Showing papers in "Cancer Chemotherapy and Pharmacology in 2005"

Endoxifen (4-hydroxy-N-desmethyl-tamoxifen) has anti-estrogenic effects in breast cancer cells with potency similar to 4-hydroxy-tamoxifen

Abstract: Tamoxifen is an effective drug for the treatment and prevention of breast cancer. It is extensively metabolized by the human cytochrome P450 enzyme system into several metabolites. Of these, 4-hydroxy-tamoxifen (4-OH-Tam) is an active metabolite, which has greater anti-estrogenic potency than the parent drug, tamoxifen. We reported recently that 4-hydroxy-N-desmethyl-tamoxifen (endoxifen) could also be active. The progesterone receptor (PR) messenger ribonucleic acid (mRNA) expression is commonly studied as a marker of estrogenic effect in breast cancer cells and PR levels in breast cancer patients are correlated with tamoxifen response. We, therefore, determined the effect of endoxifen and 4-OH-Tam on 17β-estradiol (E2)-induced PR mRNA expression in an estrogen receptor-positive human breast cancer cell line. MCF-7 cells were treated with drugs for 24 h. The total ribonucleic acid (RNA) was harvested and transcribed into complementary deoxyribonucleic acids (cDNAs). The PR mRNA level was measured by using real-time reverse transcription polymerase chain reaction (RT-PCR). The PR expression data were normalized using a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. We measured the metabolite concentrations in the cultured media by high performance liquid chromatography (HPLC) to determine whether there was conversion of one metabolite to the other. Consistent with previous reports, the dose–response of the E2 effect on the PR expression indicated an ED50 value of approximately 60 pM and the maximum induction of PR mRNA was nearly ten-fold. When 10−10 M E2 was used, induction of the PR expression was observed in 2 h and reached its maximum at 24 h. In this assay, neither endoxifen nor 4-OH-Tam alone produced any change in the PR mRNA expression. However, both endoxifen and 4-OH-Tam decreased the E2-induced PR expression with similar potency. There was very little interconversion between the two metabolites during the culture. Since endoxifen is present at greater concentrations than 4-OH-Tam in human plasma of breast cancer patients receiving chronic tamoxifen, these results provide further evidence that endoxifen is as important as, or more important than, 4-OH-Tam to the anti-estrogenic action of tamoxifen.

Population pharmacokinetics of trastuzumab in patients With HER2+ metastatic breast cancer

Abstract: Purpose: To characterize the population pharmacokinetics of trastuzumab in patients with metastatic breast cancer. Methods: A nonlinear mixed effect model was based on pharmacokinetic data from phase I, II, and III studies of 476 patients. The phase I study enrolled patients with advanced solid tumors. The phase II and III studies enrolled patients with HER2-positive metastatic breast cancer. Patients in the pivotal phase II and III studies were treated with a 4 mg/kg loading dose of trastuzumab followed by 2 mg/kg weekly for up to 840 days. The model adequately predicted observed trastuzumab concentrations. Model stability and performance were verified using bootstrap simulations. Percentiles, mean, and standard deviation of observed levels were compared with their distributions from 100 replicates of datasets simulated under the model. Results: A two-compartment linear pharmacokinetic model best described the data and accounted for the long-term accumulation observed following weekly administration of trastuzumab. Population estimates from the base model for clearance (CL) and volume of distribution of the central compartment (V1) of trastuzumab were 0.225 L/day, and 2.95 L, respectively. Estimated terminal halflife (t1/2) based on the population estimate was 28.5 days. Interpatient variabilities in clearance and volume were 43 and 29%, respectively. The number of metastatic sites, plasma level of extracellular domain of the HER2 receptor, and patient weight were significant baseline covariates for clearance, volume, or both (P<0.005). However, these covariate effects on trastuzumab exposure were modest and not clinically important in comparison with the large inter-patient variability of CL. Concomitant chemotherapy (anthracycline plus cyclophosphamide, or paclitaxel) did not appear to influence clearance. Conclusion: This population pharmacokinetic model can predict trastuzumab exposure in the long-term treatment of patients with metastatic breast cancer and provide comparison of alternative dosage regimens via simulation.

Single nucleotide polymorphisms modify the transporter activity of ABCG2

Abstract: Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P = 0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.

Imatinib pharmacokinetics in patients with gastrointestinal stromal tumour: a retrospective population pharmacokinetic study over time. EORTC Soft Tissue and Bone Sarcoma Group

Abstract: Imatinib pharmacokinetics (PK) may be affected by a number of factors that are related to the disease being treated and to the response of that disease to imatinib. Patients in the phase I and phase II trials conducted by the EORTC in patients with gastrointestinal stromal tumours (GISTs) and other sarcomas had detailed blood sampling for imatinib PK on day 1 and on day 29. Patients with GISTs also had repeat sampling, using a limited sampling strategy, after approximately 12 months on therapy. This population PK study was carried out to examine what covariates affected imatinib PK in GIST patients and what PK changes occurred over time. In the model producing the best fit, low clearance (CL) correlated with low body weight and high granulocyte count, whereas low haemoglobin correlated with low volume of distribution. For a patient with 77% of the median body weight or with 1.87 times the median granulocyte count, the apparent CL is 6.53 l/h, about 70% of the typical apparent CL of 9.33 l/h; for a patient of 84% of the typical haemoglobin level, the volume of distribution is about 70%. Total white blood cell count correlated closely with granulocyte count and there was a moderate correlation between haemoglobin and albumin (r=0.454). There was a trend towards increased imatinib clearance after chronic exposure over 12 months. The typical apparent CL increased 33% from day 1. Nevertheless, the approximate 95% confidence interval of the increase of the typical apparent CL was 33±34.6%, which contains zero. It is not yet clear whether this is a significant factor in the amelioration of imatinib toxicity that occurs with time or is related to disease control, and further work is required to confirm this observation.

Diosgenin induces cell cycle arrest and apoptosis in human leukemia K562 cells with the disruption of Ca2+ homeostasis.

Abstract: Diosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells. Cell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca2+ concentration and reactive oxygen species (ROS) generation. Cell cycle analysis showed that diosgenin caused G2/M arrest independently of p53. The levels of cyclin B1 and p21Cip1/Waf1 were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca2+ concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-xL proteins were downregulated, whereas the proapoptotic Bax was upregulated. Diosgenin inhibits K562 cell proliferation via cell cycle G2/M arrest and apoptosis, with disruption of Ca2+ homeostasis and mitochondrial dysfunction playing vital roles.

Pharmacokinetics and pharmacodynamics of 17-demethoxy 17-[[(2-dimethylamino)ethyl]amino]geldanamycin (17DMAG, NSC 707545) in C.B-17 SCID mice bearing MDA-MB-231 human breast cancer xenografts.

Abstract: 17-demethoxy 17-[[(2-dimethylamino)ethyl]amino]geldanamycin (17DMAG, NSC 707545) is a water-soluble analogue of 17-(allylamino)-17-demethoxygeldanamycin (17AAG), a compound currently in clinical trials. These preclinical studies: (1) characterized 17DMAG concentrations in plasma, normal tissues, and tumor after i.v. delivery to mice; and (2) correlated tumor and normal tissue 17DMAG concentrations with alterations in heat shock protein 90 (HSP90) and selected HSP90-chaperoned proteins. At specified times after i.v. administration of 75 mg/kg 17DMAG, SCID mice bearing s.c. MDA-MB-231 human breast xenografts were killed and plasma and tissues were retained. 17DMAG concentrations were determined by HPLC. Raf-1, heat shock protein 70 (HSP70), and HSP90 in tissues were determined by Western blotting. Peak plasma 17DMAG concentration was 15.4±1.4 μg/ml. The area under the plasma 17DMAG concentration versus time curve was 1072 μg/ml min, corresponding to a total body clearance of 70 ml/kg/min. Peak 17DMAG concentrations in liver (118.8±5.7 μg/g), kidney (122.9±10.6 μg/g), heart (81.3±8.1 μg/g), and lung (110.6±25.4 μg/g) occurred at 5–10 min, while peak concentrations in spleen (70.6±9.6 μg/g) and tumor (9.0±1.0 μg/g) occurred at 30–45 min. At 48 h, 17DMAG was detectable in tumor but not in any normal tissue. Raf-1 in tumors of 17DMAG-treated mice killed at 4, 7, 24 and 48 h was about 20% lower than in tumors from vehicle-treated mice. HSP90 and HSP70 in tumors of 17DMAG-treated animals were significantly lower than in tumors of control animals at 4, 7, and 24 h. Hepatic Raf-1 was decreased by more than 60% at all times after 17DMAG treatment; however, hepatic HSP90 was not affected. HSP70 was undetectable in livers of vehicle-treated mice or mice killed at 2 or 4 h after 17DMAG treatment, but was detected in livers at 7, 24 and 48 h. 17DMAG did not affect renal Raf-1. In contrast, renal HSP70 and HSP90 were decreased by more than 50% at 2 and 4 h after 17DMAG treatment. Renal HSP70 increased approximately twofold above that in kidneys from vehicle-treated control mice at 7 and 24 h, while HSP90 relative protein concentration was no different from that in controls. Plasma pharmacokinetics of 17DMAG in tumor-bearing mice were similar to those previously reported in nontumor-bearing mice. 17DMAG was distributed widely to tissues but was retained for longer in tumors than normal tissues. Raf-1, HSP90, and HSP70 were altered to different degrees in tumors, livers, and kidneys of 17DMAG-treated animals. These data illustrate the complex nature of the biological responses to 17DMAG.

In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative.

Abstract: To describe the preclinical basis for further development of 17-dimethyl aminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG, NSC 707545). In vitro proliferation assays, and in vivo model studies in metastatic pancreatic carcinoma and subcutaneous xenograft melanoma and small-cell lung carcinoma models. 17-DMAG emerged from screening studies as a potent geldanamycin analog, with the average concentration inhibiting the growth of the NCI anticancer cell line drug screen by 50% being 0.053 μM. “Head to head” comparison with 17-allylamino-17-demethoxygeldanamycin (17-AAG, NSC 330507) revealed 17-DMAG to possess potent activity against certain cell types, e.g., MDA-MB-231 breast carcinoma and HL60-TB leukemia which were relatively insensitive to 17-AAG. Evidence of oral bioavailability of 17-DMAG in a saline-based formulation prompted more detailed examination of its antitumor efficacy in vivo. 17-DMAG inhibited the growth of the AsPC-1 pancreatic carcinoma xenografts growing as intrahepatic metastases at doses of 6.7–10 mg/kg twice daily for 5 days administered orally under conditions where 17-AAG was without activity. 17-DMAG in an aqueous vehicle at 7.5–15 mg/kg per day for 3 days on days 1–3, 8–10 and 13–17, or 1–5 and 8–12 showed evidence of antitumor activity by the parenteral and oral routes in the MEXF 276 and MEXF 989 melanomas and by the parenteral route in the LXFA 629 and LXFS 650 adenocarcinoma and small-cell carcinoma models. The latter activity was comparable to the historical activity of 17-AAG. Taken together, the in vivo activity of 17-DMAG supports the further development of this water-soluble and potentially orally administrable geldanamycin congener.

Advanced concepts in estrogen receptor biology and breast cancer endocrine resistance: implicated role of growth factor signaling and estrogen receptor coregulators.

Abstract: Estrogen receptor (ER), mediating estrogen-signaling stimuli, is a dominant regulator and a key therapeutic target in breast cancer etiology and progression. Endocrine therapy, blocking the ER pathway, is one of the most important systemic therapies in breast cancer management, but de novo and acquired resistance is still a major clinical problem. New research highlights the role of both genomic and nongenomic ER activities and their intimate molecular crosstalk with growth factor receptor and other signaling kinase pathways in endocrine resistance. These signaling pathways, when overexpressed and/or hyperactivated, can modulate both activities of ER, resulting in endocrine resistance. Thus, these signal transduction receptors and signaling molecules may serve as both predictive markers and novel therapeutic targets to circumvent endocrine resistance. Compelling experimental and clinical evidence suggest that the epidermal growth factor/HER2/neu receptor (EGFR/HER2) pathway might play a distinct role in endocrine resistance, and especially in resistance to selective estrogen receptor modulators (SERMs) such as tamoxifen. Results from preclinical studies of treatment combinations with various endocrine therapy drugs together with several potent anti-EGFR/HER2 inhibitors are very promising, and clinical trials to see whether this new strategy is effective in patients are now ongoing.

Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: effects on Hsp90 and client proteins in melanoma models

Abstract: The heat shock protein Hsp90 is a potential target for drug discovery of novel anticancer agents. By affecting this protein, several cell signaling pathways may be simultaneously modulated. The geldanamycin analog 17AAG has been shown to inhibit Hsp90 and associated proteins. Its clinical use, however, is hampered by poor solubility and thus, difficulties in formulation. Therefore, a water-soluble derivative was desirable and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) is such a derivative. Studies were carried out in order to evaluate the activity and molecular mechanism(s) of 17DMAG in comparison with those of 17-allylamino-demethoxygeldanamycin (17AAG). 17DMAG was found to be more potent than 17AAG in a panel of 64 different patient-derived tumor explants studied in vitro in the clonogenic assay. The tumor types that responded best included mammary cancers (six of eight), head and neck cancers (two of two), sarcomas (four of four), pancreas carcinoma (two of three), colon tumors (four of eight for 17AAG and six of eight for 17DMAG), and melanoma (two of seven). Bioinformatic comparisons suggested that, while 17AAG and 17DMAG are likely to share the same mode(s) of action, there was very little similarity with standard anticancer agents. Using three permanent human melanoma cell lines with differing sensitivities to 17AAG and 17DMAG (MEXF 276L, MEXF 462NL and MEXF 514L), we found that Hsp90 protein was reduced following treatment at a concentration associated with total growth inhibition. The latter occurred in MEXF 276L cells only, which are most sensitive to both compounds. The depletion of Hsp90 was more pronounced in cells exposed to 17DMAG than in those treated with 17AAG. The reduction in Hsp90 was associated with the expression of erbB2 and erbB3 in MEXF 276L, while erbB2 and erbB3 were absent in the more resistant MEXF 462NL and MEXF 514L cells. Levels of known Hsp90 client proteins such as phosphorylated AKT followed by AKT, cyclin D1 preceding cdk4, and craf-1 declined as a result of drug treatment in all three melanoma cell lines. However, the duration of drug exposure needed to achieve these effects was variable. All cell lines showed increased expression of Hsp70 and activated cleavage of PARP. No change in PI3K expression was observed and all melanoma cells were found to harbor the activating V599E BRAF kinase mutation. The results of our in vitro studies are consistent with both 17AAG and 17DMAG acting via the same molecular mechanism, i.e. by modulating Hsp90 function. Since 17DMAG can be formulated in physiological aqueous solutions, the data reported here strongly support the development of 17DMAG as a more pharmaceutically practicable congener of 17AAG.

Potential for drug interactions in hospitalized cancer patients

Abstract: Objectives To quantify the frequency of potential drug interactions unrelated to chemotherapy in cancer patients admitted to our institution, and to define risk factors for such interactions.

Targeting BCL-2 overexpression in various human malignancies through NF-κB inhibition by the proteasome inhibitor bortezomib

Abstract: Background BCL-2 overexpression occurs in many cancer types and is associated with chemoresistance and radioresistance. The mechanisms responsible for its aberrant expression are thought to be transcriptionally mediated but remain unclear. We examined the cell type-specific mechanism of BCL-2 gene transcription in various solid organ malignancies.

Absorption and metabolism of genistein and its five isoflavone analogs in the human intestinal Caco-2 model

Abstract: The purposes of this study were to determine the effect of structural change on the intestinal disposition of isoflavones and to elucidate the mechanisms responsible for transport of phase II isoflavone conjugates. Transport and metabolism of six isoflavones (i.e., genistein, daidzein, glycitein, formononetin, biochanin A, and prunetin) were studied in the human intestinal Caco-2 model and mature Caco-2 cell lysate. Glucuronides were the main metabolites in intact Caco-2 cells for all isoflavones except prunetin, which was mainly sulfated. In addition, the 7-hydroxy group was the main site for glucuronidation whereas the 4′-hydroxy group was only one of the possible sites for sulfation. Glucuronidated isoflavones (except biochanin A) were preferably excreted to the basolateral side, whereas sulfated metabolites (except genistein and glycitein) were mainly excreted into the apical side. Polarized excretion of most isoflavone conjugates was inhibited by the multidrug resistance-related protein (MRP) inhibitor leukotriene C4 (0.1 μM) and the organic anion transporter (OAT) inhibitor estrone sulfate (10 μM). When formation and excretion rates of isoflavones were determined simultaneously, the results showed that formation served as the rate-limiting step for all isoflavone conjugates (both glucuronides and sulfates) except for genistein glucuronide, which had comparable excretion and formation rates. In conclusion, the intestinal disposition of isoflavones was structurally dependent, polarized, and mediated by MRP and OAT. Formation generally served as the rate-limiting step in the cellular excretion of conjugated isoflavones in the Caco-2 cell culture model.

Molecular mechanisms of ginsenoside Rh2-mediated G1 growth arrest and apoptosis in human lung adenocarcinoma A549 cells.

Abstract: Ginsenoside Rh2 (Rh2), a purified ginseng saponin, has been shown to have antiproliferative effects in certain cancer cell types However, the molecular mechanisms of Rh2 on cell growth and death have not been fully clarified In this study, the antiproliferative effect of Rh2 in human lung adenocarcinoma A549 cells was investigated Treatment of A549 cells with 30 μg/ml Rh2 resulted in G1 phase arrest, followed by progression to apoptosis This Rh2-mediated G1 arrest was accompanied by downregulation of the protein levels and kinase activities of cyclin-D1, cyclin-E and Cdk6, and the upregulation of pRb2/p130 In addition, Rh2-induced apoptosis was confirmed by TUNEL assay and DNA fragmentation analysis Administration of Rh2 caused an increase in the expression levels of TRAIL-RI (DR4) death receptor but did not alter the levels of other death receptors or Bcl-2 family molecules Furthermore, the Rh2-induced apoptosis was significantly inhibited by DR4:Fc fusion protein, which inhibits TRAIL-DR4-mediated apoptosis In addition, caspase-2, caspase-3 and caspase-8 were highly activated upon Rh2 treatment Inhibitors of caspase-2, caspase-3 and caspase-8 markedly prevented the cell death induced by Rh2 Inhibitor of caspase-8 significantly inhibited the activation of caspase-2, caspase-3 and caspase-8 These observations indicate that multiple G1-related cell cycle regulatory proteins are regulated by Rh2 and contribute to Rh2-induced G1 growth arrest The increase in the expression level of DR4 death receptor may play a critical role in the initiation of Rh2-triggered apoptosis, and the activation of the caspase-8/caspase-3 cascade acts as the executioner of the Rh2-induced death process

Preclinical toxicity of a geldanamycin analog, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance

Abstract: Purpose 17-DMAG is a hydrophilic derivative of the molecular chaperone inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; NSC-330507), which is currently being evaluated for the treatment of cancer in clinical trials. 17-DMAG offers a potential advantage over 17-AAG because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG. The present results are from preclinical toxicity studies evaluating 17-DMAG in rats and dogs.

Pancratistatin causes early activation of caspase-3 and the flipping of phosphatidyl serine followed by rapid apoptosis specifically in human lymphoma cells

Abstract: Recently a major research effort has been focused on the development of anticancer drugs by targeting the components of a biochemical pathway to induce apoptosis in cancerous cells. Some of the natural products (e.g. paclitaxel) have been proven to be useful in inducing apoptosis in cancer cells with limited specificity. Pancratistatin, a natural product isolated and characterized over a decade ago, has been shown to be cytostatic and antineoplastic. We investigated the specificity and biochemical mechanism of action of pancratistatin. Pancratistatin seemed to show more specificity than VP-16 or paclitaxel as an efficient inducer of apoptosis in human lymphoma (Jurkat) cells, with minimal effect on normal nucleated blood cells. Caspase-3 activation and exposure of phosphatidyl serine on the outer leaflet of the plasma membrane were earlier events than the generation of ROS and DNA fragmentation observed following pancratistatin treatment. This indicates a possible involvement of caspase-3 and plasma membrane proteins in the induction phase of apoptosis. Our results indicate that pancratistatin does not cause DNA double-strand breaks or DNA damage prior to the execution phase of apoptosis in cancer cells. Parallel experimentation with VP-16, a currently used medication for cancer treatment, indicated that VP-16 causes substantial DNA damage in normal non-cancerous blood cells, while pancratistatin does not cause any DNA double-strand breaks or DNA damage in non-cancerous cells. Taken together, our finding that pancratistatin induces apoptosis in cancer cells using non-genomic targets, and more importantly does not seem to have any affect non-cancerous cells, presents a significant platform to develop non-toxic anticancer therapies.

Oxaliplatin causes selective atrophy of a subpopulation of dorsal root ganglion neurons without inducing cell loss.

Abstract: Peripheral neuropathy is induced by multiple doses of oxaliplatin and interferes with the clinical utility of the drug in patients with colorectal cancer. In this study, we sought to determine whether cell loss or selective neuronal damage was the basis for the peripheral neuropathy caused by oxaliplatin. Adult female rats were given 1.85 mg/kg oxaliplatin twice per week for 8 weeks. Nerve conduction and L5 dorsal root ganglia (DRG) were studied 1 week after the completion of all treatment. No mortality occurred during oxaliplatin treatment, but the rate of body weight gain was reduced compared to age-matched vehicle-treated controls. Oxaliplatin slowed conduction velocity and delayed conduction times in peripheral sensory nerves, without affecting central or motor nerve conduction. In L5 DRG, total numbers of neurons were unchanged by oxaliplatin, but there were significant reductions in neuronal size distribution, ganglion volume, average cell size and the relative frequency of large cells. In addition, the relative frequency of small DRG cells was increased by oxaliplatin. Oxaliplatin significantly altered the size distribution and average cell body area of the predominantly large parvalbumin-immunoreactive DRG neurons without affecting the frequency of parvalbumin staining. On the contrary, neither the staining frequency nor the size distribution of the predominantly small substance P-immunoreactive DRG neurons was changed by oxaliplatin. In conclusion, oxaliplatin causes selective atrophy of a subpopulation of DRG neurons with predominantly large parvalbumin-expressing cells without inducing neuronal loss. Because DRG cell body size and axonal conduction velocity are positively correlated, neuronal atrophy may be the morphological basis for the development of decreased sensory nerve conduction velocity that characterizes oxaliplatin-induced peripheral neuropathy.

Clinical significance of estrogen receptor β in breast cancer

Abstract: Ever since the estrogen receptor (ER) β was discovered in 1996, we have been trying to determine its value as a prognostic and/or predictive factor in breast cancer and its potential as a novel target for pharmacological intervention. Recent progress in cellular experiments has shown that ERβ works as counter partner of ERα through inhibition of the transactivating function of ERα by heterodimerization, distinct regulation on several specific promoters by ERα or ERβ, and ERβ-specific regulated genes which are probably related to its anti-proliferative properties. Accumulated data from protein studies in breast cancer tissues indicate that positive expression of ERβ appears to correlate with a favorable prognosis. Although the number of studies is small, a positive response to tamoxifen treatment is observed in both ERα- and ERβ-positive populations. The significance of ERβ2/cx, a splicing variant of ERβ, remains controversial and needs to be analyzed in further studies. We postulate that a combined evaluation of ERβcx with progesterone receptor may help the stratification of ERα-positive breast cancer. Epidemiological studies of hormone replacement therapy and isoflavone (genistein) consumption indicate the possible contribution of ERβ-specific signaling in breast cancer prevention. A selective estrogen receptor modulator, which works as an antagonist of ERα and an agonist of ERβ, may be a promising chemo-preventive treatment.

Antitumor activity of albendazole against the human colorectal cancer cell line HT-29: in vitro and in a xenograft model of peritoneal carcinomatosis

Abstract: The peritoneal surface remains an important failure site for patients with colorectal cancer. We have recently shown that albendazole (ABZ), a safe and effective anthelmintic drug, has profound antitumor activity in hepatocellular cancer. Furthermore, albendazole also possesses unique physiochemical and pharmacokinetic properties probably making it a potential drug for use in the regional treatment of peritoneal carcinomatosis (PC). The current study was therefore designed to investigate this concept under both in vitro and in vivo conditions using human colorectal cancer cells HT-29. In cell culture, studies were conducted to investigate the effect of ABZ and its major metabolites, albendazole sulfoxide (ABZ-SO) and albendazole sulfone (ABZ-SO2) on the growth of human colorectal cell line HT-29. We also investigated the effects of ABZ on the cell cycle and the possible induction of apoptosis in these cells. Male nude mice inoculated intraperitoneally (i.p.) with HT-29 cells were treated with various schedules of ABZ given i.p. or orally for 6 weeks. Response was evaluated as the number of peritoneal tumor nodules present in animals at the end of the treatment period. In vitro, ABZ treatment of cells for 5 days led to profound inhibition of growth. 3H-Thymidine assay and trypan blue viable cell counts confirmed the dose- and time-dependency of the ABZ effect, while recovery experiments revealed the reversible nature of this inhibition. ABZ-SO and ABZ-SO2 were also evaluated in cell culture studies and compared with the parent drug. In HT-29 cells, the IC50 values were calculated to be 0.12 μM for ABZ and 2.35 μM for ABZ-SO. The other metabolite, ABZ-SO2, was completely inactive. Studies on the mechanism of ABZ action, revealed arrest of HT-29 cells at the G2/M phase of the cell cycle, while TUNEL, DNA laddering and caspase-3 activity all confirmed ABZ induced apoptosis. In nude mice with peritoneal HT-29 xenografts, ABZ profoundly inhibited peritoneal tumor growth. While alternate i.p. dosing (ABZ, 150 mg/kg) led to the highest degree of tumor growth suppression (P<0.001), schedules such as once-weekly dosing and even a single dose for the entire course of treatment (6 weeks) were also effective in reducing peritoneal tumor growth. However, no such activity was observed when ABZ was administered orally. This study shows for the first time the potent effect of regionally administered ABZ in suppressing the growth of peritoneal tumors of human colorectal origin. The effect is thought to be brought about by arresting tumor cells at the G2/M phase of the cycle and apoptosis. These findings provide evidence for potential value of ABZ in the treatment of regional PC arising from colorectal cell lines.

Antiangiogenic and antitumoral properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum

Abstract: The potential antiangiogenic and antitumoral properties of SargA, a polysaccharide extracted from the brown marine alga Sargassum stenophyllum, were studied in assays carried out in chick embryos and mice. Gelfoam plugs containing SargA (2–1500 μg/plug) implanted in vivo into fertilized 6-day-old chicken eggs induced dose-related antiangiogenic activity in the chorioallantoic membrane (CAM). By day 8, the highest dose of SargA alone decreased the vessel number in the CAM by 64%, but coadministered with hydrocortisone (156 μg/plug, which alone caused 30% inhibition) failed to potentiate its antiangiogenic effect. Combined with basic fibroblast growth factor (50 ng/plug), SargA (1500 μg/plug) abolished angiogenesis stimulated by this factor in both chick embryo CAM and in subcutaneous (s.c.) Gelfoam plugs implanted in the dorsal skin of Swiss mice (measured as plug hemoglobin content). Repeated s.c. injections of SargA (1.5 or 150 μg per animal per day for 3 days) close to B16F10 melanoma cell tumors in the dorsal skin of mice markedly decreased tumor growth in a dose-related fashion (by 40% and 80% at 2 weeks after the first injection, respectively), without evident signs of toxicity. SargA caused graded inhibitions of migration and viability of cultured B16F10 cells and also displayed antithrombotic activity in human plasma (5 mg/ml increased thrombin time 2.5-fold relative to saline). Thus, SargA exhibits pronounced antiangiogenic as well as antitumoral properties. Although the latter action of SargA might be related to the inhibition of angiogenesis, the polysaccharide also exerts cytotoxic effects on tumor cells. Because of its chemical characteristics and polyanionic constituents, we postulate that the polysaccharide SargA might modulate the activity of heparin-binding angiogenic growth factors.

A phase I and pharmacologic study of the MDR converter GF120918 in combination with doxorubicin in patients with advanced solid tumors

Abstract: Resistance to chemotherapy can partly be explained by the activity of membrane bound P-glycoprotein. Competitive inhibition of P-glycoprotein, by multidrug resistance (MDR) converters, may overcome this MDR. Previously studied MDR converters either have serious intrinsic side effects or considerably influence the pharmacokinetics of cytotoxic agents at concentrations theoretically required to convert MDR. GF120918 is a third-generation MDR converter with high affinity for P-glycoprotein and can be given orally. We performed a phase 1 study with escalating doses of GF120918 in combination with doxorubicin. The study group comprised 46 patients with advanced solid tumors. Doxorubicin was administered on day 1 (cycle 1), GF120918 on days 22–24 (cycle 2), and on days 29–33 with doxorubicin administered on day 31 (cycle 3). Pharmacokinetics of both GF120918 and doxorubicin were studied. The starting daily dose of GF120918 was 50 mg and was to be increased in subsequent cohorts until a steady state plasma level of 100 ng/ml was reached. The starting dose of doxorubicin was 50 mg/m2 and was to be increased after reaching the target dose level of GF120918. In 37 of the 46 patients, full pharmacokinetic data from the three scheduled cycles were obtained. Pharmacokinetics of GF120918 showed a less than linear increase in C max with increasing doses, with considerable interpatient variation. The target steady-state plasma level for GF120918 was exceeded in 12 out of 19 patients who received 400 mg GF120918 alone twice daily and in 12 of 17 patients who received 400 mg GF120918 twice daily in combination with doxorubicin. GF120918 pharmacokinetics were not influenced by coadministration of doxorubicin. The doxorubicin AUC was only marginally influenced by GF120918 and only at the highest dose levels. In these patients there was a significant increase in the AUC of doxorubicinol in cycle 3 as compared to cycle 1. Hematologic toxicity mainly consisted of neutropenia and was more severe in cycle 3 than in cycle 1 (13 vs 5 patients with grade 4 neutropenia, P=0.003). Neutropenic fever was the dose-limiting toxicity at a doxorubicin dose of 75 mg/m2 with 400 mg GF120918 twice daily. The toxicity of GF120918 was limited to somnolence in eight patients and occasional gastrointestinal complaints. GF120918 is an MDR converter with only minimal side effects at a dose level yielding concentrations able to convert the action of P-glycoprotein in vitro. A doxorubicin dose of 60 mg/m2 on day 3 in combination with 400 mg GF120918 twice daily on days 1–5 is an acceptable regimen for further clinical trials.

Docosahexaenoic acid enhances the susceptibility of human colorectal cancer cells to 5-fluorouracil

Abstract: Powerful growth-inhibitory action has been shown for n-3 polyunsaturated fatty acids against colon cancer cells. We have previously described their ability to inhibit proliferation of colon epithelial cells in patients at high risk of colon cancer. In the work reported here we investigated the ability of docosahexaenoic acid (DHA) to potentiate the antineoplastic activity of 5-fluorouracil (5-FU) in p53-wildtype (LS-174 and Colo 320) and p53-mutant (HT-29 and Colo 205) human colon cancer cells. When in combination with DHA, 5-FU was used at concentrations ranging from 0.1 to 1.0 μM, much lower than those currently found in plasma patients after infusion of this drug. Similarly, the DHA concentrations (≤10 μM) used in combination with 5-FU were lower than those widely used in vitro and known to cause peroxidative effects in vivo. Whereas the cells showed different sensitivity to the growth-inhibitory action of 5-FU, DHA reduced cell growth independently of p53 cellular status. DHA synergized with 5-FU in reducing colon cancer cell growth. The potentiating effect of DHA was attributable to the enhancement of the proapoptotic effect of 5-FU. DHA markedly increased the inhibitory effect of 5-FU on the expression of the antiapoptotic proteins BCL-2 and BCL-XL, and induced overexpression of c-MYC which has recently been shown to drive apoptosis and, when overexpressed, to sensitize cancer cells to the action of proapoptotic agents, including 5-FU. Our results indicate that DHA strongly increases the antineoplastic effects of low concentrations of 5-FU. Overall, the results suggest that combinations of low doses of the two compounds could represent a chemotherapeutic approach with low toxicity.

Improvement of paclitaxel therapeutic index by derivatization and association to a cholesterol-rich microemulsion: in vitro and in vivo studies.

Abstract: A cholesterol-rich microemulsion or nanoparticle termed LDE concentrates in cancer tissues after injection into the bloodstream. Here the cytotoxicity, pharmacokinetics, toxicity to animals and therapeutic action of a paclitaxel lipophilic derivative associated to LDE is compared with those of the commercial paclitaxel. Results show that LDE-paclitaxel oleate is stable. The cytostatic activity of the drug in the complex is diminished compared with the commercial paclitaxel due to the cytotoxicity of the vehicle Cremophor EL used in the commercial formulation. Competition experiments in neoplastic cultured cells show that paclitaxel oleate and LDE are internalized together by the LDL receptor pathway. LDE-paclitaxel oleate arrests the G(2)/M phase of cell cycle, similarly to commercial paclitaxel. Tolerability to mice is remarkable, such that the lethal dose (LD(50)) was ninefold greater than that of the commercial formulation (LD(50) = 326 microM and 37 microM, respectively). LDE concentrates paclitaxel oleate in the tumor roughly fourfold relative to the normal adjacent tissues. At equimolar doses, the association of paclitaxel oleate with LDE results in remarkable changes in the drug pharmacokinetic parameters when compared to commercial paclitaxel (t(1/2)=218 min and 184 min, AUC=1,334 microg h/ml and 707 microg h/ml and CL=0.125 ml/min and 0.236 ml/min, respectively). Finally, the therapeutic efficacy of the complex is pronouncedly greater than that of the commercial paclitaxel, as indicated by the reduction in tumor growth, increase in survival rates and % cure of treated mice. In conclusion, LDE-paclitaxel oleate is a stable complex and compared with paclitaxel toxicity is considerably reduced and activity is enhanced, which may lead to improved therapeutic index in clinical use.

Phase I study of green tea extract in patients with advanced lung cancer

Abstract: Epidemiologic studies suggest that consumption of green tea may have a protective effect against the development of several cancers. Preclinical studies of green tea and its polyphenolic components have demonstrated antimutagenic and anticarcinogenic activity, and inhibition of growth of tumor cell lines and animal tumor models, including lung cancer. Green tea may also have chemopreventive properties, and enhancement of cytotoxicity of chemotherapeutic agents has been demonstrated. This trial was designed to determine the maximum tolerated dose (MTD) of green tea extract (GTE) in patients with advanced lung cancer. A total of 17 patients with advanced lung cancer were registered to receive once-daily oral dosing of GTE at a starting dose of 0.5 g/m2 per day, with an accelerated dose-escalation scheme. On this schedule, the MTD of GTE was 3 g/m2 per day, and at this dose, GTE was well tolerated with no grade 3 or 4 toxicity seen. Dose-limiting toxicities were diarrhea, nausea and hypertension. No objective responses were seen in this trial. Seven patients had stable disease ranging from 4 to 16 weeks; no patient remained on therapy longer than 16 weeks due to the development of progressive disease. This study suggests that while relatively nontoxic at a dose of 3 g/m2 per day, GTE likely has limited activity as a cytotoxic agent, and further study of GTE as a single-agent in established malignancies may not be warranted. Further studies should focus on the potential chemopreventive and chemotherapy-enhancing properties of GTE.

Correlation of pharmacokinetics with the antitumor activity of Cetuximab in nude mice bearing the GEO human colon carcinoma xenograft

Abstract: Purpose: The epidermal growth factor receptor (EGFR), a protein tyrosine kinase expressed in many types of human cancers including colon and breast, has been strongly associated with tumor progression. Cetuximab, an IgG1 anti-EGFR chimeric mouse/human monoclonal antibody, has been proven to be effective in the treatment of advanced colon cancer. To date, there has not been a study to systematically evaluate the pharmacokinetics (PK) of Cetuximab in a preclinical model and to further explore any correlation of drug exposure between animal models and cancer patients. In the present study, we characterized the PK of Cetuximab in nude mice at efficacious dose levels and further compared the preclinical optimal dose and active plasma drug concentration with those determined in clinical studies. Experimental design: The antitumor activity of Cetuximab was evaluated using the GEO human colon carcinoma xenografts implanted subcutaneously in nude mice. The drug was administered ip every 3 days for five total injections (inj) (q3dx5) at dose levels ranging from 1 mg/inj to 0.04 mg/inj. The plasma PK of Cetuximab was determined at dose levels of 1.0, 0.25, and 0.04 mg/inj with a single bolus iv or ip administration in nude mice. The tumoral PK of Cetuximab was determined at dose levels of 0.25, and 0.04 mg/inj with a single bolus ip administration in nude mice bearing GEO tumor xenografts. The plasma and tumoral levels of Cetuximab were quantitated by an ELISA assay. Results: Cetuximab demonstrated a dose-dependent antitumor activity at dose levels of 0.25, 0.1, and 0.04 mg/inj, with a statistically significant tumor growth delay (in reaching a tumor target size of 1 gm) of 18 days (P<0.001), 12.3 days (P<0.01), and 10 days (P<0.01) for 0.25, 0.1, and 0.04 mg/inj, respectively. A separate study employing the same treatment schedule showed that Cetuximab was equally active at dose levels ranging from 0.25 mg/inj to 1 mg/inj. Therefore, dose levels of Cetuximab from 1 mg/inj to 0.04 mg/inj can be considered to be within the efficacious range, while dose levels of 0.25 mg/inj or higher appeared to be optimal for the antitumor activity of Cetuximab in the GEO tumor model. When Cetuximab was given iv to mice, the elimination half life (t1/2) was 39.6, 37.8, and 42.2 h for doses of 1.0, 0.25, and 0.04 mg/inj, respectively, suggesting a similar disposition kinetics of Cetuximab within this dose range. The volume of distribution (Vd) ranged from 0.062 l/kg to 0.070 l/kg, suggesting that Cetuximab is primarily confined to the plasma compartment with limited peripheral tissue distribution. Clearance (CL) was similar and no apparent PK saturation was observed across the dose ranging from 0.04 mg/inj to 1.0 mg/inj. When mice were administered with a single bolus ip administration at doses of 1, 0.25, and 0.04 mg/inj, the maximum plasma concentration (Cmax) was 407.6, 66.4, and 16.5 μg/ml. The area under the curve of plasma drug concentration (AUC) was 19212.4, 3182.4, and 534.5 μg/ml h, for 1.0, 0.25, and 0.04 mg/inj, respectively. The average steady state plasma concentration (Css avg) for the multiple dosing schedule was estimated to be 73.1 μg/ml at 0.25 mg/inj and was considered as an active plasma drug concentration. The maximum tumoral concentration of Cetuximab was 2.6 and 0.53 ng/mg-tumor while the tumoral drug exposure was 112.6 and 18.3 ng/mg h for 0.25 and 0.04 mg/inj, respectively. The EGFR was estimated to be nearly completely occupied by Cetuximab at the optimal dose of 0.25 mg/inj. Conclusion: In the present study, we compared the preclinical optimal dose and the corresponding active plasma concentration determined in mice with those being observed in cancer patients, i.e. 65–100 μg/ml. The preclinical optimal dose of 0.25 mg/inj was significantly lower than the current clinical dose. However, the active plasma concentration at 0.25 mg/inj is within the range of the active drug concentrations in cancer patients treated with Cetuximab under the current optimal dosing regimen. It appears that the active plasma drug concentration determined in preclinical model predicts better than the optimal preclinical dose for the clinical development of antibody drugs.

Magnesium depletion enhances cisplatin-induced nephrotoxicity.

Abstract: Purpose: Nephrotoxicity and magnesium (Mg)-depletion are well-known side effects to cisplatin (CP) treatment. The purpose of this present study was to investigate the role of Mg on CP induced changes in renal function. CP induced renal dysfunction was achieved by treatment with CP or vehicle (2.5 mg/kg) once weekly for 3 weeks. Since the CP-induced renal damage, including tubular reabsorption defects, is most prominent within the outer medulla (OM), changes in the expression pattern of OM aquaporins and sodium transporters including the Na,K-ATPase (α-subunit), type III Na,H-exchanger (NHE3), aquaporin 1 (AQP1) and 2 (AQP2) and the Na,K,2Cl-cotransporter (NKCC2) were investigated by semi-quantitative Western blotting. Experimental design: Rats had access to either a diet with standard Mg or to a Mg-depleted diet. Cisplatin was administered to female Wistar rats once a week for 3 weeks according to four regimens: (1) Cisplatin 2.5 mg/kg body weight i.p., to rats on a diet with standard Mg, (2) Cisplatin 2.5 mg/kg body weight i.p., to rats on a diet with low Mg, (3) Isotonic NaCl 2.5 ml/kg body weight i.p., to rats on a diet with standard Mg, (4) Isotonic NaCl 2.5 ml/kg body weight i.p., to rats on a diet with low Mg. Results: CP had no effect on plasma creatinine or urea in rats with standard Mg intake, but the expression of all five transporters was significantly reduced when compared to vehicle treated rats on standard Mg-intake. Vehicle treated rats on low Mg-intake had a significant reduction in the expression of Na,K-ATPase, NHE3 and NKCC2, but unchanged expression levels of AQP1 or AQP2 when compared to standard treated controls. Forty percent of the CP-treated rats on low Mg-intake died during the experiment and the remaining animals had marked increased plasma creatinine and urea. Furthermore, the Western blot analysis revealed an almost complete disappearance of all four transporters, suggesting a dramatic synergistic effect of CP and Mg-depletion on renal function including the expression pattern of outer medullary sodium transporters and aquaporins. Conclusions: This study indicates a substantial additive effect of Mg-depletion on cisplatin induced renal toxicity as evidenced by significant changes in plasma creatinine and urea, renal failure induced mortality and loss of renal transporters. This should give cause for concern since the nephrotoxicity observed during cisplatin treatment might be substantiated by the known Mg-loss associated with cisplatin treatment especially in patients suffering from intense gastro-intestinal side effects.

Oral bioavailability of docetaxel in combination with OC144-093 (ONT-093)

Abstract: Docetaxel given orally as monotherapy results in low bioavailability of <10%. Previous studies have indicated that the intestinal efflux pump P-glycoprotein (P-gp) prevents uptake from the gut resulting in low systemic exposure to docetaxel. The purpose of this study was to determine the degree of enhancement of the oral uptake of docetaxel on combination with orally administered OC144-093, a potent P-gp inhibitor. Furthermore, the safety of combined treatment was determined and whether known functional genetic polymorphisms of the MDR1 gene could be associated with variability in docetaxel pharmacokinetics was also investigated. A proof of concept study was carried out in 12 patients with advanced solid tumors. Patients were randomized to receive one course of 100 mg oral docetaxel combined with 500 mg OC144-093 followed 2 weeks later by a second i.v. course of docetaxel at a flat dose of 100 mg, without OC144-093, or vice versa. This was followed by standard i.v. docetaxel treatment if indicated. The apparent relative oral bioavailability of docetaxel was 26±8%. Orally administered docetaxel combined with oral OC144-093 resulted in a Cmax of 415±255 ng ml−1, an AUC0–∞ of 844±753 ng h ml−1 and kel of 0.810±0.296 h−1. These values differed significantly from those following i.v. administration of docetaxel, with a Cmax of 2124±1054 ng ml−1, an AUC0–∞ of 2571±1598 ng h ml−1 and a kel of 1.318±0.785 h−1 (P=0.003, P=0.006, P=0.016). The study medication was well tolerated and most of the adverse events possibly or probably related to OC144-093 and docetaxel were of CTC grade 1 and 2. One patient had a homozygous 3435T/T mutation, which is associated with low intestinal P-gp expression, and two other patients had a homozygous mutation on exon 21. The relative apparent bioavailability of 26% was most likely caused by a significant effect of OC144-093 on the oral uptake of docetaxel. Large intrapatient and interpatient (pharmacokinetic) variation was found after oral as well as after i.v. administration of docetaxel. Higher plasma levels were observed after 100 mg i.v. docetaxel than after 100 mg oral docetaxel plus 500 mg oral OC144-093. The safety of the oral combination was good. More patients should be evaluated to determine the effect of P-gp single nucleotide polymorphisms on oral pharmacokinetic values of docetaxel.

Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine.

Abstract: Purpose The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro and in vivo.

HERG K + channel expression-related chemosensitivity in cancer cells and its modulation by erythromycin

Abstract: Purpose Previous studies have found that the HERG K+ channel is highly expressed in some cancers. In the study reported here, we investigated HERG expression in various cancer cell lines, its correlation with chemosensitivity to vincristine, paclitaxel, and hydroxy-camptothecin, and its biochemical modulation.

Description of paclitaxel resistance-associated genes in ovarian and breast cancer cell lines.

Abstract: To identify genes involved in the paclitaxel resistance phenotype. High-density Affymetrix HG-U95Av2 microarrays were used to quantify gene expression in the resulting cell lines, SKOV-3TR, OVCAR8TR and MCF-7TR, and their drug-sensitive parental lines, SKOV-3, OVCAR8 and MCF-7. Three paclitaxel-resistant human ovarian and breast cancer cell lines were established. We identified 790 (SKOV-3TR), 689 (OVCAR8TR) and 964 (MCF-7TR) transcripts that were more than twofold overexpressed relative to their expression in the corresponding parental cell line. A comparison of these transcripts identified eight genes that were significantly overexpressed in all three drug-resistant daughter cell lines. These genes included MDR1, a gene often implicated in both in vitro and in vivo resistance to multiple chemotherapeutics, including paclitaxel. The remaining seven genes have not been previously associated with resistance to paclitaxel in human cancer. Furthermore, we identified 815 (SKOV-3TR), 430 (OVCAR8TR) and 332 (MCF-7TR) transcripts that were more than twofold decreased relative to their expression in the corresponding parental cell line. Comparison of these transcripts identified three genes that were significantly underexpressed in all three drug-resistant cell lines, none of which have been previously associated with paclitaxel resistance. Our results confirm that the paclitaxel resistance phenotype is associated with a large number of transcriptional changes. In addition, acquired paclitaxel resistance was associated with distinct transcriptional changes in each of the cell lines studied, suggesting that paclitaxel resistance is a complex phenotype that can arise through multiple mechanisms.

Biochemical modulation of cytarabine triphosphate by clofarabine

Abstract: Clofarabine has proven to be effective in the treatment of adult and pediatric acute myelogenous leukemia (AML). To investigate if clofarabine could be used with success in biochemical modulation strategies, we investigated the biochemical modulation of cytarabine triphosphate (ara-CTP) by clofarabine in a myeloid leukemia cell line and the effect of this combination on cytotoxicity. K562 cells were incubated with clofarabine and ara-C either sequentially or simultaneously to evaluate the combination effect on their phosphorylated metabolites. Clonogenic assays were used to determine the cytotoxicity of each agent alone and in combination. Deoxynucleotide analysis was performed to assess the effect of clofarabine on dNTPs. Clofarabine added either simultaneously or in sequence increased ara-CTP accumulation. The maximal modulation of ara-CTP accumulation occurred with 1 μM clofarabine. This level was achieved at the maximum tolerated dose for adult and pediatric patients with AML. With 10 μM ara-C alone, 86 μM ara-CTP had accumulated after 3 h. The optimal sequence for the drug combination, i.e., clofarabine followed 4 h later by ara-C, resulted in 248 μM ara-CTP at 3 h. Clofarabine accumulated maximally in the monophosphate form. Preincubation with ara-C did not affect the triphosphate form, but it lowered clofarabine monophosphate. Clofarabine resulted in the intracellular decrease of dATP and dGTP levels. Clonogenic assays revealed that the combination of clofarabine and ara-C produced synergistic killing of myeloid leukemia cells. These findings demonstrate that combination of clofarabine followed by ara-C results in a biochemical modulation of ara-CTP and synergistic cell kill. These studies provide a compelling rationale for clinical trials using this combination regimen for adult and pediatric patients with AML.