The cellular mechanisms that modulate the redox state of p53 tumor suppressor remain unclear, although its DNA binding function is known to be strongly inhibited by oxidative and nitrosative stresses. We show that human p53 is subjected to a new and reversible posttranslational modification, namely, S-glutathionylation in stressed states, including DNA damage. First, a rapid and direct incorporation of biotinylated GSH or GSSG into the purified recombinant p53 protein was observed. The modified p53 had a significantly weakened ability to bind its consensus DNA sequence. Reciprocal immunoprecipitations and a GST overlay assay showed that p53 in tumor cells was marginally glutathionylated; however, the level of modification increased greatly after oxidant and DNA-damaging treatments. GSH modification coexisted with the serine phophorylations in activated p53, and the thiol-conjugated protein was present in nuclei. When tumor cells treated with camptothecin or cisplatin were subsequently exposed to glutathione-enhancing agents, p53 underwent dethiolation accompanied by detectable increases in the level of p21waf1 expression, relative to the DNA-damaging drugs alone. Mass spectrometry of GSH-modified p53 protein identified cysteines 124, 141, and 182, all present in the proximal DNA-binding domain, as the sites of glutathionylation. Biotinylated maleimide also reacted rapidly with Cys141, implying that this is the most reactive cysteine on the p53 surface. The glutathionylatable cysteines were found to exist in a negatively charged microenvironment in cellular p53. Molecular modeling studies located Cys124 and -141 at the dimer interface of p53 and showed glutathionylation of either residue would inhibit p53-DNA association and also interfere with protein dimerization. These results show for the first time that shielding of reactive cysteines contributes to a negative regulation for human p53 and imply that such an inactivation of the transcription factor may represent an acute defensive response with significant consequences for oncogenesis.

The p53 tumor suppressor is a key transcription factor regulating cellular pathways such as DNA repair, cell cycle, apoptosis, angiogenesis, and senescence. It acts as an important defense mechanism against cancer onset and progression, and is negatively regulated by interaction with the oncoprotein MDM2. In human cancers, the TP53 gene is frequently mutated or deleted, or the wild-type p53 function is inhibited by high levels of MDM2, leading to downregulation of tumor suppressive p53 pathways. Thus, the inhibition of MDM2-p53 interaction presents an appealing therapeutic strategy for the treatment of cancer. However, recent studies have revealed the MDM2-p53 interaction to be more complex involving multiple levels of regulation by numerous cellular proteins and epigenetic mechanisms, making it imperative to reexamine this intricate interplay from a holistic viewpoint. This review aims to highlight the multifaceted network of molecules regulating the MDM2-p53 axis to better understand the pathway and exploit it for anticancer therapy.

In this study, we investigated the role of ubiquitination in the disposition of the inactivated O6-methylguanine-DNA methyltransferase (MGMT) protein in human (HT-29 and CEM) and murine (ts85) tumor cells. Using a combination of immunoprecipitation and immunoblotting techniques with antibodies against ubiquitin and MGMT, and anti-ubiquitin immunoaffinity chromatography, the MGMT protein was found to coexist with small amounts of its ubiquitinated species in both human and mouse tumor cells, suggesting the presence of endogenous inactivated MGMT. Further, treatment of HT-29 and CEM cells with MGMT-inactivating compounds, O6-benzylguanine (O6-BG, 20 microM) or 1,3-bis(chloroethyl)-1-nitrosourea (BCNU, 100 microM), resulted in increased levels of ubiquitinated MGMT within 1.5-3 h of drug exposure. Kinetic studies in HT-29 cells treated with O6-BG indicated a slow and gradual conversion of the inactivated MGMT to its polyubiquitinated forms over a course of 3-18 h, with a concomitant disappearance of the parent MGMT protein. We also characterized the previously reported O6-BG-induced degradation of MGMT in HT-29 cell extracts [Pegg et al. (1991) Carcinogenesis 12, 1679-1683] and showed the extracts to be active in conjugation of the MGMT protein with ubiquitin. The proteolysis of O6-BG-inactivated MGMT in HT-29 cell extracts was energy-dependent and was markedly stimulated by ATP and Mg2+ ions. Using the ts85 temperature-sensitive mutant cell line, which expresses a thermolabile ubiquitin-activating enzyme, we observed a differential stability of the inactivated MGMT protein at permissive and nonpermissive temperatures. These results provide conclusive evidence that the MGMT protein, following its inactivation, is degraded via the ubiquitin proteolytic pathway.

To improve prognosis in recurrent glioblastoma we developed a treatment protocol based on a combination of drugs not traditionally thought of as cytotoxic chemotherapy agents but that have a robust history of being well-tolerated and are already marketed and used for other non-cancer indications. Focus was on adding drugs which met these criteria: a) were pharmacologically well characterized, b) had low likelihood of adding to patient side effect burden, c) had evidence for interfering with a recognized, well-characterized growth promoting element of glioblastoma, and d) were coordinated, as an ensemble had reasonable likelihood of concerted activity against key biological features of glioblastoma growth. We found nine drugs meeting these criteria and propose adding them to continuous low dose temozolomide, a currently accepted treatment for relapsed glioblastoma, in patients with recurrent disease after primary treatment with the Stupp Protocol. The nine adjuvant drug regimen, Coordinated Undermining of Survival Paths, CUSP9, then are aprepitant, artesunate, auranofin, captopril, copper gluconate, disulfiram, ketoconazole, nelfinavir, sertraline, to be added to continuous low dose temozolomide. We discuss each drug in turn and the specific rationale for use- how each drug is expected to retard glioblastoma growth and undermine glioblastoma's compensatory mechanisms engaged during temozolomide treatment. The risks of pharmacological interactions and why we believe this drug mix will increase both quality of life and overall survival are reviewed.

A requirement for Mouse Double Minute 2 (MDM2) oncogene activation has been suggested to be associated with cancer progression and metastasis, including breast cancer. To date, most MDM2 inhibitors have been designed to block the MDM2–p53-binding interphase, and have low or no efficacy against advanced breast cancer with mutant or deficient p53. Here we use a high-throughput screening and computer-aided, structure-based rational drug design, and identify a lead compound, SP-141, which can directly bind to MDM2, inhibit MDM2 expression and induce its autoubiquitination and proteasomal degradation. SP-141 has strong in vitro and in vivo antibreast cancer activity, with no apparent host toxicity. While further investigation is needed, our data indicate that SP-141 is a novel targeted therapeutic agent that may especially benefit patients with advanced disease.

Central nervous system tumors comprising the primary cancers and brain metastases remain the most lethal neoplasms and challenging to treat. Substantial evidence points to a paramount role for inflammation in the pathology leading to gliomagenesis, malignant progression and tumor aggressiveness in the central nervous system (CNS) microenvironment. This review summarizes the salient contributions of oxidative stress, interleukins, tumor necrosis factor-α(TNF-α), cyclooxygenases, and transcription factors such as signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) and the associated cross-talks to the inflammatory signaling in CNS cancers. The roles of reactive astrocytes, tumor associated microglia and macrophages, metabolic alterations, microsatellite instability, O6-methylguanine DNA methyltransferase (MGMT) DNA repair and epigenetic alterations mediated by the isocitrate dehydrogenase 1 (IDH1) mutations have been discussed. The inflammatory pathways with relevance to the brain cancer treatments have been highlighted.

The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O(6)-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl2-DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O(6)-benzylguanine; nevertheless, the 24-36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF.

Inactivation of the tumor suppressor p53 and/or overexpression of the oncogene MDM2 frequently occur in human cancers, and are associated with poor prognosis, advanced forms of the disease, and chemoresistance. MDM2, the major negative regulator of p53, induces p53 degradation and inactivates its tumor suppressing activity. In turn, p53 regulates MDM2 expression. This MDM2-p53 negative feedback loop has been widely studied and presents an attractive target for cancer therapy, with a few of the inhibitors of this interaction already having advanced into clinical trials. Additionally, there is an increasing interest in understanding MDM2's p53-independent activities in carcinogenesis and cancer progression, which may also have implications for cancer therapy. This review aims to highlight the various roles that the MDM2-p53 interaction plays in cancer, the p53 independent oncogenic activities of MDM2 and the various strategies that may be used to target MDM2 and the MDM2-p53 interaction. We will summarize the major preclinical and clinical evidences of MDM2 inhibitors for human cancer treatment and make suggestions to further improve efficacy and safety of this interesting class of cancer therapeutics.