RETRACTED ARTICLE: IL-6 signaling promotes DNA repair and prevents apoptosis in CD133+ stem-like cells of lung cancer after radiation

A549 and H157 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 containing 10 % FBS. CD133+ CSCs were cultured in DMEM/F12 medium supplemented with 20 ng/ml EGF (Invitrogen), and 20 ng/ml FGF (Invitrogen). All cells were maintained in a humidified 5 % CO₂ environment at 37 °C.

A549 and H157 cell lines were irradiated with 6 Gy Cs-137 gamma rays and then grown as a monolayer culture for 7 days. After 7 days, the percentage of CD133+ population (CD133+ positively stained cells) in the culture was determined by flow cytometric analysis using the Canto II system (Becton-Dickinson, San Antonio, TX). The non-irradiated cells were used as control.

For incorporation of IL-6 siRNA or scramble (sc) control plasmids into A549 and H157 cells, lentivirus construct carrying either sc or IL-6 siRNA (pLenti-II vector, Applied Biological Materials Inc, Canada) was transfected into 293 T cells with a mixture of pLent-II-IL-6 siRNA, psPAX2 (virus-packaging plasmid), and pMD₂G (envelope plasmid) (4:3:2 ratio) using PolyFect Transfection reagent (Qiagen, Valencia, CA). After A549 and H157 cells were infected with virus overnight, the culture media containing the virus were removed and the infected cells were then maintained under normal cell culture media. After sub-culturing, the IL-6 knocked down cells were selected by puromycin (2 μg/ml) (Sigma) and then maintained in media containing 0.1 μg/ml puromycin.

Cells (2 × 10⁷) were detached from tissue culture plates with 5 mM EDTA, centrifuged, and incubated with magnetic microbeads conjugated with anti-CD133 antibody (Miltenyi Biotec, Cambridge, MA). The bead-bound cells (CD133+) and unbound cells (CD133-) were separated using the Quadro MACS^TM Separation Unit (Miltenyi Biotec, Cambridge, MA). The purity of isolated CD133+ cells was confirmed by flow cytometric analyses, and by qPCR analyses. The isolated CD133+ cells were then cultured in stem cell media.

For sphere formation assays, single-cell suspensions (1 × 10³ cells) were mixed with cold Matrigel (BD, Franklin Lakes) (1:1 ratio, v/v, total volume of 100 μl)) and the mixture was placed along the rim of the 24-well plates. The culture plates were placed in 37 °C incubator for 10 min to let the mixture solidify, and 500 μl medium was then added into the wells. The number of spheres with diameter greater than 50 μm was counted 7–14 days later using an Olympus light microscope. A minimum of three triplicate experiments were performed.

Cells were exposed to different doses (0, 1, 2, 4, 6, and 8 Gy) of radiation using a Cs-137 source with a dose rate of 180–205 cGy/min. After treatment, clonogenic assay was performed as previously described [14]. Cells were seeded in culture dishes with appropriate dilutions to form colonies after 7–9 days incubation. Colonies were fixed with methanol, stained with crystal violet (0.5 % w/v), and counted using a microscope. Colonies consisting of at least 50 cells were counted and the surviving fraction was calculated from the normalized plating efficiency.

Total RNAs were isolated using Trizol reagent (Invitrogen). One μg of total RNA was subjected to reverse transcription using Superscript III transcriptase (Invitrogen). qPCR was conducted using the appropriate primers and the Bio-Rad CFX96 system. SYBR green was used to determine the expression levels of mRNA from genes of interest. Expression levels were normalized to GAPDH level.

IL-6 in the supernatant of unseparated parental or isolated CD133- and CD133+ cells of A549IL-6si/sc and H157IL-6si/sc pairs was determined by the ELISA kit according to the manufacturer’s instructions (BD, Franklin Lakes). The secreted IL-6 level was normalized by cell number.

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF) and proteins (20–40 μg) were isolated and separated on 8–10 % SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies, followed by HRP-conjugated secondary antibodies, and then the proteins of interest were visualized using the Imager (Bio-Rad) and ECL (Thermo Fisher Scientific, Rochester, NY) system. Antibody of GAPDH was purchased from Abcam (Cambridge, MA) and antibodies of Mcl-1and cleaved caspase-3 were obtained from Cell Signaling (Danvers, MA). Bcl-2 antibody was obtained from Santa Cruz (Santa Cruz, CA).

Unseparated parental or isolated CD133- and CD133+ cells of A549IL-6si/sc and H157IL-6si/sc pairs (1 x 10³) were mounted on chamber slide, irradiated (6 Gy, with non-irradiated cells as control), and stained with appropriate primary antibodies. Antibodies of ATM and CHK2 were obtained from Bethyl Laboratory (Montgomery, TX), phosphorylated ATM (Ser 1981), and phosphorylated p53 (Ser 20) were from Gene Tex (Irvine CA), and γ-H2AX antibody was purchased from Trevigen (Gaithersburg, MD). Antibodies of Mcl-1 and Bcl-2 were obtained from Cell Signaling (Danvers, MA) and Santa Cruz (Santa Cruz, CA), respectively. After reaction with Alexa flour® 488 anti-goat secondary antibody (Life Technologies, Grand Island, NY), images were recorded using a fluorescent microscope (Zeiss, Germany).

Isolated CD133+ and CD133- cells of IL-6si/sc pairs were irradiated (6 Gy) and at 0 and 30 min after radiation cells were used in the assay following the procedure of Singh et al. [15] with some modifications. Briefly, cells were embedded in low-melting-point agarose in lysis buffer (10 mM Tris–HCl, pH 10, 2.5 M NaCl, 100 mM EDTA, 10 % DMSO, 1 % Triton X-100). The unwinding step was performed for 20 min at 4 °C in unwinding/electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH = 13). Electrophoresis was performed at 4 °C for 20 min in unwinding/electrophoresis buffer at electric field strength of 25 V and a current of 300 mA. The slides were then neutralized with a neutralizing buffer (0.4 Tris–HCl, pH 7.5) for 20 min, rinsed with distilled water, air-dried, stained with 30 μg/ml ethidium bromide. Images were recorded using a fluorescent microscope (Zeiss, Germany).

The data were presented as the mean ± SEM. Differences in mean values between two groups were analyzed by two-tailed Student’s t test. p ≤ 0.05 was considered statistically significant.

To investigate whether the population of CSCs in NSCLC was altered after radiation, unseparated A549 and H157 parental cells were irradiated with 6 Gy gamma rays and the percentage of CD133+ cells, before and after radiation, was analyzed by flow cytometry. The CD133 molecule was chosen because it is the most widely used surface marker for the CSC of NSCLC [4, 6]. As shown in Fig. 1a, the percentage of CD133+ cells was increased by 3.4 and 4.0 fold at 7 days after irradiation for A549 and H157 cells, respectively. Consistent with the CD133+ population data (Fig. 1a) and the published result by others [9], we also observed a higher number of spheres in the sphere formation assay (Fig. 1b) and expression of CSC markers in NSCLC cell lines following irradiation in IF staining (Fig. 1c).

Emerging evidence suggests that CSCs are likely to be more resistant to radiation than non-CSCs [7‐10, 16]. To directly compare the radiation sensitivity of CSC and non-CSC cells in NSCLC, we isolated CD133+ and CD133- cells from A549 and H157 cells by anti-CD133 antibody and the immunomagnetic separation method. Flow cytometric analyses indicated an enrichment of CD133+ cells after separation with a purity of CD133+ greater than 90 % (Fig. 2a). In order to confirm the CSC-like characteristics of the isolated CD133+ cells, we analyzed expression of CSC markers in these cells and in parental cells. The results showed a dramatic increase in CSC markers in CD133+ cells (Fig. 2b, A549 cells). The CD133+ cells were able to grow into spheres using the Matrigel-based sphere formation assay (Fig. 2c), further indicating the isolated CD133+ cells have CSC-like features. The CD133+ cells were maintained under non-adherent culture conditions with stem cell media, and the freshly isolated cells and once passaged cells were used in the entire study.

Radiation sensitivity was determined and compared between the isolated CD133+ and CD133- cells. CD133+ and CD133- cells were isolated by an immunomagnetic separation method one day prior to irradiation experiments and the CD133+ cells were kept in stem cell media. After irradiation to different doses of Cs-137 gamma rays, cells were harvested, trypsinized (in case of CD133- cells), cell numbers counted, and single cell suspensions of indicated numbers of CD133- and CD133+ cells were used in clonogenic assay at 7–9 days post-radiation. We found survival of CD133+ cells was significantly higher than that of CD133- cells in both cell lines (Fig. 2d; top, A549 cells; bottom, H157 cells), indicating that CD133+ cells are more radioresistant than CD133- cells.

In investigating the role of IL-6 in the radiation sensitivity of CSCs, the A549 and H157 cells secreted a high level of IL-6, so it was hard to observe the exogenously added IL-6 effect on survival of CD133+ cells after radiation. So, we investigated the radiation survival of CD133+ CSC-like cells obtained from parental cells whose intracellular IL-6 level was manipulated by the IL-6 siRNA in lentiviral transduction system. We have previously obtained IL-6 knocked down A549 (A549IL-6si) and H157 (H157IL-6si) cells and their corresponding scramble (sc) control (A549sc, H157sc) cells. The IL-6 knockdown efficiency was greater than 90 % (A549) and 72 % (H157) in qPCR analyses (Fig. 3a). The IL-6 knockdown in CD133- and CD133+ cells are shown in ELISA test (Fig. 3b) and IF staining (CD133+ cells, Fig. 3c). CD133+ cells were then isolated from these A549IL-6/sc and H157IL-6si/sc pairs and exposed to various doses of radiation for clonogenic cell survival assay. After exposure to radiation, we found that the survival of CD133+ cells isolated from IL-6 expressing A549sc and H157sc cells was higher than from A549IL-6si and H157IL-6si cells (Fig. 3d), suggesting that intracellular IL-6 is important for maintaining a greater level of radiation survival in CD133+ cells.

We next tested whether IL-6 was also involved in promoting the self-renewal of CD133+ cells after radiation. We irradiated the CD133+ cells isolated from A549IL-6si/sc and H157IL-6si/sc pairs and performed the sphere formation assay at 7 days after irradiation to test their self-renewal abilities. As shown in Fig. 3e, we observed a higher number of spheres from A549sc and H157sc cells than those from A549IL-6si and H157IL-6si cells. Consistent with the spheroid formation results, a higher number of Ki67 positive cells was found in CD133+ of A549sc cells following radiation exposure (Fig. 3f), indicating higher proliferating activity of IL-6 expressing CD133+ cells.The secreted IL-6 level differences in CD133+ and CD133- cells of A549IL-6si/sc and H157IL-6si/sc pairs are shown in Fig. 3g.

We then investigated whether DNA damage and repair system in CD133+ cells was affected by IL-6 signaling following irradiation. We irradiated CD133+ cells isolated from A549IL-6si/sc and H157IL-6si/sc pairs and performed the IF staining of γ-H2AX after irradiation. As shown in Fig. 4a, we detected higher levels of γ-H2AX staining in A549IL-6si and H157IL-6si cells, indicating greater amount of DNA damage still left unrepaired when IL-6 was knocked down in CD133+ cells. Different levels of post-irradiation DNA strand breaks in CD133- and CD133+ cells of A549IL-6si/sc and H157IL-6si/sc pairs were also investigated in the Comet assay. When CD133- and CD133+ cells were isolated from A549IL-6si/sc and H157IL-6si/sc pairs and irradiated at 6 Gy, we detected a similar extent of DNA damage in the CD133- and CD133+ cells of IL-6si/sc pair immediately after radiation, but lower DNA breaks in CD133+ cells of IL-6sc cell lines than those of IL-6si cell lines were detected at 30 min after radiation (Fig. 4b), suggesting higher DNA repair in CD133+ cells of IL-6sc cell lines than those of IL-6si cell lines. However, we did not observe a significant difference in DNA repair in CD133- cells of A549IL-6si/sc and H157IL-6si/sc pairs.

We then investigated whether the higher level of unrepaired DNA damage in IL-6 knocked down cells was due to lower DNA repair activity. We investigated expression of ATM [17] and its downstream molecules, including ATM, phosphorylated ATM, CHK2, and phosphorylated p53, in CD133- and CD133+ cells of A549IL-6si/sc and H157IL-6si/sc cell pairs upon radiation. The results of IF staining showed that the CD133+ cells from A549IL-6si and H157IL-6si cell lines exhibited lower expression of these molecules than those isolated from sc cell lines (Fig. 4c). However, not much difference was observed in CD133- cells, whether isolated from IL-6si or sc cell lines.

To further explore whether the ATM and CHK2 are up-regulated in IL-6 expression CD133+ cells after radiation, we compared the mRNA levels of ATM and CHK2 in A549IL-6si/sc and H157IL-6si/sc pairs following radiation exposure. Similar to the IF staining results, higher levels of ATM mRNA were observed in the IL-6 expressing and CD133+ sc cells compared to those of IL-6 knocked down cells (Fig. 4d).

Next, we investigated whether IL-6 regulates ATM at the transcriptional level in the 293HEK cell line using the ATM-luciferase constructs containing the ATM promoter region. We also used non-IL-6 expressing H1299 NSCLC cell line in this assay to observe the exogenously added IL-6 effect. We detected a dose dependent regulation of ATM-luciferase by IL-6 in these two cell lines, suggesting direct regulation of IL-6 on ATM molecule at the transcriptional level (Fig. 4e).

We then investigated whether difference in expression of apoptosis markers could be detected between IL-6 expressing (sc) and knocked down (IL-6si) CD133+ cells. As shown in Fig. 5a, we observed a higher level of cleaved caspase in A549IL-6si cells than A549sc cells, indicating more apoptosis in IL-6 knocked down cells following radiation. When we examined expression of anti-apoptotic markers Bcl-2 and Mcl-1 in the A549IL-6si/sc cell line pair, higher expression of these molecules in IL-6 expressing CD133+ cells were observed in Western blot (Fig. 5a) and IF staining (Fig. 5b) analyses. These results suggest that IL-6 in CD133+ cells modulated up-regulations of these molecules, and protected cells from apoptotic death upon irradiation.

In summary, [1] NSCLC CD133+ CSC-like cells were enriched upon radiation, [2] cell survival of NSCLC CD133+ cells after radiation was higher than that of CD133- cells, [3] cell survival of IL-6 expressing NCSLC CD133+ cells (sc) was higher than that of IL-6 knocked-down cells (IL-6si) after radiation [4] IL-6 played a role in protecting NSCLC CD133+ cells from radiation-induced DNA damage and apoptosis.