Herbimycin A, a pp60c-src tyrosine kinase inhibitor, inhibits osteoclastic bone resorption in vitro and hypercalcemia in vivo.

Since absence of expression of the c-src gene product in mice indicates that the pp60c-src tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60c-src on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A (100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-1 alpha or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp60c-src was used as a control and caused none of these effects. These data suggest that pp60c-src tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia.

Human amniotic tumor that induces new bone formation in vivo produces growth-regulatory activity in vitro for osteoblasts identified as an extended form of basic fibroblast growth factor.

Tumors occasionally stimulate bone formation and cause osteoblastic metastases. Although this occurs most frequently in widespread prostate cancer, human prostate cancer cells are difficult to grow in culture without changing their phenotype, and the few available prostatic cancer lines do not increase bone formation in vivo. To identify tumor-derived osteoblast-stimulatory factors, we studied a long-established human tumor cell line derived from human amnion that has, in the past, been reported to cause bone formation in vivo when inoculated into nude mice. Tumor cells were inoculated into nude mice and induced extensive new bone formation. To characterize osteoblast growth factors produced by these tumor cells, solid tumor was isolated from the mice and extracted at neutral pH. Biological activity, assessed by stimulation of proliferation of MG-63 osteoblast-like cells, was used to monitor purification after heparin-Sepharose column chromatography, Mono-S, and C4 reverse-phase high-performance liquid chromatography. An extend amino-terminal form of basic fibroblast growth factor (FGF) was purified by its capacity to stimulate proliferation in MG-63 cells and partially sequenced. Basic FGF is also known to stimulate proliferation in MG-63 cells and other osteoblasts in vitro and bone formation in vivo. In summary, these human tumor cells stimulate new bone formation in vivo and produce an osteoblast stimulating activity in vitro, which has been identified as a form of basic FGF.

Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells.

A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range < or = 50 microM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was > 50 microM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1-100 microM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy.

Tumor efficacy and bone marrow-sparing properties of TER286, a cytotoxin activated by glutathione S-transferase.

TER286 is a latent drug activated by human glutathione S-transferase (GST) isoforms P1-1 and A1-1 to produce a nitrogen mustard alkylating agent. M7609 human colon carcinoma, selected for resistance to doxorubicin, and MCF-7 human breast carcinoma, selected for resistance to cyclophosphamide, both showed increased sensitivity to TER286 over their parental lines in parallel with increased expression of GST P1-1. In primary human tumor clonogenic assays, the spectrum of cytotoxic activity observed for TER286 was both broad and unusual when compared to a variety of current drugs. In murine xenografts of M7609 engineered to have high, medium, or low GST P1-1, responses to TER286 were positively correlated with the level of P1-1. Cytotoxicity was also observed in several other cell culture and xenograft models. In xenografts of the MX-1 human breast carcinoma, tumor growth inhibition or regression was observed in nearly all of the animals treated with an aggressive regimen of five daily doses. This schedule resulted in a 24-h posttreatment decline in bone marrow progenitors to 60% of control and was no worse than for a single dose of TER286. These studies have motivated election of TER286 as a clinical candidate.

G-quadruplexes as targets for drug design.

G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (anthraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex-ligand complex and its effect on the dynamics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the experiments that relate the biological effects to their interactions with G-quadruplexes.

Activity of temozolomide against human tumor colony-forming units.

8-Carbamoyl-3-methylimidazo(5,1-d)-1,2,3,5-tetrazin-4(3H)-one (temozolomide) is a new imidazole tetrazinone compound with promising preclinical and clinical activity in nitrosourea-sensitive and -resistant models and manageable toxicity in Phase I and II clinical trials. In this study, we investigated the antiproliferative activity of temozolomide against a large variety of human tumors taken directly from patients in an in vitro soft agar tumor cloning system. Temozolomide was studied using a continuous exposure at final concentrations from 0.1 to 10.0 microM against a total of 222 tumor specimens, of which 101 (45%) were evaluable. A decrease in tumor colony formation was considered significant if survival of colonies treated with temozolomide was

Activity of oxaliplatin against human tumor colony-forming units.

This study was conducted to identify tumor types warranting Phase II clinical trials of oxaliplatin using the human tumor cloning assay. Oxaliplatin was tested at concentrations ranging from 0.5 to 50.0 microg/ml in 1-h and 14-day continuous exposures along with 1.4 microg/ml carboplatin and 0.2 microg/ml cisplatin for comparison. We defined in vitro response as tumor growth inhibition >50% of control. In the 1-h exposure schedule, in vitro responses were observed in 9 of 116 (8%), 18 of 115 (16%), 38 of 103 (37%), and 7 of 13 (54%) tumor specimens at concentrations of 0.5, 5.0, 10.0, and 50.0 microg/ml oxaliplatin, respectively. In the 14-day exposure schedule, in vitro responses were observed in 10 of 121 (8%), 37 of 121 (31%), 57 of 106 (54%), and 15 of 15 (100%) tumor specimens at concentrations of 0.5, 5.0, 10.0, and 50.0 microg/ml oxaliplatin, respectively. Activity was observed against colon cancer, non-small cell lung cancer, gastric cancer, and melanoma colony-forming units. In both cisplatin-resistant and cisplatin-sensitive tumors, the activity of oxaliplatin was concentration and time dependent. A 1-h exposure to 5.0 and 10.0 microg/ml oxaliplatin led to 7.4 and 23.4% in vitro responses, respectively, in specimens resistant to 1-h exposure of 0.2 microg/ml cisplatin. Moreover, 1-h exposures to 5.0 microg/ml and 10.0 microg/ml oxaliplatin showed in vitro antitumor responses in 10.2 and 24.3%, 17.2 and 34.5%, 10.0 and 20.0%, 6.7 and 16.7%, and 11.4 and 34.3% of specimens resistant to 1.4 microg/ml carboplatin, 6.0 microg/ml 5-fluorouracil, 3.0 microg/ml irinotecan, 10.0 microg/ml paclitaxel, and 0.04 microg/ml doxorubicin, respectively. The effect in those drug-resistant specimens was improved when oxaliplatin was used on the protracted exposure regimen. Our data indicate that oxaliplatin is an active drug in vitro against a large variety of human tumors. Both concentration and duration of exposure are important factors for oxaliplatin cytotoxicity. The broad spectrum of activity and the in vitro activity against some tumors primarily resistant to conventional anticancer drugs encourage further clinical investigations of oxaliplatin in patients with advanced cancer refractory to conventional chemotherapy.

Effects of hydroxyurea on extrachromosomal DNA in patients with advanced ovarian carcinomas.

PURPOSE: In vitro low concentrations of hydroxyurea eliminate double-minute chromosomes (dmins) containing amplified drug-resistance genes and oncogenes from cancer cells. This clinical trial investigated whether a noncytotoxic dose of oral hydroxyurea could reduce the number of dmins in cancer cells in patients with advanced ovarian carcinomas. EXPERIMENTAL DESIGN: The high frequency of ascites associated with ovarian cancer facilitated the monitoring of cytogenetic variations with minimal discomfort in patients who required frequent abdominal paracentesis. Sixteen patients with advanced ovarian carcinomas resistant to conventional cisplatin-based and/or paclitaxel chemotherapy and with ascites requiring frequent abdominal paracentesis were entered in this study. A course of treatment consisted of a single oral dose of 80 mg/kg hydroxyurea every 3 days for 6 weeks. Blood and i.p. levels of hydroxyurea were determined. We monitored the variations of dmins in tumor cells taken from serial abdominal paracenteses. RESULTS: The median number of courses administered to the patients was 1 (range, 1--9). In ascites, hydroxyurea concentrations were 610.3 +/- 76.3, 219.8 +/- 85.6, and 86.1 micromol/liter at 4, 24, and 30 h after oral administration, respectively. Eleven (78.6%) of 14 patient specimens contained dmins before therapy. The number of spreads with tumor cells containing dmins were reduced by more than 50% in 5 (45%) of 11 and 3 (60%) of 5 patients at the completion of the first and second course of chemotherapy, respectively. Using tumor cells taken directly from the patients and grown in soft agar, we documented that concentrations of hydroxyurea in ascites were too low to have any cytotoxic effects. No grade 3--4 hydroxyurea-related toxicities nor any objective responses were observed. However, despite the utilization of a low noncytotoxic dose of hydroxyurea, two patients had prolonged stabilization of their disease for 6 and 10 months, respectively, with concomitant decreases in the number of dmins that remained until progression. CONCLUSIONS: This study showed that, in some circumstances, a noncytotoxic dose of hydroxyurea given to patients with ovarian cancer can decrease the number of metaphase spreads containing dmins in cancer cells.

Effect of telomere and telomerase interactive agents on human tumor and normal cell lines.

Shortening of telomeres along with an up-regulation of telomerase is implicated in the immortality of tumor cells. Targeting either telomeres or telomerase with specific compounds has been proposed as an anticancer strategy. Because telomerase activity and telomeres are found in normal cells, telomere or telomerase targeting agents could induce side effects in normal tissues. We evaluated the effects of telomere and telomerase interactive agents in human tumor and normal cell lines to try to determine the potential side effects those agents might induce in patients. Toxicity of the G-quadruplex interactive porphyrins (TMPyP4, TMPyP2) and azidothymidine (AZT) were tested using a cell-counting technique against normal human cell lines (CRL-2115 and CRL-2120, fibroblasts; NHEK-Ad, adult keratinocytes; CCL-241, small intestinal cells; NCM 460, colonic mucosal epithelial cells) and human tumor cell lines (MDA-MB 231 and Hs 578T, breast cancer; SK-N-FI, neuroblastoma; HeLa, cervix cancer; MIA PaCa-2, pancreatic cancer; HT-29 and HCT-116, colon cancer; DU 145, prostatic cancer cell line). Telomerase activity of these cell lines was measured by a non-PCR-based conventional assay. The effects of TMPgammaP2, TMPyP4, and AZT were also evaluated against normal human bone marrow specimens, using a granulocyte-macrophage colony-forming assay (CFU-GM). AZT showed very low cytotoxic effects against normal and tumor cell lines, with the IC50 values above 200 microM. The IC50 values for TMPyP2 and TMPyP4 in normal human cell lines were in the range of 2.9-48.3 microM and 1.7-15.5 microM, respectively, whereas in tumor cell lines the IC50 values were 11.4-53 microM and 9.0-28.2 microM, respectively. Within the tissue types, keratinocytes were more sensitive to TMPyP4 than fibroblasts, and small intestinal cells were more sensitive than colonic mucosal epithelial cells. The IC50 for TMPyP2 and TMPyP4 in the normal marrow colony-forming assays were 19.3 +/- 5.1 microM and 47.9 +/-1.0 microM, respectively. In conclusion, the in vitro cytotoxicity of the telomere interactive agent TMPyP4 is comparable in human tumor and normal cell lines, which indicates that TMPyP4 could have effects on normal tissues.

Hormonal regulation of pp60c-src expression during osteoclast formation in vitro.

Recent studies have shown that the protooncogene c-src is required for normal osteoclastic bone resorption. In this study we examined the expression and regulation of pp60c-src in murine bone marrow cultures in which bone-resorbing multinucleated osteoclasts form over 6 days of culture. We found that both pp60c-src protein expression and pp60c-src tyrosine kinase activity correlated closely with the numbers of active osteoclasts in the cultures. PTH increased the numbers of osteoclasts, pp60c-src tyrosine kinase activity, and src protein, whereas calcitonin decreased the numbers of osteoclasts, src protein, and tyrosine kinase activity. However, when calcitonin was incubated for short periods (< 2 h) with the active osteoclasts present after 6 days of culture, there was a decrease in pp60c-src tyrosine kinase activity and the phosphorylation state, but not in total pp60c-src protein. These data suggest that pp60c-src is expressed in cultures of osteoclasts in parallel with the number of active bone-resorbing osteoclasts. They indicate that pp60c-src activity in osteoclast cultures depends on the activity and numbers of osteoclasts and is hormone regulated. As calcitonin receptors are detectable only in osteoclasts in these cultures, the inhibitory effects of calcitonin suggest that the critical site for pp60c-src expression in these cultures is in osteoclasts.

Design and synthesis of fluoroquinophenoxazines that interact with human telomeric G-quadruplexes and their biological effects.

In this study we have identified a new structural motif for a ligand with G-quadruplex interaction that results in biological effects associated with G-quadruplex-interactive compounds. Fluoroquinolones have been reported to possess weak telomerase inhibitory activity in addition to their better known bacterial gyrase poisoning. Starting with a fluoroquinobenzoxazine, which has modest potency in a human topoisomerase II assay, we have designed a more potent inhibitor of telomerase that has lost its topoisomerase II poisoning activity. This fluoroquinophenoxazine (FQP) interacts with G-quadruplex structures to inhibit the progression of Taq polymerase in a G-quadruplex polymerase stop assay. In addition, we demonstrate by 1H NMR studies that this compound interacts with telomeric G-quadruplex structures by external stacking to the G-tetrad with both the unimolecular fold-over and the parallel G-quadruplex structures. A photocleavage assay confirms the FQP interaction site, which is located off center of the external tetrad but within the loop region. Molecular modeling using simulated annealing was performed on the FQP-parallel G-quadruplex complex to determine the optimum FQP orientation and key molecular interactions with the telomeric G-quadruplex structure. On the basis of the results of these studies, two additional FQP analogues were synthesized, which were designed to test the importance of these key interactions. These analogues were evaluated in the Taq polymerase stop assay for G-quadruplex interaction. The data from this study and the biological evaluation of these three FQPs, using cytotoxicity and a sea urchin embryo system, were in accord with the predicted more potent telomeric G-quadruplex interactions of the initial lead compound and one of the analogues. On the basis of these structural and biological studies, the design of more potent and selective telomeric G-quadruplex-interactive compounds can be envisaged.

pp60 c-src expression and activity in MG-63 osteoblastic cells modulated by PTH but not required for PTH-mediated adenylate cyclase response.

A role for pp60c-src) tyrosine kinase in bone resorption was recently demonstrated in vivo. However, it is not known whether expression of this ubiquitous tyrosine kinase is essential in osteoblasts or osteoclasts. In this work, we have examined the expression of c-src in osteoblast-like cells. We found that c-src was expressed in the MG-63 osteoblastic osteosarcoma cell line as assessed by immunocytochemistry. When MG-63 cells were treated for 30 minutes with parathyroid hormone (PTH), the levels of tyrosine phosphorylation of c-src were increased in comparison with the untreated control. On the other hand, no changes in total c-src protein were observed after PTH treatment. The increase in c-src tyrosine phosphorylation due to PTH treatment was paralleled by an increase in c-src tyrosine kinase activity. Calcitonin had no effect on c-src phosphorylation, c-src protein level, or tyrosine kinase activity. To determine if c-src tyrosine kinase was required for normal bone cell function and PTH responsiveness, osteoblastic cells were isolated from the calvaria of a src-deficient mouse. The cyclic AMP response to PTH in this cell was identical to that seen in freshly isolated calvarial cells from normal mice. These results suggest that the activity of c-src in MG-63 cells is regulated by PTH as a result of tyrosine phosphorylation. Expression of src is not required for PTH responsiveness in osteoblastic cells as assessed by adenylate cyclase activation. The mode of signal transduction from the PTH receptor to nonreceptor c-src tyrosine kinase is not known at present.

Systemic administration of acidic fibroblast growth factor (FGF-1) prevents bone loss and increases new bone formation in ovariectomized rats.

There are no universally accepted agents that will substantially increase bone mass in osteoporotic patients. A number of peptides important in normal bone formation, such as members of the transforming growth factor-beta superfamily, are not satisfactory for this purpose either because their beneficial effects are predominantly local or there is systemic toxicity associated with their administration. We have examined the effects of exogenous fibroblast growth factor-1 and -2 (FGF-1 and FGF-2) on bone in vivo, since FGFs have been shown recently to be essential for normal skeletal development. FGF-1 was injected daily (0.2 mg/kg intravenously) for 28 days into the tail vein of adult female rats immediately following and 6 months after sham operation or ovariectomy (OVX). In rats treated immediately post-OVX, OVX produced more than a 30% decrease in tibial bone density, which was prevented by FGF-1 and estrogen. However, FGF-1 also had an anabolic effect. In sham-operated rats, FGF-1 increased bone density to 2-fold, whereas estrogen had no effect. In rats 6 months post-OVX, severe bone loss and disruption of trabecular microarchitecture occurred similar to that seen in patients with severe osteoporosis. In these rats, administration of FGF-1 induced extensive new woven bone formation with new trabecular-like structures filling much of the marrow spaces, and bone density in the tibial metaphysis increased 3-fold. FGF-1 and FGF-2 were also administered subcutaneously over the calvaria of mice in doses of 2-2000 microg/day for 3 days and shown to produce substantial increases in bone formation when examined morphologically. Thus, we conclude that both local and systemic FGF-1 increases new bone formation and bone density, and systemic FGF-1 also appears to restore bone microarchitecture and prevent bone loss associated with estrogen-withdrawal.

Dimethyl sulfoxide (DMSO) causes a reversible inhibition of telomerase activity in a Burkitt lymphoma cell line.

INTRODUCTION: Telomerase is an enzyme that is required for maintenance of telomeres. This enzyme has been shown to be present in germline tissues and majority of tumors and tumor cell lines. The regulation of telomerase is an area of active investigation in different models because, potentially, inhibition of this enzyme could be important in cancer therapy. To study the regulation of this enzyme in lymphoma cell lines, we used DMSO to produce a reversible G0/G1 arrest in Raji cell line, as shown earlier [Sawai M, Takase K, Teraoka H, Tsukada K. Reversible G1 arrest in the cell cycle of human lymphoid cell lines by dimethyl sulphoxide. Exp Cell Res 1990;187:4-10]. METHODS: In this study, we use a highly quantifiable conventional (non-amplified) assay to study the effect of DMSO on telomerase. In addition, we studied cellular proliferation and cell cycle profiles of the cells treated and, subsequently, released from DMSO induced blockage. RESULTS: In this model, DMSO reversibly inhibited telomerase activity that could be restored after release from the blockage. The inhibition of telomerase seems to parallel cellular proliferation and it appears that telomerase is regulated upon entry into the cell cycle. This view is consistent with other previously published views on relationship of telomerase with exit from cell cycle. CONCLUSION: Our observations demonstrate a novel effect of DMSO on cellular mechanisms in Raji cell line. It may provide an attractive model to further study regulation of telomerase in this cell line.

Exploring the mechanisms of action of FB642 at the cellular level.

FB642(methyl-2-benzimidazolecarbamate, carbendazim) is a systemic fungicide belonging to the benzimidazole family with antitumor activity against a broad spectrum of tumors both in vitro and in vivo such as pancreas, prostate, colon, and breast. Although the preclinical antitumor activity of FB642 has been well explored, its mechanism of action has not been as well delineated. Previous studies indicate that FB642 may interfere with mitosis and thus may disrupt or inhibit microtubule function resulting in apoptosis. This study seeks to determine if FB642 is a sufficiently novel agent worthy of further development by examining the effect of FB642 on apoptosis, the cell cycle, p53-positive and -negative tumors, and drug-resistant and MDR cell lines. The results of this present study indicate that FB642 increases the degree of apoptosis in all examined tumor cell lines, may induce G2/M uncoupling, may selectively kill p53 abnormal cells, and exhibits antitumor activity in drug- and multidrug-resistant cell lines. The induction of apoptosis by FB642, particularly in p53-deficient cells, its impressive in vivo activity against a broad spectrum of murine and human tumors, as well as an acceptable toxicity profile in animals, make FB642 an excellent candidate for further evaluation in clinical trials in cancer patients.

Activity of the multitargeted antifolate LY231514 in the human tumor cloning assay.

PURPOSE: This study was performed to evaluate the activity of the multitargeted antifolate (MTA or LY231514) against a broad range of human tumors taken directly from patients. MATERIALS AND METHODS: Human tumor colony-forming units were treated with MTA at concentrations of 0.1, 1.0, and 10 microg/ml in 1-h exposure studies. The responses of a limited number of specimens were also evaluated concurrently in 1-h exposures to cisplatin, fluorouracil, irinotecan, and/or paclitaxel. RESULTS: Of 358 specimens plated in the 1-h exposure studies, 148 (41%) were evaluable. Overall, responses were observed in 3% of specimens (4/144) at 0.1 microg/ml, 11% (17/148) at 1.0 microg/ml, and 23% (33/141) at 10 microg/ml. In this range of concentrations achievable clinically, there was a significant concentration-response relationship. At 10 microg/ml in the 1-h exposure studies, the response rate in colorectal cancer specimens was 32% (9/28), and the response rate in non-small-cell lung cancer was 25% (6/24). Responses were also observed in several chemoresistant tumors, including renal cell carcinoma, hepatocellular carcinoma, mesothelioma, and pancreatic carcinoma. The activity of MTA was not completely cross-resistant with that of cisplatin, fluorouracil, irinotecan, and paclitaxel. CONCLUSIONS: MTA demonstrated in vitro activity against a spectrum of tumors, including several tumors generally considered chemoresistant.

Purification and effect of chemical modifications on potato lectin activity.

Potato lectin of specific agglutinating activity of 10,000 units per mg was isolated. Chemical modifications of potato lectin with acetic and succinyl anhydrides or N-acety-limidazole result in complete loss of agglutinating activity. The lectin after reduction of disulfide bonds with dithiothreitol shows 25% of the native agglutinin activity. These results suggest that amino and phenolic groups are involved in the reaction of potato lectin with erythrocytes.

Tetracationic porphyrins inhibit angiogenesis induced by human tumor cells in vivo.

BACKGROUND: Cationic porphyrin TMPyP4, but not its isomer TMPyP2, inhibits telomerase in tumor cells in vitro and induces chromosome destabilization in vivo. MATERIALS AND METHODS: To examine the effects of these porphyrins on tumor-induced angiogenesis, 25-200 micrograms TMPyP4 or TMPyP2 were injected daily for 3 days in mice with intradermally implanted primary human tumor cells. Alternatively, tumor cells were exposed for 90 minutes to 2.5-20 microM porphyrins prior to implantation in mice. RESULTS: Either subcutaneous injections (> or = 50 micrograms/mouse) or preincubation with > or = 5 microM porphyrins significantly inhibited angiogenesis. CONCLUSION: Antiangiogenic activity is apparently unrelated to the ability of the porphyrins to inhibit telomerase.

5,6 Dihydro-5'-azacytidine (DHAC) restores androgen responsiveness in androgen-insensitive prostate cancer cells.

INTRODUCTION: The androgen resistance of some prostate cancer patients may be due to transcriptional inactivation of the androgen receptor (AR) gene catalyzed by cytosine DNA methyltransferase. MATERIALS AND METHODS: To determine if an inhibitor of cytosine DNA methyltransferase, 5,6-dihydro-5'-azacytidine (DHAC), can restore the androgen sensitivity in androgen-insensitive human prostate carcinoma cell lines in vitro, we cultured androgen-insensitive (PC3, DU-145, and TSUPrl) and androgen-responsive (LNCaP) cells with subcytotoxic concentrations (< or = IC50) of DHAC for 14 days followed by exposure to dihydrotestosterone (DHT) or to hydroxyflutamide for 7 days. RESULTS AND CONCLUSIONS: Only DHAC-treated DU-145 cells showed growth stimulation by 10(-11) to 10(-9) M DHT and a partial inhibition by 10(-5) and 10(-6) M hydroxyflutamide. However, since DU-145 is the only cell line tested that is known to have a hypermethylated AR promoter, the observed effects may be due to a partial demethylation of the AR by DHAC. Our data provide an evidence that cytosine DNA methyltransferase inhibitors can restore androgen responsiveness in androgen-refractory tumor cells, which are then sensitive to growth inhibition by antiandrogens.

5,6-Dihydro-5'-azacytidine (DHAC) affects estrogen sensitivity in estrogen-refractory human breast carcinoma cell lines.

BACKGROUND: There is little effective therapy for patients with hormone-refractory breast cancer. Hormone resistance is frequently due to the transcriptional inactivation of the estrogen receptor (ER) gene. We determined the effect of DHAC, a cytosine DNA methyltransferase (CMT) inhibitor, on the estrogen sensitivity in three human breast carcinoma cell lines with intermediate to low levels of estrogen receptor (ER) expression: MCF7 (adriamycin-sensitive), MCF7M/Adr (adriamycin-resistant), and MDA-435, and one ER+ cell line, ZR75-1. MATERIALS AND METHODS: Cells maintained in culture were exposed to DHAC or vehicle continuously for 14 days, then exposed to estradiol or tamoxifen and counted on day 21. RESULTS: Exposure to DHAC did not affect estrogen sensitivity in ZR-75-1 and MCF7M/Adr cells. DHAC treatment of MCF7 and MDA-435 cells resulted in significant (p < 0.05) growth stimulation in response to estrogen at 10(-6) M, and to growth modulation by tamoxifen at 10(-5) to 10(-7) M. CONCLUSIONS: These data suggest that DHAC can restore the estrogen sensitivity in ER-breast cancer. Thus, DHAC and other novel CMT inhibitors may have a clinical application in treating estrogen-refractory breast cancer patients by restoring the estrogen sensitivity and allowing these patients to respond again to conventional therapy with estrogen antagonists.