Patient-derived Siglec-6-targeting antibodies engineered for T-cell recruitment have potential therapeutic utility in chronic lymphocytic leukemia.

BACKGROUND: Despite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues. METHODS: We used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment-Fc and dual-affinity retargeting (DART)-Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo. RESULTS: We discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+ target cells. The RC-1 clone in the DART-Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+ primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+ fraction of primary B cells from healthy donors. The subpicomolar potency of the DART-Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days. CONCLUSION: Siglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+ leukemic and healthy B cells while sparing Siglec-6- healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy. TRIAL REGISTRATION NUMBER: NCT00923507.

Effects of Microbial Transfer during Food-Gut-Feces Circulation on the Health of Bombyx mori.

Change in habitual diet may negatively affect health. The domestic silkworm (Bombyx mori) is an economically important oligophagous insect that feeds on mulberry leaves. The growth, development, and immune-disease resistance of silkworms have declined under artificial dietary conditions. In this study, we used B. mori as a model insect to explore the relationship between changes in diet and balance of intestinal microbes due to its simpler guts compared with those of mammals. We found that artificial diets reduced the intestinal bacterial diversity in silkworms and resulted in a simple intestinal microbial structure. By analyzing the correlations among food, gut, and fecal microbial diversity, we found that an artificial diet was more easily fermented and enriched the lactic acid bacteria in the gut of the silkworms. This diet caused intestinal acidification and microbial imbalance (dysbiosis). When combined with the artificial diet, Enterococcus mundtii, a colonizing opportunistic pathogen, caused dysbiosis and allowed the frequent outbreak of bacterial diseases in the silkworms. This study provides further systematic indicators and technical references for future investigations of the relationship between diet-based environmental changes and intestinal microbial balance. IMPORTANCE The body often appears unwell after habitual dietary changes. The domestic silkworm (Bombyx mori) raised on artificial diets is a good model to explore the relationship between dietary changes and the balance of intestinal microbes. In this study, the food-gut-feces microbial model was established, and some potential key genera that could regulate the balance of intestinal microbiota were screened out. Our findings will provide a reference for future research to further our understanding of healthy silkworm development and may even be useful for similar research on other animals.

BTK inhibitors, irrespective of ITK inhibition, increase efficacy of a CD19/CD3-bispecific antibody in CLL.

Bruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb-induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

Redirecting cytotoxic T cells with chemically programmed antibodies.

T-cell engaging bispecific antibodies (T-biAbs) mediate potent and selective cytotoxicity by combining specificities for target and effector cells in one molecule. Chemically programmed T-biAbs (cp-T-biAbs) are precisely assembled compositions of (i) small molecules that govern cancer cell surface targeting with high affinity and specificity and (ii) antibodies that recruit and activate T cells and equip the small molecule with confined biodistribution and longer circulatory half-life. Conceptually similar to cp-T-biAbs, switchable chimeric antigen receptor T cells (sCAR-Ts) can also be put under the control of small molecules by using a chemically programmed antibody as a bispecific adaptor molecule. As such, cp-T-biAbs and cp-sCAR-Ts can endow small molecules with the power of cancer immunotherapy. We here review the concept of chemically programmed antibodies for recruiting and activating T cells as a promising strategy for broadening the utility of small molecules in cancer therapy.

Chemically Programmable and Switchable CAR-T Therapy.

Although macromolecules on cell surfaces are predominantly targeted and drugged with antibodies, they harbor pockets that are only accessible to small molecules and constitutes a rich subset of binding sites with immense potential diagnostic and therapeutic utility. Compared to antibodies, however, small molecules are disadvantaged by a less confined biodistribution, shorter circulatory half-life, and inability to communicate with the immune system. Presented herein is a method that endows small molecules with the ability to recruit and activate chimeric antigen receptor T cells (CAR-Ts). It is based on a CAR-T platform that uses a chemically programmed antibody fragment (cp-Fab) as on/off switch. In proof-of-concept studies, this cp-Fab/CAR-T system targeting folate binding proteins on the cell surface mediated potent and specific eradication of folate-receptor-expressing cancer cells in vitro and in vivo.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Conventional and Chemically Programmed Asymmetric Bispecific Antibodies Targeting Folate Receptor 1.

T-cell engaging bispecific antibodies (biAbs) can mediate potent and specific tumor cell eradication in liquid cancers. Substantial effort has been invested in expanding this concept to solid cancers. To explore their utility in the treatment of ovarian cancer, we built a set of asymmetric biAbs in IgG1-like format that bind CD3 on T cells with a conventional scFv arm and folate receptor 1 (FOLR1) on ovarian cancer cells with a conventional or a chemically programmed Fab arm. For avidity engineering, we also built an asymmetric biAb format with a tandem Fab arm. We show that both conventional and chemically programmed CD3 × FOLR1 biAbs exert specific in vitro and in vivo cytotoxicity toward FOLR1-expressing ovarian cancer cells by recruiting and activating T cells. While the conventional T-cell engaging biAb was curative in an aggressive mouse model of human ovarian cancer, the potency of the chemically programmed biAb was significantly boosted by avidity engineering. Both conventional and chemically programmed CD3 × FOLR1 biAbs warrant further investigation for ovarian cancer immunotherapy.

An IgG1-like bispecific antibody targeting CD52 and CD20 for the treatment of B-cell malignancies.

Bispecific antibodies (biAb) targeting two different antigens or two distinct epitopes on the same antigen have demonstrated broad therapeutic utility. CD52 and CD20 are co-expressed on the cell surface of malignant B cells in B-cell non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) and increased expression of both antigens is detected on dividing or recently divided cells ("proliferative fraction") in CLL. The CD52-targeting monoclonal antibody (mAb) alemtuzumab (atz) not only depletes malignant B cells but also healthy CD52+ B and T lymphocytes and monocytes, causing severe immunosuppression. Loss of CD20 can occur in CLL after treatment with rituximab (rtx) and other CD20-targeting mAbs. To broaden the benefit of atz and rtx, we engineered an IgG1-like biAb, atz × rtx scFv-Fc. The Fc fragment of the biAb facilitates purification by Protein A affinity chromatography and supports a longer circulatory half-life. While atz × rtx scFv-Fc retained both antigen binding specificities, it showed superior binding to CD52+CD20+ B cells compared to CD52+CD20- T cells. Moreover, atz × rtx scFv-Fc mediated potent complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in vitro and exhibited B-cell depleting but T-cell sparing activities in vivo in a CLL patient-derived xenograft model. B-cell depletion was more pronounced for cells of the proliferative fraction.

Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1.

T cell-engaging bispecific antibodies (biAbs) present a promising strategy for cancer immunotherapy, and numerous bispecific formats have been developed for retargeting cytolytic T cells toward tumor cells. To explore the therapeutic utility of T cell-engaging biAbs targeting the receptor tyrosine kinase ROR1, which is expressed by tumor cells of various hematologic and solid malignancies, we used a bispecific ROR1 × CD3 scFv-Fc format based on a heterodimeric and aglycosylated Fc domain designed for extended circulatory t1/2 and diminished systemic T cell activation. A diverse panel of ROR1-targeting scFv derived from immune and naïve rabbit antibody repertoires was compared in this bispecific format for target-dependent T cell recruitment and activation. An ROR1-targeting scFv with a membrane-proximal epitope, R11, revealed potent and selective antitumor activity in vitro, in vivo, and ex vivo and emerged as a prime candidate for further preclinical and clinical studies. To elucidate the precise location and engagement of this membrane-proximal epitope, which is conserved between human and mouse ROR1, the 3D structure of scFv R11 in complex with the kringle domain of ROR1 was determined by X-ray crystallography at 1.6-Å resolution.

A CD19/CD3 bispecific antibody for effective immunotherapy of chronic lymphocytic leukemia in the ibrutinib era.

The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of ∼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naïve patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naïve CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naïve patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Taken together, these data support investigation of CD19/CD3 bsAb's and other T cell-recruiting bsAb's as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs.

Chemically Programmed Bispecific Antibodies in Diabody Format.

Chemically programmed bispecific antibodies (biAbs) endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system. Here we report a platform of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells. Two different antibody technologies were merged together to make a novel chemically programmed biAb. This was achieved by combining the humanized anti-hapten monoclonal antibody (mAb) h38C2 with the humanized anti-human CD3 mAb v9 in a clinically investigated diabody format known as Dual-Affinity Re-Targeting (DART). We show that h38C2 × v9 DARTs can readily be equipped with tumor-targeting hapten-derivatized small molecules without causing a systemic response harming healthy tissues. As a proof of concept, we chemically programmed h38C2 × v9 with hapten-folate and demonstrated its selectivity and potency against folate receptor 1 (FOLR1)-expressing ovarian cancer cells in vitro and in vivo Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy.

Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset.

Eupolyphaga sinensis Walker Ethanol Extract Suppresses Cell Growth and Invasion in Human Breast Cancer Cells.

AIM OF THE STUDY: To examine the antiproliferation and anti-invasion of Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) on breast cancer and elucidate the underlying signaling mechanisms. METHODS: MTT and colony formation assays were used to investigate the effect of ESWE on proliferation of breast cancer cells in vitro. The xenograft mouse tumor model was used to determine the effect of ESWE on breast cancer in vivo. To investigate the underlying molecular mechanisms, we used western blotting to analyze the expression of ERK1/2, CXCR4, matrix metalloproteinase 2 (MMP2), and MMP9 pretreated with ESWE. The stromal cell-derived factor (SDF)-1α-induced migration and invasion potential of breast cancer cells were examined by wound-healing assays and Matrigel invasion chamber assays. RESULTS: ESWE effectively inhibited the proliferation of MDA-MB-435s and MDA-MB-231 cells and exhibited antitumor effects in an MDA-MB-231 xenograft mice model. Furthermore, ESWE suppressed the activity of ERK1/2, a key molecule of MAPK signaling. We also observed that ESWE treatment led to downregulation of CXCR4 expression as well as greatly reduced MMP2 and MMP9. ESWE affected CXCR4 expression partially through the modulation of autocrine vascular endothelial growth factor. However, suppression of CXCR4 expression was the result of downregulation of mRNA expression. Inhibition of CXCR4 expression by ESWE further correlated with the suppression of SDF-1α-induced migration and invasion in breast cancer cells. CONCLUSION: ESWE exerted its antiproliferation and antiinvasion by regulating MAPK signaling and related metastasis factorsand thus could be a useful therapeutic candidate for breast cancer intervention.

Ferulic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NADPH oxidase and NF-κB pathway.

Ferulic acid (FA) is a dietary phenolic acid and has a wide range of therapeutic effects, including anti-aging, antitumor activity and antihypertensive effects. The aim of present study was to evaluate the inhibitory effects of FA on cell inflammation and oxidative stress against hydrogen peroxide (H2O2)-induced injury in rat vascular smooth muscle cells (VSMCs) in vitro. VSMCs were pretreated with FA 2h before H2O2 incubation. The results suggested that FA inhibited H2O2-induced cell injury by reducing the MDA and increasing the SOD activity and GSH content. In rat VSMCs exposed to H2O2, FA increased the cell viability and restored the mitochondrial membrane depolarization. The level of ROS generation was reduced by pretreatment with FA through inhibiting the expression of NADPH oxidase and down-regulating MAPK and AKT pathways. We found that H2O2 stimulated the production of IL-6, IL-1ß, TNF-α and NO, which could be reduced by pretreatment with FA through inhibiting the p-NF-κB as well as the iNOS expression. In conclusion, our results show that FA may serve as a novel drug in the treatment of these pathologies by inhibiting NADPH oxidase and NF-κB and subsequently decreasing VSMC oxidative stress and inflammation. These suggest that the inhibitory effect of FA on VSMC inflammation and oxidative stress is partially attributed to depressing NADPH and NF-κB expressions in VSMCs, decreasing the ROS production and reducing apoptosis of VSMCs.

c-Myc plays a key role in TADs-induced apoptosis and cell cycle arrest in human hepatocellular carcinoma cells.

Cancer cell growth is complicated progression which is regulated and controlled by multiple factors including cell cycle, migration and apoptosis. In present study, we report that TADs, a novel derivative of taspine, has an essential role in resisting hepatocellular carcinoma growth (including arrest cell cycle) and migration, and inducing cell apoptosis. Our findings demonstrated that the TADs showed good inhibition on the hepatoma cell growth and migration, and good action on apoptosis induction. Using genome-wide microarray analysis, we found the down-regulated growth and apoptosis factors, and selected down-regulated genes were confirmed by Western blot. Knockdown of a checkpoint c-Myc by siRNA significantly attenuated tumor inhibition and apoptosis effects of TADs. Moreover, our results indicated TADs could simultaneously increase cyclin D1 protein levels and decrease amount of cyclin E, cyclin B1 and cdc2 of the cycle proteins, and also TADs reduced Bcl-2 expression, and upregulated Bad, Bak and Bax activities. In conclusion, these results illustrated that TADs is a key factor in growth and apoptosis signaling inhibitor, has potential in cancer therapy.

A novel compound T7 (N-{4'-[(1E)-N-hydroxyethanimidoyl]-3',5,6-trimethoxybiphenyl-3-yl}-N'-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) screened by tissue angiogenesis model and its activity evaluation on anti-angiogenesis.

A tissue model for angiogenesis that imitated new blood vessels formation in vivo had been established in the previous study. Here, it was used to screen and evaluate a series of synthesized compounds and the results indicated that compound T7 (N-{4'-[(1E)-N-hydroxyethanimidoyl]-3',5,6-trimethoxybiphenyl-3-yl}-N'-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) could effectively inhibit the blood vessels formation. Then the anti-angiogenic potential of T7 and its related molecular mechanisms against lung carcinoma in vitro and in vivo were investigated. Treatment with T7 significantly inhibited human umbilical vein endothelial cells and A549 cells proliferation and migration. T7 reduced human umbilical vein endothelial cells tube formation as well. Western blotting analysis of cell signaling molecules indicated that T7 reduced phosphorylation of KDR and its downstream signaling players AKT and ERK1/2 activation in endothelial cells and A549 cells. Moreover, T7 inhibited tumor growth in A549 xenografted model of athymic mice and reduced CD34 expression levels in tumor-bearing mice by immunohistochemistry. In sum, our findings showed that T7 was a candidate of tumor angiogenesis inhibitors, and it functioned by interrupting the autophosphorylation of KDR, AKT and ERK1/2.

Eupolyphaga sinensis walker displays inhibition on hepatocellular carcinoma through regulating cell growth and metastasis signaling.

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 PKCß 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 PKCß, 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.

Eupolyphaga sinensis Walker demonstrates angiogenic activity and inhibits A549 cell growth by targeting the KDR signaling pathway.

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 non­small 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 dose­dependent 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.