Shengfu Li

The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo.

Mesenchymal stem cells (MSCs) play an important role in the development and growth of tumor cells. The purpose of this study is to confirm the effect of MSCs on tumor cell growth in vitro and in vivo and to elucidate the mechanism. MSCs were isolated from mouse bone marrow and cocultured with murine hepatoma H22, lymphoma (YAC-1 and EL-4) and rat insulinoma INS-1 cell lines. The growth inhibitory effect of MSCs on tumor cells was tested through MTT and 3H-TdR incorporation assay. The apoptosis induction effect of MSCs on tumor cells was assessed with flow cytometry (FCM) and RT-PCR assay. MSCs were inoculated into BALB/c mice alone or coinoculated with ascitogenous hepatoma cells intraperitonealy, respectively. The tumor growth inhibition of MSCs was investigaed through the incidence and volume of ascites formation, and the immunosuppression effect was studied with splenocyte response to ConA stimulation test and T cell subsets analysis (FCM). The results showed that MSCs exhibited a number-dependent growth inhibitory effect on murine tumor cell lines in vitro and inhibited the growth of ascitogenous hepatoma cells in vivo without host immunosuppression. MSCs could upregulate tumor cells mRNA expression of cell cycle negative regulator p21 and apoptosis associated protease caspase 3. The findings of this experimental study demonstrated that MSCs had potential inhibitory effects on tumor cell growth in vitro and in vivo without host immunosuppression, by inducing apoptotic cell death and G0/G1 phase arrest of cancer cells.

An engineered multidomain bactericidal peptide as a model for targeted antibiotics against specific bacteria

We constructed a peptide consisting of a staphylococcal AgrD1 pheromone fused to the channel-forming domain of colicin Ia and named it pheromonicin. This fusion peptide had bactericidal effects against methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively), but not against Staphylococcus epidermidis or Streptococcus pneumoniae. Growth rates, vital staining and colony forming unit (CFU) counts showed that pheromonicin did not merely suppress growth but killed S. aureus cells. The specificity of pheromonicin was shown by the absence of bactericidal effects against an accessory gene regulator (agr) locus knockout of S. aureus, and a dose-dependent inhibition of the bactericidal effects of pheromonicin by competition with corresponding free AgrD pheromone. In vivo, all pheromonicin-treated mice survived administration of MRSA that was lethal to controls. No toxicity was detectable in human liver or renal cells in culture, or in livers, kidneys or spleens of pheromonicin-treated mice. The results suggest that these types of chimeric peptides may be of value as antibiotics against specific bacterial infections.

The Protective Mechanism of Ligustrazine Against Renal Ischemia/Reperfusion Injury

BACKGROUND Ischemia/reperfusion (I/R) injury is unavoidable in renal transplantation, and represents an additional risk factor for the late renal allograft failure. Our study focused on the effects of ligustrazine on oxidative stress, apoptosis, neutrophils recruitment, the expression of proinflammatory mediators and adhesion molecules caused by renal I/R injury. MATERIALS AND METHODS Renal warm I/R was induced in male C57BL/6 mice by clamping the left renal artery and vein non-traumatically. Group I was sham-operated animals; group II, nontreated animals; and group III, ligustrazine-treated animals (80 mg/kg, i.p. 30 min before I/R). Mice were sacrificed 4 and 24h post reperfusion. The effects of ligustrazine on oxidative stress, neutrophils recruitment, proinflammatory mediators, and adhesion molecules caused by renal I/R injury were assayed. RESULTS Ligustrazine pretreatment attenuated dramatically the injuries in mice kidneys caused by warm I/R (histological scores of untreated versus treated, 4.2 ± 0.4 versus 0.9 ± 0.3; P<0.01). Administration of ligustrazine significantly reduced myeloperoxidase (MPO) activity by 38.6% and decreased malondialdehye (MDA) level by 19.2%, while superoxide dismutase (SOD) activity increased by 39.6% (P<0.01), suggesting an effective reduction of oxidative stress following ligustrazine treatment. Moreover, ligustrazine also inhibited cell apoptosis, abrogated neutrophils recruitment, and suppressed the over expression of TNF-α and ICAM-1. CONCLUSIONS In conclusion, ligustrazine protects murine kidney from warm ischemia/reperfusion injury, probably via reducing oxidative stress, inhibiting cell apoptosis, decreasing neutrophils infiltration, and suppressing the overexpression of TNF-α and ICAM-1 levels.

Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury

Hypoxia/reoxygenation (H/R)‐induced injury is the key factor associated with islet graft dysfunction. This study aims to examine the effect of mesenchymal stem cells (MSCs) on islet survival and insulin secretion under H/R conditions. Islets from rats were isolated, purified, cultured with or without MSCs, and exposed to hypoxia (O2 ≤ 1%) for 8 h and reoxygenation for 24 and 48 h, respectively. Islet function was evaluated by measuring basal and glucose‐stimulated insulin secretion (GSIS). Apoptotic islet cells were quantified using Annexin V‐FITC. Anti‐apoptotic effects were confirmed by mRNA expression analysis of hypoxia‐resistant molecules, HIF‐1α, HO‐1, and COX‐2, using semi‐quantitative retrieval polymerase chain reaction (RT‐PCR). Insulin expression in the implanted islets was detected by immunohistological analysis. The main results show that the stimulation index (SI) of GSIS was maintained at higher levels in islets co‐cultured with MSCs. The MSCs protected the islets from H/R‐induced injury by decreasing the apoptotic cell ratio and increasing HIF‐1α, HO‐1, and COX‐2 mRNA expression. Seven days after islet transplantation, insulin expression in the MSC‐islets group significantly differed from that of the islets‐alone group. We proposed that MSCs could promote anti‐apoptotic gene expression by enhancing their resistance to H/R‐induced apoptosis and dysfunction. This study provides an experimental basis for therapeutic strategies based on enhancing islet function. Copyright

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery

Aim:To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells.Methods:A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models.Results:The IC50 of MG2B for cancer cells (10–15 μmol/L) was at least 10 times lower than the IC50 of unconjugated MG2 (125 μmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC50 ranging from 10 to 15 μmol/L, which was about 6–10 times lower than the IC50 for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively.Conclusion:The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.

Mechanisms of Cyclosporine-Induced Renal Cell Apoptosis: A Systematic Review

Background/Aims: Chronic cyclosporine A (CsA) nephrotoxicity (CCN) is an important cause of chronic renal dysfunction with no effective clinical intervention. To further elucidate the mechanisms of renal cell apoptosis in CCN, all relevant in vivo studies on this subject were analyzed. Methods: We searched for in vivo studies on the mechanisms of CsA-induced renal cell apoptosis in Medline (1966–July 2010), Embase (1980–July 2010) and ISI (1986–July 2010). The studies were evaluated for their quality according to a set of in vivo standards, data extracted according to PICOS, and then synthesized. Results: Renal cell apoptosis was an important feature of CCN and an important factor of renal dysfunction. First, CsA could upregulate Fas/Fas ligand, downregulate Bcl-2/Bcl-XL, and increase caspase-1 and caspase-3. Second, it could induce oxidative stress and damage the antioxidant defense system. Third, it could increase endoplasmic reticulum stress protein in a dose- and time-dependent manner. Fourth, CsA could impair the urine concentration and decrease the expression of hypertonicity-induced genes. Fifth, CsA-induced renal cell apoptosis was significantly decreased by blocking the angiotensin II type 1 receptor using losartan. Conclusions: The in vivo mechanisms for CCN are more complex than those found in vitro. CsA can induce renal cell apoptosis using five pathways in vivo and activated caspases might be the ultimate intersection of these pathways and the common intracellular pathway mediating apoptosis. These data provide new potential points for intervention and need to be confirmed by further studies.

Copper stimulates growth of human umbilical vein endothelial cells in a vascular endothelial growth factor-independent pathway

Studies in vivo have shown that dietary copper (Cu) supplementation reverses pressure overload-induced cardiac hypertrophy in a mouse model, which is vascular endothelial growth factor (VEGF)-dependent and correlates with enhanced angiogenesis. Because Cu stimulation of endothelial cell growth and differentiation would play a critical role in angiogenesis, the present study was undertaken to examine the effect of Cu on growth of human umbilical vein endothelial cells (HUVECs) in cultures. The HUVECs were treated with CuSO4 at a final concentration of 5 μmol/L Cu element in cultures or with a Cu chelator, tetraethylenepentamine (TEPA), at a final concentration of 25 μmol/L in cultures. Cell growth and Cu effect on cell cycle were determined. In addition, the effect of Cu on VEGF and endothelial nitric oxide synthase (eNOS) mRNA levels was determined, and anti-VEGF antibody and siRNA targeting eNOS were applied to determine the role of VEGF or eNOS in the Cu effect on cell growth. Cu significantly stimulated and TEPA significantly inhibited cell growth, and the TEPA effect was blocked by excess Cu. Cu increased the number of cells in the S phase and correspondingly decreased the number in the G1 phase. Interestingly, Cu did not increase the level of VEGF mRNA, but significantly increased eNOS mRNA. Furthermore, neutralizing VEGF by anti-VEGF antibody did not suppress Cu stimulation of cell growth. However, siRNA targeting eNOS completely blocked Cu reversal of TEPA inhibition of cell growth. The data demonstrate that Cu stimulation of HUVEC cell growth is VEGF-independent, but eNOS-dependent.

Chondroitin sulfate as a molecular portal that preferentially mediates the apoptotic killing of tumor cells by penetratin-directed mitochondria-disrupting peptides

The use of cell-penetrating peptides (CPPs) as drug carriers for targeted therapy is limited by the unrestricted cellular translocation of CPPs. The preferential induction of tumor cell death by penetratin (Antp)-directed peptides (PNC27 and PNC28), however, suggests that the CPP Antp may contribute to the preferential cytotoxicity of these peptides. Using PNC27 as a molecular model, we constructed three novel peptides (PT, PR9, and PD3) by replacing the leader peptide Antp with one of three distinct CPPs (TAT, R9, or DPV3), respectively. The IC50 values of PNC27 in tumor cells were 2–3 times lower than in normal cells. However, all three engineered peptides demonstrated similar cytotoxic effects in tumor and normal cells. Another three chimeric peptides containing the leader peptide Antp with different mitochondria-disrupting peptides (KLA-Antp (KGA), B27-Antp (BA27), and B28-Antp (BA28)), preferentially induced apoptosis in tumor cells. The IC50 values of these peptides (3–10 μm) were 3–6 times lower in tumor cells than in normal cells. In contrast, TAT-directed peptides (TAT-KLA (TK), TAT-B27 (TB27), and TAT-B28 (TB28)), were cytotoxic to both tumor and normal cells. These data demonstrate that the leader peptide Antp contributes to the preferential cytotoxicity of Antp-directed peptides. Furthermore, Antp-directed peptides bind chondroitin sulfate (CS), and the removal of endogenous CS reduces the cytotoxic effects of Antp-directed peptides in tumor cells. The overexpression of CS in tumor cells is positively correlated to the cell entry and cytotoxicity of Antp- directed peptides. These results suggest that CS overexpression in tumor cells is an important molecular portal that mediates the preferential cytotoxicity of Antp-directed peptides.

Do CD4+ Foxp3+ Treg cells correlate with transplant outcomes: a systematic review on recipients of solid organ transplantation.

Regulatory T cells (Tregs) are considered to be critical for the induction of transplant tolerance. Tregs counts were measured in blood, biopsy and urine sample after transplantation in many studies. Although not unanimous, some studies have suggested that Tregs is associated with better outcome and can also serve as an immune marker to predict the individual risk of rejection and identify tolerant patients. In this study, we systematically reviewed the correlation between Tregs and transplant outcomes, identifying if Tregs can predict transplant rejection and tolerance. A total of 22 articles were included and assessed, the results showed that Tregs in recipients are helpful to maintain a stable graft function, reduce acute/chronic rejection rate. And the Tregs in graft and urine, rather than in PBL, may have a better diagnostic value for transplant outcomes. However, since the low quality of included studies, results may be influenced by bias. More high quality studies with bigger sample size are still needed in future.

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis

BackgroundHypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α.ResultsQuantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells.ConclusionsOverexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.