Artonin E Induces Apoptosis via Mitochondrial Dysregulation in SKOV-3 Ovarian Cancer Cells

Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC₅₀ values of 6.5±0.5μg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell lineT1074, with IC₅₀ value of 32.5±0.5μg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.     

Estrogen induces normal murine CD5+ B cells to produce autoantibodies

Females have better humoral immune responses and are more susceptible to autoimmune diseases than males. Normal female mice (C57BL/6J, C3H/HeJ, and NZW) have significantly increased spontaneous autoimmune plaque-forming cells (APFC) to mouse erythrocytes pretreated with bromelain (Br-ME) in spleen, peritoneal exudate cell, and bone marrow compared to their male counterparts. A minor subpopulation of B cells, CD5+ B, is thought to produce this autoantibody. As determined by dual color flow cytometry, increased APFC to Br-ME in females is not due to quantitative increase of CD5+ B cells. Rather, it is due to increased numbers or percentages) of CD5+ B cells producing these autoantibodies, because CD5+ B cells from females produced greater numbers of APFC to Br-ME than equal numbers of cells derived from males. The increased autoantibody production in females can be attributed to the effect of estrogen on the immune response because this hormone markedly augments APFC to Br-ME in intact or orchidectomized males. Male hormone had little effect. Importantly, estrogen did not increase the numbers of B or CD5+ B cells but augmented the ability of B cells to produce this response. This was verified when a T cell-depleted B cell fraction or fluorescence-activated cell sorter purified CD5+ B cells from estrogen-treated mice proved more efficient in the production of APFC to Br-ME. These results suggest that the number of CD5+ B cells committed to produce autoantibodies to Br-ME is increased under the influence of estrogen. This is the first demonstration that estrogen can augment the production of natural autoantibodies in normal mice. The overall augmented humoral immune responses in females and the B cell hyperactivity in female predominant autoimmune diseases appears to be due to estrogen.     

Paradoxical Effects of All-Trans-Retinoic Acid on Lupus-Like Disease in the MRL/lpr Mouse Model

Roles of all-trans-retinoic acid (tRA), a metabolite of vitamin A (VA), in both tolerogenic and immunogenic responses are documented. However, how tRA affects the development of systemic autoimmunity is poorly understood. Here we demonstrate that tRA have paradoxical effects on the development of autoimmune lupus in the MRL/lpr mouse model. We administered, orally, tRA or VA mixed with 10% of tRA (referred to as VARA) to female mice starting from 6 weeks of age. At this age, the mice do not exhibit overt clinical signs of lupus. However, the immunogenic environment preceding disease onset has been established as evidenced by an increase of total IgM/IgG in the plasma and expansion of lymphocytes and dendritic cells in secondary lymphoid organs. After 8 weeks of tRA, but not VARA treatment, significantly higher pathological scores in the skin, brain and lung were observed. These were accompanied by a marked increase in B-cell responses that included autoantibody production and enhanced expression of plasma cell-promoting cytokines. Paradoxically, the number of lymphocytes in the mesenteric lymph node decreased with tRA that led to significantly reduced lymphadenopathy. In addition, tRA differentially affected renal pathology, increasing leukocyte infiltration of renal tubulointerstitium while restoring the size of glomeruli in the kidney cortex. In contrast, minimal induction of inflammation with tRA in the absence of an immunogenic environment in the control mice was observed. Altogether, our results suggest that under a predisposed immunogenic environment in autoimmune lupus, tRA may decrease inflammation in some organs while generating more severe disease in others.     

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.     

The survivin molecule as a double-edged sword in cellular physiologic and pathologic conditions and its role as a potential biomarker and therapeutic target in cancer

Survivin is a member of the family of apoptosis inhibitory proteins with increased expression level in most cancerous tissues. Evidence shows that survivin plays regulatory roles in proliferation or survival of normal adult cells, principally vascular endothelial cells, T lymphocytes, primitive hematopoietic cells, and polymorphonuclear neutrophils. Survivin antiapoptotic role is, directly and indirectly, related to caspase proteins and shows its role in cell division through the chromosomal passenger complex. Survivin contains many genetic polymorphisms that the role of some variations has been proven in several cancers. The −31G/C polymorphism is one of the most important survivin mutations which is located in the promoter region on a CDE/CHR motif. This polymorphism can upregulate the survivin messenger RNA. In addition, its allele C can increase the risk of cancers in 1.27-fold than allele G. Considering the fundamental role of survivin in different cancers, this protein could be considered as a new therapeutic target in cancer treatment. For this purpose, various strategies have been designed including the prevention of survivin expression through inhibition of mRNA translation using antagonistic molecules, inhibition of survivin gene function through small inhibitory molecules, gene therapy, and immunotherapy. In this study, we describe the structure, played roles in physiological and pathological states and genetic polymorphisms of survivin. Finally, the role of survivin as a potential target in cancer therapy given challenges ahead has been discussed.     

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated on day 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.     

Sex differences in the expression of lupus-associated miRNAs in splenocytes from lupus-prone NZB/W<Subscript>F1</Subscript> mice

Background

A majority of autoimmune diseases, including systemic lupus erythematosus (SLE), occur predominantly in females. Recent studies have identified specific dysregulated microRNAs (miRNAs) in both human and murine lupus, implying an important contribution of these miRNAs to lupus pathogenesis. However, to date, there is no study that examined sex differences in miRNA expression in immune cells as a plausible basis for sex differences in autoimmune disease. This study addresses this aspect in NZB/W_F1 mice, a classical murine lupus model with marked female bias, and further investigates estrogen regulation of lupus-associated miRNAs.

Methods

The Taqman miRNA assay system was used to quantify the miRNA expression in splenocytes from male and female NZB/W_F1 mice at 17–18, 23, and 30 weeks (wks) of age. To evaluate potential estrogen's effect on lupus-associated miRNAs, 6-wk-old NZB/W_F1 male mice were orchidectomized and surgically implanted with empty (placebo) or estrogen implants for 4 and 26 wks, respectively. To assess the lupus status in the NZB/W_F1 mice, serum anti-dsDNA autoantibody levels, proteinuria, and renal histological changes were determined.

Results

The sex differences in the expression of lupus-associated miRNAs, including the miR-182-96-183 cluster, miR-155, miR-31, miR-148a, miR-127, and miR-379, were markedly evident after the onset of lupus, especially at 30 wks of age when female NZB/W_F1 mice manifested moderate to severe lupus when compared to their male counterparts. Our limited data also suggested that estrogen treatment increased the expression of aforementioned lupus-associated miRNAs, with the exception of miR-155, in orchidectomized male NZB/W_F1 mice to a similar level in age-matched intact female NZB/W_F1 mice. It is noteworthy that orchiectomy, itself, did not affect the expression of lupus-associated miRNAs.

Conclusion

To our knowledge, this is the first study that demonstrated sex differences in the expression of lupus-associated miRNAs in splenocytes, especially in the context of autoimmunity. The increased expression of lupus-associated miRNA in female NZB/W_F1 mice and conceivably in estrogen-treated orchidectomized male NZB/W_F1 mice was associated with lupus manifestation. The notable increase of lupus-associated miRNAs in diseased, female NZB/W_F1 mice may be a result of both lupus manifestation and the female gender.