A novel matrine derivate inhibits differentiated human hepatoma cells and hepatic cancer stem-like cells by suppressing PI3K/AKT signaling pathways.

Matrine is an alkaloid extracted from a Chinese herb Sophora flavescens Ait, which has shown chemopreventive potential against various cancers. In this study, we evaluated the anticancer efficacy of a novel derivative of matrine, (6aS, 10S, 11aR, 11bR, 11cS)-10- methylamino-dodecahydro- 3a,7a-diazabenzo (de) (MASM), against human hepatocellular carcinoma (HCC) cells and their corresponding sphere cells in vitro and in vivo. Human HCC cell lines (Hep3B and Huh7) were treated with MASM. Cell proliferation was assessed using CCK8 and colony assays; cell apoptosis and cell cycle distributions were examined with flow cytometry. The expression of cell markers and signaling molecules was detected using Western blot and qRT-PCR analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Hep3B and Huh7 cells. The in vivo antitumor efficacy of MASM was evaluated in Huh7 cell xenograft model in BALB/c nude mice, which were administered MASM (10 mg·kg-1·d-1, ig) for 3 weeks. After the treatment was completed, tumor were excised and weighed. A portion of tumor tissue was enzymatically dissociated to obtain a single cell suspension for the spheroid formation assays. MASM (2, 10, 20 µmol/L) dose-dependently inhibited the proliferation of HCC cells, and induced apoptosis, which correlated with a reduction in Bcl-2 expression and an increase in PARP cleavage. MASM also induced cell cycle arrest in G0/G1 phase, which was accompanied by increased p27 and decreased Cyclin D1 expression. Interestingly, MASM (2, 10, and 20 µmol/L) drastically reduced the EpCAM+/CD133+ cell numbers, suppressed the sphere formation, inhibited the expression of stem cell marker genes and promoted the expression of mature hepatocyte markers in the Hep3B and Huh7 spheroids. Additionally, MASM dose-dependently suppressed the PI3K/AKT/mTOR and AKT/GSK3ß/ß-catenin signaling pathways in Hep3B and Huh7 cells. In Huh7 xenograft bearing nude mice, MASM administration significantly inhibited Huh7 xenograft tumor growth and markedly reduced the number of surviving cancer stem-like cells in the tumors. MASM administration also reduced the expression of stem cell markers while increasing the expression of mature hepatocyte markers in the tumor tissues. The novel derivative of matrine, MASM, markedly suppresses HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the treatment of hepatocellular carcinoma.

The Matrine Derivate MASM Prolongs Survival, Attenuates Inflammation, and Reduces Organ Injury in Murine Established Lethal Sepsis.

BACKGROUND: MASM, a novel derivative of matrine, has inhibitory effects on activation of macrophages, dendritic cells, and hepatic stellate cells and binds to ribosomal protein S5 (RPS5). This study was designed to evaluate the effect of MASM on murine-established lethal sepsis and its mechanisms. METHODS: Mouse peritoneal macrophages and RAW264.7 cells that were infected with recombinant lentiviruses encoding shRPS5 were incubated with lipopolysaccharide (LPS) in the absence or presence of MASM in vitro. Endotoxemia induced by LPS injection and sepsis induced by cecal ligation and puncture was followed by MASM treatment. RESULTS: MASM markedly attenuated LPS-induced release and messenger RNA expression of tumor necrosis factor α, interleukin 6, and NO/inducible NO synthase in murine peritoneal macrophages and RAW264.7 cells. Meanwhile, MASM inhibited LPS-induced activation of nuclear factor κB and MAPK pathways. Consistently, RPS5 suppressed LPS-induced inflammatory responses and at least in part mediated the antiinflammatory effect of MASM in vitro. Remarkably, delayed administration of MASM could significantly reduce mortality in mouse sepsis models, which was associated with the reduction in the inflammatory response, the attenuation in multiple organ injury, and the enhanced bacterial clearance. CONCLUSIONS: MASM could be further explored for the treatments of sepsis, especially for administration later after the onset of sepsis.

Nucleotidyl transferase TUT1 inhibits lipogenesis in osteosarcoma cells through regulation of microRNA-24 and microRNA-29a.

Osteosarcoma is the most common type of bone cancer. In the present study, by way of PCR-based microarrays, we found that TUT1, a nucleotidyl transferase, was significantly downregulated in osteosarcoma, compared with adjacent normal tissues. In the current study, we performed PCR-based microarrays using the cDNA prepared from osteosarcoma and adjacent normal tissues. The enforced expression of TUT1 was able to inhibit cell proliferation in U2OS and MG63 cells, while its knockdown using small interfering RNA (siRNA) oligos promoted cell proliferation. At the molecular level, we found that TUT1 could inhibit the expression levels of PPARgamma and SREBP-1c, two key regulators in lipogenesis, through upregulation of microRNA-24 and microRNA-29a. Therefore, our results suggest that TUT1 may act as a tumor suppressor for osteosarcoma, which might provide a novel mechanism for the tumor development.

Peripheral leukocytapheresis attenuates acute lung injury induced by lipopolysaccharide in vivo.

The mortality of acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains high and efforts for prevention and treatments have shown little improvement over the past decades. The present study investigated the efficacy and mechanism of leukocytapheresis (LCAP) to partially eliminate peripheral neutrophils and attenuate lipopolysaccharide (LPS)-induced lung injury in dogs. A total of 24 healthy male mongrel dogs were enrolled and randomly divided into LPS, LCAP and LCAP-sham groups. All animals were injected with LPS to induce endotoxemia. The serum levels of leucocytes, neutrophil elastase, arterial blood gas, nuclear factor-kappa B (NF-κB) subunit p65 in lung tissues were measured. The histopathology and parenchyma apoptosis of lung tissues were examined. We found that 7, 3, and 7 animals in the LPS, LCAP, and sham-LCAP groups, respectively, developed ALI 36 h after LPS infusion. The levels of NF-κB p65 in lung tissue, neutrophils and elastase in blood, decreased significantly following LCAP. LCAP also alleviated apoptosis, and NF-κB p65 in lung tissues. Collectively, our results show that partial removal of leucocytes from peripheral blood decreases elastase level in serum. This, in turn, attenuates lung injuries and may potentially decrease the incidence of ALI.

Matrine derivate MASM suppresses LPS-induced phenotypic and functional maturation of murine bone marrow-derived dendritic cells.

Dendritic cell (DC) maturation process is a crucial step for the development of T cell immune responses and immune tolerance. In this study, we evaluated MASM, a novel derivative of the natural compound matrine that possesses a significant anti-inflammatory and immune-regulating property, for its efficacy to inhibit lipopolysaccharides (LPS)-induced maturation of murine bone marrow-derived dendritic cells. Here we show that MASM profoundly suppresses LPS-induced phenotypic and functional DC maturation. MASM inhibited LPS-induced expression of costimulatory molecules CD80 and CD86 in a concentration-dependent manner. MASM also attenuated LPS-induced IL-12p70, TNF-α, IL-6 and NO release of DCs. The MASM-treated DCs were highly efficient at antigen capture via mannose receptor-mediated endocytosis but showed weak stimulatory capacity for allogeneic T cell proliferation. Furthermore, MASM inhibited LPS-induced PI3K/Akt, MAPK and NF-κB pathways. These novel findings provide new insight into the immunopharmacological role of MASM in impacting on the DCs.

[The role of mitochondrial damages in Helicobacter pylori-induced apoptosis of gastric cancer cells].

OBJECTIVE: To explain the role of mitochondrial pathway in the apoptosis of SGC-7901 cell line induced by concentrated Helicobacter pylori culture supernatant (CHCS). METHODS: Cytochrome oxidase (COX) I expression was detected by Western blotting. Cell apoptosis and mitochondrial membrane potential were measured by flow cytometry. RESULTS: CHCS could induce the apoptosis of SGC-7901 in a dose- and time-dependent manner. Apoptotic rates gradually enhanced followed by the concentrations increasing. The mitochondrial membrane potential (MMP) began to descend after treating CHCS for 4 h, and MMP descended most distinctly in 8 h. It descended the lowest point in 12 h, and it had no special changes in 24 h. The expression of COX I was notably lower than that of control group after CHCS treating (632.8 +/- 40.6 vs 895.1 +/- 44.2, P < 0.05). CONCLUSION: Mitochondrial pathway may play an important role in the apoptosis of SGC-7901 cells induced by CHCS.

Treatment with incomplete Freund's adjuvant and Listeria monocytogenes delays diabetes via an interleukin-17-secretion-independent pathway.

Non-obese diabetes (NOD) mice are widely used as an animal model in studies of type I diabetes (TID). Treatment with complete Freund's adjuvant (CFA) in pro-diabetic NOD mice is known to inhibit disease progression by activating CD1d-specific natural killer (NK) T cells and inducing interleukin (IL)-17 secretion in innate immune cells. The aim of the present study was to examine the effect of incomplete Freund's adjuvant (IFA) and L. monocytogenes treatment on the development of TID in NOD mice. This combined treatment of IFA and L. monocytogenes, a microbe that infects the liver and is primarily combatted by NK and cytotoxic T lymphocytes, was applied to mimic CFA treatment in pro-diabetic NOD mice. The combined IFA + L. monocytogenes treatment effectively delayed TID development in the NOD mice. In contrast to CFA, the IFA + L. monocytogenes treatment did not induce T cells or innate immune cells to secrete IL-17. However, increased levels of regulatory T cells were detected. Furthermore, IFA + L. monocytogenes mice exhibited higher levels of IgG2a, although no notable T helper 1 cell response was observed when compared with the CFA or IFA control treated mice. Therefore, combined IFA + L. monocytogenes treatment was shown to delay TID development in NOD mice via a novel mechanism, which was independent from the secretion of IL-17 by CFA-activated NKT cells.

Genetic diversity of Centella asiatica in China analyzed by inter-simple sequence repeat (ISSR) markers: combination analysis with chemical diversity.

Centella asiatica is an important plant species used in traditional Chinese medicine. To help the efficient use and conservation of this species, the genetic diversity of C. asiatica populations in China was investigated using inter-simple sequence repeat (ISSR) markers. Fourteen natural populations comprising 162 individuals were included to estimate genetic diversity. At the species level, genetic diversity was relatively high (P = 66.33%, H = 0.2183, I = 0.3305). At the population level, the genetic diversity of JH (Jinhua, Zhejiang Province, China) and JJ (Jiujiang, Jiangxi Province, China) populations was relatively high (P = 43.88%, 38.78%, H = 0.1610, 0.1301, I = 0.2376, 0.1957, respectively), whereas the genetic diversity of GA (Guang'an, Sichuan Province, China) and EM (E'mei, Sichuan Province, China) was relatively low (P = 10.2%, 5.1%, H = 0.0383, 0.0211, I = 0.0570, 0.0309, respectively). On the basis of Nei's G(st) value, more genetic differentiation among populations was determined (G(st) = 0.6573). In addition, the 14 populations were clustered into four groups in view of abundant ISSR data, which further defined the genetic relationship among populations. Interestingly, the genetic clustering result was similar to previous chemical clustering results based on high-performance liquid chromatography (HPLC) data, which would also classify the 14 populations into four groups. Thus, we combined the clustering results and compared their difference. The combined analysis and genetic diversity data provide a scientific basis for conserving populations of relatively high genetic diversity such as JH and JJ populations and establishing good agricultural practices (GAP) for C. asiatica.