Human lung cancer H2179 cells and mouse lung cancer Lewis cells were obtained from the Georgetown University(Washington DC, USA), and were maintained in Dulbecco′s modified Eagle′s medium(DMEM) (Gibco-BRL, USA) supplemented with 5% fetal bovine serum(FBS) and were cultured in an incubator at 37℃ with 5% CO₂.

The p3XFLAG-HPV16 E6 and the pcDNA3FLAG-HPV16 E7 expression plasmid has been described in our previous studies^[9]. Subconfluent proliferating cells in six-well dishes were incubated overnight with 1 μg of pcDNA3 FLAG-E7 or p3XFLAG-E6 expression in serum-free DMEM containing Lipofectamine 2000.

The transfected and untransfected cells were irradiated with various doses of 0, 2, 4, 6, 8 and 10 Gy rays using a Cs¹³⁷ irradiator at a dose rate of 1 Gy/min at room temperature.

Cells irradiated were plated in 60-mm culture dishes at a cell density of 2×10³. After 3 weeks post-irradiation, cells were fixed and stained with Giemsa solution. Colonies containing > 50 cells were counted. Colony forming efficiency=number of colonies formed/number of cells plated.

Western blot analysis was carried out as described before^[9]. Briefly, after cells were harvested and lysed, equal amounts of protein were separated using 10% SDS-PAGE gel electrophoresis, transferred onto nitrocellulose membranes and blocked with 5% skimmed milk. The membranes were blotted with primary antibodies against the FLAG epitope(M2, mouse monoclonalantibody, Sigma, 1:500 dilution), BRCA1(C-20, rabbit polyclonal, Santa Cruz Biotechnology, 1:200), and α-actin(I-19, goat polyclonal, Santa Cruz Biotechnology, USA, 1:2000), and then incubated with HRP-conjugated anti-goat or anti-rabbit IgG antibodies(SantaCruz Biotechnology, Santa Cruz, CA, USA, 1:2000). Protein bands were visualized using the enhanced chemiluminescence detection system(Amersham Life Sciences, USA). Equality of protein loading was confirmed by fast green staining of the membrane and by immunoblotting for α-actin. Colored markers(BioRad Corp., USA) were used as molecular size standards

Transfected and un-transfected cells plated on 6-well plates were exposed to a 3 Gy dose of γ-ray and were then incubated at 37℃ for 0.5, 1, 2, 4 and 24 h post-irradiation. Cells were harvested and re-suspended in PBS containing chloromethyl-2', 7'-dichlorofluorescein diacetate(50 μmol/L DCFH-DA) for 15 min at 37℃. The cells were washed and incubated with 1 mg/L ROS for another 15 min. The intensity of fluorescence was assayed using the flow cytometer at 488 nm.

The un-transfected and transfected H2179 cells seeded on 96-well plates were cultured in serum-free DMEM for 24 h, harvested the supernatants, and VEGF levels were detected with a VEGF ELISA kit(R&D Systems, Minneapolis, MN) according to the manufacturer′s instructions. There were ten samples in each group for the ELISA.

Where appropriate, statistical comparisons were made using the two-tailed Student′s t test with the statistical software SPSS 13.0(SPSS Inc., Chicago, IL, USA). A P value of less than 0.05 was considered statistically significant. The data are shown as the mean ± standard error of the mean(SEM).

Firstly, human lung cancer H2179 cells were exposed to γ-rays at different doses(0, 2, 4, 6, 8 and 10 Gy), respectively, cells survival was analyzed by colony formation assay. As shown in Fig 1, the cell survival curve of H2179 cells transfected with a HPV16 E6 or HPV16 E7 expression vector shifted to the right, indicating the cells expressing HPV16 became more radioresistance. For example, IC₉₀, a dose which inhibits 90% of cell survival, was 9.0 Gy(t=1.3145, P < 0.05) and 8.6 Gy(t=1.2106, P < 0.05) for HPV16 E6 and HPV16 E7 transfected cells respectively, compared to un-transfected control cells(6.2 Gy). Next, we also investigated the effects of HPV16 E6 and HPV16 E7 on the radiosensitivity in mouse lung cancer Lewis cells. As illustrated in Fig. 2, the cell survival curve of Lewis cells transfected with HPV16 E6 or HPV16 E7 expression vector also shifted to the right. The IC₉₀ was 2.9 Gy(t=2.1154, P < 0.05) and 4.2 Gy(t=3.3970, P < 0.05) for HPV16 E6 and HPV16 E7 transfected cells respectively, compared to un-transfected control cells(1.6 Gy). These results suggest that the HPV16 E6 and HPV16 E7 oncoproteins negatively mediate the radiosensitivity of human and mouse lung cancer cells.

Figure 1.  Negative effects of HPV16 oncoproteins E6 and E7 on radiosensitivity of human lung cancer H2179 cells. The H2179 cells were transfected with the p3XFLAG-HPV16 E6 or pcDNA3FLAG-HPV16 E7 expression plasmid as described in the "materials and methods", irradiated at indicated doses 24 h following transfection, and subjected to clonogenic survival assay. The HPV16 oncoproteins E6 or E7 are shown in the inset Western blot by using an antiFLAG antibody.

Figure 2.  Negative effects of HPV16 oncoproteins E6 and E7 on radiosensitivity of mouse lung cancer Lewis cells. The Lewis cells were transfected with a p3XFLAG-HPV16 E6 or pcDNA3FLAG-HPV16 E7 expression plasmid as described in the "materials and methods", irradiated at indicated doses 24 h following transfection, and subjected to clonogenic survival assay. The HPV16 oncoproteins E6 or E7 are shown in the inset Western blot by using an antiFLAG antibody.

To demonstrate whether transfected cells expressed the HPV16 E6 or HPV16 E7 oncoprotein after transfection, we harvested un-transfected and transfected H2179 and Lewis cells and performed Western blot assay by using an antibody against FLAG epitope^[9]. The Western blot results revealed that both H2179 and Lewis cell lines highly expressed the HPV16 E6 or HPV16 E7 oncoprotein following transfection(Figs. 1 and 2).

Intracellular ROS accumulation is an important marker for radiation-induced injury, thus we also studied the effects of HPV16 E6 and HPV16 E7 oncoprotein on the induction of ROS. As shown in Fig. 3, ROS levels in the cells transfected with the HPV16 E6 or HPV16 E7 expression vector became lower than the levels in the un-transfected control cells after irradiation in a dose-dependent manner. These results indicate that the radiosensitivity mediated by HPV16 E6 and HPV16 E7 is related to an inhibition of ROS.

Figure 3.  Effects of HPV16 oncoproteins E6 and E7 on intracellular ROS levels. H2179 cells were transfected with p3XFLAG-HPV16 E6 or pcDNA3FLAG-HPV16 E7 expression plasmid, irradiated with γ-ray after 24 h, and subjected to ROS assay as described in the "materials and methods".

VEGF is a significant mediator in radiosensitivity of lung cancer cells^[10-12]. To verify whether VEGF secretion by the lung cancer cells was affected by HPV16 E6/E7, we collected supernatants of cells for the ELISA. As shown in Fig. 4, an increase of 2.3-fold(n=10, t=2.7193, P < 0.05) and 2.6-fold(n=10, t=3.1423, P < 0.05) of secreted VEGF was respectively observed in the H2179 cells expressing HPV16 E6 and HPV16 E7 compared with the un-transfected control cells at 24 h. These findings indicate that both HPV16 E6 and HPV16 E7 enhance VEGF expression.

Figure 4.  Effect of HPV16 oncoproteins E6 and E7 on VEGF expression. H2179 cells were transfected with p3XFLAG-HPV16 E6 or pcDNA3FLAG-HPV16 E7 expression plasmid. After 24 or 48 h following transfection, the VEGF protein in the supernatants was detected by ELASA as described in the "materials and methods".

The ERK1/2 and PI3K/AKT pathways are activated in cells irradiated^[13-14]. To evaluate the involvement of ERK1/2 and AKT signaling in HPV16 E6-increased VEGF expression, we compared the phosphorylation levels of ERK1/2 and AKT protein in un-transfected and transfected cells 15 min after irradiation with 3 Gy. As shown in Fig. 5 HPV16 E6 enhanced the phosphorylation of ERK1/2 caused by radiation. An enhanced AKT phosphorylation was also observed in H2179 cells expressing the HPV16 E6 oncoprotein. Similar results was obtained in the cells expressing the HPV16 E7 oncoprotein(data not shown). These results indicate that the alteration of ERK1/2 and AKT activation may be an important contributor for HPV16 E6/E7 mediated radiosensitivity.

Figure 5.  Activation of ERK1/2 and AKT phosphorylation by HPV16 E6/E7. Western blot was performed to assess the phosphorylation of extracellular signal-regulated kinases 1/2(ERK1/2) and AKT signaling in H2179 cells after transfection with with p3XFLAG-HPV16 E6 or pcDNA3FLAG-HPV16 E7 expression plasmid and subjected to Western blot assay 15 min following irradiation at 3 Gy. A set of representative data was shown.

The HPV16/18 oncoproteins E6 and E7 are crucial for high-risk HPV-induced malignant cell transformation, such as cervical cancer. Furthermore, these oncoproteins have been proven to affect development, metastasis, chemosensitivity and radiosensitivity of human cervical cancer through different pathways^[15-17]. In this study, we showed that HPV16 oncoproteins E6 and E7 significantly decreased ROS induction and increases the secretion of VEGF in H2179 cells in a time-dependent manner. This effect is mediated by the activation of the ERK1/2 and AKT signaling pathways. Furthermore, both of HPV16 E6 and E7 caused radioresistance of lung cancer cells via the ERK1/2 and PI3K/AKT pathways. These data suggested that HPV16 E6 and E7 have the potential to induce VEGF expression and decrease susceptibility of lung cancer to radiotherapy.

It has been known that induction of intracellular ROS plays an important role for apoptosis caused by radiation^[18-19]. We found that HPV16 E6 and E7 along with radiation caused an elevation in the levels of intracellular ROS, which might indicate that the HPV16 E6 and E7 promotes the formation of ROS in lung cancer cells, thereby enhancing cell radiosensitivity.

VEGF is the most potent proangiogenic factor, and also contributes as important mediator to radiosensitivity of lung cancer cells^[20]. As a pool of VEGF, lung cells produce and secrete VEGF in response to many stimuli, including radiation^[21]. We found that the HPV16 oncoproteins E6 and E7 significantly upregulated VEGF expression in H2179 cells, and they induced VEGF expression possibly via ERK1/2 and AKT signaling, the VEGF increase last 48 h although an obvious elevation at 24 h after irradiation, this late response of VEGF expression to radiation may be that protein synthesis takes some time due to the complicated and time-consuming processes, which include DNA transcription, mRNA translation, and post-translational modification protein trafficking and secretion. The PI3K/AKT as an important signaling molecule is frequently activated in radioresistance and associated with cancer cell survival, including lung cancer^[22-23]. Consistent with these previous studies, we found that the PI3K/AKT pathway also mediated the survival of lung cancer cells as well as VEGF generation after exposure to radiation, which may be required for radioresistance mediated by the HPV16 oncoproteins E6 and E7. These data suggest that the HPV16 oncoproteins E6 and E7 may exert various roles dominantly mediated by different signaling pathways, at least including activation of ERK1/2 and PI3K/AKT pathway as well as VEGF signaling.

In summary, our present study, for the first time, show that the radiosensitivity of lung cancer cells may be modulated by the HPV16 oncoproteins E6 and E7. The mechanism involved the radioresistance might be related to ROS formation, promotion of ERK1/2 and PI3K/AKT signaling pathways and VEGF expression. These findings provide new insight into an important role of the HPV16 oncoproteins E6 and E7 as potential radiosensitivity mediators in lung cancer, excluding their roles as modifiable risk factors in lung carcinogenesis.