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Chem. Res. Toxicol. 2002, 15, 1472-1479
Metabolic Response of Normal and Malignant Tissue to Acute and Chronic Methionine Stress in Athymic Mice Bearing Human Glial Tumor Xenografts Demetrius M. Kokkinakis,* Jacquellyn B. Wick, and Qing Xian Zhou Department of Pathology and the Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received May 2, 2002
Chronic methionine (MET) stress, defined as depletion of plasma MET to levels below 5 µM, can be induced in animals with withdrawal of dietary MET, homocysteine (HCYS), and choline (CHOL) plus periodic administration of recombinant L-methionine-R-deamino-γ-lyase (rMETase) and rescue homocystine (HCYSS), given i.p. every 8 and 24 h, respectively. This study describes the effect of this MET depleting regimen (METdr) on normal and malignant tissue using athymic mice bearing human glial tumor xenografts. A 7-day METdr in athymic mice bearing SWB40 and U87 anaplastic astrocytoma xenografts reduced tumor MET to 30% of their baseline values. Although this reduction halted tumor growth, it did not induce the expected complete inhibition of mitosis or a rapid and extensive necrosis. In contrast, SWB77 and D-54 xenografts (glioblastomas) showed marked regression, widespread necrosis, and complete loss of mitotic activity when they were subjected to METdr. Levels of MET in SWB77 and D-54 did not respond to METdr as readily as those in SWB40 and U87 and remained relatively high as the tumor responded to treatment and regressed. High steady states of MET along with the absence of HCYS in high-grade gliomas indicates that transmethylation reactions may be inhibited in such tumors under modest methionine stress conditions. On the basis of these results, it is postulated that METdr in its present formulation is more effective against high-grade, more aggressive gliomas, which are resistant to chemotherapy, than against the more differentiated astrocytic tumors. This may be due to the higher requirements of high-grade gliomas for MET to maintain a state of active proliferation. Further studies are needed to identify the source of MET in glial tumors under METdr and to develop more effective regimens to deplete tumor MET, which might result in a complete and sustained regression of high-grade gliomas.
Introduction The MET-defect,1 i.e., failure of certain tumors to proliferate without an exogenous source of MET, has been a focus of research into designing anti-neoplastic therapy. It is possible to kill tumor cells by depriving them of MET, while sparing normal tissue with the administration of HCYS or HCYS thiolactone as rescue agent. The principle has been investigated against several METdependent tumor xenografts and other experimental tumors. MET and HCYS depleted diets prevent metastasis in tumor bearing animals (1). A G2 cell cycle blockade in tumors and extension of the life span of the animals bearing human tumor xenografts also has been demonstrated (2-4). Impressive tumor regressions can be induced by reduction of the plasma MET level to a steady state of under 5 µM in athymic mice bearing human tumor xenografts (5, 6). Such MET levels can be achieved with the use of recombinant rMETase during restriction of dietary MET, HCY, and choline (CHOL), combined * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: (412) 648-9850. Fax: (412) 3839822. 1 Abbreviations: AGT, O6 -alkylguanine-DNA alkyltransferase; CHOL, choline; CNS, central nervous system; CYS, cysteine; CYSS, cystine; HCYS, homocysteine; HCYSS, homocystine; MET, methionine; METdr, methionine depleting regimen; rMETase, L-methionine-Rdeamino-γ-lyase.
with rescue of normal tissue with i.p. homocystine (HCYSS). An important related observation is that, even when tumor regression is achieved with prolonged METdepletion, discontinuation of the MET-depleted state results in tumor regrowth. Since the application of METdepleting regimens is cumbersome, these observations suggest that the real potential role of methionine depleting regimens is as a part of a combined modality therapy approach (7-10). Despite the failure of MET-deprivation alone to sustain prolonged inhibition of tumor growth, such an approach has considerable potential when used in combination with chemotherapy due to the remarkable synergy between MET-depletion and DNA damaging chemotherapeutic agents such as BCNU and TMZ. Both the highly MET-dependent Daoy and D-54 xenografts did not regrow for at least 80 days following combined treatment with MET-deprivation and BCNU, despite the resistance of both tumors to BCNU (6, 11). Synergy between MET deprivation and BCNU (35 mg/m2) was also observed in the highly resistant glioblastoma xenografts SWB77. A more pronounced synergy was observed with TMZ (6), which in combination with METdepletion delayed SWB77 growth by 39 days. This was significantly longer (P < 0.005) than the delays induced by TMZ (12 days) or MET-deprivation alone (11 days). In this study we examine the effect of MET depleting regimens (MET-dr) on MET levels not only in plasma,
10.1021/tx020033n CCC: $22.00 © 2002 American Chemical Society Published on Web 10/24/2002
Glial Tumor Response to Methionine Stress
but also in glial tumors and hepatic tissue, and we correlate MET depletion with the growth of tumor and induction of pathological conditions in liver. Our data clearly demonstrate that the use of dietary means alone fails to cause significant MET-stress in glial tumors. On the other hand, a combined dietary and pharmacological approach induces a sufficient reduction of tumor MET that has been shown to trigger sensitization of tumors to genotoxic agents. Although the currently used regimens fail to cause a widespread apoptotic response of glial tumors similar to that induced in vitro (12), without the use of genotoxic drugs, it is possible that such an effect can be achieved with further reduction of MET in certain tumors using existing technology.
Materials and Methods Chemicals. Recombinant methioninase (rMETase) was prepared from an rMETase high expression clone derived from Pseudomonas putida donated by T. Nobori according to the method of Hori et al. (13). Endotoxin was removed with an Acticlean Etox (Sterogen, Arcadia, CA) column. The isolated rMETase was 98% pure by HPLC and a single band of Mr 42 720 on SDS-PAGE. The specific activity of rMETase used in this study was ∼20 units/mg of protein, and the endotoxin level was
