Photodynamic Therapy Mediated by Aloe-Emodin Inhibited Angiogenesis and Cell Metastasis Through Activating MAPK Signaling Pathway on HUVECs.

Photodynamic therapy is a clinically used, minimally invasive therapeutic procedure that involves the application of photosensitizers which can locate in target cells and so be irradiated at a corresponding wavelength. Laser light irradiation activation of photosensitizers generates free reactive oxygen species, which induces selective cytotoxic activity in target cells. Within recent years, aloe-emodin as a photosensitizer has been successfully applied in photodynamic therapy applications. Angiogenesis plays an important role in tumor growth and metastasis; thus, the development of a novel target treatment for angiogenesis is essential in order to improve treatment therapeutics for cancer treatment. An essential step in angiogenesis involves the formation of tube-like structures during matrix degradation, rearrangement, and apoptosis of endothelial cells. In the present study, we investigated the mechanisms of photocytotoxicity induced by aloe-emodin in human umbilical vein endothelial cells. Analysis of cell proliferation results noted a significant decrease in cultured cells which received various concentrations of aloe-emodin and photodynamic therapy-induced light doses. Additionally, mitochondrial mechanisms of apoptotic cell death were observed in aloe-emodin photodynamic therapy-treated cells, as tube formation assays noted angiogenesis suppression after treatment. The capacity of migration and invasion of human umbilical vein endothelial cells was measured using the transwell assay and demonstrated that aloe-emodin photodynamic therapy significantly inhibited the migration and invasion of human umbilical vein endothelial cells. The expression of p38, extracellular signal-regulated kinase, the c-Jun N-terminal kinases, and vascular endothelial growth factor suggested that the cellular metastasis was related to mitogen-activated protein kinase signal pathway. Furthermore, disorganization of F action cytoskeleton components was observed after aloe-emodin photodynamic therapy. Overall, the findings from this study suggest that aloe-emodin photodynamic therapy inhibited angiogenesis and cellular metastasis in human umbilical vein endothelial cells by activating the mitogen-activated protein kinase apoptotic signaling cell death pathway.

Enhancement of the Effect of Methyl Pyropheophorbide-a-Mediated Photodynamic Therapy was Achieved by Increasing ROS through Inhibition of Nrf2-HO-1 or Nrf2-ABCG2 Signaling.

BACKGROUND: Emerging evidence indicates that the transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays an essential role in cellular defense against oxidative stress; its activation has been related to cytoprotection. OBJECTIVE: Here, we investigated the role of Nrf2 in improving the efficacy of methyl pyropheophorbide-amediated photodynamic therapy (Mppa-PDT) via the downregulation of Nrf2. METHOD: Human ovarian cancer A2780 cells and SKOV3 cells were treated with Mppa-PDT and siRNA transfection was performed to inhibit Nrf2. After treated with siRNA and Mppa-PDT, the cell viability was examined with CCK-8 assay; cell apoptosis was detected tested by flow cytometry with Annexin V-FITC/PI; the celluar reactive oxygen species (ROS) and mitochondrial membrane potential were measured with DCFHDA and JC-1 staining; expression of protein was assessed by western blot analysis. RESULTS: We found that Nrf2 translocated from the cytoplasm to the nucleus in vitro and in vivo, and the expression of Nrf2 and P-Nrf2 increased through a possible mechanism regulated by mitogen-activated protein kinase (MAPK) after Mppa-PDT treatment. Furthermore, cytotoxicity and apoptosis induced by Mppa-PDT increased after Nrf2down-regulation. Nrf2 down -regulation increased reactive oxygen species (ROS) levels by attenuating antioxidants or pumping Mppa out of cells,which resulted from the inhibition of Nrf2-HO-1 or Nrf2- ABCG2 signaling. In addition, SKOV3 cells exhibited increased resistance to Mppa-PDT, and the expression levels of P-Nrf2 and ABCG2 were higher in SKOV3 cells than in A2780 cells, suggesting that Nrf2-ABCG2 signaling might be involved in the intrinsic resistanceto Mppa-PDT. CONCLUSION: These results provided evidence that Nrf2 down-regulation can enhance the effect of Mppa-PDT.

[Mitochondria-targeted antioxidant Mitoquinone protects post-thaw human sperm against oxidative stress injury].

OBJECTIVE: To investigate the potential protective effect of the mitochondria-targeted antioxidant Mitoquinone (MitoQ) on post-thaw human sperm. METHODS: Semen samples were collected from 60 normal fertile men, each divided into six parts of equal volume to be incubated at 37 °C in normal saline (G0, control) or in the extender with 2 nmol/L (G1), 20 nmol/L (G2), 200 nmol/L (G3), 2 µmol/L (G4), and 20 µmol/L of MitoQ (G5). After one hour of incubation, the samples were subjected to computer-assisted semen analysis (CASA) for sperm motility, flow cytometry for reactive oxygen species (ROS), thiobarbituric acid assay for the concentration of malondialdehyde (MDA), and MitoTracker fluorescent staining and flow cytometry for the sperm mitochondrial membrane potential (MMP). Then, the semen were cryopreserved with none (B0), 200 nmol/L (B1), and 2 µmol/L of MitoQ (B2), followed by detection of the changes in the ROS, MDA, and MMP of the post-thaw sperm. RESULTS: The percentage of progressively motile sperm and total rate of sperm motility were significantly higher in G3 ([30.8 ± 10.2]% and [70.6 ± 9.0]%) and G4 ([32.7 ± 13.5]% and [70.3 ± 11.9]%) than in G0 ([17.6 ± 5.0]% and [54.9 ± 11.5]%) (P < 0.05). The level of ROS dropped markedly with the increased concentration of MitoQ, 86.5 ± 31.6 in G3, 93.6 ± 42.0 in G4, and 45.1 ± 15.0 in G5, as compared with 160.8 ± 39.7 in G0 (P < 0.05). The content of MDA was remarkably lower in G3 ([0.9 ± 0.5] µmol/mg) and G4 ([0.9 ± 0.5] µmol/mg) than in G0 ([1.9 ± 1.1] µmol/mg) (P < 0.05), but not in G5 ([1.7 ± 0.7] µmol/mg), which was even higher than in G3 and G4 (P < 0.05). The MMP showed a significant reduction in G5 (1156 ± 216) in comparison with G0 (1701 ± 251) (P < 0.05) but exhibited no remarkable difference between G0 and G1 (1810 ± 298), G2 (1995 ± 437), G3 (1950 ± 334), or G4 (1582 ± 314). The percentage of progressively motile sperm and total rate of sperm motility after freezing-thawing were significantly decreased as compared with those of the fresh semen (P < 0.01), but both were remarkably higher in B1 ([3.2 ± 2.3]% and [ 43.0 ± 9.5]%) than in B0 ([0.8 ± 0.6]% and [26.5 ± 11.4]%) (P < 0.05). The ROS level was significantly lower in B1 and B2 than in B0 (34.6 ± 12. 3 and 37.0 ± 10.5 vs 56.9 ± 14.3, P < 0.05), and so was the MDA content ([1.4 ± 0.5] and [1.4 ± 0.6] µmol/mg vs [2.6 ± 1.0] µmol/mg, P < 0.05), but the MMP was markedly higher in B1 and B2 than in B0 (1010.0 ± 130.5 and 880.6 ± 128.6 vs 721.1 ± 24.8, P < 0.05). CONCLUSION: Addition of MitoQ to the freezing extender at 200 nmol/L may effectively improve the quality of human sperm and MitoQ is a good protective addictive for human sperm cryopreservation.

Exploring a Novel Target Treatment on Breast Cancer: Aloe-emodin Mediated Photodynamic Therapy Induced Cell Apoptosis and Inhibited Cell Metastasis.

Photodynamic therapy (PDT) as a clinical cancer therapy, is a mild therapy, which involves application of photosensitizers (PSs) located in target cells and then irradiated by corresponding wavelength. The activation of PSs generates radical oxygen species (ROS) to exert a selective cytotoxic activity for the target cells. Aloe-emodin (AE) has been found to be an anti-tumor agent in many studies, and has also been demonstrated as a photosensitizer, in the recent years. In order to study the mechanisms of aloe-emodin as a photosensitizer, we investigated the mechanisms of photo-cytotoxicity induced by aloe-emodin in breast cancer MCF-7 cells in the present study. Analysis of cell proliferation evidenced that there was a drastic depression after photodynamic treatment with a series of aloe-emodin concentrations and light doses. We observed changes in apoptosis and demonstrated that the mechanisms of apoptosis were involved in mitochondrial and endoplasmic reticulum death pathways. The capacity of adhesion, migration and invasion of breast cells was measured using WST8 and transwell assay and demonstrated that AE-PDT significantly inhibited adhesion, migration and invasion of MCF-7cells. The expression of MMP2, MMP9, VEGF and Nrf2 demonstrated that the metastasis was related to oxidative stress. Analysis of changes in cytoskeleton components (F-actin) evidenced cytoskeleton disorganization after treatment with AE-PDT. Taken together, the present results indicated that PDT with aloe-emodin effectively suppressed cancer development in MCF-7cells, suggesting the potential of AE as a new photosensitizer in PDT which can provide a new modility for treating cancer.

High expressions of bcl-2 and survivin, and decreased apoptosis in uterine cervical carcinosarcoma compared to cervical squamous cell carcinoma.

PURPOSES: Uterine cervical carcinosarcoma (CS) is very rare. To date, only 40 cases have been reported. It seems to have a more aggressive clinical behavior than does cervical squamous cell carcinoma (SCC). The purposes of our study were to characterize the clinicopathologic characteristics and human papillomavirus (HPV) status of the rare tumor and to analyze the molecular features in cervical CS that may account for its aggressive behavior. METHODS: Three patients were diagnosed with uterine cervical CS at West China Second Hospital of Sichuan University between 1995 and 2009. Data were retrospectively analyzed from available charts and pathological reports. Twelve patients with FIGO stage Ib-IIa cervical SCC were enrolled as the controls, and the expression profiling of p53, Ki-67, bcl-2, survivin and apoptosis index between cervical CS and SCC was compared. Immunohistochemical and apoptosis results were scored separately for the carcinomatous and sarcomatous components. RESULTS: All three patients were shown to be negative for HPV infection by Hybribio HPV genoarray assay. Expression of p53 was observed in one patient in both carcinomatous and sarcomatous components in a similar proportion; in contrast, the Ki67, bcl-2 and survivin expressions were higher in carcinomatous components than in sarcomatous components in all three cases. Compared to cervical SCC, stronger immunostaining for bcl-2, survivin and lower apoptosis was observed in cervical CS. CONCLUSIONS: Cervical CS is a peculiar tumor with many different clinicopathologic characteristics from cervical SCC. Dysregulation of apoptosis may confer tumor cells of cervical CS with survival and growth advantages, and thereby facilitate the aggressive behavior of cervical CS.

Association of an interleukin-16 gene polymorphism with the risk and pain phenotype of endometriosis.

Interleukin-16 (IL-16), a proinflammatory cytokine, plays a pivotal role in inflammatory diseases as well as in the pathogenesis of endometriosis. The aim of this study was to evaluate the association of IL-16 gene polymorphisms with the risk and clinical phenotypes of endometriosis in Chinese women. We analyzed rs4778889 T/C, rs11556218 T/G polymorphisms of the IL-16 gene in 230 patients with endometriosis and 203 controls in a Chinese population, using a polymerase chain reaction-high resolution melting analysis strategy and DNA sequencing methods. There was no significant difference in the genotype and allele frequencies of the rs11556218 T/G polymorphism between patients with endometriosis and controls (p>0.05). In contrast, the genotype and allele frequencies of the rs4778889 T/C polymorphism were statistically different between patients with endometriosis and controls, which resulted from a significantly increased proportion of TC heterozygote and CC homozygote carriers among patients with endometriosis (p=0.001 and 0.012, respectively); moreover, further subgroup analysis found that the genotype difference was more evident in patients with endometriosis who also experienced pain symptoms (p<0.001) than in patients without pain symptoms (p=0.625) when compared with controls. Our results suggest that the rs4778889 T/C polymorphism of the IL-16 gene may be associated with risk of endometriosis in the Chinese population, especially in patients with pain phenotype.

Immunological properties of recombinant Mycobacterium bovis bacillus Calmette-Guérin strain expressing fusion protein IL-2-ESAT-6.

The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides variable efficacy against adult pulmonary tuberculosis (TB). Recombinant BCG, expressing either immunodominant antigens or Th1 cytokines, is a promising strategy for developing a new TB vaccine. However, not much is known about whether the introduction of cytokine and specific antigen genes concurrently into the BCG strain could improve the immunogenicity of BCG. In this study, a recombinant BCG strain (rBCG) expressing the fusion protein human interleukin (IL)-2 and ESAT-6 (early secreted antigenic target-6 kDa) antigen of Mycobacterium tuberculosis was constructed. Six weeks after BALB/c mice (H-2d) were immunized with 106 colony forming units (CFUs) BCG or rBCG, splenocyte proliferation was determined with MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay, IL-4 and interferon (IFN)-gamma produced by splenocytes were tested by enzyme linked immunosorbent assay (ELISA,) and the cytotoxicity of splenocytes from immunized mice to P815 cells (H-2d) expressing ESAT-6 protein was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay. Compared with native BCG-vaccinated mice, rBCG induced stronger Th1 responses that were confirmed by high lymphoproliferative responses and IFN-gamma production to culture filtrate protein (CFP) or ESAT-6 protein. Moreover, rBCG induced significant enhanced CTL responses against P815-ESAT-6 cells. Results from rBCG-immunized mice demonstrated that introducing the il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6. Further investigation is needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacy against TB.