Safety and efficacy of anlotinib combined with taxane and lobaplatin in neoadjuvant treatment of clinical stage II/III triple-negative breast cancer in China (the neoALTAL trial): a single-arm, phase 2 trial.

Background: Anlotinib is a new type of tyrosine kinase inhibitor that targets vascular endothelial growth factor receptors 1/2/3, platelet-derived growth factor receptors α/ß, and fibroblast growth factor receptors 1-4 and c-Kit, with a broad spectrum of inhibitory effects on tumor angiogenesis and growth. It has been proven effective in HER2-negative metastatic breast cancer, but its efficacy in early-stage triple-negative breast cancer (TNBC) is unknown. This phase 2 study aims to evaluate the efficacy and safety of adding anlotinib to neoadjuvant chemotherapy in patients with TNBC. Methods: Patients with clinical stage II/III TNBC were treated with 5 cycles of anlotinib (12 mg, d1-14, q3w) plus 6 cycles of taxanes (docetaxel 75 mg/m2 ,d1, q3w or nab-paclitaxel 125 mg/m2, d1 and d8, q3w) and lobaplatin (30 mg/m2, d1, q3w), followed by surgery. The primary endpoint was pathological complete response (pCR; ypT0/is ypN0) and the secondary endpoints include breast pCR (bpCR), axillary pCR (apCR), residual cancer burden (RCB), objective response rate (ORR), survival, and safety. Exploratory endpoints were efficacy biomarkers based on Fudan University Shanghai Cancer Center Immunohistochemical (FUSCC IHC) classification for TNBC and next-generation sequencing (NGS) of DNA from tumor tissue and blood samples of patients with 425-gene panel. This trial is registered with www.chictr.org.cn (ChiCTR2100043027). Findings: From Jan 2021 to Aug 2022, 48 patients were assessed and 45 were enrolled. All patients received at least one dose of study treatment and underwent surgery. The median age was 48.5 years (SD: 8.7), 71% were nodal involved, and 20% had stage III. In the intention-to-treat population, 26 out of 45 patients achieved pCR (57.8%; 90% CI, 44.5%-70.3%), and 39 achieved residual cancer burden class 0-I (86.7%; 95% CI, 73.2%-94.9%). The bpCR and apCR rate were 64.4% (29/45) and 71.9% (23/32), respectively. No recurrence or metastasis occurred during the short-term follow-up. Based on the FUSCC IHC-based subtypes, the pCR rates were 68.8% (11/16) for immunomodulatory subtype, 58.3% (7/12) for basal-like immune-suppressed subtype and 33.3% (4/12) for luminal androgen receptor subtype, respectively. NGS revealed that the pCR were 77% (10/13) and 50% (14/28) in MYC-amplified and wild-type patients, respectively, and 78% (7/9) and 53% (17/32) in gBRCA1/2-mutated and wild-type patients, respectively. The median follow-up time of the study was 14.9 months (95% CI: 13.5-16.3 months). There was no disease progression or death during neoadjuvant therapy. No deaths occurred during postoperative follow-up. In the safety population (N = 45), Grade 3 or 4 treatment emergent adverse events occurred in 29 patients (64%), and the most common events were neutropenia (38%), leukopenia (27%), thrombocytopenia (25%), anemia (13%), and hypertension (13%), respectively. Interpretation: The addition of anlotinib to neoadjuvant chemotherapy showed manageable toxicity and encouraging antitumor activity for patients with clinical stage II/III TNBC. Funding: Chongqing Talents Project, Chongqing Key Project of Technology Innovation and Application Development and Chongqing Outstanding Youth Natural Science Foundation.

A multicenter single-arm trial of neoadjuvant pyrotinib and trastuzumab plus chemotherapy for HER2-positive breast cancer.

The objective of this multicenter, single-arm trial (ChiCTR1900022293) was to explore the efficacy and safety of neoadjuvant therapy with epirubicin, cyclophosphamide, and pyrotinib followed by docetaxel, trastuzumab, and pyrotinib (ECPy-THPy) in the treatment of patients with stage II-III HER2-positive breast cancer. The present study enrolled patients with stage II-III HER2-positive breast cancer. Epirubicin and cyclophosphamide were administrated for four 21-day cycles, followed by four cycles of docetaxel and trastuzumab. Pyrotinib was taken orally once per day throughout the treatment period. The primary endpoint was total pathological complete response (tpCR, ypT0/is ypN0) rate in the modified intention-to-treat (mITT) population. In total, 175 patients were included. The tpCR rate was 68.6% (95% CI, 60.7-75.8%), while the objective response rate was 89.1%. In the post-hoc subgroup analysis, no association between clinical characteristics and the tpCR rate was observed. The most common grade ≥3 adverse events were diarrhea (54.3%), followed by white blood cell count decreased (5.1%), and neutrophil count decreased (4.6%). In conclusion, the neoadjuvant regimen with ECPy-THPy showed promising pathological response and clinical benefits with an acceptable safety profile in patients with stage II-III HER2-positive breast cancer.

Construction of a ferroptosis-based prognostic model for breast cancer helps to discriminate high/low risk groups and treatment priority.

Introduction: Breast cancer is a common malignant tumor associated with high morbidity and mortality. The role of ferroptosis, a regulated form of cell death, in breast cancer development and prognosis remains unclear. This study aims to investigate the relationship between ferroptosis-related genes and breast cancer and develop a prognostic model. Methods: RNA-seq expression datasets and clinical samples of breast cancer patients were obtained from public databases. Immunity- and drug resistance-related data were integrated. A preliminary screening was performed, resulting in the identification of 73 candidate ferroptosis factors. Univariate Cox regression analysis was conducted to select 12 genes, followed by LASSO Cox regression analysis to construct a prognostic risk prediction model consisting of 10 ferroptosis-related genes. The model was further characterized by immune cell infiltration. The expression levels of ferroptosis-related genes were validated in human breast cancer cell lines, and immunohistochemical (IHC) analysis was conducted on cancer specimens to assess ferroptosis-related protein expression. Results: The study identified 10 ferroptosis-related genes that were significantly associated with breast cancer prognosis. The constructed prognostic risk prediction model showed potential for predicting the prognostic value of these genes. In addition, the infiltration of immune cells was observed to be a characteristic of the model. The expression levels of ferroptosis-related genes were confirmed in human breast cancer cell lines, and IHC analysis provided evidence of ferroptosis-related protein expression in cancer specimens. Discussion: This study provides a novel prognostic model for breast cancer, incorporating 10 ferroptosis-related genes. The model demonstrates the potential for predicting breast cancer prognosis and highlights the involvement of immune cell infiltration. The expression levels of ferroptosis-related genes and proteins further support the association between ferroptosis and breast cancer development.

The value of hematocrit for predicting bronchopulmonary dysplasia in very low birth weight preterm infants.

To determine hematocrit (HCT) and to identify independent risk factors for predicting bronchopulmonary dysplasia (BPD) in preterm infants with very low birth weight (VLBW) infants. This retrospective study included 296 premature infants with VLBW in the neonatal intensive care unit of the First Affiliated Hospital of the University of Science and Technology of China between January 2015 and December 2019. Maternal pregnant information and clinical information as well as hematological parameters of preterm babies were collected and compared. Then the maximum area under the curve of receiver operating characteristic curve was developed to estimate the predictive indicator in the blood. Finally, differential variables together with the predictive index were screened for multiple logistic regression analysis to determine independent prognostic factors for BPD. Infants were divided into a BPD group (134 cases) and a non-BPD group (162 cases). The area under the curve of HCT at postnatal 1 week was 0.737 with the sensitivity of 52.30 % and the specificity of 86.00%. Birth weight (BW) <1.12 kg, gestational age <28.4 weeks, newborn respiratory distress syndrome, mechanical ventilation ≥ 7 days, ventilation associated pneumonia, patent arterial duct, PaO2/FiO2 <300 mm Hg and HCT <0.455 at postnatal 1 week were risk factors for BPD of VLBW infants. HCT levels below 0.455 at 1 week after birth serve as a valuable indicator for the potential development of BPD.

Study on the synthesis of pine sterol esters in solvent-free systems catalyzed by Candida rugosa lipase immobilized on hydrophobic macroporous resin.

BACKGROUND: Pine sterol ester is a type of novel food source nutrient with great advantages in lowering blood cholesterol levels, inhibiting tumors, preventing prostate enlargement, and regulating immunity. Macroporous resins with large specific surface area, stable structures, and various functional groups (epoxy, amino, and octadecyl groups) have been selected for immobilization of Candida rugosa lipase (CRL) to improve its stability and efficiency in the synthesis of pine sterol esters. A solvent-free strategy using oleic acid (substrate) as an esterification reaction medium is an important alternative for avoiding the use of organic solvents. RESULTS: The immobilization conditions of CRL immobilized on several types of commercial macroporous resins were optimized. Fortunately, by adsorption (hydrophobic interaction), a high immobilization efficiency of CRL was obtained using macroporous resins with hydrophobic octadecyl groups with an immobilization efficiency of 86.5%, enzyme loading of 138.5 mg g-1 and enzyme activity of 34.7 U g-1 . The results showed that a 95.1% yield could be obtained with a molar ratio of oleic acid to pine sterol of 5:1, an enzyme amount of 6.0 U g-1 (relative to pine sterol mass) at 50 °C for 48 h. CONCLUSION: The hydrophobic macroporous resin (ECR8806M) with a large specific surface area and abundant functional groups was used to achieve efficient immobilization of CRL. CRL@ECR8806M is an efficient catalyst for the synthesis of phytosterol esters and has the potential for further large-scale applications. Therefore, this simple, green, and low-cost strategy for lipase immobilization provides new possibilities for the high-efficiency production of pine sterol esters and other food source nutrients. © 2023 Society of Chemical Industry.

Role of serum- and glucocorticoid-inducible kinase 1 in the regulation of hepatic gluconeogenesis.

Excessive hepatic gluconeogenesis partially accounts for the occurrence of type 2 diabetes mellitus. Serum- and glucocorticoid inducible-kinase 1 (SGK1) is linked to the development of metabolic syndrome, such as obesity, hypertension, and hyperglycemia. However, the regulatory role of SGK1 in glucose metabolism of liver remains uncertain. Our microarray analysis showed that SGK1 expression was strongly induced by 8-Br-cAMP and suppressed by metformin in primary mouse hepatocytes. Hepatic SGK1 expression was markedly increased in obese and diabetic mice. Metformin treatment decreased hepatic SGK1 expression levels in db/db mice. Inhibition or knockdown of SGK1 suppressed gluconeogenesis in primary mouse hepatocytes, with decreased expressions of key gluconeogenic genes. Furthermore, SGK1 silencing in liver decreased hepatic glucose production in C57BL/6 mice. Knockdown of SGK1 had no impact on CREB phosphorylation level but increased AKT and FoxO1 phosphorylation levels with decreased expressions of transcription factors including FoxO1 and hepatocyte nuclear factors. Adenovirus-mediated expression of dominant-negative AMPK antagonized metformin-suppressed SGK1 expression induced by 8-Br-cAMP. These findings demonstrate that hepatic specific silence of SGK1 might be a potential therapeutic strategy for type 2 diabetes.

Selective and Efficient Synthesis of Pine Sterol Esters Catalyzed by Deep Eutectic Solvent.

Phytosterol esters have attracted widespread academic and industrial interests due to their advantages in lowering cholesterol, as antioxidants, and in preventing or treating cancer. However, the generation of by-products limits the application of phytosterol esters in food fields. In this study, deep eutectic solvents (DESs), a series of green, nontoxic, low-cost and biodegradable solvents, were adopted as the catalyst for the synthesis of pine sterol esters. The results showed that the acidic DES which was prepared with choline chloride (ChCl) and p-toluene sulfonic acid monohydrate (PTSA) with a molar ratio of 1:3 performed best in the prescreening experiments. To further improve the efficiency of the pine sterol ester, the molar ratio of substrates, the amount of catalyst, the reaction temperature and the reaction time were optimized, and its yield was improved to 94.1%. Moreover, the by-products of the dehydration side reactions of the sterol can be efficiently inhibited. To make this strategy more universal, other fatty acids were also used as the substrate for the synthesis of pine sterol esters, and a yield of above 92.0% was obtained. In addition, the reusability of DES was also investigated in this study, and the efficiency of DES was well maintained within five recycled uses. Finally, DFT calculations suggested that the suitable H-bonds between ChCl and PTSA decreased the nucleophilic capacity and increased the steric hindrance of the latter, and further prevented the attack on ßH and reduced the generation of by-products. This study developed a reliable and eco-friendly strategy for the preparation of high-quality phytosterol esters with low-dosage catalyst usage and high selectivity.

Association of Long-term Oncologic Prognosis With Minimal Access Breast Surgery vs Conventional Breast Surgery.

Importance: Minimal access breast surgery (MABS) has been used in breast cancer management. However, long-term prognostic data associated with MABS vs conventional breast surgery (CBS) are lacking. Objective: To investigate long-term therapeutic outcomes associated with MABS vs CBS for breast cancer management. Design, Setting, and Participants: In this single-center retrospective cohort study, 9184 individuals were assessed for inclusion. After exclusions, 2412 adult female individuals were included who were diagnosed with stage 0 to III breast cancer, underwent unilateral breast surgery between January 2004 and December 2017, and had no distant metastasis or history of severe underlying disease. Propensity score matching was performed to minimize selection bias. Data were analyzed from January 1, 2004, to December 31, 2019. Exposures: MABS or CBS. Main Outcomes and Measures: Data on demographic and tumor characteristics and long-term outcomes were collected and analyzed. Results: This study included 2412 patients (100% female; median [IQR] age, 44 [40-49] years). Of these, 603 patients underwent MABS (endoscopic, endoscopy-assisted, or robot-assisted procedures in 289, 302, and 12 patients, respectively) and 1809 patients underwent CBS. The median follow-up time was 84 months (93 in the MABS group and 80 months in the CBS group). Intergroup differences were not significant for the following parameters: 10-year local recurrence-free survival (93.3% vs 96.3%; hazard ratio [HR], 1.39; 95% CI, 0.86-2.27; P = .18), regional recurrence-free survival (95.5% vs 96.7%; HR, 1.38; 95% CI, 0.81-2.36; P = .23), and distant metastasis-free survival (81.0% vs 82.0%; HR, 0.95; 95% CI, 0.74-1.23; P = .72). The 5-, 10-, and 15-year disease-free survival rates in the MABS group were 85.9%, 72.6%, and 69.1%, respectively. The corresponding rates in the CBS group were 85.0%, 76.6%, and 70.7%. The intergroup differences were not significant (HR, 1.07; 95% CI, 0.86-1.31; P = .55). The 5-, 10-, and 15-year overall survival rates in the MABS group were 92.0%, 83.7%, and 83.0%, respectively. The corresponding rates in the CBS group were 93.6%, 88.7%, and 81.0%. The intergroup differences were not significant (HR, 1.29; 95% CI, 0.97-1.72; P = .09). Post hoc subgroup analysis showed no significant intergroup differences in disease-free survival. Conclusions and Relevance: In this cohort study, long-term outcomes following MABS were not significantly different from those following CBS in patients with early-stage breast cancer. MABS may be a safe and feasible alternative in this patient population.

Cytomegalovirus-specific neutralizing antibodies effectively prevent uncontrolled infection after allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains one of the most frequent and life-threatening infectious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Herein, we comprehensively compared the immune cells of patients with uncontrolled and controlled CMV infection post-allo-HSCT and found that B-cells were extraordinarily insufficient because of impaired B-cells reconstitution in the uncontrolled infection group. Furthermore, in the controlled infection group, reconstructed B-cells showed signatures of mature B-cells, high expression of CXCR4 and IFITM1, and enrichment of CMV-associated B-cell receptors, which were lacking in the uncontrolled infection group. Consistently, sera from the uncontrolled infection group failed to inhibit CMV infection via neutralizing virus in vitro because of its lower content of anti-CMV-specific immunoglobulin G (IgG) than the controlled infection group. Overall, these results highlighted the contribution of B cells and anti-CMV-specific neutralizing IgGs to the restraint of CMV infection post-allo-HSCT, suggesting their potential as a supplementary treatment to improve outcomes.

Virtual screening analysis of natural flavonoids as trimethylamine (TMA)-lyase inhibitors for coronary heart disease.

Coronary heart disease (CHD) is defined by atherosclerosis, which can result in stenosis or blockage of the arterial cavity, leading to ischemic cardiac diseases such as angina and myocardial infarction. Accumulating evidence indicates that the gut microbiota plays a vital role in the beginning and progression of CHD. The gut microbial metabolite, trimethylamine-N-oxide (TMAO), is intimately linked to the pathophysiology of CHD. TMAO is formed when trimethylamine (TMA) is converted by flavin-containing monooxygenases in the hepatocytes. Therefore, inhibition of TMA production is essential to reduce TMAO levels. Flavonoids may reduce the risk of death from cardiovascular disease. In this article, we reviewed and evaluated twenty-two flavonoids for the therapy of CHD based on their inhibition of TMA-lyase by molecular docking. Docking results revealed that baicalein, fisetin, acacetin, and myricetin in flavonoid aglycones, and baicalin, naringin, and hesperidin in flavonoid glycosides had a good binding effect with TMA-lyase. This indicates that these chemicals were the most active and could be used as lead compounds for structural modification in the future. PRACTICAL APPLICATIONS: Flavonoids are a large class of polyphenolic compounds found in fruits, vegetables, flowers, tea, and herbal medicines, which are inexorably metabolized and transformed into bioactive metabolites by α-rhamnosidase, ß-glucuronidase, ß-glucosidase, and nitroreductase produced by the gut microbiota, which plays a beneficial role in the prevention and treatment of cardiovascular diseases. Because flavonoids protect the cardiovascular system and regulate the gut microbiota, and the gut microbiota is directly connected to TMAO, thus, reducing TMAO levels involves blocking the transition of TMA to TMAO, which may be performed by reducing TMA synthesis. Molecular docking results found that baicalein, fisetin, acacetin, and myricetin in flavonoid aglycones, and baicalin, naringin, and hesperidin in flavonoid glycosides had good binding effects on TMA-lyase, which were the most active and could be used as lead compounds for structural modification.

Lobaplatin-based neoadjuvant chemotherapy for triple-negative breast cancer: a 5-year follow-up of a randomized, open-label, phase II trial.

Purpose: We report the 5-year follow-up findings of a randomized, open-label, phase II trial of lobaplatin-based neoadjuvant chemotherapy plus adjuvant therapy for triple-negative breast cancer (TNBC). Patients and methods: This study included patients aged ⩾18 years with untreated, operable stage I-III TNBC and an Eastern Cooperative Oncology Group performance status of 0 or 1. One group of patients (TE group, n = 99) received four cycles of docetaxel (T, 75 mg/m²) plus epirubicin (E, 80 mg/m²) every 3 weeks, and another group (TEL group, n = 101) received the same treatment with the addition of lobaplatin (L, 30 mg/m2). Two cycles of the corresponding treatments were administered after surgery in both groups. The primary endpoints were total pathological complete response (tpCR) rate and overall response rate (ORR), and the secondary endpoints were disease-free survival, overall survival, and long-term safety. This trial is registered with the Chinese Clinical Trial Registry (ChiCTR-TRC-14005019). Results: The median follow-up was 48.2 months (interquartile range: 31.1-60.0). The tpCR rate was 41.4% and 17.8% in the TEL group and TE group, respectively (p < 0.001). The HR for comparison of DFS between the TEL group and TE group was 0.44 (95% CI: 0.21-0.90, P p = 0.028). The addition of lobaplatin resulted in an HR of 0.44 (95% CI: 0.18-1.02, P = 0.061) for the difference in OS between the two groups. The ORR, which included complete response and partial response, was 92.9% in the TEL group and 74.3% in the TE group (p = 0.001). The TEL group patients were more likely to develop grade III-IV anemia and thrombocytopenia. No lobaplatin-related deaths or increased risk of long-term toxicity was observed. Conclusion: Neoadjuvant lobaplatin therapy can improve the tpCR and ORR rates of TNBC with tolerable side effects and have a tendency to improve the long-term survival.

Pyrotinib-Containing Neoadjuvant Therapy in Patients With HER2-Positive Breast Cancer: A Multicenter Retrospective Analysis.

Background: Pyrotinib, a small-molecule tyrosine kinase inhibitor, has been investigated as a component of neoadjuvant therapy in phase 2 trials of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This study aimed to evaluate the effectiveness and safety of pyrotinib-containing neoadjuvant therapy for patients with HER2-positive early or locally advanced breast cancer in the real-world setting. Methods: Data of 97 patients with HER2-positive breast cancer from 21 centers across China treated with pyrotinib-containing neoadjuvant therapy were reviewed. Neoadjuvant therapy consisted of taxane/carboplatin/trastuzumab plus pyrotinib (TCbH+Py, 30 [30.9%]), anthracycline/cyclophosphamide followed by taxane/trastuzumab plus pyrotinib (AC-TH+Py) or taxane followed by anthracycline/cyclophosphamide/trastuzumab plus pyrotinib (T-ACH+Py, 29 [29.9%]), taxane/trastuzumab plus pyrotinib (TH+Py, 23 [23.7%]), and other pyrotinib-containing neoadjuvant treatment (15 [15.5%]). The primary outcome was breast pathological complete response (bpCR, ypT0/is) rate. Secondary outcomes included total pathological complete response (tpCR, ypT0/is ypN0) rate, objective response rate (ORR), and the incidence of preoperative adverse events. Results: The ORR of pyrotinib-containing neoadjuvant therapy was 87.6% (85/97). The bpCR and tpCR rates were 54.6% (95% confidence interval [CI], 44.2%-64.7%) and 48.5% [95% CI, 38.2%-58.8%], respectively. The most common grade 3 or 4 treatment-related adverse events included diarrhea (15 [15.5%]), decreased hemoglobin (nine [9.3%]), and decreased neutrophil count (eight [8.2%]). No treatment-related deaths occurred. Conclusion: Pyrotinib-containing neoadjuvant therapy for patients with HER2-positive early or locally advanced breast cancer shows favorable effectiveness with manageable toxicity in the real-world setting. Trastuzumab plus pyrotinib may be a novel option of dual HER2-targeted blockade.

Systematic investigation of the aza-Cope reaction for fluorescence imaging of formaldehyde in vitro and in vivo.

Increasing evidence has highlighted the endogenous production of formaldehyde (FA) in a variety of fundamental biological processes and its involvement in many disease conditions ranging from cancer to neurodegeneration. To examine the physiological and pathological relevance and functions of FA, fluorescent probes for FA imaging in live biological samples are of great significance. Herein we report a systematic investigation of 2-aza-Cope reactions between homoallylamines and FA for identification of a highly efficient 2-aza-Cope reaction moiety and development of fluorescent probes for imaging FA in living systems. By screening a set of N-substituted homoallylamines and comparing them to previously reported homoallylamine structures for reaction with FA, we found that N-p-methoxybenzyl homoallylamine exhibited an optimal 2-aza-Cope reactivity to FA. Theoretical calculations were then performed to demonstrate that the N-substituent on homoallylamine greatly affects the condensation with FA, which is more likely the rate-determining step. Moreover, the newly identified optimal N-p-methoxybenzyl homoallylamine moiety with a self-immolative ß-elimination linker was generally utilized to construct a series of fluorescent probes with varying excitation/emission wavelengths for sensitive and selective detection of FA in aqueous solutions and live cells. Among these probes, the near-infrared probe FFP706 has been well demonstrated to enable direct fluorescence visualization of steady-state endogenous FA in live mouse brain tissues and elevated FA levels in a mouse model of breast cancer. This study provides the optimal aza-Cope reaction moiety for FA probe development and new chemical tools for fluorescence imaging and biological investigation of FA in living systems.

[SP13786 Inhibits the Migration and Invasion of Lung Adenocarcinoma Cell A549 
by Supressing Stat3-EMT via CAFs Exosomes].

BACKGROUND: Fibroblast activation protein (FAP) is one of the surface markers of cancer-associated fibroblasts (CAFs) and is closely related to the malignant characterization of CAFs. SP13786 is a specific micromolecule inhibitor of FAP and this study is to investigate the effects and mechanism of SP13786 on the migration and invasion of A549 cells through regulating exosomes of CAFs. METHODS: CAFs and paracancerous fibroblasts (PTFs) were isolated and subcultured from freshly resected lung adenocarcinoma tissues and paracancerous normal tissues separately. MTT assay was used to detect the proliferation of CAFs incubated by different concentrations of SP13786; PTFs-exo, CAFs-exo and CAFs+SP13786-exo were extracted by polymer precipitation method. The A549 cells were divided into Ctrl group, PTFs group, CAFs group and SP13786 group and each group was incubated with DMEM, PTFs-exo, CAFs-exo and CAFs+SP13786-exo separately. Laser confocal microscope was used to observe the endocytoses of exosomes by A549 cells. The expression of alpha-smooth muscle actin (α-SMA) and FAP in PTFs and CAFs and the expression of E-cadherin, N-cadherin, Slug, Stat3 and P-Stat3 in A549 cells were detected by immunofluorescence, immunohistochemistry and Western blot. The migration and invasion ability of A549 cells were detected by cell scratch and transwell methods. RESULTS: α-SMA and FAP were expressed much higher in CAFs than that in PTFs which indicate that CAFs and PTFs were successfully obtained from lung adenocarcinoma and paracancerous tissues (P<0.05). MTT showed that the 50% inhibitory concentration (IC50) of SP13786 for CAFs was about 3.3 nmol/L. In addition, SP13786 can significantly decrease the expression of α-SMA and FAP in CAFs which means that targeted inhibition of FAP could reduce the malignant characteristics of CAFs (P<0.05). Laser confocal microscope found that exosomes from CAFs could be taken up by A549 cells and scratch and transwell tests showed that the endocytosed CAFs-exo could promote the migration and invasion of A549 cells (P<0.001), while FAP inhibitor SP13786 could inhibit the effects of CAFs-exo on A549 cells (P<0.05). Furthermore, Immunofluorescence and Western blot showed that CAFs-exo could promote EMT by decreasing E-cadherin expression and increasing N-cadherin, Slug expression in A549 cells while FAP inhibitor SP13786 could significantly supress CAFs-exo-induced epithelial-mesenchymal transition (EMT) of A549 cells (P<0.05). Moreover, the expression of P-Stat3 was obviously increased in A549 cells of CAFs group and significantly down-regulated in SP13786 group (P<0.05) whereas there was no significant difference in total Stat3 between CAFs and SP13786 groups (P>0.05). Finally, WP1066 (a specific inhibitor of Stat3) was used to comfirm whether SP13786 could influence EMT of A549 cells by inhibiting Stat3 phosphorylation via CAFs-Exo. The results showed that when the phosphorylation of Stat3 in CAFs group was inhibited by WP1066, SP13786 could not influence the P-Stat3 expression and EMT of A549 cells anymore (P>0.05). CONCLUSIONS: As a specific micromolecule inhibitor of FAP, SP13786 indirectly inhibits the migration and invasion of A549 cells by affecting exosomes of CAFs. The possible mechanism is to inhibit the phosphorylation of Stat3 and thus affect the EMT of A549 cells.

Discovery of a highly efficient nitroaryl group for detection of nitroreductase and imaging of hypoxic tumor cells.

Hypoxia is a pathological hallmark of solid tumors. Detection of hypoxia is therefore of great interest for tumor diagnosis and treatment. As a well-established biomarker of hypoxia, nitroreductase (NTR) has been widely exploited in the development of hypoxia-responsive fluorescent probes on the basis of its enzymatic activity to reduce nitroaryl groups. However, studies on the relationship between the nitroaryl structure and the probe performance for optimal probe design are still rare. Here we report a comparative investigation of nitroaryl groups and identification of the optimal nitroaryl structure for developing new fluorescent probes with extremely high efficiency in the detection of NTR and the imaging of hypoxic tumor cells. Specifically, we synthesized a series of resorufin-based fluorescent probes containing different nitroaryl groups, compared their fluorescence responses to NTR, and identified 2-nitro-N-methyl-imidazolyl as the optimal nitroaryl group that is much more efficient than the most widely used 4-nitrophenyl for NTR detection. The structure-performance relationship was then studied by theoretical molecular docking, revealing the unique features of 2-nitro-N-methyl-imidazolyl in binding and reaction with NTR. We further incorporated the 2-nitro-N-methyl-imidazolyl group into a near-infrared (NIR) hemicyanine fluorophore and developed a NIR fluorescent probe NFP-7 for the detection of NTR and hypoxic tumor cells. NFP-7 exhibits a strong fluorescence increase toward NTR in vitro with an ultrafast (within 40 seconds to fluorescence maximum) and ultrasensitive (0.2 ng mL-1 detection limit) response. NFP-7 has also been demonstrated for imaging the degree of hypoxia in live tumor cells and, more importantly, in a murine tumor model. Our study provides important insights into hypoxia probe development and new tools for hypoxia imaging.

SIRT2 ablation inhibits glucose-stimulated insulin secretion through decreasing glycolytic flux.

Rationale: Sirtuins are NAD+-dependent protein deacylases known to have protective effects against age-related diseases such as diabetes, cancer, and neurodegenerative disease. SIRT2 is the only primarily cytoplasmic isoform and its overall role in glucose homeostasis remains uncertain. Methods: SIRT2-knockout (KO) rats were constructed to evaluate the role of SIRT2 in glucose homeostasis. The effect of SIRT2 on ß-cell function was detected by investigating the morphology, insulin secretion, and metabolomic state of islets. The deacetylation and stabilization of GKRP in ß-cells by SIRT2 were determined by western blot, adenoviral infection, and immunoprecipitation. Results: SIRT2-KO rats exhibited impaired glucose tolerance and glucose-stimulated insulin secretion (GSIS), without change in insulin sensitivity. SIRT2 deficiency or inhibition by AGK2 decreased GSIS in isolated rat islets, with lowered oxygen consumption rate. Adenovirus-mediated overexpression of SIRT2 enhanced insulin secretion from rat islets. Metabolomics analysis revealed a decrease in metabolites of glycolysis and tricarboxylic acid cycle in SIRT2-KO islets compared with control islets. Our study further demonstrated that glucokinase regulatory protein (GKRP), an endogenous inhibitor of glucokinase (GCK), was expressed in rat islets. SIRT2 overexpression deacetylated GKRP in INS-1 ß-cells. SIRT2 knockout or inhibition elevated GKRP protein stability in islet ß-cells, leading to an increase in the interaction of GKRP and GCK. On the contrary, SIRT2 inhibition promoted the protein degradation of ALDOA, a glycolytic enzyme. Conclusions: SIRT2 ablation inhibits GSIS through blocking GKRP protein degradation and promoting ALDOA protein degradation, resulting in a decrease in glycolytic flux.

Protein acetylation derepresses Serotonin Synthesis to potentiate Pancreatic Beta-Cell Function through HDAC1-PKA-Tph1 signaling.

Rationale: Protein acetylation is tightly linked to transcriptional control and energy metabolism. However, the role of protein acetylation in islet function remains enigmatic. This study aims to determine how protein acetylation controls ß-cell function and explore the underlying mechanism. Methods: The gene-expression profiles were analyzed for rat islets in response to two histone deacetylase (HDAC) inhibitors. Insulin secretion, tryptophan hydroxylase 1 (Tph1) expression, and serotonin synthesis of rat islets were detected after HDAC inhibitor treatment both in vivo and ex vivo. ß-cell-specific Tph1-overexpressing transgenic rats and ß-cell-specific Tph1 knockout mice were constructed to evaluate the role of Tph1 in ß-cell function. The deacetylation of PKA in ß-cells by HDAC1 was investigated by adenoviral infection, immunoprecipitation, and western blot. Results: Inhibition of HDACs greatly potentiated pancreatic ß-cell function and reprogrammed transcriptional landscape of islets. Among the commonly up-regulated genes by two pan-HDAC inhibitors, Tph1 displayed the most prominent change. Specifically, inhibition of HDAC1 and HDAC3 by MS-275 strongly promoted Tph1 expression and endogenous serotonin synthesis in rat islets, concomitantly with enhanced insulin secretory capacity in vivo and ex vivo. ß-cell-specific Tph1-overexpressing transgenic rats exhibited improved glucose tolerance and amplified glucose-stimulated insulin secretion. On the contrary, ß-cell-specific Tph1 knockout mice displayed glucose intolerance and impaired insulin secretion with aging. Moreover, depletion of Tph1 in ß-cells abrogated MS-275-induced insulin hypersecretion. Overexpression of HDAC1, not HDAC3, inhibited Tph1 transcriptional activity and decreased MS-275-stimulated Tph1 expression. Mechanistically, HDAC1 deacetylated PKA catalytic subunit and decreased its activity, resulting in Tph1 transcriptional repression. The acetylation mimetic K62Q mutant of PKA increased its catalytic activity. HDAC1 inhibition exerted a synergistic effect with cAMP/PKA signal on Tph1 expression. Conclusions: The present findings highlight a novel role of HDAC1-PKA-Tph1 signaling in governing ß-cell functional compensation by derepressing serotonin synthesis.

Overexpression of Tumor Protein p53-regulated Apoptosis-inducing Protein 1 Regulates Proliferation and Apoptosis of Breast Cancer Cells through the PI3K/Akt Pathway.

PURPOSE: Tumor protein p53-regulated apoptosis-inducing protein 1 (TP53AIP1) functions in various cancers. We studied the effect and molecular mechanism of TP53AIP1 in breast cancer. METHODS: The degree of correlation between TP53AIP1 expression and overall survival in patients with breast cancer was obtained from the online The Cancer Genome Atlas database. Six of the TP53AIP1 levels in the tumor and adjacent non-tumor tissues randomly selected from 38 breast cancer patients were determined. Transgenic technology was used to enhance the expression of TP53AIP1 in breast cancer cell lines, MDA-MB-415 and MDA-MB-468, and to observe the effects of gene overexpression on the proliferation, cell cycle, and apoptosis of breast cancer cells. The molecular mechanism of association between cell cycle- and apoptosis-related factors and the phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) pathway was also studied. RESULTS: The messenger RNA and protein expression levels of TP53AIP1 in cancer tissues were significantly lower than those in the control group. TP53AIP1 overexpression inhibits cell viability. The mechanism of TP53AIP1 inhibition of proliferation and growth of breast cancer cells includes cell cycle arrest, apoptosis promotion (p < 0.01), promotion of the expression of cleaved-caspase-3 (p < 0.01), cleaved-caspase-9 (p < 0.01), B cell lymphoma/leukemia-2 (Bcl-2)-associated X protein, and p53 (p < 0.01), and the inhibition of Bcl-2, Ki67, and PI3K/Akt pathways (p < 0.01). CONCLUSION: TP53AIP1 may be a novel tumor suppressor gene in breast cancer and can potentially be used as an effective target gene for the treatment of breast cancer.

PPP1R3C mediates metformin-inhibited hepatic gluconeogenesis.

BACKGROUND: Metformin has been widely used to alleviate hyperglycemia in patients with type 2 diabetes mainly via suppressing hepatic gluconeogenesis. However, the underlying mechanism remains incompletely clear. Here, we aimed to explore the role of PPP1R3C in metformin-mediated inhibition of hepatic gluconeogenesis. METHODS: The differentially expressed genes in primary mouse hepatocytes incubated with 8-Br-cAMP and metformin were analyzed by microarrays. Hepatic glucose production and gluconeogenic gene expressions were detected after adenovirus-mediated overexpression or silence of PPP1R3C in vitro and in vivo. The phosphorylation level and location of transducer of regulated CREB activity 2 (TORC2) were determined by Western blot and immunofluorescence. RESULTS: Metformin and adenovirus-mediated activation of AMPK suppressed 8-Br-cAMP-stimulated Ppp1r3c mRNA expression in primary mouse hepatocytes. Overexpression of PPP1R3C in primary mouse hepatocytes or the livers of wild-type mice promoted hepatic glucose production and gluconeogenic gene expressions. On the contrary, adenovirus-mediated knockdown of PPP1R3C in primary mouse hepatocytes decreased hepatic gluconeogenesis, with the suppression of cAMP-stimulated gluconeogenic gene expressions and TORC2 dephosphorylation. Notably, Ppp1r3c expression was increased in the liver of db/db mice. After PPP1R3C silence in the livers of wild-type and db/db mice, blood glucose levels and hepatic glucose production were markedly lowered, with decreased expressions of key gluconeogenic enzymes and transcript factors as well as liver glycogen content. CONCLUSION: Metformin-activated AMPK decreases hepatic PPP1R3C expression, leading to the suppression of hepatic gluconeogenesis through blocking cAMP-stimulated TORC2 dephosphorylation. Hepatic specific silence of PPP1R3C provides a promising therapeutic strategy for type 2 diabetes.

MS-275 induces hepatic FGF21 expression via H3K18ac-mediated CREBH signal.

Fibroblast growth factor 21 (FGF21) plays an important role in the regulation of lipid and glucose metabolism. MS-275, as a class I-specific histone deacetylase (HDAC) inhibitor, has also been reported to affect energy metabolism. In this current study, we investigated the effects of MS-275 on hepatic FGF21 expression in vitro and in vivo and explored whether cAMP-responsive element-binding protein H (CREBH) was involved in the action of MS-275. Our results showed that MS-275 stimulated hepatic FGF21 mRNA and protein expressions in a dose- and time-dependent manner, as well as FGF21 secretion in primary mouse hepatocytes. Serum concentration and hepatic expression of FGF21 were elevated after injection of MS-275, along with increased expressions of genes involved in fatty acid oxidation and ketogenic production (peroxisome proliferator-activated receptor gammacoactivator1α, PGC-1α; carnitine palmitoyl-transferase 1a, CPT1a; 3-hydroxy-3-methylglutaryl-CoA synthase 2, Hmgcs2) as well as improved blood lipid profile. As a proved transcription factor of FGF21, the expression of CREBH was initiated by MS-275, with increased histone H3 lysine 18 acetylation (H3K18ac) signals and hepatocyte nuclear factor 4 alpha (HNF-4α) recruitment in CREBH promoter. Adenovirus-mediated knockdown of CREBH abolished MS-275-induced hepatic FGF21 and lipid metabolism-related gene expressions. These results suggest that MS-275 induces hepatic FGF21 by H3K18ac-mediated CREBH expression.