Mitochondrial metabolic reprograming via BRAF inhibition ameliorates senescence.

Senescence is defined as irreversible cell cycle arrest and constitutes a major driving force in diseases related to aging or premature aging. Recent studies indicate that activation of the serine/threonine protein kinase B-raf (BRAF) plays important roles in oncogene-induced senescence. However, it remains elusive whether BRAF inhibition might be effective for abrogating senescence. In this study, we assessed several BRAF inhibitors to identify compounds that ameliorate senescence and revealed SB590885 as an effective agent. Senescence-ameliorating effect upon BRAF inhibition was evident from the observation that SB590885 treatment increased cellular proliferation but diminished senescent phenotypes. Moreover, BRAF inhibition induced the mitochondrial functional recovery along with the metabolic reprogramming, which comprises two salient features that are altered in senescent cells. Furthermore, mitochondrial metabolic reprogramming via BRAF inhibition was a prerequisite for senescence amelioration. Taken together, our data revealed a novel mechanism in which senescence amelioration is mediated by mitochondrial metabolic reprogramming upon BRAF inhibition.

Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1ß.

Although strains of attenuated Salmonella typhimurium and wild-type Escherichia coli show similar tumor-targeting capacities, only S. typhimurium significantly suppresses tumor growth in mice. The aim of the present study was to examine bacteria-mediated immune responses by conducting comparative analyses of the cytokine profiles and immune cell populations within tumor tissues colonized by E. coli or attenuated Salmonellae. CT26 tumor-bearing mice were treated with two different bacterial strains: S. typhimurium defective in ppGpp synthesis (ΔppGpp Salmonellae) or wild-type E. coli MG1655. Cytokine profiles and immune cell populations in tumor tissue colonized by these two bacterial strains were examined at two time points based on the pattern of tumor growth after ΔppGpp Salmonellae treatment: 1) when tumor growth was suppressed ('suppression stage') and 2) when they began to re-grow ('re-growing stage'). The levels of IL-1ß and TNF-α were markedly increased in tumors colonized by ΔppGpp Salmonellae. This increase was associated with tumor regression; the levels of both IL-1ß and TNF-α returned to normal level when the tumors started to re-grow. To identify the immune cells primarily responsible for Salmonellae-mediated tumor suppression, we examined the major cell types that produce IL-1ß and TNF-α. We found that macrophages and dendritic cells were the main producers of TNF-α and IL-1ß. Inhibiting IL-1ß production in Salmonellae-treated mice restored tumor growth, whereas tumor growth was suppressed for longer by local administration of recombinant IL-1ß or TNF-α in conjunction with Salmonella therapy. These findings suggested that IL-1ß and TNF-α play important roles in Salmonella-mediated cancer therapy. A better understanding of host immune responses in Salmonella therapy may increase the success of a given drug, particularly when various strategies are combined with bacteriotherapy.

Branched-chain amino acid supplementation promotes aerobic growth of Salmonella Typhimurium under nitrosative stress conditions.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

In vivo efficacy of the combination of ciprofloxacin and cefotaxime against Vibrio vulnificus sepsis.

OBJECTIVES: The in vivo efficacy of a cefotaxime-ciprofloxacin combination against Vibrio vulnificus and the effects on rtxA1 expression of commonly used antibiotics are unknown. METHODS: In vitro time-kill studies were performed to evaluate synergism. Female BALB/c mice were injected subcutaneously with 1×10(7) or 1×10(8) cfu of V. vulnificus. Antibiotic therapy was initiated at 2 h after inoculation in the following four therapy groups: cefotaxime; ciprofloxacin; cefotaxime-plus-ciprofloxacin; and cefotaxime-plus-minocycline. The cytotoxicity of V. vulnificus for HeLa cells was measured using the lactate dehydrogenase assay; rtxA1 transcription was measured in a transcriptional reporter strain using a ß-galactosidase assay. RESULTS: In vitro time-kill assays exhibited synergism between cefotaxime and ciprofloxacin. In the animal experiments, the 96-h survival rate for the cefotaxime-plus-ciprofloxacin group (85%; 17/20) was significantly higher than that of the cefotaxime-plus-minocycline (35%; 7/20) and cefotaxime alone (0%; 0/20) groups (P<0.05 for both). Bacterial counts in the liver and spleen were significantly lower in the cefotaxime-plus-ciprofloxacin group 24 and 48 h after treatment, relative to the other groups. At sub-inhibitory concentrations, ciprofloxacin inhibited more effectively rtxA1 transcription and mammalian cell cytotoxicity than either minocycline or cefotaxime (P<0.05 for both). CONCLUSIONS: Ciprofloxacin is more effective at reducing rtxA1 transcription and subsequent cytotoxicity than either minocycline or cefotaxime, and the combination of ciprofloxacin and cefotaxime was more effective in clearing V. vulnificus in vivo than previously used regimens. These data suggest that the combination of ciprofloxacin and cefotaxime is an effective option for the treatment of V. vulnificus sepsis in humans.

Inappropriateness of quinolone in scrub typhus treatment due to gyrA mutation in Orientia tsutsugamushi Boryong strain.

The use of quinolone for treatment of rickettsial diseases remains controversial. Recent clinical studies suggest that quinolone is not as effective as others in patients with rickettsial diseases including scrub typhus, although the mechanism is not well understood. In this study, we evaluated the mutation in gyrA associated with quinolone resistance. We prospectively enrolled scrub typhus patients, collected blood samples and clinical data from October, 2010 to November, 2011. Among the 21 patients enrolled, one initially received ciprofloxacin for 3 days but was switched to doxycycline due to clinical deterioration. We obtained the gyrA gene of Orientia tsutsugamushi from 21 samples (20 Boryong strain, 1 Kato strain) and sequenced the quinolone resistance-determining region. All of 21 samples had the Ser83Leu mutation in the gyrA gene, which is known to be associated with quinolone resistance. This suggests that quinolones may be avoided for the treatment of serious scrub typhus.

Effect of Salmonella treatment on an implanted tumor (CT26) in a mouse model.

The use of bacteria has contributed to recent advances in targeted cancer therapy especially for its tumor-specific accumulation and proliferation. In this study, we investigated the molecular events following bacterial therapy using an attenuated Salmonella Typhimurium defective in ppGpp synthesis (ΔppGpp), by analyzing those proteins differentially expressed in tumor tissues from treated and untreated mice. CT26 murine colon cancer cells were implanted in BALB/c mice and allowed to form tumors. The tumor-bearing mice were treated with the attenuated Salmonella Typhimurium. Tumor tissues were analyzed by 2D-PAGE. Fourteen differentially expressed proteins were identified by mass spectrometry. The analysis revealed that cytoskeletal components, including vimentin, drebrin-like protein, and tropomyosin-alpha 3, were decreased while serum proteins related to heme or iron metabolism, including transferrin, hemopexin, and haptoglobin were increased. Subsequent studies revealed that the decrease in cytoskeletal components occurred at the transcriptional level and that the increase in heme and iron metabolism proteins occurred in liver. Most interestingly, the same pattern of increased expression of transferrin, hemopexin, and haptoglobin was observed following radiotherapy at the dosage of 14 Gy.

Protective effect of polygoni cuspidati radix and emodin on Vibrio vulnificus cytotoxicity and infection.

Vibrio vulnificus, a good model organism of bacterial septicemia, causes fatal septicemia manifesting a fulminating course and a high mortality rate within days. In order to identify new natural substances preventing V. vulnificus infection, a plant library was screened for inhibiting cytotoxicity to host cells by using Trypan blue staining and LDH assay. We found that Polygoni Cuspidati Radix potently suppressed the acute death of HeLa and RAW264.7 cells in a dose dependent manner. Further studies revealed that Polygoni Cuspidati Radix inhibited V. vulnificus growth and survival in HI broth and seawater, respectively. We confirmed that Polygoni Cuspidati Radix contained high level of emodin by thin layer chromatography (TLC). Emodin showed direct antibacterial activity against V. vulnificus. In addition, emodin prevented the morphologic damages and acute death of HeLa cells caused from V. vulnificus. The safety of Polygoni Cuspidati Radix and emodin to host cells was confirmed by MTT assay. Polygoni Cuspidati Radix and emodin protected mice from V. vulnificus infection.

Use of Clostridium septicum alpha toxins for isolation of various glycosylphosphatidylinositol-deficient cells.

In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.