2.1

Preparation of recombinant adenovirus

The recombinant adenoviral vectors were constructed using the AdEasy™ vector system (Qbiogene Inc.) . The construction of recombinant adenovirus Ad-p14 ^ARF , Ad-antisense-EGFR and Ad-sense-EGFR was performed according to the description of Jeffrey N. Myers . In brief, the full-length p14 ^ARF complementary was released from the p14 ^ARF -Bluescript SK +/-plasmid (Cancer Center of Johns Hopkins University kindly presented) and the 1032 bp EGFR gene fragment was obtained from the pEGFR-CXN (Dr. Danmercola, Australia, kindly presented). The resultant construct of pShuttle-CMV-p14 ^ARF and pShuttle-CMV-EGFR was linearized and transformed together with supercoiled vector plasmid (AdEasy-1) into Escherichia coli strain BJ5183 respectively. All recombinant adenoviruses were amplified in human embryonic kidney 293 (HEK293) cells (originally isolated from primary human embryo kidney cells transformed by sheared adenovirus 5 DNA). The titers of the adenoviral stocks were determined by plaque assay in HEK293 cells. Infection of Ad-p14 ^ARF , Ad-antisense-EGFR and Ad-sense-EGFR was tracked by green fluorescent protein (GFP). Then, viruses were purified by double cesium chloride gradients with adenovirus purified Kit (BD Clontech, Palo Alto, CA) and stored in phosphate buffer solution (PBS)/10% glycerol at − 80 °C. The plaques were counted under microscope, and the titers of the recombinant adenovirus were calculated.

2.2

Hep-2 cell viability and cell number assays by MTT

The Hep-2 cells (purchased from China Center for Type Culture Collection) were seeded at 1.0 × 10 ³ cells/cm ² in culture TC100 dishes and grown in Dulbecco modified Eagle medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C in a humidified atmosphere of 5% CO ₂ . When 80% cells were confluent, monoplast cell suspension was digested with 25% trypsin then divided into three TC100 dishes to be cultured. The monoplasts in exponential phase of growth were treated with 200 μl recombinant adenovirus supernatant, including Ad-p14 ^ARF , Ad-antisense-EGFR, Ad-sense-EGFR, vector control (Ad-Ctrl) and PBS control. The trypan blue dye exclusion method was used to determine cell viability. All cells were allowed to adhere and grow in culture medium prior to exposure to diosgenin for 7 days. Cell morphology was observed under the inverted microscope.

MTT assay was used to evaluate the antiproliferative effects of recombinant adenovirus on Hep-2 cells . The proliferation of Hep-2 cells treated with combination of Ad-p14 ^ARF and Ad-antisense EGFR or alone were detected by MTT based colorimetric assay at 580 nm every 24 h for 6 days. As control, Hep-2 cells were treated with Ad-sense EGFR, Ad-Ctrl and PBS, respectively. Additionally, their antiproliferative effects on Hep-2 cells were also evaluated using a cell-count assay. Cells were counted every 2 days over an 7-day period after removing the cells from the plates with 0.5 g/l trypsin.

2.3

Flow cytometry assay

Hep-2 cells were seeded in 6 culture chambers and cultured in 1.5 ml DMEM containing 10% heat-inactivated FCS, 50 units/ml of gentamycin, 100 units/ml of penicillin and 100 μg/ml of streptomycin at 37 °C in a humidified atmosphere of 5% CO ₂ . When 80% Hep-2 cells were confluent, the medium was aspirated and added 200 μl supernatant of Ad-p14 ^ARF plus Ad-antisense EGFR, Ad-p14 ^ARF , Ad-antisense EGFR, Ad-sense EGFR, Ad-Ctrl and PBS to 6 culture chambers, respectively. To obtain monoplast, all cells were harvested and digested with 0.25% parenzyme and 0.02% ethylene diamine tetraacetic acid (EDTA). Hep-2 cell suspension was centrifuged (2000 rpm/min) and fixed with 1% paraformaldehyde and 70% ethanol, stained with 50 μg/ml propidium iodide, then subjected to flow cytometric analysis (Coulter ELITEESP flow cytometer, Coulter Corp., Miami, FL). Finally, the proportion of cells in the sub-G ₀ phase, G ₁ phase, S phase, and G ₂ -M phase of the cell cycle was determined using the Cell QUEST software (Becton Dickinson, Fullerton, CA) based on the resulting DNA distributions . Approximately, 10,000 events (cells) were evaluated for each sample.

2.4

Clonogenic survival assay

The colony growth in soft agar was determined to assess the effects of combination of Ad-p14 ^ARF and Ad-antisense-EGFR, single Ad-p14 ^ARF or Ad-antisense-EGFR on Hep-2 clonogenic cells survival. To quantitate the clonogenic progenitor cells, cells were first plated in six-well plates at a density of 1 × 10 ³ cells per well. After 24 h, cells were infected with viruses as described above at multiplicity of infection (MOI) from 0 to 100. Then cells were trypsinized and resuspended in 20% FCS/Iscove’s modified DMEM (Sigma, St. Louis, MO). Quadruplicate determinations were assessed for each viral dose used. The culture plates were maintained in a humidified atmosphere of 5% CO ₂ at 37 °C for 14 days. Colonies containing more than 50 cells were scored using a 4 × objective on an inverted microscope.

2.5

Hep-2 tumor model establishment

All studies involving mice were approved by the institute’s Animal Care and Use Committee. Hep-2 models were established in immunocompetent athymic male BALB/c nude mice which were purchased from the Animal Research Centre, Sichuan University, China and housed in nude mice care facility at the Animal Centre of Sichuan University. Animal care followed the current regulations and standards of the American NIH, as well as our institutional guidelines for animal care.

2.6

Gene transfer and tumor growth inhibition study in vivo

To establish Hep-2 nude mouse xenografts in mice, 6-week-old athymic male BALB/c nude mice were injected with 2 × 10 ⁶ Hep-2 cells mixed with matrigel (Collaborative Biomedical Products, Bedford, MA) in the bilateral flank of each mouse. Treatment commenced when tumors had reached a volume of 50–100 mm ³ , the xenografts in both flanks of nude mice were randomly assigned to six independent treatment groups with 5 mice per group and all the mice were subjected to injecting into tumor directly with one of the following treatments: Ad-p14 ^ARF plus Ad-antisense EGFR, Ad-p14 ^ARF or Ad-antisense EGFR alone at MOI of 25, 50 and 100, 200, respectively; Ad-sense EGFR, Ad-Ctrl and PBS as control. Intratumoral multidirectional injections were given of the respective gene transfer per assigned treatment group. In the above experiment, the total amount of supernatant given in the combination groups was twice as much as the groups receiving only one recombinant adenovirus. The tumors were allowed to grow for over 3 weeks and were measured twice a week with linear calipers and calculated in cubic millimeters. Tumor volume (mm ³ ) was determined using the formula (length × width ² )/2 (length was the longest axis and width was the measurement at right angles to the length). Data are expressed as mean tumor volume ± standard deviation (SD) for each treatment group. This research project was approved by the Animal Ethics Committee of the Sichuan University, Huaxi Hospital. After experiments, animals of each treatment group were sacrificed, and their tumors were excised for autopsy.

2.7

Western blot analysis in vitro and in vivo

The expression levels of p14 ^ARF and EGFR in Hep-2 cells and xenografts were determined by western blot analysis. Briefly, the whole Hep-2 cell lysates from 1 × 10 ⁶ cells were prepared and scraped into a lysis buffer . Cells were then disrupted by sonication, after which they were centrifuged for 1 hour at 100,000 g to separate particles from soluble fractions for 30 minutes. Xenograft tumors were homogenized and lysed in a buffer containing 10% sodium dodecyl sulfate (SDS), 0.5 mmol/l Tris–HCl (pH 6.8), 1 mol/l dithiothreitol, 10% (v/v) glycerol and 1% bromophenol blue. Total proteins (50 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 12% gels and then electrophoretically transferred (at 40 V for 1.5 hours) to 0.45 μg nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat milk in 0.1% Tween 20 (v/v) in Tris-buffered saline, probed with rabbit polyclonal anti-EGFR (1:3000; Upstate Biotechnology, Inc., Lake Placid, NY) or mouse monoclonal anti-p14 ^ARF in 1% nonfat milk, and incubated for 60 minutes with peroxidase-conjugated mouse anti-human immunoglobulin (1:2000; Amersham, Piscataway, NJ) in 1% nonfat milk. Finally, bands of expression of p14 ^ARF and EGFR were visualized using an enhanced chemiluminescence’s detection system (ECL kit; Amersham, Arlington Heights, IL) . Histology, western blot and all animal experiments were performed in certified laboratories of Sichuan University.

2.8

Statistical analysis

The data were analyzed by analysis of variance (ANOVA) and Student’s t test using SAS software (version 10.00, SAS Institute Inc., Cary, NC). A P value < 0.05 was considered to be statistically significant. Differences in tumor growth in vivo among the treatment groups were assessed by AOV with a repeated measurement module. For the survival time of animals, Kaplan–Meier curves were established for each group, and the survivals were compared by means of the log-rank test. Differences between means or ranks as appropriate were considered significant when yielding a P value < 0.05.

2.1

Preparation of recombinant adenovirus

The recombinant adenoviral vectors were constructed using the AdEasy™ vector system (Qbiogene Inc.) . The construction of recombinant adenovirus Ad-p14 ^ARF , Ad-antisense-EGFR and Ad-sense-EGFR was performed according to the description of Jeffrey N. Myers . In brief, the full-length p14 ^ARF complementary was released from the p14 ^ARF -Bluescript SK +/-plasmid (Cancer Center of Johns Hopkins University kindly presented) and the 1032 bp EGFR gene fragment was obtained from the pEGFR-CXN (Dr. Danmercola, Australia, kindly presented). The resultant construct of pShuttle-CMV-p14 ^ARF and pShuttle-CMV-EGFR was linearized and transformed together with supercoiled vector plasmid (AdEasy-1) into Escherichia coli strain BJ5183 respectively. All recombinant adenoviruses were amplified in human embryonic kidney 293 (HEK293) cells (originally isolated from primary human embryo kidney cells transformed by sheared adenovirus 5 DNA). The titers of the adenoviral stocks were determined by plaque assay in HEK293 cells. Infection of Ad-p14 ^ARF , Ad-antisense-EGFR and Ad-sense-EGFR was tracked by green fluorescent protein (GFP). Then, viruses were purified by double cesium chloride gradients with adenovirus purified Kit (BD Clontech, Palo Alto, CA) and stored in phosphate buffer solution (PBS)/10% glycerol at − 80 °C. The plaques were counted under microscope, and the titers of the recombinant adenovirus were calculated.

2.2

Hep-2 cell viability and cell number assays by MTT

The Hep-2 cells (purchased from China Center for Type Culture Collection) were seeded at 1.0 × 10 ³ cells/cm ² in culture TC100 dishes and grown in Dulbecco modified Eagle medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 100 μg/ml streptomycin and 100 U/ml penicillin at 37 °C in a humidified atmosphere of 5% CO ₂ . When 80% cells were confluent, monoplast cell suspension was digested with 25% trypsin then divided into three TC100 dishes to be cultured. The monoplasts in exponential phase of growth were treated with 200 μl recombinant adenovirus supernatant, including Ad-p14 ^ARF , Ad-antisense-EGFR, Ad-sense-EGFR, vector control (Ad-Ctrl) and PBS control. The trypan blue dye exclusion method was used to determine cell viability. All cells were allowed to adhere and grow in culture medium prior to exposure to diosgenin for 7 days. Cell morphology was observed under the inverted microscope.

MTT assay was used to evaluate the antiproliferative effects of recombinant adenovirus on Hep-2 cells . The proliferation of Hep-2 cells treated with combination of Ad-p14 ^ARF and Ad-antisense EGFR or alone were detected by MTT based colorimetric assay at 580 nm every 24 h for 6 days. As control, Hep-2 cells were treated with Ad-sense EGFR, Ad-Ctrl and PBS, respectively. Additionally, their antiproliferative effects on Hep-2 cells were also evaluated using a cell-count assay. Cells were counted every 2 days over an 7-day period after removing the cells from the plates with 0.5 g/l trypsin.

2.3

Flow cytometry assay

Hep-2 cells were seeded in 6 culture chambers and cultured in 1.5 ml DMEM containing 10% heat-inactivated FCS, 50 units/ml of gentamycin, 100 units/ml of penicillin and 100 μg/ml of streptomycin at 37 °C in a humidified atmosphere of 5% CO ₂ . When 80% Hep-2 cells were confluent, the medium was aspirated and added 200 μl supernatant of Ad-p14 ^ARF plus Ad-antisense EGFR, Ad-p14 ^ARF , Ad-antisense EGFR, Ad-sense EGFR, Ad-Ctrl and PBS to 6 culture chambers, respectively. To obtain monoplast, all cells were harvested and digested with 0.25% parenzyme and 0.02% ethylene diamine tetraacetic acid (EDTA). Hep-2 cell suspension was centrifuged (2000 rpm/min) and fixed with 1% paraformaldehyde and 70% ethanol, stained with 50 μg/ml propidium iodide, then subjected to flow cytometric analysis (Coulter ELITEESP flow cytometer, Coulter Corp., Miami, FL). Finally, the proportion of cells in the sub-G ₀ phase, G ₁ phase, S phase, and G ₂ -M phase of the cell cycle was determined using the Cell QUEST software (Becton Dickinson, Fullerton, CA) based on the resulting DNA distributions . Approximately, 10,000 events (cells) were evaluated for each sample.

2.4

Clonogenic survival assay

The colony growth in soft agar was determined to assess the effects of combination of Ad-p14 ^ARF and Ad-antisense-EGFR, single Ad-p14 ^ARF or Ad-antisense-EGFR on Hep-2 clonogenic cells survival. To quantitate the clonogenic progenitor cells, cells were first plated in six-well plates at a density of 1 × 10 ³ cells per well. After 24 h, cells were infected with viruses as described above at multiplicity of infection (MOI) from 0 to 100. Then cells were trypsinized and resuspended in 20% FCS/Iscove’s modified DMEM (Sigma, St. Louis, MO). Quadruplicate determinations were assessed for each viral dose used. The culture plates were maintained in a humidified atmosphere of 5% CO ₂ at 37 °C for 14 days. Colonies containing more than 50 cells were scored using a 4 × objective on an inverted microscope.

2.5

Hep-2 tumor model establishment

All studies involving mice were approved by the institute’s Animal Care and Use Committee. Hep-2 models were established in immunocompetent athymic male BALB/c nude mice which were purchased from the Animal Research Centre, Sichuan University, China and housed in nude mice care facility at the Animal Centre of Sichuan University. Animal care followed the current regulations and standards of the American NIH, as well as our institutional guidelines for animal care.

2.6

Gene transfer and tumor growth inhibition study in vivo

To establish Hep-2 nude mouse xenografts in mice, 6-week-old athymic male BALB/c nude mice were injected with 2 × 10 ⁶ Hep-2 cells mixed with matrigel (Collaborative Biomedical Products, Bedford, MA) in the bilateral flank of each mouse. Treatment commenced when tumors had reached a volume of 50–100 mm ³ , the xenografts in both flanks of nude mice were randomly assigned to six independent treatment groups with 5 mice per group and all the mice were subjected to injecting into tumor directly with one of the following treatments: Ad-p14 ^ARF plus Ad-antisense EGFR, Ad-p14 ^ARF or Ad-antisense EGFR alone at MOI of 25, 50 and 100, 200, respectively; Ad-sense EGFR, Ad-Ctrl and PBS as control. Intratumoral multidirectional injections were given of the respective gene transfer per assigned treatment group. In the above experiment, the total amount of supernatant given in the combination groups was twice as much as the groups receiving only one recombinant adenovirus. The tumors were allowed to grow for over 3 weeks and were measured twice a week with linear calipers and calculated in cubic millimeters. Tumor volume (mm ³ ) was determined using the formula (length × width ² )/2 (length was the longest axis and width was the measurement at right angles to the length). Data are expressed as mean tumor volume ± standard deviation (SD) for each treatment group. This research project was approved by the Animal Ethics Committee of the Sichuan University, Huaxi Hospital. After experiments, animals of each treatment group were sacrificed, and their tumors were excised for autopsy.

2.7

Western blot analysis in vitro and in vivo

The expression levels of p14 ^ARF and EGFR in Hep-2 cells and xenografts were determined by western blot analysis. Briefly, the whole Hep-2 cell lysates from 1 × 10 ⁶ cells were prepared and scraped into a lysis buffer . Cells were then disrupted by sonication, after which they were centrifuged for 1 hour at 100,000 g to separate particles from soluble fractions for 30 minutes. Xenograft tumors were homogenized and lysed in a buffer containing 10% sodium dodecyl sulfate (SDS), 0.5 mmol/l Tris–HCl (pH 6.8), 1 mol/l dithiothreitol, 10% (v/v) glycerol and 1% bromophenol blue. Total proteins (50 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 12% gels and then electrophoretically transferred (at 40 V for 1.5 hours) to 0.45 μg nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat milk in 0.1% Tween 20 (v/v) in Tris-buffered saline, probed with rabbit polyclonal anti-EGFR (1:3000; Upstate Biotechnology, Inc., Lake Placid, NY) or mouse monoclonal anti-p14 ^ARF in 1% nonfat milk, and incubated for 60 minutes with peroxidase-conjugated mouse anti-human immunoglobulin (1:2000; Amersham, Piscataway, NJ) in 1% nonfat milk. Finally, bands of expression of p14 ^ARF and EGFR were visualized using an enhanced chemiluminescence’s detection system (ECL kit; Amersham, Arlington Heights, IL) . Histology, western blot and all animal experiments were performed in certified laboratories of Sichuan University.

2.8

Statistical analysis

The data were analyzed by analysis of variance (ANOVA) and Student’s t test using SAS software (version 10.00, SAS Institute Inc., Cary, NC). A P value < 0.05 was considered to be statistically significant. Differences in tumor growth in vivo among the treatment groups were assessed by AOV with a repeated measurement module. For the survival time of animals, Kaplan–Meier curves were established for each group, and the survivals were compared by means of the log-rank test. Differences between means or ranks as appropriate were considered significant when yielding a P value < 0.05.