FBP1-Altered Carbohydrate Metabolism Reduces Leukemic Viability through Activating P53 and Modulating the Mitochondrial Quality Control System In Vitro.

Acute myeloid leukemia (AML)-the most frequent form of adult blood cancer-is characterized by heterogeneous mechanisms and disease progression. Developing an effective therapeutic strategy that targets metabolic homeostasis and energy production in immature leukemic cells (blasts) is essential for overcoming relapse and improving the prognosis of AML patients with different subtypes. With respect to metabolic regulation, fructose-1,6-bisphosphatase 1 (FBP1) is a gluconeogenic enzyme that is vital to carbohydrate metabolism, since gluconeogenesis is the central pathway for the production of important metabolites and energy necessary to maintain normal cellular activities. Beyond its catalytic activity, FBP1 inhibits aerobic glycolysis-known as the "Warburg effect"-in cancer cells. Importantly, while downregulation of FBP1 is associated with carcinogenesis in major human organs, restoration of FBP1 in cancer cells promotes apoptosis and prevents disease progression in solid tumors. Recently, our large-scale sequencing analyses revealed FBP1 as a novel inducible therapeutic target among 17,757 vitamin-D-responsive genes in MV4-11 or MOLM-14 blasts in vitro, both of which were derived from AML patients with FLT3 mutations. To investigate FBP1's anti-leukemic function in this study, we generated a new AML cell line through lentiviral overexpression of an FBP1 transgene in vitro (named FBP1-MV4-11). Results showed that FBP1-MV4-11 blasts are more prone to apoptosis than MV4-11 blasts. Mechanistically, FBP1-MV4-11 blasts have significantly increased gene and protein expression of P53, as confirmed by the P53 promoter assay in vitro. However, enhanced cell death and reduced proliferation of FBP1-MV4-11 blasts could be reversed by supplementation with post-glycolytic metabolites in vitro. Additionally, FBP1-MV4-11 blasts were found to have impaired mitochondrial homeostasis through reduced cytochrome c oxidase subunit 2 (COX2 or MT-CO2) and upregulated PTEN-induced kinase (PINK1) expressions. In summary, this is the first in vitro evidence that FBP1-altered carbohydrate metabolism and FBP1-activated P53 can initiate leukemic death by activating mitochondrial reprogramming in AML blasts, supporting the clinical potential of FBP1-based therapies for AML-like cancers.

Establishment of a ΔF508-CF promyelocytic cell line for cystic fibrosis research and drug screening.

Cystic fibrosis (CF), one of the most common genetic disorders, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. In spite of significant improvement in patient life expectancy, the disease remains lethal and incurable. Clinically, CF lung disease claims the most morbidity and mortality, characterized by chronic bacterial infection, persistent neutrophilic inflammation, and purulent small airway obstruction. Although all these manifestations are highly associated with neutrophils, the actual role of this phagocyte in the disease pathogenesis has not been fully appreciated. One of the major obstacles impeding such progress is the lack of CF neutrophil cell lines. Taking advantage of the new CRISPR/Cas9 gene-editing technology, we have generated a homozygous ΔF508-CF promyelocytic cell line from HL-60 cells, from which unlimited CF neutrophil cells can be differentiated. The derived cells showed defective CFTR presentation, deficient phagosomal hypochlorous acid (HOCl) production, and compromised microbial killing. Such a phenotype recapitulates that of primary neutrophils from CF patients. Thus, the established human CF promyelocytic cell line should be a useful tool for future CF basic research and drug screening.

Effect of Asparagus-P on cell metabolism of cultured kidney and inflammation-mediating cells.

Asparagus-P is a traditional herbal medicinal product consisting of a combination of dried and pulverized asparagus roots and parsley leaves in equal parts. It is commonly used to support aquaretic kidney function, i.e. the increased excretion of water from the kidneys without affecting electrolyte balance. The mechanisms of this aquaretic effectiveness are widely unknown and are controversial. By using two different kidney cell lines (distal tubule-derived Madin-Darby bovine kidney (MDBK) cells and proximal tubule-derived opossum kidney (OK) cells) the study investigated whether a stimulation of basal kidney cell metabolism might be responsible for the increased excretion of water from the kidneys. The results demonstrate that Asparagus-P was able to stimulate the metabolism of both kidney cell lines in a dose-dependent manner with a maximum stimulation at the recommended daily dosage after distribution in the whole body fluid. Metabolic stimulation was much more pronounced for OK than for MDBK cells. Whether a stimulation of cell metabolism correlates with an increased water excretion is currently unknown, but in vitro data might deliver the first evidence for a better understanding of the mechanism of action. Moreover, Asparagus-P inhibited the metabolism of inflammation-mediating cells (differentiated human promyelocytes). This increases the information on the antiinflammatory efficacy of Asparagus-P as already shown by its potential to inactivate reactive oxygen radicals.

[Effects of Chinese herbs for supplementing Shen and strengthening bone on IL-6 mediated myelogenic osteoclasts formation of ovariectomized rats in early stage].

OBJECTIVE: To observe the effect of Chinese herbs for supplementing Shen and strengthening bone (HB) on myelogenic osteoclasts formation, and gene expression of interleukin-6 (IL-6), IL-6 receptor (IL-6R) and gp130 in bone marrow. METHODS: Seventy-two healthy female SD rats of 3 months, were randomly divided into three groups, 24 in the sham-operated group (A), 24 in the ovariectomized group (B) and 24 in the after ovariectomy HB treated group (C). Bone marrow cells of 6 rats from each group were respectively collected and cultured at four time points (2nd, 4th, 6th and 12th weeks after operation). After 6 days of culture, the bone marrow cells were differentiated by Wright-Giemsa stain and TRAP stain, and total RNA in them was extracted by TRIZOL. RESULTS: Beginning from the 2nd week, the osteoclasts formation in Group B was higher than that in Group A (P < 0.05), and IL-6, IL-6R gene expression significantly increased in Group B (P < 0.05 or P < 0.01). These changes reached the peak in the 4th to 6th week, with the level maintained to the 12th week. As for comparison of Group B and C, the above-mentioned changes were significantly weakened in the latter (P < 0.05 or P < 0.01). No significant change of gp130 gene expression revealed in the whole course in either group. CONCLUSION: HB could inhibit the myelogenic osteoclasts formation in ovariectomized rats, this effect may be correlated with, partially at least, its inhibitory effect on the over-expressed IL-6 and IL-6R gene expression in myelocytes after ovariectomy.

Suppressive effects of Okinawan food items on free radical generation from stimulated leukocytes and identification of some active constituents: implications for the prevention of inflammation-associated carcinogenesis.

Okinawa prefecture in Japan is a distinct area characterized by unique traditional food habits and longevity. Prolonged exposure to activated leukocytes, playing pivotal roles in chronic inflammation-associated carcinogenesis, is known to lead to oxidative and nitrosative damage to macromolecules in the body since they are primary sources of free radicals, such as superoxide anion (O(2)(-)) and nitric oxide (NO). In this study, we estimated anti-oxidative and anti-nitrosative activities of Okinawan food items by employing two cellular experimental systems: (1) phorbol ester-induced O(2)(-) generation from differentiated HL-60 human promyelocytic leukemia cells; and (2) lipopolysaccharide (LPS)-induced NO generation in RAW264.7 murine macrophages. A total of 138 food items, consisting of 42 samples unique to Okinawa and 96 common in the Japanese main island, were purchased at local markets in Okinawa and extracted with chloroform. When tested at a concentration of 100 microg/ml, 38% (16/42) of the former showed 70% or more inhibition of O(2)(-) generation while 21% (20/96) of the latter did so. In parallel, 64% (27/42) of the former showed significant NO generation suppression in contrast to 48% (46/96) of the latter . Twenty-one active species were further tested at a concentration of 20 mug/ml, and eleven species, including sugar cane, wild turmeric, and zedoary, were indicated to be most promising items with anti-oxidative and anti-nitrosative properties. In addition, some of the active constituents (chebulagic acid, a resveratrol derivative, and sesquiterpenoids) were identified. Our results suggest that food items typical in the Okinawa area have higher cancer preventive potential than those common in Japan.

Spatial pattern of sonic hedgehog signaling through Gli genes during cerebellum development.

The cerebellum consists of a highly organized set of folia that are largely generated postnatally during expansion of the granule cell precursor (GCP) pool. Since the secreted factor sonic hedgehog (Shh) is expressed in Purkinje cells and functions as a GCP mitogen in vitro, it is possible that Shh influences foliation during cerebellum development by regulating the position and/or size of lobes. We studied how Shh and its transcriptional mediators, the Gli proteins, regulate GCP proliferation in vivo, and tested whether they influence foliation. We demonstrate that Shh expression correlates spatially and temporally with foliation. Expression of the Shh target gene Gli1 is also highest in the anterior medial cerebellum, but is restricted to proliferating GCPs and Bergmann glia. By contrast, Gli2 is expressed uniformly in all cells in the developing cerebellum except Purkinje cells and Gli3 is broadly expressed along the anteroposterior axis. Whereas Gli mutants have a normal cerebellum, Gli2 mutants have greatly reduced foliation at birth and a decrease in GCPs. In a complementary study using transgenic mice, we show that overexpressing Shh in the normal domain does not grossly alter the basic foliation pattern, but does lead to prolonged proliferation of GCPs and an increase in the overall size of the cerebellum. Taken together, these studies demonstrate that positive Shh signaling through Gli2 is required to generate a sufficient number of GCPs for proper lobe growth.