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 Table of Contents  
Year : 2017  |  Volume : 3  |  Issue : 3  |  Page : 69-79

Novel molecular multilevel targeted antitumor agents

1 Department of Cancer Biology, Brain Tumor Center of Excellence, Comprehensive Cancer Center - Wake Forest Baptist Medical Center, Medical Center Boulevard, Winston-Salem, NC, USA
2 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
3 Department of Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX, USA

Date of Submission08-Mar-2017
Date of Acceptance12-Apr-2017
Date of Web Publication8-Jun-2017

Correspondence Address:
Waldemar Debinski
Department of Cancer Biology, Wake Forest Baptist Medical Center, Winston-Salem, NC 27157
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ctm.ctm_12_17

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Aim: A multifunctional fusion protein, IL-13.E13K-D2-NLS, effectively recognizes glioblastoma (GBM) cells and delivers its portion to the cell nucleus. IL-13.E13K-D2-NLS is composed of a cancer cell targeting ligand (IL-13.E13K), specialized cytosol translocation bacterial toxin domain 2 of Pseudomonas exotoxin A (D2) and SV40 Tantigen nuclear localization signal (NLS). We have now tested whether we can produce proteins that would serve as a delivery vehicle to lysosomes and mitochondria as well. Moreover, we examined whether IL-13.E13K-D2-NLS can deliver anticancer drugs like doxorubicin to their nuclear site of action in cancer cells.
Methods: We have thus constructed two novel proteins: IL-13.E13K-D2-LLS which incorporates lysosomal localization signal (LLS) of a human lysosomal-associated membrane protein 1 (LAMP-1) for targeting to lysosomes and IL-13-D2-KK2, which incorporates a pro-apoptotic peptide (KLAKLAK)2 (KK2) exerting its action in mitochondria. Furthermore, we have produced IL-13.E13K-D2-NLS and IL-13.E13K-D2-LLS versions containing a cysteine for site-specific conjugation with a modified doxorubicin, WP936.
Results: We found that single-chain recombinant proteins IL-13.E13K-D2-LLS and IL-13-D2-KK2 are internalized and localized mostly to the lysosomal and mitochondrial compartments, respectively, without major trafficking to cells' nuclei. We also determined that IL-13.E13K-D2-NLS-cys[WP936], IL-13.E13K-D2-LAMP-cys[WP936], and IL-13-D2-KK2 were cytotoxic to GBM cells overexpressing interleukin 13 receptor alpha 2, while much less cytotoxic to GBM cell lines expressing low levels of the receptor. IL-13.E13K-D2-NLS-cys[WP936] was the most potent of the tested antitumor agents including free WP936.
Conclusion: We believe that our receptor-directed intracellular organelle-targeted proteins can be employed for numerous specific and safer treatment applications when drugs have specific intracellular sites of their action.

Keywords: chemotherapy, drug conjugates, drug delivery, glioblastoma, molecular targeting

How to cite this article:
Sonawane P, Choi YA, Pandya H, Herpai DM, Fokt I, Priebe W, Debinski W. Novel molecular multilevel targeted antitumor agents. Cancer Transl Med 2017;3:69-79

How to cite this URL:
Sonawane P, Choi YA, Pandya H, Herpai DM, Fokt I, Priebe W, Debinski W. Novel molecular multilevel targeted antitumor agents. Cancer Transl Med [serial online] 2017 [cited 2020 May 28];3:69-79. Available from: http://www.cancertm.com/text.asp?2017/3/3/69/207619

  Introduction Top

Glioblastoma (GBM), a high-grade astrocytoma, is the most prevalent and aggressive primary brain tumor.[1] GBM tumors are very heterogeneous in nature, and even the multimodal therapies such as chemotherapy, radiation, surgical resection, and electric fields result in poor prognosis with a median survival of ~ 16 months postdiagnosis.[2],[3],[4],[5],[6] We have previously found that interleukin 13 receptor alpha 2 (IL-13RA2) is overexpressed in > 70% of GBM tumor specimens.[7],[8],[9] We have shown that IL-13RA2 is absent in normal adult tissue except in the testes and that IL-13RA2 biochemical moiety is a tumor-associated receptor.[10],[11] On binding to IL-13RA2, IL-13 induces receptor-mediated endocytosis.[7] Hence, wild type and mutated IL-13-based fusion cytotoxins containing a derivative of Pseudomonas exotoxin A (PE), PE38QQR possess potent antitumor activity against GBM tumors in vitro and in vivo.[7],[12] The first generation of the cytotoxin demonstrated clinical efficacy.[13],[14] An IL-13 mutant, IL-13.E13K binds specifically to IL-13RA2, but it has significantly altered affinity toward the IL-13RA1, a subunit of a normal tissue receptor for IL-13 that is shared with IL-4, IL-13RA1/IL-4A.[12],[15] This allows for much more specific targeting of tumor cells versus normal cells.

Safe delivery of effective antitumor agents is the main goal in cancer therapy. Anthracyclines, for example, doxorubicin are among the most effective therapies against hematologic malignancies and solid tumors.[16],[17],[18],[19] However, clinically effective doses of anthracyclines cause myelosuppression, cumulative cardiotoxicity, stomatitis, and extravasation.[20],[21] This is because often only a limited amount of the functional drug reaches tumor cells whereas it also acts on the normal healthy tissues resulting in serious adverse side-effects. Site-specific delivery of the anticancer agents like doxorubicin focused to tumor cells could reduce its adverse effects. One example is the antibody drug/label conjugates. Monoclonal antibodies (MAbs) and their drug conjugates have been successfully implemented in the treatment of solid tumors and blood malignancies.[22],[23],[24] This trend has been only accelerated in recent years with the approvals of Adcetris and Kadcyla antibody drug conjugates.[25],[26],[27],[28]

Our laboratory has previously developed a single-chain, recombinant proteinaceous agent that recognizes IL-13RA2 and delivers the C-terminal portion of the protein to a specific subcellular compartment (e.g., nuclei) of the GBM cells.[29] This was achieved on the basis of specific recognition of tumor cells by IL-13.E13K, internalization of the agent and proteolytic cleavage in the endocytic compartment releasing the C-terminal fragment of the protein into cell cytosol, and then subsequent transport and accumulation in cells' nuclei facilitated by the nuclear localization signal (NLS).[29],[30],[31] The protein is termed IL-13.E13K-D2-NLS and could be used for more selective delivery of chemotherapeutics, in which the site of action is in the nuclei, like doxorubicin. In the current study, we examined whether IL-13.E13K-D2-NLS has potential to deliver a thiol-reactive derivative of doxorubicin, WP936.[32],[33] Since IL-13RA2 is also overexpressed in pancreatic cancer, colorectal cancer, head and neck cancer, melanoma, and breast cancer,[34],[35],[36],[37],[38],[39],[40] drug conjugates with chemotherapeutics would be suitable for targeting this receptor in solid tumors including GBM. Our current results show that IL-13.E13K-D2-NLS-cys enters the subcellular compartment and induces cytotoxicity in the GBM cells when conjugated with a chemotherapeutic, such as WP936.

Similar to NLS, lysosomal localization sequences (LLS) can be used as a potential drug trafficking signal. In the current study, we also explored whether our delivery vectors containing a lysosomal-associated membrane protein 1 (LAMP-1) LLS could distribute fragments of targeted proteinaceous compound to the targeted organelles. Moreover, a synthetic, pro-apoptotic peptide, (KLAKLAK)2 is known to specifically act on the tumor endothelium. (KLAKLAK)2 is a 14-amino-acid amphipathic α-helical peptide.[41] Due to its cationic nature, on internalization, it selectively disrupts the anionic mitochondrial membrane, resulting in a release of cytochrome C from the electron transport chain and activation of the pro-apoptotic caspase pathway.[42] Others have fused (KLAKLAK)2 with various peptidomimetics such as DPI and MCA205[43] against murine sarcomas, and with RGD-4C against breast carcinoma cells.[42] Employing the same strategy as our other targeted proteinaceous agents, we have developed and constructed IL-13-D2-KK2 for targeting IL-13RA2-GBM cells. Here, we determined that this novel molecularly targeted and genetically engineered recombinant proteins selectively recognize tumor cells and accumulate in the intracellular compartments evoking antitumor effect.

  Methods Top

Cell culture

GBM cell lines, U87, U-251 MG, LN229, and T98G, were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown as recommended. SnB19-asIL-13RA2 are SnB19 GBM cells transfected with an anti-sense IL-13RA2 gene with subsequently diminished expression of the receptor, in contrast to the empty vector-transfected SnB19-pcDNA cells.[29] The transfected SnB19 cell lines were grown in RPMI 1640 (Hyclone, Logan, UT, USA) with 200 μg/mL of Geneticin. G48a cells were grown and maintained in RPMI 1640 (Lonza, Walkersville, MD, USA) supplemented with glucose, adjusted to 4 g/L of media and 10% fetal calf serum.

Cloning, production, and purification of targeted proteins

The IL-13 mutant recombinant constructs were made by replacing the wild-type IL-13 sequence from the parent plasmid with the mutant IL-13.E13K sequence. The IL-13-D2 plasmid was engineered by subcloning it from a previously generated IL-13-PE38QQR plasmid. Cysteine residue was added at NLS (NLS-cys) and LLS (LLS-cys) signal peptides C-terminal end to conjugate with a derivative of doxorubicin by forming disulfide bonds [Figure 1]a. A duplex primer cloning strategy was then employed wherein NLS-cys, LLS-cys and (KLAKLAK)2 5′ and 3′ sequence primers were synthesized (Invitrogen, Carlsbad, CA, USA) and made into duplex DNA (containing XhoI/BamHI ends) by incubating the primers in favorable annealing conditions. The annealed duplex was then subcloned into the IL-13.E13K-D2 containing plasmid using Xho1/BamH1 at the 3′end to produce IL-13.E13K-D2-NLS-cys or IL-13.E13K-D2-LLS-cys.
Figure 1: (a) Schemata of IL-13.E13K-D2-NLS-cys[WP936] and IL-13.E13K-D2-LLS-cys[WP936] drug conjugates. (b) Analysis of IL-13.E13K-D2-NLS-cys[WP936] drug conjugate. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein molecular weight standards (Lane 1), drug-free IL-13.E13K-D2-NLS-cys (Lane 2), and drug-conjugated IL-13.E13K-D2-NLS-cys (Lane 3). Typhoon image (Lanes 4–6) as in Lanes 1–3. (c) Analysis of IL-13.E13K-D2-LLS-cys[WP936] drug conjugate. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein molecular weight standards (Lane 1), drug-free (Lane 2), and drug-conjugated IL-13.E13K-D2-LLS-cys (Lane 3). Typhoon image (Lanes 4–6) as in Lanes 1–3. (d) Treatment of U-251-MG, G48a and T98G cells with IL-13.E13K-D2-NLS-cys[WP936] drug conjugate. Cells were visualized using phase contrast or fluorescent microscopy after 24 and 48 h of treatment. (e) Treatment of U-251-MG, G48a and T98G cells with IL-13.E13K-D2-LLS-cys[WP936] drug conjugate. Cells were visualized using phase contrast microscopy after 24 and 48 h of treatment. (f) Cytotoxic effect of IL-13.E13K-D2-NLS-cys[WP936] in U-87, G48a, and SnB19 pcDNA cells glioblastoma cells overexpressing interleukin 13 receptor alpha 2, and (g) in T98G, SnB19-A/SRA2 and LN229 cells expressing low levels of the receptor. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide cell viability assay was used. (h) Cytotoxic effect of a modified unconjugated doxorubicin, WP936, in glioblastoma cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used. (i) Cytotoxicity of IL-13.E13K-D2-LLS-cys[WP936] in glioblastoma cells

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Nuclear localization signal-cysteine primers



Molecular weight: 27510.3 TheoreticalpI: 8.69.

Lysosomal localization sequences-cysteine primers



Theoretical pI/Mw: 9.30/26118.96.

(KLAKLAK)2 duplex primers



Theoretical pI/Mw: 9.43/26361.43.

These recombinant constructs were used to transform DH5α  Escherichia More Details coli cells for amplification. All the constructs were sequenced at the DNA sequencing laboratory of the Comprehensive Cancer Center at Wake Forest University and analyzed for their inframe DNA sequence using an automated sequence analyzer before protein expression.

Protein expression was performed under the IPTG-inducible T7 promotor in an E. coli protein expression system as previously described.[44] In brief, IL-13.E13K-D2-NLS-cys, IL-13.E13K-D2-LLS-cys, and IL-13-D2-KK2 recombinant constructs were expressed in BL21 E. coli respectively, and the cells were grown in Luria-broth media supplemented with 100 μg/mL of ampicillin at 37°C shaker. When A600 of the bacteria culture media reached log phase growth (0.7–0.8), the recombinant protein expression in the cells was induced by addition of IPTG (final concentration 1 mM) and allowed to incubate for a further 90 min. The expressed proteins in the inclusion bodies were then denatured using 7 M guanidine (MP Biomedicals, Salon, OH, USA) and reduced with 1, 4-dithiothreitol (Sigma, St. Louis, MO, USA). The reduced proteins were then renatured in a 0.1 M Tris buffer containing arginine/L-glutathione oxidase (Sigma, St. Louis, MO, USA). The protein was further dialyzed and purified by SP-sepharose ion exchange liquid chromatography system (GE Healthcare, Marlborough, MA, USA). The purified proteins were subsequently run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels to identify of the isolated proteins. All of the proteins obtained were > 90% pure.

Protein conjugation with WP936, a doxorubicin derivative

A solution of protein was diluted to 100 μM in 50 mM Na2 HPO4. Tris-(2-carboxyethyl) phosphine (TCEP; Molecular Probes, Eugene, OR, USA) was added to a 10-fold molar excess. WP936 was slowly added while mixing to a final concentration of 100 μM and incubated with continuous stirring overnight at 4°C. Unreacted label was removed by dialysis with phosphate buffered saline (PBS). The efficiency of labeling on the single cysteine residue was assessed by ultraviolet–visible spectrophotometry (Nanodrop 1000 spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA).

Determination of conjugation ratio

A calibration was developed to determine the molar conjugation ratio of WP936 to protein (IL-13.E13K-D2-NLS-cys or IL-13.E13K-D2-LLS-cys) based on the absorbance spectrum of the conjugate as previously shown to evaluate the WP936 conjugation and Thermo Fisher Scientific technical tip #31.[45],[46] The extinction coefficient of doxorubicin is 8030 L mol -1 cm -1 and those of IL-13.E13K-D2-NLS-cys or IL-13.E13K-D2-LLS-cys are 20970 M - cm - and 22460 M - cm - (at 495 nm) when assuming all cysteine residues are reduced by forming disulfide bond with doxorubicin.[47]

Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxy methoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazinemethosulfate cell viability assay

Totally, 2–2.5 × 103 U87, G48a, U-251 MG, and Snb19 pcDNA GBM cells, overexpressors of IL-13RA2, T98G, Snb19 A/S RA2, and LN229 which do not overexpress the receptor were plated per well in triplicates for each concentration to be tested. After 24 h of incubation at 37°C for the cells to attach, increasing concentrations of the IL-13.E13K-D2-NLS-cys[WP936] or IL-13.E13K-D2-LLS-cys[WP936] ranging from 0.02 to 2000 nM in 0.02% F127 (Sigma-Aldrich, St. Louis, MO, USA) were added, and the plate was incubated for 48–72 h. Cells treated with cycloheximide were used as controls.

Due to WP936's overlapped excitation wavelength with 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium(MTS)/phenazinemethosulfate(PMS)(495 nm),3-(4,5- dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to analyze cell viability treated with WP936 conjugated proteins. Briefly, after the incubation with WP936 conjugated protein, MTT reagent in phenol red free RPMI 1640 (Gibco, Grand Island, NY, USA) was added to be 1/10 of culture volume. The plate was incubated at 37°C for up to 4 h in a humidified, 5% CO2 atmosphere. An equal volume of acidified isopropanol was added to stop the reaction. The absorbance was measured at 570 nm. Background absorbance at 690 nm was used as a reference wavelength and was subtracted from the absorbance at 570 nm before plotting.

The viability of cells treated with IL-13-D2-KK2 in 0.02% of F127 was measured using the MTS/PMS dye (Promega, Madison, WI, USA) as per the manufacturer's instructions. The absorbance was measured at 490 nm. Both were measured using Spectra Max 340 PC (Molecular Devices, Sunnyvale, CA, USA) microplate reader and data were plotted as a percentage of control versus concentration of the agent used.

Direct biotin labeling of IL-13.E13K-D2-LLS-cys and IL-13-D2-KK2

Biotin-XX Microscale Protein Labeling Kit (Invitrogen, Carlsbad, CA, USA) was used to label the proteins as per the manufacturer's instructions. The biotin-labeled proteins were separated on an SDS-PAGE gel, and a Western blot carried out using streptavidin-HRP (Pierce, Rockford, IL, USA) to detect for biotin-labeled proteins. The number of biotin molecules attached to the proteins was determined by the FluoReporter Biotin Quantitation assay kit (Invitrogen, Carlsbad, CA, USA) as per the manufacturer's guidelines. The fluorescent signals were measured using the BMG Optima plate reader (BMG Labtech, Ortenberg, Germany) and data were plotted as the concentration of the standard Biocytin in pmoles versus relative fluorescence units.

Immunofluorescence localization studies on interleukin 13 receptor alpha 2 positive U-251 MG or U87 cells with biotin-conjugated proteins.

Totally, 1 × 105 U-251 MG or U87 GBM cells were plated on sterilized coverslips per well in a 12-well plate. The wells were plated in duplicates for each time point. After 24 h, 1 μM/well of biotin-labeled proteins was added for the indicated incubation time. After incubation, the cells were fixed with 10% buffered formalin for 15 min at 37°C and washed 4X with PBS. The cells were then permeabilized with 0.1% Triton-X-100/0.2% bovine serum albumin-PBS for 1 min at room temperature (RT) and washed three times with 1X PBS. For colocalization studies, anti-LAMP-1 (EMD Millipore Billerica, MA, USA) or anti-ATP synthase (Thermo Fisher Scientific, Waltham, MA, USA) was applied to the cells for 1 h at RT followed by the appropriate Alexa-Fluor conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA). Location of the biotin conjugates was detected with anti-streptavidin-555 (Invitrogen, Carlsbad, CA, USA) and the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) or Topro-3 iodide (Invitrogen, Carlsbad, CA, USA). F-actin staining was performed with Phalloidin-Alexa 488 (Invitrogen, Carlsbad, CA, USA).

After wells were washed three times with 1X PBS, the coverslips were mounted with Fluoroguard (ScyTek, Logan, UT, USA). The coverslips with DAPI stained cells were analyzed under the fluorescent microscope (Olympus, IX70, Center Valley, PA and Olympus Fluoview 1200 confocal microscope) and processed using the Image-Pro plus 5.1 (Media Cybernetics, Rockville, MD, USA) and OlyVIA V2.8 software (Olympus, Waltham, MA, USA). The coverslips with Topro-3 iodide stained cells were then observed with LSM 510 Zeiss Confocal Microscope (Cellular Imaging Core, Comprehensive Cancer Center, and Wake Forest University) and the images processed using Zeiss LSM Image Browser (version 4.2).


Totally, 500 ng of each recombinant biotin-conjugated proteins were loaded onto a 12% SDS-PAGE gel and transferred to a polyvinylidenedifluoride membrane (Pierce, Rockford, IL, USA). Blots were blocked with 5% milk-PBS for 1 h at RT. Biotin-proteins were detected using streptavidin conjugated with horseradish peroxidase (Thermo Fisher Scientific, Rockford, IL, USA) diluted 1:10,000 in blocking buffer. Detection was performed using the ECL plus Western Blotting Detection System (Amersham Biosciences, UK). Membranes were exposed to autoradiographic film Kodak Biomax XR. Films were scanned at 600 dpi and images compiled using Paint Shop X2.

Flow cytometry

Cells were treated with 10 nM of the IL-13.E13K ligand and collected at various time points (0–24 h). IL-13RA2-specific primary antibody and anti-IgG-Alexa Fluor 488 secondary antibody (Molecular Probes, Eugene, OR, USA) were used for detection. An IgG isotype was used as a control. The experiments were performed using BD FACS Calibur or Accuri C6 flow cytometers (BD Biosciences, San Jose, CA, USA).

  Results Top

Persistence of interleukin 13 receptor alpha 2 on cell surface of U-251 glioblastoma cells during treatment with IL-13.E13K

In our multilevel strategy of delivering drugs to their sites of actions in specific intracellular compartments of targeted cancer cells, we effectively exploit IL-13RA2 which is overexpressed in vast majority of patients with GBM.[7],[9] To ascertain that the receptor is suitable for long-term delivery of our experimental therapeutics, we treated U-251 GBM cells with 10 nM of IL-13.E13K, which specifically recognizes IL-13RA2, and examined the levels of the receptor up to 24 h of treatment by flow cytometry [Figure 2]. The presence of IL-13RA2 on cell surface remained stable and was not down-regulated appreciably in response to treatment with its ligand, IL-13.E13K [Figure 2]b. Hence, the receptor can be suitably utilized for delivery of candidate drugs for a prolonged period.
Figure 2: (a and b) Flow cytometry for interleukin 13 receptor alpha 2 in U-251 glioblastoma cells. Isotype control (a) and (b) receptor detection at various time points. Highly purified IL-13.E13K-D2-NLS-cys (c), and IL-13.E13K-D2-LLS-cys (d). (e) Immunoblot of nonbiotinylated (Lane 1) and biotinylated (Lane 2) IL-13.E13K-D2-LLS-cys probed with streptavidin-HRP. (f and g) Internalization of biotinylated IL-13.E13K-D2-LLS-cys (1 μM) in U-251 MG cells. The cells were analyzed using anti-streptavidin Alexa Fluor 555 (red) by fluorescence microscopy. Two different fields are shown in two column panels. (h) U-251-MG cells were treated with biotin-labeled IL-13.E13K-D2-LLS-cys (1 μM), and cells were stained for the nuclei and the protein. (i) Subcellular localization of IL-13.E13K-D2-LLS-cys was monitored using Z-stack analysis. (j) Internalization and intracellular distribution of IL-13.E13K-D2-LLS-cys. U-251 MG cells were incubated for 8 h with 1 μM biotin-conjugated IL-13.E13K-D2-LLS-cys (red) with co-staining of lysosomal-associated membrane protein (green). DIC: Differential interference contrast

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Production and purification of IL-13.E13K-D2-LLS-cys and IL-13.E13K-D2-NLS-cys

After having successfully developed a recombinant, single-chain protein delivering its C-terminal portion to the nuclei of targeted cells,[29] we have now constructed a recombinant protein for potential delivery of drug loads to the lysosomal compartment of cancer cells overexpressing IL-13RA2. We used LAMP-1 LLS sequence in combination with IL-13.E13K ligand and D2 domain of PE to produce IL-13.E13K-D2-LLS-cys. We also produced IL-13.E13K-D2-NLS-cys for first in class conjugation to a cytotoxic agent, a chemotherapeutic in this case. IL-13.E13K-D2-NLS-cys and IL-13.E13K-D2-LLS-cys were produced as single-chain proteins in E. coli and purified by FPLC [Figure 2]c and [Figure 2]d. The isolated proteins were > 90% pure.

Internalization of IL-13.E13K-D2-LLS-cys protein

We biotinylated IL-13.E13K-D2-LLS-cys using biotin-XX sulfosuccinimidyl ester to form a stable biotinylated conjugate protein, which was detected using an immunoblot assay [Figure 2]e. Only the presence of biotinylated IL-13.E13K-D2-LLS-cys protein was demonstrated [Figure 2]e - Lane 2], whereas no signal was detected for the IL-13.E13K-D2-LLS-cys nonbiotinylated protein [Figure 2]e - Lane 1]. We then analyzed cell internalization and localization of biotinylated IL-13.E13K-D2-LLS-cys protein (1 μM) using anti-streptavidin Alexa Fluor-555 antibody and fluorescent microscopy [Figure 2]f. We treated U-251 MGGBM cells with biotinylated protein for 4 h; cells were also stained with phalloidin for actin labeling. We observed that the biotinylated protein effectively internalized after 4 h of incubation [Figure 2]f, which further increased with longer incubation times (8 and 24 h, respectively) [Figure 2]g. We also performed confocal microscopy and Z-stack analysis to examine the intracellular localization of the protein. IL-13.E13K-D2-LLS-cys internalized into cells with a clear omission of accumulation in the nuclei [Figure 2]h. The Z-stack analysis confirmed that IL-13.E13K-D2-LLS-cys protein internalized into the U-251 MG cells and is localized primarily in the cytosol and perinuclear regions, but never in the nuclei even after 24 h of treatment [Figure 2]i. This is in sharp contrast to the nuclei trafficking of IL-13.E13K-D2-NLS seen previously.[29]

IL-13.E13K-D2-LLS-cysco-localizes with lysosomal-associated membrane protein in glioblastoma cells

We confirmed the binding of biotinylated IL-13.E13K-D2-LLS-cys protein to the lysosomal compartment by determining its localization near LAMP-1. We first treated the U-251 MG cells at various time points (5 min, 4, 8, 12, and 24 h) with biotinylated IL-13.E13K-D2-LLS-cys followed by a double immunofluorescence staining against streptavidin and LAMP-1. The double immunofluorescence staining showed that the studied proteins localize frequently to the same regions in cells [Figure 2]j.

Conjugation of targeting proteins with a cytotoxic thiol reactive doxorubicin derivative, WP936

We next examined whether the proteins capable of delivering their portions to two different intracellular compartments of cells would cause cytotoxicity upon conjugation with a cytotoxic agent. IL-13.E13K-D2-NLS-cys was conjugated with thiol reactive doxorubicin derivative WP936[33] using TCEP, (IL-13.E13K-D2-NLS-cys[WP936]) in a way that the exact site of conjugation in both components of this antitumor agent is known [Figure 1]a. We also conjugated IL-13.E13K-D2-LLS-cys with WP936 using the same method. The schematic of both drug conjugates is shown in [Figure 1]a. The IL-13.E13K-D2-NLS-cys[WP936] and IL-13.E13K-D2-LLS-cys[WP936] conjugates were analyzed using SDS-PAGE gel and Typhoon scan [Figure 1]b and [Figure 1]c. Only the successfully WP936-conjugated proteins emitted the detectable fluorescence signals at the expected molecular sizes [Figure 1]b and [Figure 1]c.

We further analyzed the potential cytotoxic effect of IL-13.E13K-D2-NLS-cys[WP936] conjugate on U-251 MG cells that overexpress the IL-13RA2. We found that the treatment increased cell rounding/blebbing resembling pro-apoptotic cells and a diminishing number of cells using phase contrast microscopy [Figure 1]d. Fluorescent detection of WP936 showed accumulation of the conjugation treated cells, which was dose- and time-dependent [Figure 1]d. Similar effects were seen in the G48a GBM cells, also overexpressors of IL-13RA2 [Figure 1]d. In contrast, T98G cells that express low levels of the targeted receptor did not change significantly in response to treatment with the drug conjugate and little of the conjugate's accumulation was observed in cells even at a 2 μM concentration after 48 h [Figure 1]d.

We noted only a slightly present cell rounding/blebbing in the U-251 MG and G48a cells after treatment with 2 μM of IL-13.E13K-D2-LLS-cys[WP936] and even much less at a lower dose [Figure 1]e. However, the conjugate clearly accumulated in the IL-13RA2 overexpressors such as U-251 and G48a cells as evidenced by an increase in fluorescence, especially at a higher concentration of the conjugate used [Figure 1]e. No effect of IL-13.E13K-D2-LLS-cys[WP936] on cells was visible, nor the accumulation of the conjugate was observed in T98G cells expressing lower amounts of IL-13RA2 [Figure 1]e. Thus, comparatively the conjugate delivering the cytotoxic load of a modified anthracycline to cells nuclei [Figure 1]d caused a much greater effect in cells than the one directing the drug to lysosomes [Figure 1]e. Cells that are not overexpressing IL-13RA2 do not respond to such a treatment in a readily visible manner [Figure 1]d and [Figure 1]e. We have performed additional flow cytometry experiment to ascertain that the T98G cells do not overexpress IL-13RA2 while the other cells under study do [Figure 1]f.

WP936 conjugate is more cytotoxic than an unconjugated drug

We next examined the cytotoxicity of IL-13.E13K-D2-NLS-cys[WP936] on a panel of GBM cells using cell viability assay. This included cells overexpressing IL-13RA2 (G48a, SnB19 pcDNA, and U-87) [Figure 1]g and cells expressing low levels of the receptor (T98G, SnB19-A/S-RA2, and LN229) [Figure 1]h; the SnB19-A/S-RA2 are the cells transfected with an antisense IL-13RA2 gene with subsequently diminished expression of the IL-13RA2 receptor, and the SnB19 pcDNA cells are empty-vector transfected.[48] A potent cytotoxic effect was observed in cells overexpressing IL-13RA2 with increasing concentration of IL-13.E13K-D2-NLS-cys[WP936] [Figure 1]g. On the other hand, IL-13.E13K-D2-NLS-cys[WP936] showed no or little cytotoxicity on cell lines expressing low levels of IL-13RA2 [Figure 1]h. We analyzed the cytotoxic effect of unconjugated WP936 in the same panel of GBM cells. All 7 GBM cell lines were treated with unconjugated WP936 and analyzed using MTS-PMS assay. All the cells responded to the drug independent of the status of IL-13RA2, but at significantly higher concentrations from the drug conjugate [Figure 1]i. We next analyzed the cytotoxicity of IL-13.E13K-D2-LLS-cys[WP936] conjugate, on IL-13RA2 overexpressors (G48a and U87 cells), and low expressors (LN229 cells). As expected, this WP936 conjugated carrier protein was less active in affecting GBM cells overexpressing the receptor when compared to IL-13.E13K-D2-NLS-cys[WP936] and had no action on receptor-negative cells [Figure 1]j.

Cancer cell targeting fusion protein with pro-apoptotic peptide (KLAKLAK)2

(KLAKLAK)2 is a 14-amino-acid amphipathic α-helical peptide which causes apoptosis in the cancer cell by mitochondrial-dependent apoptosis. Our first step was to design, synthesize and produce IL-13-D2-KK2 protein. IL-13-D2-KK2 protein was produced in BL21 E. coli cells and then highly purified using FPLC [Figure 3]a. Further, we wanted to study the intracellular pathway and subcellular localization of IL-13-D2-KK2 fusion protein. Hence, we biotin-conjugated the IL-13-D2-KK2 at the primary amines, and the biotinylated protein was detected using HRP-streptavidin [Figure 3]b. Biotinylated IL-13-D2-KK2 protein effectively internalized into the U-251 MG cells after 24 h of incubation [Figure 3]c.
Figure 3: (a) Purified recombinant IL-13-D2-KK2 protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis stained with Coomassie blue. (b) Immunoblot of biotinylated IL-13-D2-KK2 probed with Streptavidin-HRP. (c) Cell internalization of biotinylated IL-13-D2-KK2. The protein was analyzed by using anti-streptavidin Alexa Fluor 555 and fluorescent microscopy. (d) U-251 cells were treated with biotinylated IL-13-D2-KK2 for 8 h, and the colocalization of biotinylated IL-13-D2-KK2 with mitochondrial ATP-synthase enzyme was analyzed by confocal microscopy. (e) IL-13-D2-KK2 effect on glioblastoma cell lines U-251 MG, G48a, and T98G, analyzed using phase contrast microscopy. (f) Glioblastoma cells were treated with IL-13-D2-KK2 for 72 h, and the cytotoxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-5 (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay

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ATP synthase is a large molecular weight enzyme embedded in the inner membrane of mitochondria.[49] We further confirmed the internalization and localization of IL-13-D2-KK2 protein to the mitochondria by determining the colocalization of the protein with ATP synthase. We treated the U-251 MG cells at various time points (15 min, 4, and 8 h) with IL-13-D2-KK2 biotinylated protein followed by double immunofluorescence staining against streptavidin and ATP-synthase. The double immunofluorescence staining result suggests that the biotinylated protein can localize near-mitochondrial membranes of U-251 MG cells as early as 4 h after treatment with IL-13-D2-KK2 [Figure 3]d.

We found that both U-251 MG and G48 cells showed pro-apoptotic effects such as cell rounding and blebbing after treatment with IL-13-D2-KK2 protein [Figure 3]e. T98G cells showed no such responses after treatment with the conjugated protein [Figure 3]e. Next, we compared the cytotoxicity using the MTT cell viability assay where panel of GBM cell lines were incubated with increasing concentration of IL-13-D2-KK2 protein. We found that after 72 h there was an increase in cell cytotoxicity with increasing concentration of the conjugated protein in cell lines overexpressing IL-13RA2 (U-251 and U87), whereas no effect was seen in cells expressing low levels of IL-13RA2 (T98G) [Figure 3]f.

  Discussion Top

In the current study, we designed and successfully constructed novel recombinant proteins, containing IL-13.E13K ligand targeting IL-13RA2, and either lysosomal compartment through LLS of LAMP-1, IL-13.E13K-D2-LLS, or a pro-apoptotic peptide (KLAKLAK)2, IL-13.E13K-D2-KK2, targeting mitochondrial compartment of tumor cells. We demonstrated that single-chain recombinant proteins IL-13.E13K-D2-LLS-cys and IL-13-D2-KK2 specifically recognize GBM cells, are internalized and localized in the vicinity or with the targeted lysosomal and mitochondrial compartments without any detectable protein fragments transport to cells' nuclei. For effective delivery of the cytotoxic load to tumor cells, we conjugated derivative of doxorubicin WP936 to the C-terminal cysteines introduced into our IL-13.E13K-D2-NLS-cys and IL-13.E13K-D2-LLS-cys fusion proteins. Not only did IL-13.E13K-D2-NLS-cys[WP936] and IL-13.E13K-D2-LLS-cys[WP936] bound to the targeted cells, but they were also able to deliver and accumulate WP936 within cells and their respective subcellular compartments. The cell viability assays showed that IL-13.E13K-D2-NLS-cys[WP936] inhibits growth/kills GBM cells overexpressing the targeted receptor more potently than either WP936 alone or, for example, IL-13.E13K-D2-LLS-cys[WP936].

In the previous study, we had developed a novel strategy based on genetic engineering of proteins and constructed a universal proteinaceous molecule, which recognizes cancer cells and travels intracellularly specifically to the nucleus.[29] This recombinant protein targets IL-13RA2 on GBM cells with a modified receptor ligand, IL-13.E13K and a specialized cytosol translocation bacterial toxin domain D2 of PE and consists of an NLS from the SV40 T antigen to form the IL-13.E13K-D2-NLS nuclear delivery vector. We documented directly the journey of a designer protein-based vector from the cell surface to the nucleus of GBM cells.[29] This very strategy was successfully utilized in the current study for delivery of proteins specifically to lysosomal and mitochondrial compartments, respectively. We have thus achieved the development of higher multi-specificity order of targeting drug conjugates delivering cytotoxic load in a pre-programmed way into cellular compartments. We have succeeded in targeting the lysosomal compartment of tumor cells. An earlier study investigating the presence of lysosomes in astrocytic brain tumors has suggested high amounts of lysosomes present in GBM tumors.[50] Hence, targeting of the lysosomes in which drugs could be hydrolyzed to active forms could potentially be a promising therapeutic strategy against GBM. In the present study, lysosomal targeting with WP936 served more of a purpose of contrasting control in the experiments, because the anthracycline exerts its action in the nucleus. However, some degree of hydrolysis, freeing and finding its way to the nucleus by WP936, especially during prolonged incubation and accumulation of drug conjugates in tumor cells, can be expected as our results would attest.

The cellular localization experiments strongly suggest a possibility of attaching various labels/drugs/dyes to our specific targeted proteins for efficient delivery either to nuclei or lysosomes or mitochondria. We had previously discovered a possibility of targeting cancer cells and delivery of peptides/proteins into the cell cytosol using modified PE.[30] Others have also applied this approach using Diphtheria toxin fragments.[51] In a follow-up of these original experiments, we were able to show the delivery of our subcellular compartment-directed construct, IL-13.E13K-D2-NLS, to the nucleus. Next, we conjugated IL-13.E13K-D2-NLS with a derivative of doxorubicin named WP-936, to produce IL-13.E13K-D2-N LS-cys[WP936]. A general idea behind making these novel drug conjugates was to specifically recognize and affect GBM cells while reducing the access of a chemotherapeutic to normal tissues. Our data demonstrate a significant increase in cytotoxicity with this drug conjugate construct in cells overexpressing IL-13RA2. On the other hand, WP936 conjugation with IL-13.E13K-D2-LLS-cys protein showed some cytotoxic effect on targeted cells, but less pronounced than by IL-13.E13K-D2-N LS-cys[WP936]. Both of these recombinant proteins demonstrated much lower or no cytotoxicity in tumor cells expressing low levels of IL-13RA2. Therefore, our novel drug conjugate represents a template for the development of specific cytotoxic anti-GBM agents. Using a similar approach, a MAb linked to the NLS peptide conjugated to the Auger electron emitter was able to recognize cells and cause cytotoxicity.[52],[53] More importantly, a lower dose of these protein agent-WP936 conjugates was required to achieve cancer cell cytotoxicity compared to the free WP936 drug. A major potential advantage of these agents would be that a lower dose of the WP936 would be required for chemotherapy, leading to lower systemic load, and likely reduced side-effects than free WP936.

Another emerging targeted drug delivery system in the treatment of cancer is the mitochondrial drug delivery system. (KLAKLAK)2 pro-apoptotic peptide was found to be cytotoxic to bacteria,[41] but it cannot efficiently permeate across eukaryotic plasma membrane and hence exhibits low mammalian cell cytotoxicity. However, due to the similarities between the bacterial cell membrane and eukaryotic mitochondrial membrane, once it enters the cell, it causes cytotoxicity by disrupting the mitochondrial membrane. A recent study conducted using KLA, a variant of (KLAKLAK)2 pro-apoptotic peptide, fused with a linear tumor-penetrating homing peptide iRGD demonstrated that it is internalized into cultured tumor cells.[54] Once inside the cells, the peptide leads to apoptosis through both the mitochondrial-induced apoptotic pathway and the death receptor pathway. Another study showed that the pro-apoptotic peptide (KLAKLAK)2 conjugated to penetratin led to selective inhibition of tumor cell growth.[55] To promote delivery of (KLAKLAK)2 specifically to the GBM cells, we generated a recombinant protein IL-13-D2-KK2, which targets IL-13RA2. After biotin conjugation and detection with streptavidin, we showed that IL-13-D2-KK2 was effectively internalized into the GBM cells and colocalized with ATP-synthase. We also demonstrated that IL-13-D2-KK2 has cytotoxic properties against a panel of GBM cell lines, which overexpresses IL-13RA2. Hence, our construct could potentially be used as a pro-apoptotic anticancer therapeutic agent, which will be specific for targeting mitochondria in GBM tumors.

In summary, in this proof-of-principle study, we have generated novel multi-specificity fusion recombinant proteins for targeting lysosomal and mitochondrial compartments of IL-13RA2 positive GBM tumor cells. We have also produced first-in-class drug conjugates in which a chemotherapeutic is delivered to the site of its intracellular action. Our drug candidates were able to successfully carry the WP936 cargo to the intracellular compartments of the cell and caused significant cytotoxicity. In addition, we have demonstrated that a specifically delivered pro-apoptotic peptide caused cytotoxic effect in the GBM cells. Our novel drug conjugates/fusion proteins may thus provide a highly specific and thus safer therapeutic option than the existing ones. We will test these and other chemotherapeutic drug conjugates for their stability, pharmacokinetics and potential to cross the blood-brain tumor barrier. This is in consideration that the conjugates might be delivered systemically in patients. Furthermore, recent advances could allow more efficient penetration of such drugs given systemically through an opening of the blood-brain barrier as reviewed by Rodriguez et al.[56] The drug conjugates of this type can be also administered to brain tumor patients loco-regionally, directly to tumor bed using convection-enhanced delivery.[57] Local administration can be repeated many times, and drugs can be infused for prolonged periods of time, which would represent a desirable feature of the treatment plan.[58],[59] Future preclinical studies will pave the way for clinical evaluation of the conjugates using the optimal mean of their administration.

Financial support and sponsorship

This work was supported by an NIH grant CA71745 to WD.

Conflicts of interest

There are no conflicts of interest.

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