[Show abstract][Hide abstract] ABSTRACT: Gossypol is a putative BH3 mimetic proposed to inhibit BCL2 and BCLXL based on cell-free assays. We previously demonstrated that gossypol failed to directly inhibit BCL2 in cells, or induce apoptosis in chronic lymphocytic leukemia (CLL) cells or platelets, which require BCL2 or BCLXL respectively for survival. Here, we demonstrate that gossypol rapidly increased activity of phospholipase A2 (PLA2), which led to an increase in cytoplasmic calcium, endoplasmic reticulum (ER) stress, and up-regulation of the BH3-only protein NOXA. Pre-treatment with the PLA2 inhibitor, aristolochic acid, abrogated the increase in calcium, ER stress and NOXA. Calcium chelation also abrogated the gossypol-induced increase in calcium, ER stress, and NOXA, but not the increase in PLA2 activity indicating that PLA2 is upstream of these events. In addition, incubating cells with the two products of PLA2 (lysophosphatidic acid and arachidonic acid) mimicked treatment with gossypol. NOXA is a pro-apoptotic protein that functions by binding the BCL2 family proteins MCL1 and BFL1. The BCL2 inhibitor ABT-199 is currently in clinical trials for CLL. Resistance to ABT-199 can occur from up-regulation of other BCL2 family proteins, and this resistance can be mimicked by culturing CLL cells on CD154+ stroma cells. We report here that AT-101, a derivative of gossypol in clinical trials, overcomes stroma-mediated resistance to ABT-199 in primary CLL cells suggesting that a combination of these drugs may be efficacious in the clinic.
[Show abstract][Hide abstract] ABSTRACT: A lack of strategy to counteract hypoxia (pO2 < 10-15 mmHg) and technique to repeatedly measure tumor pO2 has restricted therapeutic optimization. We report the results obtained with an innovative anti-angiogenic strategy of recurrent low-dose (metronomic) chemotherapy to modulate hypoxia and growth of the Head and Neck tumor xenografts.The FaDu tumors were established in the flank of immune deficient mice and EPR oximetry with lithium phthalocyanine crystals was used to follow the temporal changes in tumor pO2 on treatment with gemcitabine including controls for three weeks. The FaDu tumors were hypoxic with a baseline (pre-treatment) pO2 of 2-8 mmHg. A transient increase in the tumor pO2 was evident on day 3 on treatment with a conventional schedule of gemcitabine (150 mg/kg, d1, d8, d15). No significant change in the tumor pO2 on treatment with metronomic gemcitabine (25 mg/kg on d1, d3, d5 for 3 weeks) was observed. However, tumor pO2 increased significantly on d15-d18 during treatment with a metronomic schedule of 15 mg/kg gemcitabine (d1, d3, d5 for 3 weeks). A modest decrease in the tumor growth was evident on treatment with conventional gemcitabine. Notably, tumor growth was significantly inhibited by metronomic (25 and 15 mg/kg) gemcitabine treatment. The immunohistochemistry (IHC) analyses of the tumor samples indicate a decrease in HIF-1α and TSP-1 on treatment with metronomic gemcitabine.In conclusion, a significant inhibition of tumor growth on treatment with metronomic gemcitabine was observed; however, the increase in pO2 was dose dependent. EPR oximetry can be used to follow the temporal changes in tumor pO2 to identify a therapeutic window on treatment with metronomic chemotherapy for potential combination with radiotherapy.
Advances in Experimental Medicine and Biology 01/2014; 812:105-11. DOI:10.1007/978-1-4939-0620-8_14 · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The feasibility of EPR oximetry using a single-probe implantable oxygen sensor (ImOS) was tested for repeated measurement of pO2 in skeletal muscle and ectopic 9L tumors in rats. The ImOS (50 mm length) were constructed using nickel-chromium alloy wires, with lithium phthalocyanine (LiPc, oximetry probe) crystals loaded in the sensor loop and coated with AF 2400(®) Teflon. These ImOS were implanted into the skeletal muscle in the thigh and subcutaneous 9L tumors. Dynamic changes in tissue pO2 were assessed by EPR oximetry at baseline, during tumor growth, and repeated hyperoxygenation with carbogen breathing. The mean skeletal muscle pO2 of normal rats was stable and significantly increased during carbogen inhalation in experiments repeated for 12 weeks. The 9L tumors were hypoxic with a tissue pO2 of 12.8 ± 6.4 mmHg on day 1; however, the response to carbogen inhalation varied among the animals. A significant increase in the glioma pO2 was observed during carbogen inhalation on day 9 and day 14 only. In summary, EPR oximetry with ImOS allowed direct and longitudinal oxygen measurements in deep muscle tissue and tumors. The heterogeneity of 9L tumors in response to carbogen highlights the need to repeatedly monitor pO2 to confirm tumor oxygenation so that such changes can be taken into account in planning therapies and interpreting results.
Advances in Experimental Medicine and Biology 01/2014; 812:97-103. DOI:10.1007/978-1-4939-0620-8_13 · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence.
Growth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant "bolus" administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models.
MK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18-24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients' plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G2 phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval.
There are two reasons why delayed addition of MK-8776 enhances sensitivity to gemcitabine: first, there is an increased number of cells arrested in S phase; and second, the arrested cells have adequate time to initiate recombination and thereby become Chk1 dependent. These results have important implications for the design of clinical trials using this drug combination.
BMC Cancer 12/2013; 13(1):604. DOI:10.1186/1471-2407-13-604 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: S1 is a putative BH3 mimetic proposed to inhibit BCL2 and MCL1 based on cell-free assays. However, we previously demonstrated that it failed to inhibit BCL2 or induce apoptosis in chronic lymphocytic leukemia (CLL) cells, which are dependent on BCL2 for survival. In contrast, we show here that S1 rapidly increases reactive oxygen species, initiates endoplasmic reticulum stress, and upregulates the BH3-only protein NOXA. The BCL2 inhibitors, ABT-737, ABT-263, and ABT-199, have demonstrated pro-apoptotic efficacy in cell lines, while ABT-263 and ABT-199 have demonstrated efficacy in early clinical trials. Resistance to these inhibitors arises from the upregulation of anti-apoptotic factors, such as MCL1, BFL1, and BCLXL. This resistance can be induced by co-culturing CLL cells on a stromal cell line that mimics the microenvironment found in patients. Since NOXA can inhibit MCL1, BFL1, and BCLXL, we hypothesized that S1 may overcome resistance to ABT-737. Here we demonstrate that S1 induces NOXA-dependent sensitization to ABT-737 in a human promyelocytic leukemia cell line (NB4). Furthermore, S1 sensitized CLL cells to ABT-737 ex vivo, and overcame resistance to ABT-737 induced by co-culturing CLL cells with stroma.
[Show abstract][Hide abstract] ABSTRACT: Conduct a proof-of-principle study to test whether c-Jun N-terminal kinase (JNK) phosphorylation and Noxa induction occur in peripheral blood chronic lymphocytic leukemia (CLL) cells in patients receiving a vincristine infusion.
Patients with CLL received 2 mg vincristine by a 5 min intravenous infusion. Blood samples were collected at baseline and up to 6 h post vincristine infusion and assayed for JNK activation, Noxa induction and vincristine plasma concentrations.
Ex vivo treated peripheral CLL cells activated JNK in response to 10-100 nM vincristine in 6 h. Noxa protein expression, while variable, was also observed over this time frame. In CLL patients, vincristine infusion led to rapid (<1 h) JNK phosphorylation in peripheral blood CLL cells which was sustained for at least 4-6 h post vincristine infusion. Noxa protein expression was not observed in response to vincristine infusion.
This study confirmed that vincristine can activate JNK but not induce Noxa in CLL cells in vivo. The results suggest that novel JNK-dependent drug combinations with vincristine warrant further investigation.
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Cancer Research 08/2013; 73(8 Supplement):3522-3522. DOI:10.1158/1538-7445.AM2013-3522 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteins of the BCL2 family provide a survival mechanism in many human malignancies including chronic lymphocytic leukemia (CLL). The BCL2 inhibitor ABT-263 (navitoclax) is active in clinical trials for lymphoid malignancies, yet resistance is expected based on preclinical models. We recently demonstrated that vinblastine can dramatically sensitize several leukemia cell lines to ABT-737 (the experimental congener of ABT-263). The goal of these experiments was to determine the impact of vinblastine on ABT-737 sensitivity in CLL cells isolated from peripheral blood and to define the underlying mechanism. Freshly isolated CLL cells from 35 patients, as well as normal lymphocytes and platelets, were incubated with various microtubule disrupting agents plus ABT-737 to assess sensitivity to the single agents and the combination. ABT-737 and vinblastine displayed a range of sensitivity as single agents, and vinblastine markedly sensitized all CLL samples to ABT-737 within 6 h. Vinblastine potently induced the pro-apoptotic protein PMAIP1 (NOXA) in both a time- and dose-dependent manner and this was required for the observed apoptosis. Combretastatin A4, which dissociates microtubules by binding a different site, had the same effect confirming that interaction of these agents with microtubules is the initial target. Similarly, vincristine and vinorelbine induced NOXA and enhanced CLL sensitivity to ABT-737. Furthermore, vinblastine plus ABT-737 overcame stroma-mediated resistance to ABT-737 alone. Apoptosis was induced with clinically achievable concentrations, with no additional toxicity to normal lymphocytes or platelets. These results suggest that vinca alkaloids may improve the clinical efficacy of ABT-263 in patients with CLL.
Molecular Cancer Therapeutics 05/2013; 12(8). DOI:10.1158/1535-7163.MCT-12-1197 · 6.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many anticancer agents damage DNA and activate cell cycle checkpoints that permit time for the cells to repair their DNA and recover. These checkpoints have undergone intense investigation as potential therapeutic targets, and Chk1 inhibitors have emerged as promising novel therapeutic agents. Chk1 was initially recognized as a regulator of the G2/M checkpoint, but has since been demonstrated to have additional roles in replication fork stability, replication origin firing and homologous recombination. Inhibition of these pathways can dramatically sensitize cells to some antimetabolites. Current clinical trials with Chk1 inhibitors are primarily focusing on their combination with gemcitabine. Here, we discuss the mechanisms of, and emerging uses for Chk1 inhibitors as single agents and in combination with antimetabolites. We also discuss the pharmacodynamic issues that need to be addressed in attaining maximum efficacy in vivo. Following administration of gemcitabine to mice and humans, tumor cells accumulate in S phase for at least 24 h before recovering. In addition, stalled replication forks evolve over time to become more Chk1 dependent. We emphasize the need to assess cell cycle perturbation and Chk1 dependence of tumors in patients administered gemcitabine. These assessments will define the optimum dose and schedule for administration of these drug combinations.
British Journal of Clinical Pharmacology 04/2013; 76(3). DOI:10.1111/bcp.12139 · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proteasome inhibitor bortezomib (Velcade) is prescribed for the treatment of multiple myeloma. Clinically achievable concentrations of bortezomib cause less than 85% inhibition of the chymotrypsin-like activity of the proteasome, but little attention has been paid as to whether in vitro studies are representative of this level of inhibition. Patients receive bortezomib as an intravenous or subcutaneous bolus injection, resulting in maximum proteasome inhibition within one hour followed by a gradual recovery of activity. In contrast, most in vitro studies use continuous treatment so that activity never recovers. Replacing continuous treatment with 1 h-pulse treatment increases differences in sensitivity in a panel of 7 multiple myeloma cell lines from 5.3-fold to 18-fold, and reveals that the more sensitive cell lines undergo apoptosis at faster rates. Clinically achievable inhibition of active sites was sufficient to induce cytotoxicity only in one cell line. At concentrations of bortezomib that produced similar inhibition of peptidase activities a different extent of inhibition of protein degradation was observed, providing an explanation for the differential sensitivity. The amount of protein degraded per number of active proteasomes correlated with sensitivity to bortezomib. Thus, (i) in vitro studies of proteasome inhibitors should be conducted at pharmacologically achievable concentrations and duration of treatment; (ii) a similar level of inhibition of active sites results in a different extent of inhibition of protein breakdown in different cell lines, and hence a difference in sensitivity.
PLoS ONE 02/2013; 8(2):e56132. DOI:10.1371/journal.pone.0056132 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conjugated linoleic acid (CLA) is widely used as a "nutraceutical" for weight loss. CLA has anticancer effects in preclinical models, and we demonstrated in vitro that this can be attributed to the suppression of fatty acid (FA) synthesis. We tested the hypothesis that administration of CLA to breast cancer patients would inhibit expression of markers related to FA synthesis in tumor tissue, and that this would suppress tumor proliferation. Women with Stage I-III breast cancer were enrolled into an open label study and treated with CLA (1:1 mix of 9c,11t- and 10t,12c-CLA isomers, 7.5 g/d) for ≥10 days before surgery. Fasting plasma CLA concentrations measured pre- and post-CLA administration, and pre/post CLA tumor samples were examined by immunohistochemistry for Spot 14 (S14), a regulator of FA synthesis, FA synthase (FASN), an enzyme of FA synthesis, and lipoprotein lipase (LPL), the enzyme that allows FA uptake. Tumors were also analyzed for expression of Ki-67 and cleaved caspase 3. 24 women completed study treatment, and 23 tumors were evaluable for the primary endpoint. The median duration of CLA therapy was 12 days, and no significant toxicity was observed. S14 expression scores decreased (p = 0.003) after CLA administration. No significant change in FASN or LPL expression was observed. Ki-67 scores declined (p = 0.029), while cleaved caspase 3 staining was unaffected. Decrements in S14 or Ki-67 did not correlate with fasting plasma CLA concentrations at surgery. Breast tumor tissue expression of S14, but not FASN or LPL, was decreased after a short course of treatment with 7.5 g/day CLA. This was accompanied by reductions in the proliferation index. CLA consumption was well-tolerated and safe at this dose for up to 20 days. Overall, CLA may be a prototype compound to target fatty acid synthesis in breast cancers with a "lipogenic phenotype".
Breast Cancer Research and Treatment 02/2013; 138(1):175-83. DOI:10.1007/s10549-013-2446-9 · 4.20 Impact Factor