Destabilization of steroid receptors by heat shock protein 90-binding drugs: a ligand-independent approach to hormonal therapy of breast cancer.

Steroid hormone receptors have become an important target in the management of breast cancers. Despite a good initial response rate, however, most tumors become refractory to current hormonal therapies within a year of starting treatment. To address this problem, we evaluated the effects of agents that bind the molecular chaperone heat shock protein 90 (Hsp90) on estrogen receptor function in breast cancer. Unstimulated estrogen and progesterone receptors exist as multimolecular complexes consisting of the hormone-binding protein itself and several essential molecular chaperones including Hsp90. We found that interaction of the Hsp90-binding drugs geldanamycin and radicicol with the chaperone destabilizes these hormone receptors in a ligand-independent manner, leading to profound and prolonged depletion of their levels in breast cancer cells cultured in vitro. Consistent with these findings, in vivo administration of the geldanamycin derivative 17-allylaminogeldanamycin (17AAG; NSC330507) to estrogen-supplemented, tumor-bearing SCID mice resulted in marked depletion of progesterone receptor levels in both uterus and tumor. Drug administration also delayed the growth of established, hormone-responsive MCF-7 and T47D human tumor xenografts for up to 3 weeks after the initiation of therapy. We conclude that in light of their novel mechanism of anti-hormone action, consideration should be given to examining the activity of 17AAG and other Hsp90-binding agents in patients with refractory breast cancer in future clinical trials, either alone or in combination with conventional hormone antagonists.

Induction of a heat shock factor 1-dependent stress response alters the cytotoxic activity of hsp90-binding agents.

In addition to its classic role in the cellular stress response, heat shock protein 90 (Hsp90) plays a critical but less well appreciated role in regulating signal transduction pathways that control cell growth and survival under basal, nonstress conditions. Over the past 5 years, the antitumor antibiotics geldanamycin and radicicol have become recognized as selective Hsp90-binding agents (HBA) with a novel ability to alter the activity of many of the receptors, kinases, and transcription factors involved in these cancer-associated pathways. As a consequence of their interaction with Hsp90, however, these agents also induce a marked cellular heat shock response. To study the mechanism of this response and assess its relevance to the anticancer action of the HBA, we verified that the compounds could activate a reporter construct containing consensus binding sites for heat shock factor 1 (HSF1), the major transcriptional regulator of the vertebrate heat shock response. We then used transformed fibroblasts derived from HSF1 knock-out mice to show that unlike conventional chemotherapeutics, HBA increased the synthesis and cellular levels of heat shock proteins in an HSF1-dependent manner. Compared with transformed fibroblasts derived from wild-type mice, HSF1 knock-out cells were significantly more sensitive to the cytotoxic effects of HBA but not to doxorubicin or cisplatin. Consistent with these in vitro data, we found that systemic administration of an HBA led to marked increases in the level of Hsp72 in both normal mouse tissues and human tumor xenografts. We conclude that HBA are useful probes for studying molecular mechanisms regulating the heat shock response both in cells and in whole animals. Moreover, induction of the heat shock response by HBA will be an important consideration in the clinical application of these drugs, both in terms of modulating their cytotoxic activity as well as monitoring their biological activity in individual patients.

Wortmannin inhibits the growth of mammary tumors despite the existence of a novel wortmannin-insensitive phosphatidylinositol-3-kinase.

PURPOSE: Phosphatidylinositol (PtdIns) 3-kinase is an important mediator of many cellular functions. The study of PtdIns 3-kinase has been facilitated by the existence of the potent irreversible inhibitor of p110 PtdIns 3-kinase, wortmannin. The purpose of the study was to investigate the relationship between the cell growth inhibitory activity and antitumor activity of wortmannin and inhibition of PtdIns 3-kinase. METHODS: PtdIns 3-kinase activity was measured in cells and tumors and the effects of wortmannin investigated. RESULTS: Wortmannin inhibited the growth of murine C3H and human MCF-7 mammary tumors in vivo. However, the ability of wortmannin to inhibit C3H tumor growth was not related to inhibition of tumor PtdIns 3-kinase activity. The existence of wortmannin-insensitive PtdIns 3-kinase activity was demonstrated in C3H and MCF-7 cell culture lysates and solid tumors, and normal mouse tissue homogenates. In addition to being resistant to inhibition by wortmannin, MCF-7 cell lysate total PtdIns 3-kinase activity was also resistant to five additional known inhibitors of p110 PtdIns 3-kinase. Partial purification of wortmannin-insensitive PtdIns 3-kinase from MCF-7 cell lysate showed the activity to be independent of the PtdIns 3-kinase p85 regulatory subunit. CONCLUSION: The results of the current study demonstrate that wortmannin can inhibit the growth of murine and human mammary tumors despite the presence of novel wortmannin-insensitive PtdIns 3-kinases in these tissues suggesting that some other target is responsible for wortmannin's antitumor activity.

Enhancement of chemotherapy by manipulation of tumour pH.

The extracellular (interstitial) pH (pHe) of solid tumours is significantly more acidic compared to normal tissues. In-vitro, low pH reduces the uptake of weakly basic chemotherapeutic drugs and, hence, reduces their cytotoxicity. This phenomenon has been postulated to contribute to a 'physiological' resistance to weakly basic drugs in vivo. Doxorubicin is a weak base chemotherapeutic agent that is commonly used in combination chemotherapy to clinically treat breast cancers. This report demonstrates that MCF-7 human breast cancer cells in vitro are more susceptible to doxorubicin toxicity at pH 7.4, compared to pH 6.8. Furthermore 31P-magnetic resonance spectroscopy (MRS) has shown that the pHe of MCF-7 human breast cancer xenografts can be effectively and significantly raised with sodium bicarbonate in drinking water. The bicarbonate-induced extracellular alkalinization leads to significant improvements in the therapeutic effectiveness of doxorubicin against MCF-7 xenografts in vivo. Although physiological resistance to weakly basic chemotherapeutics is well-documented in vitro and in theory, these data represent the first in vivo demonstration of this important phenomenon.

Early increases in breast tumor xenograft water mobility in response to paclitaxel therapy detected by non-invasive diffusion magnetic resonance imaging.

An important goal in cancer chemotherapy is to sensitively and quantitatively monitor the response of individual patients' tumors to successful, or unsuccessful, therapy so that regimens can be altered iteratively. Currently, tumor response is monitored by frank changes in tumor morphology, yet these markers take long to manifest and are not quantitative. Recent studies suggest that the apparent diffusion coefficient of water (ADCw), measured noninvasively with magnetic resonance imaging, is sensitively and reliably increased in response to successful CTx. In the present study, we investigate the combination chemotherapy response of human breast cancer tumor xenografts sensitive or resistant to Paclitaxel by monitoring changes in the ADCw. Our results indicate that there is a clear, substantial, and early increase in the ADCw after successful therapy in drug sensitive tumors and that there is no change in the ADCw in p-glycoprotein-positive tumors, which are resistant to Paclitaxel. The mechanism underlying these changes is unknown yet is consistent with apoptotic cell shrinkage and a concomitant increase in the extracellular water fraction.

Amifostine protects normal tissues from paclitaxel toxicity while cytotoxicity against tumour cells is maintained.

The objectives of this study were to evaluate the protective effects of amifostine against paclitaxel-induced toxicity to normal and malignant human tissues. Haematopoietic progenitor colony assays were used to establish the number of CFU-GEMM and BFU-E colonies after incubation with WR-1065 alone, Amifostine alone, paclitaxel (2.5 or 5 microM) +/- WR-1065 or amifostine. MTT and alkaline elution assays evaluated the in vitro growth inhibitory and DNA damaging effects, respectively, of paclitaxel with or without amifostine against normal human fibroblasts and human non-small cell lung cancer (NSCLC) cells. This combination was also evaluated in vivo using severe combined immune deficient (scid) mouse models of early (non-palpable tumours) and advanced (palpable tumours) human ovarian cancer. Human 2780 ovarian cancer cells were inoculated subcutaneously while paclitaxel and amifostine were administered intraperitoneally. A brief exposure (15 min) to amifostine not only protected human haematopoietic progenitor colonies from paclitaxel toxicity, but stimulated the growth of CFU-GEMM and BFU-E beyond control values. Amifostine protected normal human lung fibroblasts from paclitaxel-induced cytotoxicity and DNA single-strand breaks. However, paclitaxel cytotoxicity and DNA single-strand breaks were actually enhanced by pretreatment with amifostine in the NSCLC model. Importantly, amifostine did not interfere with paclitaxel antitumour activity even with prolonged exposure (24.5 h) of the lung cancer cells to high concentrations (1.2 mM) in vitro or following five repetitive high doses (200 mg/kg) given to scid mice with human ovarian cancer xenografts. Indeed, under certain circumstances, amifostine resulted in sensitisation of tumour cells to paclitaxel. Our results confirm previous reports of the ability of amifostine to protect normal tissues from the toxic effects of chemotherapy drugs and now extend these observations to paclitaxel.

Human tumor models in the severe combined immune deficient (scid) mouse.

PURPOSE: To test a number of established human tumor cell lines and early passage breast cancer (UACC2150) and melanoma cells (UACC1273) for growth in the scid mouse and the tumors' response to conventional chemotherapeutic drugs. METHODS: Established melanoma (A375, C81-61), colon (SW480), lung (A549), lymphomoblastoid leukemia (LCL-B), promyelocytic leukemia (HL60), prostate (PC-3, DU145), and breast (MCF7) cell lines were injected at subcutaneous (s.c.), intraperitoneal (i.p.), or mammary fat pad (MFP) sites. Tumor volume growth curves and survival curves were established for the various tumor cell lines. Carmustine (BCNU), cisplatin (CDDP), cyclophosphamide (CPA), doxorubicin, dacarbazine (DTIC), tamoxifen and vincristine were injected s.c. or i.p.. The chemotherapeutic drug effects on tumor volumes and survival were determined. RESULTS: Tumor growth occurred with each cell type. After i.p. injection, 90% mortality occurred within 26 to 60 days except for the early passage melanoma cell line UACC1273 with which mortality occurred within approximately 90 days. In the MCF7 breast model, treatment with tamoxifen (P < 0.001) and CPA (P < 0.0001) resulted in significant tumor growth delay compared with control groups. BCNU and CDDP resulted in significant tumor growth delays relative to control in SW480 colon cancer (P < 0.0014) and A375 melanoma (P < 0.0001) models, respectively. CPA and doxorubicin improved survival in the HL60 leukemia model (P = 0.0018). CONCLUSIONS: These scid mouse human tumor models appear to reflect the clinical situation in that clinically active chemotherapeutic drugs are similarly active in the scid mouse models. Therefore, the scid mouse models may be useful for testing new chemotherapeutic agents against various human cancer types.

Diaryl chalcogenides as selective inhibitors of thioredoxin reductase and potential antitumor agents.

Thioredoxin reductase is a selenocysteine containing flavoenzyme that catalyzes the NADPH dependent reduction of the redox protein thioredoxin. Thioredoxin is over-expressed by a number of human tumors. Experimental studies have shown that thioredoxin is responsible for the growth and transformed phenotype of some human cancer cells. Thus, thioredoxin reductase presents an attractive target for anticancer drug development to regulate the activity of the thioredoxin system. We have examined a series of 12 organoselenium compounds and 16 organotellurium compounds, mostly of the diaryl chalcogenide type, as inhibitors of human thioredoxin reductase and have investigated the cytotoxicity and antitumor activity of some of the compounds. The organoselenium compound Ebselen was found to be a competitive inhibitor of human thioredoxin reductase (Ki 2.8 microM), while a number of organotellurium compounds were found to be noncompetitive inhibitors (Kis 2.3 to 35.2 microM). Human glutathione reductase was not appreciably inhibited by any of the compounds, except for one dinitro organotellurium compound that caused inhibition with an IC50 of 0.5 microM and an over 20-fold selectivity compared to thioredoxin reductase. The compounds inhibited the growth of human cancer cells in culture with IC50s as low as 2 microM Some organotellurium compounds when administered daily by intraperitoneal injection to mice caused up to 50% inhibition of the growth of MCF-7 human breast cancer xenografts but the relative insolubility of the compounds was a limiting factor in their use.

Transfection with human thioredoxin increases cell proliferation and a dominant-negative mutant thioredoxin reverses the transformed phenotype of human breast cancer cells.

Thioredoxin, a redox protein with growth factor activity that modulates the activity of several proteins important for cell growth, has been reported to be overexpressed in a number of human primary cancers. In the present study, the effects of stably transfecting mouse NIH 3T3 cells and MCF-7 human breast cancer cells with cDNA for wild-type human thioredoxin or a redox-inactive mutant thioredoxin, Cys32-->Ser32/Cys35-->Ser35 (C32S/C35S), on cell proliferation and transformed phenotype have been investigated. NIH 3T3 cells transfected with thioredoxin achieved increased saturation densities compared with vector alone-transfected cells, but were not transformed as assessed by tumor formation in immunodeficient mice. Thioredoxin-transfected MCF-7 cells showed unaltered monolayer growth on plastic surfaces compared with vector alone-transfected cells, but exhibited severalfold increased colony formation in soft agarose. Stable transfection of NIH 3T3 and MCF-7 cells with C32S/C35S resulted in inhibition of monolayer growth on plastic surfaces, and up to 73% inhibition of colony formation by MCF-7 cells in soft agarose. When inoculated into immunodeficient mice, thioredoxin-transfected MCF-7 cells formed tumors, although with a 38-57% growth rate compared with vector alone-transfected cells, whereas tumor formation by C32S/C35S-transfected MCF-7 cells was almost completely inhibited. The results of the study suggest that thioredoxin plays an important role in the growth and transformed phenotype of some human cancers. The inhibition of tumor cell growth by the dominant-negative redox-inactive mutant thioredoxin suggests that thioredoxin could be a novel target for the development of drugs to treat human cancer.