Differential role of hydrogen peroxide and staurosporine in induction ...

Apoptosis 2005; 10: 185–192

C 2005 Springer Science + Business Media, Inc.

Differential role of hydrogen peroxide and staurosporine in induction of cell death in glioblastoma cells lacking DNA-dependent protein kinase G. G. Chen, F. L. F. Sin, B. C. S. Leung, H. K. Ng and W. S. Poon Department of Surgery (G. G. Chen, F. L. F. Sin, B. C. S. Leung, W. S. Poon); Department of Anatomical and Cellular Pathology (H. K. Ng); The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, N.T., Hong Kong

Various DNA double-strand break repair mechanisms, in which DNA-dependent protein kinase (DNA-PK) has a major role, are involved both in the development and treatment of glioblastoma. The aim of the present study was to investigate how glioblastoma cells responded to hydrogen peroxide and staurosporine (STS) and how such a response is related to DNA-PK. Two human glioblastoma cell lines, M059J cells that lack DNA-PK activity, and M059K cells that express a normal level of DNAPK, were exposed to hydrogen peroxide or STS. The response of the cells to hydrogen peroxide or STS was recorded by measuring cell death, which was detected by three different methods—MTT, annexin-V and propidium iodide staining, and JC-1 mitochondrial probe. The result showed that both hydrogen peroxide and STS were able to induce cell death of the glioblastoma cells and that the former was mainly associated with necrosis and the latter with apoptosis. Glioblastoma cells lacking DNA-PK were less sensitive to STS treatment than those containing DNA-PK. However, DNAPK had no significant influence on hydrogen peroxide treatment. We further found that catalase, an antioxidant enzyme, could prevent cell death induced by hydrogen peroxide but not by STS, suggesting that the pathways leading to cell death by hydrogen peroxide and STS are different. We conclude that hydrogen peroxide and STS have differential effects on cell death of glioblastoma cells lacking DNA-dependent protein kinase. Such differential roles in the induction of glioblastoma cell death can be of significant value in selecting and/or optimizing the treatment for this malignant brain tumor. Keywords: apoptosis; DNA-dependent protein kinase; glioblastoma; hydrogen peroxide and staurosporine; necrosis.

Correspondence to: G. G. Chen, Department of Surgery, The Chinese University of Hong Kong, Shatin, Prince of Wales Hospital, N.T., Hong Kong. Tel.: +852-2632-3934; Fax: +852-2645-0605; e-mail: [email protected]

Introduction DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase functioning in double-strand break (DSB) repair, can be activated by DNA damage.1 Recent work has also implicated DNA-PK in a variety of processes, such as the modulation of chromatin structure, telomere maintenance and the regulation of apoptosis,2,3 all of which are known to contribute to the development of human glioma or influence the tumor therapy. Functioning as a repair protein, DNA-PK may influence gene expression by phosphorylation of certain transcription factors including Fos, Jun, Myc, Oct1, NF-kappa B, Sp1 and p53, all of which are known to participate in the regulation of cell death or growth processes. However, there is a fair amount of controversy with respect to the role of DNA-PK in the regulation of cell death. DNA-PK has been reported to protect cells from cell death via a caspase-independent pathway,4 and also the anti-apoptotic Bcl-xL appears to be positively correlated with the level of the 80 kDa regulatory component of the DNA-PK.5 On the other hand, DNA-PK promotes cell death by interacting with telomeres, forming a complex with p53, and phosphorylating Mdm2 to make it unable to inhibit p53 transctivation.3,6–8 There is also evidence showing that the status of DNAPK may not influence the percentage of apoptotic cells induced by some stimuli, such as high linear energy transfer (LET) radiation.9 However, low LET induces significantly more apoptotic cells in DNA-PK deficient cells than cells with normal DNA-PK.9 In an attempt to resolve some of the above controversies, the present study compared the response of two glioblastoma cell lines, M059J cells, that lack DNA-PK activity, and M059K cells that contain a normal level of DNA-PK,10 to cell death stimuli, hydrogen peroxide and staurosporine (STS).

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Hydrogen peroxide can be generated directly and indirectly by a plasma membrane NAD(P)H oxidase and a number of metabolic oxidases that are mostly located in peroxisomes and in the mitochondrial respiratory chain. Hydrogen peroxide has long been known to play a role in cell proliferation and growth. It can function physiologically as a cytotoxic agent in host defense, and pathologically or therapeutically as a cell death inducer to destruct cell morphology and functions. STS, a broad-range protein kinase inhibitor, is an alkaloid isolated from the culture broth of Streptomyces staurospores. STS has been shown to inhibit cell cycle progression in a variety of cell lines; enhance differentiation of cancer cells; suppress tumor cell invasion, and induce cell death. Both hydrogen peroxide and STS have been reported as therapeutic agents to suppress the growth of human glioblastoma cells.11–14 However, whether the status of DNA-PK in glioblastoma cells can influence the therapeutic efficiency of these two agents is unknown. We also examined the level of TNFalpha since both hydrogen peroxide and STS can affect the production of TNF-alpha, and TNF-alpha is one of cytokines that are known to induce cell death in glioma cells.15–17

Materials and methods Reagents Hydrogen peroxide, STS, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and catalase were purchased from Sigma (St Louis, MO). STS was dissolved in DMSO for stock and adjusted to final concentrations using complete medium. Annexin V, propidium iodine (PI) and JC-1 were obtained from Molecular Probe Inc (Eugene, OR); anti human DNA-PK antibody from Santa Cruz Biotechnology (Santa Cruz, CA); chemiluminescent detection kit (ECL system) from Amersham Pharmacia Biotech (Piscataway, NJ); and TNFalpha Immunoassay kit from R&D System (Minneapolis, MN).

Cell lines and cell culture Two human glioblastoma cell lines, M059J and M059K, were purchased from American Type Culture Collection (Rockville, MD). M059J cells lack DNA-PK activity while M059K cells express a normal level of DNA-PK. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO BRL, Grand Island, NY) with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin at 37◦ C in a humidified incubator with 95% air/5% CO2 . Media were routinely changed every 4 days. 186 Apoptosis · Vol 10 · No 1 · 2005

MTT assays MTT is a water-soluble tetrazolium salt that is metabolically reduced by viable cells to a coloured, water-insoluble formazan salt. As a consequence, it allows cell viability measurements. Following the removal of conditioned medium, cells in 96-well plates were incubated with 0.5 mg/ml MTT for 1.5 h. The medium was aspirated and cells solubilized in 200 µl DMSO. The optical density of each sample at 550 nm was measured using a Molecular Devices microplate reader. The optical density of the media was proportional to the number of viable cells.

Quantification of annexin-V and propidium iodide stained cells by Flow cytometry An early step in the process of neuronal cell death (apoptosis and necrosis) is the redistribution of phosphatidylserine (PS) from the inner layer to the outer layer of the plasma membrane, due to the loss of membrane asymmetry.18 The externalised PS can be readily visualised by incubating intact cells with a fluorescent derivative of the protein annexin-V, a phospholipid-binding protein. PI, a fluorochrome, is used to label DNA. Unlike necrotic cells, apoptotic cells do not lose their cell membrane integrity and thus they are impermeable for dyes such as PI. Therefore, the combination of annexin-V and PI staining permits the simultaneous quantification of vital, apoptotic and necrotic cells. M089K and M059J cells were washed in cold PBS and incubated with fluorescein-conjugated annexin V- and PI. Flow cytometry of annexin V- and PI-stained cells was performed on a Becton Dickinson FACSVantage machine and analyzed with CellQuest software. The vital cells should be negative for both annexinV and PI staining; apoptotic cells positive for annexin-V staining while negative for PI staining, and necrotic cells positive for both annexin-V and PI staining.

Measurement of mitochondrial membrane potential (MMP) MMP was evaluated using the dye 5,5 ,6,6 -tetrachloroiodide 1,1 ,3,3 -tetraethylbenzimidazolcarbocyanine (JC-1) based on methods described previously.19,20 JC-1, a lipophilic cation, is able to selectively enter mitochondria and exhibit potential-dependent accumulation in negatively charged mitochondria. In the presence of a low MMP, JC-1 exists mainly in a monomeric form emitting green fluorescence at 527 nm after excitation at 490 nm. At a high MMP, JC-1 forms so called J-aggregates, which are associated with a large shift in fluorescence emission and emit orange-red fluorescence at 590 nm. Thus, the color of the dye changes reversibly

Differential role of hydrogen peroxide and staurosporine

from green to orange-red as the mitochondrial membrane becomes more polarized. The dye was detected using a Becton Dickinson FACSVantage machine. After various treatments as described above, cells were incubated with 10 µg/ml JC-1 in medium at room temperature for 15 min in the dark. After incubation, the cells were washed twice, re-suspended in PBS and analyzed. Photomultiplier settings were adjusted to detect JC-1 monomers and J-aggregates fluorescence on the FL1 (530 nm) and FL2 (585 nm) detectors, respectively. The fluorescence ratio at those wavelengths was used to monitor changes in mitochondrial membrane potential.

Figure 1. Cell death measured by MTT assay. M059J and M059K cells were exposed to different concentrations of STS (A) or hydrogen peroxide (B) and incubated in a 96-well plate. After 24 h incubation, cell viability was measured by MTT assay. Percentage of cell death was calculated by formula: 100-[OD reading of STS-treated cells over OD reading of non-treated cells] × 100%. Each experiment was repeated three times. Both hydrogen peroxide and STS had significant effects on cell death (both p < 0.01, by one-way ANOVA analysis). ∗ p < 0.05, ∗∗ p < 0.01 compared with the different type cells receiving the same treatment.

TNF-alpha immunoassay The concentration of human TNF-alpha was measured in the supernatant of cell culture using an immunoassay kit from R&D System. The assay was based on the principle of quantitative sandwich enzyme immunoassay technique and the sensitivity of the assay was 0.06 pg/ml. Western blot analysis Western blot was performed according to previous description.17

Statistical analysis All values were expressed as a mean ± standard deviation. Effect of treatments was analyzed by one-way ANOVA analysis and statistical comparisons by the Student’s t-test. All statistical work was done using the computer software SPSS for Windows (Release 11.0.1, Chicago, IL). A p -value of more than 0.05 was not considered as statistically significant.

Results Expression of DNA-PK protein The expression of DNA-PK protein in M059J and M059K cells was determined by Western blot to confirm that M059J cells were deficient in DNA-PK while M059K cells contained it. The result showed that DNAPK protein was clearly present in M059K cells whereas it was undetectable in M059J cells (Data not shown).

could induce cell death of both M059J and M059K cells in a dose-dependent manner after treatment for 24 h (Figure 1), which were pre-tested to be the sub-optimized incubation period for both agents tested. It was noted that M059K cells were much more sensitive to STS stimulation at concentrations between 0.0625 and 0.25 µM than M059J cells. The difference in sensitivity between M059K and M059J cells disappeared when the concentration of STS reached 0.5 µM. There was no significant difference in cell death between M059J and M059K cells when the cells were treated with hydrogen peroxide at concentrations between 100 and 800 µM. Figure 2 showed that both STS and hydrogen peroxide induced cell death of both M059K and M059J cells in a time-dependent manner. The cell death induced by either STS or hydrogen peroxide reached a plateau after 48 h treatment.

Cell viability measured by MTT assay

Quantification of cell death by annexin V and propidium iodide staining

MTT assay was used to measure cell viability. The experiment showed that both hydrogen peroxide and STS

As described before, annexin V- and PI staining can be used to detect apoptosis and necrosis simultaneously. Apoptosis · Vol 10 · No 1 · 2005

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G. G. Chen et al. Figure 2. Cell death induced by STS or hydrogen peroxide at different time points. M059J and M059K cells were exposed to 0.125 µM STS (A) or 200 µM hydrogen peroxide (B) for 12, 24 48, 96 and 144 h in a 96-well plate. After incubation, cell viability was measured by MTT assay (please refer to Figure 1).

Using this technique, we found that cell death induced by either hydrogen peroxide or STS consisted of both apoptosis and necrosis modes. The percentage of cell death (apoptosis or necrosis) of both M059K and M059J cells treated by either hydrogen peroxide or STS was significantly higher than those without treatment (Figure 3). It appeared that hydrogen peroxide induced more cell death by necrosis than by apoptosis. In contrast to hydrogen peroxide, STS caused apoptosis and necrosis to an equal extent. Similar to the result obtained by MTT assay, M059K cells were more liable to the stimulation of STS than in M059J cells adjusted by either apoptotic or necrotic cell death. Hydrogen peroxide seemed to induce more apoptosis in M059K cells than M059J cells. However, if the percentage of cell death by apoptosis and necrosis was counted up, there was no significant difference in the percentage of total cell death induced by hydrogen peroxide between M059J and M059K cells (63 ± 8% vs. 73 ± 9%, p < 0.05), suggesting the sensitivity, in term of cell death, to hydrogen peroxide stimulation was similar between both types of cells. Cell death evaluated by mitochondrial membrane potential (MMP) The result of MMP measurement was similar to the cell death assayed by annexin V and PI staining. Both hydro188 Apoptosis · Vol 10 · No 1 · 2005

Figure 3. Quantification of cell death by annexin V and propidium iodide staining. M059J and M059K cells were treated with 200 µM hydrogen peroxide or 0.125 µM STS for 24 h. Cells were then stained with annexin V and PI. The positive cells were detected by Flow cytometry. The vital cells were negative for both annexinV and PI staining; apoptotic cells positive for annexin-V staining while negative for PI staining, and necrotic cells positive for both annexin-V and PI staining. The averages of apoptotic cells (A) and necrotic cells (B) were calculated. The total cell death (apoptosis + necrosis) was also shown (C). Each experiment was repeated three times. ∗ p < 0.05, ∗∗ p < 0.01 compared with the same type cells without treatment; + p > 0.05 compared with the same type cells treated with STS.

gen peroxide and STS could significantly decrease MMP in both M059K and M059J cells (Figure 4). M059K cells were more sensitive to MMP collapse than M059J cells when they were treated with STS. However, the percentage of MMP collapsed cells caused by hydrogen peroxide was similar between M059K and M059J cells.

Differential role of hydrogen peroxide and staurosporine Figure 4. Cell death evaluated by mitochondrial membrane potential (MMP). M059J and M059K cells were treated with 200 µM hydrogen peroxide or 0.125 µM of STS for 24 h. Cells were then were incubated with 10 mg/ml JC-1. Cells positive for the fluorescent dye of JC-1 were detected by Flow cytometry. ∗∗ p < 0.01 compared with the same type cells without treatment; ++ p > 0.01 compared with the same type cells treated with hydrogen peroxide.

effect on cell death induced by STS (0.0625–0.5 µM) in either M059J or M059K cells (Figure 5B).

TNF-alpha level Since both hydrogen peroxide and STS can affect the production of TNF-alpha in certain types of cells and TNFalpha is one of cytokines that are known to induce cell death in glioma cells,15–17 it would be interesting to know whether cell death induced by hydrogen peroxide and STS is related to the production of TNF-alpha in glioblastoma cells lacking or containing DNA-PK. However, we found that culture supernatants from both M059J and M059K cells without any treatment or treated with either hydrogen peroxide or STS, did not contain detectable TNFalpha (data not shown).

Effect of catalase on cell death induced by hydrogen peroxide and STS

Discussion

Catalase, one of the naturally occurring antioxidant enzymes, converts hydrogen peroxide to water and oxygen.21 Therefore, the effect of hydrogen peroxide on cell death could be determined by catalase treatment. Indeed, the present study showed that cell death induced by hydrogen peroxide (100–800 µM) in either M059J or M059K cells was significantly inhibited in the presence of catalase (Figure 5A). However, catalase exhibited no significant

We demonstrated that both hydrogen peroxide and STS were able to induce cell death of glioblastoma cells. However, the mechanism responsible for the effects of hydrogen peroxide and STS on cell death appears to be different. First, the cell death induced by STS can be influenced by DNA-PK, whereas cell death induced by hydrogen peroxide is independent of DNA-PK. M059J cells, which are deficient in DNA-PK, are much less sensitive to STS

Figure 5. Effect of catalase on cell death induced by hydrogen peroxide and STS M059J and M059K cells were exposure to different concentrations of hydrogen peroxide (A) or STS (B) at the presence of 1000 u/ml catalase. After 24 h incubation, cell viability was measured by MTT assay. Percentage of cell death was calculated by the formula described in Figure 1. Each experiment was repeated three times. ∗ p < 0.05, ∗∗ p < 0.01 compared with the same type cells without catalase treatment.


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stimulation, compared with DNA-PK-proficient M059K cells. However, the percentage of cell death in both types of glioblastoma cells is similar when they are treated by hydrogen peroxide. Second, the mode of cell death induced by STS mainly consists of apoptosis whereas the majority of cell death induced by hydrogen peroxide is in the form of necrosis. Third, cell death induced by hydrogen peroxide can be prevented by catalase whereas catalase has no influence on cell death induced by STS. DNA-PK is a tri-subunit DNA repair enzyme involved in the maintenance of chromosomal integrity.22 Maintaining chromosomal integrity is essential for cell growth and differentiation. Cancer cells usually have defective DNA repair mechanisms and/or DNA damage checkpoint pathways. Importantly, patients with DNA-repair deficient syndromes are often susceptible to cancer.23 There are two recent reports showing a positive correlation between deficiency of DNA-PK and tumor development. A study by Auckley et al. indicated that reduced DNA-PK repair activity was associated with risk for lung cancer.25 Ochiai et al. demonstrated that Scid mice that contained a mutated form of DNA-PK were more highly susceptible to colon carcinogenesis than the isogenic C.B-17 strain.24 It has been proposed that DNA-PK plays a role as a caretaker-type tumor suppressor gene. It is well known that the development of cancer results from an imbalance between cell growth and cell death. The rate of tumor cell accumulation can be viewed as the rate of cell proliferation minus the rate of cell death. The fact that glioblastoma cells lacking DNA-PK (M059J) are less likely to undergo cell death induced by STS, as demonstrated by the present study, is another piece of evidence to support the role of DNA-PK as a tumor suppressor molecule. The mechanism responsible for the pro-cell death role of DNA-PK is not well known. One recent study demonstrated that DNA-PK promoted cell death by interacting with telomeres.3 There are also studies showing that DNA-PK selectively regulates the p53-dependent apoptotic pathway by forming a complex with p53 and phosphorylating Mdm-2.6–8 Mice defective in DNA-PK exhibits a decreased apoptotic response and the reduction of Bax expression following exposure to ionizing irradiation, suggesting that DNA-PK serves as an unstream regulator of the p53-mediated apoptotic pathway.10 Furthermore, DNA-damage-induced apoptosis is abolished in both DNA-PK− /− and p53− /− cells.8 Apoptosis rather than necrosis plays a role in resistance to cancer development and the avoidance of apoptosis is regarded as one of the hallmarks of cancer cells.26 However, in term of cancer therapy, induction of either form of cell death may not make a significant difference, as whatever happens to the apoptotic or necrotic cell, the result is always the same: the tumor cell disappears. Our present study showed that although both forms of cell death occurred in glioblastoma cells treated with either hydrogen 190 Apoptosis · Vol 10 · No 1 · 2005

peroxide or STS, the major part of cell death associated with hydrogen peroxide was necrosis, whereas STS mainly induced apoptosis. This finding is in agreement with experiments carried out in other types of cells, including breast cancer, neuronal, oligodendroglial, squamous carcinoma and fibroblast cells.27–30 In the present study, we further observed that no matter what form of cell death was induced by STS, glioblastoma cells lacking DNA-PK (M059J) were more resistant to STS treatment than those containing DNA-PK (M059K). However, the status of DNA-PK did not affect the sensitivity of glioblastoma cells to hydrogen peroxide treatment adjusted by either apoptotic or necrotic cell death. These data indicate that DNA-PK is not universally but selectively involved in the glioblastoma cell death pathway, depending upon the cell death inducer used. Hydrogen peroxide is a well-known oxidant, which induces cell death via interacting with a variety of molecules.30,31 Although STS is not an oxidant but a broad-spectrum protein kinase inhibitor,27–29 STSinduced cell death has been reported to be related to oxidant production, as antioxidant treatment such as the over-expression of Cu/Zn-superoxide dismutases (SOD) can reduce cell death induced by STS.32 Therefore, it seems that increased oxidative stress is involved in cell death induced by hydrogen peroxide and STS. Increased oxidative stress is a common step in depolarization of mitochondria and reduction of mitochondrial membrane potential (MMP), which is a key mechanism underlying both modes of cell death: apoptosis and necrosis.33,34 Using a mitochondrial fluorescent dye, JC-1, we demonstrated that both hydrogen peroxide and STS were able to decrease MMP. It was also noted that the ability of hydrogen peroxide to collapse MMP was much greater than STS, and this finding was in line with the result obtained by other methods used in the present study, by which the percentage of cell death detected was significantly higher in the cell treated with hydrogen peroxide than that with STS. In order to verify the role of oxidative stress in cell death of glioblastoma cells treated by hydrogen peroxide and STS, we co-treated the cell with catalase. We found that catalase could almost completely block cell death induced by hydrogen peroxide. However, to our surprise, catalase did not exhibit any significant influence on cell death induced by STS. This finding suggests the following two possibilities. First, cell death induced by STS may be associated with oxidative stress molecules other than hydrogen peroxide. Second, oxidative stress may not play a main role in STS-mediated cell death pathway. In supporting the second possibility, Kelso et al. showed that mitochondrial oxidative stress was not a key step in STS-induced cell death in human osteosarcoma and Jurkat human T lymphoceytes.35 Since increased oxidant concentrations in cells is often associated with an increase in TNF-alpha level and TNF-alpha is one of cytokines that are known

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to induce cell death in glioma cells,15–17 it was thought that the production of TNF-alpha could be a cell death signal in glioblastoma cells treated by hydrogen peroxide and STS. However, we failed to detect TNF-alpha in the cell model of the present study. Therefore, hydrogen peroxide- or STS-mediated cell death in glioblastoma cells either containing or lacking DNA-PK, is independent of TNF-alpha. STS and hydrogen peroxide may similarly influence some apoptotic molecules. For example, both of them are able to increase Bax expression or its activity.36,37 However, STS and hydrogen peroxide may also affect different targets in cells, which may help to explain the different responses of glioblastoma cells to these two agents in the present study. It is known that hydrogen peroxide damages cells mainly by inducing DNA single- or/and doublestrand breaks,27,30 which can be the cause of cell death. Furthermore, hydrogen peroxide-trigged DNA damages can be repaired with comparable efficiency in both Ku80 wild type and Ku80 null cells,38 suggesting that the role of DNA-PK, if any, is minimal. On the other hand, STS appears to be ineffective in inducing DNA single- or/and double-strand breaks.39 Therefore, DNA single- or/and double-strand breaks can be a factor contributing to the different responses seen in the present study. In conclusion, our present study demonstrated that there were differential effects of hydrogen peroxide and STS on cell death of glioblastoma cells, and that such effects were related to the status of DNA-PK. DNAPK-proficient glioblastoma cells can be more sensitive to STS treatment than DNA-PK-lacking glioblastoma cells. However, this is not the case for hydrogen peroxide. Although both hydrogen peroxide and STS can induce cell death, cells treated by hydrogen peroxide mainly become necrotic while cells treated by STS are predominantly apoptotic. Catalase treatment can prevent cell death induced by hydrogen peroxide, but not by STS. Therefore, the pathways leading to cell death by hydrogen peroxide and STS are different, though both are associated with collapsed MMP.

Acknowledgment

This study was supported CUHK Direct grant 2041018.

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