Timothy Korbut

Comparison of several antiangiogenic regimens alone and with cytotoxic therapies in the Lewis lung carcinoma

Abstract The efficacy of several potential antiangiogenic agents, TNP-470, minocycline, suramin, genistein, interferon δ4, 14(sulfated)-β-cyclodextrin and tetrahydrocortisol, alone and in combination with cytotoxic therapies was examined against primary and metastatic Lewis lung carcinoma. The antiangiogenic agents when administered as single agents or in two-agent combinations were only modestly active as antitumor agents. Three antiangiogenic agent combinations, TNP-470/minocycline, TNP-470/14(SO4)βCD/THC and minocycline/14(SO4)βCD/THC, produced significant increases in tumor growth delay and decreases in the number of lung metastases when administered along with cyclophosphamide compared with cyclophosphamide alone. Two antiangiogenic agent combinations, minocycline/interferon δ4 and minocycline/14 (SO4)βCD/THC, produced significant decreases in the number of lung metastases when administered alone with adriamycin compared with adriamycin alone. The antiangiogenic combinations of TNP-470/minocycline, TNP-470/suramin, TNP-470/genistein, TNP-470/interferon δ4 and TNP-470/14(SO4)βCD/THC, resulted in increased tumor growth delays when administered along with CDDP, BCNU, fractionated radiation or 5-fluorouracil. There was not always a direct correlation between the antiangiogenic regimen that was most beneficial against the primary tumor as compared with disease metastatic to the lungs. These studies establish that a broad range of antiangiogenic therapies can interact in a positive manner with cytotoxic therapies.

Cyclooxygenase and lipoxygenase inhibitors as modulators of cancer therapies

Like many clinical non-small-cell lung cancers, the Lewis lung carcinoma produces prostaglandins. The Lewis lung carcinoma was used as a model of both primary and metastatic disease to assess the ability of cyclooxygenase inhibitors (mefenamic acid, diflunisal, sulindac, and indomethacin), the collagenase inhibitor minocycline, and the lipoxygenase inhibitor phenidone to act as modulators of cytotoxic cancer therapies. Although none of the single modulators given i.p. daily on days 4–18 altered tumor growth or the number of metastases found on day 20, modulator combinations consisting of minocycline/a cyclooxygenase inhibitor and, especially, of phenidone/a cyclooxygenase inhibitor resulted in modest tumor growth delay and a decreased number of lung metastases on day 20. The most effective modulators of cisplatin (CDDP) were phenidone/sulindac and phenidone/indomethacin, which led to 2.4- to 2.5-fold increases in the tumor growth delay produced by CDDP. The most effective modulations of cyclophosphamide resulted from administration of minocycline, minocycline/sulindac, or phenidone/sulindac and led to 2.0- to 2.1-fold increases in tumor growth delay by cyclophosphamide. The most effective modulators of melphalan produced 4.5- to 4.7-fold increases in tumor growth delay by the drug and were minocycline/sulindac, minocycline/mefenamic acid, and phenidone/sulindac. The most effective modulation of carmustine (BCNU) was obtained with minocycline/sulindac and minocycline/diflunisal leading to 2.8- to 3.1-fold increases in tumor growth delay by BCNU. Finally, the most effective modulation of radiation was obtained with minocycline/sulindac and phenidone/sulindac and resulted in 2.8- to 3.3-fold increases in tumor growth delay by radiation. The modulator combination that along with the cytotoxic therapies was most effective against metastatic disease was phenidone/mefenamic acid. There was no clear relationship between effective modulation of the cancer therapies and the degree of reduction in serum levels of prostaglandin E2 and leukotriene B4 by the agents in Lewis lung tumor bearing mice.

CAI: effects on cytotoxic therapies in vitro and in vivo.

CAI (NSC 609974; L651582), a new agent that has demonstrated antimetastatic activity in vitro and in vivo, was not very cytotoxic toward EMT-6 mouse mammary carcinoma cells in culture or toward FSaIIC fibrosarcoma cells in vivo. Coexposure of EMT-6 cells to CAI and antitumor alkylating agents under various environmental conditions did not markedly increase the cytotoxicity of cisplatin (CDDP), melphalan, or carmustine (BCNU). However, the combination of CAI and 4-hydroperoxycyclophosphamide (4-HC) produced much greater than additive killing of EMT-6 cells. CAI also increased the sensitivity of hypoxic EMT-6 cells to X-rays. CAI increased the cytotoxicity of cyclophosphamide toward FSaIIC tumor cells when animals were treated with single doses of both drugs. The effect of CAI on tumor cell killing by cyclophosphamide was greatest at high doses of the antitumor alkylating agent. CAI administration appeared to result in increased serum levels of prostaglandin E2 and leukotriene B4 in animals bearing the Lewis lung tumor. Administration of CAI on days 4–18 did not alter the growth of the Lewis lung carcinoma but did result in an increase in the tumor-growth delay produced by treatment with CDDP, cyclophosphamide, melphalan, BCNU, and fractionated radiation. Although CAI did not reduce the number of lung metastases present in Lewis lung carcinoma-bearing mice on day 20, it did appear to reduce the number of large (vascularized) metastases present on that day.

Modulation of antitumor alkylating agents by novobiocin, topotecan, and lonidamine

Topoisomerase I and topoisomerase II allow a metabolically active cell to mobilize its supercoiled chromosomal DNA and undergo replication, transcription, recombination, and repair. Several topoisomerase inhibitors have recently been shown to be active in preclinical systems. Topotecan (SK&F 104864), a water-soluble camptothecin analog, is an inhibitor of topoisomerase I. Novobiocin is an inhibitor of toposiomerase II. Lonidamine depletes cellular adenosine 5′-triphosphate (ATP) and may impede energy-dependent DNA repair, MCF-7 human breast-cancer cells were treated in vitro with topotecan, novobiocin, and lonidamine alone, in paired combinations, and in combination with CDDP and melphalan. The three enzyme inhibitors alone and in combination did not increase tumor cell sensitivity to CDDP. However, the combinations of topotecan/novobiocin and lonidamine/novobiocin did enhance the cytotoxicity of melphalan. Mice bearing the FSaII fibrosarcoma were treated in vivo with topotecan, novobiocin, and lonidamine alone, in paired combinations, and in combination with CDDP, melphalan, BCNU, and cyclophosphamide. The combination of topotecan/novobiocin had the greatest impact on tumor cell sensitivity to each cytotoxic agent tested in both tumor cell-survival and tumor growth-delay assays. This sensitization was greatest at the highest concentrations of the cytotoxic agent tested. Combinations of topoisomerase I and topoisomerase II inhibitors may be useful as modulators of antitumor alkylating agents.

Whole-body hyperthermia as an adjuvant to treatment with platinum complexes with or without etanidazole in mice bearing the Lewis lung carcinoma or the FSaLL fibrosarcoma

The response of s.c. primary and metastatic Lewis lung carcinoma to five antitumour platinum complexes with or without tolerable whole-body hyperthermia (60 min to reach temperature then 60 min at 42 degrees C) was examined. The whole-body hyperthermia treatment produced about 2.8 days of tumour growth delay in the s.c. tumours. The addition of whole-body hyperthermia to treatment with each of the platinum complexes was well tolerated by the animals and increases of 1.6-2.0-fold in tumour growth delay resulted with the combined treatment compared with the platinum complexes alone. The combination of etanidazole (1 g/kg) and the platinum complexes followed by whole-body hyperthermia produced marked increases in tumour growth delay ranging from 2.5- to 3.6-fold over the growth delays obtained with the platinum complexes alone. FSaLLC tumour cell survival and bone marrow CFU-GM experiments indicated that local hyperthermia (43 degrees C, 30 min) produced greater potentiation of the cytotoxicity of three platinum complexes than did whole-body hyperthermia (42 degrees C, 60 min). Only the complete treatments including whole-body hyperthermia/etanidazole and the platinum complexes were effective in significantly reducing the numbers of lung metastases formed from s.c. primary tumours. Serum urea nitrogen and creatinine levels were monitored over a time-course post-treatment. Although some treatment combinations caused elevations in these normal tissue parameters by day 12 post-treatment both serum urea nitrogen and serum creatinine returned to the levels of the untreated control animals.

Combination of the minor groove-binder U73-975 or the intercalator mitoxantrone with antitumor alkylating agents in MCF-7 or MCF-7/CP cells.

In an effort to improve the cytotoxicity of clinically used anticancer alkylating agents, the topoisomerase II inhibitory drugs U73-975 or mitoxantrone were added to cell cultures exposed to CDDP, carboplatin, BCNU, melphalan or thiotepa. In the MCF-7 human breast cancer cell line and in the MCF-7/CP (CDDP resistant) subline, U73-975 and mitoxantrone were both potent cytotoxic agents (IC50 0.002 microM and 0.006 microM for U73-975, respectively and 0.8 microM and 0.1 microM for mitoxantrone, respectively). As evaluated by isobologram analysis, the addition of either U73-975 or mitoxantrone to 1 h exposure to CDDP resulted in greater-than-additive killing in the MCF-7 parent cells. While U73-975 was also greater-than-additive in cytotoxicity with CDDP in the MCF-7/CP line, mitoxantrone and CDDP were only additive in cytotoxicity in these cells. In the case of carboplatin, the addition of U73-975 or mitoxantrone to treatment with the drug resulted in greater-than-additive cell killing in the MCF-7 parental cell line but in the MCF-7/CP cell line these combinations were only additive in cell killing. Addition of U73-975 to treatment with BCNU resulted in only additive cytotoxicity in both cell lines; however, the combination of mitoxantrone with BCNU resulted in greater-than-additive cell killing in both the parental and CDDP resistant cell lines. When either U73-975 or mitoxantrone was added to treatment with melphalan greater-than-additive cytotoxicity resulted in both cell lines except at low melphalan concentrations in the MCF-7/CP cell line. Finally, the addition of either modulator to treatment with thiotepa in the MCF-7 cell line produced variable interactions depending on thiotepa concentration, but in the MCF-7/CP cell line either modulator in combination with thiotepa caused greater-than-additive cell killing. These results indicate that the addition of topoisomerase II inhibitory drugs may substantially increase the cytotoxicity of some alkylating agents. In vivo experiments are necessary, however, to ascertain whether a therapeutic gain is achievable.

Cytotoxicity of antitumor platinum complexes withl-buthionine-(R, S)-sulfoximine and/or etanidazole in human carcinoma cell lines sensitive and resistant to cisplatin

Human 2008 ovarian carcinoma cells and the C13 CDDP-resistant subline and human MCF-7 breast carcinoma cells and the MCF-7/CDDP CDDP-resistant subline were exposed tol-buthionine-(S, R)-sulfoximine (50 μM) for 48 h prior to and during exposure for 1 h to the antitumor platinum complexes,cis-diamminedichloroplatinum(II), carboplatin or D,L-tetraplatin and/or to etanidazole (1 mM) for 2 h prior to and during exposure for 1 to the antitumor platinum complexes. These modulators alone did not significantly alter the cytotoxicity of CDDP toward either parental line. A twofold enhancement in cytotoxicity was observed with carboplatin in the 2008 cells and with D,L-tetraplatin in both parental lines with the single modulators. The modulator combination (buthionine sulfoximine/etanidazole) was very effective along with D,L-tetraplatin in both the MCF-7 parent and MCF-7/CDDP cell lines where at the higher platinum complex concentrations there was 1.5 to 3 logs increased killing of cells by the drug plus the modulators compared with the drug alone. Similarly, when C13 cells were exposed to CDDP (100 μM) or D,L-tetraplatin (100 μM) along with buthionine sulfoximine and etanidazole there was a 2-log increase in cell killing compared with exposure to the platinum complex alone. Treatment of each of the four cell lines with buthionine sulfoximine decreased both the non-protein and total sulfhydryl content of the cells. Treatment with the combination of modulators did not produce a further decrease in cellular sulfhydryl content compared with buthionine sulfoximine alone. The total sulfhydryl content in MCF-7 cells and 2008 cells exposed to buthionine sulfoximine and etanidazole was 58% and 31% of normal and the total sulfhydryl content of MCF-7/CDDP cells and C13 cells treated the same way was 54% and 23% of normal, respectively. DNA alkaline elution was used to assess the impact of exposure to the modulators, buthionine sulfoximine and etanidazole, alone and in combination on the cross linking of DNA by the antitumor platinum complexes in the MCF-7 and MCF-7/CDDP cell lines. Overall, the increases in DNA cross linking factors were greater in the MCF-7 cells than in the MCF-7/CDDP cells. These results indicate a possible clinical potential for this modulator combination.

Abstract 5108: The Hsp90 inhibitor ganetespib is a potent chemosensitizer in preclinical colorectal cancer models

Background: More than half of all individuals diagnosed with colorectal cancer (CRC) will develop metastases and require chemotherapy. These treatments have significantly improved overall patient survival; however, drug resistance is a frequent cause of treatment failure often due to alteration in cell cycle regulators or DNA damage repair. The molecular chaperone HSP90 plays an important role in both processes by stabilizing proteins required for pathway activation and maintenance. Here, we sought to determine if HSP90 inhibition by the investigational drug ganetespib could enhance the activity of standard chemotherapeutics utilized in CRC in order to extend responses and bypass/delay drug resistance. Results: Western blot analyses revealed that low nanomolar ganetespib treatment promoted the degradation of a number of proteins involved in proliferation (EGFR, PDGFR, PI3-K/AKT/p70 S6 kinase, cMET and MAPK), survival (MCL1), anigiogenesis (VEGFR), cell cycle check points and DNA repair (CDK1, CHK1, Survivin, WEE1) in a panel of CRC cell lines. This activity directly correlated with G0/G1 cell cycle arrest and loss of CRC cell viability (average IC 50 in 19 CRC cell lines = 43 nM). In vitro combination of ganetespib with conventional chemotherapies (cisplatin, oxaliplatin, 5-FU) augmented the accumulation of DNA damage (phospho-H2AX) and mitotic catastrophe more effectively over monotherapy alone in HCT116, HT29 and LoVo CRC cells. Ganetespib also sensitized CRC cells to ionizing radiation by inducing aberrant mitosis and enhanced DNA damage/fragmentation, resulting in a significant increase in apoptosis. In vivo, ganetespib displayed comparable antitumor activity as capecitabine, cisplatin, and bevacizumab in human CRC tumor xenografts, with treatment/control values of ∼50 indicative of slowly progressive disease. Combining ganetespib with capecitabine resulted in significant tumor regression (T/C = -52, p Conclusions: The HSP90 inhibitor ganetespib promotes the destabilization of numerous proteins essential for tumor growth and survival as well as those involved in chemotherapeutic resistance. As a result, ganetespib sensitizes colon cancer tumor models to chemotherapy and targeted agents, including VEGF inhibitors. Ganetespib is currently being evaluated in combination with capecitabine and radiation in patients with locally-advanced rectal cancer as part of an open-label, investigator-sponsored Phase 1 clinical study. Citation Format: Suqin He, Don Smith, Manuel Sequeira, John-Paul Jimenez, Chaohua Zhang, Jim Sang, Timothy Korbut, Jaime Acquaviva, Masazumi Nagai, Richard Bates, David A. Proia. The Hsp90 inhibitor ganetespib is a potent chemosensitizer in preclinical colorectal cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5108. doi:10.1158/1538-7445.AM2014-5108

Synergism between photochemical and ionizing radiation effects in MCF-7 cells in vitro

Both PtCl4 (Rh-123)2 and PtCl4 (Nile Blue)2 interact positively in trimodality therapy including each drug, x-ray, and light (400 - 800 nm) in killing human mammary carcinoma cells (MCF-7) in vitro. The combination treatment results in more than additive killing as assessed by product of the surviving fractions and a significant reduction of the shoulder of the x-ray survival curve. Both of the drugs participate in photodynamic therapy (PDT) in drug and light dose dependent manner. The primary subcellular targets for the neutral platinum complexes were indicated to be the nuclear DNA as opposed to the mitochondria for Rh-123 or the lysosomes for Nile Blue. Because the nuclear DNA is also the primary target for ionizing radiation, drug plus light plus x-ray might cause supraadditive killing. Both PtCl4 (Rh-123)2 and PtCl4 (Nile Blue)2 have been reported to be relatively non-toxic in vivo compared to the anionic or cationic compounds currently being used in photodynamic therapy. Based on these results PtCl4 (Rh-123)2 and PtCl4 (Nile Blue)2 have the potential for use in photodynamic therapy and in trimodality therapy.