A phosphoglycerate mutase 1 allosteric inhibitor overcomes drug resistance to EGFR-targeted therapy via disrupting IL-6/JAK2/STAT3 signaling pathway in lung adenocarcinoma.

Resistance to epidermal growth factor receptor (EGFR) inhibitors, from the first-generation erlotinib to the third generation osimertinib, is a clinical challenge in the treatment of patients with EGFR-mutant lung adenocarcinoma. Our previous work found that a novel allosteric inhibitor of phosphoglycerate mutase 1 (PGAM1), HKB99, restrains erlotinib resistance in lung adenocarcinoma cells. However, the role of HKB99 in osimertinib resistance and its underlying molecular mechanism remains to be elucidated. Herein, we found that IL-6/JAK2/STAT3 signaling pathway is aberrantly activated in both erlotinib and osimertinib resistant cells. Importantly, HKB99 significantly blocks the interaction of PGAM1 with JAK2 and STAT3 via the allosteric sites of PGAM1, which leads to inactivation of JAK2/STAT3 and thereby disrupts IL-6/JAK2/STAT3 signaling pathway. Consequently, HKB99 remarkably restores EGFR inhibitor sensitivity and exerts synergistic tumoricidal effect. Additionally, HKB99 alone or in combination with osimertinib down-regulated the level of p-STAT3 in xenograft tumor models. Collectively, this study identifies PGAM1 as a key regulator in IL-6/JAK2/STAT3 axis in the development of resistance to EGFR inhibitors, which could serve as a therapeutic target in lung adenocarcinoma with acquired resistance to EGFR inhibitors.

Steroid receptor coactivator-1 enhances the stemness of glioblastoma by activating long noncoding RNA XIST/miR-152/KLF4 pathway.

Glioblastoma (GBM) recurrence is attributed to the presence of therapy-resistant glioblastoma stem cells. Steroid receptor coactivator-1 (SRC-1) acts as an oncogenic regulator in many human tumors. The relationship between SRC-1 and GBM has not yet been studied. Herein, we investigate the role of SRC-1 in GBM. In this study, we found that SRC-1 expression is positively correlated with grades of glioma and inversely correlated with glioma patient's prognosis. Steroid receptor coactivator-1 promotes the proliferation, migration, and tumor growth of GBM cells. Notably, SRC-1 knockdown suppresses the stemness of GBM cells. Mechanistically, long noncoding RNA X-inactive specific transcript (XIST) is regulated by SRC-1 at the posttranscriptional level and mediates the function of SRC-1 in promoting stemness-like properties of GBM. Steroid receptor coactivator-1 can promote the expression of Kruppel-like factor 4 (KLF4) through the XIST/microRNA (miR)-152 axis. Additionally, arenobufagin and bufalin, SRC small molecule inhibitors, can reduce the proliferation and stemness of GBM cells. This study reveals SRC-1 promotes the stemness of GBM by activating the long noncoding RNA XIST/miR-152/KLF4 pathway and provides novel markers for diagnosis and therapy of GBM.

Aqp-1 Gene Knockout Attenuates Hypoxic Pulmonary Hypertension of Mice.

Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.

Gab2 Ablation Reverses the Stemness of HER2-Overexpressing Breast Cancer Cells.

BACKGROUND/AIMS: HER2 has been implicated in mammary tumorigenesis as well as aggressive tumor growth and metastasis. Its overexpression is related to a poor prognosis and chemoresistance in breast cancer patients. Although Grb2-associated binding protein 2 (Gab2) is important in the development and progression of human cancer, its effects and mechanisms in HER2-overexpressing breast cancer are unclear. METHODS: Clone formation and MTT assays were used to examine cell proliferation. To detect the effect of Gab2 on the stemness of breast cancer cells, we used flow cytometry, a sphere formation assay, real-time PCR, and western blot. An animal model was created to validate the effect of Gab2 on tumor growth in vivo. Tissue slides were analyzed by immunohistochemistry. RESULTS: Knockdown of Gab2 suppressed PI3K/AKT and MAPK/ERK pathway activity. Gab2 ablation also reduced the stemness of HER2-overexpressing breast cancer cells. In vivo, knockdown of Gab2 inhibited tumor growth. CONCLUSION: This study unveils a potential function of Gab2 in HER2-overexpressing breast cancer cells. Gab2 might be a potential target in the clinical therapy of HER2-overexpressing breast carcinoma.

Exosomal lncRNA XIST promotes perineural invasion of pancreatic cancer cells via miR-211-5p/GDNF.

Perineural invasion (PNI) is an essential form of tumor metastasis in multiple malignant cancers, such as pancreatic cancer, prostate cancer, and head and neck cancer. Growing evidence has revealed that pancreatic cancer recurrence and neuropathic pain positively correlate with PNI. Therefore, targeting PNI is a proper strategy for pancreatic cancer treatment. Exosomal lncRNA derived from pancreatic cancer cells is an essential component of the tumor microenvironment. However, whether exosomal lncXIST derived from pancreatic cancer cells can promote PNI and its exact mechanism remains to be elucidated. We show that lncXIST mediates nerve-tumor crosstalk via exosomal delivery. Our data reveal that exosomal lncXIST derived from pancreatic cancer cells is delivered to neural cells and promotes their release of glial-cell-line-derived neurotrophic factor (GDNF), essential in facilitating the PNI of pancreatic cancer. Mechanistically, microRNA-211-5p negatively regulates GDNF, and lncXIST serves as a miR-211-5p sponge. The function of exosomes in the dynamic interplay between nerves and cancer is confirmed in both in vivo and in vitro PNI models. Therefore, targeting pancreatic cancer cell-derived exosomal lncXIST may provide clues for a promising approach for developing a new strategy to combat PNI of pancreatic cancer.

GSTO1 aggravates EGFR-TKIs resistance and tumor metastasis via deglutathionylation of NPM1 in lung adenocarcinoma.

Despite significantly improved clinical outcomes in EGFR-mutant lung adenocarcinoma, all patients develop acquired resistance and malignancy on the treatment of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Understanding the resistance mechanisms is crucial to uncover novel therapeutic targets to improve the efficacy of EGFR-TKI treatment. Here, integrated analysis using RNA-Seq and shRNAs metabolic screening reveals glutathione S-transferase omega 1 (GSTO1) as one of the key metabolic enzymes that is required for EGFR-TKIs resistance in lung adenocarcinoma cells. Aberrant upregulation of GSTO1 confers EGFR-TKIs resistance and tumor metastasis in vitro and in vivo dependent on its active-site cysteine 32 (C32). Pharmacological inhibition or knockdown of GSTO1 restores sensitivity to EGFR-TKIs and synergistically enhances tumoricidal effects. Importantly, nucleophosmin 1 (NPM1) cysteine 104 is deglutathionylated by GSTO1 through its active C32 site, which leads to activation of the AKT/NF-κB signaling pathway. In addition, clinical data illustrates that GSTO1 level is positively correlated with NPM1 level, NF-κB-mediated transcriptions and progression of human lung adenocarcinoma. Overall, our study highlights a novel mechanism of GSTO1 mediating EGFR-TKIs resistance and malignant progression via protein deglutathionylation, and GSTO1/NPM1/AKT/NF-κB axis as a potential therapeutic vulnerability in lung adenocarcinoma.

Establishment of organoid models for pancreatic ductal adenocarcinoma and screening of individualized therapy strategy.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and receive effective chemotherapy have the best chance for long-term survival. Unfortunately, because of the heterogeneity of pancreatic cancer, it is difficult to find a personalized treatment strategy for patients. Organoids are ideal preclinical models for personalized medicine. Therefore, we explore the cultivation conditions and construction methods of PDAC organoid models to screen the individualized therapy strategy. METHODS: Fresh PDAC tissues from surgical resection were collected and digested with digestive enzymes; then the tumor cells were embedded in Matrigel with a suitable medium to establish the PDAC organoid models. The genetic stability of the organoids was analyzed using whole exon sequencing; hematoxylin and eosin staining and immunohistochemistry of organoids were performed to analyze their consistency with the pathological morphology of the patient's tumor tissue; After 2 days of organoid culture, we selected four commonly used clinical chemotherapy drugs for single or combined treatment to analyze drug sensitivity. RESULTS: Two cases of PDAC organoid models were successfully established, and the results of their pathological characteristics and exome sequencing were consistent with those of the patient's tumor tissue. Both PDAC organoids showed more sensitivity to gemcitabine and cisplatin, and the combined treatment was more effective than monotherapy. CONCLUSION: Both organoids better retained the pathological characteristics, genomic stability, and heterogeneity with the original tumor. Individual PDAC organoids exhibited different sensitivities to the same drugs. Thus, this study provided ideal experimental models for screening individualized therapy strategy for patients with PDAC.

Sulforaphane alleviated vascular remodeling in hypoxic pulmonary hypertension via inhibiting inflammation and oxidative stress.

Hypoxic pulmonary hypertension (HPH) is a cardiopulmonary disease featured by pulmonary vascular remodeling, which is due to abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) and dysfunction of endothelial cells (ECs). Sulforaphane (SFN) is a natural isothiocyanate extracted from cruciferous vegetables with promising anti-inflammatory and anti-oxidative activities. This study aimed to explore the effect and mechanism of SFN on HPH. Male mice were exposed to persistent chronic hypoxia for 4 weeks to induce HPH. The results demonstrated that SFN repressed the increased right ventricular systolic pressure (RVSP) and attenuated the right ventricular hypertrophy and pulmonary arteries remodeling in HPH mice. In particular, after SFN treatment, the CD68 positive cells in lung sections were reduced; TNF-α and IL-6 levels in lungs and serum declined; activation of NF-κB in PASMCs was inhibited in response to hypoxia. Besides, SFN enhanced the superoxide dismutase (SOD) activity in serum, SOD2 expression, total glutathione levels, and GSH/GSSG ratio in PASMCs, along with a decrease in malondialdehyde (MDA) contents in serum and ROS production in PASMCs after hypoxia exposure. Notably, SFN, as an Nrf2 activator, reversed the reduction in Nrf2 expression in hypoxic PASMCs. In vitro, SFN treatment inhibited hyperproliferation and promoted apoptosis of PASMCs under hypoxia conditions. SFN also prevented the apoptosis of pulmonary microvascular ECs caused by hypoxia. Therefore, these data suggested that SFN could significantly restrain the inflammation and oxidative stress, thereby inhibiting PASMCs proliferation, promoting PASMCs apoptosis, and reversing hypoxia injury in ECs to improve pulmonary vascular remodeling.

Screening of an individualized treatment strategy for an advanced gallbladder cancer using patient-derived tumor xenograft and organoid models.

Gallbladder cancer is a highly aggressive malignancy with poor sensitivity to postoperative radiotherapy or chemotherapy; therefore, the development of individualized treatment strategies is paramount to improve patient outcomes. Both patient-derived tumor xenograft (PDX) and patient-derived tumor organoid (PDO) models derived from surgical specimens can better preserve the biological characteristics and heterogeneity of individual original tumors, display a unique advantage for individualized therapy and predicting clinical outcomes. In this study, PDX and PDO models of advanced gallbladder cancer were established, and the consistency of biological characteristics between them and primary patient samples was confirmed using pathological analysis and RNA-sequencing. Additionally, we tested the efficacy of chemotherapeutic drugs, targeted drugs, and immune checkpoint inhibitors using these two models. The results demonstrated that gemcitabine combined with cisplatin induced significant therapeutic effects. Furthermore, treatment with immune checkpoint inhibitors elicited promising responses in both the humanized mice and PDO immune models. Based on these results, gemcitabine combined with cisplatin was used for basic treatment, and immune checkpoint inhibitors were applied as a complementary intervention for gallbladder cancer. The patient responded well to treatment and exhibited a clearance of tumor foci. Our findings indicate that the combined use of PDO and PDX models can guide the clinical treatment course for gallbladder cancer patients to achieve individualized and effective treatment.

Melatonin Attenuates Dasatinib-Aggravated Hypoxic Pulmonary Hypertension via Inhibiting Pulmonary Vascular Remodeling.

Dasatinib treatment is approved as first-line therapy for chronic myeloid leukemia. However, pulmonary hypertension (PH) is a highly morbid and often fatal side-effect of dasatinib, characterized by progressive pulmonary vascular remodeling. Melatonin exerts strong antioxidant capacity against the progression of cardiovascular system diseases. The present work aimed to investigate the effect of melatonin on dasatinib-aggravated hypoxic PH and explore its possible mechanisms. Dasatinib-aggravated rat experimental model of hypoxic PH was established by utilizing dasatinib under hypoxia. The results indicated that melatonin could attenuate dasatinib-aggravated pulmonary pressure and vascular remodeling in rats under hypoxia. Additionally, melatonin attenuated the activity of XO, the content of MDA, the expression of NOX4, and elevated the activity of CAT, GPx, and SOD, the expression of SOD2, which were caused by dasatinib under hypoxia. In vitro, dasatinib led to decreased LDH activity and production of NO in human pulmonary microvascular endothelial cells (HPMECs), moreover increased generation of ROS, and expression of NOX4 both in HPMECs and primary rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. Dasatinib up-regulated the expression of cleaved caspase-3 and the ratio of apoptotic cells in HPMECs, and also elevated the percentage of S phase and the expression of Cyclin D1 in primary PASMCs under hypoxia. Melatonin ameliorated dasatinib-aggravated oxidative damage and apoptosis in HPMECs, meanwhile reduced oxidative stress level, proliferation, and repressed the stability of HIF1-α protein in PASMCs under hypoxia. In conclusion, melatonin significantly attenuates dasatinib-aggravated hypoxic PH by inhibiting pulmonary vascular remodeling in rats. The possible mechanisms involved protecting endothelial cells and inhibiting abnormal proliferation of smooth muscle cells. Our findings may suggest that melatonin has potential clinical value as a therapeutic approach to alleviate dasatinib-aggravated hypoxic PH.

Development of synthetic high-density lipoprotein-based ApoA-I mimetic peptide-loaded docetaxel as a drug delivery nanocarrier for breast cancer chemotherapy.

In this study, a synthetic high-density lipoprotein (sHDL), peptide-based nanocarrier loaded with docetaxel (DTX) was constructed, against breast cancer. The thermodynamic and molecular dynamic analyses were conducted to examine the stability of nanoparticles synthesized from mimetic peptide 5 A and various types of phospholipids. Furthermore, the cellular uptake and in vivo fluorescence imaging analysis experiments, with scavenger receptor B-I (SR-BI) were carried out to examine the tumor-targeting ability of sHDL. The nanoparticles were investigated for their pharmacodynamic and cytotoxic effects to show their effectivity as anti-tumor agents. The results showed that the synthesized sHDL nanoparticles exhibited a high payload of DTX, sustained drug release properties, and excellent biocompatibility. Moreover, DTX-sHDL nanoparticles enhanced the uptake of DTX, increased the cytotoxicity against MCF-7 cells, and reduced the off-target side-effects to normal cells. Finally, experiments in 4T1 cell line-bearing mice indicate that inhibition of tumor growth by DTX-sHDL nanoparticles was superior to that of free DTX group. Thus, the sHDL nanoparticles are a promising drug delivery vehicle for improving the efficacy of anti-cancer drugs.

Designed construction of tween 60@2ß-CD self-assembly vesicles as drug delivery carrier for cancer chemotherapy.

We report a simple strategy to prepare Tween 60@2ß-CD self-assembly vesicles in aqueous solution as a new drug delivery carrier for cancer chemotherapy. The spherical shape of vesicles was confirmed by transmission electron microscopy (TEM) and mean particle sizes were about 33.7 nm, as measured by dynamic light scattering, micro-IR results indicated that the self-assembly vesicles was driven by hydrogen bonding. Hydrophilic doxorubicin (DOX) was successfully loaded into the self-assembly vesicles with drug loading content of 7.85% and loading efficiency of 42%. In addition, an in vitro cytotoxicity study and cellular uptake assays demonstrated that the DOX-loaded Tween 60@2ß-CD vesicles markedly enhanced the cellular uptake and cytotoxicity of DOX toward the Hela cells. Furthermore, when used to evaluate the in vivo therapeutic efficacy in mice bearing the breast cell line (4T1), DOX-loaded vesicles exhibited superior inhibition of tumor growth compared with the DOX solutions.

Melatonin inhibits the proliferation of breast cancer cells induced by bisphenol A via targeting estrogen receptor-related pathways.

BACKGROUND: Background: Bisphenol A (BPA) is an estrogen-like chemical widely contained in daily supplies. There is evidence that environmental exposure to BPA could contribute to the development of hormone-related cancers. As is reported in numerous studies, melatonin, an endogenous hormone secreted by the pineal gland, could markedly inhibit estrogen-induced proliferation of breast cancer (BC) cells. In this study, we intended to reveal the effects of melatonin on BPA-induced proliferation of estrogen receptor-positive BC cells. METHODS: Methods: We used methyl thiazolyl tetrazolium, luciferase reporter gene and western blotting assays to testify the effect of melatonin on BPA-mediated proliferation of MCF-7 and T47D cells. RESULTS: Methyl thiazolyl tetrazolium and colony formation assays showed that melatonin could significantly abolish BPA-elevated cell proliferation. Meanwhile, BPA-upregulated phosphorylation of ERK and AKT was decreased by melatonin treatment. Mechanistically, we found that BPA was capable of upregulating the protein levels of steroid receptor coactivators (SRC-1, SRC-3), as well as promoting the estrogen response element activity. However, the addition of melatonin could remarkably block the elevation of steroid receptor coactivators expression and estrogen response element activity triggered by BPA. CONCLUSION: Conclusions: Therefore, these results demonstrated that melatonin could abrogate BPA-induced proliferation of BC cells. Therapeutically, melatonin could be regarded as a potential medication for BPA-associated BC.

Sensitive, Selective, and Fast Detection of ppb-Level H2S Gas Boosted by ZnO-CuO Mesocrystal.

ZnO-CuO mesocrystal was prepared via topotactic transformation using one-step direct annealing of aqueous precursor solution and assembled into a H2S sensor. The ZnO-CuO mesocrystal-based sensor possesses good linearity and high sensitivity in the low-concentration range (10-200 ppb). Compared to pure CuO, the as-prepared ZnO-CuO mesocrystal sensor exhibited superior H2S sensing performance with a response ranging from 8.6 to 152 % towards H2S concentrations from 10 ppb to 10 ppm when applied at the optimized working temperature of 125 °C. The sensor showed excellent repeatability and good selectivity towards H2S gas even at a concentration four orders of magnitude lower than the interfering gases, such as H2, CO2, CO, NO2, acetone, and NH3. The improved sensitivity could be attributed partially to the effective diffusion of analyte gas through the mesocrystal surface and the abundant accessible active sites. Moreover, the nanoscale p-n junctions within the mesocrystal, which could effectively manipulate the local charge carrier concentration, are also beneficial to boost the sensing performance.

[Development of SPA-ELISA for detection of antibodies against rabies virus based on expression of main antigenic determinant of nucleoprotein].

To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000-1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.