Search

Ovarian cancer is the most lethal gynecological cancer in women. Shikonin (SHK), derived from Lithospermum eryothrorhizon, can reduce cancer activity; however, its clinical effect on type 2 ovarian cancer cells remains undetermined. Here, we studied the effects of SHK on type 2 ovarian cancer using the KURAMOCHI, OVSAHO, CP70, and ascites E04 cell lines. The proliferation curve and half-maximal inhibitory concentration of SHK for the cell lines were evaluated using the second-generation tetrazolium dye assay and the cell viability were determined by the annexin V/PI as well as TUNEL assay. The caspase dependent pathway was performed by western blotting assay with pan-caspase inhibitor Z-VAD-FMK and SHK induced miR-874-3p expression thus suppressed anti-apoptosis markers XIAP and Bcl-xL. The effect of SHK on type 2 ovarian cancer cell migration and invasion was evaluated using the wound healing and transwell assays. Quantitative RT-PCR and western blot was used to evaluate cancer stem cell (CSC)-related gene/protein (OCT4, SOX2, NANOG, ALDH1, and C-MYC) expressions, sphere formation assay was executed and a xenograft animal model for in vivo antitumor effects of SHK. Taken together, Shikonin suppressed type 2 ovarian cancer cell viability, migration, and invasion abilities; decreased CSC-related markers expression as well as the sphere colony numbers. It also reduced the tumorigenicity of KURAMOCHI ALDH+ cells and induced anti-tumor effect in a xenograft model. Thus, SHK could contribute a potential therapeutic strategy on type 2 ovarian cancer cells via multiple functions.

Mesenchymal stem cells (MSCs) are known to play a role in postnatal vasculogenesis and hold great promise for vascular regeneration. However, the mechanisms by which the endothelial differentiation and specification of MSCs remain unclear. We examined the potential role and molecular mechanisms of atypical chemokine receptor ACKR3/CXCR7 in MSC-mediated endothelial cell differentiation and specification. Here, we showed that vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) activate CXCR7 expression on MSCs through PDGF receptors, PDGFRα and PDGFRβ-mediated phosphoinositide 3-kinase (PI3K)/Akt signaling. Genetic and pharmacologic blockage of CXCR7 on MSCs suppressed the VEGF or stromal cell-derived factor 1 (SDF)-1-induced the capacity for vasculogenesis in vitro and in vivo. Moreover, CXCR7 gain of function markedly promoted vasculogenesis by MSCs in vitro and in vivo and induced endothelial differentiation along the arterial endothelial cell lineage via upregulation of Notch signaling. However, blockade of Notch signaling inhibited CXCR7-induced vasculogensis by MSCs. These results indicate CXCR7 is a critical regulator of MSC-mediated postnatal vasculogenesis and arterial specification via Notch signaling.

Additional file 1: Supplementary table S1. Q-PCR primer sequences. Supplementary table S2. shRNA target sequences. Supplemental figure 1 a Time course of cell-surface expression of CXCR7 after exposure of MSCs to osteogenic (OS) or chondrogenic ( CH) differentiation medium. The cell-surface expression of CXCR7 was determined by flow cytometry. b Changes in osteogenic (ALPL, SPP1) and chondrogenic (ACAN, COL2A1) genes in MSCs with or without CXCR7 overexpression incubated with osteogenic or chondrogenic induction medium for 21 days. Data are means ± SD (n=9). *P < 0.0001 compared with wild-type MSCs (WT). c Changes in osteogenic (ALPL, SPP1) and chondrogenic (ACAN, COL2A1) genes in MSCs with or without the CXCR7 antagonist, CCX771, or agonist, TC14012, incubated with osteogenic or chondrogenic induction medium for 21 days, respectively. Data are means ± SD (n=9). *P < 0.0001 compared with the untreated group (control). Supplemental figure 2. a Cell-surface expression of CXCR7 and CXCR4 in MSCs lentivirally transduced with control vector (CVT) or CXCR7-expressing vector (CXCR7) for 72 h. Data are means ± SD (n=9). *P < 0.001 compared with CVT- expressing MSCs. b Verification of CXCR4 knockdown in MSCs lentivirally transduced with the CXCR4 shRNA for 72 h. Data are means ± SD (n=9). *P < 0.0001 compared with wild-type MSCs (control). c Changes in osteogenic (ALPL, SPP1) and chondrogenic (ACAN, COL2A1) genes in MSCs with or without CXCR4 knockdown incubated with osteogenic or chondrogenic induction medium for 21 days. Data are means ± SD (n=9). (d and e) The quantitation of Alizarin red and Alcian blue staining in MSCs with or without CXCR4 knockdown incubated with osteogenic or chondrogenic induction medium for 21 days. Data are means ± SD co-cultured with MSCs with or without CXCR4 knockdown for 1 or 3 days. Data are means ± SD (n=9). *P < 0.001 compared with the groups on Day 1. (G) Differentiation of regulatory T (Treg, FoxP3+ cells)-like cells in Jurkat T cells co-cultured with MSCs with or without CXCR4 knockdown for 1 or 3 days. Data are means ± SD (n=9). Supplemental figure 3 The localization of PPAR-γ, β-catenin, Gli1 or NICD evaluated by cell fractionation. PPAR-γ, β-catenin, Gli1 and NICD in MSCs lentivirally transduced with control vector (CVT) or CXCR7-expressing vector (CXCR7) for 72 h were detected in both the cytoplasmic and nuclear fractions via western blot analysis. β-actin and Histone-3 were used as internal controls for protein expression in the cytoplasmic and nuclear fractions, respectively. Data are means ± SD (n=9). *P < 0.01 compared with control MSCs (CVT). Supplemental figure 4. The frenquency of CD11b+ macrophages (a) and Foxp3+ Treg cells (b) in synovial tissues derived from normal rats or rats with collagen-induced arthritis (CIA) receiving vehicle (PBS), control vector-expressing MSC (control MSCs) or CXCR7-expressing MSC (CXCR7-MSCs) treatment on day 3. The single cell suspension of synovial tissues were made by enzymatic isolation method and the frenquency of CD11b+ macrophages and Foxp3+ Treg cells were detected by flow cytometry. Data are means ± SD (n=6). *P < 0.01 compared with the normal group without CIA. # P < 0.01 compared with the control MSCs. Supplemental figure 5. Levels of osteocaicin (a and b) and type I collagen cross-linked C-telopeptide (CTX-I, c and d) in synovial tissues derived from normal rats or rats with collagen-induced arthritis (CIA) receiving vehicle (PBS), control vector-expressing MSC (control MSCs), CXCR7-expressing MSC (CXCR7-MSCs), native MSC (MSCs), vehicle-primed MSC, CCX771-primed MSC or TC14012-primed MSC treatment on Day 28. Data are means ± SD (n=6). *P < 0.01 compared with the normal group without CIA. # P < 0.01 compared with the CIA group without any treatment.

Background The major barriers to mesenchymal stem cell (MSC) therapy in rheumatoid arthritis (RA) are a low extent of tissue regeneration and insufficient immunomodulation after cell transplantation. In addition, the role of C-X-C chemokine receptor type 7 (CXCR7) and its mechanism of action in MSC-mediated osteogenic or chondrogenic differentiation and immunomodulation are unclear. Methods Gain of CXCR7 function on human MSCs was carried out by lentiviral vector-mediated CXCR7 overexpression or CXCR7 agonist, TC14012. These cells were determined the role and potential mechanisms for CXCR7-regulated MSC differentiation and immunomodulation using cellular and molecular assays. The therapeutic benefits in RA were investigated in rats with collagen-induced arthritis (CIA). Results CXCR7 was upregulated in MSCs during the induction of osteogenic or chondrogenic differentiation. Blockage of CXCR7 function inhibited osteogenic or chondrogenic differentiation of MSCs whereas gain of CXCR7 function had the opposite effects. Besides, MSCs with CXCR7 gain-of-function facilitated macrophage apoptosis and regulatory T cell differentiation in a co-culture system. Gain of CXCR7 function also promoted the production of anti-inflammatory soluble factors. A gene expression profiling assay and signaling reporter assays revealed that CXCR7 could regulate several candidate genes related to the PPAR, WNT, Hedgehog or Notch pathways, and their signaling activities, which are known to control cell differentiation and immunomodulation. Finally, MSCs with CXCR7 gain-of-function significantly reduced the articular index scores, ankle circumference, radiographic scores, histologic scores, and inflammation in rats with CIA compared with control MSCs. Conclusions CXCR7 promotes the osteogenic and chondrogenic differentiation of MSCs and MSC-mediated immunomodulation by regulating several signaling pathways and anti-inflammatory soluble factors. MSCs with CXCR7 gain-of-function significantly ameliorate arthritic symptoms in a CIA model.

Rationale: Although molecular investigations of regulator of G-protein signaling 4 (RGS4) alterations in schizophrenia patients yielded partially inconsistent findings, the previous studies suggested that RGS4 is both a positional and functional candidate gene for schizophrenia and is significantly decreased in the prefrontal cortex. However, the exact role of RGS4 in the pathophysiology of schizophrenia is unclear. Moreover, a whole genome transcription profile study showed the possibility of RGS4-regulated expression of SLC7A11(xCT), a component of cysteine/glutamate transporter or system xc-. We hypothesized that system xc- is a therapeutic target of RGS4 deficit-mediated schizophrenia. Methods: Pharmacological and genetic manipulation of RGS4 in organotypic brain slice cultures were used as an ex vivo model to investigate its role in system xc- and glutamatergic function. Lentiviral-based mouse models with RGS4 deficit in the prefrontal cortex and treatment with system xc- activator, N-acetyl cysteine (NAC), were utilized to observe their impacts on glutamatergic function and schizophrenic behaviors. Results: Genetic and pharmacological inhibition of RGS4 resulted in a significant decrease in SLC7A11 (xCT) expression and hypofunction of system xc- and reduced glutamatergic function in organotypic brain slice cultures. However, NAC restored the dysregulation of RGS4-mediated functional deficits of glutamate. Moreover, knockdown of RGS4 specifically in the prefrontal cortex caused mice to exhibit behaviors related to schizophrenia such as increased stereotypy, impaired prepulse inhibition, deficits in social interactions, working memory, and nesting behavior, while enhancing sensitivity to the locomotor stimulatory effect of MK-801. These mice displayed glutamatergic dysfunction in the prefrontal cortex, which may have contributed to the behavioral deficits. RGS4 knockdown mice that received NAC treatment had improved glutamatergic dysfunction and schizophrenia behaviors. Conclusion: Our results suggest that RGS4 deficit induces dysregulation and dysfunction of system xc-, which further results in functional deficits of the glutamatergic system and subsequently to schizophrenia-related behavioral phenotypes. Activation of system xc- offers a promising strategy to treat RGS4 deficit-mediated schizophrenia.