Robert L. Fine

Antiestrogens and steroid hormones: Substrates of the human P-glycoprotein

Multidrug-resistant (MDR) tumor cells reduce the toxicity of antineoplastic drugs by an energy-dependent active efflux mechanism mediated by the MDR1 gene product, the P-glycoprotein (Pgp). Pgp expressed in cultured Sf9 insect cells has been shown to exhibit a high capacity ATPase activity in the presence of a variety of drugs known to be transported by the Pgp (Sarkadi et al., J Biol Chem 267: 4854-4858, 1992). The strict dependence of the Pgp ATPase activity on the presence of transport substrates indicates that the drug-stimulated ATPase activity is a direct reflection of the drug transport function of the Pgp. In the present study, this system has been utilized to investigate the possibility that antiestrogens and steroid hormones are transported by the Pgp. Antiestrogens such as tamoxifen, metabolites of tamoxifen (4-hydroxytamoxifen and N-desmethyltamoxifen), droloxifen, and toremifene stimulated the Pgp ATPase activity, and the maximum stimulation obtained with these agents equalled the maximal stimulation obtained by the best known MDR chemosensitizer, verapamil. Clomifene, nafoxidine and diethylstilbestrol also stimulated the Pgp ATPase activity, with maximal activations 75, 60 and 45% of the verapamil stimulation, respectively. Different degrees of stimulation of the Pgp ATPase activity were also obtained in the presence of steroid hormones such as progesterone, beta-estradiol, hydrocortisone, and corticosterone. Among these, progesterone is a potent inducer of the Pgp ATPase activity; at 50 microM, this hormone stimulated the Pgp ATPase activity as effectively as verapamil. These results suggest that the antiestrogens and steroid hormones that are known to reverse the multidrug-resistant phenotype do so by directly interacting with Pgp, thus interfering with its anticancer drug-extruding activity.

Partial Inhibition of Multidrug Resistance by Safingol Is Independent of Modulation of P-glycoprotein Substrate Activities and Correlated with Inhibition of Protein Kinase C

Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC. We investigated the effects of safingol on antineoplastic drug sensitivity and PKC activity of MCF-7 tumor cell lines. Safingol treatment of 32P-labeled MCF-7 WT and MCF-7 DOXR cells inhibited phosphorylation of the myristoylated alanine-rich protein kinase C substrate in both cell lines, suggesting inhibition of cellular PKC. However, only in MCF-7 DOXR cells did safingol treatment increase accumulation of [3H]vinblastine and enhance toxicity of Vinca alkaloids and anthracyclines. Drug accumulation changes in MCF-7 DOXR cells treated with safingol were accompanied by inhibition of basal and phorbol 12,13-dibutyrate-stimulated phosphorylation of P-glycoprotein (P-gp). Expression of P-gp and levels of mdr1 message in MCF-7 DOXR cells were not altered by safingol treatment alone or in combination with vinblastine. Treatment of MCF-7 DOXR cell membranes with safingol did not inhibit [3H]vinblastine binding or [3H]azidopine photoaffinity labeling of P-gp. Furthermore, safingol did not stimulate P-gp ATPase activity in membranes prepared from MCF-7 DOXR cells. We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOXR cells treated with safingol are correlated with inhibition of PKC rather than competitive interference with P-gp drug binding through direct interaction with P-glycoprotein.

TOBACCO BUDWORM P-GLYCOPROTEIN : BIOCHEMICAL CHARACTERIZATION AND ITS INVOLVEMENT IN PESTICIDE RESISTANCE

Since pesticides have been shown to interact with P-glycoprotein (P-gp), the purpose of this study was to examine the possible role of P-gp in pesticide resistance in the tobacco budworm (Heliothis virescens). Using three P-gp antibodies, P-gp expression in various resistant populations of tobacco budworms was found to be 2-6-times that of the susceptible larvae. Tobacco budworm P-gp was glycosylated and localized primarily in the cuticle and fat body with little expression in the mid gut. To determine the role of P-gp in pesticide resistance, resistant tobacco budworm larvae were treated with a P-gp inhibitor, quinidine, and challenged with various doses of thiodicarb. Inhibition of P-gp decreased the LD50 for thiodicarb by a factor of 12.5. Quinidine treatment did not result in a significant inhibition of the P-450 system nor did it alter the feeding of the larvae, suggesting the potential involvement of P-gp in pesticide resistance. An age-dependent increase in P-gp expression was detected in resistant larvae as compared to control, susceptible larvae. This correlates with the reported age-dependent increase in resistance and is further evidence supporting the role of P-gp in the development of pesticide resistance.

P-Glycoprotein, Multidrug Resistance and Protein Kinase C

The multidrug resistant (MDR) phenotype is a well‐studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P‐glycoprotein plays a major role in MDR, it is not clear whether post‐translational modifications such as phosphorylation have any major impact on its modulation.

Chlorpyrifos oxon interacts with the mammalian multidrug resistance protein, P-glycoprotein.

Multidrug resistance (MDR) to chemically unrelated therapeutic anticancer agents in mammalian cells is mediated by the overexpression of an ATP-dependent 150- to 180-kD membrane glycoprotein P-glycoprotein (P-gp). Although the complete physiological role of P-gp is unknown, it is proposed to function in cellular detoxification of xenobiotics. In this study, we investigated whether the organophosphorus insecticide chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridinyl phosphorothioate) or its metabolites interact with P-gp. Immunohistochemical analysis of tissues from male Fischer 344 rats administered chlorpyrifos (7.6 mg/kg gavage) showed increased P-gp expression in the kidney, adrenal, liver, jejunum, and stomach (tissues associated with elimination of xenobiotics), compared to control tissues. The most prominent increase was detected in the large bile ducts of the liver and the proximal tubule region of the kidney. P-gp expression was increased throughout the adrenal medulla and cortex, while a moderate increase was detected in the epithelial layers of the stomach and jejunum. To examine further the interaction between chlorpyrifos and P-gp, we evaluated whether chlorpyrifos or its active metabolite, chlorpyrifos oxon, could inhibit [3H]azidopine labeling of P-gp in MDR1 baculovirus-infected insect Sf9 cells. A concentration-dependent inhibition of [3H]azidopine labeling of P-gp was detected with chlorpyrifos oxon, while significant inhibition was not detected with chlorpyrifos. To correlate the binding of chlorpyrifos oxon to P-gp with a biochemical effect, we examined its ability to stimulate P-gp-mediated ATPase activity in these Sf9 cells. Chlorpyrifos oxon stimulated P-gp ATPase activity 1.75 times that of the positive control (10 microM verapamil). Taken together, these results suggest that chlorpyrifos oxon interacts with P-gp, and support the hypothesis that P-gp may play a role in the cellular detoxification of insecticides in mammalian tissues. To our knowledge this is the first report of an organophosphorus insecticide interacting with and increasing the expression of P-gp.

A phase I/II trial of twice daily irradiation and concurrent chemotherapy for locally advanced squamous cell carcinoma of the head and neck

PURPOSEnThis study was designed to test the toxicity and efficacy of a regimen of twice daily irradiation and concurrent multiagent chemotherapy for patients with locally advanced squamous cell carcinoma of the head and neck.nnnMETHODS AND MATERIALSnThis was a prospective Phase I/II trial. Patients received 125 cGy b.i.d. to 7000 cGy with a 6 hr interfraction interval. Chemotherapy was given during weeks 1 and 6 of irradiation and consisted of a 5 day infusion of 5-fluorouracil at 600 mg/M2/day and 5 daily injections of cisplatin at 12 mg/M2/day. Two additional cycles of chemotherapy were given after the completion of radiotherapy.nnnRESULTSnForty-six patients were evaluable: 28 had technically unresectable disease and 18 had resectable tumors. All had Stage III or IV disease: 84% had T3 or T4 primaries while 53% had > or = N2 neck disease. The primary acute toxicity, confluent mucositis, was seen in 74% of patients. Late side effects occurred in four patients. Median follow-up is 36 months (range 25-44 months). Kaplan-Meier estimates of 2-year disease-free survival and overall survival are 65% and 73%, respectively, while 2-year local regional control and distant disease-free survival are 72% and 88%, respectively. Multivariate analysis revealed that resectability and receiving > 2 cycles of chemotherapy significantly influenced local regional control while age < 60 significantly influenced disease-free survival.nnnCONCLUSIONnThis form of treatment can be delivered safely. The encouraging results have led to the initiation of a Phase III trial comparing this regimen with b.i.d. radiation alone.

Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells☆

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 microM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 microM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7ADR cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.

Differential phosphorylation of sites in the linker region of P-glycoprotein by protein kinase C isozymes α, βi, βii, γ, δ, ϵ, η, and ζ

Abstract To determine whether individual protein kinase C (PKC) isozymes differentially phosphorylate sites in the linker region of human P-glycoprotein (P-gp), we used a synthetic peptide substrate, PG-2, exactly corresponding to amino acid residues spanning the region 656–689 of the multidrug resistance gene (MDR1). All tested PKC isozymes phosphorylated PG-2. The maximum phosphate incorporation by calcium-dependent PKC isozymes α, βI, βII, and γ was 3, 2, 2, and 3 mol phosphate/mol PG-2, respectively. The maximum phosphate incorporation by calcium-independent isozymes δ, ϵ, η, and ζ was 1.5, 0.5, 1.5, and 1.5 mol phosphate/mol PG-2, respectively. Two-dimensional tryptic phosphopeptide mapping indicated differential phosphorylation of the PKC consensus sites Ser-661, Ser-667, and Ser-671 by individual isozymes, which may be functionally significant. These data suggest that differential phosphorylation by PKC isoenzymes of PKC sites within the P-gp linker region may play a role in modulating P-gp activity.

Glutathione and glutathione S-transferase in benign and malignant prostate cell lines and prostate tissues.

Metastatic prostate adenocarcinoma is unresponsive to alkylator chemotherapy with virtually no prolonged remissions. Glutathione (GSH) and glutathione S-transferase (GST) have been reported to play a role in tumor resistance to alkylator therapy; however, there are no baseline studies that have investigated and compared GSH and GST in human prostate cell lines and tissues. Thus, we determined the GSH content and GST activity in benign prostate, in primary and metastatic prostate adenocarcinoma tissues, in immortal adenocarcinoma cell lines, and in primary cell cultures derived from both benign prostate and primary prostatic carcinoma tissue. The GSH content was higher in the immortal cell lines than in the fresh tissues and primary cultures. Conversely, the GST activity was significantly higher in the tissues and primary cultures than in the cell lines. The GSH content and GST activity of the primary cultured prostatic cells were similar to those of the prostate tissues. The differences between the immortal prostate cancer cell lines and prostate tissue are of sufficient magnitude to suggest that in vitro results with cell lines may not extrapolate to prostate cancer in vivo. The GSH content and GST activity in a prostate specific antigen-secreting human prostate tumor xenograft, LuCaP23, maintained in nude mice were similar to those of human prostate tissue and primary cultures. Both the xenograft and primary cultures from patients with prostate cancer may be more appropriate models than established cell lines for investigating techniques to increase the effectiveness of alkylators in prostate cancer.

N-(4-Hydroxyphenyl) Retinamide-lnduced Cytotoxicity of Cultured Human Squamous Cell Carcinoma

The role of growth factors in the development and spread of head and neck cancers has received little attention. Platelet-derived growth factor (PDGF) is a potent mitogen 9 that is released normally in wound healing but is also secreted by human malignant epithelial cells. In breast and ovarian carcinomas, elevated serum PDGF has correlated with a poorer prognosis. This preliminary study aimed to determine if PDGF is elevated in the serum of patients with carcinoma of the head and neck and to correlate these levels with tumor aggressiveness. If levels are elevated a decrease would be expected after surgery, potentiating the use of PDGF as a serum marker that could be used to detect disease recurrence. PDGF was measured by radioimmunoassay in 20 patients with head and neck cancer and in 12 normal controls. It is noteworthy that in the control group the mean level was 7.4 fmol/100 I.tl (range, 2.8 to 12.5; median, 7.6) in spite of the fact that PDGF is reportedly not measurable in normal subjects. The mean PDGF level in the patients with cancer was 19.9 fmol/100 tll (range, 4.1 to 35.7; median, 19.4), and compared with the control group the levels were significantly elevated (median 2-sample test, p <0.001). Of the 20 patients with cancer only four had levels less than 12.5 fmol/ 100 ~tl. Two of these had prior radiation therapy. PDGF levels decreased significantly after surgery. These results support the further investigation of serum PDGF levels as a prognostic indicator of tumor aggressiveness. This biomarker may also prove useful for detecting tumor recurrence.