Pharmacokinetic basis for the comparative antitumour activity and toxicity of chlorambucil,phenylacetic acid mustard and β,β-difluorochlorambucil (CB 7103) in mice

Summary This report describes the relationship between the pharmacokinetics, antitumour activity and toxicity of chlorambucil (CHL), phenylacetic acid mustard (PAAM) and ,-difluorochlorambucil (-F₂CHL) in mice. Pharmacokinetics were studied by HPLC, antitumour activity by a regrowth delay assay using the KHT murine sarcoma and toxicity by acute LD₅₀. For both antitumour activity and acute toxicity the order of potency was: PAAM>CHL>-F₂CHL. CHL and PAAM exhibited identical therapeutic indices, whereas that for -F₂CHL was somewhat improved. CHL is metabolized by mitochondrial -oxidation to the 3,4-dehydro derivative (DeHCHL) and PAAM, and the latter is further metabolized to its monodechloroethylated derivative DeC-PAAM, presumably by hepatic microsomal enzymes. Administered PAAM gave only one metabolite, DeC-PAAM. Unexpectedly, despite ,-disubstitution, -F₂CHL was also -oxidized to give DeHCHL and PAAM, but at reduced rates. Further, metabolic switching was demonstrated with the appearance in large amount of 2 new, unidentified metabolites, which may be dechlorethylation products. The pharmacokinetics of administered CHL, PAAM and -F₂CHL differ in that the plasma clearance was fastest for CHL, slowest for PAAM and intermediate for -F₂CHL. For the metabolites, CHL produced peak plasma concentrations of DeHCHL and PAAM, respectively, 7-fold and 2-fold greater than those produced by -F₂CHL. However, despite these differences, exposures to total bifunctional nitrogen mustards were similar following administration of the 3 drugs and therefore cannot account for their differential activity. In contrast, there was a good correlation between potency and PAAM exposure, which is highest after treatment with PAAM, intermediate after CHL and lowest after -F₂CHL. In plasma, 3.2% of PAAM is present as nonprotein-bound free drug, compared to 1.3% for DeHCHL, 0.9% for CHL and 0.45% for -F₂CHL. We propose the amount of free bifunctional nitrogen mustard, itself partly dependent on the extent of metabolism, to be of major importance for the in vivo potency of CHL analogues.     

Quantification of thrombospondin-1 secretion and expression of αvβ3 and α3β1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface v3 and 31 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of v3 and 31 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of v3 and 31 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/1 × 10⁶ cells/24 h (mean ± SD=626 ± 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 g. Seven of eight non-glioma cell lines secreted less than 100 0ng of TSP-1. All glioma cell lines expressed 31 integrin and syndecan-1, and seven of 10 glioma cell lines expressed v3 integrin. Treatment of the glioma cell lines with TGF-2 did not change the expression of v3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with v3 and 31 integrins, and syndecan-1.     

Beta-adrenergic receptors in DMBA-induced rat mammary tumors: Correlation with progesterone receptor and tumor growth

Summary In order to gain further knowledge about the potential role of catecholamines in mammary carcinoma, we have used the potent -adrenergic antagonist cyanopindolol (CYP) as iodinated ligand to characterize -adrenergic receptors in membranes prepared from mammary tumors induced by dimethylbenz(a)anthraene (DMBA) administration in the rat. The binding of [¹²⁵I]CYP to membrane preparations of DMBA-induced rat mammary tumors is rapid at room temperature, reaching half maximal specific binding at 30 min of incubation. Scatchard analysis of the data indicates that [¹²⁵I]CYP binds to a single class of high affinity sites (114 ± 2.1 fmoles/mg protein) at an apparent K_D value of 38.0 ± 0.3 pM. The order of potency of a series of agonists to compete for [¹²⁵I]CYP binding is consistent with interaction with a ₂-subtype receptor: zinterol > (–)isoproterenol > (–)epinephrine» (–)norepinephrine. In addition, the potency of a series of specific ₁, and ₂ synthetic compounds to displace [¹²⁵I]CYP in mammary tumors is similar to their potency in typical ₂-adrenergic tissues. The binding of [¹²⁵I]CYP to DMBA-induced rat mammary tumors shows a marked stereoselectivity, the (–)isomers of isoproterenol and propranolol being 150 and 80 times more potent, respectively, than their respective enantiomers. The autoradiographic localization of [¹²⁵I]CYP performed on frozen sections revealed the presence of specific -adrenergic receptors in all the malignant cells. Spontaneous mammary tumors of aging (18–22 months) female rats have high levels of -adrenergic receptors. Castration decreased the concentration of [¹²⁵I]CYP binding sites in DMBA-induced mammary tumors. A close correlation was observed between progressing, static, and regressing tumors after ovariectomy and -adrenergic receptor concentration. The presence of -adrenergic receptors in mammary tumors as well as the modulation of their level by ovarian hormones provides a mechanism for catecholaminergic influence in mammary cancer tissue.     

Down-regulation of transforming growth factor-β and interleukin-10 secretion from malignant glioma cells by cytokines and anticancer drugs

The effect of treatment with interleukin-1 (IL-1), interferon- (IFN-), vincristine, and etoposide was evaluated on the secretion of transforming growth factor- (TGF-) and IL-10 and the expression of major histocompatibility complex (MHC) class I, intercellular adhesion molecule-1 (ICAM-1), and CD80 molecules by malignant glioma cells. Five malignant glioma cell lines were treated with IL-1, IFN-, and/or anticancer agents (vincristine and etoposide). Combined treatment with IL-1 and IFN- caused greater inhibition of TGF- secretion compared to treatment with IFN-, and almost the same levels of inhibition as treatment with vincristine and etoposide. The greatest inhibition of TGF- secretion was achieved by treatment with all agents. Low levels of IL-10 secretion were determined in two out of five malignant glioma cell lines. This IL-10 secretion was inhibited by treatment with IL-1, IFN-, vincristine, and/or etoposide. Treatment with both cytokines and anticancer agents increased the expression of MHC class I and ICAM-1 in all tumor cell lines. The mean increase of expression of MHC class I was 50% and that of ICAM-1 was 12-fold. No tumor cell lines expressed CD80 molecules on the cell surface, and no treatment caused CD80 expression. These results suggest that TGF- and IL-10 secretion by malignant glioma cells can be suppressed by treatment with a combination of IL-1, IFN-, vincristine, and etoposide, and the treatment up-regulates MHC class I and ICAM-1 expression on tumor cells. These results have implications for immunotherapy and chemotherapy in patients with malignant tumors.     

Histamine H1 Receptor Activation Stimulates Mitogenesis in Human Astrocytoma U373 MG Cells

In human astrocytoma U373 MG cells that express histamine H₁ receptors (180 ± 6fmol/mg protein) but not H₂ or H₃ receptors, histamine stimulated mitogenesis as assessed by [³H]-thymidine incorporation (173 ± 2% of basal; EC₅₀, 2.5 ± 0.4M). The effect of 100M histamine was fully blocked by the selective H₁ antagonist mepyramine (1M) and was markedly reduced (93 ± 4% inhibition) by the phospholipase C inhibitor U73122 (10M).The activator of protein kinase C (PKC) phorbol 12-tetradecanoyl-13-acetate (TPA, 100nM) stimulated [³H]-thymidine incorporation (270 ± 8% of basal), and this response was not additive with that to 100M histamine. The incorporation of [³H]-thymidine induced by 100M histamine was partially reduced by the PKC inhibitor Ro 31-8220 (57 ± 7% inhibition at 300nM) and by the compound PD 098,059 (30M, 62 ± 14% inhibition), an inhibitor of the mitogen-activated kinase (MAPK) kinases MEK1/MEK2.These results show that histamine H₁receptor activation stimulates the proliferation of human astrocytoma U373 MG cells. The action of histamine appears to be partially mediated by PKC stimulation and MAPK activation.     

Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat

Summary Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E₁), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E₂) by 17-hydroxysteroid dehydrogenase (17-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3, 17-dihydroxy-17-ethinylestra-135(10), 14-tetraen-7-yl) undecanamide) on E₁-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 ± 7.1% of the original size, while E₁ (1.0 µg, twice daily) caused a 139 ± 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E₁ to 66.0 ± 9.0%, while the antiestrogen EM-219 (40 µg, twice daily) decreased tumor size to 70.0 ± 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 ± 7.4% of the value found in OVX animals treated with E₁. Tumor E₂ levels decreased from 1688 ± 155 pmoles/kg tissue in OVX animals receiving E₁ to 709 ± 92, 1347 ± 98, and 184 ± 11 pmoles/kg tissue in MPA-, EM-219-, and MPA + EM-219-treated OVX-E₁ animals, respectively. Treatment of OVX animals with E₁ increased by 69% the reductive activity of 17-hydroxysteroid dehydrogenase (17-HSD) while MPA abolished completely this effect of E₁. In the oxidative direction, treatment with E₁, E₁ + MPA, or E₁ + EM-219 had minimal or no significant effect on the activity of 17-HSD (vs OVX), while the combined treatment with MPA + EM-219 induced a 2-fold increase in 17-HSD activity, thus leading to an increased conversion of E₂ into E₁. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E₂ levels stimulated by E₁ than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E₂ into E₁. The present data suggest that such a combination could be a useful approach for the treatment of breast cancer, especially in post-menopausal women.     

IFN-β Gene Therapy Induces Systemic Antitumor Immunity Against Malignant Glioma

We previously demonstrated that intratumoral administration of liposomes containing the murine interferon beta (IFN-) gene [lip(pSV2muIFN-)] resulted in stronger growth-inhibitory effect on GL261 (H-2^b) mouse glioma inoculated in brains of syngeneic C57BL/6 mice than conventional exogenous IFN- administration, and histologic evaluation revealed the massive infiltration of T lymphocytes (CD8 > CD4) within the residual tumor. The present study was aimed at determining whether such tumor-infiltrating lymphocytes (TIL) have any tumor-specific cytotoxic effects. Intratumoral administration of lip(pSV2muIFN-) resulted in prolonged survival time and a 50% tumor-free incidence in the mice treated. The surviving animals were subsequently re-challenged with either subcutaneous or intracranial injection of GL261 cells, and no tumors were found to develop over a 50-day period. In vivo depletion of CD8, but not CD4 cells decreased the efficacy of lip(pSV2muIFN-). Specific cytotoxic T lymphocytes (CTL) against GL261 cells were generated from both TIL and spleen cells of the mice treated. The results of flow cytometric analysis and antibody blocking test revealed that the bulk CTL lines thus prepared were T cell receptor (TCR) , CD8 T lymphocytes with H-2^b restriction.These findings suggest that, in addition to direct growth-inhibitory effects by the IFN- gene on the tumor cells, activation of systemic cellular immunity may participate in antitumor effects in vivo, despite the fact that central nervous system is generally regarded as an immunologically privileged site.     

Transforming growth factor b as a potential tumor progression factor among hyperdiploid glioblastoma cultures: Evidence for the role of platelet-derived growth factor

Among early-passage, near-diploid gliomas in vitro, transforming growth factor type (TGF) has been previously shown to be an autocrine growth inhibitor. In contrast, hyperdiploid ( 57chromosomes/metaphase) glioblastoma multiforme (HD-GM) cultures were autocrinely stimulated by the TGF. The mechanism of this conversion from autocrine inhibitor to mitogen is not understood; previous studies have suggested that platelet-derived growth factor (PDGF) might be modulated by TGF. The similar expression of TGF types 1—3, PDGF-AA, — BB, as well as the PDGF receptor and subunits (a/PDGFR) between biopsies of the HD-GM and near-diploid, TGF-inhibited glioblastomas (GM) by immunohistochemistry did not explain the discrepancy in their regulatory responses. Flowcytometry demonstrated that TGF's mitogenic effect was selective for the aneuploid subpopulations of two of three selected HD-GM cultures,while the diploid cells were inhibited. Among the HD-GM, TGF1 induced the RNA of PDGF-A, c-sis and TGF1. The amount of PDGF-AA secreted following TGF treatment was sufficient to stimulate the proliferation of a HD-GM culture. Antibodies against PDGF-AA, -BB, -AB,PDGFR and/or PDGFR subunits effectively neutralized TGF's induction of DNA synthesis among the HD-GM cell lines, indicating that PDGF served as the principal mediator of TGF's growth stimulatory effect. By comparison, TGF induced only the RNA of PDGF-A and TGF1 among the near-diploid GM; c-sis was not expressed at all. However, the amount of PDGF-A which was secreted in response to TGF1 was insufficient to prevent TGF's arrest of the near-diploid cultures in G₁ phase. Thus, the emergence of hyperdiploidy was associated with qualitative and quantitative differences in TGF's modulation of PDGF-A and c-sis, which provided a mechanism by which the aneuploid glioma cellsmight achieve clonal dominance. We hypothesize that TGF may serve as an autocrine promoter of GM progression by providing a selective advantage to the hyperdiploid subpopulation through the loss of a tumor suppressor gene which mediates TGF's inhibitory effect.     

Pharmacokinetics and metabolism of β-2′-deoxythioguanosine and 6-thioguanine in man

Summary Resistance to the antileukemic agent 6-thioguanine (TG) inevitably develops in animal tumors. However, a new agent, -2-deoxythioguanosine (-TGdR) can overcome TG resistance in animal tumor models and is therefore of potential clinical use. The pharmacokinetics of radiolabeled TG were compared with those of -TGdR in patients with cancer after intravenous administration. [³⁵S]--TGdR (5.4 mg/kg, 200 mg/m², 200 Ci total) was administered to five patients; the radiolabel in the plasma declined with an initial half-life (t_1/2) of 14 min and a terminal t_1/2 of 19.3 h. Within 24 h, 65% of the radiolabel was excreted in the urine. In contrast, after administration of [³⁵S]-6-TG (3.4 mg/kg, 125 mg/m², 200 Ci total) the average initial t_1/2 was 40 min while the terminal phase t_1/2 was 28.9 h. Urinary excretion of the radiolabel was 75% of the dose 24 h after administration. Both thiopurines were rapidly and extensively degraded and excreted as 6-thioxanthine, inorganic sulfate, S-methyl-6 thioxanthine, and 6-thiouric acid in addition to other products. Small amounts of unchanged drug were also excreted. These studies suggest that -TGdR is merely a latent form of TG.Deceased, to whose memory this paper is dedicated     

Systemic treatment with murine recombinant interleukin-1β inhibits the growth and progression of malignant glioma in the rat

We investigated the effects of daily subcutaneous (SC) injections of 100, 200, or 400 g/kg murine recombinant interleukin-1 (rIL-1) or its excipient on normal Fischer 344 rats and ones harboring a malignant RT-2 glioma. The tumor model has a predictable course with animals dying on days 14–17 following an intracerebral inoculation of 10⁴ RT-2 glioma cells. Treatments with (rIL-1) or excipient began on day seven post-tumor inoculation and continued for 7 days. We observed no significant effect on core body temperatures although there was a significant (p < 0.05) decrease in body weight in all (rIL-1) treated animals. When tumor-bearing animals became moribund, they received an intraperitoneal injection of bromodeoxyuridine (BUdr) and were sacrificed two hours later. Blood samples were obtained prior to their sacrifice by transcardiac perfusion with a buffered aldehyde solution. Recombinant IL-4ß affected blood differentials; causing neutrophilia, lymphopenia, and slight thrombocythemia. The BUdr labeling index of glioma cells did not significantly differ between treatment groups, although tumors differed histologically at the time of necropsy. Tumors of rIL-1 treated animals had more extensive necrosis and a greater degree of leukocyte infiltration. Survival studies were conducted in which rats were given continuous daily SC injections of (rIL-1) until day of death. Overall survival between the two groups differed significantly in studies using 100 g/kg/d (p < 0.05); (rIL-1ß) treated rats had a mean survival time of 22 (± 3.0) days while excipient controls had a mean survival time of 17 (± 0.5) days. Similarly, at a dose of 200 g rIL-1(3/kg/d), mean survival was significantly (p < 0.05) increased as compared to excipient controls (18.75 ± 1.5 vs. 15.25 ± 1.7 days, respectively). Daily injections of 400 g/kg did not significantly increase the survival of glioma bearing animals, possibly as a consequence of fIL-1ß toxicity at this dose.     

Tumor necrosis factor-alpha induces interleukin-6 production via extracellular-regulated kinase 1 activation in breast cancer cells

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are frequently produced by breast cancer cells. These interleukins promote osteoclast formation and may mediate osteolysis at the site of breast cancer bone metastases. Transforming growth factor- (TGF-), tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) up-regulate IL-6 and IL-11 production in a cytokine-dependent fashion in breast cancer cells, but very little is known about their intracellular signaling pathways in breast cancer cells. To study TGF-, TNF- and IL-1 regulation of IL-6 and IL-11 production in human MDA-MB-231 breast cancer cells, we established single cell clones stably expressing dominant negative (DN) forms of the mitogen-activated protein kinases p38 (p38/AF) or ERK1 (ERK1K71R). We show here, that while basal, TGF- and IL-1 induced IL-6 production was similar in parental cells and in pcDNA3 control, ERK1K71R and p38/AF clones, TNF- induced IL-6 production was blunted in the ERK1K71R clones. TGF- and IL-1, but not TNF-, induced IL-11 production in parental MDA-MB-231 cells. Similar findings were detected in clones stably expressing p38/AF and ERK1K71R, which did not change basal IL-11 production either. In conclusion, TNF- induced IL-6 production is mediated via ERK1 activation in MDA-MB-231 cells. These observations may be helpful in designing new anti-osteolytic therapies.     

Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

Sex hormone-induced mammary carcinogenesis in female Noble rats: Expression of TGF-β1 and its receptors, TGF-α, and EGF-R in mammary carcinogenesis

We have established a Noble rat model to explore the mechanisms of hormonal mammary carcinogenesis, in which the role of androgen in promoting mammary carcinogenesis was highlighted. We have also established that stromal–epithelial interactions may be responsible for the promotional effects of testosterone in mammary carcinogenesis. Based on these understandings, in the present study we examined the expression of transforming growth factor beta-1 (TGF-₁) and its receptors (TGF- RI, TGF- RII), transforming growth factor alpha (TGF-), and epidermal growth factor receptor (EGF-R) in 'pre-malignant' mammary glands treated with different protocols of sex hormones, as well as in mammary cancers. We observed that TGF-₁ was strongly expressed in most mammary tumors, whereas TGF- RI and TGF- RII were negative in most mammary tumor cells. The results from comparative study of 'pre-malignant' glands further showed that when the animals were treated with testosterone, either alone or in combination with 17-estradiol, the mammary gland epithelial cells expressed high levels of TGF-₁. This over-expression of TGF-₁ can be blocked by flutamide, indicating that testosterone may be responsible for the expression of TGF-₁ in mammary glands. TGF- RI and TGF- RII were also expressed strongly in testosterone-treated mammary epithelial cells and only weakly detectable in 17-estradiol treated and control mammary epithelial cells. Furthermore, TGF- RI and TGF- RII were also expressed in stromal cells, both in mammary tumors and in hormone-treated mammary glands. These observations indicate that the mechanism of testosterone in mammary carcinogenesis may be through its regulation of expression of TGF-₁ and its receptors. On the other hand, TGF- was also expressed in all 39 mammary cancers, while only 81% of the cancers were EGF-R positive. TGF- was also strongly expressed in stromal cells in all three experimental groups, but only moderately expressed in epithelial cells when treated with a combination of testosterone and 17-estradiol. By contrast, EGF-R was strongly expressed in epithelial cells in the three experimental groups but negative in stromal cells. Flutamide or tamoxifen was unable to block the expression of TGF- induced by the combined sex hormone treatment. However, they were effective in blocking the expression of TGF- when the animals were treated with testosterone or 17-estradiol alone, respectively. These results suggest that both testosterone and 17-estradiol may be required for the over-expression of TGF- in the mammary carcinogenesis induced by sex hormones. To our knowledge, this is the first experimental study to explore the regulation of TGF-₁, TGF-, and their receptors by testosterone and 17-estradiol in mammary carcinogenesis.     

Inhibition of cell proliferation and glutathione S-transferase by ascorbyl esters and interferon in mouse glioma

Summary Mouse glioma-26 (G-26) cell line established in this laboratory was used in the study. Thein vitro effect of ascorbyl esters, viz., ascorbyl-palmitate (As-P), -stearate (As-S) and mouse interferon-/ (MulFN-/) on the glioma cell viability, proliferation and glutathione S-transferase (GST) activity was investigated. Cell viability and proliferation were examined by colorimetric MTT assay and [³H]-thymidine incorporation, respectively. Incubation (24 h) of G-26 cells with As-S, As-P or MulFN-/, resulted in a dose dependent decrease in cell viability (IC₅₀=125M As-S; 175M As-P and 3.6×10⁴ U/ml MulFN-/) and proliferation (IC₅₀=157M As-S; 185M As-P and 3.6×10⁴ U/ml MulFN-/). A combined exposure to 175 M As-S and 800 U/ml of MulFN-/ resulted in a greater than an additive effect on cell viability and proliferation. The inhibition of cell proliferation/viability by interferon was species specific and was observed only with homologous MulFN-/, but not with human interferon- lymphoblastoid or human interferon-. Ascorbyl esters inhibited cytosolic GST activity (1–50=15.0 M As-S and 28.5 M As-P) towards 1-chloro-2,4-dinitrobenzene in a dose dependent manner. The apparent Ki values for affinity purified GST, deduced from Dixon plots were 0.95 M and 2.0 M for As-S and As-P, respectively. Significant inhibition of GST was also observed in the cytosol isolated from G-26 cells exposed to 300 M As-S or 800 U/ml MulFN-/.     

Expression of estrogen receptor-beta in normal mammary and tumor tissues: is it protective in breast carcinogenesis?

Using messenger RNA (mRNA) in situ hybridization, we investigated estrogen receptor- (ER) mRNA levels in normal mammary, benign breast tumor (BBT), breast cancer (BC), and metastatic lymph node tissues to verify the role of ER in BC development and progression. ER expression was significantly decreased in BC and metastatic lymph node tissues compared with normal mammary and BBT tissues (p < 0.01). The intensity and extent of ER mRNA signals were also significantly lower in BC and metastatic lymph node tissues than in the normal mammary and BBT tissues (p < 0.01). An inverse relationship was found between ER mRNA level and both histologic grade (p = 0.091) and progesterone receptor expression (p = 0.052) with marginal significance, but no significant association was noted between ER expression in cancer tissues and the other clinico-pathologic data. The 3-year distant relapse-free survival probability was found to be independent of ER expression. Collectively, ER mRNA decreases in the process of BC development, but seems to be associated with poor differentiation.     

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

Summary Growth inhibitory activity of recombinant and interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant interferon showed slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant and interferon. The growth inhibitory activity of recombinant interferon on EFC-2 cells was not blocked by recombinant interferon, although recombinant and interferons shared same receptors on EFC-2 cells. Addition of DFMO (-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant interferon, and all were resistant to recombinant interferon. These results suggest differential sensitivity of EFC-2 cells to recombinant interferon, as well as a heterogeneous sensitivity to recombinant interferon among different glioma cell lines.     

Clinical pharmacokinetics of mitoxantrone after intraperitoneal administration

Summary The pharmacokinetics of intraperitoneally (i.p.) injected mitoxantrone was determined in plasma and peritoneal dialysate taken from five patients presenting with cancer confined to the peritoneal cavity over a sampling period of 1 week. The drug was given through a Tenckhoff catheter as a 15-min infusion and the peritoneal dialysate was removed after a dwell time of 4 h; the doses delivered varied between 20 and 50 mg/m². Dose-limiting local toxicity was moderate. The HPLC technique used for mitoxantrone determinations proved to be sensitive within the range of 0.3–4,000 ng/ml. Median values obtained for the pharmacokinetic parameters of mitoxantrone in peritoneal dialysate were:t _1/2 (distribution), 56.4 min (range, 16.8–235.8 min);t _1/2 (elimination), 128 h (range, 28.3–171.0 h); Vd_SS (volume of distribution at steady state), 24.8 l (range, 17.0–232.5 l); _ss (volume of distribution at steady state corrected for the body surface area in square meters), 14.4 l/m² (range, 10.6–129.2 l/m²); and clearance, 0.25 l/h (range, 0.16–0.59 l/h). For plasma the median values were:t _1/2 (absorption), 58.8 min (range, 45.6–87.0 min);t _1/2 (distribution), 2.5 h (range, 1.4–6.3 h);t _1/2 (elimination), 44.1 h (range, 9.1–91 h); Vd_SS, 2,152 l (range, 352–19,733 l); _ss, 1,345 l/m² (range, 220–11,606 l/m²); and clearance, 117 l/h (range, 51–1,609 l/h). After 168 h the median plasma concentration was 1 ng/ml. The median peak concentration in peritoneal dialysate was 490 ng/ml. Considering the moderate toxicity observed and the concentrations achieved in the peritoneal dialysate, removal of the dialysate after certain dwell times seems reasonable to be a reasonable approach for the optimization of i.p. treatment with mitoxantrone.     

Retinoic acid receptor beta2 inhibition of metastasis in mouse mammary gland xenografts

The retinoic acid receptor ₂ (RAR₂) protein is a putative tumor suppressor that inhibits proliferation and can induce apoptosis when introduced into breast, cervical, lung, and pancreatic cancer cell lines. To determine if RAR₂ suppresses proliferation of mammary-derived cancer cells in vivo, we transduced MDA-MB-435 breast cancer cells with the LXSN retroviral vector containing RAR₂ and implanted LXSN vector- or RAR₂-transduced cells into the mammary fat pads of nude and severe combined immune deficiency (SCID) mice. We analyzed the xenografts for several tumor parameters, including tumor size, inflammation, vascularity, mitoses, tumor recurrence at the primary site following resection, and metastases. We found that 19 of 52 mice inoculated with vector-transduced cells developed metastases in multiple organs while only one of 55 mice receiving RAR₂-transduced cells displayed evidence of metastases (p < 0.000001, combined experiments, two-tailed Fisher's exact test). Moreover, RAR₂-tumor cell recipient mice had a lower incidence of post-resection tumor recurrence (8/55 vs. 25/52, p=0.0004), 34% less necrosis (in three of four experiments, p=0.001), and 39% fewer mitoses in tumor tissue (p<0.000001). Our findings suggest that RAR₂ may play a role in inhibiting the metastatic cascade in a mouse mammary gland xenograft tumor model and is a potential candidate for therapeutic intervention in human breast cancer.     

Regulation of estrogen sulfotransferase by estrogen in MCF-7 human mammary cancer cells

Summary The importance of the steroid hormone microenvironment within cells is now recognised in studies on endocrine-related neoplasms such as breast cancer. This focuses attention on ezymes which control the intracellular levels of estradiol-17 (E₂). One such enzyme, estrogen sulfotransferase, which converts E₂ to inactive E₂-3 sulfate, has now been shown to be regulated by estrogen in MCF-7 human mammary cancer cells. Hydroxysteroid sulfotransferase, which sulfurylates the adrenal-derived estrogen 5-androstene-3,17-diol, is also under estrogen control. Evidence is provided which shows that one function of these enzymes may involve elimination of estrogen from the cell following processing of the ligand-charged estrogen receptor (ER).     

Sensitivities of human glioma cell lines to interferons and double-stranded RNAs individually and in synergistic combinations

Summary The antiproliferative effects of human interferons (IFNs) and double-stranded RNAs (dsRNAs) were studied in five human glioma cell lines. Dose response curves were generated over a 72 hour treatment period. The concentration of interferon or double-stranded RNA necessary to produce a 50% antiproliferative response (GI₅₀) was calculated by linear regression analysis. Two cell lines were more sensitive to IFN- than to IFN-, one cell line was more sensitive to IFN- than to IFN- and two cell lines had approximately equal sensitivities to both interferons. All cell lines showed some sensitivity to either IFN- or IFN-. IFN- had no antiproliferative effect on any of the cell lines. In addition, only one of the cell lines displayed sensitivity to dsRNA, in which the response to poly(I) · poly(C) was greater than that to a mismatched analogue of poly(I) · poly(C), r(I)n · r(C₁₂,U)_n (Ampligen). There was no correlation between the sensitivities to type I IFNs ( and ), type II IFN () or the dsRNAs. The antiproliferative effect of combinations of IFNs, or IFNs and Ampligen, was studied in one of the cell lines. A significant synergistic antitumor effect was seen with all of the IFN/Ampligen combinations (p < 0.02), including IFN-/Ampligen, even though these cells were resistant to IFN- alone. Synergy was also seen in the IFN-/IFN- (p < 0.02) and IFN-/IFN- (p < 0.05) combinations. The IFN-/IFN- combination gave an additive antitumor effect. These results indicate that IFN- and IFN- alone or combinations of type I IFNs, type II IFNs and Ampligen can be effective in inhibiting the growth of glioma cells.