ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress.

The homeostatic link between oxidative stress and autophagy plays an important role in cellular responses to a wide variety of physiological and pathological conditions. However, the regulatory pathway and outcomes remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules that regulate autophagy through ataxia-telangiectasia mutated (ATM) and cell cycle checkpoint kinase 2 (CHK2), a DNA damage response (DDR) pathway activated during metabolic and hypoxic stress. We report that CHK2 binds to and phosphorylates Beclin 1 at Ser90/Ser93, thereby impairing Beclin 1-Bcl-2 autophagy-regulatory complex formation in a ROS-dependent fashion. We further demonstrate that CHK2-mediated autophagy has an unexpected role in reducing ROS levels via the removal of damaged mitochondria, which is required for cell survival under stress conditions. Finally, CHK2-/- mice display aggravated infarct phenotypes and reduced Beclin 1 p-Ser90/Ser93 in a cerebral stroke model, suggesting an in vivo role of CHK2-induced autophagy in cell survival. Taken together, these results indicate that the ROS-ATM-CHK2-Beclin 1-autophagy axis serves as a physiological adaptation pathway that protects cells exposed to pathological conditions from stress-induced tissue damage.

SGLT2 inhibitors alleviated podocyte damage in lupus nephritis by decreasing inflammation and enhancing autophagy.

OBJECTIVES: The protective role of sodium glucose cotransporter 2 (SGLT2) inhibitors in renal outcomes has been revealed by large cardiovascular outcome trials among patients with type 2 diabetes. However, the effect of SGLT2 inhibitors on lupus nephritis (LN) and its underlying mechanisms remain unknown. METHODS: We applied empagliflozin treatment to lupus-prone MRL/lpr mice to explore the renal protective potential of SGLT2 inhibitors. An SGLT2 knockout monoclonal podocyte cell line was generated using the CRISPR/Cas9 system to examine the cellular and molecular mechanisms. RESULTS: In MRL/lpr mice treated with empagliflozin, the levels of mouse anti-dsDNA IgG-specific antibodies, serum creatinine and proteinuria were markedly decreased. For renal pathology assessment, both the glomerular and tubulointerstitial damages were lessened by administration of empagliflozin. The levels of SGLT2 expression were increased and colocalised with decreased synaptopodin in the renal biopsy samples from patients with LN and MRL/lpr mice with nephritis. The SGLT2 inhibitor empagliflozin could alleviated podocyte injury by attenuating inflammation and enhanced autophagy by reducing mTORC1 activity. Nine patients with LN treated with SGLT2 inhibitors with more than 2 months of follow-up showed that the use of SGLT2 inhibitors was associated with a significant decrease in proteinuria from 29.6% to 96.3%. Moreover, the estimated glomerular filtration rate (eGFR) was relatively stable during the treatment with SGLT2 inhibitors. CONCLUSION: This study confirmed the renoprotective effect of SGLT2 inhibitors in lupus mice, providing more evidence for non-immunosuppressive therapies to improve renal function in classic autoimmune kidney diseases such as LN.

The deacetylation of Foxk2 by Sirt1 reduces chemosensitivity to cisplatin.

In multiple types of cancer, decreased tumour cell apoptosis during chemotherapy is indicative of decreased chemosensitivity. Forkhead box K2 (FOXK2), which is essential for cell fate, regulates cancer cell apoptosis through several post-translational modifications. However, FOXK2 acetylation has not been extensively studied. Here, we evaluated the effects of sirtiun 1 (SIRT1) on FOXK2 deacetylation. Our findings demonstrated that SIRT1 inhibition increased FOXK2-induced chemosensitivity to cisplatin and that K223 in FOXK2 was acetylated. Furthermore, FOXK2 K223 deacetylation reduced chemosensitivity to cisplatin in vitro and in vivo. Mechanistically, FOXK2 was acetylated by the acetyltransferase cAMP response element binding protein and deacetylated by SIRT1. Furthermore, cisplatin attenuated the interaction between FOXK2 and SIRT1. Cisplatin or SIRT1 inhibition enhanced FOXK2 acetylation, thereby reducing the nuclear distribution of FOXK2. Additionally, FOXK2 K223 acetylation significantly affected the expression of cell cycle-related and apoptosis-related genes in cisplatin-stimulated cancer cells, and FOXK2 K223 hyperacetylation promoted mitotic catastrophe, which enhanced chemosensitivity to cisplatin. Overall, our results provided insights into the mechanisms of SIRT1-mediated FOXK2 deacetylation, which was involved in chemosensitivity to cisplatin.

Synthesis and biological evaluation of novel C6-amino substituted 4-azasteroidal purine nucleoside analogues.

Novel C6-amino substituted purine nucleoside analogues (2-12) bearing a modified pyranose-like D ring of the 4-azasteroid moiety were efficiently synthesized through nucleophilic substitution at C6 position of the steroidal nucleoside precursors (1a, b) with versatile amines. All the synthesized new compounds were evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. Among them, compounds 4b, 7b and 9b exhibited significant cytotoxicity with the IC50 values of 2.99 µM (PC-3), 2.84 µM, (PC-3) and 2.69 µM (Hela), respectively.

Immune-Stimulating Effects of Mycoleptodonoides aitchisonii (Agaricomycetes) Water Extract via TNF-α and IFN-γ.

Mycoleptodonoides aitchisonii has been used as culinary material in East Asia. We previously reported the antiasthmatic effect of M. aitchisonii; in this study we evaluated the mushroom's immune-stimulating effects. To analyze these effects, we conducted an in vitro study with splenocytes and an in vivo study with the Porsolt enforced swim test. In the in vitro study, the cell proliferation effect of the M. aitchisonii water extract (WT) was measured and the in vivo study depended on the dose of M. aitchisonii WT and the effect on swimming duration; several immune reaction-related factors were assessed, such as white blood cell count, differential cell count, the ratio of thymus or spleen weight to body weight, and concentrations of some cytokines in serum. Almost all measured factors that are related to immune stimulation were dose-dependently increased, such as the number of splenocytes and monocytes, the swimming duration, the relative weight of the thymus, and the expression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Based on the results, we conclude that M. aitchisonii WT has an immune-stimulating effect via TNF-α and IFN-γ, and that M. aitchisonii WT is an immune-stimulating agent and can be produced as a functional food.

Parentally imprinted allele typing at a short tandem repeat locus in intron 1a of imprinted gene KCNQ1.

A short tandem repeat (STR) in the intron 1a of paternally imprinted gene, KCNQ1, is evaluated as a new probe for use in parentally imprinting allele (PIA) typing. This typing can determine the inheritance of one allele from father by the methylation difference. Allelic and genotypic frequencies of the STR were determined using samples from 175 unrelated Japanese and 170 unrelated Germans. The polymorphism information contents were 0.652 and 0.634 for the Japanese and the Germans, respectively, indicating usefulness in individual identification. This method was applied to five Japanese families consisting of 19 individuals. Genomic DNA was digested by methylation-sensitive restriction endonucleases, HhaI and HapII, followed by PCR amplification using two-step sandwich primer sets and the products were analyzed on polyacrylamide gel electrophoresis. For all of the families, each child's paternal allele given by PIA typing corresponded to one of the two alleles from father, not the two from mother, that were determined by the STR genotyping. The results demonstrate that this STR probe is feasible for use in PIA typing and that its typing method can contribute to paternity testing.

Applicability of the parentally imprinted allele (PIA) typing of a VNTR upstream the H19 gene to forensic samples of different tissues.

The parentally imprinted allele (PIA) typing that we have recently developed determines parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene. The usefulness of this typing was demonstrated by its application to blood samples in paternity cases. However, its applicability to other tissue DNA remains to be tested. DNA samples from fifteen different postmortem tissues such as cerebrum, skeletal muscle and skin were examined, all of which were obtained from three autopsy cases 2-11h after death. DNA was digested with a methylation-sensitive HhaI enzyme and diluted solutions of the digests were subjected to the first PCR amplification, providing amplification of only the paternal H19 methylated allele. Subsequent VNTR typing was carried out for the amplified products to determine which allele was of paternal origin. No tissue-dependent difference was observed and all the samples examined, though degraded, were successfully used for determining the paternal allele. These results substantiate the usefulness of PIA typing in forensic examinations. Its application to two identity cases, a burned male body and a male body with adipocere formation, was also shown.

FK-3000 isolated from Stephania delavayi Diels. inhibits MDA-MB-231 cell proliferation by decreasing NF-κB phosphorylation and COX-2 expression.

The World Health Organization (WHO) has reported that cancer is one of the most prevalent diseases and a leading cause of death worldwide. Many anticancer drug development studies have been pursued over the last few decades and several viable drugs have been discovered, such as paclitaxel, topotecan and irinotecan. Previously, our research group uncovered the cytocidal and cytostatic effects of the plant Stephania delavayi Diels. In this study, we determined the active chemical to be 6,7-di-O-acetylsinococuline (FK-3000). The FK-3000 half maximal inhibitory concentration (IC50) in MDA-MB-231 breast carcinoma cells at 48 h was 0.52 µg/ml and it induced apoptosis in a dose- and time-dependent manner. FK-3000 suppressed NF-κB nuclear translocation, decreased NF-κB phosphorylation, and decreased COX-2 protein expression. MDA-MB-231 xenografted mice were treated with FK-3000, Taxol, or their combination for 21 days. The tumor size was smallest in the co-treatment group, indicating that FK-3000 may have a synergistic effect with Taxol. FK-3000 treatment showed no adverse effects on blood cell counts, serum protein levels, or pathology. These studies demonstrate that FK-3000, isolated from S. delavayi Diels., is a promising, pathway-specific anticancer agent that exhibits low toxicity.

Synthesis and biological evaluation of novel C6-cyclo secondary amine substituted purine steroid-nucleosides analogues.

Novel C6-cyclo secondary amine substituted purine steroid-nucleoside analogues (2-9) were efficiently synthesized through displacement of the C6 chloro on the purine ring of series 1 with versatile cyclic secondary amines, including pyrrolidines, piperidine, morpholine, and piperazines. All the newly-synthesized compounds were evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. Among them, compounds 5c and 6b exhibited significant cytotoxicity on PC-3 cell lines.

Synthesis and anticancer activity of novel C6-piperazine substituted purine steroid-nucleosides analogues.

Novel C6-piperazine substituted purine nucleoside analogues (2-9) bearing a modified pyranose-like D ring of the 4-azasteroid moiety were efficiently synthesized through nucleophilic substitution at C6 position of the steroid-nucleoside precursors (1) with versatile piperazines. All newly-synthesized compounds were evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. Among them, compounds 8b and 9b exhibited significant cytotoxicity on PC-3 cell lines.

Low-dose-rate californium-252 neutron intracavitary afterloading radiotherapy combined with conformal radiotherapy for treatment of cervical cancer.

PURPOSE: To study the efficacy of low-dose-rate californium-252 ((252)Cf) neutron intracavitary afterloading radiotherapy (RT) combined with external pelvic RT for treatment of cervical cancer. METHODS AND MATERIALS: The records of 96 patients treated for cervical cancer from 2006 to 2010 were retrospectively reviewed. For patients with tumors ≤4 cm in diameter, external beam radiation was performed (1.8 Gy/day, five times/week) until the dose reached 20 Gy, and then (252)Cf neutron intracavitary afterloading RT (once/week) was begun, and the frequency of external beam radiation was changed to four times/week. For patients with tumors >4 cm, (252)Cf RT was performed one to two times before whole-pelvis external beam radiation. The tumor-eliminating dose was determined by using the depth limit of 5 mm below the mucosa as the reference point. In all patients, the total dose of the external beam radiation ranged from 46.8 to 50 Gy. For (252)Cf RT, the dose delivered to point A was 6 Gy/fraction, once per week, for a total of seven times, and the total dose was 42 Gy. RESULTS: The mean ± SD patient age was 54.7 ± 13.7 years. Six patients had disease assessed at stage IB, 13 patients had stage IIA, 49 patients had stage IIB, 3 patients had stage IIIA, 24 patients had stage IIIB, and 1 patient had stage IVA. All patients obtained complete tumor regression (CR). The mean ± SD time to CR was 23.5 ± 3.4 days. Vaginal bleeding was fully controlled in 80 patients within 1 to 8 days. The mean ± SD follow-up period was 27.6 ± 12.7 months (range, 6-48 months). Five patients died due to recurrence or metastasis. The 3-year survival and disease-free recurrence rates were 89.6% and 87.5 %, respectively. Nine patients experienced mild radiation proctitis, and 4 patients developed radiocystitis. CONCLUSIONS: Low-dose-rate (252)Cf neutron RT combined with external pelvic RT is effective for treating cervical cancer, with a low incidence of complications.

Gastroprotective effect of cyanidin 3-glucoside on ethanol-induced gastric lesions in rats.

This study investigated the in vivo protective effect of cyanidin 3-glucoside (C3G) against ethanol-induced gastric lesions in rats. The experimental rats were treated with 80% ethanol after pretreatment with various doses of C3G (4 and 8 mg/kg of body weight), and the control rats received only 80% ethanol. Oral pretreatment with C3G significantly inhibited the formation of ethanol-induced gastric lesions and the elevation of the lipid peroxide level. In addition, pretreatment with C3G significantly increased the level of glutathione and the activities of radical scavenging enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, in gastric tissues. These results suggest that the gastroprotective effect of C3G removes the ethanol-induced lipid peroxides and free radicals and that it may offer a potential remedy for the treatment of gastric lesions.