Oridonin-induced ferroptosis and apoptosis: a dual approach to suppress the growth of osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is one of the most common aggressive bone malignancy tumors in adolescents. With the application of new chemotherapy regimens, finding new and effective anti-OS drugs to coordinate program implementation is urgent for the patients of OS. Oridonin had been proved to mediate anti-tumor effect on OS cells, but its mechanism has not been fully elucidated. METHODS: The effects of oridonin on the viability, clonal formation and migration of 143B and U2OS cells were detected by CCK-8, colony formation assays and wound-healing test. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore the mechanism of oridonin on OS. Western blot (WB), real-time quantitative PCR (qRT-PCR) were used to detect the expression levels of apoptosis and ferroptosis-relative proteins and genes. Annexin V-FITC apoptosis detection kit and flow cytometry examination were used to detect the level of apoptosis. Iron assay kit was used to evaluate the relative Fe2+ content. The levels of mitochondrial membrane potential and lipid peroxidation production was determined by mitochondrial membrane potential detection kit and ROS assay kit. RESULTS: Oridonin could effectively inhibit the survival, clonal formation and metastasis of OS cells. The KEGG results indicated that oridonin is associated with the malignant phenotypic signaling pathways of proliferation, migration, and drug resistance in OS. Oridonin was capable of inhibiting expressions of BAX, cl-caspase3, SLC7A11, GPX4 and FTH1 proteins and mRNA, while promoting the expressions of Bcl-2 and ACSL4 in 143B and U2OS cells. Additionally, we found that oridonin could promote the accumulation of reactive oxygen species (ROS) and Fe2+ in OS cells, as well as reduce mitochondrial membrane potential, and these effects could be significantly reversed by the ferroptosis inhibitor ferrostatin-1 (Fer-1). CONCLUSION: Oridonin can trigger apoptosis and ferroptosis collaboratively in OS cells, making it a promising and effective agent for OS therapy.

Wogonin, a Bioactive Ingredient from Huangqi Guizhi Formula, Alleviates Discogenic Low Back Pain via Suppressing the Overexpressed NGF in Intervertebral Discs.

Purpose: To investigate whether wogonin, a key bioactive ingredient of Huangqi Guizhi formula (HQGZ formula; a Traditional Chinese Medicine herbal formula) according to network pharmacology analysis, has analgesic effects on discogenic low back pain (LBP) via regulating the nerve growth factor (NGF) in intervertebral discs (IVDs). Methods: The lumbar IVDs of rats were punctured to discogenic LBP, and the therapeutic effect of orally administrated HQGZ for discogenic LBP was investigated by measuring mechanical and cold allodynia and histological analysis. A network pharmacology analysis was conducted to search for bioactive ingredients from the HQGZ formula, and wogonin was suggested to be the most possible bioactive ingredient for LBP treatment. Subsequently, the analgesic effect of wogonin was investigated in the LBP model, and the gene expression of propain peptides in the bilateral dorsal root ganglia was analyzed using RT-PCR. Finally, immunohistochemical staining was performed for NGF expression of NGF in the IVDs to determine whether wogonin treatment would ameliorate NGF-induced LBP. Results: Oral administration of HQGZ for two weeks significantly ameliorated puncture-induced IVD degeneration (IDD) and LBP. In addition, the network pharmacology analysis revealed that wogonin, quercetin, and kaempferol were the potential candidate components of HQGZ for LBP treatment. Furthermore, we proved that wogonin had significant analgesic effects in the LBP model. Finally, wogonin was demonstrated to suppress the upregulated NGF in the IVD and ameliorate NGF-induced LBP in rats. Conclusions: The HQGZ formula has significant analgesic effects for LBP. In addition, the bioactive ingredient of wogonin was extracted from HQGZ and ameliorated LBP by suppressing the overexpressed NGF in degenerated IVDs. Therefore, wogonin has potential to be alternative treatment for LBP in clinical.

Overexpression of SAPCD2 correlates with proliferation and invasion of colorectal carcinoma cells.

BACKGROUND: Suppressor anaphase-promoting complex domain containing 2 (SAPCD2) is a novel gene playing important roles in the initiation, invasion, and metastasis of several malignancies. However, its role in colorectal carcinoma (CRC) still remains unclear. METHOD: In this study, we investigated the expression and biological function of SAPCD2 in CRC. Immunohistochemistry (IHC) for SAPCD2 was performed in 410 pairs of CRC specimens and corresponding normal epithelial tissues, and in 50 adenoma tissues. Clinical pathological factors were analyzed in relation to the expression of SAPCD2. The biological functions of SAPCD2 in CRC cells and its effect on cell cycle were investigated in vitro and in vivo through gain/loss-of-function approaches. RESULTS: IHC showed that SAPCD2 expression was significantly higher in CRC tissues compared to adenoma and normal epithelium tissues and was correlated with tumor location (p = 0.018). SAPCD2 significantly promoted cell proliferation, migration, and invasion both in vitro and in vivo (p < 0.05). In addition, SAPCD2 knockdown in CRC cells was associated with reduced G1/S transition, while overexpression caused G2/M phase arrest (p < 0.05). CONCLUSIONS: In sum, SAPCD2 is overexpressed in CRC tissues and plays a critical role in CRC progression. Therefore, it might represent a promising therapeutic target for CRC treatment.

Investigation on the in vitro cytocompatibility of Mg-Zn-Y-Nd-Zr alloys as degradable orthopaedic implant materials.

Mg-Zn-Y-Nd-Zr alloy has been developed as a new type of biodegradable orthopaedic implant material by the authors' research group with its excellent mechanical properties and controllable degradation rate. In this study, the cytocompatibility of Mg-Zn-Y-Nd-Zr alloy was systematically evaluated through in vitro cell culture method. MTT assay was applied to evaluate the cytotoxicity of Mg-Zn-Y-Nd-Zr alloy and no toxic effect was observed on L929 and MC3T3-E1 cells followed the protocol of ISO 10993 standard. Considering the potential ion accumulation in the bony environment, this study further investigated the cytotoxic effect of accumulated metallic ions during the alloy degradation by extending the extract preparation time. When the extract preparation time was prolonged to 1440 h, the accumulated metallic ions leaded to severe cell apoptosis, of which the combined ion concentration was determined as 39.5-65.8 µM of Mg2+, 3.5-5.9 µM of Zn2+, 0.44-0.74 µM of Y3+, 0.3-0.52 µM of Nd3+ and 0.11-0.18 µM of Zr4+ for L929, and 65.8-92.2 µM of Mg2+, 5.9-8.3 µM of Zn2+, 0.74-1.04 µM of Y3+, 0.52-0.73 µM of Nd3+ and 0.18-0.25 µM of Zr4+ for MC3T3-E1 cells. Besides the cell viability assessment, high expression of ALP activity and calcified nodules implied that metal elements in Mg-Zn-Y-Nd-Zr alloys can promote the osteogenic differentiation. Hence, excellent cytocompatibility has equipped Mg-Zn-Y-Nd-Zr alloy as a promising candidate for orthopaedic implant application, which can remarkably guide the magnesium-based alloy design and provide scientific evidence for clinical practice in future.

The RNA-binding protein QKI suppresses cancer-associated aberrant splicing.

Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway.

Effect of hyperoside on osteoporosis in ovariectomized mice through estrogen receptor α/ITGß3 signaling pathway.

Osteoporosis is a highly prevalent bone metabolic disease in menopause due to estrogen deficiency. Hyperoside is a main compound in Semen cuscutae. Our team previously reported that Semen cuscutae has anti osteoporosis effect on ovariectomized mice by inhibiting bone resorption of osteoclasts. However, it is still unclear whether hyperoside affects osteoclast differentiation and bone resorption, and whether its anti-osteoporosis effect is related to an estrogen-like effect. This study investigates the potential mechanism of hyperoside's anti-osteoporotic effect by examining its impact on osteoclast differentiation and its relationship with the estrogen receptor. DXA, Micro-CT, TRAP staining, HE, and ELISA were used to assess the impact of hyperoside on OVX-induced osteoporosis. The effect of hyperoside on octeoclast differentiation was evaluated using TRAP activity assay, TRAP staining, F-actin staining. The activation of the estrogen receptor by hyperoside and its relationship with osteoclast differentiation were detected using dual-luciferase reporter assay and estrogen receptor antagonists. Our findings revealed that hyperoside (20-80 mg/kg) protect against OVX-induced osteoporosis, including increasing BMD and BMC and improving bone microstructure. Hyperoside inhibited osteoclast differentiation in a concentration dependent manner, whereas estrogen receptor α antagonists reversed its inhibitory effect osteoclast differentiation. Western blot results suggested that hyperoside inhibited TRAP, RANKL, c-Fos and ITG ß3 protein expression in osteoclast or femoral bone marrow of ovariectomized mice. Our findings suggest that hyperoside inhibits osteoclast differentiation and protects OVX-induced osteoporosis through the ERα/ITGß3 signaling pathway.

Potential biomarkers for the early detection of bone metastases.

The clinical manifestations of bone metastases are diversified while many sites remain asymptomatic at early stage. As the early diagnosis method is not perfect and the early symptoms of tumor bone metastasis are not typical, bone metastasis is not easy to be detected. Therefore, the search for bone metastasis-related markers is effective for timely detection of tumor bone metastases and the development of drugs to inhibit bone metastases. As a result, bone metastases can only be diagnosed when symptoms are found, increasing the risk of developing skeletal-related event (SREs), which significantly impairs the patient's quality of life. Therefore, the early diagnosis of bone metastases is of great importance for the treatment and prognosis of cancer patients. Changes of bone metabolism indexes appear earlier in bone metastases, but the traditional biochemical indexes of bone metabolism lack of specificity and could be interfered by many factors, which limits their application in the study of bone metastases. Some new biomarkers of bone metastases have good diagnostic value, such as proteins, ncRNAs, circulating tumor cells (CTCs). Therefore, this study mainly reviewed the initial diagnostic biomarkers of bone metastases which were expected to provide references for the early detection of bone metastases.

Intervertebral Disc Degeneration and Low Back Pain Depends on Duration and Magnitude of Axial Compression.

Purpose: The pathological role of axial stress in intervertebral disc degeneration (IDD) is controversial, and there was no quantified study until now. Here, we tried to clarify the correlation between IDD or low back pain (LBP) and axial stress at different duration and magnitude in vitro and in vivo. Method: In vitro, the gene expression of aggrecan, matrix metalloproteinase-3 (MMP3), calcitonin gene-related peptide (CGRP), and substance P (SP) was measured when nucleus pulposus cells (NPCs) were compressed under gradual severity. In vivo, a measurable Ilizarov-type compression apparatus was established for single coccygeal (Co) intervertebral disc (IVD) compression of Co7-8 in mouse. Gradient stress was placed at 0.4 Mpa (mild), 0.8 Mpa (moderate), and 1.2 Mpa (severe) for three days to investigate the effect of the magnitude of axial stress. Additionally, mild compression with 3, 7, and 14 days was used to determine the effect of the duration of axial stress. Subsequently, we evaluated the severity of IDD and LBP by radiological X-ray film; histological examination with H&E staining; immunohistochemical analysis with collagen II, aggrecan, and CGRP staining; and western blot analysis with collagen II, aggrecan, MMP-3, and interleukin-1ß (IL-1ß). Results: When NPCs suffered gradual increased mechanical stress, the cells exhibited gradual downregulated expression of extracellular matrix (ECM)-related gene of aggrecan, upregulated expression of IDD-related gene of MMP3, and LBP-related gene of CGRP and SP. In the meantime, with different magnitudes of axial stress, the IVD showed progressively severe IDD and LBP, with gradual narrowing intervertebral height, destruction of IVD anatomy, decreased ECM, and increased catabolic factors and proalgesic peptides. Conclusion: Axial compression is one of the critical pathological factors to cause IDD and LBP, and there was a strong positive correlation depended on the duration and magnitude of compression.

Evaluating a Panel of Autoantibodies Against Tumor-Associated Antigens in Human Osteosarcoma.

Background: The aim of this study was to identify a panel of candidate autoantibodies against tumor-associated antigens in the detection of osteosarcoma (OS) so as to provide a theoretical basis for constructing a non-invasive serological diagnosis method in early immunodiagnosis of OS. Methods: The serological proteome analysis (SERPA) approach was used to select candidate anti-TAA autoantibodies. Then, indirect enzyme-linked immunosorbent assay (ELISA) was used to verify the expression levels of eight candidate autoantibodies in the serum of 51 OS cases, 28 osteochondroma (OC), and 51 normal human sera (NHS). The rank-sum test was used to compare the content of eight autoantibodies in the sera of three groups. The diagnostic value of each indicator for OS was analyzed by an ROC curve. Differential autoantibodies between OS and NHS were screened. Then, a binary logistic regression model was used to establish a prediction logistical regression model. Results: Through ELISA, the expression levels of seven autoantibodies (ENO1, GAPDH, HSP27, HSP60, PDLIM1, STMN1, and TPI1) in OS patients were identified higher than those in healthy patients (p < 0.05). By establishing a binary logistic regression predictive model, the optimal panel including three anti-TAAs (ENO1, GAPDH, and TPI1) autoantibodies was screened out. The sensitivity, specificity, Youden index, accuracy, and AUC of diagnosis of OS were 70.59%, 86.27%, 0.5686, 78.43%, and 0.798, respectively. Conclusion: The results proved that through establishing a predictive model, an optimal panel of autoantibodies could help detect OS from OC or NHS at an early stage, which could be used as a promising and powerful tool in clinical practice.