Molecular Targeting Chemotherany for Glioblastoma

Molecular targeting chemotherany for glioblastoma

ORIGINAL ARTICLE Annals of Cancer Research and Therapy

Tatsunori Okamura*1), Masahiko Nishiyama・Katsuyuki Suzuki・

Wataru Yamamoto*2), Kaoru Kurisu*1)

We investigated the critical determinants of cytotoxic activity for cisplatin (CDDP),mitomycin C (MMC),and nimustinehydrochloride (ACNU), which are commonlyused to treat malignant glioma.In 10human cancer cell lines, glutathione S-transferase (GST)was a significantresistance factor for CDDP. MMC activity was found to be determined through the balance between activation by NADPH:quinone oxidoreductase (DTD)and inactivationby GST, while NADPH:cytochrome P450 reductase (P-450)was importantin ACNUresistance. Two glioblastoma cell lines, T98G and U-251 MG, showed remarkably high levels of these 3 enzymes and glutathione (GSH),thus being moderately sensitive to MMCand resistant to CDDPand ACNU.Inhibition of these target enzymes caused an increase of the efficacy of each drug. KW2149,a novel MMCanalogue activated by GSH, was active against T98G and U-251MGcells due to their high GSH levels. Moleculartargeting to seek the best treatment modalityor the most active drug may contribute to improvingthe effective- ness of glioma chemotherapy. Ann Cancer Res Ther 6 (1): 27～31, 1997/Received 7 July 1997, Accepted 28 July 1997 Kay words: glioblastoma, drug resisance, ACNU, cisplatin, mitomycin C

Chemotherapy has played no real role in the treatment metyrapone were obtained from Sigma, (St. Louis, MO). of malignant glioma1). Although considerable efforts have been directed towards understanding the mechanism(s) of Cell lines cellular resistance, the details remain unclear. In the Two human glioblastoma cell lines, T98G and U-251 present study, we attempted to clarify the biochemical MG, were kindly provided by Dr. S. Hama (National characteristics of gliomas in relation to drug resistance in Cancer Center Research Institute, Tokyo). A human oral order to select the most active drug and determine the epidermoid carcinoma line, KB, was kindly provided by critical factor(s) related to the cytotoxicity of cisplatin Dr. S. Akiyama (Kagoshima University). A human (CDDP), mitomycin C (MMC), and nimustine hydroch- myelogenous leukemia cell line, K562, and multidrug loride (ACNU) so as to develop ways of enhancing their resistant variant, K562/DOX, were kindly provided by actions. The investigation of molecular target(s) was Dr. T. Tsuruo (Tokyo University). Human colon focused on redox cycle-related enzymes. adenocarcinoma lines, COLO201 and COLO320DM, and the human gastric adenocarcinoma line, MKN45, were Materials and methods obtained from the Japanese Cancer Research Resources Bank. The HSC-42, HEC-46, HCC-48, and HCC-50 cell Drugs lines established by Dr. K. Yanagihara (Hiroshima All chemicals used were of analytical grade. Mitomycin University) were kindly provided by their originator. C (MMC) and 7-N-{{2-{[2-(γ-L-glutamylamino) ethyl] dithio}ethyl}} mitomycin C (KW2149) were provided Cytotoxicity assay by Kyowa Hakko Kogyo, (Tokyo, Japan). Nimustine Exponentially growing cells were concentrated to 5× hydrochloride (ACNU) was provided by Sankyo Co., 106/ml and exposed to the indicated drug concentrations (Tokyo, Japan). Cisplatin (CDDP) was provided by for 30min. After two washes in drug-free medium, cells Bristol-Myers-Squibb, (New York, NY). Ethacrynic acid were resuspended at a concentration of 2.5×104/ml in was obtained from Banyu, (Tokyo, Japan). Dichrolo- RPMI-1640 with 10% fetal bovine serum (FBS), and were phenolindophenol (DCPIP), dicumarol (DIC), and seeded into 24-well plates. After 72hr of incubation in fresh medium at 37℃, surviving cells were counted by a

Coulter counter and by trypan blue exclusion.

*1)Department of Neurosurgery , Hiroshima University School of Medi- cine Assays of enzymatic activity *2)Department of Biochemistry and Biophysics , Research Institute for Radiation Biology and Medicine, Hiroshima University NADPH: quinone oxidoreductase (DTD), NADPH: Correspondence to: Tatsunori Okamura, Department of Neurosurg- cytochrome P-450 reductase (P-450) and glutathione ery, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami- ku, Hiroshima 734, Japan. S-transferase (GST) activities were measured by the proce-

Molecular targeting of glioblastoma chemotherapy 27 dure of Belcourt et al.2) with some modifications. The protein content was measured using the Bio-Rad protein assay (Bio-Rad Laboratory, Hercules, CA).

Cytosolic fractions were prepared at 0-4℃ for measurement of DTD activity. The reaction mixture included 0.025M Tris-HCl (pH 7.4), 0.7mg/ml crystalline bovine serum albumin, 0.2mM NADPH, 0.01% Tween 20, 5μM flavin adenine dinucleotide, and 40μM DCPIP. Reac- tions were performed at 25℃ in a 3-ml volume in the presence or absence of 20μM DIC and DTD activity was measured as the DIC-sensitive reduction of DCPIP. The velocity of DCPIP reduction was measured by spectro- photometry at 600nm. P-450 was measured using cytochrome c as the electron acceptor. Exponentially growing cells (5×106) were washed twice and resuspended in 2ml Fig. 1 Resistance to CDDP in relation to NADPH: of phosphate-buffered saline (138mM NaCl/2.7mM cytochrome P450 reductase (P-450), NADPH: quinone KCl/10mM Na2HPO4/1.8mM KH2PO4, pH 7.4). Cells oxidoreductase (DTD), glutathione S-transferase (GST), were sonicated in three 10sec bursts with 1min of cooling and glutathione (GSH). Only the GST level was significant for CDDP resistance in on ice between each sonication. The reaction mixture (1 10human cancer cell lines. ml) contained 100mM phosphoric acid-potassium buffer

(pH 75), 20μM cytochrome c, 100μM NADPH as an electron donor, and 1μM rotenone. The reaction was 5'-dithiobis-(2-nitrobenzolic acid) (Sigma), and glutath- initiated by addition of NADPH at 25℃, and the reduc- ione reductase (Sigma, 0.5units/ml). The optical density tion of cytochrome c was followed spectrophotometrically at 412nm (A412) was monitored for 3min at 20-sec at 550nm for 5min. For measurement of total GST intervals, and the GSH content was calculated from the activity, exponentially growing cells (1×107) were wa- rate of change in absorbance using a standard curve for shed with phosphate-buffered saline and resuspended in each experiment.

500μl of 10mM Tris-HCl (pH 7.8) containing 0.2M

NaCl. The resuspended cells were sonicated for 30sec, Results and the solution was centrifuged at 105,000×g for 45 min to collect the supernatant. The enzyme reaction was To clarify the critical determinants of drug action, the carried out in a 1-ml cuvette. To give a final concentration activities of P-450, DTD and GST, and the total GSH of 1mM, 80mM 1-chloro-2, 4-dinitrobenzene (CDNB) content were assessed in relation to the cytotoxicity of was diluted in 0.1M potassium phosphate buffer (pH 6.5), CDDP, MMC, and ACNU. In the 10cultured human after which the sample was added. Glutathione (GSH, 2.5 cancer cell lines studied, the cellular GST activity seemed mM) was added to the warmed solution at 25℃. The to be a critical determinant of CDDP resistance (Fig. 1). optical density at 340nm (A340) was monitored and GST Only GST activity was correlated with the IC50 value of activity was calculated from the enzyme- dependent CDDP. To clarify the extent to which GST accounted for change in absorption. CDDP resistance, the effect of inhibition of this enzyme

was studied. Inhibition of GST by 3.3μM ethacrynic acid

Cellular GSH content (EA) caused a significant increase of CDDP-induced

The total cellular GSH content was measured by an cytotoxicity, while treatment with 3.3μM EA alone inhib- enzyme recycling assay3). Exponentially growing cells ited cell growth by less than 10%. These results suggested

(1-5×106) were suspended in 300μl of phosphate- that GST may be a significant factor in CDDP resistance . EDTA solution (125mM KH2PO4, 6.3mM EDTA, pH MMC activity was found to be determined through the

7.5). The resuspended cells were sonicated, and 100μl of balance between activation by DTD and inactivation by 12% 5-sulfosalicylic acid (Wako, Osaka) was added to GST. DTD activity was well related to MMC sensitivity, each sample tube. The solution was mixed and allowed to while GST activity was correlated with the degree of precipitate for 2 to 3hr on ice. After centrifugation at resistance to MMC (Fig. 2) . Inhibition of DTD activity by 10,000×g for 15min, protein-free lysates were obtained. a non-toxic concentration of DIC (50μM) significantly

The enzymatic assay of GSH was carried out at 25℃ in a decreased MMC-induced cytotoxicity in 8 of the 10cell

1-ml cuvette containing 125mM KH2PO4, 6.3mM EDTA, lines (not COLO201 and COLO320DM) . In contrast, 0.21mM NADPH (Wako, Osaka, Japan), 0.6mM 5, inhibition of GST activity by 3 .3μM EA caused a signifi-

28 Annals of Cancer Research and Therapy Vol. 6 No. 1 1997 Fig. 4 Cellular activities of NADPH: cytochrome P450 Fig. 2 Resistance to mitomycin C (MMC) in relation to reductase (P-450), NADPH: quinone oxidoreductase NADPH: cytochrome P450 reductase (P-450), NADPH: (DTD), glutathione S-transferase (GST), and glutathione quinone oxidoreductase (DTD), glutathione S-transferase (GSH) content. (GST), and glutathione (GSH). Glioblastoma T98G and U-251MG cells had relatively Cellular DTD activity was correlated with MMC-induced high levels of DTD, P-450, GST, and GSH. cytotoxicity, while GST activity was related to resistance to MMC in 10human cancer cell lines.

Fig. 5 Cellular sensitivity to mitomycin C (MMC), cis- Fig. 3 Resistance to nimustine hydrochloride (ACNU) platinum (CDDP), and nimustine hydrochloride in relation to NADPH: cytochrome P450 reductase (P- (ACNU). 450), NADPH: quinone oxidoreductase (DTD), glutath- Glioblastoma T98G and U-25IMG cells were moderately ione S-transferase (GST), and glutathione (GSH). sensitive to MMC, but were resistant to CDDP and P-450 activity was important for ACNU resistance in 10 ACNU. human cancer cell lines.

cell lines investigated. These results indicated that the cant increase of MMC activity in 9 cell lines (not K562/ resistance factors were GST for CDDP, DTD and GST DOX). Thus, DTD participates in MMC activation, while for MMC, and P-450 for ACNU. GST plays an important role in MMC resistance. Cellular P-450, DTD, and GST, and total GSH were In the case of ACNU, only P-450 was responsible for assessed in glioblastoma cells (Fig. 4). The activities of cellular resistance (Fig. 3). As P-450 activity increased, the DTD and GST as well as the GSH content were higher in ACNU concentration inhibiting 50% of cell growth (IC50) T98G and U-251MG cells than in the other 10 cancer also became higher. There was a significant correlation cells. These might be common features of brain tumors . between these two factors (r=0.970, r2=0.941). Inhibition As expected, T98G and U-251MG cells were relatively of P-450 activity by a non-toxic concentration of metyr- sensitive to MMC, but highly resistant to CDDP and apone (5mM) caused an increase of ACNU efficacy in all ACNU (Fig. 5). Resistance factors for these 3 agents may

Molecular targeting of glioblastoma chemotherapy 29 Table 1 P450 and GST targeting chemotherapy for electron reductases involved in the redox cycle. Although human glioblastoma cells these two enzymes are generally considered to be involved in drug inactivation, recent reports have suggested that they may actually activate some anticancer agents such as MMC6). Their role in the bioreductive activation of MMC, however, is still controversial. We have recently shown that MMC activity is determined through competi-

Data represent the mean values of triplicate samples, which did not tion between intracellular activation by DTD and differ by more than 10%. P450, NADPH cytochrome P450 inactivation by GSTπ7). P-450 does not play an important reductase; GST, glutathione S-transferase; ACNU, 1-(4-amino-2- role in MMC activation in most cancer cells. In the methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride present study, we showed that this is also true in glioblastoma cells. On the other hand, P-450 is a significant factor in resistance to ACNU. Although the sensitivity of T98G be common to all types of tumors including glioblastoma. and U-251MG cells to ACNU could not be explained on T98G and U-251MG cells had remarkably high levels of the basis of the P450 activity, the activity of this enzyme GST activity, but DTD activity was also high, so they correlated well with the ACNU resistance of the other were moderately sensitive to MMC. Since GST activity cancer cells. As Grant et al. have suggested, extremely was high in the glioblastoma cells, they were resistant to high levels of P450 and GST activity in glioblastoma may

CDDP. Although the degree of resistance to ACNU was play a role in ACNU inactivation8). not directly related to the P-450 activity, T98G and U-251 GST and GSH are well known to detoxify a variety of MG had high P-450 levels and thus were resistant to anticancer agents9). The present study suggested that GST ACNU. may be involved in inactivation of MMC and CDDP. To seek more effective therapy based on the biochemi- Especially for CDDP resistance, this enzyme is very cal characteristics of T98G and U-251MG cells, we important. Considering the data that glioblastoma cells modulated cellular P-450 activity using 5mM metyrapone have high GST and GSH levels, the action of CDDP on to enhance the efficacy of ACNU (Table 1). Inhibition of brain tumors must be limited without any modulation. P-45O caused a remarkable decrease in the IC50 values of We therefore attempted to inhibit GST activity using EA ACNU for T98G and U-251MG cells. Interestingly, in order to enhance CDDP activity, but the modulatory KW2149 was more effective than biochemically modulat- effect was insufficient (data not shown). KW2149 was ed ACNU. This may have been due to GSH-mediated found to be more active than biochemical modulation of activation of KW21494). CDDP using EA. As compared to P-450 inhibition These findings suggest that P-450, DTD, GST, and (ACNU and metyrapone), KW2149 was more effective GSH can be targets for glioblastoma chemotherapy and against T98G and U-251MG cells. KW2149 is a newly that the most active drug or best modality can be selected developed MMC derivative, and GSH has been suggested in relation to the biochemical heterogeneity of tumor to participate in its activation4). Although the detailed cells. mechanisms are still unclear, KW2149 may be a potent agent against malignant gliomas with high GSH levels, Discussion such as T98G and U-251MG cells. The failure of chemotherapy to eradicate glioma cells The drug resistance of cerebral gliomas is broad and from patients is due to a variety of causes. Aside from the encompasses all classes of chemotherapy agents, including biological characteristics of glioma, the blood-brain bar- alkylating agents, antimetabolites, platinum-containing rier (BBB) restricts drug delivery to tumors and this is drugs, and natural products. There are a variety of also of importance. However, the BBB has recently been mechanisms involved, such as alterations in drug targets suggested to be unlocked by modulators such as a brady- and enhanced expression of P-glycoprotein or detoxifica- kinin agonist10). Modulation of BBB permeability could tion enzymes5). selectively increase drug delivery to brain tumors . These We have previously shown that glioblastoma cells have measures may permit us to select a truly effective drug or remarkably high activities of redox cycle-related enzymes treatment adapted to the biochemical nature of a specific such as P-450, DTD, and GST, high levels of GSH. Such glioma from among the various types of anticancer agents. enzyme-directed therapy was also found to be active The relative levels of redox cycle-related enzymes in cells against T98G and U-251MG glioblastoma cells. can be demonstrated by gene analysis7) . Our present find- P-450 and DTD are respectively one-electron and two- ings about molecular targets may contribute to selecting

30 Annals of Cancer Research and Therapy Vol.6 No. 1 1997 the most active form of drug treatment for chemoresistant C derivative effective on mitomycin C-resistant tumor cells. Cancer Res, 54: 2398-2403, 1994. gliomas. 5) Hayes JD, Wolf CR. Molecular mechanisms of drug resistance. Biochemical Journal, 272: 1-15, 1990. References 6) Riley R, Workman P. DT-diaphorase and cancer chemother- 1) Nelson DF, McDonald JV, Lapham LW, Qazi R, Rubin P. apy. Biochem Pharmacol, 43: 1657-1669, 1992. Central nervous system tumors. In: Rubin S, McDonald JV, 7) Nishiyama M, Suzuki K, Kumazaki T, Yamamoto W, Toge T, Qazi R, eds. Clinical Oncology. Philadelphia; WB Saunders Okamura T, Kurisu K. Molecular targeting of mitomycin C Co., 617-644, 1992. chemotherapy. Int J Cancer, 72: 649-656, 1997. 2) Belcourt MF, Hondnick WF, Rockwell S, Sartorelli AC. 8) Grant R, Ironside JW. Glutathione S-transferases and cyto- Differential toxicity of mitomycin C and porfiromycin to chrome P450 detoxifying enzyme distribution in human cere- aerobic and hypoxic Chinese hamster ovary cells overexpress- bral glioma. J Neuro-Oncol, 25: 1-7, 1995. ing human NADPH: cytochrome c (P-450) reductase. Proc 9) Singh SV, Xu BH, Maurya AK, Mian M. Modulation of Natl Acad Sci USA, 93: 456-460, 1996. mitomycin C resistance by glutathione transferase inhibitor 3) Griffith OW. Determination of glutathione and glutathione ethacrynic acid. Biochemica et Biophysica Acta, 1137: 257- disulfide using glutathione reductase and 2-vinylpyridine. 263, 1992. Anal Biochem, 106: 207-212, 1980. 10) Elliott PJ, Hayward NJ, Dean RL, Blunt DG, Bartus RT. 4) Lee JH, Naito M, Tsuruo T. Nonenzymatic reductive activa- Intravenous RMP-7 selectively increases uptake of carboplatin tion of 7-N-{{2-{[2-(γ-L-glutamylamino) ethyl] dithio} into rat brain tumors. Cancer Res, 56: 3998-4005, 1996. ethyl}} mitomycin C by thiol molecules: A novel mitomycin

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