CD40 ligand (CD40L) gene therapy offers a potentially useful option for lung cancer due to its multiple antitumor activities. However, membrane-bound CD40L may be proteolytically cleaved to form soluble CD40L (sCD40L), which results in adverse effects. In a previous study by our group, it was demonstrated that recombinant self-complementary adeno-associated virus 5 (scAAV5) efficiently delivered genes to lung cancer cells. In the present study, an scAAV5 expressing a non-cleavable human CD40L mutant (scAAV5-CD40L-M) was generated and its direct antitumor effects in lung cancer were evaluated. Transduction with scAAV5-CD40L-M resulted in effective expression of CD40L on the cell surface with low levels of cleaved sCD40L, which significantly reduced the percentage of viable cells and promoted caspase-3-dependent apoptosis of CD40-positive lung carcinoma A549 cells, compared with scAAV5-CD40L transduction (P<0.05). Furthermore, treatment with scAAV5-CD40L-M exerted a significant antitumor effect against CD40-positive A549 xenografts by inducing apoptosis (P<0.05) with few side effects. Gene therapy using an scAAV5 vector expressing non-cleavable human CD40L mutant may therefore have direct antitumor effects against CD40-positive lung cancers. These tumoricidal effects of scAAV5-CD40L-M treatment make it a promising therapeutic technique for the treatment of lung cancer.
Lung cancer is a leading cause of global cancer-associated mortality (
Immunogene therapy using immunostimulatory molecules to enhance anti-tumor immunity offers a promising therapeutic option for lung cancer (
Several studies indicated that the precise form of the CD40 stimulus, inducing a membrane-bound and soluble version of CD40L (sCD40L), influenced the therapeutic responses in carcinoma cells. sCD40L is shed from membrane-bound forms via matrix metalloproteinase (MMP) cleavage and provides local cytokine-like amplification of CD40 signaling (
To circumvent the possible stimulating proliferative activity and adverse inflammatory effects caused by sCD40L, a membrane-stable mutant form of human CD40L (CD40L-M) was designed and engineered. As a means of delivering CD40L-M for clinical application, the transduction efficiency of various serotypes of adeno-associated virus (AAV) vectors in an NSCLC cell line were examined. The efficient recombinant self-complementary AAV5 (scAAV5) was subsequently selected as a therapeutic vehicle (
The human NSCLC cell lines A549 and H1650 and the human embryonic kidney cell line HEK293 (293T) were obtained from the Cell Resource Center (Shanghai, China). The cells were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA).
Plasmids pdsAAV-CB-CD40L and pdsAAV-CB-CD40L-M, which are self-complementary double-sequence AAV plasmids containing human wild-type CD40L cDNA and CD40L-M cDNA (containing six substitutions: Gln114 to Pro114, Lys115 to Arg115, Asp117 to Glu117, Gln118 to Glu118, Asn119 to Asp119 and Pro120 to Ser120), were constructed by Invitrogen Life Technologies (Carlsbad, CA, USA) (
PCR was performed using the SYBR Green RT-PCR kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s instructions. The PCR primer pairs used were as follows: CD40L (NM-000074, position 488–620, 132 bp fragment) forward, 5′-TGA GCA ACA ACT TGG TAA CCC TGG-3′ and reverse, 5′-CTG GCT ATA AAT GGA GCT TGA CTC G-3′; β-actin (NM-001101, position 939–1124, 205 bp fragment) forward, 5′-TGA CGT GGA CAT CCG CAA AG-3′ and reverse, 5′-CTG GAA GGT GGA CAG CGA GG-3′ (Invitrogen Life Technologies).
CD40L and CD40 expression was determined by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) using an allophycocyanin (APC)-conjugated CD154 antibody (Ab) diluted at 1:25 or phycoerythrin (PE)-conjugated CD40 Ab diluted at 1:50. Appropriate APC- or PE-conjugated isotype control was used to measure background staining. Annexin V-fluorescein isothiocyanate (FITC) and/or propidium iodide (PI) staining (TACS Annexin V-FITC kit; Trevigen, Gaithersburg, MD, USA) was used to detect apoptotic cells. Caspase-3 inhibitor (Ac-DEVD-CHO; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was employed in a competition experiment.
Cell viability was determined by MTT assay using a Cell Proliferation Kit I (Roche Diagnostics GmbH, Mannheim, Germany). Cancer cells were seeded into 96-well plates. After incubation, the medium was replaced with 50 μl MTT reagent (2 mg/ml) followed by further incubation in an incubator for 2 h. Next, the medium was removed and dimethylsulfoxide (150 μl) was added to each well. The absorbance at 560 nm was measured with a spectrophotometer (PerkinElmer, Waltham, MA, USA). A competition experiment with anti-human CD40L monoclonal antibody (100 ng/l; BioLegend, Inc., San Diego, CA, USA) was performed.
Briefly, proteins were separated by 6–15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Little Chalfont, UK). The primary antibodies used were: Rabbit anti-caspase-3 polyclonal antibody (pAb; 1:100; sc-7148) and rabbit anti-β-actin pAb (1:1,000; sc-130656; Santa Cruz Biotechnology, Inc.). The secondary antibodies were horseradish peroxidase-conjugated antibodies against rabbit immunoglobulin G (GE Healthcare, Waukesha, WI, USA). Immunoreactive bands in the western blots were visualized using enhanced chemiluminescence substrates (ECL Plus; GE Healthcare).
The activity of caspase-3 was evaluated using the Caspase-3 Activity Assay kit (C115; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions. The caspase-3 activity assay is based on spectrophotometric detection of the chromophore
The concentration of sCD40L in supernatants and serum following treatment were measured using a human sCD40L ELISA kit (ELH-CD40L-001; RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer’s instructions.
Xenografts were prepared by implanting tumor fragments derived from A549 cells [5×106 cells suspended in 100 μl phosphate-buffered saline (PBS)] subcutaneously into the back of six-week-old male BALB/c nude mice. The mice were obtained from the animal center at Nanjing Medical University (Nanjing, China). All mice were maintained on a diet of standard rodent chow and water supplied
Immunohistochemistry using the rabbit anti-CD40L antibody (ab65854; Abcam, Cambridge, UK) was employed to investigate paraffin wax-embedded tissue sections using a routine avidin-biotin-immunoperoxidase technique (Histostain-Plus kit, Zymed, San Franzisco, USA). For quantitative analysis of apoptosis, sections were assayed by the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling (TUNEL) method using a One Step TUNEL Apoptosis Assay kit (C1086; Beyotime). The number of positive cells was counted in ten random fields using microscopy (magnification, ×100; LSM 700; Carl Zeiss, Oberkochen, Germany).
The two-tailed Student’s t-test application of Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA) was used for all continuous outcome variables, including tumor size and weight. A repeated Student’s t-test was used to compare the treated groups with the control group. Values are presented as the mean ± standard deviation and P<0.05 was considered to indicate a statistically significant difference between values.
The expression cassette containing the CD40L or CD40L-M cDNA was inserted into the cloning site of pdsAAV-CB-GFP to create the expression plasmid pdsAAV-CB-CD40L or pdsAAV-CB-CD40L-M, respectively. The correct plasmids were identified by sequencing. Recombinant scAAV5-CD40L and scAAV5-CD40L-M vectors (
The direct growth-inhibitory activities of scAAV5-CD40L and scAAV5-CD40L-M were assessed by MTT assay using CD40-positive A549 and CD40-negative H1650 lung cancer cell lines. Significantly decreased A549 viability was observed (
Relative to CD40-negative H1650 cells, scAAV5-CD40L and scAAV5-CD40L-M (104 vgs/cell) were found to induce CD40-positive A549 cell apoptosis as indicated by Annexin V-FITC/PI double staining with apoptotic rates of 17.83±1.32 and 12.56±1.05%, indicating a significant difference between the two groups (P<0.05;
To investigate whether the apoptotic effect observed was associated with caspase-3 activation, western blot analysis was used to examine the expression levels of cleaved caspase-3 in A549 cells. The expression levels of cleaved caspase-3 protein in scAAV5-CD40L-M-infected cells were higher than those in scAAV5-CD40L-infected cells (104 vgs/cell;
Secretion of sCD40L following transduction was detected in supernatants to examine whether CD40L-M was resistant to cleavage on the cell surface. As indicated in
To focus on the direct tumor growth-inhibitory features of AAV5-CD40L-M,
CD40L expression was detected in the scAAV5-CD40L- and scAAV5-CD40L-M-injected tumors but not in the scAAV5-GFP-injected tumors (
The systemic toxicity of scAAV5-CD40L-M treatment was examined. Histopathological studies indicated no apparent lesions in the lung, liver, spleen or kidney. Levels of sCD40L in serial serum samples were ~0. Serum alanine transaminase, aspartate aminotransferase, serum urea nitrogen and creatinine were determined. No significant difference was detected between groups (Data not shown).
CD40L transgene expression in human CD40-positive solid carcinoma cells was found to produce a direct growth-inhibitory effect through apoptotic induction and/or cell cycle blockage (
Successful gene therapy is dependent upon the efficiency and availability of gene delivery systems. Recombinant AAVs have been widely used for gene delivery in animal models and their use in human gene therapy is currently being evaluated in clinical trials (
The results of the present study indicated that scAAV5-CD40L-M produced a more profound growth-inhibitory effect in CD40-positive cells compared with that in wild-type scAAV5-CD40L. Caspase-3 activation assay and Annexin V staining confirmed that CD40L-M was a more potent inducer of apoptosis than the wild-type CD40L, which was in agreement with previous studies by Elmetwali
In conclusion, the results of the present study demonstrated the direct antitumor effects of scAAV5 delivery of membrane-stable mutant CD40L on human CD40-positive lung carcinomas
The present study was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Jiangsu, China) and supported by grants from the International Science & Technology Cooperation Program of China (no. 2014DFA31940), the National Natural Science Foundation of China (Beijing, China; nos. 30971320, 81272602 and 81302014) and the Universities Natural Science Research Project of Jiangsu Province (Nanjing, China; no. 12KJB320004).
Maps of scAAV-CD40L-M and CD40L expression following transduction. (A) Schematic representation of the scAAV genome containing a mutated 5′ inverted terminal repeat and an actin intron from which 722 nucleotides were deleted. This construct contained the cytomegalovirus enhancer/β-actin promoter driving expression of CD40L-M, including six mutation sites. (B) scAAV5-delivered CD40L was expressed on the cell membrane of A549 cells. (C) Relative CD40L gene transcripts evaluated by quantitative polymerase chain reaction in the A549 cell line. Values are expressed as the mean ± standard deviation of triplicate samples, *P<0.05. scAAV, self-complementary adeno-associated virus; CD40L-M, CD40 ligand mutant; GFP, green fluorescent protein.
Cell viability following transduction. Cell viability was evaluated by MTT assay following transduction with recombinant scAAV5-CD40L-M vectors at a multiplicity of infection of 104 vgs/cell and 105 vgs/cell, in comparison to that of scAAV5-CD40L-treated cells. (A) CD40-positive lung carcinoma A549 cells. (B) CD40L-negative H1650 cells. Values are expressed as the mean ± standard deviation of triplicate samples, *P<0.05. scAAV5, self-complementary adeno-associated virus 5; CD40L-M, CD40 ligand mutant.
scAAV5-CD40L-M induces apoptosis in CD40-positive lung carcinoma A549 cells. (A) Flow cytometric analysis of Annexin V-FITC/PI staining in A549 cells 48 h following transduction with viral vectors at a multiplicity of infection of 104 vgs/cell. scAAV5-CD40L and scAAV5-CD40L-M induced apoptosis of CD40-positive A549 cells with apoptotic rates of 17.83±1.32 and 12.56±1.05%, respectively. (B) Cell lysates from vehicle-treated cells were evaluated by western blot analysis for the processing of caspase-3 and (C) by enzymatic assay for caspase-3 activity. Values are expressed as the mean ± standard deviation of triplicate samples, *P<0.05. scAAV5, self-complementary adeno-associated virus; FITC, fluorescein isothiocyanate; PI, propidium iodide; GFP, green fluorescent protein; CD40L-M, CD40 ligand mutant; vgs, viral genomes.
sCD40L expression was evaluated in cell-free supernatants of A549 cells post-infection by ELISA. (A) Samples were collected from the culture media 48 h post-infection at MOI 104 vgs/cell and 105 vgs/cell. (B) Samples were tested at 24, 48 and 72 h following transduction at MOI of 104 vgs/cell. Values are expressed as the mean ± standard deviation of triplicate samples. *P<0.01 vs. corresponding scAAV5-CD40L-M transductants. scAAV5, self-complementary adeno-associated virus 5; MOI, multiplicity of infection; GFP, green fluorescent protein; sCD40L, soluble CD40 ligand; M, mutant; vgs, viral genomes.
scAAV5-CD40L-M inhibits tumor growth