Suppression of VEGF-induced angiogenesis and tumor growth by Eugenia jambolana, Musa paradisiaca, and Coccinia indica extracts.

CONTEXT: Abnormal angiogenesis and evasion of apoptosis are hallmarks of cancer. Accordingly, anti-angiogenic and pro-apoptotic therapies are effective strategies for cancer treatment. Medicinal plants, namely, Eugenia jambolana Lam. (Myrtaceae), Musa paradisiaca L. (Musaceae), and Coccinia indica Wight & Arn. (Cucurbitaceae), have not been greatly investigated for their anticancer potential. OBJECTIVE: We investigated the anti-angiogenic and pro-apoptotic efficacy of ethyl acetate (EA) and n-butanol (NB) extracts of E. jambolana (seeds), EA extracts of M. paradisiaca (roots) and C. indica (leaves) with respect to mammary neoplasia. MATERIALS AND METHODS: Effect of extracts (2-200 µg/mL) on cytotoxicity and MCF-7, MDA-MB-231 and endothelial cell (EC) proliferation and in vitro angiogenesis were evaluated by MTT, 3[H]thymidine uptake and EC tube formation assays, respectively. In vivo tumour proliferation, VEGF secretion and angiogenesis were assessed using the Ehrlich ascites tumour (EAT) model followed by rat corneal micro-pocket and chicken chorioallantoic membrane (CAM) assays. Apoptosis induction was assessed by morphological and cell cycle analysis. RESULTS: EA extracts of E. jambolana and M. paradisiaca exhibited the highest cytotoxicity (IC50 25 and 60 µg/mL), inhibited cell proliferation (up to 81%), and tube formation (83% and 76%). In vivo treatment reduced body weight (50%); cell number (16.5- and 14.7-fold), secreted VEGF (∼90%), neoangiogenesis in rat cornea (2.5- and 1.5-fold) and CAM (3- and 1.6-fold) besides EAT cells accumulation in sub-G1 phase (20% and 18.38%), respectively. DISCUSSION AND CONCLUSION: Considering the potent anti-angiogenic and pro-apoptotic properties, lead molecules from EA extracts of E. jambolana and M. paradisiaca can be developed into anticancer drugs.

Combinatorial treatment with anacardic acid followed by TRAIL augments induction of apoptosis in TRAIL resistant cancer cells by the regulation of p53, MAPK and NFκß pathways.

TRAIL, an apoptosis inducing cytokine currently in phase II clinical trial, was investigated for its capability to induce apoptosis in six different human tumor cell lines out of which three cell lines showed resistance to TRAIL induced apoptosis. To investigate whether Anacardic acid (A1) an active component of Anacardium occidentale can sensitize the resistant cell lines to TRAIL induced apoptosis, we treated the resistant cells with suboptimal concentration of A1 and showed that it is a potent enhancer of TRAIL induced apoptosis which up-regulates the expression of both DR4 and DR5 receptors, which has been observed in the cellular, protein and mRNA levels. The death receptors upregulation consequent to A1 treatment was corroborated by the activation of p53 as well as phosphorylation of p38 and JNK MAP kinases and concomitant inactivation of NFκß and ERK signaling cascades. Also, A1 modulated the expression of key apoptotic players like Bax, Bcl-2 and CAD along with the abatement of tumor angiogenesis in vivo in EAT mouse model. Thus, post A1 treatment the TRAIL resistant cells turned into TRAIL sensitive cells. Hence our results demonstrate that A1 can synergize TRAIL induced apoptosis through the upregulation of death receptors and downregulation of anti-apoptotic proteins in cancer context.

Crosstalk between VEGF and MTA1 signaling pathways contribute to aggressiveness of breast carcinoma.

The expression of metastasis associated protein (MTA1) correlates well with tumor metastasis; however its role as a proangiogenic protein and the molecular mechanisms underlying the same are not fully understood. In this study the MTA1 protein was expressed and purified to evaluate its angiogenic potential. In both MCF-7 and MDA-MB-231 cells, endogenous MTA1 protein was localized in the nucleus; while added recombinant MTA1 protein was bound to cell membrane as per immunofluorescence data. MTA1 was detected both in conditioned media and in human serum samples. Recombinant MTA1 regulated cellular functions of HUVEC's such as, proliferation, tube formation, and migration. MTA1 was more potent than VEGF in inducing invasion of breast cancer cells. Analogous to VEGF, MTA1 could induce angiogenesis in both non-tumor and tumor context, as verified by rat cornea, shell less CAM and xenograft models respectively. However MTA-1 was more potent an inducer of angiogenesis. VEGF or Flt-1 gene promoter, luciferase gene reporter analysis revealed that MTA1 up regulates the expression of VEGF and its receptor Flt-1 genes. Kinetics of VEGF-induced expression of MTA1 and qPCR studies showed that there is an increased expression of MTA1 in tumor cells. VEGF induced phosphorylation of endogenous MTA1 on tyrosine residues; phosphorylation was mediated through VEGFR2 and p38-MAP kinase. Recombinant MTA1 activated signaling, in MCF-7 and MDA-MB-231 cells, involved ERK and JNK pathways. In conclusion, MTA1 is a potent angiogenic molecule and cross talk between VEGF and MTA1 protein regulates tumor angiogenesis and metastasis.

Vascular Endothelial Growth Factor Receptor, fms-Like Tyrosine Kinase-1 (Flt-1), as a Novel Binding Partner for SARS-CoV-2 Spike Receptor-Binding Domain.

Angiotensin-converting enzyme 2 (ACE2) and neuropilin 1, a vascular endothelial growth factor (VEGF) receptor, were identified to bind to the SARS-CoV-2 spike receptor-binding domain (spike RBD). In silico analysis based on 3D structure, multiple sequence alignment, and molecular docking of second domain of soluble Flt-1 (sFlt-1) and spike RBD revealed structural similarities, sequence homology, and protein-protein interaction. Interaction and binding of recombinant spike RBD (rspike RBD) and recombinant sFlt-1 (rsFlt-1) in vitro induced a conformational change, as revealed by spectrofluorimetric data, with increased fluorescence intensity in emission spectra as compared to either of the proteins alone. Results on ELISA confirmed the binding and cross-reactivity of rspike-RBD and rsFlt-1 as determined by using either specific antibodies towards each protein or immunized human serum. We found that polyclonal or monoclonal anti-spike RBD antibodies can recognize either rsFlt-1 or rspike RBD, showing cross-reactivity for the two proteins in a dose-dependent binding response. Recognition of bound rspike RBD or rsFlt-1 by anti-Flt-1 or anti-spike RBD antibodies, respectively, as observed by immunoblotting, further confirmed interaction between the two proteins. Immunoprecipitation and immunoblot analysis demonstrated the identification of rspike RBD binding to the Flt-1 receptor on A549 cells. Further, the binding of rspike RBD to Flt-1 receptor was shown using immunofluorescence on 2D-culture or 3D-spheroid of MDA-MB-231 cells, which over-express Flt-1 receptor. Together, our study concludes that the Flt-1 receptor is a novel binding partner for SARS-CoV-2 spike RBD.

Characterization and Cytotoxicity of Pseudomonas Mediated Rhamnolipids Against Breast Cancer MDA-MB-231 Cell Line.

A biosurfactant producing bacterium was identified as Pseudomonas aeruginosa DNM50 based on molecular characterization (NCBI accession no. MK351591). Structural characterization using MALDI-TOF revealed the presence of 12 different congeners of rhamnolipid such as Rha-C8-C8:1, Rha-C10-C8:1, Rha-C10-C10, Rha-C10-C12:1, Rha-C16:1, Rha-C16, Rha-C17:1, Rha-Rha-C10:1-C10:1, Rha-Rha-C10-C12, Rha-Rha-C10-C8, Rha-Rha-C10-C8:1, and Rha-Rha-C8-C8. The radical scavenging activity of rhamnolipid (DNM50RL) was determined by 2, 3-diphenyl-1-picrylhydrazyl (DPPH) assay which showed an IC50 value of 101.8 µg/ ml. The cytotoxic activity was investigated against MDA-MB-231 breast cancer cell line by MTT (4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay which showed a very low IC50 of 0.05 µg/ ml at 72 h of treatment. Further, its activity was confirmed by resazurin and trypan blue assay with IC50 values of 0.01 µg/ml and 0.64 µg/ ml at 72 h of treatment, respectively. Thus, the DNM50RL would play a vital role in the treatment of breast cancer targeting inhibition of p38MAPK.

Inhibition of ascites tumor growth in vivo by sTie-2 is potentiated by a combinatorial therapy with sFLT-1.

BACKGROUND: Inhibition of tumor angiogenesis is a promising approach for cancer therapy and the Tie-2/angiopoietin pathway appears to play an important role. In the present study, we have developed strategies to explore the therapeutic potential of blocking the Tie-2/angiopoietin pathway by sTie-2. METHODS: Ehrlich ascites tumor (EAT) cells were stably transfected to overexpress a truncated form of sTie-2. Transfectants were characterized for their in vitro growth behavior and transplanted into nude mice. Furthermore, recombinant sTie-2 produced by the baculovirus expression system was used to sequester angiopoietins in the murine ascites carcinoma model. The effect of sTie-2 treatment alone or in combination with sFLT-1 on the weight of the animal, ascites cell number and volume was studied. RESULTS: EAT cells stably transfected with a truncated form of sTie-2 showed no change in cell proliferation in vitro and colony forming in soft agar compared to control cells. However, sTie-2 transfected EAT cells transplanted into nude mice reduced tumor burden and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Recombinant sTie-2 showed angiogenic activity in the tube formation and wound healing assay in vitro. sTie-2 treatment alone or in combination with sFLT-1 in an ascites tumor mouse model resulted in reduced peritoneal angiogenesis, with a concomitant decrease in tumor cell number, volume of ascites and the number of invasive tumor cells, as assayed by CD31 staining. CONCLUSIONS: The findings of the present study demonstrate an important role for the Tie-2/angiopoietin pathway in the formation of tumor vasculature and suggest that sTie-2 might yield useful anticancer therapy.

WITHDRAWN: Anti-tumor and proapoptotic effect of withaferin A is mediated by up-regulation of Bax and inhibition NF-kappaB in Ehrlich ascites tumor cells.

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Paracrine action of sFLT-1 secreted by stably-transfected Ehrlich ascites tumor cells and therapy using sFLT-1 inhibits ascites tumor growth in vivo.

BACKGROUND: Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation. METHODS: As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. RESULTS: The sFLT-1 produced by the baculovirus system showed potent anti-angiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. CONCLUSIONS: The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy.

Antiangiogenic and proapoptotic activities of allyl isothiocyanate inhibit ascites tumor growth in vivo.

The authors investigate the antiangiogenic and proapoptotic effects of mustard essential oil containing allyl isothiocyanate (AITC) and explore its mechanism of action on Ehrlich ascites tumor (EAT) cells. Swiss albino mice transplanted with EAT cells were used to study the effect of AITC. AITC was effective at a concentration of 10 mum as demonstrated by the inhibition of proliferation of EAT cells when compared with the normal HEK293 cells. It significantly reduced ascites secretion and tumor cell proliferation by about 80% and inhibited vascular endothelial growth factor expression in tumor-bearing mice in vivo. It also reduced vessel sprouting and exhibited potent antiangiogenic activity in the chorioallantoic membrane and cornea of the rat. AITC arrested the growth of EAT cells by inducing apoptosis and effectively arrested cell cycle progression at the G1 phase. The results clearly suggest that AITC inhibits tumor growth by both antiangiogenic and proapoptotic mechanisms.

Synthesis and Screening of Pro-apoptotic and Angio-inhibitory Activity of Novel Benzisoxazole Derivatives both In Vitro and In Vivo.

BACKGROUND: Triple Negative Breast Cancer (TNBC) tends to be more aggressive than other types of breast cancer. Resistance to chemotherapy is a major obstacle hence there is a significant need for new antineoplastic drugs with multi-target potency. Numerous Benzoisoxazole moieties have been found to possess a broad spectrum of pharmacological activities. In the present study, we have synthesized 9 novel derivatives of Benzisoxazole 7(a-i) and screened them for their biological potential. METHODS: Chemical synthesis, Mass spectrometry (HRMS), cell proliferation and cytotoxicity assay, wound healing assay, flow cytometry and nuclear staining. Angio-inhibitory activity assessed by corneal micropocket assay and in vivo peritoneal angiogenesis assay. RESULTS: The Benzisoxazole derivatives 7(a-i) were synthesized and screened for their biological potency by both in vitro and in vivo experimental models. Among the series, compound 3-(1-((3-(3(Benzyloxy)-4-methoxyphenyl)- 4,5-dihydroisoxazole-5-yl)methyl)piperidine-4-yl)6-fluorobenzo[d] isoxazole (7e) was found to be most promising, with an average IC50 value of 50.36 ± 1.7 µM in MTT assay and showed 81.3% cell death. The compound 7e also showed 60-70% inhibition on a recombinant Metastasis-Associated protein (MTA1) induced proliferation and cell migration in MDAMB-231 cells, which is known to play a major role in angiogenesis. The anti-tumour studies inferred the regression of tumour activity. This was due to inhibition of neovascularization and evoking apoptosis process as assessed by corneal vascularization, peritoneal angiogenesis and apoptotic hallmarks in 7e treated cells. CONCLUSION: These findings not only show the biological efficacy of compound 7e but it is also an effective beginning to explore the mechanism of metastasis and cancer therapy strategy targeting MTA1. The observed biological activity makes compound 7e an attractive drug candidate.

A new pyrrole based small molecule from Tinospora cordifolia induces apoptosis in MDA-MB-231 breast cancer cells via ROS mediated mitochondrial damage and restoration of p53 activity.

Approximately 15% of globally diagnosed breast cancers are designated as triple negative breast cancer (TNBC). In this study, we investigated the effect of the natural compound, Bis(2- ethyl hexyl) 1H-pyrrole-3,4-dicarboxylate (TCCP), purified from Tinospora cordifolia on MDA-MB-231, a TNBC cell line. The pro-apoptotic nature of TCCP on MDA-MB-231 was determined by assessing various apoptotic markers. ROS generation, intracellular calcium, mitochondrial membrane potential (ΔΨm), MPTP, cardiolipin peroxidation and caspase activity were determined fluorometrically. BAX, BCL-2, cytochrome c, caspases, and p53 protein expressions were determined by immunoblotting. Further, the effect of TCCP on DNA and cell death was determined by DNA fragmentation assay, annexin-V staining, and cell cycle analysis. TCCP treatment caused endogenous ROS generation, increase in intracellular calcium and phosphorylation of p53 in a concentration-dependent manner, which was reverted upon pre-treatment with pifithrin-µ. This led to the downstream altered expression of Bcl-2 and Bax proteins, mitochondrial membrane depolarization, MPTP, and cardiolipin peroxidation. TCCP induced cytochrome c release into the cytosol, caspase activation, ultimately resulting in DNA fragmentation. Further, induction of apoptosis and morphological alterations were evident from the phosphatidylserine externalization and increase in sub G1 population. The in vivo Ehrlich ascites tumor (EAT) mouse study revealed the effectiveness of TCCP in reducing the tumor burden and resulted in a ~2 fold increase in mice survival with minimal hepato-renal toxicity. Overall, TCCP was shown to be efficient in inducing ROS and mitochondrial-mediated apoptosis by restoring p53 activity in MDA-MB-231 cells and also induced EAT cell death in vivo thereby inhibiting tumor proliferation.

Purification and characterization of butyrate-induced protein phosphatase involved in apoptosis of Ehrlich ascites tumor cells.

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.

Octacosanol isolated from Tinospora cordifolia downregulates VEGF gene expression by inhibiting nuclear translocation of NF-B and its DNA binding activity.

Octacosanol is a long-chain aliphatic alcohol, which is the main component of policosanol used as a normolipidemic agent. It is known that angiogenesis is involved in tumor growth and metastasis. The present study identified octacosanol isolated from the plant Tinospora cordifolia as a new antiangiogenic compound with inhibitory effects on in vivo angiogenesis assays. Our results showed that octacosanol (i) inhibits proliferation of endothelial cells and Ehrlich ascites tumor cells, (ii) inhibits neovascularization induced by angiogenic factors in chick chorioallantoic membrane and rat cornea in vivo angiogenesis assays, (iii) inhibits secretion of ascites fluid in the growing tumor cells in vivo. Concerning the mechanism of action, octacosanol inhibited secretion of vascular endothelial growth factor into ascites fluid by the tumor cells. At the molecular level octacosanol markedly inhibits activity of matrix metalloproteinases (MMPs) and translocation of transcription factor nuclear factor-B to nucleus. The mechanism of inhibition of angiogenesis by octacosanol reflects on its effect on tumor angiogenesis and metastasis.

N-Substituted-2-butyl-5-chloro-3H-imidazole-4-carbaldehyde derivatives as anti-tumor agents against Ehrlich ascites tumor cells in vivo.

A new series of N-substituted 2-butyl-5-chloro-3H-imidazole-4-carbaldehyde derivatives were synthesized by using the different bioactive heteroaralkyl halides with 2-butyl-4-chloro-1H-imidazole-5-carbaldehyde in presence of powdered potassium carbonate in DMF medium. These compounds were screened for their antitumor activity. Our results show that treatment of imidazole derivatives inhibit proliferation EAT cells, decreases the ascites volume and increases the survivability of the animals in vivo. These compounds also inhibited the cellular proliferation of HUVEC cells in vitro by MTT assay. Further, these compounds could induce apoptosis, which is evident by the nuclear condensation of imidazole derivatives treated EAT cells in vivo by the cytological analysis. We have identified that pyrrolidine substituted imidazole derivative as potent anti-tumor compound. These inhibitors could represent as promising candidates for anticancer therapies, where the formation of peritoneal malignant ascites is a major cause of morbidity and mortality.

A pyrrole-based natural small molecule mitigates HSP90 expression in MDA-MB-231 cells and inhibits tumor angiogenesis in mice by inactivating HSF-1.

Heat shock proteins (HSPs), molecular chaperones, are crucial for the cancer cells to facilitate proper functioning of various oncoproteins involved in cell survival, proliferation, migration, and tumor angiogenesis. Tumor cells are said to be "addicted" to HSPs. HSPs are overexpressed in many cancers due to upregulation of transcription factor Heat-shock factor 1 (HSF-1), the multifaceted master regulator of heat shock response. Therefore, pharmacological targeting of HSPs via HSF-1 is an effective strategy to treat malignant cancers like triple negative breast cancer. In the current study, we evaluated the efficacy of a pyrrole derivative [bis(2-ethylhexyl)1H-pyrrole-3,4-dicarboxylate], TCCP, purified from leaves of Tinospora cordifolia for its ability to suppress heat shock response and angiogenesis using MDA-MB-231 cells and the murine mammary carcinoma: Ehrlich ascites tumor model. HSP90 was downregulated by TCCP by inactivation of HSF-1 resulting in inhibition of tumor cell proliferation, VEGF-induced cell migration, and concomitant decrease in tumor burden and neo-angiogenesis in vivo. The mechanism of suppression of HSPs involves inactivation of PI3K/Akt and phosphorylation on serine 307 of HSF-1 by the activation of ERK1. HSF-1 and HSP90 and 70 localization and expression were ascertained by immunolocalization, immunoblotting, and qPCR experiments. The anti-angiogenic effect of TCCP was studied in vivo in tumor-bearing mice and ex vivo using rat corneal micro-pocket assay. All the results thus corroborate the logic behind inactivating HSF-1 using TCCP as an alternative approach for cancer therapy.

Synthesis and biological evaluation of novel isoxazolines linked via piperazine to 2- benzoisothiazoles as potent apoptotic agents.

Synthesis of 3-(4-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)piperazin-1-yl) benzoisothiazole derivatives (5a-i), which constitute a new class of isoxazolines, has been accomplished in regio-selective manner. These derivatives have been prepared by employing the reaction between substituted aldoximes (4a-i) and 3-(4-Allylpiperazin-1-yl) benzoisothiazole in presence of chloramine-T which afforded in good yields. These compounds were screened for cytotoxic activity on tumor cells. Four among the nine synthesized compounds were found to exhibit potent cytotoxic and antineoplastic activities in comparison to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) protein in mammalian cancer cells. The rest of the derivatives showed moderate activity.

Antiangiogenic and proapoptotic activity of a novel glycoprotein from U. indica is mediated by NF-kappaB and Caspase activated DNase in ascites tumor model.

We have identified a novel glycoprotein from Urginea indica bulbs with potent in vivo antitumor activity against growth of an ascites tumor, mouse mammary carcinoma. In this paper we report characterization of a 29 kDa glycoprotein from U. indica and demonstrate the mechanism of antiangiogenic and proapoptotic activity. N-terminal sequence of the high performance liquid chromatography (HPLC) pure glycoprotein showed sequence homology to an extent of 40-50% with known angiogenesis inhibitor and apoptosis-inducing protein from C. elegans and G. gallus respectively. Our results on antiangiogenic property of the glycoprotein include inhibition of in vivo angiogenesis assays, decreased micro vessel density count and CD31 antigen staining in 29 kDa glycoprotein treated mice peritoneum. In vitro inhibition of vascular endothelial growth factor induced proliferation of human umbilical vein endothelial cells (HUVECs) by the glycoprotein further supports its antiangiogenic activity. The mechanism of antiangiogenesis involved inhibition of translocation of nuclear factor kappa B to the nucleus resulting in decreased expression of vascular endothelial growth factor gene as is demonstrated by our results on quantification of vascular endothelial growth factor levels in the glycoprotein treated tumor bearing mice. Our results on activation of Caspase-3 with concomitant translocation of caspase activated DNase to the tumor cell nuclei resulting in DNA fragmentation induced by the glycoprotein in vivo clearly demonstrated a parallel proapoptotic activity of the glycoprotein.

Angiogenic and proliferative effects of the cytokine VEGF in Ehrlich ascites tumor cells is inhibited by Glycyrrhiza glabra.

Blood vessel plays a crucial role in solid tumor development. It has been suggested that blocking of angiogenesis and the action of the cytokine VEGF could be possible in cancer therapy. In a screen for naturally occurring angiogenic inhibitors, we have identified an extract from the roots of Glycyrrhiza glabra, which has potent antiangiogenic and antitumor activity. The aqueous extract inhibits the in vivo and in vitro proliferation of Ehrlich ascites tumor cells. The angioinhibitory activity of G. glabra was confirmed by its inhibition of angiogenesis in in vivo assays, peritoneal and chorioallantoic membrane assay. Reduction in the levels of the cytokine VEGF and microvessel density count in the peritoneum of mice treated with G. glabra indicated that the plant extract decreased VEGF production and the cytokine induced neovascularization. Our results suggest that the extract from the roots of G. glabra may be a potential supplemental source for cancer therapy.

Anti-metastatic action of anacardic acid targets VEGF-induced signalling pathways in epithelial to mesenchymal transition.

Anacardic acid is a major constituent of nutshell of cashew. In this study, we have isolated it from the leaves of Anacardium Occidentale L. using polarity-based fractionation and confirmed the structure using GC-MS, NMR and FT-IR. The main focus of this study is to harness the molecular mechanism of anti-metastatic action of anacardic acid (A1). We have used MCF-7, a weak metastatic and U-87, a highly metastatic, breast and glioma cell lines respectively, for our study. We have shown that VEGF increases migration and invasion activities of MCF-7 cells, upon overexpression of Twist and Snail genes. It is observed from the current study that exposure of MCF-7 cells to A1 resulted in upregulation of epithelial marker E-cadherin with a concomitant decrease in the expression of mesenchymal markers Twist and Snail gene expression besides exhibiting a strong anti-migratory and anti-invasive activity. In metastatic U-87 glioma cells, treatment with A1 decreased the phosphorylation of MAP kinases, inhibited the translocation of Sp1 and down regulated VEGF and Flt-1 gene expression. Overall, the current findings demonstrate for the first time that anacardic acid functions as a potent EMT inhibitor by targeting VEGF signaling pathway, providing a novel template for drug discovery.

Crosstalk between VEGF and novel angiogenic protein regulates tumor angiogenesis and contributes to aggressiveness of breast carcinoma.

We have identified and characterized a novel proangiogenic glycoprotein (NAP) with molecular weight of 67 kDa from synovial fluid of rheumatoid arthritis patients. Proteomic analysis of the protein revealed 29% sequence coverage with maximum identity for human retinoblastoma binding protein 2. N-terminal amino acid sequence showed no identity to recently discovered protein sequences. NAP was also identified in both normal and tumor cell lines by Western blotting. NAP is a permeability factor as verified by miles permeability assay. The proangiogenic potential of NAP was identified using shell less CAM, rat cornea and tumor on CAM assays. NAP induces expression of VEGF and Flt-1 gene as verified by promoter reporter gene analysis. Further NAP induces proliferation of endothelial cells and formation of tube like structures. NAP is also involved in migration and invasion of tumor cells. Clinical data revealed the presence of NAP in breast cancer biopsies. We have developed monoclonal antibody (mAb), and specific ELISA, which confirmed the presence of NAP in the cytosol of tumor cells. The mAb effect was evaluated with established angiogenic assays. Further, we investigated the detailed mechanism by which NAP induces angiogenesis. NAP is phosphorylated by VEGF induced activation of MAPK and JNK pathways through VEGFR2 phosphorylation. NAP involves JNK pathway predominantly with further activation of NFκB in downstream processing of VEGF activation. Together these findings establish that NAP displays angiogenic properties and promotes efficient neovascularization both in vitro and in vivo models. These observations suggest that anti-NAP-mAb can be targeted for antiangiogenic therapy of cancer.