A dual-targeting PDGFRβ/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VeGF and pDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments.The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting pDGFRβ and VeGF-A were selected for superior stability. the scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. assembly of a bsAb using stable monomeric units allowed development of an anti-pDGFRB/VeGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.Key words: bispecific, antibody, PDGFRβ, VEGF-A, stability, angiogenesis     

Chloroquine–astemizole hybrids with potent in vitro and in vivo antiplasmodial activity

A dual activity, conjugated approach has been taken to form hybrid molecules of two known antimalarial drugs, chloroquine (CQ) and the non-sedating H1 antagonist astemizole. A variety of linkers were investigated to conjugate the two agents into one molecule. Compounds 5–8 possessed improved in vitro activity against a CQ-resistant strain of Plasmodium falciparum, and examples 7 and 8 were active in vivo in mouse models of malaria.     

In vitro and in vivo antioxidant activity of exopolysaccharide fractions from Bifidobacterium animalis RH

The aim of the study was to purify the exopolysaccharides (EPS) produced from Bifidobacterium animalis RH, which was isolated from the feces of Bama centenarians in Guangxi of China, and evaluate their antioxidant activities in vitro and in vivo. 2 fractions, a neutral EPS fraction (EPSa) and an acidic EPS fraction (EPSb), were obtained and compared for antioxidative activity. In vitro, they both showed remarkable inhibition effect on lipid peroxidation and strong DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, in which the last two were measured by the electron spin resonance (ESR). In vivo, EPSa and EPSb were orally administrated for 30 days in a d-galactose induced aged mice model. As results, they both could significantly increase the activities of SOD, CAT and total antioxidant capacity (TAOC) in serums and glutathione GST in livers. They also could inhibit significantly the formation of MDA in serums and livers, and reduce the activity of MAO and lipofuscin accumulation in mice brain. Moreover, EPSb exhibited much higher antioxidant activities than EPSa in vitro and in vivo. The results suggested that EPS fractions of Bifidobacterium animalis RH had direct and potent antioxidant activities.     

Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.     

Interferon-α/β and anti-fibroblast growth factor receptor 1 monoclonal antibody suppress hepatic cancer cells in vitro and in vivo

Background

Hepatocellular carcinoma (HCC) is the most commonly occurring primary liver cancer and ranks as the fifth most frequently occurring cancer, overall, and the third leading cause of cancer deaths, worldwide. At present, effective therapeutic options available for HCC are limited; consequently, the prognosis for these patients is poor. Our aim in the present study was to identify a novel target for antibody therapy against HCC.

Methodology/Principal Findings

We used Western blot and flow cytometric and immunocytochemical analyses to investigate the regulation of FGFR1 expression by interferon-α/β in several human hepatic cancer cell lines. In addition, we tested the efficacy of combined treatment with anti-FGFR1 monoclonal antibody and interferon-α/β in a murine xenograft model of human HCC. We found that interferon-α/β induces expression of FGFR1 in human HCC cell lines, and that an anti-FGFR1 monoclonal antibody (mAb) targeting of the induced FGFR1 can effectively inhibit growth and survival of HCC cells in vitro and in vivo. Moreover, the combination of interferon-α, anti-FGFR1 mAb and peripheral blood mononuclear cells (PBMCs) exerted a significant antitumor effect in vitro.

Conclusions

Our results suggest that the combined use of an anti-FGFR1 antibody and interferon-α/β is a promising approach to the treatment of HCC.     

Docosahexaenoic acid inhibited the Wnt/β-Catenin pathway and suppressed breast cancer cells in vitro and in vivo

N-3 fatty acids (FAs) are essential FAs necessary for human health and are known to possess anticancer properties. However, the relationship between n-3 FAs and β-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, 4T1 mouse breast cancer cells were exposed to a representative n-3 FA, docosahexaenoic acid (DHA), to investigate the relationship between n-3 FAs and the Wnt/β-catenin signaling pathway in vivo and in vitro. In vitro studies showed that DHA strongly inhibited cell growth, and induced G1 cell cycle arrest both in 4T1 mouse breast cells and MCF-7 human breast cells. DHA reduced β-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity in 4T1 mouse breast cells. In addition, DHA down-regulated the expression of downstream target genes such as c-myc and cyclinD1. In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. We found that feeding mouse the 5% fish oil-supplemented diet for 30 days significantly reduced the growth of 4T1 mouse breast cancer in vivo through inhibition of cancer cell proliferation as well as induction of apoptosis. Feeding animals a 5% fish oil diet significantly induced down-regulation of β-catenin in tumor tissues with a notable increase in apoptosis. In addition, fish oil-supplemented diet decreased lung metastases of breast cancer. These observations suggested that DHA exerted its anticancer activity through down-regulation of Wnt/β-catenin signaling. Thus, our data call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of breast cancer.     

Wnt/β-catenin signaling is critical for dedifferentiation of aged epidermal cells in vivo and in vitro

Aged epidermal cells have the capacity to dedifferentiate into stem cell-like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/β-catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell-like cells that were positive for CK19 and β1 integrin but negative for CK10. In addition, Wnt/β-catenin pathway was activated during this process. There was increased expression of Wnt-1, Wnt-4, Wnt-7a, β-catenin, cyclin D1, and c-myc. Secreted frizzled-related protein 1, a Wnt/β-catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)-3β inhibitor, of Wnt/β-catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, β1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony-forming efficiency, long-term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/β-catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell-like cells.     

In vitro and in vivo pro-angiogenic effects of thymosin-β4-derived peptides

Thymosin-β4 (Tβ4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of Tβ4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) Tβ4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of Tβ4.     

In vitro and in vivo antitumor activity of a mouse CTL hybridoma expressing chimeric receptors bearing the single chain Fv from HER-2/neu- specific antibody and the γ-chain from Fc(ε) RI

To broaden the applicability of adoptive cellular immunotherapy against HER-2/neu overexpressing human cancers, we constructed a chimeric scFv/gamma gene composed of the variable regions of a HER-2/neu specific monoclonal antibody (mAb) joined to the signaling gamma-chain of the Fc(epsilon)RI receptor. The scFv(anti-HER-2/neu)/gamma chimeric gene was successfully expressed as functional surface receptor in the MD.45 cytolytic T-cell (CTL) hybridoma (MD.45-HER/gamma). Expression of the chimeric protein triggered IL-2 and IFN-gamma secretion in vitro upon encountering cell surface HER-2/neu and mediated non-major-histocompatibility-complex (MHC)-restricted HER-2/neu-specific target cell lysis. We also examined the in vivo activity of the MD.45-HER/gamma transduced cells. Severe combined immunodeficiency disease (SCID) mice that were given HER-2/neu positive (+) human tumor cell lines had significantly increased survival compared to mice treated with saline only, or with MD.45 cells transduced with a control anti-trinitrophenyl (anti-TNP) chimeric receptor gene (MD.45-TNP/gamma). These results demonstrate the feasibility of redirecting MD.45 CTL to react in vitro and in vivo with a variety of HER-2/neu(+) tumor cells by our gene transduction protocol. Moreover, they open the possibility of using the same chimeric gene for transducing primary lymphocytes and thus allowing adoptive immunotherapy against HER-2/neu(+) cancers.     

In vitro and ex vivo vanadium antitumor activity in (TGF-β)-induced EMT. Synergistic activity with carboplatin and correlation with tumor metastasis in cancer patients

Epithelial to mesenchymal transition (EMT) plays a key role in tumor progression and metastasis as a crucial event for cancer cells to trigger the metastatic niche. Transforming growth factor-β (TGF-β) has been shown to play an important role as an EMT inducer in various stages of carcinogenesis. Previous reports had shown that antitumor vanadium inhibits the metastatic potential of tumor cells by reducing MMP-2 expression and inducing ROS-dependent apoptosis. However, the role of vanadium in (TGF-β)-induced EMT remains unclear. In the present study, we report for the first time on the inhibitory effects of vanadium on (TGF-β)-mediated EMT followed by down-regulation of ex vivo cancer stem cell markers. The results demonstrate blockage of (TGF-β)-mediated EMT by vanadium and reduction in the mitochondrial potential of tumor cells linked to EMT and cancer metabolism. Furthermore, combination of vanadium and carboplatin (a) resulted in synergistic antitumor activity in ex vivo cell cultures, and (b) prompted G₀/G₁ cell cycle arrest and sensitization of tumor cells to carboplatin-induced apoptosis. Overall, the findings highlight the multifaceted antitumor action of vanadium and its synergistic antitumor efficacy with current chemotherapy drugs, knowledge that could be valuable for targeting cancer cell metabolism and cancer stem cell-mediated metastasis in aggressive chemoresistant tumors.     

DJ-1 regulates tyrosine hydroxylase expression through CaMKKβ/CaMKIV/CREB1 pathway in vitro and in vivo

Lack of dopamine production and neurodegeneration of dopaminergic neurons in the substantia nigra are considered as the major characteristics of Parkinson's disease, a prevalent movement disorder worldwide. DJ-1 mutation leading to loss of its protein functions is a genetic factor of PD. In this study, our results illustrated that DJ-1 can directly interact with Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) and modifies the cAMP-responsive element binding protein 1 (CREB1) activity, thus regulates tyrosine hydroxylase (TH) expression. In Dj-1 knockout mouse substantia nigra, the levels of TH and the phosphorylation of CREB1 Ser133 are significantly decreased. Moreover, Dj-1 deficiency suppresses the phosphorylation of CaMKIV (Thr196/200) and CREB1 (Ser133), subsequently inhibits TH expression in vitro. Furthermore, Knockdown of Creb1 abolishes the effects of DJ-1 on TH regulation. Our data reveal a novel pathway in which DJ-1 regulates CaMKKβ/CaMKIV/CREB1 activities to facilitate TH expression.     

Inhibitory effect of ribbon-type NF-κB decoy oligodeoxynucleotides on osteoclast induction and activity in vitro and in vivo

In this study we examined the effect of ribbon-type (circular-type) NF-κB decoy oligodeoxynucleotides (RNODN) on osteoclast induction and activity. We extracted bone marrow cells from the femurs of rats and incubated non-adherent cells with receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). First, transfer efficiency into osteoclasts and their precursors, resistance to exonuclease, and binding activity of decoy to NF-κB were examined. Next, to examine the effect of RNODN on osteoclast induction and activity, osteoclast differentiation and pit formation assays were performed. RNODN were injected into the ankle joints of rats with collagen-induced arthritis. Joint destruction and osteoclast activity were examined by histological study. The resistance of RNODN to exonuclease and their binding activity on NF-κB were both greater than those of phosphorothionated NF-κB decoy oligodeoxynucleotides. The absolute number of multinucleate cells scoring positive for tartrate-resistant acid phosphatase was significantly decreased in the RNODN-treated group. The average calcified matrix resorbed area was significantly decreased in the RNODN-treated group. Histological study showed marked suppression of joint destruction and osteoclast activity by intra-articular injection of RNODN. These results suggest the inhibitory effect of RNODN on the induction and activity of osteoclasts. Direct intra-articular injection of RNODN into the joints may be an effective strategy for the treatment of arthritis.     

Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimer's disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.     

Isobavachalcone and bavachinin from Psoraleae Fructus modulate Aβ42 aggregation process through different mechanisms in vitro

Spontaneous aggregation of Aβ is a key factor in the development of Alzheimer’s disease. In searching for Aβ aggregation inhibitors from traditional Chinese herbal medicines, we identified two active compounds from Psoraleae Fructus, namely isobavachalcone and bavachinin. We further demonstrated that the two compounds modulate Aβ42 aggregation process through different mechanisms. Isobavachalcone significantly inhibits both oligomerization and fibrillization of Aβ42, whereas bavachinin inhibits fibrillization and leads to off-pathway aggregation. Both of the compounds attenuated Aβ42-induced toxicity in a SH-SY5Y cell model. These findings may provide valuable information for new drug development and Alzheimer’s therapy in the future.     

AdipoRon reduces TGFβ1-mediated collagen deposition in vitro and alleviates knee stiffness in vivo

Arthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti-fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFβ1-mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFβ1-induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA-seq analysis corroborated the anti-myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle-treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.     

Escherichia coli β-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii

We have developed several protocols for the use of β-galactosidase (βGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent βGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chlorophenol red-β-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing βGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of βGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites     

7,7′-Diazaindirubin—A small molecule inhibitor of casein kinase 2 in vitro and in cells

Aza- and diaza-bisindoles were synthesized by coupling of 7-azaisatin, 7-azaoxindol, 7-azaindoxyl acetate, and their non-aza counterparts, respectively. Whereas 7,7′-diazaindigo (10) and 7,7′-diazaisoindigo (11) did not show antiproliferative activity in several human tumor cell lines up to 100 μM, 7-azaindirubin (12) and 7′-azaindirubin (13) were more active than the parent molecule, indirubin, in LXFL529L cells (human large cell lung tumor xenograft), and 7,7′-diazaindirubin (14) was exhibiting substantially enhanced growth inhibitory activity in these cells. In the NCI 60 cell line panel, 14 displayed antiproliferative activity preferentially in certain melanoma and non-small cell lung cancer cells. In contrast to the potent serine/threonine/tyrosine kinase inhibition observed for indirubins, kinase inhibition profiling of 14 in 220 kinases revealed largely a loss of kinase inhibitory activity towards most kinases, with retained inhibitory activity for just a few kinases. At 1 μM concentration, especially casein kinases CK1γ3, CK2α, CK2α2, and SIK were inhibited by more than 50%. In cell-based assays, 14 markedly affected CK2-mediated signaling in various human tumor cells. In MCF7 cells, 14 induced cell cycle arrest at G1 and G2/M and apoptosis, whereas CK2-deficient MCF7 cells were resistant. These findings reveal a novel key mechanism of action for 14, suggesting primarily CK2 inhibition to be causally related to growth inhibition of human tumor cells.