Pirfenidone inhibits TGF-ß1-induced metabolic reprogramming during epithelial-mesenchymal transition in non-small cell lung cancer.

Metastasis is an important contributor to increased mortality rates in non-small cell lung cancer (NSCLC). The TGF-ß signalling pathway plays a crucial role in facilitating tumour metastasis through epithelial-mesenchymal transition (EMT). Glycolysis, a key metabolic process, is strongly correlated with NSCLC metastasis. Pirfenidone (PFD) has been shown to safely and effectively inhibit TGF-ß1 in patients with lung diseases. Furthermore, TGF-ß1 and glycolysis demonstrate an interdependent relationship within the tumour microenvironment. Our previous study demonstrated that PFD effectively inhibited glycolysis in NSCLC cells, prompting further investigation into its potential antitumour effects in this context. Therefore, the present study aims to investigate the potential antitumour effect of PFD in NSCLC and explore the relationship among TGF-ß1, glycolysis and EMT through further experimentation. The antitumour effects of PFD were evaluated using five different NSCLC cell lines and a xenograft tumour model. Notably, PFD demonstrated a significant antitumour effect specifically in highly glycolytic H1299 cells. To elucidate the underlying mechanism, we compared the efficacy of PFD after pretreatment with either TGF-ß1 or a TGF-ß receptor inhibitor (LY2109761). The energy metabolomics analysis of tumour tissue demonstrated that PFD, a chemosensitizing agent, reduced lactate and ATP production, thereby inhibiting glycolysis and exerting synergistic antineoplastic effects. Additionally, PFD combined with cisplatin targeted TGF-ß1 to inhibit glycolysis during EMT and enhanced the chemosensitization of A549 and H1299 cells. The magnitude of the anticancer effect exhibited by PFD was intricately linked to its metabolic properties.

Lactoferrin deficiency during lactation increases the risk of depressive-like behavior in adult mice.

BACKGROUND: Lactoferrin is an active protein in breast milk that plays an important role in the growth and development of infants and is implicated as a neuroprotective agent. The incidence of depression is currently increasing, and it is unclear whether the lack of lactoferrin during lactation affects the incidence of depressive-like behavior in adulthood. RESULTS: Lack of lactoferrin feeding during lactation affected the barrier and innate immune functions of the intestine, disrupted the intestinal microflora, and led to neuroimmune dysfunction and neurodevelopmental delay in the hippocampus. When exposed to external stimulation, adult lactoferrin feeding-deficient mice presented with worse depression-like symptoms; the mechanisms involved were activation of the LPS-TLR4 signalling pathway in the intestine and hippocampus, reduced BDNF-CREB signaling pathway in hippocampus, increased abundance of depression-related bacteria, and decreased abundance of beneficial bacteria. CONCLUSIONS: Overall, our findings reveal that lactoferrin feeding deficient during lactation can increase the risk of depressive-like behavior in adults. The mechanism is related to the regulatory effect of lactoferrin on the development of the "microbial-intestinal-brain" axis.

Pirfenidone suppressed triple-negative breast cancer metastasis by inhibiting the activity of the TGF-ß/SMAD pathway.

Among breast cancer patients, metastases are the leading cause of death. Despite decades of effort, little progress has been made to improve the treatment of breast cancer metastases, especially triple-negative breast cancer (TNBC). The extracellular matrix plays an important role in tumour growth and metastasis by causing its deposition, remodelling, and signalling. As we know, the process of fibrosis results in excessive amounts of extracellular matrix being deposited within the cells. So, it will be interesting to study if the use of anti-fibrotic drugs in combination with conventional chemotherapy drugs can produce synergistic antitumor effects. In this study, we assessed the efficacy of Pirfenidone (PFD), an FDA-approved medication for the treatment of idiopathic pulmonary fibrosis, on TNBC cells as well as its anti-tumour effects in xenograft tumour model. PFD inhibited in a dose-dependent manner breast cancer cell proliferation, migration, and invasion, while promoted their apoptosis in vitro. PFD also suppressed TGF-ß-induced activation of Smad signalling pathway and expression level of EMT-inducing transcription factors (e.g. SNAI2, TWIST1, ZEB1) as well as the mesenchymal genes such as VIMENTIN and N-Cadherin. On the contrary, the expression level of epithelial marker gene E-Cadherin was up-regulated in the presence of PFD. In vivo, PFD alone exerted a milder but significant anti-tumour effect than the chemotherapy drug nanoparticle albumin-bound paclitaxel (nab-PTX) did in the breast cancer xenograft mouse model. Interestingly, PFD synergistically boosted the cancer-killing effect of nab-PTX. Furthermore, Our data suggest that PFD suppressed breast cancer metastasis by inhibiting the activity of the TGFß/SMAD pathway.

Lactoferrin Deficiency Impairs Proliferation of Satellite Cells via Downregulating the ERK1/2 Signaling Pathway.

Lactoferrin (Ltf), a naturally active glycoprotein, possesses anti-inflammatory, anti-microbial, anti-tumor, and immunomodulatory activities. Many published studies have indicated that Ltf modulates the proliferation of stem cells. However, the role of Ltf in the proliferation of satellite cells, an important cell type in muscle regeneration, has not yet been reported. Here, by using Ltf systemic knockout mice, we illustrate the role of Ltf in skeletal muscle. Results shows that Ltf deficiency impaired proliferation of satellite cells (SCs) and the regenerative capability of skeletal muscle. Mechanistic studies showed that ERK1/2 phosphorylation was significantly downregulated after Ltf deletion in SCs. Simultaneously, the cell cycle-related proteins cyclin D and CDK4 were significantly downregulated. Intervention with exogenous recombinant lactoferrin (R-Ltf) at a concentration of 1000 µg/mL promoted proliferation of SCs. In addition, intraperitoneal injection of Ltf effectively ameliorated the skeletal muscle of mice injured by 1.2% BaCl2 solution. Our results suggest a protective effect of Ltf in the repair of skeletal muscle damage. Ltf holds promise as a novel therapeutic agent for skeletal muscle injuries.

Mixolab behavior, quality attributes and antioxidant capacity of breads incorporated with Agaricus bisporus.

At present, some breads rich in edible mushrooms are more popular to meet the needs of functional bread products. In this study, different proportions of Agaricus bisporus powder (0, 2, 4, 6, 8%) were added into wheat flour to prepare bread, and their Mixolab behavior, bread quality and antioxidant capacity were studied. The results showed that the addition of Agaricus bisporus powder diluted the gluten in the flour, destroyed the dough matrix, which changed the water absorption of dough, the gelatinization and retrogradation of starch on the Mixolab. Meanwhile, the results of specific volume, texture and color measurement showed that addition of Agaricus bisporus powder reduced the specific volume of bread, deteriorated the texture and darkened the crumbs. In addition, the higher proportion of Agaricus bisporus powder, the higher total phenolics content and antioxidant ability of compound bread. However, sensory analysis showed that the overall acceptability of breads was relatively high after adding a small amount of Agaricus bisporus powder. Our results showed that breads containing about 4% Agaricus bisporus powder had higher antioxidant capacity and better sensory acceptability.

Intravoxel incoherent motion imaging used to assess tumor microvascular changes after transarterial chemoembolization in a rabbit VX2 liver tumor model.

Purpose: To evaluate the correlation between microvascular density (MVD) and intravoxel incoherent motion (IVIM) magnetic resonance imaging (MRI) parameters and the effect of glycolytic flux after transarterial chemoembolization (TACE) in a rabbit VX2 liver tumor. Materials and methods: VX2 liver tumor allografts in 15 New Zealand white rabbits were treated with sterile saline (control group, n = 5) or lipiodol-doxorubicin emulsion (experimental group, n = 10). MRI was performed 2 weeks after the procedure to evaluate IVIM parameters, including apparent diffusion coefficient (ADC), pure diffusion coefficient (D), pseudodiffusion coefficient (D*), and perfusion fraction (PF). All animal samples were taken of the tumor and surrounding liver. Immunostaining for CD31, CD34, CD105, and VEGF was used to evaluate MVD. The protein expression of Glut4, HK2, PKM2, LDHA, and MCT1 was determined using western blotting. Pearson correlation tests were used to analyze the relationship between MVD and IVIM parameters. Results: D* value in the peritumoral region was negatively correlated with CD34 (r = -0.71, P = 0.01). PF value positively correlated with CD34 (r = 0.68, P = 0.015), CD105 (r = 0.76, P = 0.004) and VEGF (r = 0.72, P = 0.008) in the peritumoral region. Glut4, HK2, PKM2, and MCT1 in the peritumoral regions were higher in the experimental group than in the control group (all P < 0.05). Conclusion: IVIM parameters were correlated with MVD in the intratumoral and peritumoral regions after TACE in a rabbit liver tumor model. The angiogenesis reflected by MVD may be related to changes of glycolytic flux.

Ultrasound-guided percutaneous implantation of rabbit VX2 carcinoma, using a coaxial technique and gelfoam pellet injection combination to establish a rabbit liver tumor model.

PURPOSE We aimed to investigate the safety and tumor seeding rate of a coaxial implantation technique combined with injection of a gelfoam pellet in establishing a VX2 liver tumor model in rabbits. METHODS A VX2 liver tumor model was established in 60 male New Zealand white rabbits, which were randomly divided into 3 groups (20 in each group) based on implantation technique (all performed under ultrasound guidance): group A, single needle only; group B, single needle with injection of a gelfoam pellet; or group C, coaxial technique with injection of a gelfoam pellet. The rates of liver tumor formation and tumor seeding to extrahepatic tissues were compared 2 weeks after implantation. Data were also collected regarding procedure time, number of punctures, occurrence of complications, and mortality rate. RESULTS A VX2 liver tumor model was established in all 60 rabbits (100%, 60/60). Ectopic implantation rate was 70% (14/20) in group A, 35% (7/20) in group B, and 5% (1/20) in group C, with significant difference among the groups (p < 0.001). Post hoc analysis showed significant difference between group A and group C (p < 0.001). However, there were no significant differences between group B and group A or group C (p = 0.027, p = 0.048, respectively). There were no significant differences among the groups in terms of procedure time (p = 0.405) or number of punctures (p = 0.612). No complications or deaths occurred. CONCLUSION A coaxial technique with injection of a gelfoam pellet is an effective and safe method for VX2 liver tumor implantation in rabbits, and this technique can reduce the risk of tumor seeding to the abdominal wall and omentum.

Intravoxel incoherent motion diffusion-weighted imaging and shear wave elastography for evaluating peritumoral liver fibrosis after transarterial chemoembolization in a VX2 rabbit liver tumor model.

In this study, we sought to evaluate changes in peritumoral fibrosis after transarterial chemoembolization (TACE) in a rabbit VX2 liver tumor model using intravoxel incoherent motion diffusion-weighted imaging (IVIM DWI) and ultrasound shear wave elastography (SWE). A total of 20 rabbits underwent implantation of VX2 tumor tissues in the left lobe of the liver. The rabbits were randomly divided into an experimental group (n = 10) or a control group (n = 10). Those in the experimental group were treated with an emulsion of lipiodol and pirarubicin through a microcatheter 2-3 weeks after implantation; those in the control group were treated with sterile water. Compared with the control group, the true diffusion coefficient (D) and pseudodiffusion coefficient (D*) values in liver tissues were significantly lower (p < 0.05 for all) and liver stiffness values (LSV) (10.58 ± 0.89 kPa) were higher in the experimental group (7.65 ± 0.86 kPa; p < 0.001). The median stage of liver fibrosis based on METAVIR scores was 1 (1,1) in the control group and 2 (2,3) in the experimental group (Z = 4.15, p < 0.001). D, D*, and LSV were significantly correlated with pathologic staining in the assessment of liver fibrosis (r = -0.54 p = 0.015; r = -0.50, p = 0.025; r = 0.91, p < 0.001; respectively). These data suggest that TACE aggravates liver injury and liver fibrosis, especially surrounding the tumor, in a rabbit VX2 liver tumor model. IVIM DWI and SWE can be used to evaluate the change in liver fibrosis.

Analysis of Risk Factors of Bleeding Complications in Percutaneous Needle Biopsy of Liver Occupying Lesions.

PURPOSE: To search for risk factors for bleeding complications after percutaneous biopsy of primary or secondary space-occupying lesions of liver guided by imaging. METHODS: Consecutive 555 patients with liver space-occupying lesions who underwent ultrasound or CT-guided percutaneous biopsy in our hospital from January 2015 to January 2021 were retrospectively analyzed. Those who cannot cooperate with breath-holding and cannot successfully complete the operation, cytology, and incomplete clinical data. After screening, a total of 502 patients were enrolled, including 313 males and 189 females. Abdominal cavity or liver subcapsular hemorrhage after procedure was used as the dependent variable, and patient's gender, age, pathological results, tumor size, preoperative platelets and international normalized ratio (INR) and hemoglobin as independent variables. All independent variables were analyzed by a single factor logistic regression analysis. The independent variables with P<0.05 were included in the regression model and analyzed by multivariate logistic regression analysis to search for the risk factors for bleeding complications of liver space-occupying lesions. RESULTS: A total of 502 patients with liver space-occupying lesions undergoing percutaneous liver biopsy guided by imaging equipment were included. Twenty-six of 502 (5.2%) patients occurred abdominal cavity or liver sub-capsule bleeding after procedure. Univariate logistic regression analysis observed that liver cirrhosis, the number of punctured tissues and the depth of puncture were related to bleeding complications after puncturing. Multivariate logistic regression analysis showed that liver cirrhosis and puncture depth were risk factors for bleeding complications (P<0.05). The ROC curve for predicting bleeding complications after needle biopsy in patients with liver cirrhosis has a sensitivity of 94.3% and a specificity of 46.2%. CONCLUSION: Liver cirrhosis and puncture depth are risk factors for bleeding complications during percutaneous biopsy of liver occupying lesions.

Lactoferrin, a Critical Player in Neonate Intestinal Development: RHLF may be a Good Choice in Formula.

Lactoferrin (LF) is a bioactive glycoprotein in human milk and has positive effects on neonates. The LF knockout mouse model was generated as a mother mouse that provided LF-free milk. The intestinal development of suckling neonates drinking normal milk and LF-free milk was studied. The results showed that the intestinal density, maturity, and barrier integrity of mice drinking LF-free milk were lower than those of mice drinking normal milk. Therefore, the importance of adding lactoferrin to the human formula is considered. Human lactoferrin (HLF), bovine lactoferrin (BLF), and recombinant HLF (RHLF) were used to compare their functional impact on Caco-2 cell lines. Cell proliferation, differentiation, the establishment of the intestinal barrier, and protective effects on lipopolysaccharide injury were detected. Our results showed that RHLF exhibited more similar functions to HLF than BLF and showed the combined advantages of HLF and BLF in promoting the establishment of the intestinal barrier. This study emphasizes the important role of LF in neonatal intestinal development and provides a theoretical basis for the availability of RHLF.

CircC16orf62 promotes hepatocellular carcinoma progression through the miR-138-5p/PTK2/AKT axis.

Circular RNA (circRNAs) functions vital in the pathogenesis and progression of hepatocellular carcinoma (HCC). However, the expressions and functions of certain circRNAs on metastasis and proliferation of that cancer is still unclear. Bioinformation analysis and qRT-PCR indicated that CircC16orf62 was prominent upregulated in HCC of which the expression level was positively associated to cancer's malignant progression. Gain or loss-of-function studies indicated that the reduction of CircC16orf62 expression promotes the proliferation, invasion, and glycolysis of HCC in vitro and in vivo. The bioinformatic analysis found that miR-138-5p and PTK2 were the downstream target of CircC16or62. Then, the FISH(Fluorescence immunoin situ hybridization) and cell nucleoplasmic separation determined that CircC16orf62 located in the cell cytoplasm. Plasmid vectors or siRNAs were used to change the expression of CircC16orf62, miR-138-5p, and PTK2 in PC cell lines. CircC16orf62 functioned as a molecular sponge for miR-138-5p, and a competitive endogenous RNA for PTK2, promoting AKT/mTOR pathway activation. Our observations lead us to conclude that CircC16orf62 functions as an oncogene in HCC progression, behaving as a competitive endogenous RNA for miR-138-5p binding, thus activating the AKT/mTOR pathway. In conclusion, CircC16orf62 is an oncogene through the miR-138-5p/PTK2/Akt axis in HCC cells, indicating CircC16orf62 can be a therapeutic target with potentiality for liver cancer and a predictive marker for people with HCC.

Malic enzyme 2 promotes the progression of hepatocellular carcinoma via increasing triglyceride production.

The incidence and mortality of hepatocellular carcinoma (HCC) are gradually increasing during the past years. Recently, some studies have reported that malic enzyme (ME) plays an important role in cancer development, while the involvement of ME2 in HCC remains still undetermined. Here, we demonstrated that ME2 played an oncogenic role in HCC. ME2 was overexpressed in HCC tissues. TCGA database showed that the ME2 transcript level was inversely associated with the survival of HCC patients. Loss-of-function and gain-of-function assays showed that ME2 promoted HCC cell growth and migration. Furthermore, the xenografted tumorigenesis of MHCC97H cells was retarded by ME2 knockdown. ME2 silencing also suppressed the cell cycle process and induced apoptosis. Mechanistically, ME2 potentiated triglyceride synthesis, inhibition of which suppressed the proliferation and migration. We propose that ME2 promotes HCC progression by increasing triglyceride production.