LPA5-Dependent signaling regulates regeneration of the intestinal epithelium following irradiation.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared with Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of Yes-associated protein (YAP), a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche.NEW & NOTEWORTHY We used mice lacking expression of the lysophosphatidic acid receptor 5 (LPA5) in intestinal epithelial cells and intestinal organoids to show that the LPA5 receptor protects intestinal stem cells and progenitors from radiation-induced injury. We show that LPA5 induces YAP signaling and regulates Paneth cells.

Efficacy and safety of Xiaoyao Pill combined with Western medicine in the treatment of schizophrenia: A Meta-analysis and Systematic review.

BACKGROUND: The efficacy and safety of Xiaoyao Pill combined with Western medicine in the treatment of schizophrenia are still inconclusive. This meta-analysis summarized relevant studies to compare the efficacy and safety of Xiaoyao Pill combined with Western medicine and Western medicine alone in the treatment of schizophrenia, aiming to provide guidance for clinical treatment. METHODS: In this meta-analysis, we searched PubMed, Embase, Cochrane Library, CNKI, Wanfang, CQVIP, and CBM databases from the establishment of the databases to August 2023. The study proposed to include studies that reported combination of Xiaoyao Pill with Western medicine and Western medicine alone in the treatment of schizophrenia, excluding published literature, unpublished literature, literature with incomplete or inadequate information, animal experiments, literature reviews and systematic studies. Data were analyzed using Review manager 5.3. RESULTS: About 9 studies (6 RCTs and 3 case-control studies) were included in this meta-analysis. The sample size ranged from 60 to 128, with a total of 779 patients, including 395 in the combined treatment group and 384 in the control group. Pooled results showed that the total effective rate of combined treatment group was significantly higher than that of Western medicine alone (OR = 4.21, 95% CI: 1.50-11.83, P = .006). Positive and Negative Syndrome Scale (PANSS) (-) (MD = -2.30, 95% CI: -3.72 ~ -0.89, P = .001) and PANSS (+) (MD = -2.60, 95% CI: -3.34 ~ -1.86, P < .00001) of combined treatment group were all significantly lower than that of Western medicine alone. Additionally, PRL levels of combined treatment group was significantly lower than that of Western medicine alone (MD = -28.78, 95% CI: -42.20 ~ -15.35, P < .0001). However, there was no significant difference in BPRS and total PANSS between combined treatment group and Western medicine alone group. Notably, pooled results showed that there was no significant difference in incidence of adverse events between combined treatment group and Western medicine alone group. CONCLUSION: The effective rate of Xiaoyao Pill combined with Western medicine in the treatment of schizophrenia is higher than that of Western medicine alone, which can effectively relieve the positive and negative symptoms of schizophrenia, and can significantly reduce the level of PRL. In the treatment of schizophrenia, clinicians can give priority to Xiaoyao Pill combined with Western medicine therapy.

Compensatory Upregulation of LPA2 and Activation of the PI3K-Akt Pathway Prevent LPA5-Dependent Loss of Intestinal Epithelial Cells in Intestinal Organoids.

Renewal of the intestinal epithelium is orchestrated by regenerative epithelial proliferation within crypts. Recent studies have shown that lysophosphatidic acid (LPA) can maintain intestinal epithelial renewal in vitro and conditional deletion of Lpar5 (Lpar5iKO) in mice ablates the intestinal epithelium and increases morbidity. In contrast, constitutive Lpar5 deletion (Lpar5cKO) does not cause a defect in intestinal crypt regeneration. In this study, we investigated whether another LPA receptor (LPAR) compensates for constitutive loss of LPA5 function to allow regeneration of intestinal epithelium. In Lpar5cKO intestinal epithelial cells (IECs), Lpar2 was upregulated and blocking LPA2 function reduced proliferation and increased apoptosis of Lpar5cKO IECs. Similar to Lpar5cKO mice, the absence of Lpar2 (Lpar2-/-) resulted in upregulation of Lpar5 in IECs, indicating that LPA2 and LPA5 reciprocally compensate for the loss of each other. Blocking LPA2 in Lpar5cKO enteroids reduced phosphorylation of Akt, indicating that LPA2 maintains the growth of Lpar5cKO enteroids through activation of the PI3K-Akt pathway. The present study provides evidence that loss of an LPAR can be compensated by another LPAR. This ability to compensate needs to be considered in studies aimed to define receptor functions or test the efficacy of a LPAR-targeting drug using genetically engineered animal models.

Survival of Stem Cells and Progenitors in the Intestine Is Regulated by LPA5-Dependent Signaling.

BACKGROUND & AIMS: Regeneration of the epithelium by stem cells in the intestine is supported by intrinsic and extrinsic factors. Lysophosphatidic acid (LPA), a bioactive lipid mediator, regulates many cellular functions, including cell proliferation, survival, and cytokine secretion. Here, we identify LPA5 receptor as a potent regulator of the survival of stem cells and transit-amplifying cells in the intestine. METHODS: We have used genetic mouse models of conditional deletion of Lpar5, Lpar5f/f;Rosa-CreERT (Lpar5KO), and intestinal epithelial cell-specific Lpar5f/f;AhCre (Lpar5IECKO) mice. Mice were treated with tamoxifen or ß-naphthoflavone to delete Lpar5 expression. Enteroids derived from these mice were used to determine the effect of Lpar5 loss on the apoptosis and proliferation of crypt epithelial cells. RESULTS: Conditional loss of Lpar5 induced ablation of the intestinal mucosa, which increased morbidity of Lpar5KO mice. Epithelial regeneration was compromised with increased apoptosis and decreased proliferation of crypt epithelial cells by Lpar5 loss. Interestingly, intestinal epithelial cell-specific Lpar5 loss did not cause similar phenotypic defects in vivo. Lpar5 loss reduced intestinal stem cell marker gene expression and reduced lineage tracing from Lgr5+ ISCs. Lpar5 loss induced CXCL10 expression which exerts cytotoxic effects on intestinal stem cells and progenitors in the intestinal crypts. By co-culturing Lpar5KO enteroids with wild-type or Lpar5KO splenocytes, we demonstrated that lymphocytes protect the intestinal crypts via a LPA5-dependent suppression of CXCL10. CONCLUSIONS: LPA5 is essential for the regeneration of intestinal epithelium. Our findings reveal a new finding that LPA5 regulates survival of stem cells and transit-amplifying cells in the intestine.

Discovery of novel aminopiperidinyl amide CXCR4 modulators through virtual screening and rational drug design.

The C-X-C chemokine receptor type 4 (CXCR4) is a potential therapeutic target for HIV infection, metastatic cancer, and inflammatory autoimmune diseases. In this study, we screened the ZINC chemical database for novel CXCR4 modulators through a series of in silico guided processes. After evaluating the screened compounds for their binding affinities to CXCR4 and inhibitory activities against the chemoattractant CXCL12, we identified a hit compound (ZINC 72372983) showing 100 nM affinity and 69% chemotaxis inhibition at the same concentration (100 nM). To increase the potency of our hit compound, we explored the protein-ligand interactions at an atomic level using molecular dynamics simulation which enabled us to design and synthesize a novel compound (Z7R) with nanomolar affinity (IC50 = 1.25 nM) and improved chemotaxis inhibition (78.5%). Z7R displays promising anti-inflammatory activity (50%) in a mouse edema model by blocking CXCR4-expressed leukocytes, being supported by our immunohistochemistry study.

MicroRNA-30c-regulated HDAC9 mediates chemoresistance of breast cancer.

PURPOSE: Although histone deacetylase (HDAC) inhibitors have been shown to effectively induce the inhibition of proliferation and migration in breast cancer, the mechanism of HDAC9's contribution to chemoresistance remains poorly understood. The aim of this study was to investigate the role of miR-30c-regulated HDAC9 in chemoresistance of breast cancer and to determine the potential of selective inhibition of HDAC9 in sensitizing resistant breast cancer cells to chemotherapy. METHODS: Expression levels of HDAC9 and miR-30c were measured in breast cancer cells and tissues using quantitative PCR analysis. The effect of selective inhibition of HDAC9 on sensitizing MDR cells to chemotherapy was assessed. MiR-30c/HDAC9 pathways' potential to mediate chemoresistance was analyzed. RESULTS: Our studies show that HDAC9 was significantly up-regulated in chemoresistant breast cancer cell lines compared to a chemosensitive cell line and was inversely correlated with the levels of miR-30c. MiR-30c mimics and HDAC9 inhibitors reversed the chemoresistance of multidrug-resistant breast cancer cells. CONCLUSIONS: These results indicate that the mechanism of chemoresistance reversal with selective HDAC inhibition was partially realized by regulating miR-30c via directly targeting HDAC9. Our findings suggest that the miR-30c/HDAC9 signaling axis could be a novel and potential therapeutic target in chemoresistant breast cancer.

A benzenesulfonamide derivative as a novel PET radioligand for CXCR4.

CXCR4 is involved in various diseases such as inflammation, tumor growth, and cancer metastasis through the interaction with its natural endogenous ligand, chemokine CXCL12. In an effort to develop imaging probes for CXCR4, we developed a novel small molecule CXCR4-targeted PET agent (compound 5) by combining our established benzenesulfonamide scaffold with a labeling component by virtue of click chemistry. 5 shows nanomolar affinity (IC50 = 6.9 nM) against a known CXCR4 antagonist (TN14003) and inhibits more than 65% chemotaxis at 10 nM in vitro assays. Radiofluorinated compound 5 ([18F]5) demonstrates a competitive cellular uptake against CXCL12 in a dose-dependent manner. Further, microPET images of [18F]5 exhibits preferential accumulation of radioactivity in the lesions of λ-carrageenan-induced paw edema, human head and neck cancer orthotopic xenograft, and metastatic lung cancer of each mouse model.

Amide-sulfamide modulators as effective anti-tumor metastatic agents targeting CXCR4/CXCL12 axis.

Breast cancer is the most frequently diagnosed malignancy and the second common cause of death in women worldwide. High mortality in breast cancer is frequently associated with metastatic progression rather than the primary tumor itself. It has been recently identified that the CXCR4/CXCL12 axis plays a pivotal role in breast cancer metastasis, especially in directing metastatic cancer cells to CXCL12-riched organs and tissues. Herein, taking the amide-sulfamide as the lead structure, the second-round structural modifications to the sulfamide structure were performed to obtain more active CXCR4 modulators against tumor metastasis. Both in vivo and in vitro experiments illustrated that compound IIIe possessed potent CXCR4 binding affinity, excellent anti-metastatic and anti-angiogenetic activity against breast cancer. More importantly, in a mouse breast cancer lung metastasis model, compound IIIe exerted a significant inhibitory effect on breast cancer metastasis. Taken together, all these positive results demonstrated that developing of CXCR4 modulators is a promising strategy to mediate breast cancer metastasis.

Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based derivatives as CXCR4 inhibitors.

The interaction between G-Protein coupled receptor CXCR4 and its natural ligand CXCL12 has been linked to inflammation experienced by patients with Irritable Bowel Disease (IBD). Blocking this interaction could potentially reduce inflammatory symptoms in IBD patients. In this work, several thiophene-based and furan-based compounds modeled after AMD3100 and WZ811-two known antagonists that interrupt the CXCR4-CXCL12 interaction-were synthesized and analyzed. Fifteen hit compounds were identified; these compounds exhibited effective concentrations (EC) lower than 1000 nM (AMD3100) and inhibited invasion of metastatic cells by at least 45%. Selected compounds (2d, 2j, 8a) that inhibited metastatic invasion at a higher rate than WZ811 (62%) were submitted for a carrageenan inflammation test, where both 8a and 2j reduced inflammation in the same range as WZ811 (40%) but did not reduce inflammation more than 40%. Select compounds were also modeled in silico to show key residue interactions. These preliminary results with furan-based and thiophene-based analogues contribute to the new class on heterocyclic aromatic-based CXCR4 antagonists.

Development of CXCR4 modulators based on the lead compound RB-108.

The CXCR4/CXCL12 axis plays prominent roles in tumor metastasis and inflammation. CXCR4 has been shown to be involved in a variety of inflammation-related diseases. Therefore, CXCR4 is a promising potential target to develop novel anti-inflammatory agents. Taking our previously discovered CXCR4 modulator RB-108 as the lead compound, a series of derivatives were synthesized structurally modifying and optimizing the amide and sulfamide side chains. The derivatives successfully maintained potent CXCR4 binding affinity. Furthermore, compounds IIb, IIc, IIIg, IIIj, and IIIm were all efficacious in inhibiting the invasion of CXCR4-positive cells, displaying a much more potent effect than the lead compound RB-108. Notably, compound IIIm significantly decreased carrageenan-induced swollen volume and paw thickness in a mouse paw edema model. More importantly, IIIm exhibited satisfying PK profiles with a half-life of 4.77 h in an SD rat model. In summary, we have developed compound IIIm as a new candidate for further investigation based on the lead compound RB-108.

Assessing Treatment Response of Glioblastoma to an HDAC Inhibitor Using Whole-Brain Spectroscopic MRI.

Histone deacetylases regulate a wide variety of cellular functions and have been implicated in redifferentiation of various tumors. Histone deacetylase inhibitors (HDACi) are potential pharmacologic agents to improve outcomes for patients with gliomas. We assessed the therapeutic efficacy of belinostat (PXD-101), an HDACi with blood-brain barrier permeability. Belinostat was first tested in an orthotopic rat glioma model to assess in vivo tumoricidal effect. Our results showed that belinostat was effective in reducing tumor volume in the orthotopic rat glioma model in a dose-dependent manner. We also tested the antidepression activity of belinostat in 2 animal models of depression and found it to be effective. Furthermore, we confirmed that myo-inositol levels improved by belinostat treatment in vitro. In a human pilot study, it was observed that belinostat in combination with chemoradiation may delay initial recurrence of disease. Excitingly, belinostat significantly improved depressive symptoms in patients with glioblastoma compared with control subjects. Finally, spectroscopic magnetic resonance imaging of 2 patient cases from this pilot study are presented to indicate how spectroscopic magnetic resonance imaging can be used to monitor metabolite response and assess treatment effect on whole brain. This study highlights the potential of belinostat to be a synergistic therapeutic agent in the treatment of gliomas.

Synthesis and evaluation of 2,5-diamino and 2,5-dianilinomethyl pyridine analogues as potential CXCR4 antagonists.

CXCR4 and its cognate ligand CXCL12 has been linked to various pathways such as cancer metastasis, inflammation, HIV-1 proliferation, and auto-immune diseases. Small molecules have shown potential as CXCR4 inhibitors and modulators, and therefore can mitigate diseases related to the CXCR4-CXCL12 pathway. We have designed and synthesized a series of 2,5-diamino and 2,5-dianilinomethyl pyridine derivatives as potential CXCR4 antagonists. Thirteen compounds have an effective concentration (EC) of 100 nM or less in a binding affinity assay and nine of these have at least 75% inhibition of invasion in Matrigel binding assay. Compounds 3l, 7f, 7j, and 7p show a minimal reduction in inflammation when carrageenan paw edema test is conducted. Overall, these compounds show potential as CXCR4 antagonist.

HDAC9 overexpression confers invasive and angiogenic potential to triple negative breast cancer cells via modulating microRNA-206.

Triple negative breast cancer (TNBC) is among the most aggressive breast cancer subtypes with poor prognosis. The purpose of this study is to better understand the molecular basis of TNBC as well as develop new therapeutic strategies. Our results demonstrate that HDAC9 is overexpressed in TNBC compared to non-TNBC cell lines and tissues and is inversely proportional with miR-206 expression levels. We show that HDAC9 selective inhibition blocked the invasion of TNBC cells in vitro and repressed the angiogenesis shown via in vivo Matrigel plug assays. Subsequent HDAC9 siRNA knockdown was then shown to restore miR-206 while also decreasing VEGF and MAPK3 levels. Furthermore, the inhibition of miR-206 neutralized the action of HDAC9 siRNA on decreasing VEGF and MAPK3 levels. This study highlights HDAC9 as a mediator of cell invasion and angiogenesis in TNBC cells through VEGF and MAPK3 by modulating miR-206 expression and suggests that selective inhibition of HDAC9 may be an efficient route for TNBC therapy.

Anti-inflammatory hybrids of secondary amines and amide-sulfamide derivatives.

The CXCR4/CXCL12 chemokine axis can chemotactically accumulate inflammatory cells to local tissues and regulate the release of inflammatory factors. Developing novel CXCR4 modulators may provide a desirable strategy to control the development of inflammation. A series of novel hybrids were designed by integrating the key pharmacophores of three CXCR4 modulators. The majority of compounds displayed potent CXCR4 binding affinity. Compound 7a exhibited 1000-fold greater affinity than AMD3100 and significantly inhibited invasion of CXCR4-positive tumor cells. Additionally, compound 7a blocked mice ear inflammation by 67% and suppressed the accumulation of inflammatory cells in an in vivo mouse ear edema evaluation. Western blot analyses revealed that 7a inhibited the CXCR4/CXCL12-mediated phosphorylation of Akt and p44 in a dose-dependent manner. Moreover, compound 7a had no observable cytotoxicity and displayed a favorable plasma stability in our preliminary pharmacokinetic study. These results confirmed that this is a feasible method to develop CXCR4 modulators for the regulation and reduction of inflammation.

HDAC inhibitor suppresses proliferation and invasion of breast cancer cells through regulation of miR-200c targeting CRKL.

Although histone deacetylase (HDAC) inhibitors have been shown to effectively induce the inhibition of proliferation and migration in breast cancer, the anticancer mechanism remains poorly understood. Our studies show that miR-200c was significantly downregulated in breast cancer cell lines compared to normal cell lines and inversely correlated with the levels of class IIa HDACs and CRKL. HDAC inhibitors and the ectopic expression of miR-200c as tumor suppressors inhibited the proliferation, invasion, and migration of breast cancer cells by downregulating CRKL. These results indicate that the anticancer mechanism of HDAC inhibitor was realized partially by regulating miR-200c via CRKL targeting. Our findings suggest that the HDAC-miR200c-CRKL signaling axis could be a novel diagnostic marker and potential therapeutic target in breast cancer.

Novel anti-inflammatory agents targeting CXCR4: Design, synthesis, biological evaluation and preliminary pharmacokinetic study.

CXCR4 plays a crucial role in the inflammatory disease process, providing an attractive means for drug targeting. A series of novel amide-sulfamide derivatives were designed, synthesized and comprehensively evaluated. This new scaffold exhibited much more potent CXCR4 inhibitory activity, with more than 70% of the compounds showed notably better binding affinity than the reference drug AMD3100 in the binding assay. Additionally, in the Matrigel invasion assay, most of our compounds significantly blocked the tumor cell invasion, demonstrating superior efficacy compared to AMD3100. Furthermore, compound IIj blocked mice ear inflammation by 75% and attenuated ear edema and damage substantially in an in vivo model of inflammation. Western blot analyses revealed that CXCR4 modulator IIj significantly blocked CXCR4/CXCL12-mediated phosphorylation of Akt. Moreover, compound IIj had no observable cytotoxicity and displayed a favourable plasma stability in our preliminary pharmacokinetic study. The preliminary structure-activity relationships were also summarized. In short, this novel amide-sulfamide scaffold exhibited potent CXCR4 inhibitory activity both in vitro and in vivo. These results also confirmed that developing modulators targeting CXCR4 provides an exciting avenue for treatment of inflammation.