Figure 1.
Construction and characterization of Ad·(ST13)·CEA·E1A(Δ24).(ST13) represents the expression cassette.
A. Schematic construction of Ad·(ST13)·CEA·E1A(Δ24) and Ad·(EGFP)·CEA·E1A(Δ24). The native E1A promoter was replaced by the CEA promoter to control the expression of the E1A gene with a 24-bp deletion. An ST13 or EGFP expression cassette under the control of the hCMV promoter was inserted between the ψ packing signal sequence and the E1A gene. B. Identification of the CEA promoter and ST13 gene in pAd·(ST13)·CEA·E1A(Δ24) by PCR. Lane M: DL2000 Marker; Lane 1: CEA promoter; Lane 2: ST13 gene. C. Detection of E1A(Δ24) and ST13 expression levels when SW620 cells were infected with Ad·(ST13)·CEA·E1A(Δ24), Ad·(EGFP)·CEA·E1A(Δ24) or the typical oncolytic virus ONYX-015 at an MOI of 5 for 48 hr. Western blot analysis was conducted to detect E1A(Δ24) and ST13 protein levels.
Figure 2.
Colorectal cancer specific antitumor effect of Ad·(ST13)·CEA·E1A(Δ24) in vitro analyzed by the MTT assay. A.
The viability of tumor cells infected with different MOIs of the various oncolytic adenoviruses. Three CRC tumor cell lines (SW620, HCT116 and HT29), and three CEA-negative cell lines (Bcap37 breast cancer, CNE Nasopharynageal carcinoma and HeLa cervical carcinoma) and two normal cells (QSG7701 and WI38) were infected with either Ad·(ST13)·CEA·E1A(Δ24), Ad·(EGFP)·CEA·E1A(Δ24), or the typical oncolytic virus ONYX-015 at a range of MOIs (0.1, 1, 5 or 10 MOI), 4 days, cell viability was determined using an MTT assay. Uninfected cells were considered to be 100% viable. Bars represent the means ± SD (n = 6). B. The influence of viral infection on cell viability at different times. Three CEA positive cell lines (SW620, HCT116, and HT29) and three CEA-negative cell lines (Bcap37, CNE and HeLa) and two normal cells (QSG7701 and WI38) were infected with either ONYX-015, Ad·(EGFP)·CEA·E1A(Δ24), or Ad·(ST13)·CEA·E1A(Δ24) at an MOI of 10. After 24, 48, 72, and 96 hours, the cell viability was measured using the MTT assay. The data are presented as the mean ± SD of triplicate experiments. C. The viability of tumor cells infected with different MOIs of Ad·(ST13)·CEA·E1A(Δ24). CEA-negative colon cancer cell line (Colo-320) and CEA-positive non-colon cancer cell line (A549, MCF-7) were infected with Ad·(ST13)·CEA·E1A(Δ24) at a range of MOIs (0.1, 1, 5 or 10 MOI), 3 days, cell viability was determined using an MTT assay. Bars represent the means ± SD (n = 6).
Figure 3.
Morphological changes and apoptosis detected by flow cytometry. A.
Morphological observations of tumor cells and normal cells infected with the various oncolytic adenoviruses as detected by microscopy. Cells were infected at an MOI of 10, and the morphological changes in the cells were observed by microscopy after 72 hours of infection. B. Detection of apoptosis in SW620 cells by FACS. SW620 cells were infected with either ONYX-015, Ad·(EGFP)·CEA·E1A(Δ24) or Ad·(ST13)·CEA·E1A(Δ24) at an MOI of 10. At 48 hours, the cells were harvested and stained with annexin V-FITC (for early-stage apoptosis) or PI (for late-stage apoptosis) and were examined by flow cytometry. C. The percentage of apoptotic cells was calculated using the Cell Quest software. The data are presented as the mean ± SD (error bars) of triplicate experiments. (**p<0.01; ***p<0.001).
Figure 4.
SW620 cells by western blot. SW620 cells were infected with either ONYX-015, Ad·(EGFP)·CEA·E1A(Δ24) or Ad·(ST13)·CEA·E1A(Δ24) at an MOI of 5, for 48 h, the apoptosis-related proteins were analyzed by western blot.
Figure 5.
The antitumor efficacy of Ad·(ST13)·CEA·E1A(Δ24) in nude mice bearing a colorectal cancer SW620 xenograft.
Tumors were established by injecting SW620 cells subcutaneously into the right flank of nude mice. When tumors reached 100–130 mm3, the mice were randomly divided into three groups (n = 8) and were treated daily with consecutive intratumoral injections four times of ONYX-015, Ad·(EGFP)·CEA·E1A(Δ24) or Ad·(ST13)·CEA·E1A(Δ24) at 5×108 PFU/day and PBS. A. The tumor size was measured with calipers, and the tumor volume was calculated using the following formula: tumor volume (mm3) = 0.5×length×width2. B. The survival curve for the animals during the observation period. The data are presented as the mean ± SD (error bars). A log-rank test has been used to analyze survival rates in the different groups. Statistical significance: a, p<0.001, compared with PBS; b, p<0.01, compared with ONYX015; c, p<0.05, compared with Ad*(EGFP)*CEA*E1A (Δ24). C. Hexon and ST13 expression in vivo. Tumor sections derived from PBS- or different adenovirus drugs treated 4 days were analyzed for Hexon and ST13 expression by immunohistochemistry. Original magnification 400x.
Figure 6.
The detection of necrosis and apoptosis in vivo. A.
Tumor sections of HCT116 CRC were analyzed for necrosis by hematoxylin–eosin (H&E) staining, for apoptosis by TdT-mediated dUTP-biotin nick-end labeling (TUNEL), and for morphological observations by transmission electron microscope (TEM) analysis. (B1), Apoptosis in tumor cells. (B2), For clarity, a single tumor was observed for apoptosis.
Figure 7.
Possible mechanisms of the antitumor effect of Ad·(ST13)·CEA·E1A(Δ24) in vivo.
After Ad·(ST13)·CEA·E1A(Δ24) infection, on the one hand, the phosphorylated P38, ATF2 and upregulation of CHOP expression were detected. On the other hand, executioner caspase-3 was activated.