Bone morphogenetic protein 9 (BMP9) has been demonstrated to robustly induce osteogenic differentiation of mesenchymal stem cells (MSCs), offering substantial potential for advancements in bone tissue engineering. The purinergic receptor P2X7 has emerged as a crucial modulator of bone formation and bone metabolism. However, the precise role of P2X7 in BMP9-induced osteogenic differentiation of MSCs and the associated molecular mechanisms remain partially understood. This study aims to ascertain the exact function of P2X7 in BMP9-induced osteogenic differentiation of MSCs, and to unravel the relevant molecular mechanism. Transcriptome sequencing, quantitative real-time polymerase chain reaction, Western blot, and chromatin immunoprecipitation assays were initially performed to validate the up-regulation of P2X7 by BMP9. Subsequently, the influence of P2X7 on BMP9-induced osteogenic differentiation of MSCs was assessed through ALP determination, calcium deposition analysis, Western blotting, ectopic bone formation model, and skull defect model. Finally, the mechanism through which P2X7 modulated BMP9-induced osteogenic differentiation of MSCs was investigated using intracellular Ca2+ imaging, Western blot, molecular docking, immunoprecipitation and immunofluorescence staining. BMP9 was confirmed to upregulate P2X7 expression via Smad signaling. Activation of P2X7 significantly potentiated BMP9-induced osteogenic differentiation of MSCs and enhanced BMP9-promoted ectopic bone formation, then enhanced the repair of bone defect by BMP9. Conversely, inhibition of P2X7 elicited a contrary effect. Mechanistically, P2X7 activation promoted calcium influx, subsequently leading to the activation of CaMKII by phosphorylation. The activated CaMKII then interacted with GSK-3β, facilitating the inhibitory phosphorylation of GSK-3β at Serine 9 (Ser 9) residue. This may stabilize β-catenin and increase its nuclear translocation, thus finally mediating the osteogenic differentiation of MSCs induced by BMP9. This study clarifies that P2X7 may mediate BMP9-induced osteogenic differentiation of MSCs through the CaMKII/GSK-3β/β-catenin axis, providing novel insights into the molecular mechanism of BMP9-induced osteogenesis and a potential target for bone defect treatment.

Bladder cancer (BC) is an aggressive and treatment-resistant malignancy with a high recurrence rate. Current first-line therapies only provide limited relief due to chemoresistance and toxicity. Natural products are emerging as promising chemotherapeutic agents due to their multi-target mechanisms and excellent safety profile. In this study, we evaluated the anti-BC effects of echinocystic acid (EA), a pentacyclic triterpenoid, on BC cells and investigated the underlying mechanism. We found that EA effectively inhibited proliferation, triggered G1-phase cell cycle arrest, and simultaneously suppressed cellular invasion and migration capabilities in vitro. Although EA did not obviously induce apoptosis in BC cells at the doses that were effective in suppressing their growth, it successfully triggered ferroptosis within these cells. Moreover, EA suppressed xenograft tumor growth of BC cells in vivo and demonstrated a favorable safety profile. Mechanistically, EA interacted with AKT1, reducing its phosphorylation and thereby inhibiting the AKT1/GSK3β/β-Catenin signaling pathway. Furthermore, EA exhibited synergistic effects with gemcitabine in inhibiting BC cells in vitro and in vivo. Overall, our results suggest that EA may exert its anti-BC effects, at least in part, by inhibiting the AKT1/GSK3β/β-Catenin signaling pathway. When combined with the clinically approved anti-tumor drug gemcitabine, EA may produce a synergistic anti-BC effect. Most importantly, this study identifies EA as a novel inducer of ferroptosis in BC and underscores its potential for clinical application.

Osteosarcoma (OS) is a highly aggressive tumor with a propensity for early metastasis. Current treatment methods, such as chemotherapy, often bring significant side effects, affecting patients' quality of life. Veratramine (VER), an alkaloid derived from the American lily plant, has shown potential in cancer treatment. This study looks at the effects and mechanisms of VER on osteosarcoma. VER's impact was assessed using a variety of procedures, including crystal violet staining, the CCK-8 assay, and the colony formation assay, which measured cell proliferation. Wound healing assay and transwell assay were employed to evaluate the migration and invasion of osteosarcoma cells. Hoechst33258 staining, flow cytometry, and transmission electron microscopy were used to investigate apoptosis. Protein expression was assessed using western blotting and immunofluorescence. Blood tests and hematoxylin-eosin staining were used to establish VER's in vivo safety, and its effectiveness was proven using an orthotopic tumor model. The results showed that VER greatly decreased osteosarcoma cell growth, migration, and invasion while inducing apoptosis. Animal tests confirmed these findings, confirming VER's high efficacy and safety in vivo. VER might function by inhibiting the PI3K/AKT signaling pathway. To sum up, VER shows promise in treating osteosarcoma by exhibiting significant anti-tumor activity in laboratory and animal studies, likely through the regulation of the PI3K/AKT signaling pathway.

Bone morphogenetic protein 9 (BMP9) has remarkable potential to induce the differentiation of mesenchymal stem cells (MSCs) towards the osteoblastic lineage. Additionally, research suggests that certain growth factors have the ability to potentiate BMP9-induced osteogenic differentiation of MSCs. Sonic Hedgehog (Shh) plays an indispensable role in the regulation of skeletal development. The objective of this research was to assess the potential influence of Shh on BMP9-induced osteogenic differentiation of MSCs. Our findings indicated that Shh effectively enhanced BMP9-induced early and late osteogenic differentiation of MSCs, and increased BMP9-induced expression/transcriptional activity of osteogenesis-related transcription factors. Besides, it was observed that Shh promoted BMP9-induced ectopic bone formation of MSCs in vivo. Moreover, BMP9 was able to facilitate the repair of bone defects in rats, while Shh further accelerated this reparative process. Mechanistically, Shh enhanced the activation of the Smad1/5/8 signaling pathway which was induced by BMP9. Furthermore, GANT-61, an inhibitor of Gli1 and Gli2, attenuated the enhancing effect of Shh on BMP9-induced osteogenic differentiation of MSCs. Collectively, the co-administration of BMP9 and Shh may present a promising therapeutic approach for the treatment of fracture nonunion, delayed fracture healing, and bone defects.

Hepatocellular carcinoma (HCC) ranks among the most prevalent cancers, with both a high incidence and a significant mortality rate. Clinical medications are highly toxic to patients and prone to resistance. Natural products are highly valued in the development of antitumour drugs. This study aimed to elucidate the anti-HCC ability and potential mechanism of Echinatin (Ecn), a natural existed flavonoid. Our findings revealed that Ecn suppressed the growth, migration, and invasion of HCC cells and demonstrated a superior inhibitory impact on the development of xenograft tumors. Moreover, Ecn was less toxic to mice and had a good drug safety. Mechanistically, Ecn was found to activate p38 and JNK signaling pathways. Accordingly, the suppressive effect of Ecn on HCC cells was attenuated by the introduction of p38 blocker SB203580 and JNK blocker SP600125. Collectively, our research suggests that Ecn might have anti-HCC properties through the activation of p38 and JNK signaling.

Isothermal amplification (IA) techniques have emerged as promising alternatives to polymerase chain reaction (PCR), enabling rapid and efficient nucleic acid amplification at constant temperatures. Meanwhile, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas‐based technology has revolutionized molecular diagnostics by harnessing collateral cleavage for programmable biomarker detection. Especially, the combination of IA techniques with CRISPR/Cas systems establishes a synergistic framework for next‐generation diagnostic platforms, addressing the sensitivity‐specificity trade‐offs and challenges posed by resource‐limited settings inherent in conventional methods. In this review, we trace the developmental milestones of IA techniques and elucidate the fundamental mechanisms of relevant IA techniques and CRISPR/Cas systems. Synergistic strategies for coupling IA techniques with CRISPR/Cas systems were summarized. Subsequently, personalized applications including point‐of‐care testing devices, droplet microfluidics platforms for rare biomarker detection, and IA/CRISPR/Cas‐based nanosensors were introduced. Finally, several challenges and perspectives in the field are discussed.

Osteosarcoma is a primary solid bone malignancy, and surgery + chemotherapy is the most commonly used treatment. However, chemotherapeutic drugs can cause a range of side effects. Casticin, a polymethoxyflavonoid, has anti-tumor therapeutic effects. This study is aim to investigate the anti-osteosarcoma activity of casticin and explore the mechanism. Crystal violet staining, MTT assay, colony formation assay, wound healing assay, transwell assay, hoechst 33,258 staining, and flow cytometry analysis were used to investigate the effects of casticin on proliferation, migration, invasion, and apoptosis of osteosarcoma cells in vitro. The intracellular Fe 2 + , ROS, MDA, GSH/GSSG content changes were detected using the corresponding assay kits. The mRNA sequencing + bioinformatics analysis and western blot were used to detect the possible mechanism. We found that casticin caused G2/M phase cell cycle arrest in human osteosarcoma cells, inhibited the migration and invasion, and induced cell apoptosis and ferroptosis. Mechanistic studies showed the ferroptosis pathway was enriched stronger than apoptosis. Casticin up-regulated the expression of HMOX1, LC3 and NCOA4, meanwhile it activated MAPK signaling pathways. Animal experiments proved that casticin also inhibited the growth and metastasis of osteosarcoma cell xenograft tumor in vivo. In conclusion, casticin can induce ferroptosis in osteosarcoma cells through Fe2+ overload and ROS production mediated by HMOX1 and LC3-NCOA4. This provides a new strategy for osteosarcoma treatment.

Exosomal microRNAs (exomiRNAs) have emerged as promising biomarkers for the early clinical diagnosis of osteoporosis. However, their limited abundance and short length in peripheral blood present significant challenges for the accurate detection of exomiRNAs. Herein, we have designed and implemented an efficacious fluorescence-based biosensor for the highly sensitive detection of exomiRNA associated with osteoporosis, leveraging the enhancing 3D DNA walker-induced CRISPR/Cas12a technology. The engineered DNA walker is capable of efficiently transforming target exomiRNA into amplifying DNA strands, thereby enhancing the sensitivity of the developed biosensor. Concurrently, the liberated DNA strands serve as activators to trigger Cas12a trans-cleavage activity, culminating in a significantly amplified fluorescent signal for the highly sensitive detection of exomiRNA-214. Under optimal conditions, the devised technology demonstrated the capacity to detect target exomiRNA-214 at concentrations as low as 20.42 fM, encompassing a wide linear range extending from 50.0 fM to 10.0 nM. Moreover, the fluorescence-based biosensor could accurately differentiate between healthy individuals and osteoporosis patients via the detection of exomiRNA-214, which was in agreement with RT-qPCR results. As such, this biosensing technology offers promise as a valuable tool for the early diagnosis of osteoporosis.

Bladder cancer (BC) is the most common malignant tumor in urinary system. Although chemotherapy is one of the most important adjuvant treatments for BC, drug resistance, non-specific toxicity and severe side effects are the major obstacles to BC chemotherapy. Natural products have always been a leading resource of antitumor drug discovery, with the advantages of excellent effectiveness, low toxicity, multi-targeting potency and easy availability.In this study, we evaluated the potential anti-tumor effect of securinine (SEC), a natural alkaloid from Securinega suffruticosa, on BC cells in vitro and in vivo, and delineated the underlying mechanism. We found that SEC inhibited the proliferation, migration and invasion, induced the apoptosis of BC cells in vitro, and retarded the xenograft tumor growth of BC cell in vivo. Notably, SEC had a promising safety profile because it presented no or low toxicity on normal cells and mice. Mechanistically, SEC inactivated Wnt/β-catenin signaling pathway while activated p38 and JNK signaling pathway. Moreover, β-catenin overexpression, the p38 inhibitor SB203580 and the JNK inhibitor SP600125 both mitigated the inhibitory effect of SEC on BC cells. Furthermore, we demonstrated a synergistic inhibitory effect of SEC and gemcitabine (GEM) on BC cells in vitro and in vivo.Taken together, our findings suggest that SEC may exert anti-BC cell effect at least through the activation of p38 and JNK signaling pathways, and the inhibition of Wnt/β-catenin signaling pathway. More meaningfully, the findings indicate that GEM-induced BC cell killing can be enhanced by combining with SEC.

Osteosarcoma (OS) is an aggressive primary tumor with the highest incidence in children and adolescents. Natural plant compounds (NPCs) have long been promising resources in the field of antitumor drug discovery because of their high efficacy and low toxicity. Here, the aim of this study is to investigate the potential inhibitory effects of Cyclovirobuxine D (CVB-D), a natural bioactive isolated from the traditional Chinese medicinal herb Huangyang, on OS cells. We showed that CVB-D reduced OS cell growth in vitro and in vivo. Mechanistically, CVB-D inhibited PI3K-AKT-mTOR pathway and initiated autophagy. On the other hand, CVB-D induced lysosomal over-acidification by interacting with the V-type proton ATPase 116 kDa subunit a1 (ATP6V0A1), ultimately leading to autophagy flux arrest which might be related to the inhibitory effect of CVB-D on OS cells. Conclusively, our results propose a potential foundation for CVB-D to be developed into an anti-OS drug and an autophagy inhibitor.

Bladder cancer (BC) is one of the most common malignant tumors in the urinary system. Due to the poor prognosis and high mortality rate of the disease, it is urgent to develop new drugs with high efficacy and low toxicity to treat BC. Echinatin (Ecn) is a bioactive natural flavonoid oflicorice that has attracted special attention for its promising anti-tumor potential. Herein, we explored the inhibitory effects of Echinatin on BC cells and probed the possible molecular mechanism. We found that Ecnin vitro inhibited the proliferation, migration, and invasion, arrested the cell cycle at the G2/M phase, and promoted apoptosis in BC cells. Besides, Ecn had no notable cytotoxicity towards human normal cells. We subsequently confirmed that Ecn restrained xenograft tumor growth and metastasis of BC cells in vivo. Mechanistically, Ecn activated the p38 signaling pathway but inactivated the Wnt/β-catenin signaling pathway, while over-expression of β-catenin and the p38 inhibitor both attenuated the inhibitory effects of Ecn on BC cells. Remarkably, Ecn combined with cisplatin (DDP) or gemcitabine (Gem) had synergistic inhibitory effects on BC cells. In summary, our results validate that Ecn inhibits the tumor growth of human BC cells via p38 and Wnt/β-catenin signaling pathways. More meaningfully, our results suggest a potential strategy to enhance DDP- or Gem-induced inhibitory effects on BC cells by combining with Ecn.

[This corrects the article DOI: 10.1016/j.gendis.2020.07.014.].

[This retracts the article DOI: 10.2147/OTT.S204574.].

Subsequently to the publication of the above paper, a concerned reader drew to the authors' attention that there were a number of overlapping data panels featured in the cellular images shown in Fig. 2C on p. 1644, and Figs. 3D and 4 on p. 1645, such that data that were allegedly obtained under different experimental conditions appeared to have been derived from some of the same original sources. Given the number of errors that had been made during the compilation of the figures in this article, the Editor of International Journal of Molecular Medicine has decided that this article should be retracted from the publication owing to a lack of overall confidence in the presented data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience that might result from the retraction of this article. [International Journal of Molecular Medicine 35: 1641‑1650, 2015; DOI: 10.3892/ijmm.2015.2172].

Diseases, such as bone nonunion with bone defects, osteoporosis, etc, seriously endanger people's quality of life, and bone tissue engineering based on mesenchymal stem cells is an effective method to solve such problems. Several studies have shown that BMP9 can effectively promote osteogenic differentiation of MSCs, but the underlying molecular mechanisms are still unclear. Gli1 and Gli2 were important transcription factors and play an important role in the Hedgehog signaling pathway. In this study, we investigated the role of Gli1 and Gli2 in BMP9-induced osteogenic differentiation of MSCs. We found that inhibition of Gli1 and Gli2 weakened BMP9-induced osteogenic differentiation of MSCs, and early osteogenic markers (alkaline phosphatase, ALP), late osteogenic markers (calcium salt deposition), the expression of pivotal osteogenic markers were attenuated, and inhibition of Gli1 and Gli2 weakened the expression of p-Smad1/5/8 and p-p38 induced by BMP9. In conclusion, our study shows that Gli1 and Gli2 play an important role in BMP9-induced osteogenic differentiation.

Osteosarcoma (OS) is a primary malignant tumor of bone. Chemotherapy is one of the crucial approaches to prevent its metastasis and improve prognosis. Despite continuous improvements in the clinical treatment of OS, tumor resistance and metastasis remain dominant clinical challenges. Macropinocytosis, a form of non-selective nutrient endocytosis, has received increasing attention as a novel target for cancer therapy, yet its role in OS cells remains obscure. Benzethonium chloride (BZN) is an FDA-approved antiseptic and bactericide with broad-spectrum anticancer effects. Here, we described that BZN suppressed the proliferation, migration, and invasion of OS cells in vitro and in vivo, but simultaneously promoted the massive accumulation of cytoplasmic vacuoles as well. Mechanistically, BZN repressed the ERK1/2 signaling pathway, and the ERK1/2 activator partially neutralized the inhibitory effect of BZN on OS cells. Subsequently, we demonstrated that vacuoles originated from macropinocytosis and indicated that OS cells might employ macropinocytosis as a compensatory survival mechanism in response to BZN. Remarkably, macropinocytosis inhibitors enhanced the anti-OS effect of BZN in vitro and in vivo. In conclusion, our results suggest that BZN may inhibit OS cells by repressing the ERK1/2 signaling pathway and propose a potential strategy to enhance the BZN-induced inhibitory effect by suppressing macropinocytosis.