mtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft.

BACKGROUND: Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in eukaryotic cellular function. However, the effects mitochondrial DNA (mtDNA) levels have on the nuclear transcriptome have not been defined under physiological conditions. In order to address this issue, we characterized the gene expression profiles of A549 lung cancer cells and their mtDNA-depleted rho0 counterparts grown in culture and as tumor xenografts in immune-deficient mice. RESULTS: Cultured A549 rho0 cells were respiration-deficient and showed enhanced levels of transcripts relevant to metal homeostasis, initiation of the epithelial-mesenchymal transition, and glucuronidation pathways. Several well-established HIF-regulated transcripts showed increased or decreased abundance relative to the parental cell line. Furthermore, growth in culture versus xenograft has a significantly greater influence on expression profiles, including transcripts involved in mitochondrial structure and both aerobic and anaerobic energy metabolism. However, both in vitro and in vivo, mtDNA levels explained the majority of the variance observed in the expression of transcripts in glucuronidation, tRNA synthetase, and immune surveillance related pathways. mtDNA levels in A549 xenografts also affected the expression of genes, such as AMACR and PHYH, involved in peroxisomal lipid metabolic pathways. CONCLUSION: We have identified mtDNA-dependent gene expression profiles that are shared in cultured cells and in xenografts. These profiles indicate that mtDNA-depleted cells could provide informative model systems for the testing the efficacy of select classes of therapeutics, such as anti-angiogenesis agents. Furthermore, mtDNA-depleted cells grown culture and in xenografts provide a powerful means to investigate possible relationships between mitochondrial activity and gene expression profiles in normal and pathological cells.

Synthesis and biologic properties of hydrophilic sapphyrins, a new class of tumor-selective inhibitors of gene expression.

BACKGROUND: Sapphyrin analogues and related porphyrin-like species have attracted attention as anticancer agents due to their selective localization in various cancers, including hematologic malignancies, relative to surrounding tissues. Sapphyrins are electron affinic compounds that generate high yields of singlet oxygen formation. Although initially explored in the context of photodynamic therapy, sapphyrins have intrinsic anticancer activity that is independent of their photosensitizing properties. However, the mechanisms for their anticancer activity have not been fully elucidated. RESULTS: We have prepared a series of hydrophilic sapphyrins and evaluated their effect on proliferation, uptake, and cell death in adherent human lung (A549) and prostate (PC3) cancer cell lines and in an A549 xenograft tumor model. PCI-2050, the sapphyrin derivative with the highest in vitro growth inhibitory activity, significantly lowered 5-bromo-2'-deoxyuridine incorporation in S-phase A549 cells by 60% within eight hours and increased levels of reactive oxygen species within four hours. The growth inhibition pattern of PCI-2050 in the National Cancer Institute 60 cell line screen correlated most closely using the COMPARE algorithm with known transcriptional or translational inhibitors. Gene expression analyses conducted on A549 plateau phase cultures treated with PCI-2050 uncovered wide-spread decreases in mRNA levels, which especially affected short-lived transcripts. Intriguingly, PCI-2050 increased the levels of transcripts involved in RNA processing and trafficking, transcriptional regulation, and chromatin remodeling. We propose that these changes reflect the activation of cellular processes aimed at countering the observed wide-spread reductions in transcript levels. In our A549 xenograft model, the two lead compounds, PCI-2050 and PCI-2022, showed similar tumor distributions despite differences in plasma and kidney level profiles. This provides a possible explanation for the better tolerance of PCI-2022 relative to PCI-2050. CONCLUSION: Hydrophilic sapphyrins were found to display promise as novel agents that localize to tumors, generate oxidative stress, and inhibit gene expression.

Motexafin gadolinium and zinc induce oxidative stress responses and apoptosis in B-cell lymphoma lines.

There is an emerging appreciation of the importance of zinc in regulating cancer cell growth and proliferation. Recently, we showed that the anticancer agent motexafin gadolinium (MGd) disrupted zinc metabolism in A549 lung cancer cells, leading, in the presence of exogenous zinc, to cell death. Here, we report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines. We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptosis. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1)-regulated and hypoxia-inducible transcription factor-1 (HIF-1)-regulated genes. Cultures cotreated with MGd and zinc acetate displayed further increases in the levels of MTF-1- and HIF-1-regulated transcripts as well as additional transcripts regulated by NF-E2-related transcription factor 2. These data provide insights into the molecular changes that accompany the disruption of intracellular zinc homeostasis and support a role for MGd in treatment of B-cell hematologic malignancies.

Motexafin gadolinium disrupts zinc metabolism in human cancer cell lines.

To gain a better understanding of the mechanism of action of the metal cation-containing chemotherapeutic drug motexafin gadolinium (MGd), gene expression profiling analyses were conducted on plateau phase human lung cancer (A549) cell cultures treated with MGd. Drug treatment elicited a highly specific response that manifested in elevated levels of metallothionein isoform and zinc transporter 1 (ZnT1) transcripts. A549 cultures incubated with MGd in the presence of exogenous zinc acetate displayed synergistic increases in the levels of intracellular free zinc, metallothionein transcripts, inhibition of thioredoxin reductase activity, and cell death. Similar effects were observed in PC3 prostate cancer and Ramos B-cell lymphoma cell lines. Intracellular free zinc levels increased in response to treatment with MGd in the absence of exogenous zinc, indicating that MGd can mobilize bound intracellular zinc. These findings lead us to suggest that an important component of the anticancer activity of MGd is related to its ability to disrupt zinc metabolism and alter cellular availability of zinc. This class of compounds may provide insight into the development of novel cancer drugs targeting control of intracellular free zinc and the roles that zinc and other metal cations play in biochemical pathways relevant to cancer.

Tumor localization and antitumor efficacy of novel sapphyrin compounds.

Sapphyrins are pentapyrrolic metal-free expanded porphyrins with potential medical use as anticancer agents. The novel sapphyrin derivative, PCI-2050, functionalized with 2-[2-(2-methoxyethoxy)ethoxy]ethoxy groups to enhance solubility and a modified bipyrrole moiety was found to be more potent in inducing apoptosis than the previously described sapphyrin PCI-2000. Because some sapphyrins may localize to tumors, we took advantage of the intrinsic fluorescence of these compounds to develop a flow cytometry-based assay to track sapphyrin biodistribution in tumor-bearing mice. Ex vivo analysis of sapphyrin-injected animals revealed that PCI-2050 preferentially localized to tumor, whereas PCI-2000 distributed into normal tissues rather than tumor. PCI-2050 uptake in xenograft tumor cells and resultant tumor cell cytotoxicity was dose dependent. To investigate structure-activity relationships, we focused on PCI-2050 and three derivatives that differ by their alkyl substituents on the bipyrrole moiety: PCI-2051, PCI-2052, and PCI-2053. Treatment of Ramos cells in culture or treatment of Ramos xenograft-bearing animals with each of the sapphyrins followed by ex vivo growth of tumor cells revealed the same pattern of cytotoxicity: PCI-2050 > PCI-2052 > PCI-2051 > PCI-2053. Thus, subtle changes in the alkyl substituents on the bipyrrole moiety result in significant changes in antitumor activity. PCI-2050 displayed significant antitumor efficacy in both Ramos and RKO xenograft models without hematologic, hepatic, or renal abnormalities as assessed by complete blood counts and serum chemistries. On the basis of these findings, it is concluded that the sapphyrin PCI-2050 warrants further evaluation as a potential anticancer agent due to its intrinsic proapoptotic activity and tumor localization ability.

Sapphyrins induce apoptosis in hematopoietic tumor-derived cell lines and show in vivo antitumor activity.

Sapphyrins are pentapyrrolic, metal-free, expanded porphyrins. In the present study, the activity of sapphyrins as anticancer agents in hematopoietic-derived tumor cells was explored. It was found that a dihydroxylated water-soluble sapphyrin derivative (PCI-2000) is a potent inducer of apoptosis in a wide variety of tumor cell lines including lymphoma (Ramos, DHL-4, and HF-1), leukemia (Jurkat and HL-60), and myeloma (8226/S, 1-310, C2E3, and 1-414). PCI-2000 triggers an apoptotic pathway in these tumor cells as shown by release of cytochrome c from mitochondria; activation of caspases 9, 8, and 3; cleavage of the caspase substrate poly(ADP-ribose) polymerase; and Annexin V binding. Apoptosis can be partially inhibited by overexpression of the antiapoptotic protein Bcl-2 or treatment with benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone, a cell-permeable caspase inhibitor. Both PCI-2000 and PCI-2010, a tetrahydroxy bis-carbamate derivative of PCI-2000, result in increased levels of phosphorylated p38 mitogen-activated protein kinase. Inhibition of p38 mitogen-activated protein kinase phosphorylation resulted in a synergistic increase of PCI-2000 cytotoxicity. PCI-2010 showed less toxicity in mice than PCI-2000 and was active in slowing the growth of Ramos and HL-60 tumor xenografts in nude mice. These results provide preclinical rationale for the further study of sapphyrins for potential use in the treatment of hematopoietic-derived tumors.

Antiproliferative and proapoptotic activities of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells.

Interest in exploiting traditional medicines for prevention or treatment of cancer is increasing. Extracts from the herb Tripterygium wilfordii hook F have been used in China for centuries to treat immune-related disorders. Recently it was reported that triptolide (PG490), a purified compound from Tripterygium, possessed antitumor properties and induced apoptosis by p53-independent mechanisms in a variety of malignant cell lines. This property of triptolide attracted our attention because we have found that primary cultures of human prostatic epithelial cells derived from normal tissues and adenocarcinomas are in general extremely resistant to apoptosis. Furthermore, the function of wild-type p53 is impaired in these cells such that drugs that require p53 activity to induce cell death are ineffective. Therefore, the properties of triptolide and the recent approval of its water-soluble form (PG490-88) for entry into Phase I clinical trials suggested that this drug was a promising candidate to test for antitumor activity against prostate cells. Experiments presented here demonstrated that triptolide had dose-dependent effects on both normal and cancer-derived primary cultures of human prostatic epithelial cells. Low concentrations of triptolide inhibited cell proliferation and induced a senescence-like phenotype. Higher concentrations of triptolide induced apoptosis that was unexpectedly associated with nuclear accumulation of p53. Paradoxically, levels of the p53 target genes, p21(WAF1/CIP1) and hdm-2, were reduced, as was bcl-2. Our preclinical studies suggest that triptolide might be an effective preventive as well as therapeutic agent against prostate cancer and that triptolide may activate a functional p53 pathway in prostate cells.

Motexafin gadolinium reacts with ascorbate to produce reactive oxygen species.

Motexafin gadolinium (MGd) oxidizes ascorbate, in neutral buffer and in cell culture, forming reactive oxygen species and a coordination polymer with oxalate.

Targeting hypoxic cells through the DNA damage response.

Exposure to hypoxia-induced replication arrest initiates a DNA damage response that includes both ATR- and ATM-mediated signaling. DNA fiber analysis was used to show that these conditions lead to a replication arrest during both the initiation and elongation phases, and that this correlated with decreased levels of nucleotides. The DNA damage response induced by hypoxia is distinct from the classical pathways induced by damaging agents, primarily due to the lack of detectable DNA damage, but also due to the coincident repression of DNA repair in hypoxic conditions. The principle aims of the hypoxia-induced DNA damage response seem to be the induction of p53-dependent apoptosis or the preservation of replication fork integrity. The latter is of particular importance should reoxygenation occur. Tumor reoxygenation occurs as a result of spontaneous changes in blood flow and also therapy. Cells experiencing hypoxia and/or reoxygenation are, therefore, sensitive to loss or inhibition of components of the DNA damage response, including Chk1, ATM, ATR, and poly(ADP-ribose) polymerase (PARP). In addition, restoration of hypoxia-induced p53-mediated signaling may well be effective in the targeting of hypoxic cells. The DNA damage response is also induced in endothelial cells at moderate levels of hypoxia, which do not induce replication arrest. In this situation, phosphorylation of H2AX has been shown to be required for proliferation and angiogenesis and is, therefore, an attractive potential therapeutic target.

ATM activation and signaling under hypoxic conditions.

The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G(2) phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxia-induced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.

Effects of motexafin gadolinium in a phase II trial in refractory chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) cells are susceptible to oxidative stress. The expanded porphyrin, motexafin gadolinium (MGd), reacts with intracellular reducing metabolites and protein thiols to generate reactive oxygen species (ROS). A phase II trial administered MGd 5 mg/kg/day IV for 5 days every 3 weeks until disease progression to patients with previously treated CLL and small lymphocytic lymphoma. Thirteen patients (median age 66 years) with a median of four prior therapies (range 2-9) were enrolled. Modest anti-tumor activity was seen in three patients, with improvement in lymphocytosis, lymphadenopathy and/or splenomegaly, but no patient achieved a partial or complete response by NCI 96 criteria. Flow cytometry confirmed tumor uptake of MGd. Serial increase in AKT phosphorylation in patient samples following MGd treatment was not observed, suggesting intracellular generation of ROS was not optimal. Therefore, this schedule of administration achieved MGd uptake into primary tumor cells in vivo, but clinical activity was modest.

Synthesis and anticancer properties of water-soluble zinc ionophores.

Several water-solubilized versions of the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT), synthesized as part of the present study, have been found both to increase the intracellular concentrations of free zinc and to produce an antiproliferative activity in exponential phase A549 human lung cancer cultures. Gene expression profiles of A549 cultures treated with one of these water-soluble zinc ionophores, PCI-5002, reveal the activation of stress response pathways under the control of metal-responsive transcription factor 1 (MTF-1), hypoxia-inducible transcription factor 1 (HIF-1), and heat shock transcription factors. Additional oxidative stress response and apoptotic pathways were activated in cultures grown in zinc-supplemented media. We also show that these water-soluble zinc ionophores can be given to mice at 100 micromol/kg (300 micromol/m(2)) with no observable toxicity and inhibit the growth of A549 lung and PC3 prostate cancer cells grown in xenograft models. Gene expression profiles of tumor specimens harvested from mice 4 h after treatment confirmed the in vivo activation of MTF-1-responsive genes. Overall, we propose that water-solubilized zinc ionophores represent a potential new class of anticancer agents.

Motexafin gadolinium generates reactive oxygen species and induces apoptosis in sensitive and highly resistant multiple myeloma cells.

Motexafin gadolinium (MGd), an expanded porphyrin, is a tumor-selective redox-mediator that reacts with many intracellular reducing metabolites. Because redox mechanisms mediate apoptosis in multiple myeloma, we hypothesized that disruption of redox balance by MGd would result in cellular cytotoxicity in myeloma. We examined the effects of MGd on cellular cytotoxicity, apoptosis, reactive oxygen species (ROS) production, and intracellular drug uptake in dexamethasone-sensitive (C2E3), dexamethasone-resistant (1-310 and 1-414) chemotherapy-sensitive (8226-RPMI) and highly chemotherapy-resistant (DOX-10V) myeloma cells. We found complete inhibition of proliferation and cytotoxicity in each sensitive and resistant cell line with 24-hour exposure to clinically relevant concentrations of 50 muM MGd and 50 to 100 microM ascorbate, which was required for the effect. The mechanism of cytotoxicity was related to induction of apoptosis as demonstrated by alteration in mitochondrial membrane potential and elevated annexin V expression. This was accompanied by depletion of intracellular glutathione and increased ROS production. Moreover, catalase substantially abrogated MGd-induced cell death. Using fluorescence microscopy and flow cytometry, we found intracellular uptake of MGd and intracellular ROS production. MGd also induced apoptosis in fresh malignant cells from patients with multiple myeloma. These studies provide a rationale for clinical investigation of this novel redox-mediating agent in patients with multiple myeloma and related disorders.

Leptomycin B stabilizes and activates p53 in primary prostatic epithelial cells and induces apoptosis in the LNCaP cell line.

BACKGROUND: Previous studies showed that primary cultures of normal and malignant human prostatic epithelial cells are defective in their ability to upregulate the tumor suppressor protein p53 in response to DNA damage. This dysfunctional regulation of p53 may be relevant to both the high incidence of prostate cancer and its resistance to chemotherapy. Leptomycin B (LMB) has recently been found to increase the protein level and transcriptional activity of p53 by interfering with nucleocytoplasmic export and subsequent degradation by the proteasome. We investigated the ability of LMB to activate p53 in prostatic epithelial cells. METHODS: Primary cultures and the cell lines LNCaP and DU 145 were treated with LMB. p53 protein was evaluated in Western blots and by immunocytochemistry. Induction of downstream targets of p53 was evaluated in Western and Northern blots. Growth inhibition, cell cycle arrest, and apoptosis in response to LMB were measured in clonal growth assays, by flow cytometry, and by Hoescht/propidium iodide staining, respectively. RESULTS: Treatment of prostatic epithelial cells with LMB led to post-translational stabilization of p53, activation of downstream target genes, and induction of cell cycle arrest in primary cultures and apoptosis in LNCaP (with wild-type p53) but not DU 145 (with mutant p53) cells. CONCLUSIONS: p53 in primary cultures of normal and malignant prostate cells, although dysfunctional in that it is not responsive to DNA damage, is activated by LMB. The ability of LMB to stabilize p53 and induce expression of p53-responsive growth inhibitory genes may be a useful lead in the development of chemopreventive or therapeutic small molecules that can modulate p53 function in prostatic epithelial cells.