Janet A. Houghton

Efficacy of topoisomerase I inhibitors, topotecan and irinotecan, administered at low dose levels in protracted schedules to mice bearing xenografts of human tumors

The efficacy of protracted schedules of therapy of the topoisomerase I inhibitors 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan) and 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (irinotecan; CPT-11) were evaluated against a panel of 21 human tumor xenografts derived from adult and pediatric malignancies. Tumors included eight colon adenocarcinomas, representing an intrinsically chemorefractory malignancy, six lines derived from childhood rhabdomyosarcoma (three embryonal, three alveolar) representing a chemoresponsive histiotype, sublines of rhabdomyosarcomas selected in vivo for resistance to vincristine and melphalan, and three pediatric brain tmors. All tumors were grown at the subcutaneous site. Topotecan was administered by oral gavage 5 days per week for 12 consecutive weeks. The maximum tolerated dose (MTD) was 1.5 mg/kg per dose. Irinotecan was given by i.v. administration daily for 5 days each week for 2 weeks [(d×5)2] (one cycle of therapy), repeated every 21 days. The MTD for three cycles was 10 mg/kg per dose. Treatment was started against advanced tumors. Topotecan caused a high frequency of objective regressions in one of eight colon tumor lines, whereas irinotecan caused complete regressions (CR) of all tumors in three colon lines and a high frequency of CRs in three additional lines. Both drugs demonstrated similar activity against rhabdomyosarcoma xenografts. Topotecan caused CR of all tumors in four of six lines, and irinotecan in five of six lines evaluated. Both agents retained full activity against tumors selected for primary resistance to vincristine, but only irinotecan retained activity against a tumor selected for primary resistance to melphalan. Both agents demonstrated good activity against brain tumor xenografts with irinotecan causing CR in two of three lines and topotecan inducing CR in one of three lines. Results indicate that low-dose protracted schedules of daily administration of these topoisomerase I inhibitors is either equi-effective or more efficacious than more intense shorter schedules of administration reported previously.

Evaluation of 9-dimethylaminomethyl- 10-hydroxycamptothecin against xenografts derived from adult and childhood solid tumors

The topoisomerase I inhibitor 9-dimethylaminomethyl-10-hydroxycamptothecin (topotecan) was evaluated against a panel of xenografts comprising four lines of adult colon adenocarcinoma, three colon tumors derived from adolescents, six childhood rhabdomyosarcomas from previously untreated patients as well as sublines selected in vivo for resistance to vincristine and melphalan, and three lines of childhood osteogenic sarcoma. Efficacy was determined at maximal tolerated dose levels using intermittent i.p. administration [every 4 days for 4 doses (q4d×4)] or daily p.o. or i. p. administration 5 days per week for up to 20 courses. On a q4d×4 schedule, the maximum tolerated dose (MTD) was 12.5 mg/kg per administration, which caused marked weight loss and lethality in ≈5% of the tumor-bearing mice. This schedule caused significant growth inhibition (but no tumor regression) in advanced adult colon adenocarcinomas. The minimal treated/control (T/C) ratios were 0.49, 0.54, and 0.3 for three of the tumor lines and were achieved at 18–21 days after the initiation of treatment. In contrast, rhabdomyosarcomas were considerably more sensitive, with T/C ratios being <0.1 for three lines, whereas topotecan was less active against two other rhabdomyosarcoma xenografts (minimal T/C ratios, 0.17 and 0.14). As inhibitors of topoisomerase I have been demonstrated to have activity in the replication phase of the cell cycle (S-phase-specific), prolonged administration schedules were examined. Mice received topotecan 5 days per week for 3 weeks either by i.p. injection or by oral gavage (p.o.). In selected experiments, p.o. administration was continued for up to 20 weeks. Oral administration for 3 weeks (2 mg/kg per dose) resulted in complete regression of all six lines of rhabdomyosarcoma, with two lines demonstrating no regrowth during the period of observation (≥84 days). Similar results were obtained after i.p. administration, suggesting significant schedule dependency for these tumors. For colon tumors, the daily administration schedule (i.p. or p.o.) demonstrated some advantage over the intermittent schedule, resulting in partial regressions and significant inhibition of the growth of several colon adenocarcinoma lines. In rhabdomyosarcoma Rh 12 and VRC5 colon adenocarcinoma, both of which demonstrated intermediate sensitivity to topotecan, and in osteosarcoma OS33, protracted p.o. administration for 13–20 weeks (1.0–1.5 mg/kg per dose given daily x 5 days) caused complete regression without regrowth in Rh12 and OS33 tumors and partial regression of all VRC5 tumors. No toxicity was observed using this schedule of administration. Topotecan demonstrated significant activity against all three osteosarcoma xenografts examined, with optimal schedules causing complete regression in two lines. Topotecan demonstrated similar activity against KB 3-1 and KB 8-5 multidrug-resistant cells in culture, and the Rh 12/VCR an Rh 18/VCR xenografts selected for vincristine (VCR) resistance in vivo were as sensitive as their parental lines. However, Rh 28/L-PAM, selected for resistance to melphalan, was cross-resistant to topotecan. Plasma pharmacokinetics studies were carried out at the respective MTD for oral (2 mg/kg) or i.p. (1.75 mg/kg) administration. During oral administration the maximal plasma concentration (of the active lactone) was achieved at 0.25 h (Cmax 41.7 ng/ml) and thet1/2α andt1/2β values were 0.55 and 2.8 h, respectively. Administration i.p. resulted in peak plasma levels of 523 ng/ml, witht1/2α andt1/2β elimination rates being 0.29 and 2.5 h, respectively. Although i.p. administration resulted in a 3-fold increase in AUC as compared with oral dosing, similar antitumor activity was observed against most xenograft lines. These results suggest that topotecan may have significant activity against several human cancers and that its efficacy may be schedule-dependent. Topotecan may have a particular role to play in the treatment of childhood solid tumors such as rhabdomyosarcoma and osteosarcoma.

Shared Pathways: Death Receptors and Cytotoxic Drugs in Cancer Therapy *

Death ligands (TNF, FasL, TRAIL) and their respective death receptor signaling pathways can be used to induce tumor cells to undergo apoptosis. Chemotherapeutic drugs can induce apoptosis and the upregulation of death ligands or their receptors. Downstream events following cytotoxic stressinduced DNA damage and the signaling pathways that lead to the induction of apoptosis may be either dependent or independent of death receptor signaling. The involvement of the Fas signaling pathway in chemotherapy-induced apoptosis has been the most extensively studied, with the current emergence of information on the TRAIL signaling pathway. Fas-mediated and chemotherapy-induced apoptosis can converge at the level of the receptor, FasL, DISC formation, activation of the initiator caspase-8, at the level of the mitochondria, or at the level of downstream effector caspase activation. Convergence is influenced by the specific form of DNA damage, the cellular environment, and the specific pathway(s) by which death receptor-mediated or drug-mediated apoptosis are induced. This review discusses the different levels of interaction between signaling pathways in the different forms of cell death.

Hedgehog Signaling Drives Cellular Survival in Human Colon Carcinoma Cells

Aberrant activation of Hedgehog (HH) signaling is implicated in many human cancers. Classical HH signaling is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which transcriptionally regulate target genes. A small molecule inhibitor of Gli1 and Gli2, GANT61, was used to block HH signaling in human colon carcinoma cell lines that express HH signaling components. GANT61 administration induced robust cytotoxicity in 5 of 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In comparison, the classical Smo inhibitor, cyclopamine, induced modest cytotoxicity. Further, GANT61 treatment abolished the clonogenicity of all six human colon carcinoma cell lines. Analysis of the molecular mechanisms of GANT61-induced cytotoxicity in HT29 cells showed increased Fas expression and decreased expression of PDGFRα, which also regulates Fas. Furthermore, DR5 expression was increased whereas Bcl-2 (direct target of Gli2) was downregulated following GANT61 treatment. Suppression of Gli1 by shRNA mimicked the changes in gene expression observed in GANT61-treated cells. Overexpression of dominant-negative FADD (to abrogate Fas/DR5-mediated death receptor signaling) and/or Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued GANT61-induced cytotoxicity in HT29 cells. Thus, activated GLI genes repress DR5 and Fas expressions while upregulating Bcl-2 and PDGFRα expressions to inhibit Fas and facilitate cell survival. Collectively, these results highlight the importance of Gli activation downstream of Smo as a therapeutic target in models of human colon carcinoma.

Myc regulates aggresome formation, the induction of Noxa, and apoptosis in response to the combination of bortezomib and SAHA

The histone deacetylase inhibitor SAHA enhances cell death stimulated by the proteasome inhibitor bortezomib (BZ) by disrupting BZ-induced aggresome formation. Here we report that Myc regulates the sensitivity of multiple myeloma (MM) cells to BZ + SAHA-induced cell death. In MM cells, Myc expression directly correlated with intracellular ER content, protein synthesis rates, the percentage of aggresome-positive cells, and the sensitivity to BZ + SAHA-induced cell death. Accordingly, Myc knockdown markedly reduced the sensitivity of MM cells to BZ + SAHA-mediated apoptosis. Furthermore, activation of Myc was sufficient to provoke aggresome formation and thus sensitivity to BZ + SAHA, and these responses required de novo protein synthesis. BZ + SAHA-mediated stimulation of apoptosis includes the induction of the proapoptotic BH3-only protein Noxa as well as endoplasmic reticular stress, a disruption of calcium homeostasis, and activation of capase-4. Finally, knockdown studies demonstrated that both caspase-4 and Noxa play significant roles in Myc-driven sensitivity to BZ + SAHA-induced apoptosis. Collectively, our results establish a mechanistic link between Myc activity, regulation of protein synthesis, increases in HDAC6 expression and aggresome formation, induction of Noxa, and sensitivity to BZ + SAHA-induced apoptosis. These data suggest that MM patients with elevated Myc activity may be particularly sensitive to the BZ + SAHA combination.

Therapeutic efficacy of the topoisomerase I inhibitor 7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxy-camptothecin against pediatric and adult central nervous system tumor xenografts

Abstract Therapy of patients with malignant central nervous system tumors is frequently unsuccessful, reflecting limitations of current surgical, radiotherapeutic, and pharmacotherapeutic treatments. The camptothecin derivative irinotecan (CPT-11) has been shown to possess antitumor activity in phase II trials for patients with carcinoma of the lung, cervix, ovary, colon, or rectum and for patients with non-Hodgkin’s lymphoma. The current study was designed to test the efficacy of the drug against a panel of human tumor xenografts derived from adult and pediatric central nervous system malignancies. Tumors included childhood high-grade gliomas (D-212 MG, D-456 MG), adult high-grade gliomas (D-54 MG, D-245 MG), medulloblastomas (D341 Med, D487 Med), ependymomas (D528 EP, D612 EP), and a rhabdomyosarcoma (TE-671), as well as sublines with demonstrated resistance to busulfan (D-456 MG (BR)), cyclophosphamide (TE-671 CR), procarbazine (D-245 MG (PR)) or melphalan (TE-671 MR), growing subcutaneously and intracranially in athymic nude mice. In replicate experiments, CPT-11 was given at a dosage of 40 mg/kg per dose via intraperitoneal injection in 10% dimethylsulfoxide on days 1–5 and 8–12, which is the dosage lethal to 10% of treated animals. CPT-11 produced statistically significant (P<0.001) growth delays in all subcutaneous xenografts tested, including those resistant to busulfan, cyclophosphamide, procarbazine, and melphalan, with growth delays ranging from 21.3 days in D487 Med to 90+ days in several tumor lines. Further, tumor regression was evident in every treated animal bearing a subcutaneous tumor, with some xenografts yielding complete tumor regression. Statistically significant (P<0.001) increases in survival were demonstrated in the two intracranial xenografts – D341 EP (73.0% increase) and D-456 MG (114.2% increase) – treated with CPT-11. These studies demonstrate that, of over 40 drugs evaluated in this laboratory, CPT-11 is the most active against central nervous system xenografts and should be advanced to clinical trial as soon as possible.

Phase II testing of melphalan in children with newly diagnosed rhabdomyosarcoma: a model for anticancer drug development.

We describe events that led to successful testing of melphalan, one of the nitrogen mustard compounds, in children with newly diagnosed, poor-risk rhabdomyosarcoma (RMS). Preclinical studies with xenografts of human RMS, growing in the flanks of immune-deprived mice, had indicated superior oncolytic activity by melphalan compared with other agents commonly used to treat this tumor. However, in a conventional phase II trial, melphalan failed to produce partial responses in 12 of 13 heavily pretreated patients with recurrent tumors. Subsequent comparison of the drugs pharmacokinetics in mice and patients indicated that its poor clinical performance was not the result of interspecies differences in drug disposition. Therefore, we elected to retest melphalan in untreated patients, before they were enrolled in a phase III study. Of 13 children who received the drug for 6 weeks, ten had partial responses, confirming the significant antitumor activity seen in the xenograft system. These findings illustrate the inherent limitations of phase II drug trials in previously treated patients and suggest a useful paradigm for the development of antineoplastic drugs.

Blocking Hedgehog Survival Signaling at the Level of the GLI Genes Induces DNA Damage and Extensive Cell Death in Human Colon Carcinoma Cells

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

Casein kinase II (CK2) enhances death-inducing signaling complex (DISC) activity in TRAIL-induced apoptosis in human colon carcinoma cell lines.

Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli, as well as being elevated in many human cancers examined. We have demonstrated that CK2 is a key survival factor that protects human colon carcinoma cells from TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. We determined that inhibition of CK2 phosphorylation events by DRB (5,6-dichlorobenzimidazole) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis, in the absence of effects in normal cells. Sensitization was caspase dependent, and independent of regulation via NF-κB. Further, inhibition of phosphorylation by CK2 did not modify the expression level of antiapoptotic proteins. Analysis of TRAIL-induced death-inducing signaling complex (DISC) formation demonstrated enhanced formation of the DISC, enhanced cleavage of caspase-8 and cleavage of Bid in the presence of DRB, thereby facilitating the release of proapoptotic factors from the mitochondria with subsequent downregulation of the expression of XIAP and c-IAP1. Further, silencing of CK2α in HT29 cells following transfection of CK2α shRNA abrogated CK2 kinase activity while simultaneously increasing TRAIL sensitivity. These findings demonstrate that CK2 plays a critical antiapoptotic role by conferring resistance to TRAIL at the level of the DISC.

Reactive Oxygen Species Regulate Caspase Activation in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Human Colon Carcinoma Cell Lines

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.