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.
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 regarding the role of autophagy in angiogenesis and cancer cell death. Although some studies have shown that the autophagy is a different form of cell death [
26,
27], others have reported that it is a protective mechanism against chemotherapy related toxicity [
28,
29]. In our studies we used the natural compound derivative BA145, a robust autophagy inducer, to investigate the complex association between autophagy, angiogenesis, and cell fate. We are reporting for the first time that BA145 induced autophagy in cancer cells is cytoprotective in nature, and confers protection against the cytotoxic and anti-angiogenic properties of BA145. Using different
in vitro,
ex vivo and experimental mouse models, we have shown that BA145 triggered autophagy promotes cytoprotection and angiogenesis. We further show that combinatorial treatments involving BA145 and autophagy inhibitors can be exploited for enhanced tumor cytotoxicity.
BA145 triggered the apoptotic cell death of PC-3 cells and HUVECs based on an observed increase in SubG1 cell populations, a loss in MMP, PARP-1 cleavage, and activation of caspases. BA145 also exhibited anti-angiogenic properties by suppressing VEGF signaling both in vitro and in vivo. This included the expression of the pro-angiogenic drivers VEGFR-1 and VEGFR-2 as well as several downstream signaling components like HIF-1α, HIF-1β, Src, and FAK. It was further observed that BA145 triggered robust autophagy in PC-3 cells and HUVECs as observed by microscopy, flow cytometry, and the expression of key autophagy proteins. While the role of autophagy in cell death and survival is controversial, our findings conducted in PC-3 cells and HUVECs demonstrated that BA145 induced autophagy is cytoprotective and its inhibition through siRNA or pharmacological inhibitors (e.g. ammonium chloride, 3-MA, or LY294002) augmented the anti-angiogenic and proapoptotic effects of BA145. We further demonstrated that the consequences of BA145 triggered autophagy on cell fate was cell line specific. In the case of HCT116 and COLO205 colon cancer cells, MCF-7 breast cancer cells, and SH-SY5Y neuroblastoma cells, BA145 induced autophagy remained cytoprotective in nature and cytotoxicity was enhanced by autophagy inhibitors. However, in PANC-1 and Mia PaCa-2 cancer cells, autophagy inhibition did not show any significant effect on BA145 mediated cytotoxicity. In MDA-MB-231 breast cancer cells, autophagy inhibition by LY294002 actually decreased BA145 induced cytotoxicity.
Inhibition of autophagy in BA145 treated PC-3 cells enabled significant downregulation of VEGF induced angiogenic signaling compared to BA145 alone. Furthermore, the expression levels of intracellular and extracellular VEGF in PC-3 cells was also diminished. Stat-3 is a point of convergence for many signaling pathways involved in cell proliferation, tumor growth, and angiogenesis [
30], and its expression correlates with the expression of VEGF in human cancer cell lines [
31]. Activated Stat-3 also mediates the up-regulation of HIF-1α by increasing its stability and transcriptional activity [
31]. Interestingly, it was also observed that under conditions of hypoxia, the expression of HIF-1, Stat-3 and VEGF proteins were significantly reduced by combinatorial treatments of BA145 and autophagic inhibitors as compared to BA145 alone. This indicates that autophagy can promote the expression of these pro-survival and pro-angiogenic proteins. The addition of autophagy inhibitors significantly enhanced the anti-angiogenic effects of BA145 as evidenced by reduced capillary like structures, microvessel sprouting and migration in HUVECs. Autophagy inhibition in PC-3 cells enhanced the inhibitory effect of BA145 on colony formation. In mouse models, autophagy inhibition by CQ cooperated with BA145 to completely block vascular formation. And in highly aggressive prostate cancer xenografts in NOD-SCID mice, treatment with BA145 and CQ suppressed tumor growth by 58%, an over 2-fold enhancement relative to BA145 treatment alone. Expression analysis of tumor samples for key angiogenic and autophagy factors revealed that autophagy inhibition by CQ significantly potentiated the negative regulation of BA145 on VEGFR-2, HIF-1α, and HIF-1β, and increased pro-apototic signals. Similarly, inhibiting autophagy in sunitinib treated PC-3 cells and HUVECs greatly enhanced the antiproliferative activity of sunitinib against cancer cells. Consistent with previous studies, it was found that sunitinib treatment increased VEGFR-2 and HIF-1α expression in PC-3 cells [
32‐
34]. This enhanced expression of HIF-1α may be due to the creation of hypoxia, which plays a critical role in the development of angiogenic drug resistance. Our results further demonstrated that the inhibition of autophagy with ammonium chloride in sunitinib treated PC-3 cells led to the supression of angiogenesis as evidenced by the significant decrease in VEGFR-2, HIF-1α, and HIF-1β expression.
Methods
Reagents and Antibodies
RPMI-1640, MEM, propidium iodide (PI), rhodamine-123 (Rh-123), acridine orange, 3-(4, 5- dimethylthiazole-2-yl)-2,5 diphenyltetrazolium bromide (MTT), phenylmethanesulfonyl fluoride (PMSF), 3-methyl adenine (3-MA), chloroquinone (CQ), ammonium chloride, bafilomycin, cobalt chloride, sodium fluoride, kanamycin, streptomycin, fetal bovine serum (FBS), β-mercaptoethanol, and human VEGF were purchased from Sigma-Aldrich, Missouri, USA. Growth factor reduced Matrigel were from BD Biosciences, New Jersey, USA. Antibodies for casapse-8, caspase-9, caspase-3, PARP-1, and β-Actin were from Santa Cruz Biotechnology, Texas, USA. All other antibodies and LY294002 were purchased from Cell Signaling Technology, Massachusetts, USA. Electrophoresis reagents, reagents for protein estimation, and protein molecular weight markers were from Bio-Rad Laboratories, California, USA.
Cell Culture and Treatments
The human prostate cancer cell line PC-3, the colorectal carcinoma cell lines HCT116 and COLO205, the human breast cancer cell lines MCF-7 and MDA-MB-231, the human pancreatic cancer cell lines Panc-1 and Mia PaCa-2, the human neuroblastoma cell line SH-SY5Y and primary human umbilical vein endothelial cells (HUVECs) were purchased from ECACC, England. Cancer cells were grown in RPMI/MEM/McCOY/DMEM growth media containing 10% FBS, 100U penicillin G, and 100 μg streptomycin per ml. HUVECs were grown in EndoGRO-LS complete media from Millipore. Cells were grown in a CO2 incubator (Thermocon Electron Corporation, Texas, USA) at 37°C with 95% humidity. BA145, bafilomycin, and LY294002 were dissolved in DMSO (final concentration <0.2%); ammonium chloride, CQ, and 3-MA were solubilized in Milli-Q water.
Cell Proliferation Assay
Cells were seeded in 96 well plates. When at 70-75% confluency, cells were treated with different concentrations of BA145 for 24 and 48 h. All inhibitors used in the experiment were added 1 h before the treatment of BA145. Cell proliferation was assessed by using an MTT assay as previously described [
35].
Mitochondrial Membrane Potential (MMP) Loss
Cells were treated with or without inhibitors at different concentrations for 24 h. Mitochondrial membrane potential loss (Ψmt) was analyzed by using the fluorescent probe rhodamine 123 as previously described [
11].
Wound Healing Migration Assay
Cells were seeded in 6 well plates. When at 80% confluency, a micro tip was used to draw a scratch across the center of the culture plate to produce a clean wound area. Cells were incubated with BA145 for 8-10 h, and cell migration was examined under the microscope (Olympus Imaging) by manual counting.
Cells were treated with BA145 in the presence or absence of autophagic inhibitors at different concentrations for 24 h. Cells were then trypsinized, viable cells were counted, and 1000 cells were seeded in 60 mm dishes for 15 days to determine the effect of these compounds on clonogenic survival. The colonies were fixed in 4% formaldehyde for 15-20 min, stained with 1% crystal violet, and the colonies counted.
Enzyme-linked Immunosorbent Assay (ELISA) for VEGF
PC-3 cells were seeded in 60 mm dishes. When at 90-95% confluency, cells were treated with BA145 in the presence or absence of autophagic inhibitors and incubated under hypoxic or normoxic conditions for 16 h. Supernatants from the media were collected and the VEGF concentration was determined according to manufacturer guidelines using a VEGF ELISA kit from R&D Systems (#DVE00), Minnesota, USA.
A tube formation assay was performed by using an in vitro angiogenesis assay kit (Millipore, #ECM 625) according to manufacturer guidelines. Briefly, the extracellular matrix was diluted with diluents buffer before use at 4°C, and 50 μl of this solution was transferred to each well of a pre-cooled 96 well plate. The plate was placed in an incubator at 37°C for at least 1 h to allow the solidification of the matrix solution. HUVECs were trypsinized, and 5000–8000 cells were seeded in each matrix coated well, followed by overnight incubation at 37°C. Cells were treated with BA145 and autophagic inhibitors for 10 h. Tube formation was observed under the light microscope at 10X magnification equipped with a digital camera (Olympus Imaging), and were counted manually.
Aortic Ring Assay
A rat aortic ring assay was performed as previously described [
36]. Briefly,
Sprague Dawley rats were obtained from the central animal facility of the institute. Rats were sacrificed by cervical dislocation and aortas were isolated, precleaned from periadventitial fat, and cut into rings at 1 to 1.5 mm in circumference under aseptic conditions. The aortic rings were embedded in Matrigel with or without VEGF (20 ng/ml), followed by treatment with BA145 (10 μM) alone or in combination with LY294002 (10 μM), 3-MA (5 mM) and ammonium chloride (10 mM) for 4 days. Microvessel sprouts were fixed, photographed using an Olympus IX70 inverted microscope, and counted using Image J software [
36].
In VivoAngiogenesis Assay
A Matrigel plug assay was performed as previously described [
37]. Briefly, 0.5 ml ice cold Matrigel (BD Biosciences) was injected subcutaneously along with VEGF-A (150 ng/mL), BA145 (50 mg/Kg body weight (BW), and/or CQ (50 mg/kg BW) into C57/BL6J male mice (20-25 g, 4-6 weeks old). After 10 days, mice were sacrificed in order to remove the Matrigel plugs and photographs were taken. The neovascularization of the Matrigel plugs was quantified using Drabkin’s reagent and the absorbance was measured spectrophotometerically at 540 nm in order to estimate hemoglobin (Hb) where Hb (g/dl) = absorbance of sample/absorbance of standard × concentration of standard. All mice were obtained from the central animal facility of the institute. Mice were housed and cared under standard conditions and animal studies were performed according to experimental protocols approved by the Institutional Animal Ethics Committee (IAEC).
Tumor Studies
The antitumor efficacy of BA145 and the autophagic inhibitor CQ were examined against human prostate cancer xenografts in mice. Male NOD.SCID mice (18-23 g) were procured from the animal facility of the institute and were housed in a sterile microenvironment. The number of animals and the protocols used in this study were approved by the IAEC. PC-3 M-
luc2 cells (3 × 10
6) were suspended in PBS, mixed with an equal volume of Matrigel, and injected subcutaneously into the right flanks of each animal on day 0. Tumor growth was monitored on every alternate day and animals bearing 50-100 mg tumor mass were selected for the experiment on the staging day (day 18). Mice were randomly distributed into 5 different groups with 6 mice in each group. Group I was administered 0.2 ml normal saline (i.p.), and served as an untreated control. Group II was treated with BA145 (75 mg/kg i.p.) on every alternate day. Group III was administered BA145 (75 mg/kg i.p.) and CQ (50 mg/kg i.p.) on every alternate day. Group IV was administered flutamide (25 mg/kg p.o.), and served as a positive control. Group V was administered with CQ (50 mg/kg i.p.) on every alternate day. Tumors volumes were calculated as described earlier [
38]. On day 46, all animals were sacrificed and tumors were excised. Cell lysates were prepared from the harvested tumors using RIPA buffer for western blotting.
Immunofluorescence and Confocal Microscopy
PC-3 cells were grown on coverslips and treated with BA145 at different concentrations for 24 h. Cells were fixed, stained, and analyzed under the fluorescent microscope as previously described [
11].
Preparation of Cell Lysates and Western Blot Analysis
The preparation of cell lysates and protein specific immunoblotting was performed as previously described [
11].
Statistical Analysis
Data are presented as means of three similar experiments and the error bars represent the standard deviation (SD) between the experiments. Statistical analysis was done using the Bonferroni method and a p value <0.05 was considered to be significant (***p < 0.001, **p < 0.01, *p < 0.05).
Acknowledgements
We are thankful to the Council of Scientific and Industrial Research (CSIR), India for financial assistance to carry out this research work, including a research fellowship to ASP. We are also grateful to Dr. PR Sharma for his help in microscopy and Mr. Girish Mahajan for his support during the in vivo studies.
Financial Support
This work was supported by grants (BSC-205 and P81-113) from the Council of Scientific and Industrial Research (CSIR), India.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
ASP performed the experiments. FM and ASP planned and designed the experiments. ZAW and DMM performed the mice xenograft model experiments. SKG and SK performed the in vivo Matrigel plug assay. BKC performed the H&E staining. FM, ASP and HK analyzed and interpreted the data. HK and DS helped in refining the manuscript. All authors read and approved the final manuscript.