Synergistic suppression effect on tumor growth of acute myeloid leukemia by combining cytarabine with an engineered oncolytic vaccinia virus

Abstract

Background: In consideration of the drug resistance and side effects associated with cytarabine, one of the most effective drugs for the treatment of acute myeloid leukemia (AML), there is a need for safer and effective strategies.

Methods: In the present investigation, we fabricated a new oncolytic vaccinia virus (oVV-ING4), which expresses the inhibitor of growth family member 4 (ING4) and explored its antitumor activity individually and in combination with cytarabine in AML cells.

Results: The experiments confirmed that oVV can efficiently and specifically infect leukemia cells, and augment the ING4 gene expression. Flow cytometry and western blot demonstrated that oVV-ING4 enhances apoptosis and G2/M phase arrest in AML cells, and causes remarkable cancer cell death. In addition, the synergistic efficiency of oVV-ING4 and cytarabine was investigated in vitro and in vivo; the combination significantly inhibited the survival of leukemia cells in vitro and xenografted KG-1 AML tumor growth in vivo.

Conclusion: In brief, oVV-ING4 can increase the sensitivity of leukemia cells to cytarabine and induce cell apoptosis in vitro and in vivo. Thus, oVV-ING4 may be a promising therapeutic candidate for leukemia and in combination with cytarabine represents a potential antitumor therapy.

Keywords: ING4; acute myeloid leukemia; combination therapy; cytarabine; oncolytic vaccinia virus.

Figure 1

Construction and characterization of oVV-ING4. Notes: (A) Schematic structure of recombinant oVV and oVV-ING4. All viruses were constructed through homologous recombination between pCB-transgene and wild-type vaccinia virus (VACV) in HEK293A cells. T7 promoter and gpt gene work as promoter and screen gene. The ING4 expression cassette was introduced into the TK region of the vaccinia virus. (B) Infectious efficiency of oVV by fluorescence microscopy. Leukemia cell lines were treated with oVV-GFP at multiple doses for 48 hours and then observed under the inverted fluorescence microscope. (C) ING4 expression was determined by Western blotting 48 hours post-infection (MOI =10). Data shown (mean ± SD) are representative of three experiments. GAPDH was used as a protein-loading control. Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; TK, thymidine kinase; MOI, multiplicity of infection; GFP, green fluorescent protein.

Figure 2

ING4 is specific to AML cells and is safe for normal cells. Notes: (A) Leukemia cells infected with oVV and oVV-ING4 at the indicated MOI (Lg-3 to Lg3) for 48 hours. Then, MTS viability assay used to examine the cell viability (real lines indicate the infection with oVV, broken lines indicate the infection with oVV-ING4). (B) IC₅₀ values of different cell lines to oVV or oVV-ING4. (C) Human normal peripheral blood mononuclear cells (PBMCs) which treated with similar ways were used as the control to normalize the results. Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection.

Figure 2

ING4 is specific to AML cells and is safe for normal cells. Notes: (A) Leukemia cells infected with oVV and oVV-ING4 at the indicated MOI (Lg-3 to Lg3) for 48 hours. Then, MTS viability assay used to examine the cell viability (real lines indicate the infection with oVV, broken lines indicate the infection with oVV-ING4). (B) IC₅₀ values of different cell lines to oVV or oVV-ING4. (C) Human normal peripheral blood mononuclear cells (PBMCs) which treated with similar ways were used as the control to normalize the results. Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection.

Figure 3

oVV-ING4-induced cell apoptosis in leukemia cells. Notes: (A) Leukemia cells were infected with oVV or oVV-ING4 (10 MOI) for 48 hours. Cell apoptosis was assessed by FACS analysis of Annexin V-FITC/PI double staining. (columns: apoptotic cell number expressed as the mean ± SD of three separate experiments; bars, SD. ^**P<0.01, ^***P<0.001, one-way ANOVA and multiple comparisons). (B) Cells were treated as indicated above, and then cell lysates were immunoblotted for the detection of activation of caspase pathway and inhibition of NF-κB pathway. GAPDH was used as a loading control. Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection; FACS, fluorescence activated cell sorter.

Figure 3

oVV-ING4-induced cell apoptosis in leukemia cells. Notes: (A) Leukemia cells were infected with oVV or oVV-ING4 (10 MOI) for 48 hours. Cell apoptosis was assessed by FACS analysis of Annexin V-FITC/PI double staining. (columns: apoptotic cell number expressed as the mean ± SD of three separate experiments; bars, SD. ^**P<0.01, ^***P<0.001, one-way ANOVA and multiple comparisons). (B) Cells were treated as indicated above, and then cell lysates were immunoblotted for the detection of activation of caspase pathway and inhibition of NF-κB pathway. GAPDH was used as a loading control. Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection; FACS, fluorescence activated cell sorter.

Figure 4

oVV-ING4-induced cell cycle arrest in leukemia cell lines. Notes: (A) Detection of cell cycle distribution. Flow cytometry analysis of PI-stained cells was conducted at 48 hours post-infection at an MOI of 10. The percentages of cells in G1, S, and G2/M phase were scored and presented graphically (^*P<0.05, ^**P<0.01, and ^***P<0.001, one-way ANOVA and multiple comparisons. The results are presented as the mean ± SD of three separate experiments). (B) The expression of cell cycle-related protein was determined by Western blotting. The cells were treated as same as (A), and GAPDH was served as an internal reference. Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection.

Figure 4

oVV-ING4-induced cell cycle arrest in leukemia cell lines. Notes: (A) Detection of cell cycle distribution. Flow cytometry analysis of PI-stained cells was conducted at 48 hours post-infection at an MOI of 10. The percentages of cells in G1, S, and G2/M phase were scored and presented graphically (^*P<0.05, ^**P<0.01, and ^***P<0.001, one-way ANOVA and multiple comparisons. The results are presented as the mean ± SD of three separate experiments). (B) The expression of cell cycle-related protein was determined by Western blotting. The cells were treated as same as (A), and GAPDH was served as an internal reference. Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection.

Figure 5

Synergistic effects of cytarabine and oVV-ING4 in leukemia cells. Notes: AML cells were seeded at 5,000 cells/well into 96-well plates. (A–C) Cell lines are treated with Ara-C at multiple doses (0.01–50 µM) with or without oVV-ING4 (MOI =1 or 2). Cell viability was measured by MTS after 3 days. Data shown are representative of three independent experiments (a). The potential synergistic effect of cytarabine combined with oVV-ING4 on AML cells was assessed by Chou–Talalay CI analysis using CalcuSyn software. The middle curved line stands for the simulated CI values, which was expressed as the log10 (CI)±1.96 SD, encircled by two lines of algebraic evaluation of the 95% confidence intervals. The log10 (CI) values attained at given fractional affects represent an additive efficiency when equal to 0 and a synergism when <0. It was quantified by CIN analysis and expressed as CIN vs fraction affected. Where calculable, 95% CI are shown (b). Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection; CIN, combination index.

Figure 6

Combined treatments with oVV-ING4 and Ara-C showed an enhanced effect of inhibition tumor growth in vivo. Notes: (A) Schematic representation of the treatment. BALB/c athymic nude mice bearing KG-1 AML tumors (0.2–0.3 cm³) were intratumorally injected with oVV-ING4 (1×107 pfu), oVV (1×107 pfu), or PBS (100 µL) every day for a total of seven times and intraperitoneally injected with a dose of cytarabine (50 mg/kg) on alternative days for four times or coinjection of cytarabine and oVV-ING4. (B) Tumor volume was measured at different times after treatment (a). Twenty-five days after injection, the mice were sacrificed and the tumors were excised to weight (b) and photograph (c). Flow cytometry was used to assess tumor cell apoptosis in vivo (C and D) the safety of oVV-ING4. The treated hematopoietic stem cells were assessed by FACS analysis of anti-CD34-APC staining. (E) Histopathological analysis. The effect of apoptosis induced in tumor tissues was detected by H&E staining, TUNEL assay and immunohistochemical analysis of ING4 and caspase-3 (magnification 400×). (Data are presented as means ± SD [n=6]) (^*P<0.05, and ^***P<0.001). Abbreviations: oVV, oncolytic vaccinia virus; ING4, inhibitor of growth family member 4; MOI, multiplicity of infection; CI, combination index; H&E, hematoxylin & eosin; FACS, fluorescence activated cell sorter; NS, non-significant.