Growth inhibitory effect of a new camptothecin analog, DX-8951f, on various drug-resistant sublines including BCRP-mediated camptothecin derivative-resistant variants derived from the human lung cancer cell line PC-6

DX-8951f, a new water-soluble camptothecin (CPT) derivative, has been reported to show potent antitumor effects against various tumors in vitro and in vivo. We further evaluated the cytotoxic effect of DX-8951f against eight drug-resistant sublines derived by stepwise exposure of human oat cell carcinoma PC-6 to various drugs. In paclitaxel-, adriamycin-, vincristine- and etoposide-resistant cells, overexpression of P-glycoprotein (P-gp) and a correlative reduction in drug accumulation and typical drug-sensitivity pattern were confirmed. The etoposide-resistant line with the highest P-gp level was cross-resistant also to SN-38, CPT-11 and topotecan (TPT), but not to 9-aminocamptothecin (9-AC), CPT and DX-8951f. SN-38- and CPT-11-resistant cells, of which topoisomerase I activities and levels were similar to those of the parent cells, showed cross-resistance clearly to TPT, 9-AC and mitoxantrone, but hardly to DX-8951f. In these two resistant sublines, the intracellular topotecan level was significantly lower than that in parental PC-6 and the reduced accumulation was found to be mediated by breast cancer resistant protein (BCRP). The cisplatin-resistant variant, which had a 2-fold increase in glutathione content, showed no cross-resistance and the 5-fluorouracil-resistant variant, which had a 50% decrease in glutathione content, exhibited collateral sensitivity to most of the other anticancer agents including DX-8951f. We concluded that DX-8951f showed a potent cytotoxic effect on various types of drug-resistant cells.     

Comparison of DX-8951f and topotecan effects on tumor colony formation from freshly explanted adult and pediatric human tumor cells.

DX-8951f, which shows great therapeutic potential, was tested in the human tumor cloning system in adult and pediatric tumor types against which topotecan has been active. In 47 tumors from adults, DX-8951f had definite cytotoxic activity in a concentration-dependent manner with both 1 h and continuous exposures. Topotecan was minimally effective using a 1 h exposure and showed concentration-dependent inhibition with continuous exposure. In head-to-head comparisons at 1 h exposure against adult tumors, DX-8951f was significantly more effective at 0.1 and 1.0 microg/ml than topotecan. In head-to-head comparisons (continuous exposure), 1.0 microg/ml DX-8951f was more effective than topotecan at 1.0 microg/ml in adult tumors, including three of four head and neck, one of two kidney, two of five liver, six of 10 non-small cell lung, five of eight ovarian, four of eight prostate tumors, and in single specimens of breast, mesothelioma, colon and small cell lung tumors. With continuous exposure, DX-8951f and topotecan were equally effective at equimolar concentrations. The maximum tolerated dose for DX-8951f is 3 times that of topotecan, so higher doses of DX-8951f could be administered to patients. DX-8951f is a promising new antineoplastic agent with significant activity against tumors taken directly from patients.     

Antitumour activity of DX-8951f: a new camptothecin derivative

DX-8951f is a water-soluble camptothecin analogue with a unique hexacyclic structure. Compared to other current camptothecin derivatives, DX-8951f is the most effective topoisomerase I (topo I) inhibitor and has the most potent cytotoxic activity against various tumour cell lines in vitro. Of particular interest is DX-8951f’s significant effect on certain tumour cell lines resistant to other camptothecin derivatives, as well as on multi-drug resistant variants that overexpress P-glycoprotein. In addition, in in vivo xenograft systems using nude mice, DX-8951f strongly inhibits the growth of human solid tumours, including resistant tumours. Its antitumour effects and resulting life prolongation in tumour-bearing mice have also been confirmed in several metastasis models. DX-8951f provides greater therapeutic efficacy and broader effective dose ranges using multiple injections than with a bolus injection and simple intermittent applications. The in vivo effects of the compound are superior to those of CPT-11 and SK&F104864, suggesting that DX-8951f is a promising therapeutic agent for the treatment of cancer patients. Phase I clinical trials are ongoing in Europe, the USA and Japan.     

Antitumour activity of DX-8951f: a new camptothecin derivative

DX-8951f is a water-soluble camptothecin analogue with a unique hexacyclic structure. Compared to other current camptothecin derivatives, DX-8951f is the most effective topoisomerase I (topo I) inhibitor and has the most potent cytotoxic activity against various tumour cell lines in vitro. Of particular interest is DX-8951f's significant effect on certain tumour cell lines resistant to other camptothecin derivatives, as well as on multi-drug resistant variants that overexpress P-glycoprotein. In addition, in in vivo xenograft systems using nude mice, DX-8951f strongly inhibits the growth of human solid tumours, including resistant tumours. Its antitumour effects and resulting life prolongation in tumour-bearing mice have also been confirmed in several metastasis models. DX-8951f provides greater therapeutic efficacy and broader effective dose ranges using multiple injections than with a bolus injection and simple intermittent applications. The in vivo effects of the compound are superior to those of CPT-11 and SK&F104864, suggesting that DX-8951f is a promising therapeutic agent for the treatment of cancer patients. Phase I clinical trials are ongoing in Europe, the USA and Japan.     

Variation in the kinetics of caspase-3 activation, Bcl-2 phosphorylation and apoptotic morphology in unselected human ovarian cancer cell lines as a response to docetaxel

Paclitaxel is able to cause cell death through the induction of apoptosis. Cell death characteristics for docetaxel have not yet been described in detail. We investigated four unselected human ovarian cancer cell lines for the sensitivity to a 1hr exposure to docetaxel and calculated the concentrations inhibiting 50% (IC(50)) and 90% (IC(90)) of cell growth. Of the cell lines A2780, H134, IGROV-1 (all wild-type p53) and OVCAR-3 (mutant, mt p53) A2780 was most sensitive and OVCAR-3 least sensitive. Equitoxic drug concentrations representing IC(90) values (25-510nM) were applied for 1hr to measure cell cycle distribution, DNA degradation, and to count apoptotic cell bodies and cells with multifragmented nuclei at various time-points after drug exposure. H134, IGROV-1 and OVCAR-3 showed a continued mitotic block up to at least 72hr and prolonged presence of cells with multifragmented nuclei. High percentages of apoptosis were calculated at 48hr and at later time-points. In contrast, A2780 cells accumulated in the S-phase of the cell cycle and apoptosis was hardly present. The changes in the expression levels of p53, p21/WAF1, Bax and Bcl-2, were not predictive for docetaxel-induced apoptosis. Caspase-3 activation occurred only in cells with accumulation in the G2/M phase starting as early as 8hr in OVCAR-3. Prolonged Bcl-2 phosphorylation was evident in OVCAR-3, visible at 24hr in H134 and IGROV-1, while this phenomenon did not occur in A2780. The mitogen-activated protein kinase pathway (JNKs/SAPKs or c-Jun N-terminal kinases/stress-activated protein kinases, JNK1/2; extracellular response kinase, ERK1/2; p38) did not seem to be directly involved in Bcl-2 phosphorylation or apoptosis. We conclude that docetaxel is able to activate caspase-3, induce Bcl-2 phosphorylation and apoptosis in cells that show a prolonged G2/M arrest, but cells may also die by a caspase-3-independent cell death mechanism.     

Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2

Background and purpose:Paclitaxel is highly efficacious in the treatment of breast, head and neck, non-small cell lung cancers and ovarian carcinoma. For malignant gliomas, paclitaxel is prevented from reaching its target by the presence of the efflux pump P-glycoprotein (P-gp) at the blood-brain barrier. We investigated the utilization of a new drug delivery system to increase brain delivery of paclitaxel.Experimental approach:Paclitaxel molecules were conjugated to a brain peptide vector, Angiopep-2, to provide a paclitaxel-Angiopep-2 conjugate named ANG1005. We determined the brain uptake capacity, intracellular effects and antitumour properties of ANG1005 in vitro against human tumour cell lines and in vivo in human xenografts. We then determined ANG1005 activity on brain tumours with intracerebral human tumour models in nude mice.Key results:We show by in situ brain perfusion that ANG1005 enters the brain to a greater extent than paclitaxel and bypasses the P-gp. ANG1005 has an antineoplastic potency similar to that of paclitaxel against human cancer cell lines. We also demonstrate that ANG1005 caused a more potent inhibition of human tumour xenografts than paclitaxel. Finally, ANG1005 administration led to a significant increase in the survival of mice with intracerebral implantation of U87 MG glioblastoma cells or NCI-H460 lung carcinoma cells.Conclusions and implications:These results demonstrate the antitumour potential of a new drug, ANG1005, and establish that conjugation of anticancer agents with the Angiopep-2 peptide vector could increase their efficacy in the treatment of brain cancer.British Journal of Pharmacology (2008) 155, 185-197; doi:10.1038/bjp.2008.260; published online 23 June 2008.     

Synthesis and biological evaluation of novel A-ring modified hexacyclic camptothecin analogues

Eleven A-ring modified hexacyclic analogues of camptothecin (CPT) containing a 1,4-oxazine ring were synthesized from 10-hydroxycamptothecin (11a) and 7-ethyl-10-hydroxycamptothecin (3) (SN-38) in four to five steps and were subjected to the biological tests such as cytotoxicity, topoisomerase I (Topo I) inhibitory activity, acetylcholinesterase (AChE) inhibition, and stability in human plasma. Four compounds 15a, 15b, 16a, and 16c were about 2-fold more potent than topotecan and as potent as CPT toward human cancer cell lines A549, H128, WiDr, MKN45, SK-OV-3, and SK-BR-3 in vitro, even though the most active compound 15b was slightly less potent than SN-38. The potency of Topo I inhibition of these compounds showed relatively good correlation with their cytotoxicity. Most of the compounds exhibited AChE inhibitory activity weaker (9 +/- 2 to 20 +/- 3%) than CPT (23 +/- 5%) or topotecan (20 +/- 4%) and similar to SN-38 (13 +/- 2%), indicating that they might have little effect on causing early diarrhea. The stability of lactone forms of these compounds in human plasma seemed to be much higher than that of CPT and similar to that of topotecan but lower than that of SN-38. Among the new hexacyclic CPT analogues, compound 15b showed higher antitumor activity against human tumor xenograft, WiDr, in nude mice compared to that of SN-38. The most promising compound 15b has been selected for further development.     

A phase II clinical and pharmacokinetic study of intravenous exatecan mesylate (DX-8951f) in patients with untreated metastatic gastric cancer

Purpose: To determine the anti-tumor activity DX-8951f when administered as a 30-minute infusion daily for 5 days every 3 weeks to patients with previously untreated metastatic gastric cancer, and to evaluate toxicities and pharmacokinetics (PK) of DX-8951f in this patient population. Patients and methods: Forty-one patients were enrolled. All had previously untreated metastatic gastric cancer. DX-8951f was administered until disease progression or unacceptable toxicity. Responses were assessed after every 2 courses using RECIST criteria. Results: Thirty-nine patients were evaluable. Two patients achieved a partial response (PR) and 18 achieved stable disease (SD), including five patients with unconfirmed PR. A total of 141 courses of therapy were delivered (median 3, range 1–10). The most common drug-related toxicity was neutropenia. Non-hematologic toxicities were mostly mild to moderate; the most common were nausea, vomiting and anorexia. Plasma concentrations of DX-8951 (the anhydrous form of DX-8951f) were well described using a linear 2-compartment PK model. All concentrations and dose events were simultaneously modeled and explained by the population PK model. There was no evidence of non-linearity in the elimination PK, auto-inhibition or induction of DX-8951 clearance over the five days of administration. Conclusions: DX-8951f had modest activity against metastatic gastric cancer and its PK was dose-proportional. The toxicity profile was predictable and manageable. Further development of this agent is warranted.Supported by a contract from Daiichi Pharmaceutical Corporation, Montvale, New Jersey.     

Synthesis and evaluation of (2-(4-methoxyphenyl)-4-quinolinyl)(2-piperidinyl)methanol (NSC23925) isomers to reverse multidrug resistance in cancer

Development of multidrug resistance (MDR) during chemotherapy is a fundamental obstacle associated with cancer care. Prior studies have identified (2-(4-methoxyphenyl)-4-quinolinyl)(2-piperidinyl)methanol (5) (NSC23925) to be a small molecule agent that reverses MDR in cancer cells. We synthesized all four isomers of 5 and analyzed them by liquid chromatography-mass spectrometry (LCMS). Structure-activity relationships for reversing MDR were evaluated. Isomer 11 demonstrated the most potent activity. 11 reversed MDR in several drug-resistant cell lines expressing Pgp, including ovarian, breast, colon, uterine, and sarcoma cancer. 11 resensitized these cell lines to paclitaxel, doxorubicin, mitoxantrone, vincristine, and trabectedin with no effect on cell sensitivity to cisplatin, topotecan, and methotrexate. 11 significantly enhanced in vivo antitumor activity of paclitaxel in MDR xenograft models, without increasing the level of paclitaxel toxicity. In conclusion, 11 and derivatives of this compound may hold therapeutic value in the treatment of MDR-dependent cancers.     

In vitro sensitivity of human endometrial cancer cell lines to paclitaxel or irinotecan (CPT-11) in combination with other aniticancer drugs

We have evaluated the growth inhibitory effects of paclitaxel alone or irinotecan (CPT-11) alone and their combined effects with other drugs on human endometrial cancer cell lines. IC50 doses of paclitaxel (Tx), SN-38 (active metabolite of CPT-11; 7-ethyl-10-hydroxycamptothecin) and cisplatin, including other drugs which have been used for treatment of patients with endometrial cancer, were examined using five human endometrial cancer cell lines (Ishikawa, HEC-1A, HEC-50B, HEC-59 and HEC-108). When in vitro sensitivity was defined IC50 lower than 10% of the peak plasma concentration (PPC), all endometrial cancer cell lines were sensitive to paclitaxel and three of five endometrial cancer cell lines were sensitive to SN-38, whereas cisplatin was not active against any endometrial cancer cell lines used in this study. Regarding the other drugs, aclarubicin (ACR) and actinomycin D (ACD) were active against four of five endometrial cancer cell lines, etoposide (VP-16) and pirarubicin (THP) against two, and 5-fluorouracil (5-FU) against only one, while ifosfamide (4-OHIFO) was not active against any endometrial cancer cell lines. When combined effects of paclitaxel or SN-38 with other one drug were determined by the median-effect analysis, paclitaxel followed by cisplatin resulted in synergistic effects to all endometrial cancer cell lines. Paclitaxel followed by SN-38 also had synergistic effects to four cell lines. Sequential but not simultaneous administration of taxol and THP-adriamycin showed synergistic effects to three cell lines. In combinations of SN-38 with other drugs, simultaneous administration of SN-38 and cisplatin resulted in synergistic effects to all cell lines. It is noteworthy that ACD followed by SN-38 showed synergistic effects to all cell lines, and simultaneous treatment of ACD and SN-38 or SN-38 followed by ACD also resulted in synergistic effects to three cell lines. THP-adriamycin followed by SN-38 had synergistic effects to four cell lines. The present quantitative data analysis for synergism provides a basis for a rational design of clinical protocols for combination chemotherapy in patients with endometrial cancer of the uterus.     

Antitumor activity of 7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin, CKD602, as a potent DNA topoisomerase I inhibitor

We developed a novel water-soluble camptothecin analogue, CKD602, and evaluated the inhibition of topoisomerase I and the antitumor activities against mammalian tumor cells and human tumor xenografts. CKD602 was a nanomolar inhibitor of the topoisomerase I enzyme in the cleavable complex assay. CKD602 was found to be 3 times and slightly more potent than topotecan and camptothecin as inhibitors of topoisomerase, respectively. In tumor cell cytotoxicity, CKD602 was more potent than topotecan in 14 out of 26 human cancer cell lines tested, while it was comparable to camptothecin. CKD602 was tested for thein vivo antitumor activity against the human tumor xenograft models. CKD602 was able to induce regression of established HT-29, WIDR and CX-1 colon tumors, LX-1 lung tumor, MX-1 breast tumor and SKOV-3 ovarian tumor as much as 80, 94, 76, 67, 87% and 88%, respectively, with comparable body weight changes to those of topotecan. Also the therapeutic margin (R/Emax: maximum tolerance dose/ED₅₈) of CKD602 was significantly higher than that of topotecan by 4 times. Efficacy was determined at the maximal tolerated dose levels using schedule dependent i.p. administration in mice bearing L1210 leukemia. On a Q4d×4 (every 4 day for 4 doses) schedule, the maximum tolerated dose (MTD) was 25 mg/kg per administration, which caused great weight loss and lethality in <5% tumor bearing mouse. This schedule brought significant increase in life span (ILS), 212%, with 33% of long-term survivals. The ex vivo antitumor activity of CKD602 was compared with that of topotecan and the mean antitumor index (ATI) values recorded for CKD602 were significantly higher than that noted for topotecan. From these results, CKD602 warrants further clinical investigations as a potent inhibitor of topoisomerase I.     

Sensitization of ABCB1 overexpressing cells to chemotherapeutic agents by FG020326 via binding to ABCB1 and inhibiting its function

The effectiveness of chemotherapeutic treatment is usually limited by the overexpression of adenosine triphosphate binding cassette (ABC) transporters, which mediate multidrug resistance (MDR) by acting as efflux pumps to remove chemotherapeutic agents from MDR cancer cells. Thus, the inhibition of ABC transporters may represent a promising strategy to reverse MDR. This study was to characterize the actions of FG020326, a newly synthesized triaryl-substituted imidazole derivative, to reverse MDR in vitro and in vivo. FG020326 significantly potentiated the cytotoxicity of paclitaxel, doxorubicin, and vincristine in the ABCB1 (P-glycoprotein, P-gp) overexpressing cells KBv200 and MCF-7/adr, but not in the ABCB1 negative parental cell lines KB and MCF-7. However, FG020326 did not alter the cytotoxicity of the aforementioned drugs in ABCC1 (MRP1), ABCC4 (MRP4), ABCG2 (BCRP) and LRP overexpressing cell lines, KB-CV60, NIH3T3/MRP4-2, S1-M1-80 and SW1573/2R120, respectively. FG020326, following p.o. administration, was present in concentrations sufficient for reversal of MDR in mice. The co-administration of FG020326 with paclitaxel or vincristine significantly enhanced the antitumor activity of these drugs without significantly increasing toxicity in the mice bearing the KBv200 cell xenografts. In addition, FG020326, at concentrations that reversed MDR, did not significantly affect the activity of CYP3A4 or alter the pharmacokinetic profile of paclitaxel after co-administration with paclitaxel. FG020326 produced a significant concentration-dependent displacement of [³H]azidopine and inhibition of efflux of drug from cells. Furthermore, FG020326 was co-localized with ABCB1 in cell membranes. Hence, FG020326 is characterized as a third generation MDR modulator that holds great promise for the treatment of cancer patients with ABCB1-mediated MDR.     

Distribution of breast cancer resistance protein (BCRP/ABCG2) mRNA expression along the human GI tract

Human breast cancer resistance protein (BCRP/ABCG2) is an ABC-transporter that is present on the luminal membrane of intestinal epithelial cells and restricts absorption of anticancer drugs such as methotrexate, topotecan, mitoxantrone, and doxorubicin. The exact anatomic distribution of BCRP along the gastrointestinal (GI) tract, however, has not been determined before. The aim of this study was, therefore to investigate BCRP mRNA expression pattern along the GI tract in 14 healthy subjects. Furthermore, BCRP duodenal mRNA expression was compared with MDR1/ABCB1 mRNA. Additionally, BCRP mRNA expression was investigated in two human intestinal cell lines (Caco-2 and LS180). Since previous animal studies have suggested sex specific differences in BCRP expression, we analyzed intestinal BCRP expression with respect to sex. Biopsies were taken from different gut segments (duodenum, terminal ileum and ascending, transverse, descending and sigmoid colon). Gene expression was assessed by quantitative real-time PCR (Taqman). BCRP mRNA expression was maximal in the duodenum and decreased continuously down to the rectum (terminal ileum 93.7%, ascending colon 75.8%, transverse colon 66.6%, descending colon 62.8%, and sigmoid colon 50.1% compared to duodenum, respectively). BCRP expression in the duodenum was comparable to MDR1/ABCB1 gene expression. Caco-2 cells showed a comparable expression of BCRP as human duodenal tissue. Gender specific differences in BCRP expression were not observed. These findings represent the first systematic site-specific analysis of BCRP expression along the GI tract. This information might be helpful to develop target strategies for orally administered anticancer drugs.     

SL-01, an oral gemcitabine derivative, inhibited human cancer growth more potently than gemcitabine

SL-01, an oral gemcitabine derivative, was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl)pyrazine-2-carbonyl at the N4-position on the cytidine ring of gemcitabine. Our goal in this study was to evaluate the efficacy of SL-01 on the growth of human cancers with gemcitabine as control. Experiments were performed on human non-small cell lung cancer NCI-H460 and colon cancer HCT-116 both in vitro and in vivo. In vitro assays, SL-01 significantly inhibited the growth of cancer cells as determined by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Further studies indicated that SL-01 induced the cancer cells to apoptosis showing chromatin condensation and externalization of phosphatidylserine. In in vivo studies, we evaluated the efficacy of SL-01 in nude mice bearing human cancer xenografts. SL-01 effectively delayed the growth of NCI-H460 and HCT-116 without significant loss of body weight. Molecular analysis indicated that the high efficacy of SL-01 was associated with its ability to induce apoptosis as evidenced by increase of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining cells, activation of caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) in tumor tissues. SL-01 also increased Bax/Bcl-2 ratio in cancer cells. These biological activities of SL-01 were more potential than that of gemcitabine. Based on these in vitro and in vivo results, SL-01 is proposed as a potent oral anticancer agent that may supplant the use of gemcitabine in the clinic.     

DE-310, a macromolecular prodrug of the topoisomerase-I-inhibitor exatecan (DX-8951), in patients with operable solid tumors

Background: DE-310 is composed of the topoisomerase-I-inhibitor DX-8951 (exatecan) and a biodegradable macromolecular carrier, which are covalently linked by a peptidyl spacer. In pre-clinical studies, high levels and prolonged retention of conjugated DX-8951 (carrier-bound DX-8951) have been observed in tumor tissues following DE-310 administration. This phenomenon is explained as the enhanced permeability and retention (EPR) effect. DX-8951 and G-DX-8951 (glycyl-DX-8951) exerting anti-tumor activity in vivo are released from DE-310 by enzymatic cleavage of the spacer. Methods: To quantify the concentration of conjugated DX-8951, DX-8951 and G-DX-8951 in human tissues, six patients with different solid tumor types received 6.0 mg/m² of DE-310 (as equvalent of DX-8951) as a single three-hour infusion administered 7 days (±2 days) prior to scheduled tumor resection. Drug concentrations were then determined in the resected tissues. To evaluate the plasma PK of DE-310, plasma samples were taken up to 42 days post dosing. Results: There were no severe side effects of the DE-310 infusion. Concentrations of conjugated DX-8951, DX-8951 and G-DX-8951 were in general similar in tumor and relevant normal tissue samples and preferential accumulation of DE-310, DX-8951 and G-DX-8951 in human tumor tissues was not observed. Conclusions: These data indicate that there is distribution of DE-310 into tissue and that DX-8951 and G-DX-8951 are released slowly over an extended period from DE-310 providing prolonged exposure similar to a continuous infusion. However, the similarity in the concentrations in tumor and relevant normal tissues does not support the EPR concept in the studied human cancers.     

Mitochondria-mediated apoptosis operating irrespective of multidrug resistance in breast cancer cells by the anticancer agent prodigiosin

Prodigiosin (PG) is a red pigment produced by Serratia marcescens with pro-apoptotic activity in haematopoietic and gastrointestinal cancer cell lines, but no marked toxicity in non-malignant cells. Breast cancer is the most frequent malignancy among women in the European Union and better therapies are needed, especially for metastatic tumors. Moreover, multidrug resistance is a common phenomenon that appears during chemotherapy, necessitating more aggressive treatment as prognosis worsens. In this work, we extend our experiments on PG-induced apoptosis to breast cancer cells. PG was potently cytotoxic in both estrogen receptor positive (MCF-7) and negative (MDA-MB-231) breast cancer cell lines. Cytochrome c release, activation of caspases-9, -8 and -7 and cleavage of poly (ADP-ribose) polymerase protein typified the apoptotic event and caspase inhibition revealed that PG acts via the mitochondrial pathway. In a multidrug-resistant subline of MCF-7 cells that over-expresses the breast cancer resistance protein, the cytotoxic activity of PG was slightly reduced. However, flow-cytometry analysis of PG accumulation and efflux in MCF-7 sublines showed that PG is not a substrate for this resistance protein. These results suggest that PG is an interesting and potent new pro-apoptotic agent for the treatment of breast cancer even when multidrug resistance transporter molecules are present.