VEGFR affects miR-3200-3p-mediated regulatory T cell senescence in tumour-derived exosomes in non-small cell lung cancer.

Numerous studies have demonstrated that regulatory T (Treg) cells play an important role in the tumour microenvironment (TME). The aim of this study was to investigate whether VEGFR2 affects the expression of miR-3200-3p in exosomes secreted by tumour cells, thereby influencing Treg senescence in the TME. The results showed that VEGFR2 expression level was the highest in Calu-1 cells, and after transfection with si-VEGFR2, the exosomes secreted from Calu-1 cells were extracted and characterised with no significant difference from the exosomes of the untransfected group, but the expression of miR-3200-3p in the exosomes of the transfected si-VEGFR2 group was elevated. The Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM) results suggested that exosomes highly expressing miR-3200-3p could inhibit Treg cell viability and promote apoptosis levels when treated with Treg cells. Detection of the senescence-associated proteins p16 INK4A and MMP3 by western blot (WB) revealed that exosomes highly expressing miR-3200-3p were able to elevate their protein expression levels. Tumour xenograft experiments demonstrated that exosomes with high miR-3200-3p expression promoted Treg cell senescence and inhibited subcutaneous tumour growth in nude mice. Dual-luciferase reporter assays and RNA pull-down assays showed that miR-3200-3p could be linked with DDB1. Overexpression of DDB1 reverses changes in DCAF1/GSTP1/ROS protein expression caused by exosomes with high miR-3200-3p expression. In conclusion, inhibition of VEGFR2 expression in tumour cells promotes the expression of miR-3200-3p in exosomes secreted by tumour cells. miR-3200-3p enters the TME through exosomes and acts on DDB1 in Treg cells to promote senescence of Treg cells to inhibit tumour progression.

Plasma sPD-L1 and VEGF levels are associated with the prognosis of NSCLC patients treated with combination immunotherapy.

The clinical significance of plasma soluble programmed cell death ligand 1 (sPD-L1) and vascular endothelial growth factor (VEGF) for non-small cell lung cancer (NSCLC) treated with the combination of anti-angiogenic therapy and anti-PD-L1 antibody (Ab) remain unknown. This study aimed to explore the association between plasma sPD-L1 and VEGF levels and the prognosis of NSCLC patients treated with the combination of Envafolimab and Endostar. Peripheral blood samples were collected from 24 NSCLC patients at baseline and after 6 weeks of treatment and were detected for sPD-L1 and VEGF levels. Both baseline and posttreatment sPD-L1 were significantly higher in progressive disease (PD) group than in controlled disease (CD) group (median: 77.5 pg/ml vs. 64.6 pg/ml, P  = 0.036, median: 8451 pg/ml vs. 5563 pg/ml, P  = 0.012). In multivariate analysis, lower baseline sPD-L1 levels were significantly associated with longer progression-free survival (PFS) (HR = 6.834, 95% CI: 1.350-34.592, P  = 0.020). There were significantly higher posttreatment VEGF levels in PD group compared with CD group (median: 323.7 pg/ml vs. 178.5 pg/ml, P  = 0.009). Higher posttreatment VEGF levels were significantly associated with shorter PFS in multivariate analysis (HR = 5.911, 95% CI: 1.391-25.122, P  = 0.016). Plasma sPD-L1 and VEGF levels are associated with the clinical response and prognosis of NSCLC patients treated with the combination of PD-L1 inhibitors and anti-angiogenetic therapy.

Safety and efficacy of PD-1 blockade-activated multiple antigen-specific cellular therapy alone or in combination with apatinib in patients with advanced solid tumors: a pooled analysis of two prospective trials.

BACKGROUND: The lethal effects of multiple antigen-specific cellular therapy (MASCT) may be enhanced by blocking PD-1 in vitro and vascular endothelial growth factor receptor 2 inhibitor (apatinib). We analyzed the pooled data from our phase I/II trials to determine the toxicity and efficacy of PD-1 blockade (SHR-1210)-activated MASCT (aMASCT) alone or in combination with apatinib in advanced solid tumors. METHODS: Patients with advanced solid tumors received aMASCT alone (n = 32) or aMASCT plus apatinib (500 mg q.d., n = 38) after standard treatment. The safety profile was the primary end point. The secondary end points were antitumor response, progression-free survival (PFS), and overall survival (OS). The circulating T cells were quantified before and after aMASCT infusion. RESULTS: Treatment-related adverse events (AEs) occurred in 18/32 (56.3%) and 25/38 (65.8%) patients in the aMASCT and aMASCT plus apatinib groups, respectively. No serious AEs were reported, and apatinib did not increase immunotherapy-related toxicity. The objective response rate (34.2% and 18.8%) and PFS (median 6.0 and 4.5 months, P = 0.002) were improved in the aMASCT plus apatinib group compared with the aMASCT group; however, the OS was not improved (median 10.0 and 8.2 months, P = 0.098). Multivariate analyses indicated that two or more cycles of aMASCT treatment was an independent and favorable prognostic factor of PFS and OS. The circulating T cells increased and Tregs decreased in both groups after one cycle of aMASCT treatment. CONCLUSIONS: Treatment with aMASCT plus apatinib was safe and effective for the management of advanced solid tumors.

Non-immune functions of B7-H3: bridging tumor cells and the tumor vasculature.

B7-H3 (CD276), an immune checkpoint molecule, is overexpressed in various types of cancer and their tumor vasculature, demonstrating significant associations with adverse clinical outcomes. In addition to its well-known immune functions, B7-H3 exhibits dual co-stimulatory/co-inhibitory roles in normal physiology and the tumor microenvironment. The non-immune functions of B7-H3 in tumor cells and the tumor vasculature, including promoting tumor cell anti-apoptosis, proliferation, invasion, migration, drug resistance, radioresistance, as well as affecting cellular metabolism and angiogenesis, have increasingly gained attention from researchers. Particularly, the co-expression of B7-H3 in both tumor cells and tumor endothelial cells highlights the higher potential and clinical utility of therapeutic strategies targeting B7-H3. This review aims to summarize the recent advances in understanding the non-immune functions of B7-H3 in tumors and provide insights into therapeutic approaches targeting B7-H3, focusing on its co-expression in tumor cells and endothelial cells. The aim is to establish a theoretical foundation and practical reference for the development and optimization of B7-H3-targeted therapies.

Case report: Envafolimab combined with Endostar in the treatment of advanced non-small cell lung cancer with malignant pleural effusion.

Malignant pleural effusion (MPE) is one of the common complications of lung cancer. The quality of life and prognoses for MPE patients are significantly compromised. Controlling the production of MPE can relieve patients' symptoms, improve their quality of life, and prolong their survival. This article presents a case of advanced non-small cell lung cancer (NSCLC) with MPE and negative driver genes. The patient received envafolimab and Endostar in combination, resulting in a complete reduction of MPE and durable clinical benefits. The exploratory use of this treatment method improved the quality of life of this patient and has the potential to prolong the survival of this patient.

Long noncoding RNA lnc-SNAPC5-3:4 inhibits malignancy by directly upregulating miR-224-3p in non-small cell lung cancer.

The mounting body of evidence demonstrates the growing importance of long noncoding RNAs in the advancement of tumors. This study aimed to investigate the molecular mechanism of lnc-SNAPC5-3:4 in non-small cell lung cancer (NSCLC). We investigated the expression of miR-224-3p and lnc-SNAPC5-3:4 in clinical NSCLC samples and NSCLC cell lines using reverse transcription polymerase chain reaction (RT-PCR). In vitro studies, A549 cell growth was estimated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EDU), and flow cytometry assays. In vivo studies, NSCLC tumorigenesis was determined using xenograft tumor mouse models, tumor growth was evaluated using antigen Kiel 67 (Ki67) staining, and tumor apoptosis was detected through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The relationship between lnc-SNAPC5-3:4 and miR-224-3p was determined by luciferase reporter gene assay. Results indicated that the expression of lnc-SNAPC5-3:4 was observed to be downregulated in NSCLC tissues and cell lines. After overexpression of lnc-SNAPC5-3:4 in cultured A549 cells, proliferation decreased and apoptosis increased. Furthermore, the expression of miR-224-3p was targeted and negatively regulated by lnc-SNAPC5-3:4. The lnc-SNAPC5-3:4 upregulation inhibited cell proliferation and promoted apoptosis, which was partially blocked by miR-224-3p overexpression in A549 cells. In addition, we constructed a subcutaneous inoculation model using BALB/c nude mice, and the results indicated that lnc-SNAPC5-3:4 overexpression restrained the growth of subcutaneous tumors, decreased Ki67 expression levels, and increased apoptosis, as indicated by TUNEL staining in nude mice. However, miR-224-3p transfection resulted in the reversal of the inhibitory effect of lnc-SNAPC5-3:4 on tumor growth. In conclusion, our study revealed that lnc-SNAPC5-3:4 inhibits tumor progression in NSCLC by targeting miR-224-3p. This study provides a potential therapeutic target for inhibiting NSCLC progression.

The role of plasma exosomal lnc-SNAPC5-3:4 in monitoring the efficacy of anlotinib in the treatment of advanced non-small cell lung cancer.

PURPOSE: Anlotinib is an oral small-molecule multitarget tyrosine kinase inhibitor that hampers neovascularization thus providing antitumor effect. The ALTER 0303 trial was a multicenter, double-blind, phase 3 randomized clinical study to evaluate the efficacy of anlotinib in patients with advanced non-small cell lung cancer (NSCLC). The ALTER 0303 results showed that patients in the anlotinib group had a median progression-free survival of 5.4 months, a significant improvement compared with 1.4 months in the placebo group; however, median overall survival was only extended by 3.3 months (9.6 vs 6.3 months). The problem of anlotinib resistance cannot be ignored, and an in-depth exploration of biomarkers of anlotinib treatment response is urgently needed to further improve the efficacy of anlotinib in the treatment of NSCLC. This study aimed to identify plasma exosome markers that could be used to monitor the efficacy of anlotinib. METHODS: We enrolled 5 patients with advanced NSCLC, and 15 blood samples were collected before anlotinib treatment, when the treatment was effective, and when the treatment was ineffective. The plasma exosomal RNA profiles were analyzed by whole-transcriptome sequencing at three different stages. The expression of dysregulated exosomal RNAs in 43 additional patients was also verified by real-time quantitative PCR. RESULTS: In the plasma exosomal RNA profiles of the 5 patients with advanced NSCLC during treatment with anlotinib, 7 miRNAs, 3 lncRNAs, and 83 mRNAs were significantly dysregulated. The regulation trend was opposite when the treatment was effective and ineffective, showing dynamic changes. After validation, we finally found that plasma exosomal lnc-SNAPC5-3:4 was significantly upregulated when anlotinib treatment was effective, and it was significantly downregulated when the treatment failed (p < 0.05). Thus, it can be used as a potential biomarker for monitoring the efficacy of anlotinib. CONCLUSION: Our results demonstrate the potential of plasma exosomal lnc-SNAPC5-3:4 as a biomarker for monitoring anlotinib efficacy.

NEAT 1 knockdown enhances the sensitivity of human non-small-cell lung cancer cells to anlotinib.

Anlotinib treatment of non-small cell lung cancer (NSCLC) is hindered by drug insensitivity. Downregulation of long non-coding RNA (lncRNA) NEAT1 can suppress the proliferation and invasion by NSCLC cells. This study explored the role of the combination of anlotinib with NEAT1 knockdown on NSCLC progression. A549 and NCI-H1975 cells were used to evaluate the effect of anlotinib with NEAT 1 knockdown on NSCLC cells in vitro. The proliferation, invasion, migration, and apoptosis of NSCLC cells were evaluated with CCK-8 assays, EdU staining, Transwell assays, and flow cytometry. The antitumor effect of anlotinib with NEAT 1 knockdown was further explored in a mouse xenograft model. NEAT 1 knockdown enhanced the inhibitory effect of anlotinib on NSCLC cell proliferation, migration, and invasion. NEAT 1 knockdown also increased the pro-apoptotic and cytotoxic effects of anlotinib through downregulation of the Wnt/ß-catenin signaling pathway. The inhibitory effect of anlotinib on tumor growth was boosted in the presence of NEAT 1 knockdown in vivo. NEAT 1 knockdown promoted NSCLC cell sensitivity to anlotinib in vitro and in vivo. Thus, combined treatment of anlotinib with NEAT 1 knockdown may provide a new combined therapeutic approach for NSCLC patients.

Exosomal miR-136-5p Derived from Anlotinib-Resistant NSCLC Cells Confers Anlotinib Resistance in Non-Small Cell Lung Cancer Through Targeting PPP2R2A.

BACKGROUND: Anlotinib resistance is a challenge for advanced non-small cell lung cancer (NSCLC). Understanding the underlying mechanisms against anlotinib resistance is of great importance to improve prognosis and treatment of patients with advanced NSCLC. METHODS: RT-qPCR assay was used to assess the level of miR-136-5p in anlotinib-resistant NSCLC cells and exosomes derived from anlotinib-resistant NSCLC cells. In addition, miR-136-5p level in tumor tissues from patients who exhibited a poor response to anlotinib therapy and patients who were therapy naïve or patients who exhibited a positive response to anlotinib therapy was detected by RT-qPCR assay. RESULTS: In this study, we found that high levels of plasma exosomal miR-136-5p is correlated with clinically poor anlotinib response. In addition, anlotinib-resistant NSCLC cells promoted parental NSCLC cell proliferation via transferring functional miR-136-5p from anlotinib-resistant NSCLC cells to parental NSCLC cells via exosomes. Moreover, exosomal miR-136-5p could endow NSCLC cells with anlotinib resistance by targeting PPP2R2A, leading to the activation of Akt pathway. Furthermore, miR-136-5p antagomir packaging into anlotinib-resistant NSCLC cell-derived exosomes functionally restored NSCLC cell anlotinib sensitivity in vitro. Animal studies showed that A549/anlotinib cell-derived exosomal miR-136-5p agomir promoted A549 cell anlotinib resistance in vivo. CONCLUSION: Collectively, these findings indicated that anlotinib-resistant NSCLC cell-derived exosomal miR-136-5p confers anlotinib resistance in NSCLC cells by targeting PPP2R2A, indicating miR-136-5p may act as a potential biomarker for anlotinib response in NSCLC.

Effects of microRNA regulation on antiangiogenic therapy resistance in non-small cell lung cancer.

Antiangiogenic drugs have become a standard therapeutic regimen for advanced non-small cell lung cancer (NSCLC); however, many issues remain to be solved. Identifying specific markers to predict patient response to antiangiogenic drugs to ensure therapeutic efficacy would increase their clinical benefit. MicroRNAs (miRNAs) are involved in the process of resistance to antiangiogenic therapy, as they regulate various key signaling pathways. Therefore, miRNAs may be used as targets for reversing tumor resistance to antiangiogenic therapy. This article reviews the molecular mechanisms of antiangiogenic therapy resistance and the specific mechanisms of miRNA regulation of resistance. Signal transducer and activator of transcription 3 (STAT3) is one of multiple target genes of miRNAs, and is closely related to antiangiogenic research. Thus, it is described separately in this review article.

Apatinib sensitizes chemoresistant NSCLC cells to doxetaxel via regulating autophagy and enhances the therapeutic efficacy in advanced and refractory/recurrent NSCLC.

The prognosis of advanced non­small cell lung cancer (NSCLC) is poor; therefore, identifying novel treatment strategies for patients with NSCLC is important. The present study aimed to investigate the efficacy of apatinib plus docetaxel vs. docetaxel alone, as well as their effects on regulating autophagy markers in patients with advanced NSCLC. Furthermore, it was evaluated whether apatinib sensitized chemoresistant NSCLC cells to docetaxel via regulating autophagy. A total of 39 patients with advanced NSCLC were consecutively enrolled and treated with apatinib plus docetaxel (n=19) or docetaxel alone (n=20) for four treatment cycles. The treatment response, adverse events and expression levels of autophagy markers [(light chain 3 α (LC3A) and Beclin­1] were evaluated in tumor samples, which were obtained via biopsy, before treatment and after 2­cycle treatment. In addition, in a mechanistic in vitro experiment, apatinib, docetaxel, the autophagy activator rapamycin and the autophagy inhibitor 3­methyladenine (3­MA) were used to treat docetaxel­resistant A549 (A549/DTX) cells alone or in various combinations. The expression levels of LC3A, Beclin­1, poly (ADP) ribose polymerase (PARP) and phosphorylated (p)­AKT were detected via western blotting, while the cell apoptosis rate was detected with an Annexin V/PI assay. The overall remission rate (37 vs. 10%; P=0.047) and disease control rate (84 vs. 45%; P=0.011) were increased in the Apatinib plus docetaxel group compared with the Docetaxel group. Most of the adverse events were mild and tolerable, and there was no difference between the two groups except for total hypertension and hand­foot syndrome, which were higher in the Apatinib plus docetaxel group). Compared with the levels prior to treatment, Beclin­1 and LC3A remained unchanged post­treatment in the Apatinib plus docetaxel group, while they were increased in the Docetaxel group. Docetaxel increased LC3A, Beclin­1 and p­AKT expression levels, PARP cleavage and the cell apoptosis rate in A549/DTX cells, and rapamycin further enhanced, while 3­MA reduced these effects of docetaxel. Moreover, apatinib repressed LC3A, Beclin­1, p­AKT expression levels and promoted the cell apoptosis rate in A549/DTX cells and docetaxel­treated A549/DTX cells. In conclusion, apatinib synergize the effect of docetaxel in treating patients with advanced NSCLC and chemoresistant NSCLC cells via inhibiting autophagy.

Apatinib Combined with Local Irradiation Leads to Systemic Tumor Control via Reversal of Immunosuppressive Tumor Microenvironment in Lung Cancer.

PURPOSE: This study aimed to investigate the potential systemic antitumor effects of stereotactic ablative radiotherapy (SABR) and apatinib (a novel vascular endothelial growth factor receptor 2 inhibitor) via reversing the immunosuppressive tumor microenvironment for lung carcinoma. MATERIALS AND METHODS: Lewis lung cancer cells were injected into C57BL/6 mice in the left hindlimb (primary tumor; irradiated) and in the right flank (secondary tumor; nonirradiated). When both tumors grew to the touchable size, mice were randomly divided into eight treatment groups. These groups received normal saline or three distinct doses of apatinib (50 mg/kg, 150 mg/kg, and 200 mg/kg) daily for 7 days, in combination with a single dose of 15 Gy radiotherapy or not to the primary tumor. The further tumor growth/regression of mice were followed and observed. RESULTS: For the single 15 Gy modality, tumor growth delay could only be observed at the primary tumor. When combining SABR and apatinib 200 mg/kg, significant retardation of both primary and secondary tumor growth could be observed, indicated an abscopal effect was induced. Mechanism analysis suggested that programmed death-ligand 1 expression increased with SABR was counteract by additional apatinib therapy. Furthermore, when apatinib was combined with SABR, the composition of immune cells could be changed. More importantly, this two-pronged approach evoked tumor antigen-specific immune responses and the mice were resistant to another tumor rechallenge, finally, long-term survival was improved. CONCLUSION: Our results suggested that the tumor microenvironment could be managed with apatinib, which was effective in eliciting an abscopal effect induced by SABR.

Effects of combined inhibition of STAT3 and VEGFR2 pathways on the radiosensitivity of non-small-cell lung cancer cells.

PURPOSE: The goals of this study were to determine the effects of combined inhibition of STAT3 and vascular endothelial growth factor receptor 2 (VEGFR2) pathways on the radiosensitivity of non-small-cell lung cancer (NSCLC) cells, and to assess the underlying mechanisms. METHODS: The expressions of VEGFR2, STAT3, related signaling molecules, hypoxia-inducible factor 1-alpha (HIF-1α), and cyclin D1 were determined by Western blotting. Radiosensitivity was assessed using the colony-forming assay, and cell cycle and cell death were analyzed by flow cytometry. A nude mouse xenograft tumor model of Calu-1 cells was established. The hepatorenal toxicity of the above-mentioned treatment on tumor-bearing mice was observed by H&E staining. The expression of STAT3, VEGFR2, HIF-1α, and cyclin D1 of the transplanted tumor tissues was detected by immunohistochemistry. Apoptosis of tumor tissues was evaluated by TUNEL staining. RESULTS: In vitro, we selected two cell lines with high expression levels of STAT3, including Calu-1 cells that exhibit high VEGFR2 expression and A549 cells that exhibit low VEGFR2 expression. When apatinib treatment was combined with S3I-201, the expression of VEGFR2, STAT3, and their downstream signaling molecules was significantly decreased (P<0.01). There was an increase in cell death and G2/M phase arrest after treatments, with the most significant changes occurring upon dual inhibition of STAT3 and VEGFR2 (P<0.01). In vivo, combined treatment of radiotherapy and dual inhibition of VEGFR2 and STAT3 was well tolerated and did not deliver additional toxicity. Compared with the control group and the radiation treatment (RT) + apatinib or RT + S3I-201 duplex group, the expression level of STAT3, p-STAT3, VEGFR2, HIF-1α, and cyclin D1 in the triple group (RT + apatinib + S3I-201) was the lowest, and the proportion of apoptotic cells was the highest (P<0.05). CONCLUSION: The combined inhibition of VEGFR2 and STAT3 is effective in enhancing radiosensitizing effects in NSCLC cells.

miR­497 suppresses malignant phenotype in non­small cell lung cancer via targeting KDR.

The aim of the present study was to evaluate the expression of microRNA­497 (miR­497) in non­small cell lung cancer (NSCLC) tissues and cell lines, and to investigate possible mechanisms associated with its regulatory role on cell behaviors. The expression level of miR­497 was evaluated in 15 cases of NSCLC tissues and 8 adjacent normal tissues, and in 8 NSCLC cell lines, including H1975, A549, H358, H1650, H460, Calu­1, H1299 and H292, by reverse transcription­quantitative polymerase chain reaction. Effects of miR­497 overexpression on cell proliferation, invasion, apoptosis and radiosensitivity were examined with a Cell Counting Kit­8 assay, Matrigel assay, flow cytometry and a clone formation assay in vitro, respectively, and in an in vivo ectopic tumor nude mice model. A dual luciferase reporter assay was employed for interaction between miR­497 and its target gene kinase insert domain receptor (KDR). A significantly decreased level of miR­497 was determined in NSCLC tissues, compared with adjacent normal tissues, and Calu­1 and H1975 exhibited the lowest miR­497 expression among the 8 NSCLC cell lines. miR­497 overexpression could inhibit cell proliferation and invasion, promote cancer cell apoptosis and decrease cell clone formation following radiation treatment in vitro, and decrease tumor growth in vivo. Furthermore, a dual luciferase reporter assay revealed that KDR as the target gene for miR­497. It was demonstrated that miR­497 was downregulated in NSCLC specimens. Additionally, miR­497 directly targeted and downregulated KDR expression, and inhibited malignant behaviors of NSCLC cells. These data indicated that miR­497 could serve as a tumor suppressor gene involved in NSCLC pathogenesis.

Autophagy inhibition potentiates the anti-angiogenic property of multikinase inhibitor anlotinib through JAK2/STAT3/VEGFA signaling in non-small cell lung cancer cells.

BACKGROUND: The efficacy and safety of multikinase inhibitor anlotinib have been confirmed in the treatment of advanced non-small cell lung cancer (NSCLC). However, the direct functional mechanisms of tumor lethality mediated by anlotinib were not fully elucidated, and the underlying mechanisms related to resistance remain largely elusive. METHODS: Cell viability, colony formation, apoptosis and tumor growth assays were performed to examine the effect of anlotinib on lung cancer cells in vitro and in vivo. The punctate patterns of LC3-I/II were detected by confocal microscopy. HUVECs motility was detected using Transwell and scratch wound-healing assay. To visualize the microvessels, tubular formation assay was performed. The expression of LC3-I/II and beclin-1 and the changes of JAK2/STAT3/VEGFA pathway were detected by western blotting. The VEGFA levels in tumor supernatant were measured by ELISA. RESULTS: Anlotinib treatment decreased cell viability and induced apoptosis in Calu-1 and A549 cells. Moreover, anlotinib induced human lung cancer cell autophagy in a dose- and time-dependent manner. Blocking autophagy enhanced the cytotoxicity and anti-angiogenic ability of anlotinib as evidenced by HUVECs migration, invasion, and tubular formation assay. Co-administration of anlotinib and chloroquine (CQ) further reduced VEGFA level in the tumor supernatant, compared with that of anlotinib or CQ treatment alone. When autophagy was induced by rapamycin, the JAK2/STAT3 pathway was activated and VEGFA was elevated, which was attenuated after deactivating STAT3 by S3I-201. Further in vivo studies showed that anlotinib inhibited tumor growth, induced autophagy and suppressed JAK2/STAT3/VEGFA pathway, and CQ enhanced this effect. CONCLUSION: Anlotinib induced apoptosis and protective autophagy in human lung cancer cell lines. Autophagy inhibition further enhanced the cytotoxic effects of anlotinib, and potentiated the anti-angiogenic property of anlotinib through JAK2/STAT3/VEGFA signaling.

Role of the NRP-1-mediated VEGFR2-independent pathway on radiation sensitivity of non-small cell lung cancer cells.

PURPOSE: To determine if inhibiting neuropilin-1 (NRP-1) affects the radiosensitivity of NSCLC cells through a vascular endothelial growth factor receptor 2 (VEGFR2)-independent pathway, and to assess the underlying mechanisms. METHODS: The expression of VEGFR2, NRP-1, related signaling molecules, abelson murine leukemia viral oncogene homolog 1 (ABL-1), and RAD51 were determined by RT-PCR and Western blotting, respectively. Radiosensitivity was assessed using the colony-forming assay, and the cell apoptosis were analyzed by flow cytometry. RESULTS: We selected two cell lines with high expression levels of VEGFR2, including Calu-1 cells that have high NRP-1 expression, and H358 cells that have low NRP-1 expression. Upon inhibition of p-VEGFR2 by apatinib in Calu-1 cells, the expression of NRP-1 protein and other related proteins in the pathway was still high. Upon NRP-1 siRNA treatment, the expression of both NRP-1 and RAD51 decreased (p < 0.01; p < 0.05). Upon ABL-1 siRNA treatment, the expression of NRP-1 was increased and the expression of RAD51 was unchanged. Calu-1 cells treated with NRP-1 siRNA exhibited significantly higher apoptosis and radiation sensitivity in radiation therapy compared to Calu-1 cells treated with apatinib alone (p < 0.01; p < 0.01). The apoptosis and radiation sensitivity in H358 cells with NRP-1 overexpression was similar to the control group regardless of VEGFR2 inhibition. CONCLUSIONS: We demonstrated that when VEGFR2 was inhibited, NRP-1 appeared to regulate RAD51 expression through the VEGFR2-independent ABL-1 pathway, consequently regulating radiation sensitivity. In addition, the combined inhibition of VEGFR2 and NRP-1 appears to sensitize cancer cells to radiation.

The role and significance of VEGFR2+ regulatory T cells in tumor immunity.

Tumor development is closely related to angiogenesis, and VEGFR2 plays an important role in tumor angiogenesis. It is broadly expressed in the blood vessels, especially in the microvessels of tumor tissues. Furthermore, VEGFR2 is detected on the surface of the cell membrane in various immune cells, such as dendritic cells, macrophages, and regulatory T cells (Tregs). Tregs, which are one of the key negative regulatory factors in tumor immune microenvironments, show high-level expression of VEGFR2 which participates in the regulation of immunosuppressive function. VEGFR2+ Tregs play a potent suppressive role in the formation of immunosuppressive microenvironments. A large number of reports have proven the synergistic effects between targeted therapy for VEGFR2 and immunotherapy. The depression of VEGFR2 activity on T cells can significantly reduce the infiltration of Tregs into the tumor tissue. Targeted therapy for VEGFR2+ Tregs also provides a new choice for the clinical treatment of malignant solid tumors. In this paper, the role and significance of VEGFR2+ Tregs in tumor immunity in recent years are reviewed.

Apatinib concurrent gemcitabine for controlling malignant ascites in advanced pancreatic cancer patient: A case report.

RATIONALE: Malignant ascites (MA) is one of the poor prognostic factors for advanced pancreatic cancer and can bring about serious symptoms. The improvement of quality of life for patients is priority. However, there is no standard method for the treatment for pancreatic cancer-mediated MA. Apatinib is a novel and highly selective tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor-2. There are no reports of concurrent apatinib with gemcitabine in patients with pancreatic cancer-mediated MA. PATIENT CONCERNS: Herein, we presented a 64-year-old man patient who visited hospital due to abdominal pain for 1 month. DIAGNOSES: He was initially diagnosed with pancreatic cancer and his first symptom was MA. INTERVENTIONS: After failing in tube drainage and gemcitabine therapy, the patient received gemcitabine combined apatinib orally and after administrated 1 month, the MA was evaluated as nearly clear response according to the RECIST 1.1 standard, and without further need of paracentesis. The CEA and CA199 reached the lowest level after administrating for 2.5 months during the treatment process. OUTCOMES: 10.5 months following apatinib administration, the patient achieved a progression-free survival for more than 11 months. Hypertension (grade IV), hand-foot syndrome (grade I) and proteinuria (grade II) were observed. LESSONS: It indicated that apatinib concurrent gemcitabine may be a superior choice for pancreatic cancer-mediated MA. Further clinical trials required to confirm its efficacy and safety.

Selenite inhibits glutamine metabolism and induces apoptosis by regulating GLS1 protein degradation via APC/C-CDH1 pathway in colorectal cancer cells.

Glutaminolysis is important for metabolism and biosynthesis of cancer cells, and GLS is essential in the process. Selenite is widely regarded as a chemopreventive agent against cancer risk. Emerging evidence suggests that it also has chemotherapeutic potential in various cancer types, but the mechanism remains elusive. We demonstrate for the first time that supranutritional dose of selenite suppresses glutaminolysis by promoting GLS1 protein degradation and apoptosis. Mechanistically, selenite promotes association of APC/C-CDH1 with GLS1 and leads to GLS1 degradation by ubiquitination, this process is related to induction of PTEN expression. In addition, GLS1 expression is increased in human colorectal cancer tissues compared with normal mucosae. Our data provide a novel mechanistic explanation for the anti-cancer effect of selenite from a perspective of cell metabolism. Moreover, our results indicate that glutaminolysis especially GLS1 could be an attractive therapeutic target in colorectal cancer.

Selenite-induced autophagy antagonizes apoptosis in colorectal cancer cells in vitro and in vivo.

In the present study, we aimed to investigate the relationship between autophagy and apoptosis in selenite­treated colorectal cancer (CRC) cells. The effects of selenite on HCT116 and SW480 cell apoptosis were investigated with an Annexin V/propidium iodide (PI) double staining kit by flow cytometry. The punctate of LC3 protein following treatment with selenite was observed by a laser scanning confocal microscope and by transmission electron microscopy. Using western blot assays, we detected the apoptotic and autophagic markers in both CRC cells and mouse xenograft tumor models. We found that sodium selenite induced autophagy in the two CRC cell lines. Consistent with the in vitro results, we observed that the expression of autophagy marker LC3 was increased. Finally, we discovered that modulation of reactive oxygen species by MnTMPyP inhibited autophagy, while H2O2 activated autophagy. These results help to elucidate the anticancer effect of selenium, providing further evidence to exploit novel anticancer drugs targeting selenium.