Research ArticleExperimental Studies

Enhancement of Radiation-induced DNA Damage and Inhibition of Its Repair by a Novel Camptothecin Analog

GE HUANG, HUIJUAN WANG and LI-XI YANG
Anticancer Research March 2010, 30 (3) 937-944;

Abstract

A novel camptothecin derivative (TLC388) with higher efficacy and reduced toxicity has been synthesized and tested as a novel chemoradiosensitizing agent. This study investigated the mechanisms of the chemoradiosensitizing effects of TLC388 on H23 human non-small cell lung cancer (NSCLC) cells. Using the TUNEL assay, a significantly higher percentage of apoptotic cells was observed in the group treated with TLC388 plus X-ray radiation than those in groups treated with drug or radiation alone. The sensitizer enhancement ratio (SER) was 1.91. Apoptosis increased with drug concentration and radiation dose, exhibiting dose-dependent pattern. The results suggested that apoptosis could be a main mode of cell death that might underlie the increased chemoradio-sensitization of TLC388. Treatment with 30 nM of TLC388 plus 4 Gy X-ray also produced up to 42% of necrotic cells that were measured by trypan blue exclusion assay, but with TLC388 alone or 4 Gy radiation alone 9.8% or 11.1% necrotic cells were detected, respectively. An immunofluorescent staining method was employed to determine the levels of γ-H2AX (phosphorylated H2AX, a variant of the H2A protein family, which is a component of the histone octomer in nucleosomes and is phosphorylated by kinases like ATM and ATR in the PI3K pathway, as the first step in recruiting and localizing DNA repair proteins) as a molecular biomarker of DNA double strand breaks (DSBs) in cells treated with TLC388 ±radiation, or radiation alone. The formation of γ-H2AX foci was observed after TLC388 or radiation exposure and when the cells were treated with 30 nM TLC388 plus radiation at a dose of 2 Gy, the percentage of cells containing γ-H2AX foci increased significantly. Even more interesting, a markedly higher percentage (65.4%) of mitotic cells displayed γ-H2AX foci after treatment with 30 nM TLC388 plus 0.5 Gy radiation, compared to only 5.9% or 26.1% of the M-phase cells treated with 30 nM TLC388 alone or 0.5 Gy radiation alone, respectively. It is suggested that mitotic cells become very sensitive to the production of DSBs after TLC388-radiation combined treatment and the formation of DSBs is strongly suggested to lead to the induction of apoptosis at doses lower than 4 Gy and to some necrosis at doses of 4 Gy or above. TLC388 enhances the production of DSBs and inhibits their repair, which contributes to the elucidation of the mechanisms of chemoradiosensitization of TLC388 and its development as a novel chemoradiosensitizing drug for improved radiotherapy.

Camptothecin (CPT) derivatives (topotecan and irinotecan [CPT11]) are active anticancer drugs developed in the 1990's (1-4). Many other CPT derivative compounds have been synthesized and are undergoing preclinical and clinical trials. CPT has been long regarded as a topoisomerase I (Topo I) inhibitor leading to an increase of DNA-Topo I complex. Some of the derivatives have been proved to radiosensitize cancer cells in vitro and in vivo. For example, topotecan could enhance the cytotoxic effects of ionizing radiation on glioblastoma cells (GBM) which express a low level of topoisomerase I (Topo I) and are resistant to topotecan alone (5). The active metabolite of CPT-11 (SN-38: 7-ethyl-10-hydroxycamptothecin) at 2.5 μg/ml greatly increases the lethal effects of radiation on HT-29 (human colon adenocarcinoma cells) spheroids (multicellular tumor spheroids with several biologic characteristic in common with in vivo tumors). The radiosensitizing effects are dependent upon the drug dose, schedule, timing, cell line type, and cell cycle phase (6). Moreover, CPT-11 has been tested in clinical trials as a chemoradiosensitizer (7-9). The preliminary results have shown that CPT-11 improved local control and disease-free survival when combined with radiotherapy. The combination of CPT-11 with radiotherapy is feasible and yields a high complete response rate. However, the toxicity of all CPT derivatives in normal tissues remains very high (10, 11). There exists a great need for the development of lessr toxic and more potent CPT derivatives as potential chemoradiosensitizing agents for improved radiotherapy.

It is generally believed that the intact lactone E ring of CPT molecule is most critical for antitumor activity. The opening of the lactone E ring is the cause for reduced potency and higher toxic side-effects to normal tissues. The intrinsic instability of the lactone E ring is a common feature of all existing CPT derivatives which forms an inactive ring-opened carboxylate (12). To overcome this problem and develop a second-generation CPT derivative with higher efficacy and reduced toxicity, a novel compound (TLC388) has been synthesized in our laboratory (unpublished data). Many preclinical studies have shown that this newly synthesized camptothecin derivative is remarkably effective in the chemoradiosensitization of cancer cells. TLC388 is significantly less toxic to normal animals than topotecan. In this study, the chemoradiosensitizing effects of TLC388 were evaluated in H23 human non-small cell lung cancer (NSCLC) cells and the possible mechanisms of its action were investigated.

Materials and Methods

Cell cultures and treatment. H23 human non-small cell lung cancer cells were obtained from the NCI (National Cancer Institute). The cells were cultured in modified McCoy's supplemented with 10% fetal bovine serum, streptomycin sulfate (100 μg/ml) and penicillin (100 U/ml). The incubations were conducted at 37°C in an atmosphere of 5% CO2 and 95% air. The cells were plated at a density of 1×105 per dish (60 mm). After 5 h incubation, the cells were treated with various concentrations of TLC388 for 24 h. To evaluate the effect of a combination of TLC388 and X-ray radiation, the cells were first treated with TLC388 for 24 h, and then exposed to different doses of radiation.

Reagents. Mouse monoclonal anti-β tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and FITC (Fluorescein isothiocyanate) conjugated anti-mouse IgG and rhodamine conjugated anti-rabbit IgG from Biomeda Corp (Foster City, CA, USA). The in situ cell death detection kit, AP (alkaline phosphotase) for the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) assay kits was purchased from Roche Diagnostics Corporation (Indianapolis, IN, USA). TLC388 was synthesized by TLC Biopharmaceuticals Inc (South San Francisco, CA, USA). Stock solutions of the drug were prepared in DMSO and then diluted in sterile distilled water before use.

Irradiation. Cells were plated on poly-lysine coated glass coverslips at a density of 1.3×104/cm2. The cells were allowed to attach for 24 h and then irradiated with doses of 2-8 Gy at room temperature using a Varian CL21-EX accelerator (Varian Medical Systems, Palo Alto, CA) (dose rate: 4 Gy/min).

Topoisomerase I relaxation assay. Topo I inhibitory activity of TLC388 was assayed by supercoiled DNA relaxation assay (13). Supercoiled pBR322 (the first artificial plasmid, created in 1977, it was named eponymously after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez) DNA (TopoGEN, FL, USA) was incubated at 37°C for 30 min with purified human Topo I (TopoGEN, FL, USA) which was pre-mixed with TLC388 or topotecan (as control) each at 10, 40, or 80 μM, then terminated with proteinase K/SDS stop buffer, and loaded on to 1% agarose gel. The standard reaction system contained: 0.25 μg DNA, 10 mM Tris-HCl, pH 7.9, 1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin, 0.1 mM spermidine, and 1 U Topo I. The samples were electrophoresed in a horizontal 1% agarose gel in TAE (tris base, acetic acid and EDTA) buffer at 1.5 V/cm overnight at 4°C. DMSO concentrations in each reaction were maintained at 1.5% by the addition of serially diluted drug stocks so as not to produce solvent mediated inhibition of Topo I activity. The gels were stained with ethidium bromide, destained in water and photographed under UV illumination. The relative inhibition rate was measured by microdensitometry of the supercoiled DNA band using the formula: (S−S0)(Scontrol−S0)×100(%) Where Scontrol is the percentage of supercoiled DNA in the control (without enzyme and test compounds), S0 is the percentage of supercoiled DNA in the sample with enzyme without test compounds, and S the percentage of supercoiled DNA in the sample with enzyme and test compounds.

TUNEL assay. To determine whether TLC388 ±radiotherapy could induce apoptosis in H23 cells, the TUNEL assay was employed to detect apoptotic cells. H23 cells were treated with or without TLC388 for 24 h, irradiated or not and then incubated for 5 days. After the incubation, both detached cells and attached cells were collected and resuspended in serum-free medium. Five microliters of the cell suspension were dropped onto a Colorfrost* Plus microscope slide (Fisher Scientific). The cells were dried in air and fixed with a freshly prepared paraformaldehyde solution (4% in PBS, pH 7.4) for 1 h at room temperature. Slides were rinsed with 3× PBS for 5 min and incubated with 1× PBS for 5 min. The PBS was discarded and fresh PBS was added to the slides. The slides were incubated at 4°C for 5 more min, and then dehydrated through a series of 5 min incubations in 50% ethanol, 70% ethanol, 95% ethanol, and then 100% ethanol. The slides were completely dried in air and stored at −70°C. The slides were rinsed twice with 0.1 M PBS at room temperature for 15 min each time and incubated in permeabilization solution (0.1% triton X-100, 0.1% sodium citrate) for 4 min at 4°C and then treated with the reagents included in an in situ cell death detection kit (Roche Diagnostics Corporation, Indianapolis, IN, USA) following all detailed procedures provided with the kit. Positive apoptotic cells were counted by eye with blind assessment under a light microscope. The apoptotic index was calculated as the percentage of apoptotic positive cells in the total of 200 counted cells. The experiments were repeated three times. Sensitizer enhancement ratio (SER) = slope of the curve of TLC388 with X-rays/slope of the curve of TLC388 alone or X-rays alone.

Cell viability assay. Cells treated with or without TLC388were collected at different incubation time-points with or without X-rays after irradiation and resuspended in 0.9 ml of PBS. To the cell suspension (0.9 ml), 0.1 ml of 0.4% trypan blue in PBS was added and mixed. The number of blue stained cells among 500 cells was counted using a hemocytometer, within 4 min. The percentage of stained blue cells/total cells was calculated. The experiments were repeated three times.

Figure 1.
Figure 1.

Inhibitory effects of TLC388 on activity of topoisomerase I, investigated by relaxation DNA assay in vitro. The relative topoisomerase I inhibition rate was calculated from microdensitometry measurements of the supercoiled DNA band.

Immunofluorescent staining. To determine whether TLC388 induced an increase in X-ray–mediated DNA damage, double-strand breaks (DSBs) were monitored using histone γ-H2AX as a marker (14). Histone H2AX, one of several variants of the nucleosome core histone H2A, undergoes phosphorylation on Ser-139 in response to the induction of DSBs. The phosphorylation affects the H2AX molecules that flank the DSBs in the chromatin and covers a domain of ~1 Mb. After the induction of DSBs, the Ser-139-phosphorylated H2AX, defined as γ-H2AX, can be detected immunocytochemically in the form of discrete nuclear foci. Each focus is presumed to represent a single DSB (15). Cells grown on coverslips were fixed with 100% methanol at −20°C for 10 min, then washed in PBS 3×5 min. The cells were blocked with PBS contain 10% bovine serum (Hyclone, Logan, UT, USA) for 1 h at 37°C, then incubated with the primary γ-H2AX antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:400 in PBS overnight at 4°C. The cells were washed with PBS for 5 min twice and incubated in the dark for 2 h at room temperature with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody at 1:100 (Biomeda, Foster City, CA, USA). The secondary antibody solution was then aspirated, and the cells were washed three times in PBS for 5 min and then were incubated in the dark with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml) in PBS for 5 min (14). The coverslips were mounted and the cells viewed with a Nikon fluorescence microscope. The γ-H2AX foci were counted by eye in at least 300 cells from the stored images with blind assessment.

Statistical analysis. All the experiments were performed in triplicates. Statistical significance was determined by using the Student's t-test, ANOVA or Chi-square test. Differences were deemed significant if the calculated p-value was <0.05 or 0.01.

Results

Topoisomerase inhibitory activity of TLC388. Strong inhibitory activity of TLC388 against Topo I was detected, in a dose-dependent manner, as illustrated in Figure 1. Compared to topotecan as a positive control, TLC388 proved to be about 1.8-fold, 2.3-fold and 2.1-fold more effective in inhibition of Topo I at concentrations of 10 μM, 40 μM and 160 μM, respectively.

Figure 2.
Figure 2.

Induction of apoptosis and necrosis in H23 cells treated with TLC388 and/or radiation. A and B: Apoptosis assessed by TUNEL assay; C: induction of necrotic cells assessed by trypan blue.

Induction of apoptosis and necrosis by TLC388 plus X-ray. Treatment with TLC388 alone produced apoptotic cells (Figure 2B) and when combined with radiation, TLC388 significantly induced apoptosis (p<0.01). The sensitizer enhancement ratio (SER) was 1.91. The percentage of apoptotic cells increased with radiation dose (Figure 2A) or drug dose (Figure 2B), showing dose-dependent patterns.

To test the possibility that necrosis might be elicited by treatment with this novel CPT analog ±radiation, the trypan blue staining assay was employed. The results showed that 30 nM TLC388 produced necrotic cells. Radiation alone also generated a small percentage of necrotic cells. The combination of the drug with radiation produced a significant amount of necrotic cells, since the dead cells stained with trypan blue were mainly necrotic cells (Figure 2C). The SER value was 4.85 for TLC388.

Figure 3.
Figure 3.

H23 cells γ-H2AX foci formation in response to TLC388 plus X-ray treatment. A: row 1, Control; row 2, TLC388; row 3, X-ray; row 4, TLC388 plus X-ray. Yellow cycled cells in Merge column are γ-H2AX positive cells. B: Dependence of TLC388 radiosensitization on X-ray dose (**p<0.01). C: Inhibition of DSB repair by TLC388. γ-H2AX foci in the two treatment groups (p<0.01, Chi-square test) during the period of 1-5 h after treatment.

γ-H2AX foci formation in response to TLC388 plus X-ray treatment. As shown by the micrographs in Figure 3, in the absence of DNA DSBs, the control H23 cells (neither TLC388 nor X-ray treated) had a small number of dim nuclear γ-H2AX foci (1.6%, 3A, Row 1). Treatment with 30 nM of TLC388 alone had a slight effect on the γ-H2AX foci, 24 h after TLC388 treatment, 2.9% of the cells showed nuclear γ-H2AX foci staining (3A, Row 2). Typical nuclear γ-H2AX foci (3A, Row 3) were observed 2 h after 2 Gy X-ray radiation, and 20% of the cells contained γ-H2AX foci. When the H23 cells were treated with 30 nM of TLC388 for 24 h and were then exposed to 2 Gy X-rays, γ-H2AX foci could be clearly distinguished 2 h after irradiation. The number of foci per cell were increased, the foci was enlarged, the fluorescence intensity of the foci was enhanced and neighboring foci joined together to form big, bright pan-foci (3A, Row 4). The percentage of cells containing γ-H2AX foci was dramatically increased to 57.9%, (p<0.01, Chi-square test) for the TLC388 plus X-ray treatment compared with TLC388 alone or radiation alone.

Time effects of TLC388 plus radiation on γ-H2AX foci. As shown in Figure 3C, when the H23 cells were exposed to X-ray alone, 24% of cells showed nuclear γ-H2AX staining at 1 h after radiation. The number of γ-H2AX-positive cells decreased to 20%, 15.4%, 9.1% and 1.3% at 2 h, 5 h, 24 h and 72 h after radiation, respectively. When the H23 cells were treated with 30 nM of TLC388 for 24 h and were then exposed to 2 Gy of X-ray, approximately 32.4% of the cells showed nuclear γ-H2AX staining 1 h after radiation. Surprisingly the percentage of γ-H2AX-positive cells with the combination treatment continued to increase until it reached a peak of 57.9% 2 h after radiation (Figure 3C). Even 5 h after radiation, the level of γ-H2AX foci was still 41.4% signifying that only 28.5% of the DNA damaged cells had been repaired 5 h after the combination treatment, whereas more than 35.8% of the DNA damaged cells had been repaired with only radiation treatment. Compared to the cells with only radiation treatment, the combination treated cells had a significantly higher level of γ-H2AX foci (p<0.01) during the period 1-5 h after radiation. Seventy-two hours later, the percentage of γ-H2AX-positive cells decreased to 4% in the combination treated cells, which still showed a higher level than that in the radiation alone cells.

Figure 4.
Figure 4.

Radiosensitization of M-phase cells by TLC388. A: γ-H2AX foci shown by fluorescence microscopy. B: Quantification of γ-H2AX incidence dose (**p<0.01). C: Cellular location of γ-H2AX foci.

Chemoradiosensitization of TLC388 with various X-ray doses. In clinical radiotherapy, repeated small doses are better tolerated by normal tissues than single large doses so that tumors are typically exposed to 2 Gy daily doses. To test the chemoradiosensitization of TLC388 with low doses of X-ray radiation in vitro, the H23 cells were treated with TLC388 at 30 nM for 24 h, and then exposed to 0.5 Gy, 1 Gy or 2 Gy X-ray radiation. Compared with X-ray treatment alone, the TLC388 treatment significantly increased the level of 2 Gy induced γ-H2AX foci in interphase cells (Figure 3B), but, it did not show an effect on the X-ray-mediated γ-H2AX foci when the radiation dose was lower than 2 Gy. These results suggested that radiosensitization by TC388 in H23 cells would be X-ray-dose dependent. and was most effective when the cancer cells were treated with TLC388 combined with clinically relevant 2 Gy of X-ray irradiation.

TLC388 chemoradiosensitization of M-phase cells. Using the spindle as a marker, γ-H2AX foci were hardly observed in the M-phase cells which were unirradiated (Figure 4A, Row 1) or treated by TLC388 alone (5.9%) (Figure 4A, Row 2). After exposure to 0.5 Gy of X-ray, chromosomally located γ-H2AX foci appeared in 26.1% of the M-phase cells (Figure 4A, Row 3). The percentage of γ-H2AX-positive M-phase cells rapidly increased to 65.4% after 30 nM of TLC388 was combined with 0.5 Gy of X-ray treatment (Figure 4A, Row 4). The percentage of M-phase cells with γ-H2AX foci was significantly higher after the combined treatment than in the cells treated with TLC388 alone or X-ray alone (p<0.01, Chi-square test) (Figure 4B). The intensely stained γ-H2AX foci on chromosomes were mostly localized on the metaphase plate (Figure 4A, Row 4). The fluorescence was so strong that it was very easy to distinguish DNA damaged cells from normal M-phase cells. Surprisingly, some discrete γ-H2AX foci were dispersed in the cytoplasm, but this only occurred in the M-phase cells with the combination treatment. This indicated that the DSBs caused a detachment of damaged DNA segments from the spindle fibers (Figure 4C, top). When the γ-H2AX foci were seen in telophase cells, they were seen in both nuclei of a dividing cell (Figure 4C, center). The multi-polar spindle was another common phenotype in the M-phase cells with combination treatment (Figure 4C, bottom).

Discussion

It is widely accepted that DNA is the major target of ionizing radiation. Radiation-induced DNA damage includes base damage, sugar damage, single-strand breaks (SSBs) and DSBs (16-19), of which radiation-induced DSBs are lethal to cells. The moderate increase of radiation-induced DSBs has been demonstrated as one of the mechanisms of radiosensitizing effects of topotecan (20-24). For topotecan combined with radiotherapy, it has been hypothesized that the interaction between the SSBs generated by inhibition of Topo I and radiation-induced SSBs has been hypothesized to lead to the formation of DSBs. In this study, using γ-H2AX as a molecular biomarker of DSBs, the combination of TLC388 with radiation remarkably induced DSBs in the H23 lung cancer cells and implied that radiosensitization with TLC388 could be due to the enhancement of X-ray-induced DSBs. This was consistent with the apoptosis and necrosis data. Apoptosis could be the main mechanism responsible for the remarkable chemoradiosensitization of TLC388 and TLC388 plus radiation could cause necrosis which would be another cell death pathway for killing cancer cells at dose of 4 Gy. Apparently, the enhancement of DSBs would underlie the mechanism involved and might be ascribed to a unique chemical property of TLC388. It would be very interesting to explore the detailed mechanisms of the induction of DSBs with or without radiation in future studies.

Lamond et al. reported that cells were not able to carry out potentially lethal damage repair (PLDR) after topotecan treatment and suggested that CPT-11 derivatives such as topotecan inhibited PLDR by the inhibition of Topo I (25, 26). The Topo I inhibition data suggested that TLC388 might be more effective than topotecan. It is generally accepted that the molecular basis of PLDR is the repair of DSBs (27, 28), thus.the radiosensitizing effects of topotecan might result from alteration of the repair of DSBs. Analysis of the kinetics of γ-H2AX clearance after radiation reveals the persistence of unrepaired DNA damage. Recent genetic and biochemical studies suggested that γ-H2AX plays an important role in the recruitment and/or retention of DNA repair and checkpoint proteins. After DSBs are rejoined and chromatin is repackaged, γ-H2AX is dephosphorylated and cell proliferation resumes. However, residual foci can be detected a long time after irradiation and these foci may represent sites of misrepair or incomplete repair (29, 30). In the present study, the repair of DSBs was significantly slower in the group treated with TLC388 plus X-ray radiation than in the group treated with radiation alone, indicating that TLC388 might indeed act as an inhibitor of DSB repair. The inhibitory effects of TLC388 on DSB repair could constitute one of the molecular bases for its strong chemoradiosensitization of H23 NSCLC cells. Many molecular studies will be required to address exactly how TLC388 inhibits the repair process of radiation-induced DSBs and the elucidation of this mechanism would significantly contribute to the preclinical/clinical development of TLC388 as a useful chemoradiosensitizing agent.

CPT derivatives selectively target Topo I by trapping the catalytic intermediate of the Topo I-DNA reaction (the cleavage complex) (1). The up-regulated levels of Topo I expression in cancer cells provide the molecular basis for selective targeting in cancer therapy (31-33). TLC388 has proved to be a potent Topo I inhibitor. The development of improved CPT derivatives for treating cancer with high levels of Topo I holds great promise. In cells that express a low level of Topo I and might be resistant to topotecan, such as glioblastoma cells (5), radiosensitization was reported to occur with prolonged exposure to topotecan, but not after short-term drug treatment. The results suggested that the radiosensitizing effects of topotecan could result from its inhibition of radiation-induced PLDR. Apparently, for the treatment of those tumors with lower levels of Topo I, the combination of CPT derivatives with radiotherapy would be an effective modality. The detection of Topo I levels in clinical tumor samples could guide the therapeutic decisions.

It is well known that CPT derivatives are selectively toxic to S-phase cells (34, 35), due to the formation of DSBs when advancing replication forks collide with CPT derivative-Topo I-DNA complexes. In contrast, G2/M-phase cells are most sensitive to ionizing radiation killing (36). The present study revealed that TLC388 dramatically increased the percentage of γ-H2AX positive M-phase cells when combined with a low dose of radiation and the TLC388-radiation-induced DSBs in the M-phase cells often occurred as small DNA fragments detached from the spindles. It seems likely that these DSBs in M-phase might be difficult to repair, and might be inherited from parent cells (Figure 4C), then or result in genomic instability causing cells to undergo apoptosis and/or die through necrosis. TLC388 might enhance the killing effects of X-ray radiotherapy even at low doses of irradiation by a new mechanism of induction of DSBs in sensitive M-phase cancer cells. Thus TLC388 not only killed S-phase cells, but also sensitized M-phase cells to radiation treatment. Obviously, these are unique features of TLC388 and could underlie its substantial chemoradiosensitization. However, the molecular mechanisms involved in the enhancement of DSBs in M-phase are unknown at the present time and future studies in this area could significantly contribute to the development of TLC388 as an effective chemoradiosensitizing drug.

Acknowledgements

This work was supported in part by a grant from the Tobacco-Related Disease Research Program, University of California, U.S.A.

Footnotes

    • Received August 21, 2009.
    • Revision received February 11, 2010.
    • Accepted February 18, 2010.

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Enhancement of Radiation-induced DNA Damage and Inhibition of Its Repair by a Novel Camptothecin Analog
GE HUANG, HUIJUAN WANG, LI-XI YANG
Anticancer Research Mar 2010, 30 (3) 937-944;
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