Clinical Evaluation of Liposome Encapsulated Doxorubicin and the Modulation of Multidrug Resistance in Cancer Cells

Abstract

Doxorubicin is the cornerstone of some widely used combination chemotherapy regimens because of its high anticancer activity in a number of human neoplasms. However, its clinical use is highly compromised because of treatment-limiting acute and chronic toxicities of which cardiotoxicity has the most debilitating effect. Our laboratories have demonstrated that liposome encapsulated doxorubicin (LED) provides important advantages in regards to the attenuation of cardiotoxicity in rodents by altering pharmacokinetics and pharmacodynamics of the drug, provides effective protection from immunotoxicity and maintains full therapeutic activity of the drug in liposomes. A Phase I clinical trial of LED in cancer patients has establish the maximum tolerated dose of 90 mg/m² with granulocytopenia being the major treatment-limiting toxicity. We have performed a Phase II trial of LED in 20 recurrent breast cancer patients at a dose of 75 mg/m² as an intravenous infusion every three weeks. Objective responses were observed in 9/20 patients of which 5 demonstrated a complete response. Hematologic toxicity with LED consisted of only grade 1-2 granulocytopenia in some patients, whereas gastrointestinal toxicity, mucositis and venous sclerosis were markedly reduced. Alopecia was complete in all patients. Twelve patients received cumulative LED doses of more than 400 mg/m² and 8 of them received doses of over 500 mg/m². Five of these patients were followed by endomyocardial biopsies and 4 of them were found to be Billingham Grade 0 whereas one of them had Billingham Grade 1 toxicity (cumulative dose of 750 mg/m²). This Phase II trial demonstrates higher therapeutic efficacy of LED than free doxorubicin in recurrent breast cancer patients with strong indication of cardiotoxicity protection at doses of 500-800 mg/m².

The emergence of tumor cells resistant to major classes of cytotoxic agents is a predominant obstacle in cancer treatment. This resistance is frequently related to the expression of a plasma membrane P-glycoprotein (pgp) of 170 Kd that is encoded by a family of MDR genes. Support for the involvement of pgp in MDR has been shown by transaction of sensitive cells with an expression vector containing full length cDNA of the MDR1 gene, which results in the appearance of pgp and the sensitive cells convert to the drug-resistant phenotype. Our studies demonstrate that LED modulates very effectively the MDR phenotype in LZ cells, a Chinese hamster cell line made resistant to doxorubicin and the cellular drug uptake was 2 to 3 fold higher with LED exposure than with free drug. This modulation of drug resistance and enhanced cellular drug uptake is effected by the direct binding of liposomes to pgp on the surfaces of MDR phenotype cells. LED completely inhibited the photoaffinity labeling of pgp by azidopine in membrane vesicles of HL-60/VCR cells and in KB-GSV2 cells transfected with human MDR gene. These studies demonstrate that LED has unique effectiveness in overcoming MDR phenotype in cancer cells and appears to be a potentially attractive modality of treatment of human cancers.     

Identification of members of the P-glycoprotein multigene family.

Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, we found that the hamster P-glycoprotein gene family consists of three genes. We also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. We propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgp1 and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. We speculate that the hamster pgp1 and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.     

Studies on low-level MDR cells

Acquired or spontaneous resistance is a major clinical problem in the treatment of cancer. Low levels of MDR gene expression or P-glycoprotein have been correlated with a high level of drug resistance in vitro and a poor response to chemotherapy in some tumors. A strong correlation between MDR mRNA, P-glycoprotein levels and degree of drug resistance has not been found in several resistant model tumor cell lines. In some cell lines at low and high level of resistance different mechanisms seem to be involved.     

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.     

Purification and reconstitution of functional human P-glycoprotein

The overexpression of the P-glycoprotein, theMDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [³H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.     

Functional expression of human P-glycoprotein in Schizosaccharomyces pombe

Human MDR1 cDNA was introduced into the human cultured cells KB-3-1 and Schizosaccharomyces pombe pmdI null mutant KN3. The drug sensitivity of KB-G2 and KN3/pgp, expressing human P-glycoprotein, was examined. KB-G2 was resistant to the peptide antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype. KN3/pgp was resistant to valinomycin and actinomycin D, but not to adriamycin. The ATP-hydrolysis-deficient mutant did not confer KN3 resistance to these antibiotics. Human P-glycoprotein expressed in S. pombe seemed to lack N-glycosylation. The N-glycosylation-deficient mutant, however, conferred a typical MDR phenotype on KB-3-1. These results suggest that human P-glycoprotein functions as an efflux pump of valinomycin and actinomycin D in the membrane of S. pombe.     

Repair synthesis and induction of thymidine-resistant variants in mouse lymphoma cells of different radiosensitivity

An assay system has been characterised using excess thymidine (TdR) as a selective agent, and the dose-response curve for X-ray induced variants resistant to thymidine has been compared with that for X-ray induced variants resistant to 5-iodo-2-deoxyuridine (IUdR) in P388 lymphoma cells. Dose-response curves showed a linear and a dose squared component in this cell line and were similar in both selective systems. A comparison has been mae of the dose-response curves for X-ray induced thymidine resistant (TdR⁺) variants in four other lymphoma cell lines of differing radiosensitivity. When induced frequencies were compared either at the same dose or at the same survival level the most sensitive line L5178YS was found to be most mutable.Repair replication levels were measured in all cell lines but no correlation between observed levels of repair replication and mutability in the 5 cell lines was found. The data are discussed in relation to the possible involvement of repair processes in mutation induction by X-rays in mammalian cells.     

Isolation and characterization of Drosophila multidrug resistance gene homologs.

Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp. The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances. This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene. These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins. Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism. This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.     

Synthesis of a dual functional anti-MDR tumor agent PH II-7 with elucidations of anti-tumor effects and mechanisms

Multidrug resistance mediated by P-glycoprotein in cancer cells has been a major issue that cripples the efficacy of chemotherapy agents. Aimed for improved efficacy against resistant cancer cells, we designed and synthesized 25 oxindole derivatives based on indirubin by structure-activity relationship analysis. The most potent one was named PH II-7, which was effective against 18 cancer cell lines and 5 resistant cell lines in MTT assay. It also significantly inhibited the resistant xenograft tumor growth in mouse model. In cell cycle assay and apoptosis assay conducted with flow cytometry, PH II-7 induced S phase cell cycle arrest and apoptosis even in resistant cells. Consistently revealed by real-time PCR, it modulates the expression of genes related to the cell cycle and apoptosis in these cells, which may contributes to its efficacy against them. By side-chain modification and FITC-labeling of PH II-7, we were able to show with confocal microscopy that not only it was not pumped by P-glycoprotein, it also attenuated the efflux of Adriamycin by P-glycoprotein in MDR tumor cells. Real-time PCR and western blot analysis showed that PH II-7 down-regulated MDR1 gene via protein kinase C alpha (PKCA) pathway, with c-FOS and c-JUN as possible mediators. Taken together, PH II-7 is a dual-functional compound that features both the cytotoxicity against cancer cells and the inhibitory effect on P-gp mediated drug efflux.     

Identification of a 170 kDa membrane kinase with increased activity in KB-V1 multidrug resistant cells

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of ³²P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723–20729, 1989; Mol Cell Biol 10:3020–3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca⁺⁺, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr^R and murine melanoma B16/Adr^R. cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.     

Immunological characterization of hypoxanthine-guanine phosphoribosyl transferase mutants of mouse L cells: Evidence for mutations at different loci in the HGPRT gene

A large collection (105) of mouse L cell mutants lacking hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT; E. C. 2.4.2.8) were analyzed for the presence of serologically cross reacting material (CRM). Antibody directed against highly purified mouse liver HGPRT was used for detecting CRM activity by two methods: (1) the standard precipitation-inhibition assay; and (2) a radioimmune-precipitation assay. The latter assay proved to have far greater sensitivity for the detection of altered forms of HGPRT. Approximately 40% of the HGPRT⁻ cell lines contain CRM activity (i.e., were CRM⁺). This indicates that a minimum of 40% of the HGPRT⁻ clones arose as a result of mutations in the HGPRT structural gene. The CRM⁺ cell lines were shown to contain different levels of CRM activity. Measurements of the heat sensitivity of CRM in the different HGPRT⁻ cell lines showed a broad spectrum of CRM heat inactivation kinetics. These latter two observations provide strong evidence that the mutations giving rise to the HGPRT⁻CRM⁺ phenotype occurred at different sites in the HGPRT structural gene.     

Detection of recombinant P-glycoprotein in multidrug resistant cultured cells

The MDR1 multidrug resistance gene encodes a high molecular weight membrane-spanning cell surface protein, P-glycoprotein, that confers multidrug resistance by pumping various cytotoxic drugs, including vinblastine, doxorubicin or paclitaxel, out of cells. Overexpression of P-glycoprotein in human tumors has been recognized as a major obstacle for successful chemotherapy of cancer. Thus, P-glycoprotein represents an important drug target for pharmacological chemosensitizers. Initially, cell culture models to study the multidrug resistance phenotype were established by selecting drug-sensitive cells in step-wise increasing, sublethal concentrations of chemotherapy agents. P-glycoprotein was found to be overexpressed in many of these models. Multidrug resistant cells can also be generated by transfection of cultured cells with the MDR1 gene, followed by selection with cytotoxic drug at a concentration that kills all untransfected host cells. Transfectants expressing wild-type or mutant recombinant P-glycoprotein have significantly contributed to our understanding of the structure of P-glycoprotein and its molecular and cellular functions. Additionally, the MDR1 gene has also been used as a selectable marker for the transfer and coexpression of non-selectable genes. This article details means for detection of P-glycoprotein in DNA-transfected or retrovirally transduced, cultured cells. Different experimental approaches are described that make use of specific antibodies for detection of P-glycoprotein. Strategies to visualize P-glycoprotein include metabolic labeling using ³⁵S-methionine, labeling with a radioactive photoaffinity analog, and non-radioactive immunostaining after Western blotting.     

Abundance and state of phosphorylation of the retinoblastoma susceptibility gene product in human colon cancer

In an effort to understand the possible role of Rb in cellular growth control, we have investigated the abundance and the state of phosphorylation of Rb protein (pRb) in normal and colon tumor cell lines as well as in matched colon tumors, adenomas and adjoining normal colonic mucosa. Resting normal human fibroblast cell lines were found to have only unphosphorylated pRb and phosphorylation of pRb occurred when the cells entered G1-S phase. In general, the colon tumor tissues had atleast 1.5–2.0 fold increase in the abundance of pRb and 1.5–2.5 fold increase in the percentage of its phosphorylation as compared to the corresponding normal colonic mucosa. Whereas, the adenomas had similar pRb level and its phosphorylation status as observed in the normal colonic mucosa. The actively growing tumor cell lines had approximately two fold higher total pRb than normal cell lines. Although, the percentage of phosphorylated form in growing tumor cell lines as well as normal cell lines were almost equal, it was still considerably higher than normal colonic mucosa. Moreover, DNA binding assay revealed reduced binding affinity of pRb from colon tumor cell line SW480 as compared to the normal cell line W138. These results suggest that the abundance of pRb and its phosphorylation level may have a role in the cellular growth control in human colonic epithelium.