Robust Anticancer Efficacy of a Biologically Synthesized Tumor Acidity

Subscriber access provided by READING UNIV

Article

Robust Anticancer Efficacy of a Biologically Synthesized Tumor Acidity-Responsive and Autophagy-Inducing Functional Beclin 1 Guo-Bin Ding, Junqing Sun, Gengfeng Wu, Binchun Li, Peng Yang, Zhuoyu Li, and Guangjun Nie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17454 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Robust Anticancer Efficacy of a Biologically Synthesized Tumor Acidity-Responsive and Autophagy-Inducing Functional Beclin 1

Guo-Bin Ding,*,†,‡,# Junqing Sun,†,‡,# Gengfeng Wu,†,‡ Binchun Li,† Peng Yang,†,‡ Zhuoyu Li*,†,‡,∥ and Guangjun Nie*,§

†

Institute of Biotechnology, the Key Laboratory of Chemical Biology and Molecular

Engineering of Ministry of Education, Shanxi University, Taiyuan 030006, China ‡

Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, China

∥

§

School of Life Science, Shanxi University, Taiyuan 030006, China

CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National

Center for Nanoscience and Technology, Beijing 100190, China

#

These authors contributed equally to this work.

E-mail: [email protected] (G.B. Ding); [email protected] (Z. Li); [email protected] (G. Nie)

KEYWORDS: functional Beclin 1, pHLIP, biological synthesis, autophagy induction, anticancer efficacy

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT As a potent autophagy inducer, Beclin 1 is essential for initiation of autophagic cell death, and triggering extensive autophagy by targeted delivery of Beclin 1 to tumors has enormous potential to inhibit tumor growth. Yet the therapeutic application of Beclin 1 is hampered by its inability to internalize into cells and non-selective biodistribution in vivo. To tackle this challenge, we employed a novel Beclin 1 delivery manner by constructing a functional protein (Trx-pHLIP-Beclin 1, TpB) composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP) and an evolutionarily conserved motif of Beclin 1. This protein could effectively transport Beclin 1 to breast and ovarian cancer cell lines under weakly acidic conditions (pH 6.5), markedly inhibit tumor cell growth and proliferation, and induce obvious autophagy. Furthermore, the in vivo antitumor efficacy of the functional Beclin 1 against an SKOV3 xenograft tumor mouse model was tested via intravenous injection. TpB preferentially accumulated in tumors and exhibited significantly higher tumor growth inhibition than the non-targeted Beclin 1 control, while no overt side effects were observed. Taken together, this study sheds light on the potential application of TpB as a highly efficient yet safe antitumor agent for cancer treatment.

2 ACS Paragon Plus Environment

Page 2 of 35

Page 3 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION Autophagy is an elementary and highly conservative intracellular degradation process for clearance of dysfunctional organelles and damaged macromolecules.1,2 Aberrant autophagy is closely implicated in diverse pathologic processes such as neurodegenerative disorders, autoimmune disease, cardiac disease, diabetes, infectious disease, inflammation and cancer.3–6 Current chemotherapy and radiotherapy suffer from low efficacy due to the fact that many cancers acquire resistance to apoptosis (type I programmed cell death), thus therapeutic strategies mediated by alternative cell death mechanisms are urgently needed.7,8 There is now considerable evidence demonstrating that over-activation of autophagy can induce autophagic cell death (ACD, type II programmed cell death) and suppress the initiation and progression of tumors.9,10 Therefore, ACD-dependent therapeutic strategy has attracted ever-growing attention over the past few years and may provide an ideal cancer treatment option especially for autophagy-deficient tumors. Beclin 1 is a core component of a class III phosphatidylinositol 3-kinase (class III PI3K, also named Vps34) complex that induces autophagosomes formation.11–13 As a haploinsufficient tumor-suppressor gene, Beclin 1 is monoallelically deleted in ovarian cancers (up to 75%), breast cancers (~50%) and prostate cancers (about 40%).14–16 Accumulating evidence indicate that decreased Beclin 1 function promotes tumor initiation,17 upregulation of Beclin 1 strengthens the death of breast cancer cells and suppresses tumor progression.18,19 Thus, elevating the Beclin 1 level in some certain tumor cells may open new avenues for the therapy of Beclin 1-deficient cancers. Nonetheless, the clinical application of Beclin 1 peptide is hampered by its inability to enter cells, poor stability and low specificity.20 3 ACS Paragon Plus Environment


Unfortunately, there are very few reports concerning the targeted delivery of Beclin 1 to tumors.20,21 It has long been observed that the tumor extracellular pH (pHe) (6.2–6.8) is more acidic than that of healthy tissues (7.2–7.4) due to the Warburg effect. Tumor acidity is a hallmark of tumor fast growth and can be exploited for targeted cancer therapy.22,23 The ability to target acidic tumor microenvironment shows great superiority over conventional passive and active targeting in several aspects.24,25 pH low insertion peptide (pHLIP) is a 36-aa, water-soluble and reasonably hydrophobic polypeptides originated from the bacteriorhodopsin C helix.26–28 As a bionanosyringe, pHLIP could translocate cell-impermeable cargoes that are coupled to its C terminus across a plasma membrane via forming a stable transmembrane α-helix under acidic environment.29 pHLIP’s exceptional low-pH targeting property has been employed for the effective delivery of various theranostic substances to acidic tumors.30–35 In the work, a functional Beclin 1 denoted as Trx-pHLIP-Beclin 1 (TpB) consisted of three distinct components with different functions - a thioredoxin (Trx) tag, a pHLIP (AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT) and a 20-aa conserved motif of Beclin 1 (CGTNVFNATFHIWHSGQFGT) (Figure 1a), was biologically synthesized using the genetic engineering method for the first time. As schematically illustrated in Figure 1a, when injected into the tumor-bearing mice via tail vein, TpB could specifically enter tumor cells with the aid of pHLIP, triggering massive autophagic cell death and thereby suppressing the tumor growth. The antiproliferative effect and autophagy-inducing potency of the TpB were determined in two cancer cell lines. We then exploit the potential of TpB as an efficient and safe autophagy-induction agent for ovarian cancer treatment in an SKOV3 xenograft 4 ACS Paragon Plus Environment

Page 4 of 35

Page 5 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tumor mice model.

Figure 1. Schematic demonstration of the composition, mechanism of action and in vitro characterization of functional Beclin 1 (TpB). (a) TpB is composed of three different component which endow it with some unique characteristics. TpB could specifically accumulate in weakly acidic tumors (pH 6.5) and effectively deliver Beclin 1 to cancer cells via forming an α-helix across the plasma membrane. TpB can trigger massive autophagic cell death and thereby suppress the tumor growth. (b) SDS-PAGE of the Trx and recombinant TpB after purification by nickel affinity column chromatography. Lane M: protein molecular weight marker; Lane 1: purified Trx; Lane 2: purified TpB.

2. EXPERIMENTAL SECTION 2.1. Materials: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, in chloroform) was purchased from Avanti Polar Lipids, Inc. (USA). Acridine orange (AO) and MTT were obtained from Sigma-Aldrich. BCA assay kit, carboxyfluorescein diacetate succinimidyl ester 5 ACS Paragon Plus Environment


Page 6 of 35

(CFDA-SE) cell proliferation assay kit and Ad-mCherry-GFP-LC3B adenovirus were purchased from Beyotime Institute of Biotechnology (China). The conserved motif of Beclin 1 (CGTNVFNATFHIWHSGQFGT, ≥ 98%) was synthesized by Bankpeptide Biological Technology Co. Ltd. (Hefei, China). Primary antibodies against p62, LC3, Ki-67 and Beclin-1 were purchased from Abcam (UK). 2.2. Biosynthesis and Characterization of Trx-pHLIP-Beclin 1 (TpB): The pHLIP-Beclin 1 gene sequence was synthesized by Sangon Biotech Co., Ltd (Shanghai, China). The primer sequence used for amplification of pHLIP-Beclin 1 gene by PCR were as follows: 5′-CCGCGTGGATCCGCGGCGGAACAGAACCCGA-3′

(5′-primer),

5′-GGTCGACTCGAGTTAGGTGCCAAACTGGCCG-3′ (3′-primer). The pHLIP-Beclin 1 gene was subcloned into pET32a vector at the BamHI and XhoI sites, and the recombinant expression vector pET32a-pHLIP-Beclin 1 was obtained. This vector was transformed into the competent E. coli BL21 (DE3) followed by stimulating with IPTG (1 mM) at 16 °C for 12 h, the cells were collected, lysed and centrifuged. The supernatant was loaded onto Ni-NTA resin, washed with PBS, and eluted with 300 mM imidazole (pH 8.0). The protein solution was dialyzed against water to remove the imidazole and concentrated by ultrafiltration. The thioredoxin (Trx) tag was obtained by transforming empty pET32a vector into E. coli BL21 (DE3). The purity of the recombinant TpB and Trx was monitored based on densitometry analysis using ImageJ software and the production yields were determined using BCA assay. The purified proteins were stored as freeze-dried powder at -20 °C for following use. 2.3. In Vitro Stability of TpB: The in vitro stability of TpB was assessed using a circular dichroism (CD) spectrometer. TpB solution (in 5 mM phosphate buffer) was incubated at 6 ACS Paragon Plus Environment

Page 7 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37 °C for different times (0, 12, 24, 36 and 48 h). Then POPC liposomes (200 µM) and TpB (10 µM) were mixed in 5 mM phosphate buffer (pH 6.5) and incubated for another 1 h at 37 °C. The CD spectra of different samples were recorded on a Chirascan spectrometer. 2.4. MTT Cell Viability Assay: Human breast cancer cell line MCF-7 and human ovarian cancer cell line SKOV3 were seeded onto 96-well plates (5×103 cells/well) in RPMI-1640 and DMEM medium respectively supplemented with 10% FBS, and incubated overnight. Then fresh medium with a pH of 7.4 or 6.5 containing varying final concentrations (10, 50 and 100 µM) of Trx, Beclin 1 and TpB was added for 24 h. the cell viability was determined by MTT assay and analyzed on a microplate reader at 570 nm. 2.5. Crystal Violet Staining for Cell Viability Assay: Crystal violet assay was carried out following the procedures in previous report.36 Briefly, MCF-7 and SKOV3 were seeded into 96-well plates (5×103 cells/well) and incubated overnight. The culture medium was aspirated, replaced by fresh medium (pH 7.4 or 6.5) supplemented with Trx, Beclin 1 and TpB of different concentrations (10, 50 and 100 µM) and incubated for 24 h. Then crystal violet staining solution (0.5%) was added to each well and incubated for 20 min followed by observation using a Cytation 5 Cell Imaging Multi-Mode Reader. For quantitation of cell viability, 200 µL of methanol was added and incubated for another 20 min, the cell viability was analyzed on a microplate reader at 570 nm. 2.6. CFDA-SE Labeled Proliferation Assay: MCF-7 and SKOV3 cells were trypsinized and stained with CFDA-SE for 10 min. The CFDA-SE labeled cells were seeded into 6-well plates, incubated overnight and exposed to 10 µM Trx, Beclin 1 or TpB for 24 h at pH 7.4 or 6.5. Then the cells were collected, washed two times with PBS and subjected to flow cytometry 7 ACS Paragon Plus Environment


Page 8 of 35

analysis (BD LSRFortessa X-20, USA) to determine the intensity of CFDA-SE. 2.7. Acridine Orange (AO) Staining: MCF-7 and SKOV3 were incubated with 10 µM Trx, Beclin 1 and TpB at pH 7.4 or 6.5 for 12 h, and stained with AO (1 µg/mL) for a period of 10 min. After washing with PBS for three times, the cells were observed by the Deltavision Elite Microscopy Imaging Systems (GE Healthcare, USA) and analyzed by flow cytometry. 2.8. Ad-mCherry-GFP-LC3B Transfection: MCF-7 and SKOV3 cells were seeded into 12-well

plates

and

cultured

to

60%–70%

confluence

and

subjected

to

Ad-mCherry-GFP-LC3B adenovirus transfection at an MOI of 5 for 24 h at 37 °C. The infected cells was treated with 10 µM Trx, Beclin 1 and TpB at pH 7.4 or 6.5 for 12 h, expression of mCherry and GFP was visualized by the Deltavision Elite Microscopy Imaging Systems (GE Healthcare, USA). Autophagy flux was assessed by calculating the number of yellow and red puncta. 2.9. Transmission Electron Microscopy (TEM): MCF-7 and SKOV3 cells were exposed to 10 µM Trx, Beclin 1 and TpB at pH 7.4 or 6.5 for 12 h. The treated cells were fixed successively with glutaraldehyde (2%) and osmium tetroxide (1%), followed by ethanol dehydration and resin embedding. Then the ultrathin sections (50 nm) were stained with uranyl acetate and lead citrate, and examined using a JEM-1011 transmission electron microscope to observe the formation of autophagosomes. 2.10. Western Blotting Analysis: MCF-7 and SKOV3 cells were harvested from cultured dishes after treatment with 10 µM Trx, Beclin 1 and TpB at pH 7.4 or 6.5 for 12 h and lysed. Samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The blotted membranes were blocked, incubated consecutively with primary antibodies and 8 ACS Paragon Plus Environment

Page 9 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


appropriate secondary antibodies. The immunoreactive bands were visualized using ECL solution. 2.11. Animals and Tumor Model: Four-week old female BALB/c mice were purchased from National Institutes for Food and Drug Control and were housed in a Specific Pathogen Free (SPF) facility with routine and comprehensive health monitoring. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanxi University. To establish an ovarian cancer xenograft model, about 5×106 SKOV3 cells in 100 µL saline were subcutaneously inoculated into armpits of right anterior limbs. 2.12. In Vivo Tumor Suppression Study: The SKOV3 tumor-bearing mice were randomly divided into four groups (n = 7): control, Trx, Beclin 1 and TpB. And treatment was initiated two days later, taken as day 2. Trx, Beclin 1 and TpB dissolved in saline were administered via tail vein injection at a dose of 10 µmol/kg every two days. The control group only received saline (5 mL/kg). Tumor volume and body weight were monitored every two days for a period of 22 days. The following equation was used to calculate the tumor growth inhibition ratio: (Vc-Vt)/Vc×100%, where Vc and Vt are the average tumor volume of the control group and treated group respectively.37 All mice were sacrificed two days after last treatment, the tumors and main organs (heart, liver, spleen, lung and kidney) were excised, fixed in 4% formaldehyde, embedded in paraffin and sectioned into 5 µm slices. Then, the slices were stained with hematoxylin and eosin (H&E). Tumor sections dissected from four groups of mice were subjected to immunohistochemical staining for detection of p62, LC3 and Ki-67. Furthermore, western blotting was conducted to monitor the level of autophagy-associated protein p62, LC3-I 9 ACS Paragon Plus Environment


LC3-II in tumors of various groups. 2.13. In Vivo Biodistribution of TpB: The excised tumors and normal tissues were subjected to western blotting and immunohistochemical staining for analysis of Beclin 1 to detect the in

vivo distribution of TpB. 2.14. Statistical Analysis: Results for cell viability, autophagic flux assay, relative protein expression levels, body weight and tumor volume and weight were presented as the mean ± standard deviation (SD), and were statistically analyzed using Student’s t-test or one-way analysis of variance (ANOVA). p

Robust Anticancer Efficacy of a Biologically Synthesized Tumor Acidity

Recommend Documents