Synergic effect of PD-1 blockade and endostar on the PI3K/AKT/mTOR-mediated autophagy and angiogenesis in Lewis lung carcinoma mouse model.
Wu Jing,Zhao Xiaogang,Sun Qifeng,Jiang Yunfeng,Zhang Weiquan,Luo Junwen,Li Yixin
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
BACKGROUND:Immunotherapy has been shown to be effective as a first-line treatment option for non-small cell lung cancer (NSCLC) patients. Unfortunately, it has failed to acquire an anticipant anti-tumour effect for relatively lower clinical benefit rates. It is therefore important to identify novel strategies for improving immunotherapy. Endostar is a novel recombinant human endostatin that exerts its anti-angiogenic effects via vascular endothelial growth factor (VEGF)-related signalling pathways. Anti-programmed death receptor 1 (PD-1) antibody is an immune checkpoint inhibitor that was developed to stimulate the immune system. In this study, the synergy of PD-1 blockade and endostar was assessed in a lung carcinoma mouse model. METHODS:Lewis lung carcinoma (LLC)-bearing mice were randomly assigned into three groups: controls, anti-PD-1 and anti-PD-1+endostar. The levels of cytokines such as interleukin (IL)-17, transforming growth factor-β1 (TGF-β1) and interferon-γ (IFN-γ) were measured with enzyme-linked immune sorbent assay (ELISA). The expression of VEGF, CD34 and CD31 was assessed with immunohistochemistry (IHC). The proportion of mature dendritic cells (mDC) and myeloid-derived suppressor cells (MDSC) was analysed with flow cytometry. The major proteins in PI3K/AKT/mTOR and autophagy were quantified with Western blot. RESULTS:Anti-PD-1 combined with endostar dramatically suppressed tumour growth in LLC mouse models. This synergistic effect resulted in decreased pro-inflammatory cytokine IL-17 and immunosuppressive factor TGF-β1 levels, increased IFN-γ secretion, reduced myeloid-derived suppressor cell (MDSC) accumulation, and reversed CD8 + T cell suppression. The expression of VEGF, CD34 and CD31 was significantly down-regulated, while tumour cell apoptosis and PI3K/AKT/mTOR-mediated autophagy was up-regulated. CONCLUSION:The combination of anti-PD-1 and endostar has a remarkably synergic effect on LLC tumour growth by means of improving the tumour microenvironment and activating autophagy.
Experimental study of Endostar injection concomitant with cryoablation on lung adenocarcinoma A549 xenografts.
Ma Chun-Hua,Jiang Rong,Li Jin-Duo,Wang Bin,Sun Li-Wei,Lv Yuan
Asian Pacific journal of cancer prevention : APJCP
OBJECTIVE:To explore the inhibiting effect and mechanism of Endostar injection concomitant with cryoablation on lung adenocarcinoma A549 xenografts in nude mice. MATERIALS AND METHODS:A total of 24 nude mice with subcutaneous xenografts of the A549 cell line were established and divided into 4 groups when the maximal diameters of tumors became 1 cm: control group, Endostar group, cryoablation group and combination group (Endostar concomitant with cryoablation). The nude mice were sacrificed after 21-days treatment, tumour tissues were removed to measure their volume, in situ test of TdT-mediated dUTP nick end labeling (TUNEL) was adopted to determine the cellular apoptosis around freezing injury zones, and immunohistochemical SP test was applied for the detection of micro-vessel density (MVD) and vascular endothelial growth factor (VEGF) expression levels. RESULTS:At 21-days after treatment, the growth velocities of control group, Endostar group, cryoablation group and combination group were 236.7 ± 51.2%, 220.0 ± 30.6%, 159.5 ± 29.3% and 103.3 ± 25.5% (P<0.01), while cellular apoptosis rates of tumors were 21.7 ± 2.34%, (22.17 ± 1.47)%, 38.3 ± 1.37% and 49.2 ± 1.72%, (P<0.01), respectively, according to the immunohistochemical test. MVD and VEGF expression levels in the combination group were both lower than in other groups (P<0.01), also being positively related (r=0.925, P<0.01). CONCLUSIONS:Endostar can significantly improve the inhibitory effects of cryoablation on xenografts of lung adenocarcinoma A549, and the mechanism is probably associated with its function as an inhibitor of tumour neo-angiogenesis through down-regulating VEGF expression.
Enhanced antitumor and anti-angiogenic effects of metronomic Vinorelbine combined with Endostar on Lewis lung carcinoma.
Qin Rong-Sheng,Zhang Zhen-Hua,Zhu Neng-Ping,Chen Fei,Guo Qian,Hu Hao-Wen,Fu Shao-Zhi,Liu Shan-Shan,Chen Yue,Fan Juan,Han Yun-Wei
BACKGROUND:Conventional chemotherapy is commonly used to treat non-small cell lung cancer (NSCLC) however it increases therapeutic resistance. In contrast, metronomic chemotherapy (MET) is based on frequent drug administration at lower doses, resulting in inhibition of neovascularization and induction of tumor dormancy. This study aims to evaluate the inhibitory effects, adverse events, and potential mechanisms of MET Vinorelbine (NVB) combined with an angiogenesis inhibitor (Endostar). METHODS:Circulating endothelial progenitor cells (CEPs), apoptosis rate, expression of CD31, vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 (HIF-1α) were determined using flow cytometry, western blot analysis, immunofluorescence staining and Enzyme-linked immunosorbent assay (ELISA) analysis. And some animals were also observed using micro fluorine-18-deoxyglucose PET/computed tomography (F-FDG PET/CT) to identify changes by comparing SUVmax values. In addition, white blood cell (WBC) counts and H&E-stained sections of liver, lungs, kidney, and heart were performed in order to monitor toxicity assessments. RESULTS:We found that treatment with MET NVB + Endo was most effective in inhibiting tumor growth, decreasing expression of CD31, VEGF, HIF-1α, and CEPs, and reducing side effects, inducing apoptosis, such as expression of Bcl-2, Bax and caspase-3. Administration with a maximum tolerated dose of NVB combined with Endostar (MTD NVB + Endo) demonstrated similar anti-tumor effects, including changes in glucose metabolism with micro fluorine-18-deoxyglucose PET/computed tomography (F-FDG PET/CT) imaging, however angiogenesis was not inhibited. Compared with either agent alone, the combination of drugs resulted in better anti-tumor effects. CONCLUSION:These results indicated that MET NVB combined with Endo significantly enhanced anti-tumor and anti-angiogenic responses without overt toxicity in a xenograft model of human lung cancer.
Endostar, a modified endostatin inhibits non small cell lung cancer cell in vitro invasion through osteopontin-related mechanism.
Ni Qinggui,Ji Hui,Zhao Zhihui,Fan Xin,Xu Chengyun
European journal of pharmacology
In this study, we studied the inhibition of non small cell lung cancer (NSCLC) cells invasion by a recombinant human Endostar, a modified endostatin and the possible osteopontin-related mechanism. The results showed that Endostar significantly inhibited highly metastatic NSCLC (NCI-H460) cells in vitro invasion. ELISA demonstrated that reduction of osteopontin level in the medium by Endostar may be responsible for the inhibition of invasion. RT-PCR assay and western blot analysis revealed that the reduction of osteopontin was due to under-regulation of osteopontin expression. Furthermore, Endostar also inhibited osteopontin-induced less metastatic NSCLC (A549) cells invasion, indicating that Endostar may have other different osteopontin-related mechanism. In an adhesion assay, we found that Endostar reduced NCI-H460 cells binding to osteopontin. Flow cytometric analysis suggested that the reduction of adhesion may be related to under-regulation of its receptors (CD44v6 and alpha(V)beta(3) integrin) expression. Additionally, we found, via gelatin zymographic analysis, that osteopontin-induced the expression and activation of pro-matrix metalloproteinase (MMP)-2 and pro MMP-9 secreted from A549 cells were blocked upon Endostar treatment, indicating that Endostar may block osteopontin-mediated signal transduction pathways through MMP families. The above results indicate that Endostar may have an intrinsic non-angiogenesis-related antitumor activity through osteopontin-related mechanism against NSCLC, including osteopontin change and osteopontin signal transduction blockade. Tumor cell invasion is important for tumor metastasis, our findings suggest that it is probably a good strategy to put Endostar into treatment of NSCLC metastasis.
Enhanced anti-tumor and anti-angiogenic effects of metronomic cyclophosphamide combined with Endostar in a xenograft model of human lung cancer.
Wang Rui,Qin Shukui,Chen Yuqing,Li Yumei,Chen Changjie,Wang Zishu,Zheng Rongsheng,Wu Qiong
Standard chemotherapy for advanced NSCLC has reached a therapeutic plateau. More effective strategies must be explored. The purpose of this study was to evaluate the role of metronomic chemotherapy combined with an angiogenesis inhibitor in non-small cell lung cancer (NSCLC). A total of 114 BALB/c nude mice were inoculated subcutaneously with human NSCLC cells (A549), and when xenograft tumors were palpable, mice were randomly injected with saline as controls (Ctrl), or treated with metronomic cyclophosphamide (MET CPA), recombinant human endostatin, Endostar (Endo), MET CPA combined with Endostar (MET CPA+Endo) or maximum tolerance dose of CPA (MTD CPA), respectively. The growth of xenograft tumors and mouse survival were monitored. The frequency of peripheral blood circulating endothelial cells (CECs), microvessel density (MVD) and pericyte coverage was determined using flow cytometry and immunofluorescence staining. In comparison with the controls, treatment with either drug significantly inhibited the growth of xenograft tumors in mice. Treatment with MET CPA or Endostar, but not with MTD CPA, significantly reduced the frequency of peripheral blood total and viable CECs and the value of MVD. Endostar also considerably reduced pericyte coverage in xenograft tumors. Moreover, MET CPA combined with Endostar further reduced the frequency of peripheral blood CECs, the value of MVD, and pericyte coverage, with concomitant delay in tumor growth and extension of mouse survival. Our results indicate that MET CPA combined with Endostar results in enhanced anti-tumor and anti-angiogenic effects in a xenograft model of human lung cancer. Combined therapy with metronomic chemotherapy and an angiogenesis inhibitor may serve as a promising treatment strategy for patients with advanced NSCLC.
Effect of endostar combined with cisplatin on expression of VEGF and Sema3A of Lewis lung cancer rats.
Feng Ping,Zhang Zhi-Lin,Zhang Zhi-Hua,Zhang Xiu-Long,Xiang Feng,Tang Jian-Hua,Xiang Bao-Li
Asian Pacific journal of tropical medicine
OBJECTIVE:To evaluate the therapeutic effect of endostar (ED) combined with cisplatin(DDP) on model of C57BL/6 rats, and to further investigate the inhibiting mechanism of endostar from tumor angiogenesis. METHODS:Lewis lung cancer cells were inoculated in C57BL/6 mouse, then the mouse were randomized into control group (group A), ED (group B), DDP (group C) and ED/DDP (group D). They were treated according to the plan. And the expressions of VEGF and Sema3A were evaluated by immunhistochemisty. RESULTS:The weight of tumor increased in group A and B. It was decreased in group C and D. The tumor volume was increased in all the 4 groups. The VEGF expression of group D was obviously lower than the other group 3, but the Sema3A expressed of group D was significantly strengthener than the other group 3. The VEGF expression of group B and group D were obviously low especially in the 4th-8th days. Pearson correlated analysis showed that the expression VEGF and Sema3A were negatively correlated (r=-0.72, P<0.05). CONCLUSIONS:ED combined with DDP could control the tumor growth effectively, and avoid weight loss. ED could reduce VEGF expression, and enhance Sema3A expression. Tumor vessel presents transient normalization. It is easy for DDP perfusion, and to kill tumor cells.
[Inhibitory effect of endostar on lymphangiogenesis in non-small cell lung cancer and its effect on circulating tumor cells].
Shang Liqun,Zhao Jie,Wang Wei,Xiao Wang,Li Jun,Li Xuechang,Song Weian,Liu Junqiang,Wen Feng,Yue Caiying
Zhongguo fei ai za zhi = Chinese journal of lung cancer
BACKGROUND AND OBJECTIVE:It is unclear how could endostatin effect tumor lymphangiogenesis? The aim of this study is to explore inhibitory effect of recombinant human endostatin injection (endostar) on lymphangiogenesis in non-small cell lung cancer (NSCLC) tissue and its effect on circulating tumor cells (CTC) in peripheral blood. METHODS:Tumor-bearing model nude mice were divided into eight groups randomly (n=7), including control group, cisplatin group, several concentration endostar groups and endostar plus cisplatin groups. Continuous administration of Endostar for two weeks, observed one week after the end of administration. Using HE staining and immunohistochemical staining to diagnose the tumor tissue and suspect metastasis lymph nodes, detected vascular endothelial growth factor (VEGF)-C, VEGF-D, VEGF receptor (VEGFR)-3 expression level and microlymphatic vessel density (MLVD) of tumor tissue. Enrichment of circulating tumor cells in peripheral blood used immunomagnetic negative selection strategy, used immunofluorescence staining to diagnose and count CTCs. RESULTS:Microlymphatic vessel density and the positive expression rate of VEGF-C, VEGF-D, VEGFR-3 in three endostar groups and three endostar plus cisplatin groups were significantly less than those in control group and cisplatin group. Microlymphatic vessel density and the positive expression rate of VEGF-C, VEGF-D, VEGFR-3 in endostar plus cisplatin group and endostar group with high endostar concentration were significantly less than those with low endostar concentration; There was a significant positive correlation between microlymphatic vessel density and the positive expression rate of VEGF-C, VEGF-D, VEGFR-3. The number of circulating tumor cells in endostar plus cisplatin groups were significantly less than that of endostar or cisplatin alone. CONCLUSIONS:Endostar could inhibit tumor lymphangiogenesis and reduce tumor cells into the bloodstream through the lymphatic. Inhibitory effect concerned with drug concentrationwith a dose-dependant.
Comparison of efficacy and toxicity of bevacizumab, endostar and apatinib in transgenic and human lung cancer xenograftzebrafish model.
Jin Yinghua,Wei Lingxiao,Jiang Qiuying,Song Xiaowei,Teng Chong,Fan Chengjuan,Lv Yanju,Liu Ying,Shen Weixi,Li Li,Huang Dayong,Xin Tao
The poor prognosis in non-small-cell lung cancer has driven the development of novel targeted therapies. Vascular endothelial growth factor is the most potent force in mediating tumor angiogenesis, and many angiogenesis inhibitors have been developed for oncology treatment. We performed a study to characterize the efficacy, safety and tumor suppression of three lung cancer related anti-angiogenic drugs (bevacizumab, endostar and apatinib) using transgenic zebrafish embryo and human lung cancer xenotransplantation model. All three drugs demonstrated remarkable angiogenesis and tumor inhibition effect in the zebrafish model, within the nonlethal dose range. Endostar and bevacizumab showed competitive anti-tumor efficacy. The anti-tumor performance of apatinib was hamstrung by its elevated toxicity at 35 °C. The addition of pemetrexed to anti-angiogenesis therapy had no obvious additional benefit in tumors.
Endostar regulates EMT, migration and invasion of lung cancer cells through the HGF-Met pathway.
Shen Yuyao,Chen Qingwen,Li Lihong
Molecular and cellular probes
AIM:Though Endostar (ES) could inhibit tumor growth by inhibiting tumor angiogenesis, other possible mechanisms have been less reported. This study aims to investigate the role of ES in the treatment of lung cancer from the perspective of macrophage-mediated epithelial mesenchymal transformation (EMT). METHODS:THP1 cells were induced to polarized macrophages (MΦ). A549 and H1795 cells were separately treated with MΦ conditioned medium, ES (12.5 μg/ml) and HGF (5 ng/ml) for 24 h at 37 °C. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of CCL17, CD163, hepatocyte growth factor (HGF), Epidermal Growth Factor (EGF), transforming growth factor (TGF)-β1 and interleukin (IL)-6. Western blot was carried out to detect the p-MET, MET and EMT-related proteins (E-cadherin, N-cadherin, Snail and vimentin). Fibroblast-like A549 and H1975 cells were observed by a microscope. Cell invasion and migration were observed and analyzed by transwell and scratch assays. RESULTS:The expression levels of CCL17 and CD163 were significant higher in MΦ. ES significantly inhibited the expression of HGF in MΦ. Moreover, ES could restore the abnormal expressions of EMT-related proteins and inhibit MΦ-induced and HGF-induced fibroblast-like lung cancer cells. Furthermore, ES suppressed the MΦ-induced and HGF-induced migration and invasion of lung cancer cells. ES was also found to down-regulate HGF-Met signaling in HGF-treated lung cancer cells. CONCLUSION:ES suppresses lung cancer progression by down-regulating HGF-Met signaling, revealing the possible mechanism of ES in the process of treating lung cancer patients.