Total Methionine Restriction Treatment of Cancer.

This chapter reviews how total methionine (MET) restriction (MR) of a human brain tumor xenograft, effected by the combination of recombinant L-methionine-α-deamino-γ-lyase (rMETase) and a MET-free diet, greatly potentiates standard chemotherapy for brain tumors in mouse models. The growth of human brain tumor Daoy, SWB77, and D-54 xenografts in nude mice was arrested after the depletion of mouse plasma methionine (MET) with a combination of an MR diet and rMETase and homocysteine to rescue normal cells and tissues. MET was depleted to below 5 µm by this treatment. MR for 10-12 days inhibited tumor growth, but did not prevent tumor regrowth after treatment cessation. A single dose of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), which was ineffective alone, was administered at the end of the MR regimen, and caused a more than 80-day growth delay for Daoy and D-54 and a 20-day growth delay for SWB77. The total MR treatment regimens also increased the efficacy of temozolomide (TMZ) against the SWB77 xenograft when administered at the end of the MET regimen.

Mitotic arrest, apoptosis, and sensitization to chemotherapy of melanomas by methionine deprivation stress.

Methionine deprivation stress (MDS) eliminates mitotic activity in melanoma cells regardless of stage, grade, or TP53 status, whereas it has a negligible effect on normal skin fibroblasts. In most cases, apoptosis accounts for the elimination of up to 90% of tumor cells from the culture within 72 hours after MDS, leaving a scattered population of multinucleated resistant cells. Loss of mitosis in tumor cells is associated with marked reduction of cyclin-dependent kinase (CDK) 1 transcription and/or loss of its active form (CDK1-P-Thr(161)), which is coincident with up-regulation of CDKN1A, CDKN1B, and CDKN1C (p21, p27, and p57). Expression of the proapoptotic LITAF, IFNGR, EREG, TNFSF/TNFRSF10 and TNFRSF12, FAS, and RNASEL is primarily up-regulated/induced in cells destined to undergo apoptosis. Loss of Aurora kinase B and BIRC5, which are required for histone H3 phosphorylation, is associated with the accumulation of surviving multinucleated cells. Nevertheless, noncycling survivors of MDS are sensitized to temozolomide, carmustin, and cisplatin to a much greater extent than normal skin fibroblasts possibly because of the suppression of MGMT/TOP1/POLB, MGMT/RAD52/RAD54, and cMET/RADD52, respectively. Sensitivity to these and additional genotoxic agents and radiation may also be acquired due to loss of cMET/OGG1, reduced glutathione reductase levels, and a G(2)-phase block that is a crucial step in the damage response associated with enhancement of drug toxicity. Although the genes controlling mitotic arrest and/or apoptosis in response to low extracellular methionine levels are unknown, it is likely that such control is exerted via the induction/up-regulation of tumor suppressors/growth inhibitor genes, such as TGFB, PTEN, GAS1, EGR3, BTG3, MDA7, and the proteoglycans (LUM, BGN, and DCN), as well as the down-regulation/loss of function of prosurvival genes, such as NFkappaB, MYC, and ERBB2. Although MDS targets several common genes in tumors, mutational variability among melanomas may decide which metabolic and signal transduction pathways will be activated or shutdown.

Aberrant Wnt/beta-catenin signaling in pancreatic adenocarcinoma.

Wnt/beta-catenin signaling plays an important role in normal development. However, its aberrant activation is associated with several cancers. The aim of this study is to examine the Wnt/beta-catenin pathway in patients with advanced pancreatic adenocarcinoma (n = 31). Paraffin sections from tumors (n = 16) and normal pancreata (n = 3) were used to determine the localization of beta-catenin. An additional 15 frozen tumors, adjacent normal pancreata (n = 5), or normal pancreata (n = 4) were utilized for protein isolation. Tumors were also examined for mutations in exon 3 of the CTNNB1 gene. More than 65% of the tumors showed an increase in total beta-catenin, consistent with its enhanced membranous, cytoplasmic, and nuclear localization, but only two showed mutations in CTNNB1. The majority of the remaining tumors demonstrated concurrent increases in Wnt-1 and frizzled-2 (positive regulators) and a decrease in Ser45/Thr41-phospho-beta-catenin. Electrophoretic mobility shift assay demonstrated beta-catenin-T-cell factor binding in tumors only. Adenomatous polyposis coli and axin, which are both negative regulators, remained unchanged. Unexpectedly, total glycogen synthase kinase-3beta protein was elevated in these tumors. Elevated levels of E-cadherin were also observed, although E-cadherin-beta-catenin association in tumors remained unaffected. Thus, Wnt/beta-catenin activation was observed in 65% of pancreatic adenocarcinomas, independently of beta-catenin gene mutations in most tumors.

Methionine-stress: a pleiotropic approach in enhancing the efficacy of chemotherapy.

Malignant cells fail to utilize homocysteine (HCYS) in place of methionine (MET) and they are dependent on exogenous MET for growth. In animals, reduction of plasma MET to <5 microM can be induced by combined dietary restriction of MET and administration of L-methionine-alpha-deamino-gamma-lyase (methioninase). This treatment, termed as MET-stress, inhibits the growth of brain tumor xenografts in athymic mice and enhances the efficacy of DNA alkylating chemotherapeutic agents. The response of tumors to MET-stress depends on their mutational status, however, it always involves inhibition of CDK1 and in most cases the upregulation of p21, p27, GADDs and 14-3-3sigma in response to upregulation of TGF-beta, IRF-1, TNF-alpha, Rb and/or MDA-7 and the downregulation of PI3K, RAS and NF-kappaB. Although inhibition of the cell cycle and mitosis is not necessarily dependent on the tumor's p53 status, the expression of p21, GADD45 and apoptosis related genes (BAX, BCL-2) are regulated by wt-p53, in addition to their regulation by TGF-beta or MDA-7 in mutated p53 tumors. Mutational variability determines the mode of death (mitotic catastrophe versus apoptosis) in tumor cells subjected to MET-stress. The increase of the efficacy of alkylating agents is related to marked inhibition of O6-methylguanine-DNA methyltransferase (MGMT) expression, the induction of cell cycle check points and the inhibition of pro-survival pathways by MET-stress.

Modulation of cell cycle and gene expression in pancreatic tumor cell lines by methionine deprivation (methionine stress): implications to the therapy of pancreatic adenocarcinoma.

The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

Physiology and gene expression characteristics of carcinogen-initiated and tumor-transformed glial progenitor cells derived from the CNS of methylnitrosourea (MNU)-treated Sprague-Dawley rats.

Glial progenitors from the brain of normal adult Sprague-Dawley rats were compared to their initiated and malignant counterparts that were isolated from apparently normal brains of animals exposed to methylnitrosourea (MNU). Fibroblast growth factor-2 (FGF-2) or platelet-derived growth factor (PDGF)-A or -B induced differentiation of normal progenitors to a pro-astrocytic or oligodendrocytic morphology, respectively, whereas the combination of these factors resulted in their terminal differentiation to oligodendrocytes and senescence. In contrast, initiated progenitors did not exit the cell cycle when stimulated with PDGF and/or FGF-2. cDNA oligoarray analysis and RT-PCR verification showed an early upregulation/ induction of growth factor/receptors, PDGF-A, PDGFR-beta, IGFR-1, IGF-1 and -2, IL-6, MEGF-5, FRAG-1, IRS-2, HSPG, and FGFR-1, followed by a late increase in the expression IGFBP-6, PDGF-alpha, FGFR-4A, c/ERB-A, and FGFR-4, 2, and 1 during the tumorigenic progression. Western blot analyses demonstrated that MNU exposure caused progressive reduction of p21 protein levels, an increase of Rb phosphorylation, activation of AKT and CDK2, and upregulation of FGF receptors. Double immunofluorescence labeling showed progressive increase in nuclear colocalization of FGFR1, 2, and 4, which peaked in malignant lines. It is postulated that transition of normal rat glial progenitors to an initiated state is driven by IGF-1 and 2, IL-6, and the upregulation of the receptors PDGFR-beta and FGFR-1, 2, and 4. Deregulation of the cell cycle in this state involves reduction of p21 protein, concomitant upregulation of CDC2, and an increase in Rb phosphorylation that favors expression and nuclear translocation of FGFR-4 and FRAG-1 and 2. These events are associated with progressive activation of AKT and RAS. Malignant transformation is enhanced by near elimination of p21 and PC3, induction of AP-1 (upregulation of JUN-B, c-JUN, FRA-1), activation of the NF-kB pro-survival pathway, and inhibition of the TGF-beta pro-apoptotic pathway possibly in response to changes in the expression of nerve growth factor (NGF) I-A and NGFI-B. These data demonstrate that the events leading to malignancy in the rat brain in response to MNU treatment are to a great extent similar to those described for secondary glial malignancies in humans.

Modulation of gene expression in human central nervous system tumors under methionine deprivation-induced stress.

Methionine deprivation imposes a metabolic stress, termed methionine stress, that inhibits mitosis and induces cell cycle arrest and apoptosis. The methionine-dependent central nervous system tumor cell lines DAOY (medulloblastoma), SWB61 (anaplastic oligodendroglioma), SWB40 (anaplastic astrocytoma), and SWB39 (glioblastoma multiforme) were compared with methionine-stress resistant SWB77 (glioblastoma multiforme). The cDNA-oligoarray analysis and reverse transcription-PCR verification indicated common changes in gene expression in methionine-dependent cell lines to include up-regulation/induction of cyclin D1, mitotic arrest deficient (MAD)1, p21, growth arrest and DNA-damage-inducible (GADD)45 alpha, GADD45 gamma, GADD34, breast cancer (BRCA)1, 14-3-3sigma, B-cell CLL/lymphoma (BCL)1, transforming growth factor (TGF)-beta, TGF-beta-induced early response (TIEG), SMAD5, SMAD7, SMAD2, insulin-like growth factor binding protein (IGFBP7), IGF-R2, vascular endothelial growth factor (VEGF), TNF-related apoptosis-inducing ligand (TRAIL), TNF-alpha converting enzyme (TACE), TRAIL receptor (TRAIL-R)2, TNFR-related death receptor (DR)6, TRAF interacting protein (I-TRAF), IL-6, MDA7, IL-1B convertase (ICE)-gamma, delta and epsilon, IRF1, IRF5, IRF7, interferon (IFN)-gamma and receptor components, ISG15, p65-NF-kappaB, JUN-B, positive cofactor (PC)4, C/ERB-beta, inositol triphosphate receptor I, and methionine adenosyltransferase II. On the other hand, cyclins A1, A2, B1 and B2, cell division cycle (CDC)2 and its kinase, CDC25 A and B, budding uninhibited by benzimidazoles (BUB)1 and 3, MAD2, CDC28 protein kinase (CKS)1 and 2, neuroepithelial cell transforming gene (NET)1, activator of S-phase kinase (ASK), CDC14B phosphatase, BCL2, TGF-beta activated kinase (TAK)1, TAB1, c-FOS, DNA topoisomerase II, DNA polymerase alpha, dihydrofolate reductase, thymidine kinase, stathmin, and MAP4 were down-regulated. In the methionine stress-resistant SWB77, only 20% of the above genes were affected, and then only to a lesser extent. In addition, some of the changes observed in SWB77 were opposite to those seen in methionine-dependent tumors, including expression of p21, TRAIL-R2, and TIEG. Despite similarities, differences between methionine-dependent tumors were substantial, especially in regard to regulation of cytokine expression. Western blot analysis confirmed that methionine stress caused the following: (a) a marked increase of GADD45alpha and gamma in the wt-p53 cell lines SWB61 and 40; (b) an increase in GADD34 and p21 protein in all of the methionine-dependent lines; and (c) the induction of MDA7 and phospho-p38 in DAOY and SWB39, consistent with marked transcriptional activation of the former under methionine stress. It was additionally shown that methionine stress down-regulated the highly active phosphatidylinositol 3'-kinase pathway by reducing AKT phosphorylation, especially in DAOY and SWB77, and also reduced the levels of retinoblastoma (Rb) and pRb (P-ser780, P-ser795, and P-ser807/811), resulting in a shift in favor of unphosphorylated species in all of the methionine-dependent lines. Immunohistochemical analysis showed marked inhibition of nuclear translocation of nuclear factor kappaB under methionine stress in methionine-dependent lines. In this study we show for the first time that methionine stress mobilizes several defined cell cycle checkpoints and proapoptotic pathways while coordinately inhibiting prosurvival mechanisms in central nervous system tumors. It is clear that methionine stress-induced cytotoxicity is not restricted by the p53 mutational status.

O6-benzylguanine suppression of O6-alkylguanine-DNA alkyltransferase in anaplastic gliomas.

The purpose of the study was to determine the dose of O(6)-benzylguanine (BG) that would suppress O(6)-alkylguanine-DNA alkyltransferase (AGT) activity to undetectable levels in > 90% of anaplastic gliomas, as measured 6 h after a 1-h BG infusion. Subjects who were scheduled for surgical resection of a known or presumed anaplastic glioma received a 1-h infusion of BG. Tumor tissue was surgically removed approximately 6 h after the end of the infusion and was analyzed for AGT activity. The BG dose was escalated until at least 11 of 14 subjects had no detectable AGT activity. An additional cohort of patients received the identified effective dose of BG approximately 18 h before tumor resection in order to compare our results with an earlier study using the longer time interval. In the 79 subjects who were enrolled, there was no significant toxicity that was attributed to the BG. A dose-response relationship was determined between the BG dose and the percentage of subjects with undetectable AGT. A dose of 120 mg/m(2) suppressed AGT to less than detectable levels in 17 of 18 patients when the drug-resection interval was 6 h. With an 18-h interval, only 5 of 11 subjects had undetectable AGT at the 120-mg/m(2) dose. We conclude that a BG dose of 120 mg/m(2) given 6 h before an alkylating drug would be effective in suppressing AGT and possibly potentiating the cytotoxic effects of the drug.

Sensitization of pancreatic tumor xenografts to carmustine and temozolomide by inactivation of their O6-Methylguanine-DNA methyltransferase with O6-benzylguanine or O6-benzyl-2'-deoxyguanosine.

Adenocarcinoma of the pancreas is refractory to chemotherapeutic agents, including BCNU and streptozotocin. We have previously shown that drugs, which adduct the O(6)- position of guanine, are ineffective against pancreatic tumor cell lines because of high expression of O(6)-methylguanine-DNA methyltransferase (MGMT). The effect of MGMT inactivation on the resistance of pancreatic tumors to carmustine (BCNU) and to temozolomide (TMZ) was examined in five human pancreatic tumor xenografts in athymic mice. Tumor-bearing mice were treated: (a) with a single i.p. injection of BCNU or TMZ at the maximum-tolerated doses of 75 and 340 mg/m(2), respectively; and (b) with O(6)-benzylguanine (BG) or O(6)-benzyl-2'-deoxyguanosine (dBG) in combination with BCNU or TMZ. Pretreatment with the MGMT inactivators BG or dBG reduced the maximum-tolerated doses of BCNU and TMZ to 35 and 170 mg/m(2), respectively. MIA PaCa-2, CFPAC-1, PANC-1, CAPAN-2, and BxPC-3 having MGMT levels of 890, 1680, 680, 900, and 330 fmol/mg protein, respectively, were unresponsive to BCNU. MIA PaCa-2 and CFPAC-1 were also unresponsive to TMZ, whereas CAPAN-2 responded with a tumor delay of 32 days. BG or dBG sensitized all tumors to both BCNU and TMZ. BG plus BCNU treatment of MIA PaCa-2, CFPAC-1, PANC-1, CAPAN-2, and BxPC-3 induced tumor delays of 18, 16, 12, 14, and 16 days, respectively. In comparison, dBG plus BCNU at doses that were equitoxic to BCNU plus BG yielded tumor delays of 30, 19, 16, 21, and 22 days, respectively. The pancreatic tumors tested displayed functional mismatch repair that, however, may not be always sufficiently restrictive to prevent mutations under alkylation stress. Treatments with either BCNU or TMZ resulted in some degree of mutation in recurring tumors with the exception of CAPAN-2, the only wt-p53 xenograft. dBG, a weak MGMT inactivator in vitro as compared with BG, was markedly more effective than the latter in enhancing the efficacy of BCNU against pancreatic tumor xenografts. Both BG and dBG also enhanced the efficacy of TMZ against pancreatic tumors, possibly because of the repression of MGMT, which cannot be achieved with TMZ treatments alone. These results suggest that pancreatic tumors, which are resistant to DNA alkylating agents, may be sensitized to such agents when pretreated with MGMT inactivators.

Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits growth of PC-3 human prostate cancer xenografts in vivo.

We have shown previously that allyl isothiocyanate (AITC), a constituent of cruciferous vegetables, significantly inhibits survival of PC-3 and LNCaP human prostate cancer cells in culture, whereas proliferation of a normal prostate epithelial cell line is minimally affected by AITC even at concentrations that are highly cytotoxic to the prostate cancer cells. The present studies were designed to test the hypothesis that AITC administration may retard growth of human prostate cancer xenografts in vivo. Bolus i.p. injection of 10 micromol AITC, three times per week (Monday, Wednesday and Friday) beginning the day of tumor cell implantation, significantly inhibited the growth of PC-3 xenograft (P < 0.05 by two-way ANOVA). For example, 26 days after tumor cell implantation, the average tumor volume in control mice (1025 +/- 205 mm3) was approximately 1.7-fold higher compared with AITC-treated mice. Histological analysis of tumors excised at the termination of the experiment revealed a statistically significant increase in number of apoptotic bodies with a concomitant decrease in cells undergoing mitosis in the tumors of AITC-treated mice compared with that of control mice. Western blot analysis indicated an approximately 70% reduction in the levels of anti-apoptotic protein Bcl-2 in the tumor lysate of AITC-treated mice compared with that of control mice. Moreover, the tumors from AITC-treated mice, but not control mice, exhibited cleavage of BID, which is known to promote apoptosis. Statistically significant reduction in the expression of several proteins that regulate G2/M progression, including cyclin B1, cell division cycle (Cdc)25B and Cdc25C (44, 45 and 90% reduction, respectively, compared with control), was also observed in the tumors of AITC-treated mice relative to control tumors. In conclusion, the results of the present study indicate that AITC administration inhibits growth of PC-3 xenografts in vivo by inducing apoptosis and reducing mitotic activity.

Metabolic response of normal and malignant tissue to acute and chronic methionine stress in athymic mice bearing human glial tumor xenografts.

Chronic methionine (MET) stress, defined as depletion of plasma MET to levels below 5 microM, can be induced in animals with withdrawal of dietary MET, homocysteine (HCYS), and choline (CHOL) plus periodic administration of recombinant L-methionine-alpha-deamino-gamma-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.

Multifaceted resistance of gliomas to temozolomide.

PURPOSE AND EXPERIMENTAL DESIGN: The contributions of O6-methylguanine-DNA-methyltransferase(MGMT), p53 status, mismatch repair, and apoptotic response to the resistance of glial tumors to temozolomide (TMZ) were tested using seven established human glial tumor cell lines in culture and xenografts in athymic mice. RESULTS: Resistance to TMZ was only marginally dependent on MGMT activity, because subtoxic doses of TMZ easily eliminated MGMT reserves for at least 18 h after treatment. Resistance to TMZ varied most notably with the p53 status of the tumor. Tumors with wild-type (wt) p53 and a functional p53 response to DNA damage (SWB40 and SWB61) were most sensitive. The p21-related cell cycle arrest was intimately linked to TMZ toxicity because tumors with wt p53 but lacking a robust increase in p21 protein level (D-54) were resistant to TMZ. In contrast, tumors with a dysfunctional p53 cycle and a weak cell cycle response to DNA damage (SWB39 and SWB77) were extremely unresponsive to treatment even with the aid of MGMT inactivators. Notable exceptions to the above were observed with the p53 mutated tumors SWB33 and SWB95, which were arrested by TMZ in G1-S and consequently underwent apoptosis despite their failure to express p21. CONCLUSIONS: By testing a limited number of glial tumors in cell culture and also as xenografts, we have shown that mobilization of the p53 in response to TMZ damage is likely to induce a cell cycle arrest and apoptosis in glial tumors. Additional pathways linking cell cycle arrest and apoptosis contribute to the efficacy of TMZ against p53 mutated glial tumors. The unusual resistance of tumors, of which the cell cycle was not arrested in response to TMZ treatment, was associated with allelic losses during regrowth of treated tumors. Nevertheless such resistance was not related to dysfunctional mismatch repair.