Inhibiting tumor growth by targeting tumor vasculature with galectin-1 antagonist anginex conjugated to the cytotoxic acylfulvene, 6-hydroxylpropylacylfulvene.

Targeted delivery of therapeutic drugs promises to become the norm to treat cancer. Here, we conjugated the cytotoxic agent 6-hydroxypropylacylfulvene (HPAF) to anginex, a peptide that targets galectin-1, which is highly expressed in endothelial cells of tumor vessels. In a human ovarian cancer model in mice, the conjugate inhibited tumor growth better than equivalent doses of either compound alone. Immunofluorescence on tumor tissue demonstrated that the conjugate, like parent anginex, selectively targeted tumor vasculature and inhibited tumor angiogenesis. Increased activity from the conjugate further suggests that HPAF retains at least some of its normal cytotoxic activity when linked to anginex. More importantly perhaps is the observation that the conjugate abrogates apparent systemic toxicity from treatment with HPAF. This work contributes to the development of tumor vascular targeting agents against cancer in the clinic.

Ovarian tumor growth regression using a combination of vascular targeting agents anginex or topomimetic 0118 and the chemotherapeutic irofulven.

Combination of chemotherapeutic agents and angiogenesis inhibitors is now commonly employed in the clinic to treat cancer. Here, we used angiostatic agents anginex and 0118, in combination with the chemotherapeutic irofulven, to treat human ovarian tumor xenografts in mice. General linear mixed models were used to statistically analyze tumor growth curves. Overall, combination of a low, non-toxic dose of irofulven with either angiogenesis inhibitor was more effective at inhibiting tumor growth than any of the single agent therapies. For example, the anginex/irofulven and 0118/irofulven combinations inhibited tumor growth relative to controls by 92% (p<0.0001) and 96% (p<0.0001), respectively, with the 0118/irofulven combinations yielding 100% complete responses. This study suggests that combination therapy of 0118 or anginex and irofulven may be highly effective in the clinical setting.

Characterizations of irofulven cytotoxicity in combination with cisplatin and oxaliplatin in human colon, breast, and ovarian cancer cells.

PURPOSE: This study assessed the cytotoxic effects of irofulven in combination with oxaliplatin and cisplatin in a panel of human cancer cell lines. METHODS: Growth inhibition studies were performed using the human HT29 colon cancer cell line, irofulven-resistant derivative HT29/IF2, breast cancer cell line MCF7, and ovarian cancer line CAOV3. Irofulven-oxaliplatin combinations were compared with irofulven-cisplatin combinations in the same cell lines using similar experimental settings. Cells were exposed for 1 h to irofulven and then for 24 h to oxaliplatin or cisplatin and vice versa. RESULTS: Single agent irofulven displayed cytotoxic effects against human colon HT29 cells, human breast cancer cell lines including MCF7, SKBR3, and ZR-75-1, and human ovarian cancer cell lines CAOV3, OVCAR3, and IGROV1, with OVCAR3 being the most sensitive cancer cell line (IC50: 2.4 microM). In all tested cell lines the oxaliplatin-irofulven combination led to clear evidence of synergistic activity. In HT29 and HT29/IF2, the sequence oxaliplatin followed by irofulven appears to be the most effective whereas in MCF7 cells, irofulven given prior to or simultaneously with oxaliplatin is more effective than the other schedule. The combination displays additive activity toward CAOV3 ovarian cells when irofulven was administered prior to or simultaneously with oxaliplatin and partially synergistic when oxaliplatin was followed by irofulven. In most of the cell lines, the sequence oxaliplatin followed by irofulven appears to be the most effective as compared to other schedules. A combination of irofulven with cisplatin has the same efficacy as with oxaliplatin for the same cell lines. Cell cycle studies show that irofulven increases the proportion of cells in the S phase. Cisplatin-irofulven and oxaliplatin-irofulven combinations block cells in G1/S and potently induce apoptosis. CONCLUSION: Irofulven displays synergistic antiproliferative and pro-apoptotic effects when combined with oxaliplatin over a broad range of concentrations in human colon and breast cancer cells. Acquired resistance to irofulven has limited impact on the effects of cisplatin-irofulven and oxaliplatin-irofulven combinations. Based on these data, irofulven-oxaliplatin and cisplatin-irofulven combinations will be further explored in clinical trials, favoring the use schedules of oxaliplatin given prior to irofulven in patients with cancer.

Caspase-mediated apoptosis and caspase-independent cell death induced by irofulven in prostate cancer cells.

Irofulven (hydroxymethylacylfulvene) is a novel antitumor drug, which acts by alkylating cellular macromolecular targets. The drug is a potent inducer of apoptosis in various types of tumor cells, whereas it is nonapoptotic in normal cells. This study defined molecular responses to irofulven involving mitochondrial dysfunction and leading to death of prostate tumor LNCaP-Pro5 cells. Irofulven caused early (2-5 hours) translocation of the proapoptotic Bax from cytosol to mitochondria followed by the dissipation of mitochondrial membrane potential and cytochrome c release at 4 to 12 hours. These effects preceded caspase activation and during the first 6 hours were not affected by caspase inhibitors. Processing of caspase-9 initiated the caspase cascade at approximately 6 hours and progressed over time. The activation of the caspase cascade provided a positive feedback loop that enhanced Bcl-2-independent translocation and cytochrome c release. General and specific caspase inhibitors abrogated irofulven-induced apoptotic DNA fragmentation with the following order of potency: pan-caspase > or = caspase-9 > caspase-8/6 > caspase-2 > caspase-3/7 > caspase-1/4. Abrogation of caspase-mediated DNA fragmentation failed to salvage irofulven-treated cells from growth inhibition and loss of viability, demonstrating a substantial contribution of a caspase-independent cell death. Monobromobimane, an inhibitor of alternative caspase-independent apoptotic pathway that is mediated by mitochondrial permeability transition, antagonized both apoptosis, measured as phosphatidylserine externalization, and cytotoxicity of irofulven. Collectively, the results indicate that irofulven-induced signaling is integrated at the level of mitochondrial dysfunction. The induction of both caspase-dependent and caspase-independent death pathways is consistent with pleiotropic effects of irofulven, which include targeting of cellular DNA and proteins.

Cell cycle effects and induction of premitotic apoptosis by irofulven in synchronized cancer cells.

Unlike postmitotic cell death, direct premitotic apoptosis diminishes the risk of clonal selection and allows for the elimination of slowly growing cancer cells. This study characterized the ability to induce premitotic apoptosis by irofulven (hydroxymethylacylfulvene), a novel alkylating drug which targets cellular DNA and proteins. Irofulven effects were examined in HeLa-derived BH2 cancer cells with conditional overexpression of antiapoptotic Bcl-2. Cells were synchronized in either early S or in G(1). Following 12 h exposure to irofulven, cells that were originally in early S accumulated in late S or remained in early S phase (at 0.5 and 2.5 muM drug, respectively). Drug treatment of cells in the G(1) cohort prevented their entry into the S phase. Significant apoptosis was detected based on the appearance of sub-G(1) particles and cells with DNA strand breaks in both G(1) and S cohorts. Apoptotic cells were mostly recruited from the G(1)/S border ("G(1)" cohort) and from the S phase ("early S" cohort). All the cell cycle and apoptotic effects were only marginally affected by Bcl-2 overexpression. Similar results were obtained with irofulven-treated synchronized cultures of leukemic CEM cells. Collectively, these observations indicate that irofulven-treated cells become committed to death early. Neither active DNA replication nor traverse through mitosis are necessary for irofulven-induced cell death. The ability to promote direct premitotic apoptosis is likely to play a role in the consistently potent apoptotic effects of irofulven and its ability to cause tumor regression in vivo.

ATM-dependent CHK2 activation induced by anticancer agent, irofulven.

Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser(1981), which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven.

Antitumor activity of irofulven monotherapy and in combination with mitoxantrone or docetaxel against human prostate cancer models.

BACKGROUND: Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is a novel antitumor agent currently undergoing clinical trials in hormone-refractory prostate cancer. This report examines the efficacy of irofulven alone or in combination with mitoxantrone or docetaxel against androgen-independent prostate cancer cell lines. METHODS: To elucidate the activity of irofulven monotherapy and in combination, PC-3 and DU-145 cell lines were utilized in cellular viability assessments and tumor growth inhibition studies. RESULTS: Viability assays with irofulven and mitoxantrone show additive to synergistic activity. Furthermore, irofulven and mitoxantrone in combination exhibit enhanced antitumor activity against PC-3 and DU-145 xenografts. Additive combination effects are also observed when irofulven and docetaxel were tested against PC-3 xenografts and curative activity (8/10 CR) is observed in DU-145 xenografts. CONCLUSIONS: These studies demonstrate that irofulven displays strong activity as monotherapy and in combination with mitoxantrone or docetaxel against androgen-independent prostate cancer in vitro and in vivo; thus, supporting the clinical investigation of irofulven against hormone-refractory prostate cancer.

Activity of irofulven against human pancreatic carcinoma cell lines in vitro and in vivo.

BACKGROUND: Irofulven (MGI 114), a novel antitumor agent synthesized from the natural product illudin S, has a unique mechanism of action involving macromolecule adduct formation, S-phase arrest and induction of apoptosis. MATERIALS AND METHODS: This study utilized MiaPaCa pancreatic xenografts to demonstrate irofulven antitumor activity using either a daily or intermittent dosing schedule. Additionally, irofulven and gemcitabine were tested in vitro and in vivo to assess the anticancer activity of the combination. RESULTS: Both dosing regimens of irofulven demonstrated curative activity against the MiaPaCa xenografts. Similar activity of irofulven on the intermittent schedule was observed at lower total doses compared to the daily dosing schedule. Furthermore, enhanced antitumor activity was observed when irofulven and gemcitabine were combined compared to single agent activity. CONCLUSION: These results support further clinical characterization of intermittent irofulven dosing schedules and suggest that irofulven combined with gemcitabine may have activity in patients with pancreatic tumors.

Enhanced antitumor activity of irofulven in combination with 5-fluorouracil and cisplatin in human colon and ovarian carcinoma cells.

Irofulven (6-hydroxymethylacylfulvene, MGI-114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product obtained from the Omphalotus mushroom. Irofulven has demonstrated potent activity against a broad range of solid tumors in both cellular and xenograft models and has shown promising activity in clinical trials. To guide the clinical use of irofulven, the present study used the MTT viability assay to examine the cytotoxic effects obtained by combining irofulven with two other anticancer agents: cisplatin and 5-fluorouracil (5-FU). The study was carried out with HT-29 and HCT-116 colorectal and A2780 ovarian carcinoma cells as well as with their irofulven- (HT-29/IF2, HCT-116/IF27) or cisplatin-resistant (A2780/CP70) variants. The combinations showed strong sequence specificity. Simultaneous exposure to cisplatin and irofulven was at least additive for four cell lines including the cisplatin-resistant A2780/CP70 ovarian cells which exhibit a multifactorial resistance phenotype. Cisplatin followed by irofulven was additive for parental HCT-116 and A2780 cells whereas irofulven followed by cisplatin was antagonistic in all cellular models. Simultaneous exposure to 5-FU and irofulven was at least additive for all six cell lines. 5-FU followed by irofulven was additive for the parental HT-29 and A2780 cells and synergistic for the irofulven-resistant HCT-116 cell line. Irofulven followed by 5-FU was synergistic for the two ovarian cell lines and additive for the two parental colon cell lines. These studies demonstrate that simultaneous exposure to irofulven and cisplatin is at least additive for most cell lines whereas simultaneous exposure to irofulven and 5-FU is additive to synergistic for all the cell lines tested, including the irofulven- and cisplatin-resistant variants. The enhanced cytotoxicity of irofulven in combination with cisplatin and 5-FU support the clinical application of these regimens.

Marked activity of irofulven toward human carcinoma cells: comparison with cisplatin and ecteinascidin.

PURPOSE: To characterize the activities of irofulven, a novelanticancer agent derived from the mushroom natural productilludin S toward human cancer cells. EXPERIMENTAL DESIGN: We have determined the activity spectrum of irofulven toward a human tumor cell panel comprised of 10 different tumor types in comparison with cisplatin and ET-743. We have also evaluated the influence of major resistance mechanisms, such as expression of multidrug resistance-associated drug efflux pumps, cisplatin resistance, loss of p53 function, and absence of mismatch repair on the cytotoxic activity of irofulven. RESULTS: The activity spectrum of irofulven is clearly different from that of ET-743 and cisplatin. Irofulven shows excellent cytotoxicity toward the majority of human carcinoma cell lines tested, but lesser activity toward sarcoma and leukemia cell lines. The cytotoxic activity of irofulven was particularly pronounced toward head and neck, non-small cell lung, colon, and ovary carcinoma cells, as well as toward malignant glioma cell lines. In addition, irofulven displayed good activity toward poorly differentiated, androgen-independent prostate cancer cells and cell lines expressing high levels of the detoxifying enzymes glutathione S-transferase and gamma-glutamyl cysteine synthetase. The cytotoxicity of irofulven was not affected by loss of p53 or mismatch repair function, and the drug was not a substrate for multidrug transporters, such as the P-glycoprotein and multidrug resistance protein 1. CONCLUSIONS: Irofulven has an unusual activity spectrum with strong activity toward tumor cells of epithelial origin. Furthermore, irofulven is not or only marginally affected by resistance mechanisms limiting the efficacy of other alkylating agents.

Apoptosis induction by the dual-action DNA- and protein-reactive antitumor drug irofulven is largely Bcl-2-independent.

The overexpression of Bcl-2 is implicated in the resistance of cancer cells to apoptosis. This study explored the potential of irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863), a novel DNA- and protein-reactive anticancer drug, to overcome the anti-apoptotic properties of Bcl-2 in HeLa cells with controlled Bcl-2 overexpression. Irofulven treatment resulted in rapid (12hr) dissipation of the mitochondrial membrane potential, phosphatidylserine externalization, and apoptotic DNA fragmentation, with progressive changes after 24hr. Bcl-2 overexpression caused marginal or partial inhibition of these effects after treatment times ranging from 12 to 48hr. Both Bcl-2-dependent and -independent responses to irofulven were abrogated by a broad-spectrum caspase inhibitor. Despite the somewhat decreased apoptotic indices, cell growth inhibition by irofulven was unaffected by Bcl-2 status. In comparison, Bcl-2 overexpression drastically reduced apoptotic DNA fragmentation by etoposide, acting via topoisomerase II-mediated DNA damage, but had no effect on apoptotic DNA fragmentation by helenalin A, which reacts with proteins but not DNA. Irofulven retains its pro-apoptotic and growth inhibitory potential in cell lines that have naturally high Bcl-2 expression. Collectively, the results implicate multiple mechanisms of apoptosis induction by irofulven, which may differ in time course and Bcl-2 dependence. It is possible that the sustained ability of irofulven to induce profound apoptosis and to block cell growth despite Bcl-2 overexpression may be related to its dual reactivity with both DNA and proteins.

Irofulven (6-hydroxymethylacylfulvene, MGI 114)-induced apoptosis in human pancreatic cancer cells is mediated by ERK and JNK kinases.

BACKGROUND: Pancreatic carcinoma resists chemotherapeutic mediation of apoptosis. Irofulven (MGI 114, 6-hydroxymethylacylfulvene) is a novel illudin S analogue that we have shown to induce caspase-mediated apoptosis in pancreatic carcinoma cell lines. MATERIALS AND METHODS: Westem blot analysis and kinase assays were used to demonstrate the activation of Erk 1/2 and JNK1 kinases following Irofulven administration in the presence and absence of selective kinase inhibitors. RESULTS: Irofulven activates JNK1 and Erk1/2, but not p38. The addition of the MAPK inhibitors, SB202190 and PD98059 (targeting JNK1 and Erk1/2 activation, respectively), prevents kinase activation and blocks Irofulven-induced activation of caspases -3, -7, -8 and -9. Blockade of either JNK1 or Erk1/2 results in a 50% decrease in apoptosis in MiaPaCa-2 cells treated with Irofulven. CONCLUSION: Our data demonstrated that JNK1 and Erk1/2 are activated by Irofulven treatment and that blockade of either MAPK subfamily decreases apoptosis by rendering Irofulven incapable of inducing caspase activation.