RESEARCH Open Access The anti-angiogenic and cytotoxic

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RESEARCH Open Access The anti-angiogenic and cytotoxic

Transcript Of RESEARCH Open Access The anti-angiogenic and cytotoxic

Pathania et al. Molecular Cancer 2015, 14:6 http://www.molecular-cancer.com/content/14/1/6

RESEARCH

Open Access

The anti-angiogenic and cytotoxic effects of the boswellic acid analog BA145 are potentiated by autophagy inhibitors
Anup S Pathania1,2, Zahoor A Wani1, Santosh K Guru1, Suresh Kumar1,2, Shashi Bhushan1, Hasan Korkaya3, Darren F Seals5, Ajay Kumar1, Dilip M Mondhe1, Zabeer Ahmed4, Bal K Chandan4 and Fayaz Malik1,2*

Abstract
Background: While angiogenesis inhibitors represent a viable cancer therapy, there is preclinical and clinical data to suggest that many tumors develop resistance to such treatments. Moreover, previous studies have revealed a complex association between autophagy and angiogenesis, and their collective influence on tumorigenesis. Autophagy has been implicated in cytoprotection and tumor promotion, and as such may represent an alternative way of targeting apoptosis-resistant cancer cells. This study explored the anti-cancer agent and boswellic acid analog BA145 as an inducer of autophagy and angiogenesis-mediated cytoprotection of tumor cells.
Methods: Flow cytometry, western blotting, and confocal microscopy were used to investigate the role of BA145 mediated autophagy. ELISA, microvessel sprouting, capillary structure formation, aortic ring and wound healing assays were performed to determine the relationship between BA145 triggered autophagy and angiogenesis. Flow cytometery, western blotting, and microscopy were employed to examine the mechanism of BA145 induced cell death and apoptosis. Live imaging and tumor volume analysis were carried out to evaluate the effect of BA145 triggered autophagy on mouse tumor xenografts.
Results: BA145 induced autophagy in PC-3 cancer cells and HUVECs significantly impeded its negative regulation on cell proliferation, migration, invasion and tube formation. These effects of BA145 induced autophagy were observed under both normoxic and hypoxic conditions. However, inhibition of autophagy using either pharmacological inhibitors or RNA interference enhanced the BA145 mediated death of these cells. Similar observations were noticed with sunitinib, the anti-angiogenic properties of which were significantly enhanced during combination treatments with autophagy inhibitors. In mouse tumor xenografts, co-treatment with chloroquinone and BA145 led to a considerable reduction in tumor burden and angiogenesis compared to BA145 alone.
Conclusion: These studies reveal the essential role of BA145 triggered autophagy in the regulation of angiogenesis and cytoprotection. It also suggests that the combination of the autophagy inhibitors with chemotherapy or anti-angiogenic agents may be an effective therapeutic approach against cancer.
Keywords: Autophagy, angiogenesis, hypoxia, vascular endothelial growth factor receptor-2 (VEGFR-2), light chain protein 3 (LC3), BA145, Chloroquinone (CQ)

* Correspondence: [email protected] 1Department of Cancer Pharmacology, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Jammu and Kashmir 180001, India 2Academy of Scientific and Innovative Research (AcSIR), New Delhi 110001, India Full list of author information is available at the end of the article
© 2015 Pathania et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Background Angiogenesis is the physiological process by which new blood vessels are made from a pre-existing vasculature. While angiogenesis plays an important role in tissue repair and in the formation of the placenta during pregnancy, it is also utilized by malignant cells for their growth and metastasis [1,2]. Suppressing neoangiogenesis in cancer has been considered an attractive therapeutic option in the era of target based drug discovery, and several anti-angiogenic agents are in clinical development for a number of human malignancies [3,4]. Despite the anticipated benefits, angiogenesis inhibitors have failed to produce sustained clinical responses in most patients because of resistance towards these inhibitors. It has been proposed that the activation of cytoprotective autophagy accompanies anti-angiogenic drug resistance [5]. Thus, modulating autophagy may enhance the therapeutic activity of anti-angiogenic drugs.
Autophagy removes the unnecessary and dysfunctional cellular components of the cell via a lysosomal degradation pathway. It protects cells and promotes survival during nutrient starvation, infection, or metabolic stress. There is a complex relationship between autophagy and angiogenesis in different types of cancers. Some studies have revealed that autophagy inhibits the angiogenic vasculature [6,7], while others have suggested that autophagy promotes it [8,9].
To better understand the cross-talk between angiogenesis and autophagy, we focused on the boswellic acid analog BA145. Our group had recently shown that BA145 is the most potent anti-cancer analogue of boswellic acid, and induces robust apoptosis in human leukemia HL-60 cells [10,11]. Other studies have reported the anti-metastatic and anti-angiogenic properties of boswellic acids [12,13]. Here we used a well-studied angiogenesis cell model involving human umbilical vein endothelial cells (HUVECs), the highly aggressive and metastatic human prostate cancer cell line PC-3, as well as mouse models of angiogenesis and cancer to better appreciate the role of autophagy on cell survival, angiogenesis, and tumor progression. Our studies indicate that autophagy plays an important role in cytoprotection by inducing an angiogenic signaling cascade, and that autophagic inhibitors in combination with cytotoxic agents overcome this cytoprotection, thus revealing a previously unknown utility of boswellic acids in cancer therapies.
Results
BA145 exhibits antiproliferative and anti-angiogenic properties To determine its antiproliferative potential, both PC-3 cells and HUVECs were treated with different concentrations of BA145 for 24 and 48 h. Proliferation was monitored by an MTT assay. BA145 treatment inhibited the proliferation of PC-3 cells with IC50 values of 40

and 18 μM after 24 and 48 h, respectively (Figure 1A). Similarly, treatment of HUVECs with BA145 indicated IC50 values of 8 and 4 μM after 24 and 48 h, respectively. FACS analysis of the BA145 treated PC-3 cells and HUVECs displayed concentration-dependent increases in a SubG1 apoptotic cell population as well as a loss in mitochondrial membrane potential (MMP) (Figures 1B and 1C). The proapoptotic potential of BA145 was further confirmed by observing a dose dependent increase in cleavage of procaspase-3 and PARP-1 in both PC-3 cells and HUVECs (Additional file 1: Figure S1A).
To evaluate the anti-angiogenic potential of BA145 in vivo, a Matrigel plug assay was performed in C57/BL6J mice and functional blood vessels were quantified spectrophotometrically by using Drabkin’s reagent. BA145 treatment inhibited VEGF induced blood vessel formation at a dose of 50 and 100 mg/kg when given subcutaneously for 9 days (Figure 1D). RAD001 (5 mg/kg) was used as a positive control. Furthermore, in a wound healing assay it was observed that various concentrations of BA145 inhibited HUVEC and PC-3 cell migration (Figure 1E).
BA145 inhibits proliferative and angiogenic signaling in PC-3 cells VEGF plays a vital role in angiogenesis. VEGF binds to the cell surface receptors VEGFR-1 and VEGFR-2 and activates downstream signaling leading to proliferation, migration, and survival [14]. Hypoxia in tumor tissues induces hypoxia inducible factor-1 (HIF-1) expression, which acts as a transcription factor of genes involved in hypoxic adaptation, promotion of local neovascularisation, and angiogenesis [15,16]. BA145 treatment significantly inhibited VEGF induced expression of VEGFR-1/R-2 and HIF-1α/1β in PC-3 cells in a dose dependent manner (Figure 2A). Since PI3K/Akt plays a vital role in VEGF mediated angiogenesis [17], we determined whether BA145 was also able to suppress the activation of this signaling pathway. Indeed, treatment of PC-3 cells with BA145 led to downregulation of Akt, Raptor, mTOR, and its downstream substrates p70S6 Kinase and eIF4E (Figure 2A).
BA145 induces robust autophagy in HUVECs and cancer cells During autophagy LC3-II is processed from cytosolic LC3-I and expressed on autophagosome membranes along with simultaneous degradation of p62. It was observed that various treatments of BA145 in PC-3 cells and HUVECs for 24 h led to significant increases in LC3-II expression and p62 degradation compared to untreated cells (Figure 2B). Time dependent analysis of BA145 treated PC-3 and HUVECs showed that LC3-II accumulation took place after 2 h along with attendant degradation of p62 (Figure 2F). Acridine orange staining of BA145 treated cells also showed increased formation of acidic

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Figure 1 (See legend on next page.)

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(See figure on previous page.) Figure 1 BA145 exhibits antiproliferative and anti-angiogenic properties. (A) MTT cell viability assay of PC-3 cells and HUVECs treated with different concentrations of BA145 for 24 and 48 h. (B) FACS analysis of SubG1 cell populations following BA145 treatment of PC-3 cells and HUVECs at the indicated concentrations for 24 h. (C) Flow cytometric analysis of MMP loss following BA145 treatment of PC-3 cells and HUVECs at the indicated concentrations for 24 h. (D) Effect of BA145 on VEGF induced angiogenesis in vivo. Six week old C57/BL6J mice were injected with Matrigel containing 50 and 100 mg/kg BA145 along with 100 ng of VEGF into the ventral area. After 10 days, animals were sacrificed to remove Matrigel plugs and were photographed. The neovascularization of the Matrigel plugs were quantified spectrophotometrically using Drabkin’s reagent. (E) VEGF induced chemotactic motility in PC-3 cells and HUVECs following treatment with the indicated concentrations of BA145 for 10 h. Migrated cells were counted manually, and the percent inhibition of cell migration was calculated as shown. Columns, mean; bars, SD; with ***p < 0.001, **p < 0.01, *p < 0.05 versus control.

vesicles in the cytoplasm (Figure 2C, Additional file 1: Figure S1B). Furthermore, BA145 treatment caused a significant increase in the punctate distribution of LC3-II in PC-3 cells, supporting the notion that LC3-II was localized to autophagososmes (Figure 2D). In PC-3 cells, autophagy initiation by BA145 treatment was confirmed by the increased capture of red fluorescence emitted by the acridine orange dye through flow cytometry (Figure 2E). These experiments collectively demonstrated the BA145 dependent induction of autophagic flux in PC-3 cells and HUVECs. We simultaneously confirmed that BA145 triggers autophagy associated increases in LC3-II expression and acridine orange positive vesicles among the colorectal cancer cell lines HCT116 and COLO205 (Additional file 1: Figures S2A and S2B).
Inhibition of autophagy accentuates the cytotoxic properties of BA145 and sunitinib External stimuli or chemically induced stress in tumor cells often triggers autophagy causing cellular dormancy and developmemt of chemoresistance [18]. Inhibitors of such prosurvival autophagy can improve the efficacy of anti-cancer agents when used in combination. BA145 induced autophagy is protective in nature as inhibition of autophagy potentiates BA145 mediated cytotoxicity in PC-3 cells and HUVECs. The combination of BA145 with various autophagy inhibitors, including ammonium chloride (an inhibitor of autophagosome-lysosome fusion) and LY294002 (an inhibitor of the class III PI3kinase activity required for autophagosome formation), significantly enhanced the antiproliferative potential of BA145 in PC-3 cells (Figure 3A) and HUVECs (Additional file 1: Figure S2C) as evidenced by an increase in SubG1 cell populations, MMP loss, and cleavage of procaspase-3 and PARP1 (Figures 3B, 3C, and 3D, Additional file 1: Figures S2D and S2E). Surprisingly, the combinatorial effect of 3-MA with BA145 did not show any significant alteration on cell survival, though the cleavage of caspase-3 and PARP-1 was considerably increased (Figure 3). To further explore the cytoprotective role of autophagy, we tried to inhibit the expression of key autophagy mediator LC3 in PC-3 cells through siRNA. LC3 silencing promoted the apoptotic effect of BA145 as evidenced by increased procaspase-3 and PARP-1 cleavage when compared to a scrambled siRNA

control (Figure 3E). In order to generalize our findings with other anti-angiogenic agents, we treated PC-3 cells and HUVECs with sunitinib. It was observed that sunitinib treatment decreased cell viability, and simultaneously induced autophagy (Additional file 1: Figures S3A, S3B, and S3C). Moreover, inhibition of autophagy with ammonium chloride and bafilomycin greatly enhanced sunitinib induced cytotoxicity. Note that all the pharmacological inhibitors of autophagy were used at noncytotoxic concentrations during these experiments (Figure 3 and Additional file 1: Figure S3).
BA145 has diverse effects on autophagy among different cancer cell lines In the colorectal cancer cell lines HCT116 and COLO205, autophagy induced by BA145 is cytoprotective in nature as co-treatment of BA145 with autophagic inhibitors potentiated its cytotoxicity (Additional file 1: Figures S4A and S4B). This was also true in the MCF-7 breast cancer cell line where autophagy inhibition with LY294002 significantly enhanced BA145 mediated cytotoxicity (Additional file 1: Figures S4D and S4E). In contrast, MDA-MB-231 breast cancer cells respond to LY294002 pre-treatment with decreased cytotoxic effects. In the pancreatic cancer cell lines Mia PaCa-2 and PANC-1, co-treatment of BA145 and LY294002 did not show any significant effect on its cytotoxicity. However, LY294002 further enhanced LC3-II accumulation in BA145 treated Mia PaCa-2 cells. Moreover, LY294002 co-treatment in SH-SY5Y neuroblastoma cells was found to enhance the antiproliferative effect of BA145 (Additional file 1: Figures S4D and S4E). Thus, while the impact of BA145 on autophagy can vary among different cancer cell lines, cancers of widely different origins may also respond similarly to BA145 and thereby be favorable targets for combinatorial treatment with autophagic inhibitors.
Autophagy inhibition potentiates the anti-angiogenic signaling effects of BA145 under both normoxic and hypoxic conditions One of the limitations in anti-angiogenic therapies is the creation of hypoxic tumor microenvironments that favor cytoprotective autophagy [19]. To explore the role of autophagy in promoting angiogenesis, we treated PC-3

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Figure 2 (See legend on next page.)

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(See figure on previous page.) Figure 2 BA145 triggers autophagy and suppresses VEGFR signaling in cancer cells. (A) Western blot analysis of the indicated proteins in VEGF activated PC-3 cells with or without BA145 treatment for 24 h. (B) Western blot analysis of the expression of the autophagy marker proteins LC3 and p62 in BA145 treated PC-3 cells and HUVECs after 24 h. (C) Detection of acidic autophagic vesicles in PC-3 cells and HUVECs. After 24 h treatment with BA145, cells were stained with acridine orange (1 μg/ml) in serum free media for 15 min and fluorescent micrographs were obtained by microscopy. Autophagy is indicated by the red fluorescence while the untreated control cells are green. (D) Detection of LC3-II protein in PC-3 cells by immunofluorescent microscopy. (E) Quantification of acridine orange positive cells by flow cytometry. PC-3 cells were treated with BA145 at the indicated concentrations for 24 h, stained with acridine orange (1 μg/ml) for 15 min, and analyzed with the FL3-H (red color intensity) and FL1-H (green color intensity) channels of the flow cytometer. (F) Time dependent accumulation of LC3-II and loss of p62 in BA145 treated PC-3 cells and HUVECs.

cells with BA145 along with autophagic inhibitors. The co-treatment of BA145 with ammonium chloride, 3-MA, or LY294002 accentuated the anti-angiogenic properties of BA145 in PC-3 cells. Autophagy inhibitors augmented the inhibition of VEGFR-1/R-2 by BA145, and also augmented the inhibition of downstream signaling molecules like HIF-1α/1β, Src, and FAK (Figure 4A). This was

despite the lack of any significant effect of autophagy inhibitors alone on VEGF signaling in PC-3 cells (Additional file 1: Figure S5A). Silencing of the autophagic gene LC3 also accentuated the anti-angiogenic effects of BA145 in PC-3 and HUVECs (Figure 4B). In contrast, the addition of VEGF (20 ng/ml) to BA145 treated PC-3 cells increased the expression of LC3-II as compared to BA145 alone,

Figure 3 Effect of autophagic inhibitors on the antiproliferative properties of BA145 in PC-3 cells. (A) PC-3 cells were co-treated with BA145 (30 μM) and/or the autophagy inhibitors ammonium chloride (10 mM), 3-MA (5 mM), or LY294002 (20 μM) for 24 h. Cell viability was assessed by an MTT assay. (B, C) Analysis of SubG1 cell populations and MMP loss in BA145 treated PC-3 cells. Cells were pretreated with autophagy inhibitors for 1 h followed by addition of BA145 (30 μM) for the next 24 h. PI and Rh-123 dyes were used for the analysis of cell cycle and MMP loss, respectively using flow cytometry. (D) Effect of autophagic inhibitors on the cleavage of procaspase-3 and PARP-1 in BA145 (30 μM) treated PC-3 cells after 24 h. (E) Effect of BA145 on procaspase-3 and PARP-1 cleavage in LC3 silenced cells.

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Figure 4 Autophagy inhibition potentiates the anti-angiogenic effects of BA145. (A) PC-3 cells were treated with BA145 (30 μM) along with autophagic inhibitors for 16 h. Whole cell protein lysates were prepared for western blotting, and the detection of the indicated proteins. (B) LC3 specific siRNA were used to silence the expression of LC3 in PC-3 cells and HUVECs that were further treated with BA145 for 12 h. Protein lysates were analyzed by western blotting for the indicated proteins. In the case of HUVECs, prior treatment of VEGF (20 ng/ml) for about 40 min was given before the addition of BA145 (7 μM). For HIF-1α expression in PC-3 cells, VEGF was added 30 min before the BA145 treatment as PC-3 cells have less endogenous HIF-1α expression. (C) Effect of BA145 on VEGF production in PC-3 cells. Cells were seeded in 6 well plates, grown to 90% confluency, and then exchanged with fresh media containing BA145 for 16 h. Secreted VEGF in the culture media as well as intracellular VEGF were measured by ELISA and western blot, respectively. Columns, mean; bars, SD; with ***p < 0.001, **p < 0.01, *p < 0.05 versus control. (D) Effect of autophagic inhibitors on VEGF production in BA145 treated PC-3 cells under hypoxic and normoxic conditions. Hypoxia was created by treating cells with 100 μM cobalt chloride (CoCl2) for 24 h. After incubation, the media was replaced and cells were treated with BA145 (30 μM) and autophagic inhibitors for the next 16 h. Extracellular VEGF in the media was measured by ELISA. Columns, mean; bars, SD; with **p < 0.01 versus BA145 alone. (E) Western blot analysis of the indicated proteins following treatment of PC-3 cells with BA145 and autophagy inhibitors under normoxic and hypoxic conditions.

and this was followed by a decrease in the SubG1 cell population, and in procaspae-3 and PARP-1 cleavage (Additional file 1: Figures S5B, S5C, and S5D).
To generalize our findings with other anti-angiogenic agents, we found that the autophagy triggered by sunitinib treatment of PC-3 cells was accompanied by increased expression of VEGFR-2 and HIF-1α. However, co-treatment of sunitinib with ammonium chloride

significantly downregulated the increased expression of these proteins and further potentiated the inhibitory effect of sunitinib on HIF-1β expression as well (Additional file 1: Figure S6).
In order to simulate actual tumor conditions, we next determined the consequences of BA145 mediated autophagy under hypoxic microenvironments. Cobalt chloride was added to the media of PC-3 cells to create hypoxic

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conditions, and this was followed by treatments with BA145 in combination with inhibitors of autophagy. As shown in Figure 4C, BA145 inhibited cellular and secreted VEGF expression in PC-3 cells. This was further potentiated by co-treatment with autophagy inhibitors (Figure 4D). The switching of normoxic to hypoxic conditions in cancers leads to transcriptional activation of the HIF-1 gene, which is responsible for the induction of genes that adapt cancer cells under oxygen starved conditions [20]. PC-3 cells produce low levels of VEGF and HIF-1α proteins under normoxia, while the expression of these proteins increases under hypoxic conditions (Figure 4E). The treatment of PC-3 cells with BA145 inhibited HIF-1α/1β expression under hypoxic conditions and co-treatment with autophagy inhibitors further augmented this effect. Moreover, the effect of autophagy inhibition was also observed on the expression of Signal transducer and activation of transcription-3 (Stat-3), the constitutive activation of which enhances HIF-1α expression and triggers glycolytic metabolism via the Warburg effect [21,22]. Our results thus demonstrate that autophagy inhibition in BA145 treated PC-3 cells is able to block this Stat-3 activation under hypoxic conditions (Figure 4E).
Autophagy inhibition accentuates the inhibitory effects of BA145 on angiogenesis as well as on tumor cell colony formation We used ex vivo and in vitro models to explore the role of BA145 triggered autophagy in the regulation of angiogenesis. First, we found in an aortic ring assay that autophagy inhibitors enhanced the BA145 mediated suppression of microvessel sprouting (Figures 5A and 5C). Secondly, in a capillary tube formation assay, it was observed that BA145 abolished VEGF induced tube formation by HUVEC cultures and co-treatment with autophagy inhibitors intensified this effect (Figures 5B and 5C). And thirdly, in a wound healing cell assay, it was demonstrated that the effect of BA145 in the inhibition of VEGF induced HUVECs migration was potentiated by autophagy inhibitors (Additional file 1: Figure S7). BA145 treatment also decreased the colony forming ability of PC-3 cells, and the numbers of colonies were significantly decreased during the combinatorial treatment with ammonium chloride, LY294002, or 3-MA (Figure 5D). Importantly, individual treatments with autophagy inhibitors showed no effect on colony formation (Additional file 1: Figure S8A).
Autophagy inhibition promotes the anti-angiogenic effects of BA145 in vivo Based on the previously described in vitro experiments, we next wanted to determine whether a similar role exists for autophagy inhibitors in BA145 regulation of angiogenesis in vivo using the Matrigel plug assay. As shown in Figure 6A, Matrigel plugs containing VEGF are dark red

in color due to the formation of a functional vasculature. As expected, treatment with BA145 inhibited angiogenesis, as indicated by the faint color of the Matrigel plugs. Chloroquinone (CQ), an antimalarial drug is known to inhibit autophagy by preventing the acidification of lysosomes, which itself impairs the activity of lysosomal proteases and hence the autophagic degradation process [23]. Co-treatment with CQ and BA145 was able to prominently block Matrigel vasculature formation beyond that of BA145 alone (Figure 6A). H&E staining of the formed vasculature in the Matrigel plugs further confirmed that autophagy inhibition by CQ greatly enhanced the antiangiogenic activity of BA145 (Figures 6B and C).
Autophagy inhibitors potentiate the antitumor effect of BA145 Given the success of using CQ to augment the antiangiogenic properties of BA145, we next determined whether CQ could similarly augment the therapeutic efficacy of BA145 against aggressive PC-3 M-luc2 xenografts in NOD.SCID mice. It was observed that the mean tumor volume increased in control mice from 69.0 ± 18.5 mm3 on day 18 to 984.16 ± 100.62 mm3 on day 46. This was compared to the 28 day treatment with BA145 that resulted in a tumor volume increase of 60.0 ± 14.68 mm3 to 710 ± 77.48 mm3 (28% inhibition), and the combination treatment of BA145 and CQ that resulted in a tumor volume increase of 64.0 ± 13.7 mm3 to 410.8 ± 36 mm3 (58% inhibiton) (Figures 6D and E). Additionally, the average weight of the excised tumors was 0.85 ± 0.15 g in control mice, 0.65 ± 0.14 g in BA145 treated mice, and 0.22 ± 0.1 g in mice treated with both BA145 and CQ (Figure 6E). Interestingly, it was observed that BA145 and CQ cotreatment did not have any significant effect on the body weight of mice (Additional file 1: Figure S8B). Flutamide, an anti-androgen used in the treatment of prostate cancers, was used as a positive control group during the experiment, while CQ at a dose of 50 mg/kg was non-toxic to mice (Figure 6D).
Inhibition of autophagy enhances the suppression of pro-angiogenesis factors and associated tumor burden in BA145 treated mice To explore the mechanism by which CQ improved the anti-tumor efficacy of BA145, we examined the changes in angiogenic and apoptotic signals in tumor tissues. Hence, tumor tissues were harvested and lysed in order to monitor the expression of angiogenic and apoptotic factors by western blot analysis. As shown in Figure 6F, BA145 inhibited the activation of VEGFR-2/HIF-1α/ HIF-1β in prostate xenografts while the addition of CQ potentiated this effect. Moreover, co-treatment with CQ and BA145 enhanced the cleavage of procaspase-3 and PARP-1 in tumor tissues.

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Figure 5 Autophagy inhibition significantly enhances BA145 mediated anti-angiogenic activity. (A) Aortic segments were isolated from Sprague-Dawley rats and placed in Matrigel-covered wells. BA145 (10 μM) was added to the wells along with ammonium chloride (N; 10 mM), 3-MA (5 mM), or LY294002 (10 μM) for 4 days. Microvessel sprouting was quantitated with Image J software. (B) Effect of autophagy inhibition on BA145 mediated suppression of VEGF-induced tube formation. HUVECs were seeded in 24-well plates coated with Matrigel and co-treated with BA145 (10 μM) and autophagy inhibitors for 8 h. Cells were fixed and tubular structures were photographed. (C) Percentage inhibition of microvessel sprouting and tube formation of endothelial cells by BA145 along with autophagy inhibitors. (D) Colony formation assay of PC-3 cells. Cells were treated with BA145 (30 μM) along with the indicated autophagy inhibitors for 24 h. Cells were trypsinized and 1000 viable cells were seeded in 60 mm dishes. Cells were allowed to form colonies for 15 days, stained with 1% crystal violet, and counted manually. Columns, mean; bars, SD; with ***p < 0.001, **p < 0.01, *p < 0.05 versus BA145 alone.

Discussion Over the past decade, our understanding of angiogenesis and its role in cancer has increased to a great extent leading to the approval of anti-angiogenic drugs for the treatment of cancer [1,4]. However, many tumors develop drug resistance with progression of the disease occurring after

just a few months of treatment [24,25]. The molecular mechanism of resistance is not well understood as there are many factors which may play role in this process. Several studies have demonstrated that autophagy plays a crucial role in cell survival and resistance to external stress. Regarding tumors, there are several contentious reports

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Figure 6 (See legend on next page.)
AutophagyHuvecsCellsAngiogenesisAutophagy Inhibitors