Rob Robey

Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression

MCF‐7 human breast cancer cells selected in Adriamycin in the presence of verapamil developed a multidrug resistant phenotype, which was characterized by as much as 100,000‐fold resistance to mitoxantrone, 667‐fold resistance to daunorubicin, and 600‐fold resistance to doxorubicin. Immunoblot and PCR analyses demonstrated no increase in MDR‐1 or MRP expression in resistant cells, relative to parental cells. This phenotype is similar to one previously described in mitoxantrone‐selected cells. The cells, designated MCF‐7 AdVp, displayed a slower growth rate without alteration in topoisomerase IIα level or activity. Increased efflux and reduced accumulation of daunomycin and rhodamine were observed when compared to parental cells. Depletion of ATP resulted in complete abrogation of efflux of both daunomycin and rhodamine. No apparent alterations in subcellular daunorubicin distribution were observed by confocal microscopy. No differences were noted in intracellular pH. Molecular cloning studies using DNA differential display identified increased expression of the alpha subunit of the amiloride‐sensitive sodium channel in resistant cells. Quantitative PCR studies demonstrated an eightfold overexpression of the alpha subunit of the Na+ channel in the resistant subline. This channel may be linked to the mechanism of drug resistance in the AdVp cells. The results presented here support the hypothesis that a novel energy‐dependent protein is responsible for the efflux in the AdVp cells. Further identification awaits molecular cloning studies. J. Cell. Biochem. 65:513–526.

Inhibitors of histone deacetylases alter kinetochore assembly by disrupting pericentromeric heterochromatin.

The kinetochore, a multi-protein complex assembled on centromeric chromatin in mitosis, is essential for sister chromosome segregation. We show here that inhibition of histone deacetylation blocks mitotic progression at prometaphase in two human tumor cell lines by interfering with kinetochore assembly. Decreased amounts of hBUB1, CENP-F and the motor protein CENP-E were present on kinetochores of treated cells. These kinetochores failed to nucleate and inefficiently captured microtubules, resulting in activation of the mitotic checkpoint. Addition of histone deacetylase inhibitors prior to the end of S-phase resulted in decreased HP1-? on pericentromeric heterochromatin in S-phase and G2, decreased pericentromeric targeting of Aurora B kinase, resulting in decreased pre-mitotic phosphorylation of pericentromeric histone H3(S10) in G2, followed by assembly of deficient kinetochores in M-phase. HP1-?, Aurora B and the affected kinetochore proteins all were present at normal levels in treated cells; thus, effects of the inhibitors on mitotic progression do not seem to reflect changes in gene expression. In vitro kinase activity of Aurora B isolated from treated cells was unaffected. We propose that the increased presence in pericentromeric heterochromatin of histone H3 acetylated at K9 is responsible for the mitotic defects resulting from inhibition of histone deacetylation.

Phase I Study of Infusional Paclitaxel in Combination With the P-Glycoprotein Antagonist PSC 833

PURPOSE PSC 833 (valspodar) is a second-generation P-glycoprotein (Pgp) antagonist developed to reverse multidrug resistance. We conducted a phase I study of a 7-day oral administration of PSC 833 in combination with paclitaxel, administered as a 96-hour continuous infusion. PATIENTS AND METHODS Fifty patients with advanced cancer were enrolled onto the trial. PSC 833 was administered orally for 7 days, beginning 72 hours before the start of the paclitaxel infusion. Paclitaxel dose reductions were planned because of the pharmacokinetic interactions known to occur with PSC 833. RESULTS In combination with PSC 833, maximum-tolerated doses were defined as paclitaxel 13.1 mg/m(2)/d continuous intravenous infusion (CIVI) for 4 days without filgrastim, and paclitaxel 17.5 mg/m(2)/d CIVI for 4 days with filgrastim support. Dose-limiting toxicity for the combination was neutropenia. Statistical analysis of cohorts revealed similar mean steady-state concentrations (C(pss)) and areas under the concentration-versus-time curve (AUCs) when patients received paclitaxel doses of 13.1 or 17.5 mg/m(2)/d for 4 days with PSC 833, as when they received a paclitaxel dose of 35 mg/m(2)/d for 4 days without PSC 833. However, the effect of PSC 833 on paclitaxel pharmacokinetics varied greatly among individual patients, although a surrogate assay using CD56+ cells suggested inhibition of Pgp was complete or nearly complete at low concentrations of PSC 833. Responses occurred in three of four patients with non-small-cell lung cancer, and clinical benefit occurred in five of 10 patients with ovarian carcinoma. CONCLUSION PSC 833 in combination with paclitaxel can be administered safely to patients provided the paclitaxel dose is reduced to compensate for the pharmacokinetic interaction. Surrogate studies with CD56+ cells indicate that the maximum-tolerated dose for PSC 833 gives serum levels much higher than those required to block Pgp. The variability in paclitaxel pharmacokinetics, despite complete inhibition of Pgp in the surrogate assay, suggests that other mechanisms, most likely related to P450, contribute to the pharmacokinetic interaction. Future development of combinations such as this should include strategies to predict pharmacokinetics of the chemotherapeutic agent. This in turn will facilitate dosing to achieve comparable CPss and AUCs.

A Phase I Study of the P-Glycoprotein Antagonist Tariquidar in Combination with Vinorelbine

Purpose: P-glycoprotein (Pgp) antagonists have had unpredictable pharmacokinetic interactions requiring reductions of chemotherapy. We report a phase I study using tariquidar (XR9576), a potent Pgp antagonist, in combination with vinorelbine. Experimental Design: Patients first received tariquidar alone to assess effects on the accumulation of 99mTc-sestamibi in tumor and normal organs and rhodamine efflux from CD56+ mononuclear cells. In the first cycle, vinorelbine pharmacokinetics was monitored after the day 1 and 8 doses without or with tariquidar. In subsequent cycles, vinorelbine was administered with tariquidar. Tariquidar pharmacokinetics was studied alone and with vinorelbine. Results: Twenty-six patients were enrolled. Vinorelbine 20 mg/m2 on day 1 and 8 was identified as the maximum tolerated dose (neutropenia). Nonhematologic grade 3/4 toxicities in 77 cycles included the following: abdominal pain (4 cycles), anorexia (2), constipation (2), fatigue (3), myalgia (2), pain (4) and dehydration, depression, diarrhea, ileus, nausea, and vomiting, (all once). A 150-mg dose of tariquidar: (1) reduced liver 99mTc-sestamibi clearance consistent with inhibition of liver Pgp; (2) increased 99mTc-sestamibi retention in a majority of tumor masses visible by 99mTc-sestamibi; and (3) blocked Pgp-mediated rhodamine efflux from CD56+ cells over the 48 hours examined. Tariquidar had no effects on vinorelbine pharmacokinetics. Vinorelbine had no effect on tariquidar pharmacokinetics. One patient with breast cancer had a minor response, and one with renal carcinoma had a partial remission. Conclusions: Tariquidar is a potent Pgp antagonist, without significant side effects and much less pharmacokinetic interaction than previous Pgp antagonists. Tariquidar offers the potential to increase drug exposure in drug-resistant cancers.

Fr901228 causes mitotic arrest but does not alter microtubule polymerization

FR901228, a natural cyclic depsipeptide, shows high cytotoxicity against human cancer cell lines (low nM IC50 values). Cells exposed to FR901228 arrest with G1 or G2/M DNA content; S phase is depleted. G2/M cells include cells arrested in mitosis. We wished to understand the mitotic arrest by this compound. Mitotic arrest is often due to interference with microtubules and COMPARE testing in the NCI drug screen indicated a possible taxane-like mechanism. Testing of FR901228 for tubulin binding or alteration of in vitro MT assembly failed to reveal any effect. Likewise, examination of cellular microtubules following exposure to FR901228 did not reveal any change. Similar G2/M accumulation was observed in MCF7, MCF10 and PC3 cells. About 50% of G2/M cells were mitotic and contained microtubule spindles. Mitotic cells peaked at about 14-16 h drug exposure and declined to near 0% by 24-30 h. The block was at prometaphase, with numerous chromosomes unattached to the spindle. We conclude that FR901228 induces formation of aberrant spindles probably by interfering with chromosome attachment, causing mitotic accumulation without affecting mitotic microtubules.

Clinical Reversal of Multidrug Resistance

Reversal of drug resistance offers the hope of increasing the efficacy of conventional chemotherapy. We tested dexverapamil as a P‐glycoprotein antagonist in combination with EPOCH chemotherapy in refractory non‐Hodgkins lymphoma. In a cross‐over design, dexverapamil was added to EPOCH after disease stabilization or progression occurred. Objective responses were observed in 10 of 41 assessable patients. Biopsies for mdr‐1 were obtained before EPOCH treatment and at the time of cross‐over to dexverapamil. Levels of mdr‐1 were low before EPOCH, but increased four‐fold or more in 42% of patients in whom serial samples were obtained. Pharmacokinetic analysis revealed median peak concentrations of dexverapamil and its metabolite, nor‐dexverapamil, of 1.66 μmol/l and 1.58 μmol/l, respectively. Since both are comparable antagonists, a median peak total reversing concentration of 3.24 μmol/l was achieved. Pharmacokinetic analysis of doxorubicin and etoposide levels confirmed a delay in the clearance of doxorubicin ranging from 5% to 24%; no change in the pharmacokinetics of etoposide was observed. This study provides sufficient rationale for testing dexverapamil in a randomized clinical trial.

Differential Gene and MicroRNA Expression between Etoposide Resistant and Etoposide Sensitive MCF7 Breast Cancer Cell Lines

In order to develop targeted strategies for combating drug resistance it is essential to understand it’s basic molecular mechanisms. In an exploratory study we have found several possible indicators of etoposide resistance operating in MCF7VP cells, including up-regulation of ABC transporter genes, modulation of miRNA, and alteration in copy numbers of genes.

Abstract 3399: Resistance to the histone deacetylase inhibitor romidepsin is associated with degradation of Bim following MAPK pathway activation.

Inhibition of histone deacetylase (HDAC) enzymes represents a promising therapeutic approach in clinical oncology, as aberrant gene expression and alterations in histone acetylation due to HDACs have been implicated in tumor development and progression. Even though several histone deacetylase inhibitors (HDIs) are currently in clinical trials, so far only the HDIs romidepsin and vorinostat have been approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (CTCL). During clinical trials with romidepsin in CTCL, disease progression was noted in some patients who initially responded to therapy, while the disease in other patients did not respond to therapy suggesting that both de novo and acquired resistance to romidepsin were observed. To identify molecular determinants of resistance, we selected HuT78 CTCL cells with romidepsin in the presence of inhibitors of P-glycoprotein (Pgp) to prevent upregulation of Pgp as a mechanism of resistance. Resistant sublines were approximately 250- to 385-fold resistant to romidepsin; the Pgp inhibitor tariquidar did not significantly reverse resistance. The sublines also exhibited resistance to apoptosis following treatment with the HDIs apicidin, belinostat, entinostat, panobinostat, and vorinostat. A custom gene-expression array detected elevated expression of insulin receptor (INSR) in romidepsin resistant cells compared to parental cells. Immunoblot analysis of downstream effectors of the IR pathway demonstrated a 4- to 8-fold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation. Even though resistant cells did not respond to 48 h treatment with inhibitors of the insulin receptor, they exhibited exquisite sensitivity to treatment with as little as 1 nM of the MEK inhibitor PD0325901. Sensitivity to MEK inhibition in resistant cells was associated with restoration of the pro-apoptotic protein Bim. Combined treatment of romidepsin with MEK inhibitors also significantly yielded greater apoptosis in resistant cells compared to romidepsin and MEK inhibitor treatment alone. Gene expression analysis of circulating tumor samples obtained from patients with CTCL enrolled on the NCI 1312 Phase II romidepsin study suggested interaction of romidepsin with the MAPK pathway, indicated by altered expression of genes demonstrated to be under its control. These findings implicate activation of MEK as a resistance mechanism to romidepsin, and suggest combination of romidepsin with MEK inhibitors in clinical trials. Citation Format: Arup R. Chakraborty, Rob Robey, Zhirong Zhan, Victoria Luchenko, Michael Gottesman, Nathan Collie, Jean-Pierre Gillet, Richard Piekarz, Andrew Kossenkov, Louise Showe, Susan Bates. Resistance to the histone deacetylase inhibitor romidepsin is associated with degradation of Bim following MAPK pathway activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3399. doi:10.1158/1538-7445.AM2013-3399