Informed by Cancer Stem Cells of Solid Tumors: Advances in Treatments Targeting Tumor-Promoting Factors and Pathways.

Cancer stem cells (CSCs) represent a subpopulation within tumors that promote cancer progression, metastasis, and recurrence due to their self-renewal capacity and resistance to conventional therapies. CSC-specific markers and signaling pathways highly active in CSCs have emerged as a promising strategy for improving patient outcomes. This review provides a comprehensive overview of the therapeutic targets associated with CSCs of solid tumors across various cancer types, including key molecular markers aldehyde dehydrogenases, CD44, epithelial cellular adhesion molecule, and CD133 and signaling pathways such as Wnt/ß-catenin, Notch, and Sonic Hedgehog. We discuss a wide array of therapeutic modalities ranging from targeted antibodies, small molecule inhibitors, and near-infrared photoimmunotherapy to advanced genetic approaches like RNA interference, CRISPR/Cas9 technology, aptamers, antisense oligonucleotides, chimeric antigen receptor (CAR) T cells, CAR natural killer cells, bispecific T cell engagers, immunotoxins, drug-antibody conjugates, therapeutic peptides, and dendritic cell vaccines. This review spans developments from preclinical investigations to ongoing clinical trials, highlighting the innovative targeting strategies that have been informed by CSC-associated pathways and molecules to overcome therapeutic resistance. We aim to provide insights into the potential of these therapies to revolutionize cancer treatment, underscoring the critical need for a multi-faceted approach in the battle against cancer. This comprehensive analysis demonstrates how advances made in the CSC field have informed significant developments in novel targeted therapeutic approaches, with the ultimate goal of achieving more effective and durable responses in cancer patients.

Study on the therapeutic mechanism of HJ granules in a rat model of urinary tract infection caused by Escherichia coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Urinary tract infections (UTIs) are globally prevalent infectious diseases, predominantly caused by uropathogenic Escherichia coli (UPEC). The misuse of antibiotics has led to the emergence of several drug-resistant strains. Traditional Chinese Medicine (TCM) has its own advantages in the treatment of UTIs. HJ granules is a herbal formula used for the treatment of UTIs. However, its mechanism of action is not clear. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic efficacy and mechanism of action of HJ granules in a rat model of UTI caused by Escherichia coli (E coli) CFT073. MATERIALS AND METHODS: SD rats were selected to establish a rat UTI model by injecting UPEC strain CFT073 into the bladder using the transurethral placement method. HJ granules were administered to rats after modelling and the efficacy of HJ granule was investigated by measuring urinary decanalogue, inflammatory factors in bladder tissue and pathological changes in the bladder after 3d of administration. Expression of sonic hedgehog (SHH), NOD-like receptor thermoprotein domain 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and activation of cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by western blotting and immunofluorescence staining in rat bladder tissue. NLRP3, ASC and caspase-1, a cysteine-containing aspartic protein, were expressed and activated. RESULTS: The results showed that infection of rats with UPEC resulted in increased pH and erythrocytes in bladder irrigation fluid; increased expression of IL-1ß, IL-6 and SHH and decreased expression of IL-10 in bladder tissue; and significant upregulation of the expression of both SHH and NLRP3 inflammasom and significant activation of NLRP3 inflammasom. HJ granules significantly increased the concentration of IL-10 in the bladder, inhibited the expression of SHH and NLRP3 inflammasom in bladder tissue, and suppressed the activation of NLRP3 inflammasom, thereby reducing inflammatory lesions in bladder tissue. CONCLUSION: HJ granules may improve bladder injury and treat UTIs by inhibiting the expression and activation of NLRP3 inflammasom.

Targeted inhibition of the HNF1A/SHH axis by triptolide overcomes paclitaxel resistance in non-small cell lung cancer.

Paclitaxel resistance is associated with a poor prognosis in non-small cell lung cancer (NSCLC) patients, and currently, there is no promising drug for paclitaxel resistance. In this study, we investigated the molecular mechanisms underlying the chemoresistance in human NSCLC-derived cell lines. We constructed paclitaxel-resistant NSCLC cell lines (A549/PR and H460/PR) by long-term exposure to paclitaxel. We found that triptolide, a diterpenoid epoxide isolated from the Chinese medicinal herb Tripterygium wilfordii Hook F, effectively enhanced the sensitivity of paclitaxel-resistant cells to paclitaxel by reducing ABCB1 expression in vivo and in vitro. Through high-throughput sequencing, we identified the SHH-initiated Hedgehog signaling pathway playing an important role in this process. We demonstrated that triptolide directly bound to HNF1A, one of the transcription factors of SHH, and inhibited HNF1A/SHH expression, ensuing in attenuation of Hedgehog signaling. In NSCLC tumor tissue microarrays and cancer network databases, we found a positive correlation between HNF1A and SHH expression. Our results illuminate a novel molecular mechanism through which triptolide targets and inhibits HNF1A, thereby impeding the activation of the Hedgehog signaling pathway and reducing the expression of ABCB1. This study suggests the potential clinical application of triptolide and provides promising prospects in targeting the HNF1A/SHH pathway as a therapeutic strategy for NSCLC patients with paclitaxel resistance. Schematic diagram showing that triptolide overcomes paclitaxel resistance by mediating inhibition of the HNF1A/SHH/ABCB1 axis.

Shh-Gli2-Runx2 inhibits vascular calcification.

BACKGROUND: In patients with chronic kidney disease (CKD), vascular calcification (VC) is common and is associated with a higher risk of all-cause mortality. Shh, one ligand for Hedgehog (Hh) signaling, participates in osteogenesis and several cardiovascular diseases. However, it remains unclear whether Shh is implicated in the development of VC. METHODS: Inorganic phosphorus 2.6 mM was used to induce vascular smooth muscle cells (VSMCs) calcification. Mice were fed with adenine diet supplement with 1.2% phosphorus to induce VC. RESULTS: Shh was decreased in VSMCs exposed to inorganic phosphorus, calcified arteries in mice fed with an adenine diet, as well as radial arteries from patients with CKD presenting VC. Overexpression of Shh inhibited VSMCs ostosteoblastic differentiation and calcification, whereas its silencing accelerated these processes. Likewise, mice treated with smoothened agonist (SAG; Hh signaling agonist) showed alleviated VC, and mice treated with cyclopamine (CPN; Hh signaling antagonist) exhibited severe VC. Additionally, overexpression of Gli2 significantly reversed the pro-calcification effect of Shh silencing on VSMCs, suggesting that Shh inhibited VC via Gli2. Mechanistically, Gli2 interacted with Runx2 and promoted its ubiquitin proteasomal degradation, therefore protecting against VC. Of interest, the pro-degradation effect of Gli2 on Runx2 was independent of Smurf1 and Cullin4B. CONCLUSIONS: Our study provided deeper insight to the pathogenesis of VC, and Shh might be a novel potential target for VC treatment.

Dauricine attenuates Oct4/sonic hedgehog co-activated stemness and induces reactive oxygen species-mediated mitochondrial apoptosis via AKT/ß-catenin signaling in human neuroblastoma and glioblastoma stem-like cells.

Neuroblastoma and glioblastoma are primary malignant tumors of the nervous system, with frequent relapse and limited clinical therapeutic drugs. The failure of their treatment is due to the tumor cells exhibiting cancer stem-like cells (CSLCs) properties. Octamer binding transcription factor 4 (Oct4) is involved in mediating CSLCs, our previous work found that Oct4-driven reprogramming of astrocytes into induced neural stem cells was potentiated with continuous sonic hedgehog (Shh) stimulation. In this study, we aimed to study the importance of Oct4 and Shh combination in the stemness properties induction of neuroblastoma and glioblastoma cells, and evaluate the anti-stemness effect of dauricine (DAU), a natural product of bis-benzylisoquinoline alkaloid. The effect of Oct4 and Shh co-activation on cancer stemness was evaluated by tumor spheres formation model and flow cytometry analysis. Then the effects of DAU on SH-SY5Y and T98-G cells were assessed by the MTT, colony formation, and tumor spheres formation model. DAU acts on Oct4 were verified using the Western blotting, MTT, and so on. Mechanistic studies were explored by siRNA transfection assay, Western blotting, and flow cytometry analysis. We identified that Shh effectively improved Oct4-mediated generation of stemness in SH-SY5Y and T98-G cells, and Oct4 and Shh co-activation promoted cell growth, the resistance of apoptosis. In addition, DAU, a natural product, was found to be able to attenuate Oct4/Shh co-activated stemness and induce cell cycle arrest and apoptosis via blocking AKT/ß-catenin signaling in neuroblastoma and glioblastoma, which contributed to the neuroblastoma and glioblastoma cells growth inhibition by DAU. In summary, our results indicated that the treatment of DAU may be served as a potential therapeutic method in neuroblastoma and glioblastoma.

Molecular mechanism of angiogenesis for cerebral infarction rats by acupuncture intervention based on sonic hedgehog signaling pathway.

BACKGROUND: To study the factors of the Sonic Hedgehog (Shh) signaling pathway after permanent cerebral ischemic and the effects by acupuncture. METHODS: Male Wistar rats were divided into Electro-acupuncture (EA) group, Model Control (MC) group, and blank control (Control) group. EA and MC were divided into 9 phases, namely 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 3 d, 7 d, and 12 d after the operation. The neurological deficits and permanent cerebral ischemic volume were observed. The immunofluorescence method was used to examine the angiogenesis. (Polymerase Chain Reaction) PCR and (Immunohistochemistry) IHC were used to test the changes in Shh, Ptch, Smo, and Gli2 mRNA and proteins. RESULTS: The neurological severity scores (NSS) of the Control was 0, the score of the EA group was less than that of the MC. The cerebral permanent ischemic volume of the Control was 0 %, and the EA group's was smaller than that of the MC. The expression of copositive cells in the EA group was higher than the MC's from 12 h to 12 d, and the EA group had more peripheral blood vessels. The rat brain expressions of Shh, Ptch, Smo and Gli2 mRNA and proteins in the MC was higher than that of the Control, the rat brain expression of the EA group was higher than that of the MC. CONCLUSIONS: EA can upregulate the expression of the Shh signaling pathway factors, thereby promoting angiogenesis.

Surgical management of giant skin tumor - A case report.

INTRODUCTION: Malignant non-melanoma skin cancers (NMSC) are of two main types: basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). In rare occasions, some of the malignant skin lesions show histopathological characteristics of both BCC and SCC and are known as basosquamous carcinomas (BSC). In some cases of large tumors, extensive reconstructive surgery might be needed to correct the skin defect after the primary excision. PRESENTATION OF CASE: We report a case of a 76-year-old Bulgarian male patient who presented with a neglected giant cutaneous tumor with more than a 15-year history of a growing mass in the right deltoid area. On physical exam an enormous exophytic ulcerated and crusted skin lesion measuring around 11×11cm was found. Wide local excision of the lesion with 10-mm resection margins and partial resection of the underlying deltoid muscle were performed due to signs of infiltration. A full-thickness total skin graft from the left inguinal area was harvested to cover the skin defect. Final histopathological examination showed metatypical carcinoma with mixed characteristics of SCC and BCC - BSC, with infiltration of the fatty tissue, deltoid muscle and clear margins of resection, staged as T4R0. Two and a half years after surgery there are no signs of upper arm motor dysfunction and no evidence of local recurrence and distant metastasis on a follow-up PET/CT. DISCUSSION: Following current National Comprehensive Cancer Network's guidelines for primary treatment of BCC, surgical candidates should undergo standard excision with wider surgical margins, postoperative margin assessment and second intention healing, linear repair, or skin graft. Therapeutic strategy for non-operable cases includes administration of radiotherapy or system therapy in the face of Hedgehog pathway inhibitors and programmed cell death protein 1 inhibitor. They can provide an alternative solution to unresectable or difficult-to-treat locally advanced cases of BSC. CONCLUSION: Similarly to BCC and SCC, the first-line treatment option for BCS is surgical excision, but surgical margins should be wider than those for low-risk BCC due to the infiltrative growth pattern of this tumor. Favorable esthetic outcome requires precise planning of the reconstructive technique.

Genome-wide DNA methylation analysis of Astragalus and Danshen on the intervention of myofibroblast activation in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF), a chronic progressive interstitial lung disease of unknown etiology, is characterized by continuous damage to alveolar epithelial cells, abnormal repair of alveolar tissue, and alveolar wall scar formation. Currently, the recommended treatment for IPF in Western medicine is relatively limited. In contrast, traditional Chinese medicine and compound prescriptions show advantages in the diagnosis and treatment of IPF, which can be attributed to their multi-channel and multi-target characteristics and minimal side-effects. The purpose of this study was to further corroborate the effectiveness and significance of the traditional Chinese medications Astragalus and Danshen in IPF treatment. METHODS: We performed whole-genome methylation analysis on nine rat lung tissue samples to determine the epigenetic variation between IPF and non-fibrotic lungs using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and quantitative reverse transcription polymerase chain reactions. RESULTS: We identified differentially methylated regions and 105 associated key functional genes in samples related to IPF and Chinese medicine treatment. Based on the methylation levels and gene expression profiles between the Chinese medicine intervention and pulmonary fibrosis model groups, we speculated that Astragalus and Salvia miltiorrhiza (traditionally known as Danshen) act on the Isl1, forkhead box O3, and Sonic hedgehog genes via regulation at transcriptional and epigenetic levels during IPF. CONCLUSIONS: These findings provide novel insights into the epigenetic regulation of IPF, indicate the effectiveness of Astragalus and Danshen in treating IPF, and suggest several promising therapeutic targets for preventing and treating IPF.

["Huayu Tongluo" moxibustion improves learning-memory ability and promotes myelin regeneration by regulating Sonic Hedgehog signaling pathway in vascular dementia rats].

OBJECTIVE: To investigate the effect of "Huayu Tongluo" (resolving blood stagnation to dredge meridian-collaterals) moxibustion on remyelination and Sonic Hedgehog (Shh) signaling pathway in the corpus callosum of vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. METHODS: Male Wistar rats were randomized into sham-operation, model, medication and moxibustion groups, with 12 rats in each group.The VD model was established by bilateral common carotid artery occlusion. Moxibustion was applied to "Shenting"(GV24), "Baihui"(GV20) and "Dazhui"(GV14) for 20 min once a day, 7 d as a treatment course, for 3 courses, with one day's rest between every two courses. Rats of the medication group were treated by gavage of 10 mg/kg of chloromastine solution once a day, and the course of treatment was the same as that of the moxibustion group. The rat's learning-memory ability was assessed by Morris water maze test (escape latency). The neurological deficits were evaluated by using Longa's scale.The mRNA and protein expressions of Shh and Gli1 in the corpus callosum were measured by quantitative real-time fluorescence PCR and Western blot, separately. The ultrastructure of the myelin sheath and myelinated axons was observed under transmission electron microscopy (TCM). RESULTS: Compared with the sham-operation group, the neurologic score and escape latency were significantly increased and prolonged (P<0.01), and the mRNA and protein expression levels of Shh and Gli1 and the number of myelinated axons were obviously decreased in the model group (P<0.01). In comparison with the model group, the escape latency was apparently shortened (P<0.05), while the mRNA and protein expression levels of Shh and Gli1 as well as the number of myelinated axons were strikingly increased in both moxibustion and medication groups (P<0.01). Results of TCM showed that in the model group, the arrangement of myelin coil structures was sparse and fuzzy, and some structures were bulged and disbanded. The oligodendrocytes were irregular, and the number of myelin sheath was rare. These situations were relatively milder in both moxibustion and medication groups. CONCLUSION: "Huayu Tongluo" moxibustion can promote the differentiation and maturation of oligodendrocyte precursor cells after cerebral ischemia by regulating the expressions of Shh and Gli1 in Shh signaling pathway, thus promoting the regeneration of cerebral white matter myelin sheaths in VD rats, which may contribute to improving learning-memory ability.

Lobetyolin Alleviates Ferroptosis of Skeletal Muscle in 5/6 Nephrectomized Mice via Activation of Hedgehog-GLI1 Signaling.

BACKGROUND: Muscle wasting increases morbidity and mortality and is related to chronic kidney disease (CKD) and dialysis. It is still unclear whether ferroptosis occurs during this progression and whether it is a potential intervention target for the treatment of CKD-related muscle injury. PURPOSE: The objective is to identify potential compounds for treating ferroptosis and muscle wasting and explore the potential mechanisms in vivo/in vitro. METHODS: Initially, we explored whether ferroptosis is present in the skeletal muscle of 5/6 nephrectomized (NPM) mice via RNA-Seq analysis, TUNEL staining, Oil red O staining, MDA/GSH/GSSG level detection and real-time quantitative PCR (qPCR). Subsequently, utilizing our established molecular phenotyping strategy, we screened potential traditional Chinese herb-derived compounds for alleviation of muscle wasting and ferroptosis. HE staining, Oil red O staining, TUNEL staining, immunofluorescence staining, MDA/GSH/GSSG level detection, Fe level detection, western blotting and qPCR were applied to assess the effects of the identified compound on muscle wasting and ferroptosis and explore the potential mechanism. Furthermore, RNA-Seq analysis, ChIP-Seq analysis and further experiments in vitro were performed to determine the role of Hedgehog signaling in the effect of Lobetyolin (LBT) on ferroptosis. RESULTS: In NPM mice, skeletal muscle dysfunction, lipogenesis, reduced GSH/GSSG ratio, decreased GSH content, increased MDA production and and higher levels of ferroptosis markers were observed. LBT treatment (30 mg/kg or 50 mg/kg) significantly alleviates skeletal muscle injury by inhibiting ferroptosis. Additionally, in an in vitro investigation, C2C12 cells exposed to Indolyl sulfate (IS) induced ferroptosis and LBT treatment (20 µM and 50 µM) protected C2C12 from such injury, consistent with the results from the in vivo analysis. Furthermore, it was found LBT increased the levels of protein involving Hedgehog signaling pathway (SMO and GLI1), and rescue analysis revealed that this pathway played a crucial role in the regulation of ferroptosis. Further experiments demonstrated that LBT upregulated a series of suppressors of ferroptosis by activating Gli1 transcription. CONCLUSION: LBT alleviates CKD-induced muscle injury by inhibiting ferroptosis through activation of the Hedgehog signaling pathway.

Ginsenoside Rb1 for overcoming cisplatin-insensitivity of A549/DDP cells in vitro and vivo through the dual-inhibition on two efflux pumps of ABCB1 and PTCH1.

BACKGROUND: The multi-drug resistance is an inherent weakness in the chemotherapeutics of non-small cell lung cancer occurring frequently all over the world. Clinically, ginseng and Chinese medicinal prescriptions including ginseng usually used as anti-tumor adjuncts due to its characteristic of qi-invigorating, which could improve the curative effect of chemotherapy drugs and reduce their toxic side effects. Triterpenoid saponins are the crucial active ingredients in Panax ginseng, and Ginsenoside Rb1 is of the highest quantities. However, the research on the tumor drug-resistance reversal effect and mechanism of ginsenoside Rb1 is still not clear. PURPOSE: This study aimed to systematically estimate the reversal activity of Ginsenoside Rb1 on cisplatin-insensitivity of A549/DDP cells and to reveal its prospective molecular mechanism. METHODS: MTT assay were conducted to evaluate the reversal activity on cisplatin-insensitivity of A549/DDP cells of Ginsenoside Rb1in vitro, and the behavior was also studied by establishing a subcutaneous transplanted tumor model of A549/DDP in BALB/c-nu mice. In addition, P-gp ATPase activity assay, cisplatin accumulation assay, Annexin V-FITC apoptosis assay, real-time qPCR analysis and western blotting analysis were used to clarify the potential mechanism. RESULTS: Ginsenoside Rb1 could effectively reverse the cisplatin-resistance of A549/DDP in vitro and vivo. And after the co-treatment of Ginsenoside Rb1 plus cisplatin, the accumulation of cisplatin increased in A549/DDP cells, which was accompanied with the down-regulation of the mRNA and protein expression levels of ABCB1, SHH, PTCH1 and GLI2. Besides, the apoptosis-inducing ability of cisplatin improved by the relative regulation on the protein expression level of Bax and Bcl-2. Far more importantly, the changes of CYP3A4 mRNA and protein levels were not significant. CONCLUSION: Ginsenoside Rb1 could increase the concentration of intracellular cisplatin and improve the insensitivity for cisplatin on A549/DDP cells. Even better, there was perhaps no unpredictable CYP3A4-mediated pharmacokinetic interactions after the combination of Ginsenoside Rb1 plus cisplatin. Ginsenoside Rb1 was a probable reversal agent for the cisplatin-insensitivity of A549/DDP cells, with a bifunction of inhibiting the efflux of two drug pumps (P-gp and PTCH1) by targeting ABCB1 and Hedgehog (Hh) pathway. In general, this research laid the groundwork for the development of a new reversal agent for the cisplatin-insensitivity of NSCLC.

Berberine is a suppressor of Hedgehog signaling cascade in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a malignant affliction that burdens people globally. Overactivated Hedgehog signal is highly implicated in CRC pathogenesis. Phytochemical berberine exerts strong potency on CRC, with molecular mechanism elusive. PURPOSE: We sought to study berberine's anti-CRC action and explore its underlying mechanism based on Hedgehog signaling cascade. METHODS: In CRC HCT116 cells and SW480 cells treated with berberine, the proliferation, migration, invasion, clonogenesis, apoptosis and cell cycle were measured, with determination of Hedgehog signaling pathway activity. Following establishment of mouse model of HCT116 xenograft tumor, the efficacies of berberine on carcinogenesis, pathological manifestation and malignant phenotypes of CRC were examined, with analysis of Hedgehog signaling axis in HCT116 xenograft tumor tissues. Additionally, toxicological study of berberine was conducted on zebrafish. RESULTS: Berberine was discovered to suppress the proliferation, migration, invasion and clonogenesis of HCT116 cells and SW480 cells. Furthermore, berberine caused cell apoptosis and blockaded cell cycle at phase G0/G1 in CRC cells, with dampened Hedgehog signaling cascade. In HCT116 xenograft tumor of nude mice, berberine inhibited tumor growth, alleviated pathological score, and promoted apoptosis and cell cycle arrest in tumor tissues, through constraining Hedgehog signaling. The toxicological study of berberine on zebrafish indicated that berberine incurred damage to the liver and heart of zebrafish at high dosage and prolonged administration. CONCLUSIONS: Taken together, berberine may inhibit the malignant phenotypes of CRC through diminishing Hedgehog signaling cascade. However, the potential adverse reactions should be taken into account upon abuse of berberine.

Weierning, a Chinese patent medicine, improves chronic atrophic gastritis with intestinal metaplasia.

ETHNOPHARMACOLOGICAL RELEVANCE: Weierning tablet (WEN) is a traditional Chinese patent medicine widely used in clinical for chronic atrophic gastritis (CAG) therapy for years. However, the underlying mechanisms of WEN on anti-CAG are still unveiled. AIM OF THE STUDY: The present study aimed to elucidate the characteristic function of WEN on anti-CAG and to illuminate its potential mechanism. METHODS: The CAG model was established by gavage rats with a modeling solution (consisting of 2% sodium salicylate and 30% alcohol) with irregular diets and free access to 0.1% ammonia solution for two months on end. An enzyme-linked immunosorbent assay was used to measure the serum levels of gastrin, pepsinogen, and inflammatory cytokines. qRT-PCR was applied to measure mRNA expressions of IL-6, IL-18, IL-10, TNF-α, and γ-IFN in gastric tissue. Pathological changes and the ultrastructure of gastric mucosa were examined by hematoxylin and eosin staining and transmission electron microscopy, respectively. AB-PAS staining was applied to observe the intestinal metaplasia of gastric mucosa. Immunohistochemistry and Western blot were used to measure the expression levels of mitochondria apoptosis-related proteins and Hedgehog pathway-related proteins in gastric tissues. Expressions of Cdx2 and Muc2 protein were determined by immunofluorescent staining. RESULTS: WEN could dose-dependently lower the serum level of IL-1ß and the mRNA expressions of IL-6, IL-8, IL-10, TNF-α, and γ-IFN in gastric tissue. Also, WEN significantly alleviated the collagen deposition in gastric submucosa, regulated the expressions of Bax, Cleaved-caspase9, Bcl2, and Cytochrome c to reduce the apoptosis of gastric mucosa epithelial cells, and maintained the integrity of the gastric mucosal barrier. Moreover, WEN could reduce protein expressions of Cdx2, Muc2, Shh, Gli1, and Smo, and reverse intestinal metaplasia of gastric mucosa to block the progress of CAG. CONCLUSION: This study demonstrated a positive effect of WEN on improving CAG and reverse intestinal metaplasia. These functions were related to the suppression of gastric mucosal cells' apoptosis and the inhibition of Hedgehog pathways' activation.

B13, a well-tolerated inhibitor of hedgehog pathway, exhibited potent anti-tumor effects against colorectal carcinoma in vitro and in vivo.

Abnormal activation of Hedgehog (Hh) signaling pathway mediates the genesis and progression of various tumors [1]. Currently, three drugs targeting the Hh signaling component Smoothened (Smo) have been marketed for the clinical treatment of basal cell tumors or acute myeloid leukemia. However, drug resistance is a common problem in those drugs, so the study of Smo inhibitors that can overcome drug resistance has important guiding significance for clinical adjuvant drugs. MTT assay, clone formation assay and EdU assay were used to detect the proliferation inhibitory activity of the drugs on tumor cells. The effect of B13 on cell cycle and apoptosis were detected by flow cytometry. An acute toxicity test was used to detect the toxicity of B13 in vivo, and xenograft tumor model was used to detect the efficacy of B13 in vivo. The binding of B13 to Smo was studied by BODIPY-cyclopamine competitive binding assay and molecular docking. The effect of B13 on the expression and localization of downstream target gene Gli1/2 of Smo was investigated by Western Blot and immunofluorescence assay. SmoD473H mutant cell line was constructed to study the effect of B13 against drug resistance. (1) B13 had the strongest inhibitory activity against colorectal cancer cells. (2) B13 can effectively inhibit the clone formation and EdU positive rate of colon cancer cells. (3) B13 can block the cell cycle in the G2/M phase and cell apoptosis. (4) B13 has low toxicity in vivo, and its efficacy in vivo is better than that of the Vismodegib. (5) Molecular docking and BODIPY-cyclopamine experiments showed that B13 could bind to Smo protein. (6) B13 can inhibit the protein expression of Gli1, the downstream of Smo, and inhibit its entry into the nucleus. (7) B13 could inhibit the expression of Gli1 in the HEK293 cells with SmoD473H, and the molecular docking results showed that B13 could bind SmoD473H protein. B13 with the best anti-tumor activity was screened out by MTT assay. In vitro, pharmacodynamics experiments showed that B13 could effectively inhibit the proliferation and metastasis of colorectal cancer cells, induce cell cycle arrest, and induce cell apoptosis. In vivo pharmacodynamics experiments showed that B13 was superior to Vismodegib in antitumor activity and had low toxicity in vivo. Mechanism studies have shown that B13 can bind Smo protein, inhibit the expression of downstream Gli1 and its entry into the nucleus. Notably, B13 overcomes resistance caused by SmoD473H mutations.

Manipulation of Signal Gradient and Transcription Factors Recapitulates: Multiple Hypothalamic Identities.

During development, the hypothalamus emerges from the ventral diencephalon and is regionalized into several distinct functional domains. Each domain is characterized by a different combination of transcription factors, including Nkx2.1, Nkx2.2, Pax6, and Rx, which are expressed in the presumptive hypothalamus and its surrounding regions, and play critical roles in defining each area. Here, we recapitulated the molecular networks formed by the gradient of Sonic Hedgehog (Shh) and the aforementioned transcription factors. Using combinatorial experimental systems of directed neural differentiation of mouse embryonic stem (ES) cells, as well as a reporter mouse line and gene overexpression in chick embryos, we deciphered the regulation of transcription factors by different Shh signal intensities. We then used CRISPR/Cas9 mutagenesis to demonstrate the mutual repression between Nkx2.1 and Nkx2.2 in a cell-autonomous manner; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region. Our findings suggest that Shh signaling and its downstream transcription network are required for hypothalamic regionalization and establishment.

Total flavonoid extract from Dracocephalum moldavica L. improves pulmonary fibrosis by reducing inflammation and inhibiting the hedgehog signaling pathway.

Dracocephalum Moldavica L. is a traditional herb for improving pharynx and relieving cough. However, the effect on pulmonary fibrosis is not clear. In this study, we explored the impact and molecular mechanism of total flavonoid extract from Dracocephalum moldavica L. (TFDM) on bleomycin-induced pulmonary fibrosis mouse model. Lung function testing, lung inflammation and fibrosis, and the related factors were detected by the lung function analysis system, HE and Masson staining, ELISA, respectively. The expression of proteins was studied through Western Blot, immunohistochemistry, and immunofluorescence while the expression of genes was analyzed by RT-PCR. The results showed that TFDM significantly improved lung function in mice, reduced the content of inflammatory factors, thereby reducing the inflammation. It was found that expression of collagen type I, fibronectin, and α-smooth muscle actin was significantly decreased by TFDM. The results further showed that TFDM interferes with hedgehog signaling pathway by decreasing the expression of Shh, Ptch1, and SMO proteins and thereby inhibiting the generation of downstream target gene Gli1 and thus improving pulmonary fibrosis. Conclusively, these findings suggest that TFDM improve pulmonary fibrosis by reducing inflammation and inhibition of the hedgehog signaling pathway.

Baicalin attenuates dexamethasone-induced apoptosis of bone marrow mesenchymal stem cells by activating the hedgehog signaling pathway.

BACKGROUND: Perturbations in bone marrow mesenchymal stem cell (BMSC) differentiation play an important role in steroid-induced osteonecrosis of the femoral head (SONFH). At present, studies on SONFH concentrate upon the balance within BMSC osteogenic and adipogenic differentiation. However, BMSC apoptosis as well as proliferation are important prerequisites in their differentiation. The hedgehog (HH) signaling pathway regulates bone cell apoptosis. Baicalin (BA), a well-known compound in traditional Chinese medicine, can affect the proliferation and apoptosis of numerous cell types via HH signaling. However, the potential role and mechanisms of BA on BMSCs are unclear. Thus, we aimed to explore the role of BA in dexamethasone (Dex)-induced BMSC apoptosis in this study. METHODS: Primary BMSCs were treated with 10 -6 mol/L Dex alone or with 5.0 µmol/L, 10.0 µmol/L, or 50.0 µmol/L BA for 24 hours followed by co-treatment with 5.0 µmol/L, 10.0 µmol/L, or 50.0 µmol/L BA and 10 -6 mol/L Dex. Cell viability was assayed through the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining followed by flow cytometry. The imaging and counting, respectively, of Hochest 33342/PI-stained cells were used to assess the morphological characteristics and proportion of apoptotic cells. To quantify the apoptosis-related proteins (e.g., apoptosis regulator BAX [Bax], B-cell lymphoma 2 [Bcl-2], caspase-3, and cleaved caspase-3) and HH signaling pathway proteins, western blotting was used. A HH-signaling pathway inhibitor was used to demonstrate that BA exerts its anti-apoptotic effects via the HH signaling pathway. RESULTS: The results of CCK-8, Hoechst 33342/PI-staining, and flow cytometry showed that BA did not significantly promote cell proliferation (CCK-8: 0 µmol/L, 100%; 2.5 µmol/L, 98.58%; 5.0 µmol/L, 95.18%; 10.0 µmol/L, 98.11%; 50.0 µmol/L, 99.38%, F   =  2.33, P   >  0.05), but it did attenuate the effect of Dex on apoptosis (Hoechst 33342/PI-staining: Dex+ 50.0 µmol/L BA, 12.27% vs. Dex, 39.27%, t  = 20.62; flow cytometry: Dex + 50.0 µmol/L BA, 12.68% vs. Dex, 37.43%, t  = 11.56; Both P  < 0.05). The results of western blotting analysis showed that BA reversed Dex-induced apoptosis by activating the HH signaling pathway, which down-regulated the expression of Bax, cleaved-caspase 3, and suppressor of fused (SUFU) while up-regulating Bcl-2, sonic hedgehog (SHH), and zinc finger protein GLI-1 (GLI-1) expression (Bax/Bcl-2: Dex+ 50.0 µmol/L BA, 1.09 vs. Dex, 2.76, t  = 35.12; cleaved caspase-3/caspase-3: Dex + 50.0 µmol/L BA, 0.38 vs . Dex, 0.73, t  = 10.62; SHH: Dex + 50.0 µmol/L BA, 0.50 vs . Dex, 0.12, t  = 34.01; SUFU: Dex+ 50.0 µmol/L BA, 0.75 vs . Dex, 1.19, t  = 10.78; GLI-1: Dex+ 50.0 µmol/L BA, 0.40 vs . Dex, 0.11, t  = 30.68. All P  < 0.05). CONCLUSIONS: BA antagonizes Dex-induced apoptosis of human BMSCs by activating the HH signaling pathway. It is a potential candidate for preventing SONFH.

A drug screening to identify novel combinatorial strategies for boosting cancer immunotherapy efficacy.

BACKGROUND: Chimeric antigen receptor (CAR) T cells and immune checkpoint blockades (ICBs) have made remarkable breakthroughs in cancer treatment, but the efficacy is still limited for solid tumors due to tumor antigen heterogeneity and the tumor immune microenvironment. The restrained treatment efficacy prompted us to seek new potential therapeutic methods. METHODS: In this study, we conducted a small molecule compound library screen in a human BC cell line to identify whether certain drugs contribute to CAR T cell killing. Signaling pathways of tumor cells and T cells affected by the screened drugs were predicted via RNA sequencing. Among them, the antitumor activities of JK184 in combination with CAR T cells or ICBs were evaluated in vitro and in vivo. RESULTS: We selected three small molecule drugs from a compound library, among which JK184 directly induces tumor cell apoptosis by inhibiting the Hedgehog signaling pathway, modulates B7-H3 CAR T cells to an effector memory phenotype, and promotes B7-H3 CAR T cells cytokine secretion in vitro. In addition, our data suggested that JK184 exerts antitumor activities and strongly synergizes with B7-H3 CAR T cells or ICBs in vivo. Mechanistically, JK184 enhances B7-H3 CAR T cells infiltrating in xenograft mouse models. Moreover, JK184 combined with ICB markedly reshaped the tumor immune microenvironment by increasing effector T cells infiltration and inflammation cytokine secretion, inhibiting the recruitment of MDSCs and the transition of M2-type macrophages in an immunocompetent mouse model. CONCLUSION: These data show that JK184 may be a potential adjutant in combination with CAR T cells or ICB therapy.

Enhanced yield of cholinergic neurons from induced pluripotent stem cells (iPSC): A two-step induction protocol.

BACKGROUND: Cholinergic neurons, a type of neurons found in central nervous system, play a vital role in muscle movement and activities. Cholinergic neurons degeneration is the main pathological symptom of neurodegenerative diseases. Among a variety of stem cells, iPSCs have emerged as a promising candidate for transplantation to improve the repair of neuronal lesion sites. However, the establishment of an appropriate induction method to yield large numbers of cholinergic neurons has yet to be determined. Here, we studied the differentiation potential of iPSCs to generate cholinergic neurons by developing a new optimized differentiation protocol. METHODS: The iPSCs were harvested on 6-well matrigel-coated plate and incubated with serum­free DMEM/F12 with 2 % B27 supplement, 20 ng/ml the basic fibroblast growth factor and 20 ng/ml epidermal growth factor for 48 hours. Then, the pre-induced cells were treated in neuronal induction medium supplemented with all-trans retinoic acid, sonic hedgehog, 100 ng/ml glial-derived neurotrophic factor and 200 ng/ml brain-derived neurotrophic factor for 7 days. Cell viability during induction stages was tested by MTT assay. Differentiated cells were evaluated with crystal violet staining, immunocytochemistry and real­time PCR. RESULTS: Our results showed that the survival rate of iPSCs leveled out and was similar to that in the control group following the differentiation process. Immunochemistry results revealed that the expression of ChAT was observed in cells in both pre­induction and induction stages with a significantly higher expression level at the induction stage as compared to the pre-induction stage. However, none of these markers was expressed in the iPSCs. Cresyl violet staining confirmed the neuronal phenotype of differentiated cells. The induction group significantly expressed the higher levels of Islet1, Olig2 and HB9, whereas pluripotency markers including those of Oct4 and Nestin plunged. CONCLUSION: Our investigation represents a highly efficient protocol for iPSCs differentiation toward cholinergic neurons which could be used for further preclinical transplantation studies (Tab. 1, Fig. 5, Ref. 35). Text in PDF www.elis.sk Keywords: induced pluripotent stem cells, cholinergic neurons, neurotrophic factors, induction protocol, preclinical transplantation.

SAG-dihydrochloride enhanced the expression of germ cell markers in the human bone marrow- mesenchymal stem cells (BM-MSCs) through the activation of GLI-independent hedgehog signaling pathway.

Different studies indicated that the enhancing the expression of germ cell markers improved the efficiency of stem cells in the generation of germ line cells. The aim of the present study was to investigate the effect of SAG-dihydrochloride on the expression of germ cell markers in the human bone marrow-mesenchymal stem cells (BM-MSCs). For this purpose, the human BM-MSCs were cultured in the medium containing different concentrations of SAG-dihydrochloride (10, 20 and 30 µM). After RNA extraction and cDNA synthesis, the expression level of PTCH1, GLI1, PLZF, DDX4 and STRA8 genes were determined by using SYBR Green Real time PCR. The analysis of the results obtained from PTCH1 and GLI1 expression indicated that SAG-dihydrochloride had the ability to enhance the expression of germ cell markers in a Gli-independent manner. Furthermore, the significant increased expression of STRA8 was observed in the BM-MSCs treated by 10 µM SAG-dihydrochloride for 4 and 6 days (p < 0.05). There was also the up-regulation of DDX4 in the BM-MSCs following treatment with 20 µM SAG-dihydrochloride for 4 and 6 days. The obtained results suggested that treatment with SAG-dihydrochloride increased the expression of germ cell markers in the human BM-MSCs through the activation of non-canonical sonic hedgehog signaling pathway.