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Aim: Docetaxel is a microtubule-stabilizing drug used for the treatment of several cancers, including prostate cancer. Resistance to docetaxel can either occur through intrinsic resistance or develop under therapeutic pressure, i.e., acquired resistance. A possible explanation for the occurrence of acquired resistance to docetaxel is increased drug efflux via P-glycoprotein (P-gp) drug transporters. Methods: We have generated docetaxel-resistant cell lines DU-145DOC10 and 22Rv1DOC8 by exposing parental cell lines DU-145DOC and 22Rv1 to increasing levels of docetaxel. Gene expression levels between DU-145DOC10 and 22Rv1DOC8 were compared with those of their respective originator cell lines. Both parental and resistant cell lines were treated with the taxane drugs docetaxel and cabazitaxel in combination with the P-gp/CYP3A4 inhibitor ritonavir and the P-gp inhibitor elacridar. Results: In the docetaxel-resistant cell lines DU-145DOC10 and 22Rv1DOC8, the ABCB1 (P-gp) gene was highly up-regulated. Expression of the P-gp protein was also significantly increased in the docetaxel-resistant cell lines in a Western blotting assay. The addition of ritonavir to docetaxel resulted in a return of the sensitivity to docetaxel in the DU-145DOC10 and 22Rv1DOC8 to a level similar to the sensitivity in the originator cells. We found that these docetaxel-resistant cell lines could also be re-sensitized to cabazitaxel in a similar manner. In a Caco-2 P-gp transporter assay, functional inhibition of P-gp-mediated transport of docetaxel with ritonavir was demonstrated. Conclusion: Our results demonstrate that ritonavir restores sensitivity to both docetaxel and cabazitaxel in docetaxel-resistant cell lines, most likely by inhibiting P-gp-mediated drug efflux., Competing Interests: van der Putten E is a full-time employee of Modra Pharmaceuticals B.V.; Wosikowski K has received consult fees from Modra Pharmaceuticals B.V.; Beijnen JH is a shareholder of Modra Pharmaceuticals B.V.; Imre G has received consult fees from Modra Pharmaceuticals B.V.; Freund CR is a full-time employee of Modra Pharmaceuticals B.V.., (© The Author(s) 2024.)

Objective: TGF-β2 (TGF-β, transforming growth factor beta), the less-investigated sibling of TGF-β1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-β2 in biliary-derived liver diseases., Design: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-β2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels., Results: TgfB2 -induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2 -directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3 , Ccl4 , Ccl5 , Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue., Conclusions: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-β2 silencing and provide a direct rationale for TGF-β2-directed drug development., Competing Interests: Competing interests: Isarna Therapeutics GmbH supported this study financially. The company develops TGF-β isoform specific antisense oligonucleotides for therapeutic approaches. However, the company did not influence experimental design and data interpretation. KW, HK and MJ were employed by Isarna Therapeutics at the time of contribution. The position of AD was funded by Isarna Therapeutics., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To evaluate the safety and tolerability of intravitreal ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2), in patients with primary open angle glaucoma (POAG) undergoing trabeculectomy (TE; glaucoma filtration surgery)., Methods: In this prospective phase I trial glaucoma patients scheduled for TE with mitomycin C (MMC) received a single intravitreal injection of ISTH0036 at the end of surgery in escalating total doses of 6.75 μg, 22.5 μg, 67.5 μg or 225 μg, resulting in calculated intraocular ISTH0036 concentrations in the vitreous humor of approximately 0.3 μM, 1 μM, 3 μM or 10 μM after injection, respectively. Outcomes assessed included: type and frequency of adverse events (AEs), intraocular pressure (IOP), numbers of interventions post trabeculectomy, bleb survival, visual acuity, visual field, electroretinogram (ERG), slit lamp biomicroscopy and optic disc assessment., Results: In total, 12 patients were treated in the 4 dose groups. Main ocular AEs observed were corneal erosion, corneal epithelium defect, or too high or too low IOP, among others. No AE was reported to be related to ISTH0036. All other safety-related analyses did not reveal any toxicities of concern, either. The mean medicated preoperative IOP at decision time-point for surgery was 27.3 mmHg +/- 12.6 mmHg (SD). Mean IOP (±SD) for dose levels 1, 2, 3, and 4 were at Day 43 9.8 mmHg ± 1.0 mmHg, 11.3 mmHg ± 6.7 mmHg, 5.5 mmHg ± 3.0 mmHg and 7.5 mmHg ± 2.3 mmHg SD; and at Day 85 9.7 mmHg ± 3.3 mmHg, 14.2 mmHg ± 6.5 mmHg, 5.8 mmHg ± 1.8 mmHg and 7.8 mmHg ± 0.6 mmHg, respectively. In contrast to IOP values for dose levels 1 and 2, IOP values for dose levels 3 and 4 persistently remained below 10 mmHg throughout the observation period., Conclusion: This first-in-human trial demonstrates that intravitreal injection of ISTH0036 at the end of TE is safe. Regarding IOP control, single-dose ISTH0036 administration of 67.5 μg or 225 μg at the time of TE resulted in IOP values persistently < 10 mmHg over the three month postoperative observation period.

Background/purpose: Acute bronchitis (AB) is a common lung condition characterized by inflammation of the large bronchi in response to infection. Bronchipret(®) syrup (BRO), a fixed combination of thyme and ivy extracts has been effectively used for the treatment of AB. Combining in vivo and mechanistic in vitro studies we aimed to provide a better understanding of the therapeutic potential of BRO on key aspects of AB and to identify potential mechanisms of action., Methods: Bronchoalveolitis in rats was induced by intratracheal LPS instillation. BRO was administered p.o. once daily at 1- to 10-fold equivalents of the human daily dose. Animals were sacrificed 24-72 h post LPS challenge to analyze leukocyte numbers in lung tissue, bronchoalveolar lavage fluid (BALF) and blood as well as goblet cells in bronchial epithelium. Inhibitory effects of BRO analogue on leukotriene (LT) production were determined in human neutrophils and monocytes as well as on isolated 5-lipoxygenase (5-LO)., Results: BRO significantly reversed the LPS-induced increase in leukocyte numbers in lung tissue, BALF and blood as well as goblet cell numbers in bronchial epithelium. In vitro, BRO analogue suppressed cellular release of LTB4 (IC50 = 36 µg⋅ml(-1)) and cysLT (IC50 = 10 µg⋅ml(-1)) and inhibited the activity of isolated 5-LO (IC50 = 19 µg⋅ml(-1))., Conclusion: BRO exerts significant anti-inflammatory effects and attenuates goblet cell metaplasia in LPS-induced bronchoalveolitis in vivo potentially via interference with 5-LO/LT signaling. These effects may contribute to its observed clinical efficacy in AB., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The literature on TGF-ß in cancer including data on the expression or activation of TGF-ß pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-ß pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-ß isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-ß1, TGF-ß2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-ß isoform directed therapies. Of note, TGF-ß antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-β isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-ß directed antisense therapies based upon TGF-β immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.

Unlabelled: The active metabolite (JM118) of the oral platinum analog satraplatin (JM216) was investigated for potential synergism with erlotinib, an epidermal growth factor receptor (EGFR) inhibitor. JM118 sensitivity of 7 cancer cell lines (ovarian: 2008, A2780; colon: Lovo92, WiDr; lung: A549, SW1573; epidermoid: A431), was enhanced most pronounced when JM118 preceded erlotinib, which was associated with increased formation of DNA-platinum adducts. The combination increased G2/M phase accumulation and enhanced apoptosis. JM118 increased the phosphorylation of the cell cycle proteins CDK2 and CHK1 after 24 hr exposure. JM118/erlotinib enhanced Erk and Akt phosphorylation after 2 hr. JM118 significantly decreased the phosphorylation of PTEN, VEGFR, EPHA1, ERBB4, FGF-R, andSTAT3 by 20 (PTEN) to >90% (STAT3)., Conclusion: Erlotinib enhanced the effects of JM118, even in cells with mutations in Ras. The mechanism of synergy involved a combination of effects on platinum-DNA adduct formation, cell cycle distribution and signaling.

Purpose: Satraplatin is an orally available platinum analog. The purpose of this study was to better characterize satraplatin's preclinical antitumor efficacy in a variety of sensitive and resistant human tumor cell lines and in a prostate cancer xenograft model and to evaluate the effect of satraplatin on PSA expression and/or secretion in a prostate cancer cell line., Methods: Satraplatin and its primary metabolite JM-118 were preclinically tested for their cytotoxic activity in a range of cancer cells including: human prostate, those forming the NCI drug screening panel, and those resistant to anti-cancer drugs. Also, the antiproliferative efficacy of satraplatin was tested in vivo in a human prostate cancer model. The effect of satraplatin and JM-118 on PSA transcription was measured by quantitative real time PCR., Results: Satraplatin and JM-118 inhibited in vitro and in vivo the growth of prostate cancer cells in a dose-dependent fashion. The IC50 cytotoxicity values for satraplatin ranged from 1 to 3 microM for androgen-insensitive cells and was 11 microM for the androgen-sensitive cell line. Interestingly, JM-118 was up to 16-fold more potent than satraplatin. Oral administration of satraplatin to nude mouse PC-3 xenograft models inhibited the growth of these human tumors. Satraplatin had no direct effect on PSA transcription and the observed decrease in secreted PSA correlated with a decrease in cell number. When evaluated in the NCI drug-screening panel, satraplatin was most active in leukemia and small cell lung cancer cell lines. Both satraplatin and JM-118 were tested on cells resistant to chemotherapeutic agents. Satraplatin and JM-118 were equally active in the cisplatin-resistant A129cp80 ovarian carcinoma cell line, with activity comparable to that observed in the parent line. Neither expression of MDR1, BCRP, MRP1, nor altered tubulin or topoisomerase I were found to mediate resistance to satraplatin or JM-118. Although these resistance mechanisms contribute to drug resistance for a number of chemotherapeutics, they do not appear to play a role in satraplatin resistance., Conclusions: These results demonstrate that satraplatin and JM-118 have preclinical antitumor activity in human prostate cancer and other tumor types as well, including several cell lines displaying drug resistance to cisplatin, docetaxel and mitoxantrone. In addition, the results suggest that PSA should be further evaluated as a relevant marker of clinical response in patients with prostate cancer treated with satraplatin.

The overall survival rate of patients suffering from carcinomas has remained poor and nearly unchanged over the last decades. This is mainly due to the so-called minimal residual disease, i.e., remaining tumor cells that overcome surgery and/or radiotherapy and are the cause of locoregional and distant metastases. To metastasize, tumor cells take advantage of proteases to invade and remodel surrounding tissues. Here, we analyzed the efficiency of WX-UK1, a novel 3-amidinophenylalanine-based inhibitor of the uPA system, at inhibiting the invasive capacity of carcinoma cells. First, uPAR expression was characterized in different carcinoma cell lines, including SCCHN, breast and cervical carcinoma. Thereafter, the invasive potential of these cell lines was determined using Matrigel invasion chambers and a spheroid cocultivation model with human fibroblasts. uPAR expression levels correlated positively with invasion capacity, which could be significantly inhibited by WX-UK1. A decrease of tumor cell invasion by up to 50% was achieved in both models with the SCCHN line FaDu and the cervical carcinoma line HeLa after treatment with WX-UK1. Thus, our results demonstrate the potential of WX-UK1 in vitro as a promising adjuvant antimetastatic therapy of carcinomas., (Copyright 2004 Wiley-Liss, Inc.)

To avoid systemic toxicity of the cytotoxic drug methotrexate (MTX) and to improve tumor selectivity, MTX was bound to human serum albumin (HSA) as a drug carrier. To understand more about the mechanism of action of MTX conjugated to HSA (MTX-HSA), the uptake of MTX-HSA into the cell was determined as well as the effect of MTX-HSA on thymidylate synthase (TS), cell cycle distribution, and cell proliferation. Different uptake kinetics were observed for [(3)H]MTX and [(3)H]MTX-HSA. However, similar uptake kinetics were measured for (125)I-HSA and (125)I-MTX-HSA (2.1 and 1.8 pmol/10(7) cells/h when cells were treated with 10 micro M (125)I-HSA and (125)I-MTX-HSA, respectively), suggesting that MTX-HSA enters the cells by albumin-mediated endocytosis. We observed no effect of MTX-HSA on TS when folate receptor-expressing KB cells were treated for 4 h (IC(50), >50 micro M). However, 24 h after incubation, MTX-HSA inhibited TS with an IC(50) of 6.9 micro M. In addition, we found that MTX-HSA had a delayed effect on the cell cycle compared with MTX and that this effect could be inhibited with the lysosomal inhibitor methylamine, suggesting that MTX-HSA activity is dependent on lysosomal processes. The proliferation of different wild-type and MTX-resistant tumor cell lines was inhibited at IC(50) concentrations between 2 and 78 micro M, respectively. MTX-HSA accumulates in vivo in the tumor tissue. Local concentrations of 18-29 micro M were measured, which are effective antiproliferative concentrations as determined in vitro. We also investigated the antitumor activity of MTX-HSA in vivo in different human tumor xenografts grown s.c. in nude mice. Fourteen tumors from eight different tissues were tested. Nine of 14 tumors (64%) showed a clear response with tumor inhibition, stasis, or regression; 5 of 14 (36%) gave a moderate response with tumor growth delay or no response. In conclusion, MTX-HSA is effectively taken up by the cells via albumin receptor- or folate receptor-mediated endocytosis and time-dependently released as an active compound into the cytosol to exert an inhibiting effect on TS and to induce cell cycle alterations. In vivo, effective concentrations of MTX-HSA were reached in tumor tissue to exhibit antitumor activity.

We recently developed a class of novel antitumor agents that elicit a potent growth-inhibitory response in many tumor cells cultured in vitro. WK175, a member of this class, was chosen as a model compound that showed strong in vitro efficacy. WK175 interferes with the intracellular steady-state level of NAD(+), resulting in a decreased cellular NAD(+) concentration. We found that WK175 induces apoptotic cell death without any DNA-damaging effect. The apoptotic death signaling pathway initiated by WK175 was examined in detail: mitochondrial membrane potential, cytochrome c release, caspase 3 activation, caspase 3 and poly(ADP-ribose) polymerase cleavage, and the appearance of a sub-G(1) cell cycle population were determined in time course studies in THP-1 (a human monocytic leukemia cell line) cells. We found activation of this cascade after 24 h of treatment with 10 nM WK175. Induction of apoptosis was prevented by bongkrekic acid, Z-Asp-Glu-Val-Asp-fluoromethylketone, and Z-Leu-Glu-His-Asp-fluoromethylketone, inhibitors of the mitochondrial permeability transition and of caspase 3 and 9, respectively, but not by Ac-Tyr-Val-Ala-Asp-CHO, a specific caspase 1 inhibitor, suggesting the involvement of the permeability transition pore, caspase 3, and caspase 9 in the WK175-induced apoptotic cascade. These results imply that decreased NAD(+) concentration initiates the apoptotic cascade, resulting in the antitumor effect of WK175.

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.

It is increasingly recognized that drug-resistant cells undergo transitions not directly linked to "classical" drug resistance. We examined the expression of growth factors, growth factor receptors, and the estrogen receptor in 17 drug-resistant and 2 revertant human breast cancer sublines to provide an understanding of the phenotypic changes that occur and how these changes could affect the biology of the cell. These sublines were derived from five parental human breast cancer cell lines (MCF-7, ZR75B, T47D, MDA-MB-231, and MDA-MB-453). The expression of estrogen receptor was absent or decreased in 6 of the 15 resistant MCF-7, ZR75B, and T47D sublines. Increases of as much as 49-fold compared to parental levels were observed in transforming growth factor alpha, epidermal growth factor receptor, c-erbB2, and/or c-erbB3 mRNA expression in 14 of the 17 resistant sublines. Altered amphiregulin and insulin-like growth factor-I receptor expression was observed in nine and four drug-resistant sublines, respectively. No major alterations were observed in epidermal growth factor and c-erbB4 expression. Few alterations were observed in two sublines derived from estrogen receptor-negative cells. Higher levels of phosphotyrosine residues were detected in a subset of the resistant sublines, indicating an increased tyrosine kinase activity in these cells. Interestingly, decreased growth rates were observed in all of the sublines, despite up-regulated growth factor-related gene expression. Taken together, these data suggest that loss of estrogen receptor, increased expression of growth factor pathway genes, and decreased growth rate regularly occur in drug-resistant breast cancer cells. Although we do not know whether the altered expression of growth factor pathway genes is linked as a cause or a consequence of the reduced growth rate, it is well established that decreased growth rate confers drug resistance. These phenotypic changes in drug-resistant human breast cancer cells could serve to initiate, support, or extend the drug resistance.

Background: Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database., Methods: The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity., Results: EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM., Conclusions: Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.

Brain-derived neurotrophic factor (BDNF) and its receptors are necessary for the survival and development of many neuronal cells. Because BDNF and TrkB are expressed in many poor-prognosis neuroblastoma (NB) tumors, we evaluated the role of BDNF in affecting sensitivity to chemotherapeutic agents. We investigated the effects of activation of the BDNF-TrkB signal transduction pathway in two NB cell lines, 15N and SY5Y. 15N cells lack the high-affinity receptor p145TrkB and express BDNF; 15N cells were used along with 15N-TrkB cells, a subline transfected with a TrkB expression vector. In cytotoxicity assays, 15N-TrkB cells were consistently 1.4-2 fold more resistant to vinblastine than 15N cells. Drug accumulation assays showed a 50% reduction in[3H]vinblastine accumulation in 15N-TrkB cells compared with control 15N cells. Addition of 30 ng/ml BDNF resulted in a reduction to 46% of control in 15N cells and a reduction to 28% of control in 15N-TrkB cells. SY5Y cells were chosen as a second model because they lack both endogenous BDNF and TrkB expression. p145TrkB expression is induced by 1 nM retinoic acid. Vinblastine accumulation was not significantly affected by 1 nM retinoic acid in SY5Y cells. Addition of 30 ng/ml BDNF decreased [3H]vinblastine accumulation to 58% of control in SY5Y cells and decreased [3H]vinblastine accumulation to 62% of control in TrkB-expressing SY5Y cells. Although an increase in BDNF expression in seen in multidrug-resistant sublines of SY5Y and BE(2)-C NB cells, the protective effect of BDNF in vinblastine toxicity may be unrelated to mdr-1, because the activity of other agents transported by P-glycoprotein was not affected. There was no increase in mdr-1 expression in 1 nM RA SY5Y cells and 15N-TrkB cells, as assessed by Northern blot analysis. In addition to the effects of BDNF on vinblastine cytotoxicity and accumulation, there was an inhibition in the ability of vinblastine to depolymerize tubulin in BDNF-treated cells. Thus, BDNF and TrkB may partially rescue NB cells from vinblastine toxicity and thereby may contribute to a more chemoresistant phenotype.

Loss of or mutations in p53 protein have been shown to decrease both radio- and chemosensitivity. The present study assessed the p53 gene status, ability to arrest in G1 of the cell cycle, the functionality of the p53 transduction pathway, and apoptosis following treatment with radiation in a series of drug-resistant human breast cancer cells to determine whether p53 alterations occur during the development of drug resistance. We used 13 sublines derived from MCF-7, ZR75B, and T47D cells, which were resistant to doxorubicin, paclitaxel, vinblastine, cisplatin, etoposide, and amsacrine. Eleven of 12 drug-resistant sublines retained the parental p53 gene status, as determined by sequence analysis and functional yeast assay; only one subline was found to have acquired a mutation in the p53 gene. The MCF-7 TH subline was found to both acquire mutated p53 and to have major changes in p53 protein expression and function. In 12 other drug-resistant sublines, the G1 checkpoint was conserved or only slightly impaired. A normal accumulation of p53, p21Cip1/Waf1, and Mdm2 proteins and hypophosphorylation of Rb protein occurred in response to radiation with only small differences noted in the kinetics of p53 and p21Cip1/Waf1 induction. Increased susceptibility to apoptosis was found in the ZR75B drug-resistant sublines, whereas no evidence for apoptosis was observed in the ZR75B, MCF-7, and T47D parentals and the MCF-7 and T47D drug-resistant sublines. This effect could not be explained by alterations in bcl-2 or bax expression. Our results demonstrate that alterations in: (a) p53 gene status; (b) ability to arrest in G1; (c) induction of p53 protein and p53-dependent genes; and (d) decreased activation of apoptosis is not a requirement for the onset of drug resistance. The function of p53 appears to be dissociated from drug resistance in our model system.

Human breast cancer cells selected for multidrug resistance frequently overexpress ligands and receptors in the epidermal growth factor (EGF) receptor family. To determine whether this overexpression contributes to the drug resistant phenotype, EGF receptor transfected ZR75B human breast cancer cells were examined. Two EGF receptor overexpressing clones were evaluated: clone 11 with > 1 x 10(6) sites, and clone 13 with 310,000 receptor sites/cell. These were compared with clone 2-neo, which was transfected with the neomycin gene only and contained 43,000 receptor sites/cell. The EGF receptor overexpressing clones and the neo transfected control clone displayed comparable growth rates. Cytotoxicity analyses were performed with doxorubicin, vinblastine, cisplatin and 5-fluorouracil to determine the sensitivity of the clones to antineoplastic drugs. The EGF receptor overexpressing clones were found to be 1.5-5.6 times more resistant to the four drugs tested. This increase in the IC50 conferred a selective advantage when grown in the presence of 2, 3 and 6 ng/ml doxorubicin. Clone 13 cells overtook a mixed population which began with clone 2-neo comprising 95% of the cells. Clone 2-neo remained the dominant clone in the absence of drug. Finally, after long-term selection of the clones with 6 ng/ml doxorubicin, clone 2-neo became fourfold more resistant than the unselected clone 2-neo, a level which was comparable to that found in the EGF receptor overexpressing clones 11 and 13. No additional increase in resistance was observed for these clones, suggesting that clone 2-neo had developed additional resistance mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

In breast cancer, hormone dependency is inversely correlated with the number of surface epidermal-growth-factor (EGF) receptors on the tumor cells. In vitro, EGF stimulated only hormone-dependent immortalized human breast cancer cells to grow with an increased rate whereas hormone-independent cells were not affected by EGF. The number of EGF surface receptors is about 5-10-times smaller on hormone-dependent cells than on hormone-independent cells. Two cell lines representing the two cell types were used to demonstrate the signal-transduction capabilities of the EGF receptors. The two cell lines were the hormone-dependent MCF-7 cells and the hormone-independent MDA-MB-231 cells. Incubation at 37 degrees C for 15 min with 10(-8) M EGF increased the surface EGF-receptor density substantially on MCF-7 cells (50%) and reduced the number of these receptors on MDA-MB-231 cells to about 65% of the control. Both cell lines internalized a fluorescein-isothiocyanate-labeled EGF with similar kinetics. EGF triggered tyrosine phosphorylation of several targets in isolated MCF-7 cell membranes. One of these targets was shown by immunoprecipitation to be the EGF receptor. In MDA-MB-231 cell membranes, the EGF receptor was demonstrated to be the main target for tyrosine phosphorylation. The mRNA expression of the immediate early proto-oncogene c-fos was stimulated by EGF only in MCF-7 cells. In contrast, the mRNA of the EGF receptors was stimulated by EGF in both cell lines. These results demonstrate that, although EGF-binding sites are present on both cell lines, their signal-transduction capacity and activities are substantially different and resulted in a divergent response of the two cell types to EGF.

We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug-resistant subline of MCF-7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer. The drug-resistant subline was derived from MCF-7 cells by selection with Adriamycin in the presence of the P-glycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype. The Adriamycin-resistant cells retain estrogen-binding, estrogen-responsive monolayer growth, and estrogen-dependent tumorigenesis. Estrogen-binding studies demonstrate 1.4 x 10(6) sites per cell with unaltered affinity when compared to parental MCF-7 cells, which have 2.7 x 10(5) sites per cell. An increase in expression of EGF receptor, eight to 12-fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression. Partially drug-sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression. We postulate that activation of growth factor pathways in drug-resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure.

Recently, it was reported that the anti-estrogen tamoxifen not only inhibits estradiol-stimulated growth of MCF-7 cells but also significantly reduces the proliferation rate of cells stimulated by growth factors. We have confirmed this finding and also shown that the new anti-estrogen droloxifene inhibits the proliferation of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I)-stimulated MCF-7 cells. The growth-factor-induced proliferation was inhibited in a dose-dependent manner by the anti-estrogens in the complete absence of estrogen and FCS. Of the anti-estrogens, droloxifene was considerably more potent than tamoxifen. Because the expression of the proto-oncogenes c-fos and c-myc has been considered a key event in development of the mitogenic response, we examined the effects of anti-estrogens on c-myc and c-fos gene expression. We included in these investigations the steroidal anti-estrogen ICI 164,384 because this compound has no or very little estrogenic activity. The studies revealed that all 3 anti-estrogens transiently induced c-myc mRNA expression. However, the anti-estrogens inhibited estradiol-induced c-myc mRNA expression, although with different potencies. Pre-incubation of MCF-7 cells with droloxifene and tamoxifen resulted in elevated levels of growth-factor-induced c-myc mRNA expression. In contrast, the anti-estrogens did not induce c-fos mRNA or affect the expression of c-fos mRNA induced by growth factors. In conclusion, non-steroidal anti-estrogens inhibit growth-factor-stimulated proliferation of MCF-7 cells without inhibiting growth-factor-induced c-myc or c-fos mRNA expression.

Insulin-like growth factor I (IGF-I) was 3 times more potent in pagating MCF-7 cell proliferation than epidermal growth factor (EGF). IGF-I stimulated c-fos mRNA expression about 5 times less than EGF. Both growth factors were equipotent in inducing c-jun and c-myc mRNA expressions. The protein level of c-Myc correlated with the mRNA level. IGF-I and EGF stimulated the transcriptional activity dependent on the phorbol 12-myristate 13-acetate-responsive element (TRE) to the same extent, when measured by the chloramphenicol acetyl transferase activity of a transiently transfected multiple TRE construct. These results strongly indicate that the expression levels of the measured proto-oncogenes do not correlate with the increase of growth stimulation by IGF-I and EGF and are not growth rate limiting for the human MCF-7 breast cancer cells.

Pharmacologic investigations with droloxifene in vitro and in vivo revealed that droloxifene is a more efficient antiestrogen than tamoxifen. Droloxifene differs from tamoxifen in the following ways: it has a more than 10-fold higher binding affinity to the estrogen receptor; it shows lower estrogenic and higher antiestrogenic effects on rat uterus, indicating a higher therapeutic index; it more potently inhibits growth of various human ER-positive mammary carcinoma cell lines; short-term exposures with clinically relevant concentrations of droloxifene produce long-term growth inhibition of human ER-positive cancer cells and are more effective than continuous treatment with tamoxifen; it more effectively reduces S-phases and arrests ER-positive cells in G1-phase of the cell cycle; it antagonizes estrogen independent, growth factor stimulated proliferation of MCF-7 cells with higher efficiency; it blocks estrogen activated c-myc expression better than tamoxifen; it more effectively inhibits growth of various experimental tumors of animal (R 3230, DMBA) and human (T61) origin. Therefore, in all experimental systems, it was found that droloxifene is a more potent antiestrogen than tamoxifen.