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The oncogenic role of ephrin type-B receptor 4 (EPHB4) has been reported in many types of tumors, including chronic myeloid leukemia (CML). Here, we found that CML patients have a higher EPHB4 expression level than healthy subjects. EPHB4 knockdown inhibited growth of K562 cells (a human immortalized myelogenous leukemia cell line). In addition, transient transfection of EPHB4 siRNA led to sensitization to imatinib. These growth defects could be fully rescued by EPHB4 transfection. To identify an EPHB4-specific inhibitor with the potential of rapid translation into the clinic, a pool of clinical compounds was screened and vandetanib was found to be most sensitive to K562 cells, which express a high level of EPHB4. Vandetanib mainly acts on the intracellular tyrosine kinase domain and interacts stably with a hydrophobic pocket. Furthermore, vandetanib downregulated EPHB4 protein via the ubiquitin-proteasome pathway and inhibited PI3K/AKT and MAPK/ERK signaling pathways in K562 cells. Vandetanib alone significantly inhibited tumor growth in a K562 xenograft model. Furthermore, the combination of vandetanib and imatinib exhibited enhanced and synergistic growth inhibition against imatinib-resistant K562 cells in vitro and in vivo. These findings suggest that vandetanib drives growth arrest and overcomes the resistance to imatinib in CML via targeting EPHB4.

Breast cancer is one leading cause of death in females, especially triple‐negative breast cancer (TNBC). Hypoxia is a key feature leading to tumour progression driven by hypoxia‐inducible factor (HIF)‐1α. The aim is to investigate the mechanism of HIF‐1α and signal transducer and activator of transcription‐3 (STAT3) interaction and discover a compound to disrupt the interaction in breast cancer cells. The regulation pattern of HIF‐1α and STAT3 was analysed in hypoxic TNBC cells and patient samples. The effects of a natural alkaloid, sanguinarine, on HIF‐1α and STAT3 colocalization and interaction were evaluated in vitro and mouse xenograft models. We observed strong colocalization of HIF‐1α, p‐STAT3‐Tyr and p‐STAT3‐Ser in TNBC patient samples. Sanguinarine could inhibit the nuclear colocalization and interaction of HIF‐1α with p‐STAT3‐Tyr and p‐STAT3‐Ser in vivo and in vitro. Our results may bring insights to the HIF‐1α/STAT3 interaction in breast cancers and suggest sanguinarine as a promising candidate for HIF‐α/STAT3 inhibition.

Background Hepatocellular carcinoma (HCC) is the third leading cause of cancer‐related deaths worldwide. Overexpression of pleomorphic adenoma gene like‐2 (PLAGL2) is associated with tumorigenesis. However, its function in HCC is unclear, and there are currently no anti‐HCC drugs that target PLAGL2. Drug repositioning may facilitate the development of PLAGL2‐targeted drug candidates. Methods The expression of PLAGL2 in HCC clinical tissue samples and HCC cell lines was analyzed by western blotting. The constructed HCC cell models were used to confirm the underlying function of PLAGL2 as a therapeutic target. Multiple in vitro and in vivo assays were conducted to determine the anti‐proliferative and apoptosis‐inducing effects of selenium sulfide (SeS2), which is clinically used for the treatment of seborrheic dermatitis and tinea versicolor. Results PLAGL2 expression was higher in HCC tumor tissues than in normal adjacent tissues. Its overexpression promoted the resistance of HCC cells of mitochondrial apoptosis through the regulation of the downstream C‐MET/STAT3 signaling axis. SeS2 exerted significant anti‐proliferative and apoptosis‐inducing effects on HCC cells in a PLAGL2‐dependent manner. Mechanistically, SeS2 suppressed C‐MET/STAT3, AKT/mTOR, and MAPK signaling and triggered Bcl‐2/Cyto C/Caspase‐mediated intrinsic mitochondrial apoptosis both in vitro and in vivo. Conclusions Our data reveal an important role of PLAGL2 in apoptosis resistance in HCC and highlight the potential of using SeS2 as a PLAGL2 inhibitor in patients with HCC., The C‐MET/STAT3 signaling axis as a novel downstream target of pleomorphic adenoma gene like‐2 (PLAGL2) contributed to PLAGL2‐induced mitochondrial apoptosis resistance in HCC. Selenium sulfide (SeS2) induced cell proliferation inhibition and apoptosis on HCC cells by inhibiting the activity of PLAGL2 and restraining the novel PLAGL2/C‐MET/STAT3, AKT/mTOR and MAPK signaling pathways.

Epithelial–mesenchymal transition (EMT) plays a crucial role in hepatocellular carcinoma (HCC) progression. Hypoxia and excessive transforming growth factor-β (TGF-β) have been identified as inducers and target for EMT in HCC. Here, we show hypoxia inducible factor-1α (HIF-1α) and TGF-β form a feed-forward loop to induce EMT in HCC cells. Further mechanistic study indicates under both hypoxia and TGF-β stimulation, Smad and PI3K-AKT pathways are activated. We show sanguinarine, a natural benzophenanthridine alkaloid, impairs the proliferation of nine kinds of HCC cell lines and the colony formation of HCC cells. In hypoxic and TGF-β cell models, sanguinarine inhibits HIF-1α signaling and the expression of EMT markers, translocation of Snail and activation of both Smad and PI3K-AKT pathways. Sanguinarine could also inhibit TGF-β-induced cell migration in HCC cells. In vivo studies reveal that the administration of sanguinarine inhibits tumor growth and HIF-1α signaling, inhibits the expression changes of EMT markers as well as Smad and PI3K-AKT pathway proteins. Our findings suggest that sanguinarine is a promising candidate targeting HIF-1α/TGF-β signaling to improve the treatment for HCC patients.

Background: Skin cutaneous melanoma (SKCM) is considered one of the most aggressive and lethal cancers of the skin. G-protein coupled receptor 143 (GPR143), which has been reported to cause congenital nystagmus, belongs to the superfamily of G protein-coupled receptors. Methods and Results: We analyzed the expression of GPR143 and survival of SKCM patients in SKCM via Gene Expression Profiling Interactive Analysis (GEPIA). Then, logistic regression and multivariate cox analysis was used to analyze the influence of GPR143 expression on clinicopathological elements and survival. We explored the immune response of 22 TIICs in SKCM via CIBERSORT and used TIMER to assess the correlation of GPR143 expression and immune infiltration level. Finally, we used gene set enrichment analysis (GSEA) to assess the TCGA dataset. The outcomes suggest that GPR143 expression in tumor samples is remarkedly higher than in normal samples and high GPR143 expression is associated with poorer prognosis. The result of multivariate analysis indicated that increased GPR143 expression is an independent prognostic factor for prognosis. We found GPR143 expression level has prominent negative correlations with infiltrating levels of B cell, CD8+ T cells, etc. GSEA indicated that pigment metabolic process, pigment biosynthetic process and other pathways were identified as differentially enriched pathways in Gene Ontology (GO). Oxidative phosphorylation, Parkinson’s disease and other pathways were showed significantly differential enrichment in GPR143 high expression phenotype in Kyoto Encyclopedia of Genes and Genomes (KEGG).Conclusions: In conclusion, GPR143 may be a novel prognostic biomarker and associated with immune infiltrates in SKCM.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Loss of WW-domain containing oxidoreductase (WWOX) has been proven to be associated with malignant metastasis in patients with HCC. In this study, by using a non-biased CRISPR knockout genetic screen targeting 19,050 human genes, we found that toosendanin (TSN) is a novel druggable WWOX candidate agonist for metastatic HCC patients. We also found that TSN exhibited significant anti-proliferative and anti-metastatic effects on HCC cells in a WWOX-dependent manner. Overexpression and knockdown of WWOX in vitro and in vivo confirmed that the suppression of HCC by TSN involved WWOX. TSN regulated Stat3, DVL2, and GSK3β by transforming their interactions with WWOX as demonstrated by a Co-IP assay. TSN accelerated the degradation of β-catenin by promoting the function of APC, AXIN1, CK1, and GSK3β complex. Nuclear translocation of p-Stat3 Y705 and β-catenin was impeded by the TSN-induced blockade of JAK2/Stat3 and Wnt/β-catenin signaling, accompanied by the inhibition of MMPs and C-MYC.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson’s disease (PD) and contribute to sporadic PD. Common genetic variation in LRRK2 modifies susceptibility to immunological disorders including Crohn’s disease and leprosy. Previous studies have reported that LRRK2 is expressed in B lymphocytes and macrophages, suggesting a role for LRRK2 in immunological functions. In this study, we characterized the LRRK2 protein expression and phosphorylation using human lymphoblasts. Lipopolysaccharide (LPS), a proinflammatory agent, induced the increase of LRRK2 expression and kinase activities in human lymphoblasts in a time-dependent manner. Moreover, LPS activated the Toll-like receptor (TLR) signaling pathway, increased TRAF6/LRRK2 interaction, and elevated the phosphorylation levels of MAPK (JNK1/2, p38, and ERK1/2) and IkBα. Treatment with LRRK2 inhibitor 68 reduced LPS-induced TRAF6/LRRK2 interaction and MAPK and IkBα phosphorylation, thereby reducing TNF-α secretion. These results indicate that LRRK2 is actively involved in proinflammatory responses in human lymphoblasts, and inhibition of GTP binding by 68 results in an anti-inflammation effect against proinflammatory stimuli. These findings not only provide novel insights into the mechanisms of LRRK2-linked immune and inflammatory responses in B-cell-like lymphoblasts, but also suggest that 68 may also have potential therapeutic value for LRRK2-linked immunological disorders.

Background Skin cutaneous melanoma is one of the most aggressive types of cancers worldwide. Therefore, the identification of skin cutaneous melanoma (SKCM) biomarkers is of great importance. NLRP3 inflammasome complex nodelike receptor protein 3 (NLRP3) is one of the most characteristic inflammasomes belonging to the NLR protein family. This is the first time to use TCGA data to study NLRP3 expression in SKCM patients and its prognostic value, potential biological function, and impact on the immune system. Methods The expression of NLRP3 in SKCM was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA). We assessed the impact of NLRP3 on SKCM patient survival through the survival module and then downloaded the SKCM data set from TCGA. Logistic regression was used to analyze the correlation between clinical data and NLRP3 expression. Univariate survival rate and multivariate Cox analysis were used to compare several clinical features and survival rates. We also used CIBERSORT to investigate the association between NLRP3 and cancer immune infiltration. We used TIMER to investigate NLRP3 expression and collection of immune infiltration levels in SKCM, as well as cumulative survival in SKCM. Gene set enrichment analysis (GSEA) was performed using the TCGA dataset. Besides, data from The Human Protein Atlas (HPA) was used to validate the results. Results Univariate Logistic regression analysis showed that increased NLRP3 expression was significantly correlated with age, stage, and tumor status. Specifically, NLRP3 expression level had significant positive correlations with infiltrating levels of B cell, CD4+ T cells, CD8+ T cells, Macrophages, Neutrophils, and DCs in SKCM. GSEA revealed that NLRP3 was closely correlated with pathways in cancer. HPA showed that in tumor tissues, NLRP3 had higher levels of expression compared to normal tissues. Conclusions The discovery of NLRP3 as a new biomarker of SKCM helps to elucidate how changes in the immune environment promote the occurrence of cutaneous melanoma. Further analysis suggested that NLRP3 might be a predictor of SKCM prognosis.

Targeted anti-tumor small molecules are considered to be promising candidates for cancer treatment. The novel diphenyl urea derivative (DUD) was synthesized by the molecular docking based on the structure optimization of Taspine (a natural product). In this study, we explored the anti-metastatic potential of DUD for NSCLC in vitro. DUD significantly suppressed A549 cell migration by reversing EMT. The inhibition was reflected on upregulation of E-cadherin and downregulation of N-cadherin, vimentin, Snail and HIF-1α. Meanwhile, DUD inhibited the β-catenin nuclear translocation by upregulating Axin and downregulating the expression of APC, CK1 and phosphorylation of GSK3β, and simultaneously decreasing MMP9 and MMP13 expression. Moreover, it was associated with the downregulation of the PI3K/Akt/mTOR signaling. Furthermore, we used XAV939, an β-catenin inhibitor, to verify the mechanism of DUD. These results suggested that DUD inhibited A549 cells migration by reversing EMT via Wnt/β-catenin and PI3K/Akt signaling. DUD might be a potential therapeutic drug candidate for NSCLC treatment.

Hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. The tyrosine kinase receptor EphB4 promotes oncogenesis and tumor development and progression. Its inhibition is regarded as an effective strategy for the treatment of solid tumors. In the present study, we identified cantharidin as a novel EphB4 inhibitor for HCC treatment and evaluated the underlying molecular pharmacological mechanisms of action. We observed increased expression levels of EphB4 in HCC patients and a positive correlation between EphB4 and p-JAK2 levels in HCC patient samples. Knockdown of EphB4 using small interfering RNA decreased the expression levels of p-JAK2 and p-STAT3 in HCC cells, suggesting JAK2/STAT3 being a novel downstream signaling target of EphB4. Cell viability experiments revealed that the anti-cancer effect of cantharidin was positively correlated with EphB4 expression levels in HCC cell lines. We confirmed the potent antiproliferative activity of cantharidin on HepG2 cells with high expression of EphB4 and tumor xenograft. Molecular docking assay, immunoblotting assay and quantitative reverse transcription PCR assay indicated that cantharidin bound to EphB4, and thereby resulted in EphB4 suppression at mRNA and protein levels. Hep3B and SMMC-7721 cells were with low expression of EphB4. In EphB4-/HepG2, EphB4+/HepG2, and EphB4+/Hep3B cells, EphB4 knockdown alleviated the cantharidin-induced decrease in cell viability and colony formation ability and increase in apoptosis in HepG2 cells, while its overexpression exacerbated these effects in Hep3B cells and increased the apoptosis of HepG2 cells. In nude mouse models, cantharidin suppressed tumor growth more effectively in EphB4+/SMMC-7721 xenografts than in wild-type SMMC-7721 xenografts. Underlying mechanistic study showed that by targeting EphB4, cantharidin blocked a novel target, the downstream JAK2/STAT3 pathway, and the previously known target, the PI3K/Akt signaling, resulting in intrinsic apoptosis. These results indicated that cantharidin may be a potential candidate for HCC treatment by regulating the EphB4 signaling pathway.

Hepatocellular carcinoma (HCC) is a biologically complex disease. Combination chemotherapy is a good strategy after surgery treatment. In this study, we report that berberine combined with HMQ1611 (BCH) had a good synergistic effect on the HCC. Our findings concluded that BCH showed good inhibition on the HCC proliferation and colony formation, which attributed to cell cycle arrest by BCH at G1 phase through impairing the expression of cyclinD1, cyclinE, and cdc2 and downregulated the phosphorylation of Akt, mTOR, and ERK. Moreover, BCH negatively regulated Wnt signaling pathway by upregulating the Axin and inhibiting the nuclear translocation of β-catenin. BCH suppressed the phosphorylation of LRP5/6, GSK3β, the expression of Wnt5a, Frizzled8, CK1, and APC, as well as the nucleus protein included MMP2, MMP3, MMP9, and c-myc. The above data of Wnt signaling regulators contributed to inhibition by BCH on cell migration. In vivo studies, BCH significantly suppressed the growth of SMMC-7721 xenograft tumors through downregulating Ki67 and β-catenin, as well as upregulating Axin and p-β-catenin. In conclusion, the results revealed that BCH exhibited potential antitumor activities against human liver cancer in vitro and in vivo, and the potential mechanism underlying these activities depended on the inhibition of the Wnt/β-catenin signaling pathway.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer worldwide and it is necessary to discover and develop novel preventive strategies and therapeutic approaches for HCC. Herein, we report that EphrinB2 expression is correlated with liver cancer progression. Moreover, by using phosphorylated proteomics array, we reveal a pro‐apoptosis protein whose phosphorylation and activation levels are up‐regulated upon EphrinB2 knockdown. These results suggest that EphrinB2 may act as an anti‐apoptotic protein in liver cancer cells. We also explored the therapeutic potential of HMQ‐T‐B10 (B10), which was designed and synthesized in our laboratory, for HCC and its underlying mechanisms in vitro and in vivo. Our data demonstrate that B10 could bind EphrinB2 and show inhibitory activity on human liver cancer cells. Moreover, induction of human liver cancer cell apoptosis by B10 could be augmented upon EphrinB2 knockdown. B10 inhibited HCC cell growth and induced HCC cell apoptosis by repressing the EphrinB2 and VEGFR2 signalling pathway. Growth of xenograft tumours derived from Hep3B in nude mice was also significantly inhibited by B10. Collectively, these findings highlight the potential molecular mechanisms of B10 and its potential as an effective antitumour agent for HCC.

Receptor tyrosine kinases (RTKs), especially VEGFR-2, TIE-2, and EphB4, play a crucial role in both angiogenesis and tumorigenesis. Moreover, complexity and heterogeneity of angiogenesis make it difficult to treat such pathological traits with single-target agents. Herein, we developed two classes of multi-target RTK inhibitors (RTKIs) based on the highly conserved ATP-binding pocket of VEGFR-2/TIE-2/EphB4, using previously reported BPS-7 as a lead compound. These multi-target RTKIs exhibited considerable potential as novel anti-angiogenic and anticancer agents. Among them, QDAU5 displayed the most promising potency and selectivity. It significantly suppressed viability of EA.hy926 and proliferation of several cancer cells. Further investigations indicated that QDAU5 showed high affinity to VEGFR-2 and reduced the phosphorylation of VEGFR-2. We identified QDAU5 as a potent multiple RTKs inhibitor exhibiting prominent anti-angiogenic and anticancer potency both in vitro and in vivo. Moreover, quinazolin-4(3H)-one has been identified as an excellent hinge binding moiety for multi-target inhibitors of angiogenic VEGFR-2, Tie-2, and EphB4.

Colorectal cancer is a common gastrointestinal malignancy worldwide and it is a lethal and aggressive malignancy with a dismal prognosis. In the present study, we investigated the effect of taspine derivative 12k on human colorectal cancer targeted at EphrinB2 and its PDZ. The results indicated that 12k could bind to EphrinB2 and showed a higher suppressive effect on EphrinB2/HEK293 than on HEK293 cells. Caco‑2 cells were screened for high expression of EphrinB2. We found that 12k not only significantly decreased Caco‑2 cell viability and colony formation but impaired migration. Meanwhile, 12k effectively inhibited blood vessel formation in a tissue model of angiogenesis. Mechanistic studies revealed that 12k significantly reduced the phosphorylation of EphrinB2 and PDZ protein PICK1. Accordingly, it was associated with the downregulation by 12k of the PI3K/AKT/mTOR and MAPK signaling pathways which were downstream of VEGFR2, yet it had no effect on VEGFR3. Moreover, the expression of CD34, CD45 and HIF‑1α were downregulated in the Caco‑2 cells. In conclusion, our findings showed that 12k had an inhibitory effect on the growth of Caco-2 cells, and it functioned by interrupting the phosphorylation of EphrinB2 and its related signaling pathway.

Epithelial‑mesenchymal transition (EMT) is closely related to tumor metastasis, and offers insight into novel strategies for cancer treatment. HMQ‑T‑F2 (F2) is a taspine derivative, which has excellent anticancer activity in human cervical cancer. The present study aimed to evaluate the effect of F2 on in vitro migration of HeLa cells. The present data demonstrated that F2 inhibited migration of HeLa cells by negatively regulating the Wnt signaling pathway and reversing EMT. F2 not only mediated Frizzled8, p‑LRP6 and LRP6 expression, but also downregulated the phosphorylation of GSK3β, and concurrently decreased the nucleus protein expression of MMP2, MMP3, MMP7, MMP9, and c‑Myc. In addition, the expression of N‑cadherin, vimentin, Snail and HIF‑1α were downregulated and that of E‑cadherin was upregulated after F2 treatment. F2 was also associated with the downregulation of the PI3K/Akt/mTOR signaling pathways. Notably, F2 induced HeLa cell accumulation at the S phase and cell apoptosis. These results provide evidence that F2 inhibits HeLa cell migration, proliferation and promotes apoptosis. It also reverses EMT, potentially via the PI3K/Akt signaling pathway. Therefore, F2 may be a potential therapeutic reagent against cervical cancer.

Taspine has been indicated to be a potential anti‑carcinogenic agent. The present study investigated the effects of TAS9, a modified taspine derivative, on the proliferation and migration of the SMMC‑7721 human liver cancer cell line. First, the effects of TAS9 on SMMC‑7721 cell growth were examined using MTT and colony formation assaya. In vivo Transwell and wound healing assays were then performed to assess the inhibitory effects of TAS9 on cell invasion and migration, respectively. The expression of cell proliferation‑ and migration‑associated signaling molecules was investigated by western blot analysis. The results indicated that TAS9 inhibited SMMC‑7721 cell growth by downregulating the signaling molecules protein kinase Cβ (PKCβ), Akt, mammalian target of rapamycin, mitogen‑activated protein kinase kinase 2, RAF and c‑Jun N‑terminal kinase‑1, and inhibiting SMMC‑7721 cell migration by suppressing the expression of matrix metalloproteinase (MMP)‑2, MMP‑9, chemokine (C‑X‑C motif) receptor 4, nuclear factor κB, p38 and p53. Small interfering RNA‑mediated knockdown of PKCβ in the SMMC‑7721 cells significantly attenuated the tumor inhibitory effects of TAS9. In conclusion, the results of the present study suggested that TAS9 may have inhibitory effects on the proliferation and migration of SMMC‑7721 cells, and may serve as a potential candidate for cancer treatment.

Background Drug therapy plays an important role in the treatment of cervical cancer, which is one of the most common solid tumors in women. Therefore, it is important to seek more effective and less toxic therapies. Purpose The aim of this study is to investigate the therapeutic potential of HMQ-T-F5 (1-(4-(2-aminoquinazolin-7-yl)phenyl)-3-(2‑bromo‑5-(trifluoromethoxy) phenyl)thiourea) (F5) for cervical cancer and explore the related mechanism. Methods By performing MTT assay, colony formation assay, flow cytometry, wound-healing assay, transwell assay, immunofluorescent staining and siRNA assay, we study the effect of F5 on human cervical HeLa cells. The mechanism of F5 was also investigated. Results We found that F5 significantly inhibited HeLa cell proliferation, led to accumulation of cells in the S phase, and induced apoptosis and inhibited migration. Mechanistically, F5 inhibited HeLa cell growth and migration through repressing the expression and nuclear translocation of β-catenin, enhancing Axin expression, inhibiting the phosphorylation of LRP5/6 and GSK3β, as well as downregulating the Wnt downstream targeted proteins. Knockdown of a checkpoint β-catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of β-catenin, was used to treat HeLa cells and the results demonstrated that F5 inhibited proliferation and migration via the inhibition of the Wnt/β-catenin pathway. Conclusion Our findings demonstrated that F5 can target β-catenin potentially and is useful in the treatment of cervical cancer.

We recently reported that TPD7 suppressed tumor cell proliferation, and inhibited invasion, through the suppression of C-X-C chemokine receptor type 4 (CXCR4). In the present study, we investigated the anticancer effect of TPD7 on apoptosis and invasion of cervical cancer HeLa cells. Cell cycle analysis revealed that TPD7 decreased cyclin-dependent kinase (CDK)1 and cyclin D1 expression, and increased cyclin A expression, following S phase blockade. TPD7 induced chromatin condensation and significantly elevated the number of apoptotic cells, suggesting that its inhibitory effect on HeLa cells was due to the induction of cell cycle blockade and apoptosis. Mechanistically, TPD7 altered the extrinsic apoptosis pathway by upregulating Fas expression, and the intrinsic pathway by modulating Bcl-2 family proteins, p53, and NF-κB p65, leading to enhanced apoptosis. TPD7 inhibited HeLa cell invasion by downregulating the expression of matrix metalloproteinase (MMP)-9 and CXCR4 proteins. In vivo experiments revealed that TPD7 inhibited tumor growth in HeLa cell xenografted mice. These findings indicated that TPD7 may be a potential chemoprevention agent for the management of cervical carcinoma.

Eupolyphaga sinensis Walker has been reported to have anticoagulation, antithrombotic, liver protective and antitumor effects. In the present study, the inhibitory effects on proliferation of A549 human non‑small cell lung cancer cells and the underlying mechanisms were examined. Firstly, three solvents, 70% ethanol, distilled water and 95% ethanol, were used to extract Eupolyphaga sinensis Walker. The MTT assay results demonstrated that the 70% ethanol extract more potently reduced the growth of A549 cells and it was therefore adopted in the subsequent experiments. Eupolyphaga sinensis Walker 70% ethanol extract significantly inhibited A549 cell migration in a time‑ and dose‑dependent manner and inhibited human umbilical vein endothelial cell proliferation, migration and tube formation. Furthermore, Eupolyphaga sinensis Walker 70% ethanol extract effectively inhibited blood vessel formation in the established tissue model for angiogenesis. In addition, Eupolyphaga sinensis Walker 70% ethanol extract was demonstrated to inhibit the autophosphorylation of KDR, and downregulate the subsequent activation of AKT and extracellular signal regulated kinase (ERK)1/2 in A549 cells. In conclusion, these findings demonstrated that the antitumor mechanism of Eupolyphaga sinensis Walker 70% ethanol extract was through inhibiting angiogenesis. It functioned by interrupting the autophosphorylation of KDR and subsequently, AKT and ERK1/2.

In the present study, we investigated the antitumor activity of HMQ1611, a novel synthetic taspine derivative, in vivo and evaluated associated potential antiangiogenesis mechanisms. The proliferation of A549 cells was examined by WST-1 assay in vitro. Tube formation and lung tissue vessel models were used to observe the antiangiogenic activity of HMQ1611. In addition, vascular enodthelial growth factor (VEGF) secretion and KDR kinase activities were measured by ELISA and the HTRF(®)KinEASE(™)-TK assay. In vivo, the antitumor activity was assessed by implantation of A549 cells in athymic mice. The results showed that HMQ1611 inhibited A549 cell proliferation and VEGF secretion, while it significantly inhibited tube formation and tissue vascularization. Furthermore, HMQ1611 inhibited A549 xenograft tumor growth. In conclusion, the results of our study suggest that HMQ1611 has latent properties for the inhibition of angiogenesis which are involved in its antitumor activity.

Tumor growth and metastasis are responsible for most cancer patients' deaths. Here, we report that eupolyphaga sinensis walker has an essential role in resisting hepatocellular carcinoma growth and metastasis. Compared with proliferation, colony formation, transwell assay and transplantable tumor in nude mouse in vitro and vivo, eupolyphaga sinensis walker extract (ESWE) showed good inhibition on the SMMC-7721 cell growth and metastasis. Using genome-wide microarray analysis, we found the down-regulated growth and metastasis factors, and selected down-regulated genes were confirmed by real-time PCR. Knockdown of a checkpoint PKCβ by siRNA significantly attenuated tumor inhibition and metastasis effects of ESWE. Moreover, our results indicate ESWE inhibits HCC growth by not only downregulating the signaling of PKCβ, Akt, m-TOR, Erk1/2, MEK-2, Raf and JNK-1, but also increasing cyclin D1 protein levels and decreasing amount of cyclin E, cyclin B1 and cdc2 of the cycle proteins. At the same time, ESWE reduced MMP2, MMP9 and CXCR4, PLG, NFκB and P53 activities. Overall, our studies demonstrate that ESWE is a key factor in growth and metastasis signaling inhibitor targeting the PKC, AKT, MAPK signaling and related metastasis signaling, having potential in cancer therapy.

A novel tissue model for angiogenesis (TMA) is established for effective evaluation of angiogenesis inhibitors or promoters in vitro. Lung tissues were cultured in fibrinogen "sandwich" structure which resembled the formation of neovessels in vivo. The cells and capillary-like structures grew from the lung tissues were identified as endothelial cells and neovessels. Both immunohistochemisty and western blot results indicated that autocrine VEGF bound to the KDR and induced KDR autophosphorylation that could induce the proliferation of endothelial cells and their migration as well as the formation of microvessels on the lung tissue edge. With addition of the TMA, the murine VEGF and cultured medium produced by A549 tumor cells apparently promoted the increase of neovessels. Sorafenib as a tumor angiogenesis inhibitor and Tongxinluo as an angiogenesis promoter were both used to evaluate the TMA performance and they exhibited a good effect on neovessels in the TMA. The model established imitated angiogenesis in vivo and could well serve as an effective method in evaluating the angiogenesis inhibitors or promoters, and could also be practical for screening small molecules that affect blood vessel formation.

Eupolyphaga sinensis Walker is not only used as a food to enhance immunity, but is used as a traditional Chinese medicine and is known as the "preferred drug to regulate blood flow". Previous studies have reported its potential biological activities including anticoagulation, antithrombotic, liver protective effect and antitumor effects. Our results indicated that E. sinensis Walker 70% ethanol extract exhibited anti-tumor effects on S180 (murine sarcoma cell line) cells implanted mice. It effectively inhibited K562 (human chronic myeloid leukemia cell line) cells proliferation and induced G(2)-M phase arrest accompanying through up-regulation of cyclin B1, cdc2 and down-regulation of cyclin D1, cyclin E1, cdc25c and p53. In addition, it inhibited EGF secretion and EGFR kinase activity. Western blotting analysis indicated that it also inhibited the phosphorylation EGFR and activation of its downstream signaling molecules AKT and ERK. These results suggested that the antitumor mechanism of E. sinensis Walker involved altering the cell cycle and inhibiting EGFR phosphorylation in the EGFR signaling pathway.

In order to investigate the anti-angiogenesis potential and related mechanisms of Ta1722 (a novel taspine derivative compound), a series of experiments in vivo and in vitro were carried out. The proliferation on human cell lines of SMMC-7721, A549, MCF-7, Lovo, and ECV304 was examined by MTT. Angiogenesis inhibition was examined by chick embryo chorioallantoic membrane (CAM) angiogenesis and tube formation assays. Related angiogenesis proteins and their mRNA expression were determined by western blotting and RT-PCR. In addition, the SMMC-7721 nude mouse xenotransplant model was used to evaluate the inhibition of tumor growth. The results showed that Ta1722 inhibited cell proliferation, angiogenesis of CAM and tube formation, and downregulated related positive angiogenesis proteins. The above indicated Ta1722 could serve as a promising candidate of angiogenesis inhibitors by interrupting the VEGF/VEGFR-2 pathway.

EGFR, as a critical signaling pathway in many human tumors, has become an important target of cancer drug design. Taspine has shown meaningful angiogenesis activity in previous studies. This paper is to investigate the antitumor action of taspine by modulating the EGFR signaling pathway. The study determined the expression of key signaling molecules of EGFR (EGFR, Akt, p-Akt, Erk, and p-Erk) by Western blot and real-time PCR and analyzed their correlations with subsequent reactions. In addition, the cell proliferation, migration, and EGF production were examined by MTT, transwell system, and ELISA. The antitumor activity in vivo was carried out by xenograft in athymic mice. The results showed that taspine could inhibit A431 and Hek293/EGFR cell proliferation and A431 cell migration as well as EGF production. Compared to the negative control, EGFR, Akt, and phosphorylation of Akt were significantly inhibited by taspine treatment in A431 and HEK293/EGFR cells. Consistent with the inhibition of Akt activity, Erk1/2 and its phosphorylation were reduced. Moreover, taspine inhibited A431 xenograft tumor growth. These results suggest that EGFR activated by EGF and its downstream signaling pathways proteins could be downregulated by taspine in a dose-dependent manner. The antitumor mechanism of taspine through the EGFR pathway lies in the ability to inhibit A431 cell proliferation and migration by reducing EGF secretion. This occurs through the repression of EGFR which mediates not only MAPK (Erk1/2) but also Akt signals.

Colorectal cancer represents the fourth commonest malignancy, and constitutes a major cause of significant morbidity and mortality among other diseases. However, the chemical therapy is still under development. Angiogenesis plays an important role in colon cancer development. We developed HMQ18-22 (a novel analog of taspine) with the aim to target angiogenesis. We found that HMQ18-22 significantly reduced angiogenesis of chicken chorioallantoic membrane (CAM) and mouse colon tissue, and inhibited cell migration and tube formation as well. Then, we verified the interaction between HMQ18-22 and VEGFR2 by AlphaScreen P-VEGFR assay, screened the targets on angiogenesis by VEGF Phospho Antibody Array, validated the target by western blot and RNAi in lovo cells. We found HMQ18-22 could decrease phosphorylation of VEGFR2(Tyr(1214)), VEGFR1(Tyr(1333)), Akt(Tyr(326)), protein kinase Cα (PKCα) (Tyr(657)) and phospholipase-Cγ-1 (PLCγ-1) (Tyr(771)). Most importantly, HMQ18-22 inhibited proliferation of lovo cell and tumor growth in a human colon tumor xenografted model of athymic mice. Compared with normal lovo cells proliferation, the inhibition on proliferation of knockdown cells (VEGFR2, VEGFR1, Akt, PKCα and PLCγ-1) by HMQ18-22 decreased. These results suggested that HMQ18-22 is a novel angiogenesis inhibitor and can be a useful therapeutic candidate for colon cancer intervention.