Radiosensitizing effect of aCD4 on Hela cells
γ-radiation dose-response curves were generated for each tumor cell line. The cervical carcinoma cell line Hela was used as our tumor model (Fig. 1A). Based on this data, a suboptimal γ-radiation dose of 4 Gy, which yielded 27.3% growth inhibition, was selected for use in aCD4 presensitization experiments.
To investigate whether aCD4 could sensitize Hela cells to enhance the effect of γ-radiation, Hela cells were co-cultured with indicated doses of aCD4 for 2 days, then γ-irradiated with 4 Gy. Cell viability was determined using WST-1 assay on Day 5. Hela cell growth was inhibited by aCD4 in a dose dependent manner (Fig. 1B). Radiation alone resulted in 31.1 ± 2.1% cell growth inhibition. aCD4 alone, at a dose of 5 × 104 or 1 × 105/ml, caused 4.8 ± 3.5% and 10.1 ± 1.9% cell growth inhibition, respectively; while presensitization with 5 × 104 or 1 × 105 aCD4/mL CM (hereafter called 5 × 104 or 1 × 105 aCD4), followed by γ-irradiation resulted in 37 ± 1.9% (P < 0.05 versus single agent, irradiation or aCD4 alone) and 53.3 ± 1.7% (P < 0.001 versus single agent) cell growth inhibition, respectively.
To investigate the contribution of cell-cell contact versus soluble factor(s) in the radiosensitization of tumor cells, a permeable transwell system was used to avoid the contact between tumor cells and aCD4. Similar degrees of growth inhibition were seen using the transwell system. In this system, 4 Gy irradiation alone, 5 × 104 aCD4 alone, or 1 × 105 aCD4 alone caused 25.8 ± 3.3%, 11.8 ± 2.6% and 31.5 ± 11.3% cell growth inhibition, respectively; while presensitization with 5 × 104 or 1 × 105 aCD4 via transwell, followed by γ-irradiation resulted in 32.2 ± 2.2% and 60.5 ± 1.3% cell growth inhibition, respectively. Compared with 4 Gy irradiation alone, a significant decrease in percentage of viable cells was seen in the 1 × 105 aCD4 plus irradiation group (P < 0.01) (Fig. 1C). To confirm that soluble factors were responsible for the observed activity, we examined the radiosensitizing effect of cell-free supernatants from aCD4 on Hela cells. Cell-free supernatants were collected from aCD4 after 48 h (hereafter called aCD4S). Hela cells were treated with aCD4S for 2 days, followed with 4 Gy γ-irradiation, and plated in a 96-well plate for cell viability assay on Day 5. Similar results were obtained as those in aCD4 and Hela cell-cell direct contact or in a transwell system. 30.8 ± 6.9%, 17.3 ± 7.1% and 32.9 ± 6.8% cell growth inhibition were seen in the 4 Gy irradiation alone, 5 × 104 or 1 × 105 aCD4S alone groups, respectively, while 53.4 ± 10.4% and 62.6 ± 1.0% cell growth inhibition were obtained when Hela cells were presensitized with 5 × 104 aCD4S or 1 × 105 aCD4S, respectively, followed by 4 Gy irradiation. Compared with 4 Gy of irradiation alone, a significant decrease in cell viability was seen in the combination of 5 × 104 or 1 × 105 aCD4S and irradiation groups (P < 0.05 and P < 0.01, respectively, versus single agent) (Fig. 1D). These data confirmed the role of aCD4, particularly soluble factors released from these cells, in sensitizing tumor cells to greatly augment the antitumor activity of γ-irradiation.
aCD4 radio-sensitizing activity is irradiation dose dependent
The data from Fig. 1 demonstrated the aCD4 dose dependent effect on the observed activity. Here we also demonstrated that the observed activity was also dependent on γ-irradiation doses. The survival fractions of Hela cells treated with 4 Gy, 6 Gy, or 8 Gy γ-irradiation alone were 88.51 ± 4.33%, 61.6 ± 5.9%, and 39.1 ± 4.1%, respectively; while those of Hela cells presensitized with 1 × 105 aCD4S, followed by the same doses of γ-irradiation were 53.8 ± 6.8%, 35.5 ± 5.5%, 26.1 ± 1.3%, respectively. The difference between irradiation alone and irradiation plus aCD4S was significant at each irradiation doses (P < 0.01) (Fig. 2).
Confirmation of the radiosensitizing effect of aCD4 on the radioresistant glioma cell line LN229
To assess whether the radiosensitizing activity of aCD4 was unique to Hela cervical cancer cell line, similar experiments were carried out using the human glioma cell line LN229. Based on radiation dose response curve (Fig. 3A) and optimal presensitization condition for LN229, 6 Gy γ-irradiation was used instead of 4 Gy. This dose alone caused 14.6 ± 2.9% cell growth inhibition. LN229 cells were presensitized with aCD4 cells in a cell-cell direct contact manner, or via transwell system, or as cell-free conditioned medium (aCD4S) for 2 days, followed by 6 Gy γ-irradiation. After irradiation, the cells were cultured in CM containing 25% aCD4S for 5 days, and cell viability was measured using WST-1 reagent. In a cell-cell direct contact system, 6 Gy γ-irradiation alone, 5 × 104 aCD4 alone or 1 × 105 aCD4 alone caused 20.5 ± 6.0%, 10.0 ± 3.2% and 15.4 ± 3.4% cell growth inhibition, respectively; while presensitization with 5 × 104 or 1 × 105 aCD4, followed by 6 Gy γ-irradiation resulted in 32.7 ± 9.0% and 52.0 ± 0.8% cell growth inhibition, respectively. There was significant difference between the 1 × 105 aCD4 plus irradiation versus single agent, irradiation or aCD4 alone (P < 0.001 for both) (Fig. 3B). In the transwell system, 6 Gy γ-irradiation alone, 5×104 aCD4 alone or 1 × 105 aCD4 alone caused 16.3 ± 6.1%, 13.4 ± 7.2% and 14.9 ± 1.3% cell growth inhibition, respectively; while presensitization with 5 × 104 or 1 × 105 aCD4 via transwell, followed by 6 Gy γ-irradiation resulted in 26.7 ± 6.4% and 44.7 ± 6.7% cell growth inhibition, respectively. Compared single agent, irradiation or aCD4 alone, the 1 × 105 aCD4 plus irradiation groups significantly reduced LN229 cell viability (P < 0.01 for both) (Fig. 3C). In the aCD4S system, 6 Gy γ-irradiation alone, 5 × 104 aCD4S alone or 1 × 105 aCD4S alone caused 20.5 ± 6.0%, 23.4 ± 2.3% and 47.4 ± 2.9% cell growth inhibition, respectively; while presensitization with 5 × 104 or 1 × 105 aCD4S, followed by γ-irradiation resulted in 57.1 ± 2.3% and 71.7 ± 0.8% cell growth inhibition, respectively (P < 0.001 versus single agent for both) (Fig. 3D). The data demonstrated that aCD4, whether as whole cells cocultured directly with LN229 tumor cells (Fig. 3B) or via transwell membrane (Fig. 3C), or as cell free conditioned medium (aCD4S) (Fig. 3D), significantly enhanced the cell growth inhibition effect of γ-irradiation.
Role of cytokines in the radiosensitization of tumor cells
To understand the soluble factors involved in the observed activity, the aCD4 supernatants (aCD4S) were screened for the presence of 13 common Th1 and Th2 pro-inflammatory and anti-inflammatory cytokines as described in materials and methods, and were found to contain various concentrations of IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-13, IFN-γ, TNF-α and GM-CSF (data not shown). Presensitization of Hela cells with the combination of these cytokines, at concentrations equivalent to those in the supernatants of 1 × 105 aCD4S, significantly enhanced the growth inhibition effect of γ-irradiation (Fig. 4).
Of the 10 cytokines tested, only IFN-γ was found to significantly enhance the radiosensitivity of irradiation on Hela cells. We used IFN-γ-free cytokine combination as well as IFN-γ blockade experiments in the presensitization process to further confirm the essential role of IFN-γ in the observed activity. 4 Gy of γ-irradiation alone, IFN-γ alone, combination of all 10 cytokines previously described (Cyt), or 1 × 105 aCD4S alone caused 18.2 ± 3.5%, 21.4 ± 2.2%, 26.6 ± 6.1%, and 9.0 ± 1.1% cell growth inhibition, respectively. When Hela cells were presensitized with IFN-γ, Cyt, or aCD4S, followed by 4 Gy γ-irradiation, the cell growth inhibition was significantly increased to 46.3 ± 0.8% (P < 0.001 versus single agent), 52.3 ± 0.7% (P < 0.001 and P < 0.01 versus irradiation and aCD4S, respectively), and 45.9 ± 0.5% (P < 0.001 versus single agent).
In order to confirm the principal role of IFN-γ, it was omitted from Cyt (Cyt-IFN-γ), and the radiosensitization effect of the cytokine combination was lost, with 15.9 ± 3.6% cell growth inhibition observed versus 18.2 ± 3.5% for 4 Gy γ-irradiation alone (P > 0.05) (Fig. 4). To confirm our results, anti-IFN-γ monoclonal antibody (anti-IFN-γ) was used to block IFN-γ's activity in aCD4S, with IgG1 isotype antibody as control (Isotype). Treatment of Hela cells with 1 × 105 aCD4S pre-incubated with anti-IFN-γ, followed by 4 Gy γ-irradiation, abolished the radiosensitization effect of 1 × 105 aCD4S, resulting in 12.9 ± 4.4% cell growth inhibition versus 18.2 ± 3.5% for 4 Gy γ-irradiation alone (P > 0.05). The cell growth inhibition was 43.7 ± 0.5% for 1 × 105 aCD4S pre-incubated with Isotype plus 4 Gy γ-irradiation and 45.9 ± 0.5% for 1 × 105 aCD4S plus 4 Gy γ-irradiation (for both, P < 0.01 versus single agent alone) (Fig 4).
Enhancement of radiosensitizing effect of IFN-γ by TNF-α
For these experiments, 80 U/ml of IFN-γ and 5.6 ng/ml of TNF-α, concentrations equivalent to those in 1 × 105 aCD4S, were used. TNF-α by itself had no radiosensitizing activity. The treatment of Hela cells with TNF-α, followed by 4 Gy irradiation resulted in growth inhibition of 34.3 ± 5.1% vs. 41.6 ± 2.5% for irradiation alone (P > 0.05). In contrast, treatment with IFN-γ, followed by 4 Gy irradiation resulted in growth inhibition of 58.0 ± 2.1% (P < 0.01 versus irradiation or IFN-γ alone). Interestingly, when combined with IFN-γ, TNF-α significantly augmented the radiosensitizing effect of IFN-γ in Hela cells. Cell growth inhibition for IFN-γ + TNF-α followed by irradiation was 78.5 ± 0.6% versus 41.6 ± 2.5% for irradiation alone versus 25.8 ± 5.7% for IFN-γ + TNF-α alone (P < 0.001 for both). The difference between the IFN-γ + TNF-α followed by irradiation group and the IFN-γ followed by irradiation group was statistically significant (P < 0.001). The combination of all 10 cytokines as described previously (Cyt) and aCD4S were used as positive controls (P < 0.001 versus single agent treatment for both) (Fig. 5).
Effect of aCD4 and cytokine presensitization on the redistribution of cell cycle phases and apoptosis of Hela cells
To investigate the mechanism underlying cell growth inhibition in our model, flow cytometric analysis was performed to assess the redistribution of cell cycle phases and apoptosis of Hela cells. Cell cycle analysis indicated IFN-γ, IFN-γ/TNF-α combination, or aCD4S shifted Hela cells into S-phase; while TNF-α alone had no effect, and TNF-α did not contribute to the activity seen with the IFN-γ/TNF-α combination. 4 Gy of γ-irradiation induced G2/M-phase arrest in Hela cells. While the presensitization of Hela cells with IFN-γ, TNF-α, or IFN-γ/TNF-α combination before the γ-irradiation did not affect γ-radiation induced G2/M-phase arrest, presensitization of Hela cells with aCD4S increased the γ-irrradiation-induced G2/M-phase arrest from 28.2% to 40.8% (Fig. 6).
Apoptosis analysis was performed two days after irradiation (as opposed to five days for cell viability assay). As shown in Fig. 7, the percentage of apoptosis was 7.15 ± 0.63% with 4 Gy γ-irradiation alone, 4.70 ± 0.57%, respectively, with 5 × 104 aCD4 alone, with 1 × 105 aCD4 alone, while the numbers increased to 10.30 ± 1.56% in 5 × 104 aCD4 plus 4 Gy of irradiation, and 15.65 ± 4.60% in 1 × 105 aCD4 plus irradiation. The differences were significant (p < 0.05) for 1 × 105 aCD4 plus 4 Gy group versus irradiation alone or aCD4 alone (Fig. 7).
aCD4 presensitization followed by irradiation enhanced Bax expression
To further investigate the mechanisms underlying the radiosensitizing activity of aCD4, western blot analysis was performed to evaluate the expression of the pro-apoptotic protein Bax. There was a slight increase in Bax expression when Hela cells were treated with aCD4S alone, compared with no treatment or γ-irradiation alone groups. However, Bax expression was highly upregulated in Hela cells presensitized with aCD4S followed by γ-irradiation (Fig. 8).
General Role of aCD4 or IFN-γ/TNF-α combination in the radio-sensitization of cancer cells
To assess the generality of aCD4 or IFN-γ/TNF-α combination as a radiosensitizer, we tested our approach on the cervical carcinoma cell line Caski, prostate carcinoma cell line DU145 and glioma cell line U373. Radiation dose response curves were generated for each cell line (data not shown). aCD4S or the combination of 160 U/ml of IFN-γ and 11.2 ng/ml of TNF-α were used to presensitize tumor cells for 48 hrs, followed by irradiation. Significant enhancement of cell growth inhibition was seen in each case (Fig. 9). In Caski cells, 4 Gy of γ-irradiation alone, 2 × 105 aCD4S alone or IFN-γ/TNF-α alone caused 26.2 ± 7.3%, 28.6 ± 6.3% and 61.3 ± 8.2% cell growth inhibition, respectively; while presensitization with 2 × 105 aCD4S or IFN-γ/TNF-α, followed by γ-irradiation resulted in 46.0 ± 4.7% (P < 0.05 versus irradiation alone) and 73.6 ± 4.4% (P < 0.001 versus irradiation alone) cell growth inhibition, respectively (Fig. 9A). In Du145 cells, 4 Gy of γ-irradiation alone, 2 × 105 aCD4S alone or IFN-γ/TNF-α alone caused 42.9 ± 7.9%, 6.0 ± 2.6% and 10.9 ± 0.1% cell growth inhibition, respectively; while presensitization with 2 × 105 aCD4S or IFN-γ/TNF-α, followed by γ-irradiation resulted in 54.1 ± 6.8% and 50.4 ± 6.3% cell growth inhibition, respectively. Compared with irradiation alone, aCD4S alone and IFN-γ/TNF-α alone treatments, aCD4S and IFN-γ/TNF-α combined with irradiation clearly inhibited the cell growth (P < 0.05) (Fig. 9B). In U373 cells, 8 Gy of γ-radiation alone, 2 × 105 aCD4S alone and IFN-γ/TNF-α alone caused 33.2 ± 5.2%, 15.2 ± 3.8% and 25.0 ± 0.3% cell growth inhibition, respectively; while presensitization with 2 × 105 aCD4S and IFN-γ/TNF-α, followed by γ-irradiation resulted in 59.3 ± 6.8% and 60.3 ± 7.5% cell growth inhibition. Compared with irradiation alone, aCD4S alone and IFN-γ/TNF-α alone treatments, aCD4S and IFN-γ/TNF-α combined with irradiation markedly inhibited the cell growth (P < 0.01 for both) (Fig. 9C). The results showed either aCD4S or the combination of IFN-γ/TNF-α could increase the radiosensitivity of these cancer cell lines to γ-irradiation.