In vitro and clinical studies of gene therapy with recombinant human adenovirus-p53 injection for oral leukoplakia.
ABSTRACT Oral leukoplakia is a well-recognized precancerous lesion of squamous cell carcinoma. When accompanied with abnormal p53 expression, it suffered a higher risk of canceration. The present study was carried out to test whether the recombinant human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia cells and induce cell cycle arrest and apoptosis.
We select p53(-) oral dysplastic keratinocyte POE-9n, to observe the growth inhibition, cell cycle change, apoptosis-induced effects, and elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of multipoints intraepithelial injections of recombinant adenovirus-p53 in 22 patients with dysplastic oral leukoplakia.
Exogenous p53 could be successfully transduced into POE-9n cells by recombinant adenovirus-p53. The optimal infecting titer in this study was multiplicity of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell proliferation, induce apoptosis, and arrest cell cycle in stage G(1) in POE-9n cells by inducing p21(CIP/WAF) and downregulating bcl-2 expression. In the posttreatment patients, p53 protein and p21(CIP/WAF) protein expression were significantly enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical response to the treatment, and 14 patients showed obvious histopathologic improvement.
Intraepithelial injections of recombinant human adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic oral leukoplakia.
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ABSTRACT: In this study, a combination of recombinant adenoviral p53 (rAd-p53) gene therapy and intra-arterial delivery of chemotherapeutic agents for treatment of oral squamous cell carcinoma was evaluated. In total, 99 patients with stage III or IV oral carcinoma who had refused or were ineligible for surgery were enrolled in a randomized, placebo-controlled, double-blind, phase III clinical trial. They were randomly assigned to group I (n = 35; intra-arterial infusion of rAd-p53 plus chemotherapy), group II (n = 33; intra-arterial infusion of rAd-p53 plus placebo chemotherapy), or group III (n = 31; intra-arterial infusion of placebo rAd-p53 plus chemotherapy). The median length of follow-up was 36 months (range, 3 to 86 months). During follow-up, 16 patients in group I, 20 in group II, and 22 in group III died. Group I (48.5%) had a higher complete response rate than groups II (16.7%) and III (17.2%) (P = 0.006). The rate of non-responders in group I was significantly lower than that in groups II and III (P < 0.020). A log-rank test for survival rate indicated that group I had a significantly higher survival rate than group III (P = 0.019). The survival rate of patients with stage III but not stage IV oral cancer was significantly higher in group I than in group III (P = 0.015, P = 0.200, respectively). The survival rate of patients with stage IV did not differ significantly among the three groups. Or the 99 patients, 63 patients experienced adverse events of either transient flu-like symptoms or bone marrow suppression, while 13 patients had both these conditions together. No replication-deficient virus was detected in patient serum, urine, or sputum. rAd-p53 treatment increased Bax expression in the primary tumor of 80% of patients, as shown by immunohistochemical staining. Intra-arterial infusion of combined rAd-p53 and chemotherapy significantly increased the survival rate of patients with stage III but not stage IV oral cancer, compared with intra-arterial chemotherapy. Intra-arterial infusion of combined rAd-p53 and chemotherapy may represent a promising alternative treatment for oral squamous cell carcinoma.Trial registration: ChiCTR-TRC-09000392(Date of registration: 2009-05-18;BMC Medicine 01/2014; 12(1):16. · 6.68 Impact Factor
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ABSTRACT: Premalignancy and chemoprevention studies in head and neck cancer typically focus on the oral cavity. Avoiding or cessation of alcohol and smoking, early detection of potentially malignant disorders or cancer, and early detection of recurrent and/or second primary tumor form the basis of prevention of oral cancer. Analysis of tissue prospectively collected in evaluation of retinoids for chemoprevention trials allowed identification of molecular biomarkers of risk to develop oral cancer, loss of heterozygosity being the most validated one. Improving risk assessment and identification of new targets for chemoprevention represent the main challenges in this field.Otolaryngologic Clinics of North America 08/2013; 46(4):579-97. · 1.46 Impact Factor
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ABSTRACT: A randomized, open-label, phase II, multicenter clinical trial was conducted to evaluate the efficacy and safety of the addition of a recombinant human endostatin adenovirus (E10A) to cisplatin and paclitaxel in patients with advanced head and neck squamous cell carcinoma (HNSCC) or nasopharyngeal carcinoma (NPC). Patients with locally advanced or metastatic HNSCC or NPC not suitable for operation or radiotherapy were randomly assigned to receive E10A plus chemotherapy every 3 weeks for a maximum of 6 cycles or to receive chemotherapy only. 136 eligible patients were randomly assigned. The addition of E10A did not significantly improve the objective response rate (29.9% vs. 39.7%, p=0.154). However, patients who received endostatin had longer progression-free survival (7.03 vs. 3.60 months, p=0.006; hazard ratio, 0.55). The combination of E10A with chemotherapy benefited prior chemotherapy-treated patients and those who received 3-4 treatment cycles (6.50 vs. 3.43 months, p=0.003; 8.27 vs. 4.27 months, p=0.018; respectively). The overall disease control rate significantly increased from 80.6% in the control group to 92.6% in the test group (p=0.034). Except for fever, no adverse events were associated with the E10A treatment. In summary, E10A plus chemotherapy is a safe and effective therapeutic approach in patients with advanced HNSCC or NPC.Molecular Therapy (2014); doi:10.1038/mt.2014.53.Molecular Therapy 03/2014; · 7.04 Impact Factor
In vitro and Clinical Studies of Gene Therapy with
Recombinant Human Adenovirus-p53 Injection for Oral
Clin Cancer Res 2009;15(21) November 1, 2009
Yi Li,1 Long-Jiang Li,1,2 Song-Tao Zhang,1 Li-Juan Wang,3 Zhuang Zhang,1
Ning Gao,1 Yuan-Yuan Zhang,1 and Qian-Ming Chen1
Authors' Affiliations: 1State Key Laboratory of Oral Diseases, 2Department of Head
and Neck Oncology, West China College of Stomatology, and 3West China Health
Hospital, Sichuan University, Chengdu, P.R. China
Purpose: Oral leukoplakia is a well-recognized precancerous lesion of squamous cell
carcinoma. When accompanied with abnormal p53 expression, it suffered a higher
risk of canceration. The present study was carried out to test whether the recombinant
human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia
cellsand induce cell cycle arrest and apoptosis.
Experimental Design: We select p53(-) oral dysplastic keratinocyte POE-9n, to
observe the growth inhibition, cell cycle change, apoptosis-induced effects, and
elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on
POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of
multipoints intraepithelial injectionsof recombinant adenovirus-p53 in 22 patientswith
dysplastic oral leukoplakia.
Results: Exogenous p53 could be successfully transduced into POE-9n cells by
recombinant adenovirus-p53. The optimal infecting titer in thiss tudy wasmultiplicity
of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell
proliferation, induce apoptosis, and arrest cell cycle in stage G1 in POE-9n cellsby
inducing p21CIP/WAF and downregulating bcl-2 expression. In the posttreatment
patients, p53 protein and p21CIP/WAF protein expression were significantly
enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical
response to the treatment, and 14 patients showed obvious histopathologic
improvement. Conclusion: Intraepithelial injectionsof recombinant human
adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic
Oral leukoplakia (OLK) is defined as a white patch or plaque that cannot be
clinically diagnosed as any other disease (1). Histologically, OLK is the epithelial
hyperkeratosis accompanied with pure proliferation or epithelial dysplasia. It is the
most common premalignant lesion of mucosa and 43% OLK develop into oral
squamous cell carcinoma (2, 3), which is the sixth common malignant carcinoma in
the world (4). It has been shown by previous studies (5, 6) that abnormal p53
expression was detected in 33% to 76% of oral squamous cell carcinoma patients and
20% of OLK ones, and OLK accompanied with abnormal p53 expression suffered a
higher risk of canceration.Therefore, the effective treatment toward OLK becomes
one of the preventive methods against oral squamous cell carcinoma.
The wild-type human tumor suppressor p53 (wt-p53) gene is a primary mediator
of cell cycle arrest, DNA repair, and apoptosis and is intimately involved in tumor
development. The mutation and inactivation of p53 is a critical event in the formation
and progression of head and neck carcinoma (7, 8). Transduction of the wt-p53 gene
into OLK cells is expected to restore the tumor suppressor functions and prevent the
Reintroduction of wt-p53 has been accomplished with recombinant human
adenovirus-p53 (rAd-p53), a replicationincompetent human type 5 adenovirus in
which the E1 region has been replaced with an expression cassette containing the
human wt-p53 cDNA (10). Adenovirus delivery of the wt-p53 gene results in strong
p53 protein expression in tumor cells with minimal toxicity to the hematopoietic
system (11–15). Clinical trials results showed that rAd-p53 is effective against a
variety of malignancies, including colon, glioma, lung, ovarian, and head and neck
tumors (11–17). In the clinical trials, the routes of administration typically used for
rAd-p53 were intratumoral injection (17), perfusion (18), and i.v. infusion (19).
These in vitro and clinical studies evaluate for the first time a novel strategy to
treat oral leukoplakia: gene therapy with recombinant human adenovirusp53 via the
multipoint intraepithelial injection. The results show that exogenous p53 can be
successfully transduced and inhibit cell proliferation, induce apoptosis, and arrest cell
cycle in stage G1, and the clinical method was both safe and effective. We conclude
that intraepithelial injection of recombinant adenovirus-p53 is an effective new
strategy for treating oral leukoplakia. Meanwhile, the present study provided a
feasible method to block the progression of the pre-malignant lesions.
In this article, we selected rAd-p53 as the intervention drug and POE-9n, an OLK
cell line with negative p53 expression as the intervention object. Aiming to explore
the molecular mechanism of rAd-p53, we observed changes of biological behaviors of
preinfection and postinfection cells and analyzed gene expressions relating with the
functional pathway of p53. Moreover, to evaluate the feasibility of rAd-p53 as a
clinical therapy, 22 patients with dysplastic OLK received multipoint intraepithelial
injections of rAd-p53. The results of in vitro and clinical studies are reported in this
Materials and Methods
POE-9n cell line was purchased from the Institute of Medical Research, Harvard
University. This p53 expression deletion cell line was derived from OLK lesion with
severe epithelial dysplasia.POE-9n cells were cultured in D-keratinocyte serum–free
medium supplemented with 5 ng/mL epidermal growth factor, 50 μg/mL bovine
pituitary extractive (Invitrogen-Life Technologies), 100 IU/mL penicillin, and 100
μg/mL streptomycin. Cells were amplificated by using routine cell culture techniques
at 37°C in 5% CO2.
A total of 22 patients with dysplastic OLK were enrolled in the clinical trial in
West China Hospital of Stomatology, Sichuan University. All the cases were
confirmed by clinical and pathologic diagnosis. The average area of the lesions was
4.1 cm2 with the largest of 7 cm2 and the smallest of 2 cm2. All the cases were
classified as mild (nine cases), moderate (eight cases), or severe dysplasia (five cases).
The patients were excluded of systemic diseases and did not receive any treatments
within 3 mo. The informed consents were signed by all patients before the therapy.
This study has been approved by our Institutional Review Board. The Declaration of
Helsinki protocols were followed during the whole study.
rAd-p53 (Shenzhen Sibiono Genetech Co. Ltd) was diluted by 0.9% saline solution
to the concentration of 4 × 109 vp/mL before using. The whole course of treatment
included 15 d, and multipoint intraepithelial injections were done on the 1st, 4th, 7th,
10th and 13th day under the local block anesthesia. With the identified best infection
titer of rAd-p53 (MOI = 100) and the reported maximum single tolerant dose (20, 21),
the injection specification was determined as 2 × 109 vp/cm2, i.e., one injection point
per square centimeter and 0.5 mL rAd-p53 solution for one point. The injection
needles should be pricked into mucosa at a 30- to 45-degree angle and the depths
were 2 to 3 mm according to the thickness of the lesion, to inject the solution
intraepithelially. On the 15th day of the course, samples were collected for biopsy
under local anesthesia. Therapeutic response of all the patients was monitored during
therapeutic course and 30 d after the therapy. All patients were followed up for 24 mo
and the therapeutic effects were recorded. All of the patients did not receive any
therapy during the follow-up except for recurrence and cancerization.
Infection efficiency of rAd-GFP. POE-9n cells in logarithmic growth phase were
seeded onto six-well plate with the concentration of 1 × 105 per well. After 24h,
rAd-GFP solution were added into the medium with the amount of MOI = 0, 25, 50,
100, 200, and 500. Cultured for another 72 h, fluorescence-activated cell sorting was
done for quantitation of infection efficiency.
POE-9n cells in logarithmic growth phase were seeded onto 25-mL culture flask.
After 24h of culture, rAd-p53 of MOI = 100 was added into medium, and then the
cells were cultured for another 72 h. The POE-9n infected with rAd (Shenzhen
Sibiono Genetech Co. Ltd, China) of MOI = 100, which does not harbor p53, was set
as the blank control. The cell morphology was observed under an inverted
Cell growth inhibition ratio
POE-9n cells were seeded onto wells of 96-well plates at 5 × 104 cells per well
and cultured for 24h. rAd-p53 virus solution were added into wells in the amount of
MOI = 0, 25, 50,100, 200, and 500. The control group was established with rAd of the
same MOI. For each concentration, three paralleled holes were set up. Continue the
cultivation for another 24, 48, 72, 96, and 120 h before the culture medium was
abandoned. The proliferative activity was determined every 24h by MTT assay at 570
nm. Growth inhibition ratio was calculated by the formula of (ODcontrol -
ODtreated)/ODcontrol × 100%.
Cell cycle and apoptosis assay
After rAd-p53 (MOI = 100) infection for 24 and 72 h, cell cycle and apoptosis
was detected by flow cytometric analysis. The POE-9n infected with rAd (MOI = 100)
for 72 h was set as the control. Quantification of apoptotic cells was done by terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using
a DeadEnd Fluorometric TUNEL System(Promega, Roche). In clinical samples,
similar methods were used to evaluate cell apoptosis of pre-rAd-p53 and
post-rAd-p53–treated patients. The correlation analysis was conducted between
TUNEL staining and p53 protein staining.
Reverse transcription-PCR and Western blotting analysis
After rAdp53 and rAd infection (MOI = 100) for 24 and 72 h, POE-9n cells were
detected at transcript and protein level. Total RNA was extracted with TRIzol reagent
(Invitrogen). First-strand cDNA was prepared fromtotal RNA by reverse transcriptase
using oligo (dT) primers, and β-actin was set as the internal control. The primers of
wt-p53, p21CIP/WAF, bcl-2, and β-actin were synthesized for PCR as following:
p53: 620 bp, upstream: 5′-TACTCCCCTGCCCTCAACAAGA-3′; downstream:
p21CIP/WAF: 495 bp, upstream: 5′-TTAGGGCTTCCTCTTGGAGAAGAT-
3′ downstream: 5′-ATGTCAGAACCGGCTGGGGATGTC-3′
Bcl-2: 318 bp, upstream: 5′-CGACGACTTCTCCCGCCGCTACCGC-3′
β-actin: 548 bp, upstream: 5′-GTGGGGCGCCCCAGGCACCA-3′ downstream:
Messenger RNA expression of p53, p21CIP/WAF, and bcl-2 were investigated
using reverse transcription-PCR. PCR products were separated with 1% agarose gel
electrophoresis, and the results were recorded and analyzed by gel imaging system.
Cells (1 × 107) were solubilized at 4°C in lysis buffer consisting of 0.125 mol/L
Tris-Cl (pH 6.8), 2% SDS, 2.5%-mercaptoethanol, and 10% glycerol. The protein
levels of p53, p21CIP/WAF, and bcl-2 were detected by Western blotting, and β-actin
was used as inner control.
POE-9n cells were cultured for 24h, and then rAd-p53 and rAd (MOI = 100) were
added into the medium and cultured for another 48 h. Immunohisto- chemical staining
was carried out to detect p53 expression according to the instruction of PV-9000
universal histostain TM-plus kits (ZYMED). The expressions of p53, p21CIP/WAF,
and bcl-2 protein in samples of pre– and post–rAdp53 treatment were detected using
the same method. The criterion of positive expression refers to Shimizu
semiquantitative grade method (22). This scoring procedure included two parts: one
was based on the staining intensity, in which no, mild, moderate, and strong staining
were scored as 0, 1, 2, 3; the other part was based on the rate of positive cells, in
which 0 to 5%, 6 to 30%, 31 to 70%, and 71 to 100% were scored as 0, 1, 2, and 3;
and the total score of 0 to 2, 3 to 4, and ≥5 were labeled as negative (-), positive (+),
and strong positive (++).
Statistical analyses were done using SPSS 15.0 statistics software (SPSS). The
statistical differences between two groups were evaluated by t test. And a χ2/Fisher
exact test was used for categorical variables. Spearman test was done to analyze the
correlation of different groups. All statistical assessments were two sided and
evaluated at the 0.05 level of significant difference.
Infection efficiency of rAd-GFP. POE-9n cells were sensitive to adenovirus. Ten
to 12 hours after rAd-GFP infection, green fluorescence could be detected under
fluorescence microscope, and the fluorescence intensity reached its peak 72 hours
after infection. Infection efficiencies were 36.1 ± 1.56%, 72.7 ±1.40%, and 95.6 ±
2.76%, respectively, when MOI were 25,50, and 100. A positive statistical
correlations existed between efficiency and titer (r = 0.849; P < 0.010). When MOI
were 200 and 500, the infection efficiencies were 97.1 ± 4.12% and 98.9 ± 1.19%, and
the elevation of infection efficiency was less obvious (r = 0.124; P > 0.05).
Cell morphology. Under a phase-contrast microscope, POE-9n cells were