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. 2009 May 27;16(1):49.
doi: 10.1186/1423-0127-16-49.

Combination of apigenin treatment with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects

Chi-Mu Chuang  1 , Archana Monie, Annie Wu, Chien-Fu Hung
Affiliations

Combination of apigenin treatment with therapeutic HPV DNA vaccination generates enhanced therapeutic antitumor effects

Chi-Mu Chuang et al. J Biomed Sci. .

Abstract

Background: It is important to develop innovative therapies for advanced stage cancers in addition to the conventional therapies including chemotherapy, radiation and surgery. Antigen-specific immunotherapy has emerged as a novel alternate therapy for advanced stage cancers, which may be employed in conjunction with conventional therapies.

Methods: In the current study, we tested the effect of treatment with the chemotherapeutic agent, apigenin in combination with DNA vaccines encoding the HPV-16 E7 antigen linked to heat shock protein 70 (HSP70) in the control of the E7-expressing tumor, TC-1.

Results: We observed that treatment with apigenin rendered the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic CD8+ T cells. Furthermore, treatment of TC-1 tumor cells with apigenin was found to enhance apoptotic tumor cell death in vitro in a dose-dependant manner. We showed that TC-1 tumor-bearing mice treated with apigenin combined with E7-HSP70 DNA generate highest frequency of primary and memory E7-specific CD8+ T cells, leading to potent therapeutic anti-tumor effects against E7-expressing tumors.

Conclusion: Thus, apigenin represents a promising chemotherapeutic agent, which may be used in combination with immunotherapy for the treatment of advanced stage cancers. The clinical implications of the current strategy are discussed.

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Figures

Figure 1
Figure 1
In vitro cytotoxicity assay. Luciferase-expressing TC-1 tumor cells were added to 24-well plates at a dose of 1 × 105/well. TC-1/luc tumor cells were (A & B) treated with solvent DMSO (0.1%) as a control or treated with different concentrations of apigenin (20, 40, 80 μM) or (C & D) treated with E7-specific cytotoxic T cells at different E:T ratios (1:1, 1:5) with or without treated 40 mM apigenin. Bioluminescence imaging was performed on D0, D1, D2 and D3. The degree of CTL-mediated killing of the tumor cells was indicated by the decrease of luminescence activity using the IVIS luminescence imaging system series 200. Bioluminescence signals were acquired for 10 seconds. (A & C) Representative luminescence images of 24-well plates showing lysis of the tumor cells by (A) different concentrations of apigenin or by (C) E7-specific T cells and apigenin. (B & D) Bar graph depicting the quantification of luminescence intensity in tumor cells treated with (B) different concentrations of apigenin or treated with (D) apigenin and/or E7-specific cytotoxic T cells (mean ± SD). P values less than 0.05 are considered to be statistically significant. Data shown are representative of two experiments performed.
Figure 2
Figure 2
Flow cytometry analysis to determine the number of apoptotic cells induced by apigenin treatment. TC-1 cells were co-incubated with various concentrations of apigenin (20, 40, 80 μM). Cells treated with solvent DMSO (0.1%) alone were used as controls. The cells were then stained with PE-conjugated annexin V antibody (BD, San Diego) and 7-AAD to detect the expression of annexin V and 7-AAD. Flow cytometry analysis was performed to characterize the expression of annexin V+ 7-AAD+ (apoptotic) cells among the TC-1 cells treated with apigenin. A & B. Representative flow cytometry data demonstrating the percentage of apoptotic cells at (A) 6 hours after incubation or (B) 48 hours after incubation with different concentrations of apigenin. C & D. Bar graphs depicting in the percentage of apoptotic cells at (C) 6 hours after incubation or (D) 48 hours after incubation with different concentrations of apigenin. Data shown are representative of two experiments performed (mean ± SD).
Figure 3
Figure 3
Intracellular cytokine staining followed by flow cytometry analysis to determine the number of primary and memory E7-specific CD8+ T cells in tumor-bearing mice treated with apigenin and/or E7-HSP70 DNA vaccine. Groups of C57BL/6 mice (5 per group) were challenged subcutaneously with 1 × 104/mouse of TC-1 tumor cells. Mice were treated with apigenin alone, E7-HSP70 DNA vaccine alone, the combination of apigenin and the E7-HSP70 DNA vaccine. Apigenin was administered intraperitoneally at the dose of 25 mg/kg after TC-1 inoculation and continued for 10 days. Mice were vaccinated with 2 μg/mouse of E7-HSP70 via gene gun, 3 days before TC-1 inoculation and receive a booster dose 7 days after the first vaccination. Untreated tumor challenged mice were used as negative controls. 14 days (for primary immune response) and 42 days (for memory immune response) after tumor challenge, splenocytes from mice were harvested and stained for CD8 and intracellular IFN-γ and then characterized for E7-specific CD8+ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. A & B. Representative data of intracellular cytokine stain followed by flow cytometry analysis showing the number of E7-specific IFNγ+ CD8+ T cells in mice treated with apigenin and/or DNA vaccine at (A) 14 days or (B) 42 days after tumor challenge. C & D. Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8+ T cells per 3 × 105 pooled splenocytes at (C) 14 days or (D) 42 days after tumor challenge. Data shown are representative of two experiments performed (mean ± SD).
Figure 4
Figure 4
Intracellular cytokine staining followed by flow cytometry analysis to determine the memory recall response of E7-specific CD8+ T cells in tumor-bearing mice treated with apigenin and/or E7-HSP70 DNA vaccine. Groups of C57BL/6 mice (5 per group) were challenged subcutaneously with 1 × 104/mouse of TC-1 tumor cells. Mice were treated with apigenin alone, E7-HSP70 DNA vaccine alone, or the combination of apigenin and the E7-HSP70 DNA vaccine as described in Figure 3. Untreated tumor challenged mice were used as negative controls. Sixty days after the last vaccination, the mice were vaccinated twice with E7-HSP70 DNA vaccine to generate the memory recall response. Ten days after the recall vaccination, splenocytes were harvested and stained for CD8 and intracellular IFN-γ and then characterized for E7-specific CD8+ T cells using intracellular IFN-γ staining followed by flow cytometry analysis. (A) Representative data of intracellular cytokine stain followed by flow cytometry analysis showing the memory recall response of E7-specific IFNγ+ CD8+ T cells in mice treated with apigenin and/or DNA vaccine. (B) Bar graph depicting the numbers of E7-specific IFN-γ-secreting CD8+ T cells per 3 × 105 pooled splenocytes.
Figure 5
Figure 5
In vivo tumor treatment experiments. Groups of C57BL/6 mice (5 per group) were subcutaneously challenged with 5 × 104/mouse of TC-1 tumor cells. Tumor challenged mice were treated with apigenin and/or E7-HSP70 DNA vaccine as indicated in Figure 3. Untreated TC-1 tumor-bearing mice were used as a control. (A) Line graph depicting the tumor volume in TC-1 tumor bearing mice treated with apigenin and/or E7-HSP70 DNA. (B) Kaplan & Meier survival analysis of TC-1 tumor challenged mice treated with apigenin and/or E7-HSP70 DNA. Data shown are representative of two experiments performed (mean ± SD).
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