Self-Stimulated Photodynamic Nanoreactor in Combination with CXCR4 Antagonists for Antileukemia Therapy.

The treatment of acute myeloid leukemia (AML) remains unsatisfactory, owing to the absence of efficacious therapy regimens over decades. However, advances in molecular biology, including inhibiting the CXCR4/CXCL12 biological axis, have introduced novel therapeutic options for AML. Additionally, self-stimulated phototherapy can solve the poor light penetration from external sources, and it will overcome the limitation that traditional phototherapy cannot be applied to the treatment of AML. Herein, we designed and manufactured a self-stimulated photodynamic nanoreactor to enhance antileukemia efficacy and suppress leukemia recurrence and metastasis in AML mouse models. To fulfill our design, we utilized the CXCR4/CXCL12 biological axis and biomimetic cell membranes in conjunction with self-stimulated phototherapy. This nanoreactor possesses the capability to migrate into the bone marrow cavity, inhibit AML cells from infiltrating into the visceral organ, significantly enhance the antileukemia effect, and prolong the survival time of leukemic mice. Therefore, this nanoreactor has significant potential for achieving high success rates and low recurrence rates in leukemia treatment.

A highly selective and potent CXCR4 antagonist for hepatocellular carcinoma treatment.

The CXC chemokine receptor type 4 (CXCR4) receptor and its ligand, CXCL12, are overexpressed in various cancers and mediate tumor progression and hypoxia-mediated resistance to cancer therapy. While CXCR4 antagonists have potential anticancer effects when combined with conventional anticancer drugs, their poor potency against CXCL12/CXCR4 downstream signaling pathways and systemic toxicity had precluded clinical application. Herein, BPRCX807, known as a safe, selective, and potent CXCR4 antagonist, has been designed and experimentally realized. In in vitro and in vivo hepatocellular carcinoma mouse models it can significantly suppress primary tumor growth, prevent distant metastasis/cell migration, reduce angiogenesis, and normalize the immunosuppressive tumor microenvironment by reducing tumor-associated macrophages (TAMs) infiltration, reprogramming TAMs toward an immunostimulatory phenotype and promoting cytotoxic T cell infiltration into tumor. Although BPRCX807 treatment alone prolongs overall survival as effectively as both marketed sorafenib and anti-PD-1, it could synergize with either of them in combination therapy to further extend life expectancy and suppress distant metastasis more significantly.

Dissecting the role of the CXCL12/CXCR4 axis in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is the most common adult acute leukaemia with the lowest survival rate. It is characterised by a build-up of immature myeloid cells anchored in the protective niche of the bone marrow (BM) microenvironment. The CXCL12/CXCR4 axis is central to the pathogenesis of AML as it has fundamental control over AML cell adhesion into the protective BM niche, adaptation to the hypoxic environment, cellular migration and survival. High levels of CXCR4 expression are associated with poor relapse-free and overall survival. The CXCR4 ligand, CXCL12 (SDF-1), is expressed by multiple cells types in the BM, facilitating the adhesion and survival of the malignant clone. Blocking the CXCL12/CXCR4 axis is an attractive therapeutic strategy providing a 'multi-hit' therapy that both prevents essential survival signals and releases the AML cells from the BM into the circulation. Once out of the protective niche of the BM they would be more susceptible to destruction by conventional chemotherapeutic drugs. In this review, we disentangle the diverse roles of the CXCL12/CXCR4 axis in AML. We then describe multiple CXCR4 inhibitors, including small molecules, peptides, or monoclonal antibodies, which have been developed to date and their progress in pre-clinical and clinical trials. Finally, the review leads us to the conclusion that there is a need for further investigation into the development of a 'multi-hit' therapy that targets several signalling pathways related to AML cell adhesion and maintenance in the BM.

Surface Plasmon Resonance-Based Membrane Protein-Targeted Active Ingredients Recognition Strategy: Construction and Implementation in Ligand Screening from Herbal Medicines.

Membrane proteins (MPs) are playing important roles in several biological processes. Screening new candidate compounds targeting MPs is important for drug discovery. However, it remains challenging to characterize the interactions between MPs and small-molecule ligands in a label-free method. In this study, a surface plasmon resonance (SPR)-based membrane protein-targeted active ingredients recognition strategy was constructed. This strategy contains two major modules: affinity detection module and ligand screening module. Through the combination of these two functional modules, it is feasible to screen small molecular ligands targeting MPs from herbal medicines. First, we have constructed high/low comparative C-X-C chemokine receptor type 4 (CXCR4)-expressed lentiviral particles (LVPs) models and characterized the expression levels. Then we immobilized LVPs on CM5 chips and detected the affinity between AMD3100 and CXCR4 by using affinity detection module. The KD of AMD3100 was 32.48 ± 3.17 nM. Furthermore, the suitability and robustness of the ligand screening module were validated by using AMD3100 as a positive compound. Subsequently, this module was applied in the screening of CXCR4 small molecular ligands from herbal medicine extracts. Senkyunolide I was screened out from Chuanxiong extract. The affinity constant between senkyunolide I and CXCR4 was 2.94 ± 0.36 µM. The Boyden chamber assay revealed that senkyunolide I could inhibit cell migration process. In conclusion, an SPR-based small molecular ligand recognition strategy combined with virus-based membrane protein stabilization method was constructed. The SPR-based membrane protein-targeted active ingredients recognition strategy will be an effective tool to screen target components from complex systems acting on MPs.

Electroacupuncture Alleviates Mechanical Allodynia in a Rat Model of Complex Regional Pain Syndrome Type-I via Suppressing Spinal CXCL12/CXCR4 Signaling.

Complex regional pain syndrome (CRPS) results in chronic and excruciating pain in patients. Conventional therapies lack effectiveness, rendering it one of the most difficult to treat neurological conditions.. Electroacupuncture (EA) is an effective alternative therapy for pain relief. Here, we investigated whether EA exerts analgesic effect on a rat model of CRPS type-I (CRPS-I) and related mechanisms. The rat chronic postischemic pain (CPIP) model was established to mimic CRPS-I. 100Hz EA exerted robust and persistent antiallodynic effect on CPIP model compared with 2 Hz EA or sham EA. EA markedly suppressed the overexpression of CXCL12/CXCR4 in spinal cord dorsal horn (SCDH) of CPIP model, leading to substantial decrease in neuronal and glial cell activities in SCDH. Pharmacological blocking CXCR4 mimicked EA-induced antiallodynic effect and related cellular events in SCDH, whereas exogenous CXCL12 abolished EA's effect. CXCR4 signaling resulted in ERK activation in SCDH, contributing to mechanical allodynia of CPIP model rats, whereas EA markedly reduced ERK activation. Therefore, we demonstrated that EA interferes with CXCL12/CXCR4 signaling in SCDH and downstream ERK pathway to exert robust antiallodynic effect on an animal model of CRPS-I. Our work suggests that EA may be a potential therapeutic option for CRPS-I in clinic. PERSPECTIVE: Our work identified that EA exerts robust antiallodynic effect on an animal model of CRPS-I, via mechanisms involving inhibition of CXCL12/CXCR4 signaling. EA further attenuates downstream neuronal and glial cell activation and ERK pathway in SCDH. This work suggests that EA may be a potential therapeutic option for CRPS-I management in clinic.

Inhibitory potential of Hydroxychavicol on Ehrlich ascites carcinoma model and in silico interaction on cancer targets.

Hydroxychavicol (HC), a major phenolic derivative isolated from the leaves of Piper betle L. is well known for its antibacterial, antifungal and antimutagenic properties. The present study evaluated the in vivo antitumor activity of HC against Ehrlich Ascites Carcinoma (EAC) cells in Swiss albino mice and in silico interaction of HC with the receptors involved in the cancer. Hydroxychavicol (200 and 400 mg/kg bw) was orally administered for 21 consecutive days and was effective in inhibiting the tumor growth in ascitic mouse model. HC consistently reduced the tumor volume, viable cell count, lipid peroxidation and elevated the life span of HC treated mice. Besides the hematological profiles, SGOT and SGPT levels reverted back to normal and oxidative stress markers GSH, SOD and CAT also increased in HC treated groups. In silico docking analysis revealed that HC possessed potent antagonist activity against all the cancer targets demonstrating its inhibitory activity.

Silibinin efficacy in a rat model of pulmonary arterial hypertension using monocrotaline and chronic hypoxia.

BACKGROUND: C-X-C chemokine receptor type 4 (CXCR4) may be involved in the development of pulmonary arterial hypertension (PAH). CXCR4 inhibitor AMD3100 was described to have a positive effect on the prevention of pulmonary arterial muscularization in PAH models. Silibinin is a traditional medicine that has an antagonistic effect on CXCR4. We investigated the effect of silibinin using rat models of PAH. METHODS: PAH was induced by a single subcutaneous injection of monocrotaline. The rats were maintained in a chronic hypoxic condition (10% O2) with or without silibinin. To evaluate the efficacy of silibinin on PAH, right ventricular systolic pressure (RVSP), Fulton index (weight ratio of right ventricle to the left ventricle and septum), percent medial wall thickness (% MT), and vascular occlusion score (VOS) were measured and calculated. Immunohistochemical analysis was performed targeting CXCR4 and c-Kit. Reverse transcription-quantitative polymerase chain reaction was performed for the stem cell markers CXCR4, stromal cell derived factor-1 (SDF-1), c-Kit, and stem cell factor (SCF), and the inflammatory markers monocyte chemoattractant protein 1 (MCP1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα). Statistical analyses were performed using t-test and one-way analysis of variance with Bonferroni's post hoc test. RESULTS: Silibinin treatment for 1 week reduced RVSP and Fulton index. Treatment for 2 weeks reduced RVSP, Fulton index, % MT, and VOS, as well as downregulating the expression of CXCR4, SDF-1, and TNFα in pulmonary arteries. In contrast, treatment for 3 weeks failed to ameliorate PAH. The time-course study demonstrated that RVSP, Fulton index, % MT, and VOS gradually increased over time, with a decrease in the expression of CXCR4 and TNFα occurring after 2 weeks of PAH development. After 3 weeks, SDF-1, c-Kit, and SCF began to decrease and, after 5 weeks, MCP1 and IL-6 gradually accumulated. CONCLUSIONS: The CXCR4 inhibitor silibinin can ameliorate PAH, possibly through the suppression of the CXCR4/SDF-1 axis, until the point where PAH becomes a severe and irreversible condition. Silibinin results in reduced pulmonary arterial pressure and delays pulmonary arteriolar occlusion and pulmonary vascular remodeling.

Penicillixanthone A, a marine-derived dual-coreceptor antagonist as anti-HIV-1 agent.

Marine micro-organisms have been proven to be excellent sources of bioactive compounds against HIV-1. Several natural products obtained from marine-derived Aspergillus fungi were screened for their activities to inhibit HIV-1 infection. Penicillixanthone A (PXA), a natural xanthone dimer from jellyfish-derived fungus Aspergillus fumigates, displayed potent anti-HIV-1 activity by inhibiting infection against CCR5-tropic HIV-1 SF162 and CXCR4-tropic HIV-1 NL4-3, with IC50 of 0.36 and 0.26 µM, respectively. Molecular docking study was conducted to understand the possible binding mode of PXA with the CCR5/CXCR4. The results revealed that, the marine-derived PXA, as a CCR5/CXCR4 dual-coreceptor antagonist, presents a new type of potential lead product for the development of anti-HIV therapeutics.

The CXCL12 (SDF-1)/CXCR4 chemokine axis: Oncogenic properties, molecular targeting, and synthetic and natural product CXCR4 inhibitors for cancer therapy.

Chemokine 12 (CXCL12), also known as stromal cell derived factor-1 (SDF-1) and a member of the CXC chemokine subfamily, is ubiquitously expressed in many tissues and cell types. It interacts specifically with the ligand for the transmembrane G protein-coupled receptors CXCR4 and CXCR7. The CXCL12/CXCR4 axis takes part in a series of physiological, biochemical, and pathological process, such as inflammation and leukocyte trafficking, cancer-induced bone pain, and postsurgical pain, and also is a key factor in the cross-talking between tumor cells and their microenvironment. Aberrant overexpression of CXCR4 is critical for tumor survival, proliferation, angiogenesis, homing and metastasis. In this review, we summarized the role of CXCL12/CXCR4 in cancer, CXCR4 inhibitors under clinical study, and natural product CXCR4 antagonists. In conclusion, the CXCL12/CXCR4 signaling is important for tumor development and targeting the pathway might represent an effective approach to developing novel therapy in cancer treatment.

Design, Synthesis, and Pharmacological Evaluation of Second-Generation Tetrahydroisoquinoline-Based CXCR4 Antagonists with Favorable ADME Properties.

CXCR4 is a G-protein-coupled receptor that interacts with its cognate ligand, CXCL12, to synchronize many physiological responses and pathological processes. Disruption of the CXCL12-CXCR4 circuitry by small-molecule antagonists has emerged as a promising strategy for cancer intervention. We previously disclosed a hit-to-lead effort that led to the discovery of a series of tetrahydroisoquinoline-based CXCR4 antagonists exemplified by the lead compound TIQ15. Herein, we describe our medicinal-chemistry efforts toward the redesign of TIQ15 as a result of high mouse-microsomal clearance, potent CYP2D6 inhibition, and poor membrane permeability. Guided by the in vitro ADME data of TIQ15, structural modifications were executed to provide compound 12a, which demonstrated a reduced potential for first-pass metabolism while maintaining CXCR4 potency. Subsequent SAR studies and multiparameter optimization of 12a resulted in the identification of compound 25o, a highly potent, selective, and metabolically stable CXCR4 antagonist possessing good intestinal permeability and low risk of CYP-mediated drug-drug interactions.

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4.

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor's function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

Curcumin protects against hepatic stellate cells activation and migration by inhibiting the CXCL12/CXCR4 biological axis in liver fibrosis：A study in vitro and in vivo.

The C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/ CXCR4) biological axis plays an important role in the pathogenesis of liver fibrosis. Curcumin is known to have an anti-fibrosis effect, but the specific mechanism needs to be elucidated. There is currently no evidence illustrating a connection between curcumin and the CXCL12/CXCR4 axis in liver fibrosis. Here, we investigated the contribution of curcumin on CXCL12/ CXCR4 biological axis in liver fibrosis. Our results showed that curcumin remarkably improved hepatic function and liver fibrosis, and the effects are similar as silymarin. The alleviation of liver fibrosis with curcumin treatment was associated with a reduction of CXCL12, CXCR4, α-SMA and RhoA. In addition, curcumin markedly inhibited the proliferation and migration of HSC-T6 cells. This study indicates that curcumin could protect against hepatic stellate cells activation and migration by inhibiting the CXCL12/CXCR4 biological axis in liver fibrosis.

Inhibitors of HIF-1α and CXCR4 Mitigate the Development of Radiation Necrosis in Mouse Brain.

PURPOSE: There is mounting evidence that, in addition to angiogenesis, hypoxia-induced inflammation via the hypoxia-inducible factor 1α (HIF-1α)-CXC chemokine receptor 4 (CXCR4) pathway may contribute to the pathogenesis of late-onset, irradiation-induced necrosis. This study investigates the mitigative efficacy of an HIF-1α inhibitor, topotecan, and a CXCR4 antagonist, AMD3100, on the development of radiation necrosis (RN) in an intracranial mouse model. METHODS AND MATERIALS: Mice received a single-fraction, 50-Gy dose of hemispheric irradiation from the Leksell Gamma Knife Perfexion and were then treated with either topotecan, an HIF-1α inhibitor, from 1 to 12 weeks after irradiation, or AMD3100, a CXCR4 antagonist, from 4 to 12 weeks after irradiation. The onset and progression of RN were monitored longitudinally via noninvasive, in vivo magnetic resonance imaging (MRI) from 4 to 12 weeks after irradiation. Conventional hematoxylin-eosin staining and immunohistochemistry staining were performed to evaluate the treatment response. RESULTS: The progression of brain RN was significantly mitigated for mice treated with either topotecan or AMD3100 compared with control animals. MRI-derived lesion volumes were significantly smaller for both of the treated groups, and histologic findings correlated well with the MRI data. By hematoxylin-eosin staining, both treated groups demonstrated reduced irradiation-induced tissue damage compared with controls. Furthermore, immunohistochemistry results revealed that expression levels of vascular endothelial growth factor, CXC chemokine ligand 12, CD68, CD3, and tumor necrosis factor α in the lesion area were significantly lower in treated (topotecan or AMD3100) brains versus control brains, while ionized calcium-binding adapter molecule 1 (Iba1) and HIF-1α expression was similar, though somewhat reduced. CXCR4 expression was reduced only in topotecan-treated mice, while interleukin 6 expression was unaffected by either topotecan or AMD3100. CONCLUSIONS: By reducing inflammation, both topotecan and AMD3100 can, independently, mitigate the development of RN in the mouse brain. When combined with first-line, antiangiogenic treatment, anti-inflammation therapy may provide an adjuvant therapeutic strategy for clinical, postirradiation management of tumors, with additional benefits in the mitigation of RN development.

Zerumbone targets the CXCR4-RhoA and PI3K-mTOR signaling axis to reduce motility and proliferation of oral cancer cells.

BACKGROUND: The CXCR4-RhoA and PI3K-mTOR signaling pathways play crucial roles in the dissemination and tumorigenesis of oral squamous cell carcinoma (OSCC). Activation of these pathways have made them promising molecular targets in the treatment of OSCC. Zerumbone, a bioactive monocyclic sesquiterpene isolated from the rhizomes of tropical ginger, Zingiber zerumbet (L.) Roscoe ex Sm. has displayed promising anticancer properties with the ability to modulate multiple molecular targets involved in carcinogenesis. While the anticancer activities of zerumbone have been well explored across different types of cancer, the molecular mechanism of action of zerumbone in OSCC remains largely unknown. PURPOSE: Here, we investigated whether OSCC cells were sensitive towards zerumbone treatment and further determined the molecular pathways involved in the mechanism of action. METHODS: Cytotoxicity, anti-proliferative, anti-migratory and anti-invasive effects of zerumbone were tested on a panel of OSCC cell lines. The mechanism of action of zerumbone was investigated by analysing the effects on the CXCR4-RhoA and PI3K-mTOR pathways by western blotting. RESULTS: Our panel of OSCC cells was broadly sensitive towards zerumbone with IC50 values of less than 5 µM whereas normal keratinocyte cells were less responsive with IC50 values of more than 25 µM. Representative OSCC cells revealed that zerumbone inhibited OSCC proliferation and induced cell cycle arrest and apoptosis. In addition, zerumbone treatment inhibited migration and invasion of OSCC cells, with concurrent suppression of endogenous CXCR4 protein expression in a time and dose-dependent manner. RhoA-pull down assay showed reduction in the expression of RhoA-GTP, suggesting the inactivation of RhoA by zerumbone. In association with this, zerumbone also inhibited the PI3K-mTOR pathway through the inactivation of Akt and S6 proteins. CONCLUSION: We provide evidence that zerumbone could inhibit the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways leading to the reduced cell viability of OSCC cells. Our results suggest that zerumbone is a promising phytoagent for development of new therapeutics for OSCC treatment.

Platelet releasate promotes breast cancer growth and angiogenesis via VEGF-integrin cooperative signalling.

BACKGROUND: Selective platelet release of pro- or anti-angiogenic factors distinctly regulated angiogenesis. We hypothesised that selective release of platelet angiogenic factors could differently regulate tumour growth. METHODS: Breast cancer cell proliferation, cancer cell-induced endothelial tube formation in vitro, and tumour growth in vivo were studied in the presence of protease-activated receptor 1-stimulated platelet releasate (PAR1-PR; rich in pro-angiogenic factors) or PAR4-PR (rich in anti-angiogenic factors). RESULTS: The PAR1-PR and PAR4-PR supplementation (10%) similarly enhanced cell proliferation of MCF-7 and MDA-MB-231 breast cancer cells. The cancer cells triggered capillary-like tube formation of endothelial cells that was further enhanced by pro-angiogenic factor-rich PAR1-PR. The VEGF, but not SDF-1α, receptor blockade abolished PAR1-PR/PAR4-PR-enhanced cancer cell proliferation. Integrin blockade by RGDS had identical effects as VEGF inhibition. The Src and ERK inhibition diminished, whereas PI3K and PKC blockade abolished platelet releasate-enhanced cancer cell proliferation. Using a model of subcutaneous implantation of MDA-MB-231 cells in nude mice, PAR1-PR enhanced tumour growth more markedly than PAR4-PR, and seemed to achieve the exaggeration by promoting more profound tumour angiogenesis. CONCLUSIONS: Platelet releasate increases breast cancer cell proliferation through VEGF-integrin cooperative signalling. Pro-angiogenic factor-rich platelet releasate enhances cancer cell-induced angiogenesis more markedly, and thus exaggerates tumour growth in vivo.

The brain-penetrating CXCR4 antagonist, PRX177561, increases the antitumor effects of bevacizumab and sunitinib in preclinical models of human glioblastoma.

BACKGROUND: Glioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. It has been demonstrated that anti-VEGF/VEGFR therapies control the invasive phenotype and that relapse occurs through the increased activity of CXCR4. We therefore hypothesized that combining bevacizumab or sunitinib with the novel CXCR4 antagonist, PRX177561, would have superior antitumor activity. METHODS: The effects of bevacizumab, sunitinib, and PRX177561 were tested alone or in combination in subcutaneous xenografts of U87MG, U251, and T98G cells as well as on intracranial xenografts of luciferase tagged U87MG cells injected in CD1-nu/nu mice. Animals were randomized to receive vehicle, bevacizumab (4 mg/kg iv every 4 days), sunitinib (40 mg/kg po qd), or PRX177561 (50 mg/kg po qd). RESULTS: The in vivo experiments demonstrated that bevacizumab and sunitinib increase the in vivo expression of CXCR4, SDF-1α, and TGFß1. In addition, we demonstrate that the co-administration of the novel brain-penetrating CXCR4 antagonist, PRX177561, with bevacizumab or sunitinib inhibited tumor growth and reduced the inflammation. The combination of PRX177561 with bevacizumab resulted in a synergistic reduction of tumor growth with an increase of disease-free survival (DSF) and overall survival (OS), whereas the combination of PRX177561 with sunitinib showed a mild additive effect. CONCLUSIONS: The CXC4 antagonist PRX177561 may be a valid therapeutic complement to anti-angiogenic therapy, particularly when used in combination with VEGF/VEGFR inhibitors. Therefore, this compound deserves to be considered for future clinical evaluation.

Onbaekwon Suppresses Colon Cancer Cell Invasion by Inhibiting Expression of the CXC Chemokine Receptor 4.

Cysteine X cysteine (CXC) chemokine receptor 4 (CXCR4) and C-X-C motif chemokine 12 (CXCL12) were originally identified as chemoattractants between immune cells and sites of inflammation. Since studies have validated an increased level of CXCL12 and its receptor in patients with colorectal cancers, CXCL12/CXCR4 axis has been considered as a valuable marker of cancer metastasis. Therefore, identification of CXCR4 inhibitors has great potential to abrogate tumor metastasis. Onbaekwon (OBW) is a complex herbal formula that is derived from the literature of traditional Korean medicine Dongeuibogam. In this study, we demonstrated that OBW suppressed CXCR4 expression in various cancer cell types in a concentration- and time-dependent manner. Both proteasomal and lysosomal inhibitors had no effect to prevent the OBW-induced suppression of CXCR4, suggesting that the inhibitory effect of OBW was not due to proteolytic degradation but occurred at the transcriptional level. Electrophoretic mobility shift assay further confirmed that OBW could block endogenous activation of nuclear factor kappa B, a key transcription factor that regulates the expression of CXCR4 in colon cancer cells. Consistent with the aforementioned molecular basis, OBW abolished cell invasion induced by CXCL12 in colon cancer cells. Together, our results suggest that OBW, as a novel inhibitor of CXCR4, could be a promising therapeutic agent contributing to cancer treatment.

Identification of anti-cancer chemical compounds using Xenopus embryos.

Cancer tissues have biological characteristics similar to those observed in embryos during development. Many types of cancer cells acquire pro-invasive ability through epithelial-mesenchymal transition (EMT). Similar processes (gastrulation and migration of cranial neural crest cells [CNCC]) are observed in the early stages of embryonic development in Xenopus during which cells that originate from epithelial sheets through EMT migrate to their final destinations. The present study examined Xenopus embryonic tissues to identify anti-cancer compounds that prevent cancer invasion. From the initial test of known anti-cancer drugs, AMD3100 (an inhibitor of CXCR4) and paclitaxel (a cytoskeletal drug targeting microtubules) effectively prevented migration during gastrulation or CNCC development. Blind-screening of 100 synthesized chemical compounds was performed, and nine candidates that inhibited migration of these embryonic tissues without embryonic lethality were selected. Of these, C-157 (an analog of podophyllotoxin) and D-572 (which is an indole alkaroid) prevented cancer cell invasion through disruption of interphase microtubules. In addition, these compounds affected progression of mitotic phase and induced apoptosis of SAS oral cancer cells. SAS tumors were reduced in size after intratumoral injection of C-157, and peritoneal dissemination of melanoma cells and intracranial invasion of glioma cells were inhibited by C-157 and D-572. When the other analogues of these chemicals were compared, those with subtle effect on embryos were not tumor suppressive. These results suggest that a novel chemical-screening approach based on Xenopus embryos is an effective method for isolating anti-cancer drugs and, in particular, targeting cancer cell invasion and proliferation.

CXCR4 Antagonist AMD3100 Suppresses the Long-Term Abnormal Structural Changes of Newborn Neurons in the Intraventricular Kainic Acid Model of Epilepsy.

Abnormal hippocampal neurogenesis is a prominent feature of temporal lobe epilepsy (TLE) models, which is thought to contribute to abnormal brain activity. Stromal cell-derived factor-1 (SDF-1) and its specific receptor CXCR4 play important roles in adult neurogenesis. We investigated whether treatment with the CXCR4 antagonist AMD3100 suppressed aberrant hippocampal neurogenesis, as well as the long-term consequences in the intracerebroventricular kainic acid (ICVKA) model of epilepsy. Adult male rats were randomly assigned as control rats, rats subjected to status epilepticus (SE), and post-SE rats treated with AMD3100. Animals in each group were divided into two subgroups (acute stage and chronic stage). We used immunofluorescence staining of BrdU and DCX to analyze the hippocampal neurogenesis on post-SE days 10 or 74. Nissl staining and Timm staining were used to evaluate hippocampal damage and mossy fiber sprouting, respectively. On post-SE day 72, the frequency and mean duration of spontaneous seizures were measured by electroencephalography (EEG). Cognitive function was evaluated by Morris water maze testing on post-SE day 68. The ICVKA model of TLE resulted in aberrant neurogenesis such as altered proliferation, abnormal dendrite development of newborn neurons, as well as spontaneous seizures and spatial learning impairments. More importantly, AMD3100 treatment reversed the aberrant neurogenesis seen after TLE, which was accompanied by decreased long-term seizure activity, though improvement in spatial learning was not seen. AMD3100 could suppress long-term seizure activity and alter adult neurogenesis in the ICVKA model of TLE, which provided morphological evidences that AMD3100 might be beneficial for treating chronic epilepsy.

Role of Berberine on molecular markers involved in migration of esophageal cancer cells.

Berberine is an isoquinoline alkaloid found in several plant species like famous chinese herb, Rhizoma coptidis which has been used locally as a strong gastrointestinal remedy for thousands of years. The inhibitory effects of berberine on tumor progression properties have been reported before. In this study, we investigated the effect of berberine on an esophageal cancer cell line, KYSE-30 with emphasis on its effects on the expression of certain chemokine receptors. The cytotoxic effect of berberine on KYSE-30 cells was analyzed by MTT assay. In vitro cell migration assay was also applied to the treated cells and the expression levels of the selected chemokine receptors (CXCR4 and CCR7) was measured at mRNA level. A retarded growth, associated with increasing concentrations of berberine, was obvious. On the other hand, the migration rate of the cells was decreased when they were treated with different concentrations of berberine and the expression levels of the two chemokine receptors, involved in the migration and metastasis of esophageal cancer cells, were decreased following the same treatments. With these results, we tend to conclude that berberine might be a proper candidate for further investigations, by targeting the chemokine receptors, and possible applications as anti-metastatic agent in cancer studies.