyses. The groups of downregulated genes included neuropeptides (Cck), kinases (Prkcb, Camk2a), transcription factors (Tcf4), and transporters (Aqp4), and these have been suggested to be associated with psychiatric disorders and/or PVN function. The results of the present study indicated that ECS treatment could modulate PVN functions through altered gene expression, which may contribute to its antidepressant effects.

Electroconvulsive seizures activate anorexigenic signals in the ventromedial nuclei of the hypothalamus.

The ventromedial nucleus of the hypothalamus (VMH) plays an important role in feeding and energy homeostasis. Electroconvulsive seizure (ECS) therapy is highly effective in the treatment of several psychiatric diseases, including depression, but may also have beneficial effects in other neurological diseases. Although it has been reported that the neurons of the VMH are strongly activated by ECS stimulation, the specific effects of ECS in this hypothalamic subnucleus remain unknown. To address this issue, we investigated the changes in gene expression in microdissected-VMH samples in response to ECS in mice, and examined the behavioral effects of ECS on feeding behavior. ECS significantly induced the expression of immediate-early genes such as Fos, Fosb, and Jun, as well as Bdnf, Adcyap1, Hrh1, and Crhr2 in the VMH. Given that signals of these gene products are suggested to have anorexigenic roles in the VMH, we also examined the effect of ECS on food intake and body weight. Repeated ECS had a suppressive effect on food intake and body weight gain under both regular and high-fat diet conditions. Furthermore, gold-thioglucose-induced hypothalamic lesions, including the VMH and the arcuate nucleus, abolished the anorexigenic effects of ECS, indicating the requirement for the activation of the hypothalamus. Our data show an effect of ECS on increased expression of anorexigenic factors in the VMH, and suggest a role in the regulation of energy homeostasis by ECS.

Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling.

To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2(-/-) cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix- (ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE(2)-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions.

A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis.

Mammalian spermatogenesis produces numerous sperm for a long period based on a highly potent stem cell system, which relies on a special microenvironment, or niche, that has not yet been identified. In this study, using time-lapse imaging of green fluorescent protein-labeled undifferentiated spermatogonia (A(undiff)) and three-dimensional reconstitution, we revealed a biased localization of A(undiff) to the vascular network and accompanying Leydig and other interstitial cells, in intact testes. Differentiating spermatogonia left these niche regions and dispersed throughout the basal compartment of the seminiferous epithelium. Moreover, rearrangement of A(undiff) accompanied the vasculature alteration. We propose that the mammalian germline niche is established as a consequence of vasculature pattern formation. This is different from what is observed in Drosophila or Caenorhabditis elegans, which display developmentally specified niche structures within polarized gonads.

The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage.

Mammalian spermatogenesis is maintained by a continuous supply of differentiating cells from self-renewing stem cells. The stem cell activity resides in a small subset of primitive germ cells, the undifferentiated spermatogonia. However, the relationship between the establishment of this population and the initiation of differentiation in the developing testes remains unclear. In this study, we have investigated this issue by using the unique expression of Ngn3, which is expressed specifically in the undifferentiated spermatogonia, but not in the differentiating spermatogonia or their progenitors, the gonocytes. Our lineage analyses demonstrate that the first round of mouse spermatogenesis initiates directly from gonocytes, without passing through the Ngn3-expressing stage (Ngn3- lineage). By contrast, the subsequent rounds of spermatogenesis are derived from Ngn3-positive undifferentiated spermatogonia, which are also immediate descendents of the gonocytes and represent the stem cell function (Ngn3+ lineage). Thus, in mouse spermatogenesis, the state of the undifferentiated spermatogonia is not an inevitable step but is a developmental option that ensures continuous sperm production. In addition, the segregation of gonocytes into undifferentiated spermatogonia (Ngn3+ lineage) or differentiating spermatogonia (Ngn3- lineage) is topographically related to the establishment of the seminiferous epithelial cycle, thus suggesting a role of somatic components in the establishment of stem cells.