Acetylation of transcription factor BpTCP20 by acetyltransferase BpPDCE23 modulates salt tolerance in birch.

Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors function in abiotic stress responses. However, how TCPs confer salt tolerance is unclear. Here, we characterized a TCP transcription factor, BpTCP20, that responds to salt stress in birch (Betula platyphylla Suk). Plants overexpressing BpTCP20 displayed increased salt tolerance, and Bptcp20 knockout mutants displayed reduced salt tolerance relative to the wild-type (WT) birch. BpTCP20 conferred salt tolerance by mediating stomatal closure and reducing reactive oxygen species (ROS) accumulation. Chromatin immunoprecipitation sequencing showed that BpTCP20 binds to NeuroD1, T-box, and two unknown elements (termed TBS1 and TBS2) to regulate target genes. In birch, salt stress led to acetylation of BpTCP20 acetylation at lysine 259. A mutated BpTCP20 variant (abolished for acetylation, termed BpTCP20259) was overexpressed in birch, which led to decreased salt tolerance compared with plants overexpressing BpTCP20. However, BpTCP20259-overexpressing plants still displayed increased salt tolerance relative to untransformed WT plants. BpTCP20259 showed reduced binding to the promoters of target genes and decreased target gene activation, leading to decreased salt tolerance. In addition, we identified dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (BpPDCE23), an acetyltransferase that interacts with and acetylates BpTCP20 to enhance its binding to DNA motifs. Together, these results suggest that BpTCP20 is a transcriptional regulator of salt tolerance, whose activity is modulated by BpPDCE23-mediated acetylation.

A reverse chromatin immunoprecipitation technique based on the CRISPR-dCas9 system.

DNA-protein interaction is one of the most crucial interactions in biological processes. However, the technologies available to study DNA-protein interactions are all based on DNA hybridization; however, DNA hybridization is not highly specific and is relatively low in efficiency. RNA-guided DNA recognition is highly specific and efficient. To overcome the limitations of technologies based on DNA hybridization, we built a DNA-binding protein capture technology based on the clustered regularly interspaced palindromic repeats (CRISPR)-dead Cas9 (dCas9) system and transient genetic transformation, termed reverse chromatin immunoprecipitation based on CRISPR-dCas9 system (R-ChIP-dCas9). In this system, dCas9 was fused with Strep-Tag II to form a fusion protein for StrepTactin affinity purification. Transient transformation was performed for the expression of dCas9 and guide RNA (gRNA) to form the dCas9-gRNA complex in birch (Betula platyphylla) plants, which binds to the target genomic DNA region. The dCas9-gRNA-DNA complex was crosslinked, then the chromatin was sonicated into fragments, and purified using StrepTactin beads. The proteins binding to the target genomic DNA region were identified using mass spectrometry. Using this method, we determined the upstream regulators of a NAM, ATAF, and CUC (NAC) transcription factor (TF), BpNAC090, and 32 TFs potentially regulating BpNAC090 were identified. The reliability of R-ChIP-dCas9 was further confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assays, and yeast one-hybrid. This technology can be adapted to various plant species and does not depend on the availability of a stable transformation system; therefore, it has wide application in identifying proteins bound to genomic DNA.

Amphichdiral enhancement on singlet oxygen generation and stable thallium immobilization using iron-driven copper oxide.

Thallium (Tl) as a prominent priority contaminant in aquatic environment necessitates rigorous regulation. However, limited horizon devotes the impact of selective oxidation on the process of thallium purification. In this study, selective active radical of singlet oxygen (1O2) was continually generated for Tl(Ⅰ) oxidation accomplished with efficient Tl(Ⅲ) immobilization using iron-driven copper oxide (CuFe)/peroxymonosulfate (PMS). Fe-doping changed the active center of electronic structure for enhancing the catalytic and adsorptive reactivities, and installed magnetism for solid-liquid separation. Rapid reaction rate (0.253 min-1) coupled with vigorous elimination efficiency (98.32%) relied on electrostatic attraction, surface complexation, and H-bond interaction. EPR and XPS analyses demonstrated that the synergistic effects of ≡ Cu(Ⅰ)/≡Cu(Ⅱ) and ≡ Fe(Ⅲ)/≡Fe(Ⅱ) redounded to the sustained generation of 1O2 through the pathway of PMS → •O2- → 1O2, and 1O2 exploited an advantage to selectively oxidize Tl(Ⅰ) to Tl(Ⅲ). 3D isosurface cubic charts revealed that the immobilizing ability of Tl(Ⅲ) hydrate for CuFe was notably superior to that of Tl(Ⅲ) hydrate for CuO and Tl(Ⅰ) hydrate for CuO/CuFe, which further attested surface reactivity promoted stable immobilization form. This work develops the continuous generation of 1O2 and stable immobilization with the goal of efficiently cleansing Tl-containing wastewater.

Anlotinib plus icotinib as a potential treatment option for EGFR-mutated advanced non-squamous non-small cell lung cancer with concurrent mutations: final analysis of the prospective phase 2, multicenter ALTER-L004 study.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation and concurrent mutations have a poor prognosis. This study aimed to examine anlotinib plus icotinib as a first-line treatment option for advanced NSCLC carrying EGFR mutation with or without concurrent mutations. METHODS: This phase 2, single-arm, multicenter trial (ClinicalTrials.gov NCT03736837) was performed at five hospitals in China from December 2018 to November 2020. Non-squamous NSCLC cases with EGFR-sensitizing mutations were treated with anlotinib and icotinib. The primary endpoint was progression-free survival (PFS). Secondary endpoints included the objective response rate (ORR), disease control rate (DCR), overall survival (OS), and toxicity. RESULTS: Sixty participants were enrolled, including 31 (52%) and 29 (48%) with concurrent mutations and pathogenic concurrent mutations, respectively. The median follow-up was 26.9 (range, 15.0-38.9) months. ORR and DCR were 68.5% and 98.2%, respectively. Median PFS was 15.1 (95%CI: 12.6-17.6) months which met the primary endpoint, median DoR was 13.5 (95%CI: 10.0-17.1) months, and median OS was 30.0 (95%CI: 25.5-34.5) months. Median PFS and OS in patients with pathogenic concurrent mutations were 15.6 (95%CI: 12.5-18.7) months and not reached (95%CI: 17.46 months to not reached), respectively. All patients experienced TRAEs, including 26 (43%) and 1 (1.7%) who had grade ≥ 3 and serious treatment-related adverse events (TRAEs). CONCLUSIONS: Anlotinib combined with icotinib was effective and well-tolerated as a first-line treatment option for EGFR mutation-positive advanced NSCLC with or without concurrent mutations. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03736837.

Unlocking the Full Potential of Cas12a: Exploring the Effects of Substrate and Reaction Conditions on Trans-Cleavage Activity.

The trans-cleavage activity of Cas12a has been widely used with various applications. Here, we report that the trans-cleavage activity of Cas12a can be significantly affected by the fluorescent probe length and reaction buffer. The optimal probe length for Cas12a is found to be 15 nucleotides, and the optimal buffer is NEBuffer 4. Compared to the popularly used reaction conditions, the activity of Cas12a is improved by about 50-fold. In addition, the detection limit of Cas12a for DNA targets has been reduced by nearly three orders of magnitude. Our method provides a powerful tool for Cas12a trans-cleavage activity applications.

Assessment of a novel aminated magnetic adsorbent with excellent adsorption capacity for dyes and drugs.

Dyes and drugs with high toxicity and low biodegradability pose risk to human health and ecological security, and should be purified efficiently from effluents before discharge. Traditional adsorbents are limited by the insufficient active adsorption sites and low stability. In this study, a novel aminated magnetic adsorbent (MCTs) was fabricated via two cross-linking steps using chitosan and triethylenetetramine to fill the gaps between current adsorbent and performance requirements. The morphological and physicochemical characteristics of the as-prepared MCTs were determined and identified with the aid of several characterization techniques. The adsorption performance of dyes and drugs was also investigated and represented by their adsorption capacities. In particular, the adsorption capacities of Congo Red, Chicago Sky Blue, Reactive Brilliant Red, and Ibuprofen were 583.11, 465.01, 403.12, and 291.71 mg/g, respectively. They also remained at around 80% after four reuse cycles. MCTs were adsorbed via a monolayer spontaneous chemical reaction, and hydrogen bonding and electrostatic interaction were the dominant adsorption mechanisms. These results demonstrated that the preparation of MCTs via two cross-linking steps enhanced the adsorbents' adsorption capacity, reusability, and stability. They provided a new perspective for the preparation of high-efficient adsorbents and the purification of dye- and drug-polluted wastewater.

Arabidopsis heat shock transcription factor HSFA7b positively mediates salt stress tolerance by binding to an E-box-like motif to regulate gene expression.

Plant heat shock transcription factors (HSFs) are involved in heat and other abiotic stress responses. However, their functions in salt tolerance are little known. In this study, we characterized the function of a HSF from Arabidopsis, AtHSFA7b, in salt tolerance. AtHSFA7b is a nuclear protein with transactivation activity. ChIP-seq combined with an RNA-seq assay indicated that AtHSFA7b preferentially binds to a novel cis-acting element, termed the E-box-like motif, to regulate gene expression; it also binds to the heat shock element motif. Under salt conditions, AtHSFA7b regulates its target genes to mediate serial physiological changes, including maintaining cellular ion homeostasis, reducing water loss rate, decreasing reactive oxygen species accumulation, and adjusting osmotic potential, which ultimately leads to improved salt tolerance. Additionally, most cellulose synthase-like (CSL) and cellulose synthase (CESA) family genes were inhibited by AtHSFA7b; some of them were randomly selected for salt tolerance characterization, and they were mainly found to negatively modulate salt tolerance. By contrast, some transcription factors (TFs) were induced by AtHSFA7b; among them, we randomly identified six TFs that positively regulate salt tolerance. Thus, AtHSFA7b serves as a transactivator that positively mediates salinity tolerance mainly through binding to the E-box-like motif to regulate gene expression.

Tumor necrosis factor superfamily 15 promotes lymphatic metastasis via upregulation of vascular endothelial growth factor-C in a mouse model of lung cancer.

Lymphatic metastasis is facilitated by lymphangiogenic growth factor vascular endothelial growth factor-C (VEGFC) that is secreted by some primary tumors. We previously identified tumor necrosis factor superfamily 15 (TNFSF15), a blood vascular endothelium-derived cytokine, in lymphatic endothelial cells, as a key molecular modulator during lymphangiogenesis. However, the effect of TNFSF15 on tumor lymphatic metastasis and the underlying molecular mechanisms remain unclear. We report here that TNFSF15, which is known to inhibit primary tumor growth by suppressing angiogenesis, can promote lymphatic metastasis through facilitating lymphangiogenesis in tumors. Mice bearing tumors induced by A549 cells stably overexpressing TNFSF15 exhibited a significant increase in densities of lymphatic vessels and a marked enhancement of A549 tumor cells in newly formed lymphatic vessels in the primary tumors as well as in lymph nodes. Treatment of A549 cells with TNFSF15 results in upregulation of VEGFC expression, which can be inhibited by siRNA gene silencing of death domain-containing receptor-3 (DR3), a cell surface receptor for TNFSF15. In addition, TNFSF15/DR3 signaling pathways in A549 cells include activation of NF-κB during tumor lymphangiogenesis. Our data indicate that TNFSF15, a cytokine mainly produced by blood endothelial cells, facilitates tumor lymphangiogenesis by upregulating VEGFC expression in A549 cells, contributing to lymphatic metastasis in tumor-bearing mice. This finding also suggests that TNFSF15 may have potential as an indicator for prognosis evaluation.

[Analysis of correlation factors for occurrence and progression-free survival of cavitating lung cancer in 947 cases].

OBJECTIVE: This study was designed to investigate the correlation factors for occurrence and progression-free survival of patients with cavitating lung cancer. METHODS: We collected the clinical data of 947 lung cancer patients. Tumor cavitation was observed in 51 patients at baseline and in 23 patients after treatment, while was not discovered in other 873 patients. Multifactor logistic regression was performed to analyze the correlation factors for occurrence. The independent predictors of PFS were analyzed with Cox proportional regression. Survival curves were constructed with the Kaplan-Meier product limit method and compared using the log-rank test. RESULTS: In the 947 cases, the proportion of cases with baseline cavitation was 5.4% and the incidence of cavitation after treatment was 2.6%. Multivariate logistic regression analysis revealed that the occurrence of baseline cavitation is related to age, history of diabetes, history of drinking, pathologic types, tumor location, tumor diameter and distant metastasis (P < 0.05). Multifactor logistic regression analysis revealed that the occurrence of post-therapeutic cavitation is related to sex, pathologic types and tumor diameter (P < 0.05).The median PFS of patients with baseline cavitation (7.3 months) was significantly longer than the cases without it (5.2 months) (P = 0.002). While there was no significant difference between the median PFS of patients with post-therapeutic cavitation and patients without it (5.1 months vs. 5.3 months, P = 0.060). Cox proportional regression analysis revealed that cyfra21-1 is related to PFS of patients with baseline cavitaion (P < 0.05) and smoking history is related to PFS of patients with post-therapeutic cavitaion (P < 0.05). CONCLUSIONS: Patients with baseline and post-therapeutic cavitation present different clinical features and progression-free survivals. The PFS of patients with baseline cavitation is longer than that of the cases without it. On the contrary, PFS of patients with post-therapeutic cavitation is shorter than the patients without it.

[Screening for prodromes of chemotherapy-induced vomiting and correlation between prodromes and chemotherapy-induced vomiting in lung cancer patients].

OBJECTIVE: To explore prodromes of chemotherapy-induced vomiting (CIV) and their association with CIV in lung cancer patients. METHODS: The prodromes of CIV in 250 lung cancer patients were analyzed. Logistic regression was used to determine the symptoms most likely correlated with CIV. One hundred fifty-seven patients received medical interventions. The development of correlative symptoms and occurrence of CIV between the intervention and non-intervention groups was analyzed. RESULTS: Among the 250 patients with the prodromes of CIV, the incidence rate of CIV was 67.2%. Logistic regression indicated that nausea, constipation, insomnia, hiccups, anorexia, and history of drinking were correlated with CIV (P < 0.05 for all). Among the 20 symptoms observed in this study, the incidence rates of relatively common symptoms were nausea (72.0%), anorexia (68.4%), taste changes (48.8%), constipation (45.6%), abdominal distension (45.6%), stomach distension(40.4%), and insomnia (40.0%). The incidence rats of all symptoms except hiccups before and after intervention had significant difference (P < 0.05 for all). The incidence rates of CIV were 30.0% in the intervention group and 50.6% in the non-intervention group, with a significant difference between the two groups (P = 0.009). CONCLUSIONS: Prodromes of CIV are closely related to the occurrence of CIV. Timely intervention for prodromes of CIV can reduce the incidence rate of CIV during chemotherapy in lung cancer patients.

The homeodomain (PHD) protein, PdbPHD3, confers salt tolerance by regulating squamosa promoter binding protein PdbSBP3 in Populus davidiana × P. bolleana.

Plant homeodomain (PHD) proteins are a family of zinc finger transcription factors that play roles in abiotic stress tolerance. However, their mechanisms in conferring salt tolerance are largely unknown. In this study, we characterized a PHD gene, PdbPHD3, from Populus davidiana × P. bolleana (Shanxin poplar) in response to salt stress. PdbPHD3 is a nuclear protein that is strongly induced by salt and abscisic acid (ABA) treatments. Overexpression of PdbPHD3 conferred salt tolerance, while silencing of PdbPHD3 increased sensitivity to salt. PdbPHD3 could enhance the activities of superoxide dismutase and peroxidase to reduce the abundance of reactive oxygen species, and enhance the osmotic potential by increasing the proline content. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that PdbPHD3 could bind to various DNA motifs, including the G-box ("CACGTG"), PALBOXAPC ("GGACGG"), and POLLEN1LELAT52 ("TTTCTT"). ChIP-seq combined with RNA sequencing identified a transcription factor gene, squamosa promoter binding protein 3 (PdbSBP3), which is directly regulated by PdbPHD3. Overexpression and silencing of PdbSBP3 improved and decreased salt tolerance, respectively. PdbSBP3 could also regulate all the physiological changes associated with salt tolerance, similar to PdbPHD3. These results suggest that PdbPHD3 confers salt tolerance by regulating PdbSBP3 to reduce ROS accumulation and increase proline content. Therefore, the regulatory axis of PdbPHD3 and PdbSBP3 confers salt tolerance in Shanxin poplar.

Birch WRKY transcription factor, BpWRKY32, confers salt tolerance by mediating stomatal closing, proline accumulation, and reactive oxygen species scavenging.

Plant WRKY transcription factors (TFs) play important roles in abiotic stress responses. However, how WRKY facilitate physiological changes to confer salt tolerance still needs to be studied. Here, we identified a WRKY TF from birch (Betula platyphylla Suk), BpWRKY32, which is significantly (P < 0.05) induced by salt stress. BpWRKY32 binds to W-box motif and is located in the nucleus. Under salt stress conditions, fresh weights (FW) of OE lines (BpWRKY32 overexpression lines) are increased by 66.36% than that of WT, while FW of knockout of BpWRKY32 (bpwrky32) lines are reduced by 39.49% compared with WT. BpWRKY32 regulates the expression of BpRHC1, BpNRT1, and BpMYB61 to reduce stomatal, and width-length ratio of the stomatal aperture in OE lines are reduced by 46.23% and 64.72% compared with in WT and bpwrky32 lines. BpWRKY32 induces P5CS expression, but inhibits P5CDH expression, leading to the proline content in OE lines are increased by 33.41% and 97.58% compared with WT and bpwrky32 lines. Additionally, BpWRKY32 regulates genes encoding SOD and POD family members, which correspondingly increases the activities of SOD and POD. These results suggested that BpWRKY32 regulates target genes to reduce the water loss rate, enhance the osmotic potential, and reduce the ROS accumulation, leading to improved salt tolerance.

Gene point mutation information translation and detection: Leveraging single base extension and CRISPR/Cas12a.

Gene point mutations play a significant role in the development of cancer. Therefore, developing a sensitive, specific, and universally applicable method for detecting gene point mutation is crucial for clinical diagnosis, prognosis, and cancer treatment. Recently, gene point mutation detection methods based on CRISPR/Cas12a detection have emerged. However, existing methods generally lack universality and specificity. In this study, we have developed a CRISPR/Cas12a-based method that combines improved allele-specific polymerase chain reaction and single base extension to translate the point mutation information in the target dsDNA into length information in ssDNA activators to overcome the limitations associated with PAM sequences in the CRISPR/Cas12a system. Our method achieved a detection limit of 0.002% for clinically significant EGFR T790M mutation. The CRISPR/Cas12a system we constructed demonstrates high sensitivity, specificity, and universality in detecting gene point mutations, making it a promising tool for clinical cancer screening.

Circulating endothelial cells and tumor blood volume as predictors in lung cancer.

The current criteria for evaluating antiangiogenic efficacy is insufficient as tumor shrinkage occurs after blood perfusion decreases. Tumor blood volume (BV) in computed tomography perfusion imaging and circulating endothelial cells (CEC) might predict the status of angiogenesis. The present study aimed to validate their representation as feasible predictors in non-small-cell lung carcinoma (NSCLC). A total of 74 patients was categorized randomly into two arms undergoing regimens of vinorelbine and cisplatin (Navelbine and platinum [NP]) with rh-endostatin or single NP. The response rate, perfusion imaging indexes and activated CEC (aCEC) during treatment were recorded. Progression-free survival (PFS) was determined through follow up. Correlations among the above indicators, response and PFS were analyzed: aCEC increased significantly in cases of progressive disease after single NP chemotherapy (P = 0.024). Tumor BV decreased significantly in cases with a clinical benefit in the combined arm (P = 0.026), whereas inverse correlations existed between ∆aCEC (post-therapeutic value minus the pre-therapeutic value) and PFS (P = 0.005) and between ∆BV and PFS (P = 0.044); a positive correlation existed between ∆aCEC and ∆BV. Therefore, both aCEC and tumor BV can serve as predictors, and detection of both indicators can help evaluate the chemo-antiangiogenic efficacy in NSCLC more accurately.

Transformation of tetracycline by multipurpose Fe-Mn-Cu nano-composite oxide: Dual-synergies and dual-mechanisms.

The interaction between tetracycline (TTC) and mixed metallic oxides remains unclear, and even complexation usually is ignored. This study firstly distinguished the triple functions of adsorption, transformation and complexation in presence of Fe-Mn-Cu nano-composite metallic oxide (FMC) on TTC. Rapid adsorption and faint complexation initiated the transformation that dominated the entire reactions at 180 min, which completed TTC removal (up to 99.04%) synergistically within 48 h. Environmental factors (dosage, pH and coexisting ions) had small influence on TTC removal, which primarily depended on the stable transformation characteristics of FMC. Kinetic models incorporating pseudo-second-order kinetics and transformation reaction kinetics demonstrated that the surface sites of FMC promoted electron transfer process through chemical adsorption and electrostatic attraction. ProtoFit program coupled with characterization methods concluded that Cu-OH was the main reaction site of FMC where the protonated surface favored to generate·O2-. Meanwhile, three metal ions developed simultaneous mediated transformation reactions on TTC in liquid phase, and·O2- induced the production of·OH. The transformed products were subjected to toxicity assessment, which had lost antimicrobial properties toward Escherichia coli. Insights gained from this study can refine the dual mechanisms of multipurpose FMC in solid and liquid phases underlying TTC transformation.

Mechanism and potential treatments for gastrointestinal dysfunction in patients with COVID-19.

The global coronavirus disease 2019 (COVID-19) has become one of the biggest threats to the world since 2019. The respiratory and gastrointestinal tracts are the main targets for severe acute respiratory syndrome coronavirus 2 infection for they highly express angiotensin-converting enzyme-2 and transmembrane protease serine 2. In patients suffering from COVID-19, gastrointestinal symptoms have ranged from 12% to 61%. Anorexia, nausea and/or vomiting, diarrhea, and abdominal pain are considered to be the main gastrointestinal symptoms of COVID-19. It has been reported that the direct damage of intestinal mucosal epithelial cells, malnutrition, and intestinal flora disorders are involved in COVID-19. However, the underlying mechanisms remain unclear. Thus, in this study, we reviewed and discussed the correlated mechanisms that cause gastrointestinal symptoms in order to help to develop the treatment strategy and build an appropriate guideline for medical workers.

Hesperidin Inhibits Lung Cancer In Vitro and In Vivo Through PinX1.

New drugs or active leads with high efficiency and low toxicity are needed in the treatment of lung cancer. Natural products are an important source of anti-tumor drugs. At present, there are many molecular-targeted anti-tumor drugs derived from natural products or their derivatives for tumor treatment or in clinical trials. Hesperidin is a flavanone isolated from the Rutaceae plant lime Citrus aurantium L. or Citrus sinensis Osbeck. It has been considered to inhibit cancer cell viability in vitro. However, the effect of hesperidin on lung cancer and its underlying mechanism remain unclear. In this study, we found that the pinX1 expression level is closely related to overall survival and plays an important role in regulating lung cancer cell proliferation, migration, invasion, and senescence. More importantly, hesperidin significantly increased pinX1 protein expression, and knockdown pinX1 by its specific siRNA blocked the protective effects of hesperidin. Moreover, we also assessed that hesperidin at 100 mg/kg is safe in vivo. These findings showed that hesperidin is a potential therapeutic candidate for preventing the progression of lung cancer.

Anlotinib Downregulates RGC32 Which Provoked by Bevacizumab.

Background: Bevacizumab is the representative drug in antiangiogenic therapy for lung cancer. However, it induced resistance in some neoplasm. Anlotinib, a novel multi-target tyrosine kinase inhibitor which has an inhibitory action on both angiogenesis and malignancy, is possible to reverse the resistance. Methods: Transwell migration and invasion experiments of bevacizumab with or without anlotinib were conducted to verify the activated/inhibited ability of lung adenocarcinoma cells. We sequenced A549 cells with enhanced migration and invasion abilities after bevacizumab treatment, screened out the differentially expressed gene and further confirmed by western blot and q-PCR assays. We also investigated immunohistochemical staining of tumor tissue in mice and human lung adenocarcinoma. Results: Bevacizumab facilitated migration and invasion of lung adenocarcinoma cells. Differentially expressed gene RGC32 was screened out. Bevacizumab upregulated the expression of RGC32, N-cadherin, and MMP2 through ERK-MAPK and PI3K-AKT pathways. Anlotinib downregulated their expression and reversed the effect of bevacizumab on A549 cells. In vivo experiments confirmed that higher-dose bevacizumab facilitated metastasis in tumor-bearing nude mice and upregulated the expression of RGC32, N-cadherin, and MMP2, whereas anlotinib abrogated its effect. Expression of both RGC32 and N-cadherin positively correlated with lymph node metastasis and stage in lung adenocarcinoma was found. Survival analysis revealed that higher expressions of RGC32 and N-cadherin were associated with poor progression-free survival and overall survival. Conclusions: Bevacizumab may promote invasion and metastasis of lung adenocarcinoma cells by upregulating RGC32 through ERK-MAPK and PI3K-AKT pathways to promote epithelial-mesenchymal transition, whereas anlotinib reverses the effect. RGC32 and N-cadherin are independent prognostic factors in lung adenocarcinoma.

[Efficacy of endostar combined with chemotherapy in multi-cycle treatment of patients with advanced non-small cell lung cancer].

OBJECTIVE: To observe the correlation between long term efficacy/safety and treatment cycles of rh-endostatin (endostar) combined with TP (paclitaxel plus cisplatin/carboplatin) or NP (navelbine plus cisplatin/carboplatin) regimens in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Twenty-five patients with advanced NSCLC confirmed by histopathology and/or cytology were enrolled in this study. Twenty-one patients underwent endostar combined with NP regimen and other four patients underwent endostar combined with TP regimen (all repeated 21 days) treatment. The therapeutic effects, quality of life (QOL) and adverse effects were evaluated according to RECIST criteria, Karnofsky performance scores and WHO grading of adverse effects, respectively. Our intention was to make knowledge of the therapeutic effects, median time to progression, one-year survival rate, median overall survival and adverse reactions. The amount of circulating endothelial cells (CEC) in peripheral blood was measured by flow cytometry. RESULTS: All the 25 patients were evaluable for efficacy and safety. They were comprised of 5 cases of PR, 14 cases of SD and 6 cases of PD. Of the 25 cases, RR was obtained in 5 cases (20.0%), CBR in 19 cases (76.0%), mTTP was 8 months and mOS was 19 months. Of the 14 patients with short treatment cycles (< 4), PR was obtained in 2 cases, SD in 6 cases and PD in 6 cases, RR was 14.3%. Of the 8 patients who obtained PR or SD, the median TTP was 6 months and median overall survival was 18 months. Of the 11 patients with long treatment cycles (≥ 4), PR was obtained in 3 cases, SD in 8 cases, RR was 27.3%, mTTP was 17 months and mOS was 26 months. After treatment, the amount of activated CECs was increased by (293 ± 12)/10(5) in patients with short treatment cycles, and decreased by (243 ± 181)/10(5) in patients with long treatment cycles. A positive correlation was found between the changes of activated CECs after therapy, time to progression (TTP) and treatment cycles (r = 0.970, P = 0.001; r = 0.829, P = 0.042, respectively). The quality of life (QOL) was improved in 12 cases (48.0%), stable in 10 cases (40.0%), and decreased in 3 cases (12.0%). Grade 3 and 4 toxicities were mainly related with chemotherapeutics, including neutropenia in 4 cases (16.0%), vomiting in 3 cases (12.0%) and arrhythmia in 1 case. No hypertension was observed. All the adverse reactions did not affect the following treatment, and there was no significant difference in incidence rate of grade 3 and 4 adverse events between the patients treated with long-term and short-term cycles. CONCLUSIONS: Endostar combined with TP or NP regimen chemotherapy is effective and safe in the treatment of advanced NSCLC, especially in patients with long term treatment cycles which can effectively prolong TTP and reach long term survival, but not increase adverse events. The QOL of patients can be improved or remain stable. The changes of CECs may be used as a useful maker in predicting the efficacy of the combination treatment.

XPO1 inhibition synergizes with PARP1 inhibition in small cell lung cancer by targeting nuclear transport of FOXO3a.

Patient mortality rates have remained stubbornly high for the past decades in small cell lung cancer (SCLC) because of having no standard targeted therapies with confirmed advantages at present. Poly [ADP-ribose] polymerase (PARP) inhibitors have shown promise in preclinical models but have had unsatisfactory clinical results in SCLC. By RNA-seq and isobaric tags for relative and absolute quantification (ITRAQ), we revealed that PARP1 inhibition led to the relocalization of forkhead box-O3a (FOXO3a) from nuclear to cytoplasm. By performing co-Immunoprecipitation (co-IP) and CRISPR-Cas9-mediated knockout plasmid we showed that FOXO3a was subject to exportin 1 (XPO1)-dependent nuclear export. We demonstrated the effects of the PARP inhibitor BMN673 on apoptosis and DNA damage were markedly enhanced by simultaneous inhibition of XPO1 in vitro. The combination of BMN673 and the XPO1 inhibitor selinexor inhibited primary SCLC cell proliferation in mini-patient-derived xenotransplants (miniPDXs) and markedly inhibited tumor growth without significant toxicity in xenograft models. The efficacy was enhanced for more than 2.5 times, compared to the single agent. Based on these findings, we further designed a novel dual PARP-XPO1 inhibitor and showed its effectiveness in SCLC. In this work, we illustrated that combining a PARP inhibitor with an XPO1 inhibitor is associated with significantly improved efficacy and tolerability. Dual PARP-XPO1 inhibition restored the FOXO3a balance and activity in SCLC. Collectively, targeting PARP1 and XPO1 opens new avenues for therapeutic intervention against SCLC, warranting further investigation in potential clinical trials.