This study is focused on the use of the extract because of combining the effects of the compounds in EENL which could have multitargeted approach for regulation of multiple signaling pathways in cancer progression. Cytotoxic and antitumor effects of neem leaf extracts have been reported in a panel of cancer cell lines [21
]. In our previous study, we have demonstrated antiproliferative activity of EENL in vitro
in prostate cancer cell lines and antitumor activity in vivo
in prostate cancer xenograft models [15
]. In this study we evaluated the effect of EENL on angiogenesis by assessing the tube formation of endothelial cells using a matrigel culture system. We observed that EENL inhibited VEGF-induced in vitro
tube formation of HUVECs in a dose-dependent manner (). Moreover, we have shown that EENL is able to block development of vasculature in vivo
induced by angiogenic factors which is essential for new vessel development that leads to tumor cell proliferation and invasion (). These results suggest that antiangiogenesis activity of EENL is associated with inhibition of VEGF activity and EENL can be recognized as a therapeutic candidate of anticancer drugs.
We further explored the effect of EENL on proliferation, invasion, and migration of endothelial cells. The extent of HUVECs growth inhibition was measured by MTS assay which was used to determine the number of viable cells in proliferation. EENL treatment significantly inhibited the growth of HUVECs (). Furthermore, migration and invasion assays showed that HUVECs treated with EENL showed markedly attenuated migration and invasion in a concentration-dependent manner (). Mitochondria are key organelles in conversion of energy, regulation of cellular signaling, and amplification of programmed cell death. A growing body of evidence shows changes in mitochondrial shape are related to its altered metabolic state affecting the regulation of cellular functions [23
]. Ultrastructural changes in the HUVECs observed by TME suggest that EENL plays a vital role in altering the cellular functions (). Further studies are required to elucidate the mechanism of action of EENL on the inhibition the cell growth.
In order to identify the molecular targets involved in mediating the effect of EENL in endothelial cells, we used genome-wide gene expression microarray analysis. HUVECs showed a significant upregulation of 1182 genes and downregulation of 1247 genes after treatment with 20.0μ
g/mL of EENL, and 3169 upregulated genes and 1893 downregulated genes with 40.0μ
g/mL of EENL. We further validated the expression of 30 upregulated and 30 downregulated genes by quantitative real-time PCR. There was a significant upregulation of the genes ALDH3A2, ALOX5, ATF3, CLU, EGF, EGR1, FOXC1, GCLM, HMOX1, JDP2, LY96, PEG3, S100P, and SPRR1A. These genes are associated with kidney failure, renal disease, and urological disease [24
]. Significant up-regulation was also confirmed for AKR1B10, AKR1C1, AKR1C2, AKR1C3, CHAC1, CSTA, DDIT3, DMRT1, GPNMB, ID2, LAMP3, SESN2, SPINK1, TRIM16, and TUBA1A. These genes are involved in cellular development and cell death functions [25
]. The majority of the downregulated genes, including ANKRD12, ASPM, CDC25A, CDCA4, CENPE, CHEK1, DLGAP5, DPP4, DTL, EBP, E2F8, FBXO5, FOLH1, HIST1H4C, HSP90B1, ITGAV, KIF14, MAD2L1, METAP2, NRIP1, POLA1, PRIM2, SKP2, TOP2A, TOP2B, TPR, and UHRF1, are associated with cell cycle function and cancer development [26
]. All these validated upregulated and downregulated genes have been shown to be down-regulated and up-regulated, respectively (Tables (a) and (b)), in various cancer tissues, as shown in Oncomine microarray data base [28
]. This again suggests EENL could induce an inhibitory effect on cancer growth and should be further considered for the prevention and treatment of cancer.
To correlate gene expression changes with protein levels, we selected 3 upregulated genes HMOX1, ATF3, and EGR1 for western blot analysis. HMOX1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions [29
]. Although the expression of HMOX1 is low in most tissues, a large number of plant extracts and pharmacological compounds (e.g., green tea, curcumin, and statins) have been demonstrated to induce HMOX1 [30
]. Pharmacologic inhibition of HMOX1 induces marked leukocyte infiltration and enhances VEGF-induced angiogenesis [33
]. Overexpression of HMOX1 in prostate cancer cells downregulated the MMP9 expression and decreased the invasive potential [34
]. The inhibition of invasion and migration of HUVECs following EENL treatment could be the result of a highly significant increase in the RNA and protein expression levels of HMOX1 ( and ). Under prolonged expression of HMOX1 the released iron, carbon monoxide, biliverdin, and bilirubin from the HMOX1 activity may attenuate the stimulatory effects of VEGF and exert antiangiogenic effect [35
]. The induction of HMOX1 by EENL treatment could be the possible mechanism to attenuate the excess formation of blood vessels in inflammatory angiogenesis of the cancer.
ATF3 is a novel stress-activated regulator of p53 protein stability and function [36
]. It provides the cell with a means of responding to a wide range of environmental insult, thus maintaining DNA integrity and protecting the cell against transformation [37
]. ATF3 has been demonstrated to play a role in apoptosis and proliferation, two cellular processes critical for cancer progression [38
]. Enforced expression of ATF3 can restore p53 activity and induce apoptosis of cells [39
]. EENL treatment increased the expression of ATF3 in HUVECs. Targeting ATF3 expression through EENL could be a promising strategy for cancer therapy. EGR-1 is an immediate early gene that is rapidly and transiently induced by many stimuli, including hypoxia, shear stress, and injury [40
]. EGR-1 controls the expression of a wide variety of genes, many of which play a pivotal role in the regulation of cell growth, differentiation, and apoptosis [41
]. However, although several studies have shown EGR-1 promotes cancer progression, there is increasing evidence that EGR-1 may also exerts tumor suppression [42
]. In leukemia, EGR-1 has been implicated in the apoptosis of myeloma cells via interaction with c-JUN while it behaves as a tumor suppressor against the oncogenes E2F-1 and c-MYC [44
]. EGR-1 activation by EGF inhibited MMP9 expression and lymphoma growth [45
]. EENL induced significant upregulation of EGR-1 expression in endothelial cells ( and ) suggesting that EGR1 might have negative effects on endothelial cells. Further studies are needed to elucidate the role of EGR-1 upregulation on the endothelial cell function.
Analysis of the compounds in EENL by LC/TOF-MS including sodium acetate in the mobile phase revealed 4 major peaks which suggest 2′,3′-dehydrosalannol, nimbolide, 6-desacetyl nimbinene, and nimolinone as the major compounds in the EENL (). The formation of an even electron sodium adduct in the source was utilized to provide increased selectivity for the tetranortriterpenoids. Compared to the formation of the protonated adduct as presented earlier [15
], the present data reveals that addition of the basic ion can pick up nimbolide present in the EENL. Further testing revealed that nimbolide and 2′,3′-dehydrosalannol inhibited HUVECs proliferation (), suggesting that these compounds contribute to the antiproliferative activity of EENL.
In conclusion, these findings suggest that EENL containing the compounds nimbolide, 2′,3′-dehydrosalannol, 6-desacetyl nimbinene, and nimolinone inhibits proliferation, migration, invasion and angiogenesis response of HUVECs. We used genome-wide profiling approach to identify the genes associated with multiple biological pathways as potential targets of EENL, and demonstrated the upregulation of the HMOX1, ATF3, and EGR-1 protein expression in HUVECs. Over-stimulation of HMOX1 production could be one of the contributing factor for inhibition of VEGF induced angiogenesis. Our study indicates the importance for further validation of the antiangiogenic potential in preclinical models and in clinical trials, for successful translation of nontoxic neem treatment into the clinic to prevent tumor progression.