DMBA In Rats Research Articles

Effects of Tomato and Carrot-Supplemented Diets on Physical and Haemalogical Characteristics of Wistar Rats Exposed to Breast Carcinogen (Dimethylbenz-[A]-Anthracene)

Cancer, particularly of the breast, is one of the most common cases of morbidity and mortality in women globally. This research aimed at exploring the potentials of tomato and/or carrot-supplemented diets in mitigating against breast cancer carcinogen, DMBA in rats. Forty-eight female rats were randomly distributed into 8 groups. Groups 1 and 2 comprised of unexposed rats fed standard diet and DMBA-administered group fed standard diet respectively. Groups 3-5 were DMBA-administered groups fed diets containing 20% tomato, 20% carrot, and 20% of an equally mixed tomato and carrot respectively. For groups 6-8, unexposed rats fed diets containing 20% tomato, carrot, and 20% of an equally mixed tomato and carrot respectively. The feeding was for a period of 10 weeks. Feed intake, changes in weight and hematological changes were determined. The exposed rats fed supplemented diets had significantly (p<0.05) higher feed intake and percentage weight gain compared with the exposed group fed standard diet. Similarly, the unexposed groups fed supplemented diets had significantly (p<0.05) higher feed intake and percentage weight gain in comparison with the unexposed group fed standard diet. DMBA- Exposed group and unexposed group fed 20% tomato-supplemented diet had significantly higher WBC and hemoglobin compared with the control. In conclusion, supplementation of diets with either carrot or tomato improves feed intake in normal rats and antagonizes appetite-suppressing effects of cancer, and ameliorate weight loss in rats exposed to breast carcinogen.

Effect and mechanism of Yueju Pills on breast cancer induced by DMBA in rats

This paper aims to explore the inhibitory effect of Yueju Pills on breast cancer and decipher the underlying mechanism. A total of 92 SPF-grade SD female rats were involved in this study, and 14 of them were randomly selected into control group. The remaining 78 rats were administrated with 7,12-dimethylbenzanthracene(DMBA) by gavage to establish the breast cancer model. The modeled rats were randomized into model, tamoxifen(1.9 mg·kg~(-1)·d~(-1)), and low-and high-dose(17, 34 g·kg~(-1)·d~(-1)) Yueju Pills groups. The mental state, food intake, and activities of the rats were observed daily, and the body weight was measured on alternate days. After 12 weeks of administration, the rats were sacrificed and the tumor weight was measured. The serum estrogen and progeste-rone levels were determined by enzyme-linked immunosorbent assay. The histopathological changes of the breast and tumor were observed by hematoxylin-eosin staining. Western blot was employed to measure the protein levels of glucose transporter 1(GLUT1), lactate dehydrogenase A(LDHA), phosphofructokinase muscle(PFKM), pyruvate kinase isozyme type M2(PKM2), hexokinase 2(HK2), nuclear factor-kappaB(NF-κB), and phosphorylated NF-κB. The intestinal microbiome was examined by 16S rRNA high-throughput sequencing. The results showed that compared with the model group, high and low-dose Yueju Pills showed the tumor inhibition rate of 15.8% and 64.5%, respectively, and the low dose group had stronger inhibitory effect. Compared with the control group, the model group presented elevated the levels of estrogen and progesterone in serum. The administration of Yueju Pills lowered such ele-vation, and the low-dose group showed stronger lowering effect(P<0.05). Compared with the model group, Yueju Pills reduced the glands with increased breast tissue, the degree of breast duct expansion, the number and area of acinar cavity, the secretions, and the layers of mammary epithelial cells. Furthermore, Yueju Pills down-regulated the expression of GLUT1, LDHA, PFKM, PKM2, HK2, and NF-κB(P<0.05) and altered the diversity, composition, structure, and abundance of intestinal flora. The results showed that Yueju Pills could inhibit breast cancer by regulating the secretion of estrogen and progesterone, glycolysis, inflammatory cytokines, and intestinal flora.

Apple polyphenol phloretin complexed with ruthenium is capable of reprogramming the breast cancer microenvironment through modulation of PI3K/Akt/mTOR/VEGF pathways

Our recent investigation directed to synthesize a novel ruthenium–phloretin complex accompanied by the study of antioxidant in addition to DNA binding capabilities, to determine the chemotherapeutic activity against breast carcinoma in vitro and in vivo. Ruthenium–phloretin complex was synthesized and characterized by different spectroscopic methods. The complex was further investigated to determine its efficacy in both MCF-7 and MDA-MB-231 human carcinoma cell lines and finally in an in vivo model of mammary carcinogenesis induced by DMBA in rats. Our studies confirm that the chelation of the metal and ligand was materialize by the 3-OH and 9-OH functional groups of the ligand and the complex is found crystalline and was capable of intercalating with CT-DNA. The complex was capable of reducing cellular propagation and initiate apoptotic events in MCF-7 and MDA-MB-231 breast carcinoma cell lines. Ruthenium-phloretin complex could modulate p53 intervene apoptosis in the breast carcinoma, initiated by the trail of intrinsic apoptosis facilitated through Bcl2 and Bax and at the same time down regulating the PI3K/Akt/mTOR pathway coupled with MMP9 regulated tumor invasive pathways. Ruthenium-phloretin chemotherapy could interrupt, revoke or suspend the succession of breast carcinoma by altering intrinsic apoptosis along with the anti-angiogenic pathway.

Abstract PS8-13: Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer

Abstract Background: Hereditary breast cancer is common. With enhanced awareness and the recent introduction in affordable multi-gene germ line testing, an estimated 0.5-1 million women will learn that they carry a considerable risk to develop breast cancer. Thus, a rapidly increasing number of women, many of them very young, will be in need of effective strategies for breast cancer prevention. Current options to prevent breast cancer in women at high risk include bilateral mastectomies or systemic anti-estrogen therapy. Both options, while effective, may have a detrimental impact on the physical and emotional well-being of the patient. Localized delivery of an established anti-estrogen to breast tissue only may thus offer an attractive alternative for cancer prevention and may replace systemic therapy for ductal carcinoma in situ and early stage breast cancer with minimal risk for metastases. Methods: As such, we have sought to develop a silastic-silicone device, which when placed under breast tissue, will deliver the anti-estrogen fulvestrant directly to the target tissue. Sustained slow release of fulvestrant from a silastic-silicone device directly into the mammary tissue will provide the risk reducing benefits of systemic hormonal therapy while minimizing systemic exposure and the resulting poor compliance due to adverse effects. Results: Using a combination of in vitro and in vivo studies, we show that fulvestrant can be delivered through a silastic-silicone device. Implanted adjacent to mammary tissue, this drug delivery device provides sustained high levels of fulvestrant to inhibit estrogen receptor signaling and breast cancer cell proliferation. In a MCF-7 breast cancer xenograft model we have shown that silastic-silicone delivers fulvestrant selectively to mouse mammary tissue for more than 1 year with anti-tumor effects similar to those achieved with systemic fulvestrant exposure. Using the Sprague-Dawley rat DMBA spontaneous breast cancer induction model, we further demonstrate that fulvestrant delivered by silastic-silicone devices implanted adjacent to mammary tissue significantly delays time to first tumor compared to control animals (n=90, HR = 0.42, 95% CI 0.21-0.83) with minimal systemic exposure (plasma average 1.1 ng/mL, SD ±1.5 ng/mL). Initial large animal safety and toxicity studies in female sheep (ewes, n=2) support the surgical strategy to place the device between breast tissue (i.e. udder) and the chest wall (abdominal wall in ewes). Following 1 month of fulvestrant release, gross and histopathological analysis found no adverse effects or implant related toxicity. Bioanalytical analysis of mammary tissues suggested that drug was found highest near the implant (max 341 ng/g, udder average 58.4 ng/g, SD ±17.1 ng/g), with diminishing levels distal from the implant. Systemic exposure was low, with plasma levels 1.2 ng/mL, SD ±0.22 ng/mL, comparing favorably to clinical plasma levels achieved by intramuscular fulvestrant of 500 mg: 28.0 ng/mL, SD ±27.9 ng/mL (FDA package insert). Subsequent large animal studies will focus on the safety and pharmacokinetics of device delivered fulvestrant in longer studies (e.g. 3 to 6 months). Conclusions: The greater awareness and genetic identification of individuals at risk for breast cancer brings about an increased need for novel approaches to breast cancer prevention. The development of a silastic-silicone based device for sustained and localized drug delivery with an approved and effective anti-estrogen should allow rapid transition into clinical testing. This strategy will provide an alternative option to mastectomies and allow breast conservation for women identified to have a more than 40% lifetime chance of developing breast cancer, as well as provide an alternative to systemic hormonal therapy in women with ductal carcinoma in situ or stage I breast cancer. Citation Format: Pamela N Munster, Pujan K Desai, Nela Pawlowska, Elysia Roche, Romain Pacaud, Maxim A Daud, Emily Hsu, David Gingrich, Amelia Deitchman, Fran Aweeka, Scott Thomas. Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-13.

The effect of zinc and melatonin supplementation on immunity parameters in breast cancer induced by DMBA in rats

Objective: The aim of the study was to determine the effects of zinc and melatonin supplements on the immunity parameters of female rats with breast cancer induced by DMBA.Methods: Group 1; Control, Group 2; 7,12-dimethylbenz[a]anthracene (DMBA), Group 3; DMBA + zinc, Group 4; DMBA + melatonin, Group 5; DMBA + zinc + melatonin. The rats’ breast cancer was induced by DMBA 80 mg/kg. Groups 3–5 received daily 5 mg/kg doses of zinc, melatonin, and zinc + melatonin, respectively. Lymphocyte rates, T-lymphocyte subgroups, B-lymphocyte and natural killer cells (NK), and natural killer T (NKT) were evaluated.Results: The most significant increase in lymphocyte, T-lymphocyte, and CD4 lymphocyte rates was found in Group 5. The highest NKT cell rates were found in Group 3.Conclusions: Findings show that zinc and melatonin supplements have led to an increase in the immunity parameters of rats with breast cancer.

Evaluation of the Black Cumin Seed Oil Role (BCSO) on a Decline in eNOS Expression and Plasma NO Levels: Initial Studies Kemopreventive BCSO for Lung Cancer

BCSO is effective as an anti-aging agent in cultures of endothelial and macrophage exposed 7.12, dimethyl-benzantracene (DMBA). Reactive radicals damage endothelial tissue, cause inflammatory reactions and have a role in carcinogenesis. Excessive eNOS expression linked to chronic inflammation and carcinogenesis of lung cancer. The research objective was to determine the effect of BCSO on the expression of eNOS and NO levels of macrophage SD rats exposed DMBA. A study used 36 rats SD that divided into 6 groups, given BCSO (0,01, 0,05 and 0.1 ml / day), positive, negative and normal control. Variables were serum levels of NO and eNOS expression in endothelial tissue. The results showed that eNOS expression in DMBA group is higher than the normal group (P <0.05). BCSO will decrease eNOS expression and NO levels. ENOS expression and NO levels in group BCSO 0,01 and 0,05 ml / kg body weight / day lower than the DMBA group (p 0.05). Conclusion: a dose BCSO can degrade endothelial eNOS expression and NO levels of macrophage culture supernatant SD rat DMBA induced

Expression of DNA-repair proteins and their significance in pancreatic cancer and non-cancerous pancreatic tissues of Sprague–Dawley rats

BackgroundTo establish a model of pancreatic cancer induced by 7,12-dimethylbenzantracene (DMBA) in Sprague–Dawley (SD) rats, and detect the expression of DNA-repair proteins (MGMT, ERCC1, hMSH2, and hMLH1) and their significance in pancreatic cancer and non-cancerous pancreatic tissues of SD rats.MethodsDMBA was directly implanted into the parenchyma of rat pancreas (group A and group B), and group B rats were then treated with trichostatin A (TSA). The rats in both groups were executed within 3 to 5 months, and their pancreatic tissues were observed by macrography and under microscopy. Meanwhile, the rats in the control group (group C) were executed at 5 months. Immunohistochemistry was used to assay the expression of MGMT, ERCC1, hMSH2, and hMLH1.ResultsThe incidence of pancreatic cancer in group A within 3 to 5 months was 48.7% (18/37), including 1 case of fibrosarcoma. The incidence of pancreatic cancer in group B was 33.3% (12/36), including 1 case of fibrosarcoma. The mean of maximal diameters of tumors in group A was higher than that in group B (P <0.05). No pathological changes were found in pancreas of group C and other main organs (except pancreas) of group A and group B. No statistical differences were found among the positive rates of MGMT, ERCC1, hMSH2, and hMLH1 in ductal adenocarcinoma and non-cancerous pancreatic tissues of group A (P >0.05). The positive rates of MGMT, ERCC1, hMSH2, and hMLH1 were significantly lower in ductal adenocarcinoma than those in non-cancerous tissues of group B (P ≤0.05). All pancreas of group C had positive expression of MGMT, ERCC1, hMSH2, and hMLH1 and two cases of fibrosarcoma showed a negative expression.ConclusionsDMBA, directly implanted into the parenchyma of pancreas, creates an ideal pancreatic cancer model within a short time. TSA might restrain DNA damage related to the genesis and growth of pancreatic cancer in rats. The DNA-repair proteins, including MGMT, ERCC1, hMSH2, and hMLH1, might play an important role in the genesis of pancreatic cancer induced by DMBA in rats.

P4-02-02: The Natural Fetal Estrogen Estetrol (E4), Causes Anti-Estrogenic Effects in Women with Endocrine-Responsive Positive Early Breast Cancer.

Abstract Background: Estetrol (E4) is a natural fetal estrogen which exerts estrogenic effects on reproductive organs and on the bone, and effectively reduces menopausal smyptoms. In contrast to other estrogens, however, E4 has estrogen-antagonistic effects on breast cancer cell lines in vitro and in the rat DMBA model, which would make it a suitable Hormone Replacement Therapy (HRT) in breast cancer patients, particularly in women who are being treated with aromatase inhibitors. Patients and Methods: We have investigated the effect of 14 days preoperative treatment with 20 mg E4 per day on tumor proliferation, apoptosis, ER-receptors, PgR receptor and several endocrine parameters in a prospective, randomised, double-blind, placebo-controlled, neo-adjuvant study in 15 pre- and 14 postmenopausal women with estrogen-receptor positive early breast cancer. Results: Estetrol induced a significant increase of SHBG, a significant decrease of FSH in postmenopausal women and no increase of gonadotrophins in premenopausal women. Estetrol had no effect on Ki67 expression and on apoptosis-related Bax and Bcl-2, but the apoptosis index in tumor tissue increased significantly. Systemic IGF-1 levels decreased significantly. Surprisingly the intratumoral epithelial ER-alpha expression decreased significantly, whereas the ER-beta expression showed a trend to increase. Conclusion: Our data suggest that E4 may be suitable and safe for HRT in women with spontaneous or induced menopausal symptoms, since apoptosis increases, IGF-1 decreases and no unfavourable effects are observed on Ki67, Bax and Bcl-2. The decrease of ER-alpha and the increase of ER-beta suggest a mechanism of action, explaining why the natural fetal estrogen E4 has estrogen-antagonistic effects on breast cancer tissue. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-02.

Antiestrogenic effects of the natural fetal estrogen estetrol (E4) in women with estrogen-receptor-positive early breast cancer.

280 Background: Estetrol (E4) is a fetal estrogen which exerts estrogenic effects on vaginal epithelium, hot flushes and bone, but has estrogen-antagonistic effects on breast cancer cell lines in vitro and in the rat DMBA model. Therefore E4 may be suitable for Hormone Replacement Therapy (HRT) in women with breast cancer including women treated with aromatase inhibitors. Methods: We have investigated the effect of 14 days pre-operative treatment with 20 mg E4 per day on proliferation, apoptosis, ER-receptors and sex steroid levels in a prospective, randomised, double-blind, placebo-controlled, neo-adjuvant study in 15 pre- and 15 postmenopausal women with estrogen-receptor positive early breast cancer. Results: Estetrol induced a significant increase of SHBG, a significant decrease of FSH in postmenopausal women and no increase of gonadotrophins in premenopausal women. Estetrol had no effect on Ki67 expression and on apoptosis-related Bax and Bcl-2, but the apoptosis index in tumor tissue increased significantly. Systemic IGF-1 levels decreased significantly. Surprisingly the intratumoral epithelial ER-alpha expression decreased significantly, whereas the ER-beta expression showed a trend to increase. Conclusions: This data show that E4 has estrogenic endocrine effects. The data support the hypothesis that E4, may be suitable and safe for HRT in women with spontaneous or induced menopausal symptoms, since apoptosis increases, IGF-1 decreases and no unfavourable effects are observed on Ki67, Bax and Bcl-2. The decrease of ER-alpha and the increase of ER-beta suggest a mechanism of action, explaining why the natural fetal estrogen E4 has estrogen-antagonistic effects on breast cancer tissue.

Grape Seed Oil plus DMBA Has a Hepatorenal Protective Effect against Damage Induced By DMBA in Rats

Grape Seed Oil plus DMBA Has a Hepatorenal Protective Effect against Damage Induced By DMBA in Rats

Abstract DD01-01: NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase

Abstract DD01-01: NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase

Effects of 4-n-Octylphenol on the Induction of Mammary Tumors Induced by 7,12-Dimethylbenz[a]anthracene in Rats

We previously reported that a neonatal administration of diethylstilbestrol or 17beta-estradiol affected the mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats. The aim of this present study was to investigate the effects of 4-n-octylphenol (OP), a weak estrogenic disruptor, on the induction of mammary carcinomas (MC) and benign proliferative lesions (PL) induced by DMBA in rats. All female rats were administered at 0, 0.1, 10, 100, and 1,000 microg OP once at birth, given 10 mg DMBA at 50 days after birth, and thereafter underwent necropsy at 351 days after birth. All male rats were given 10 mg DMBA at 28, 42, and 56 days after birth, and 0, 10, 100, and 1,000 ppm OP fed from day 70-153; they underwent necropsy at 153 days after birth. Neonatal single administration of OP in female rats showed no effects such as persistent estrus, anovulatory ovaries, or PL. A slight increase in numbers of rats with MC occurred at the highest dosage. Feeding a large dose of OP for a long period in male rats induced atrophy of testes and slightly increased numbers of affected MC but not increased numbers of males while it showed no effects on PL. These results suggested that administration of a large dose of OP for a long period may have had a minimal effect on mammary carcinogenesis in male rats.

The Investigation of the Antioxidative Properties of the Novel Synthetic Organoselenium Compounds in Some Rat Tissues

DMBA (7,12-dimethylbenz[a]anthracene) is a polycyclic aromatic hydrocarbon (PAH) known to cause tumors in rats. Selenium is an essential element with physiological non-enzymatic antioxidant properties. Because of the health problems induced by many environmental pollutants, many efforts have been undertaken in evaluating the relative antioxidant potential of selenium and synthetic organoselenium compounds. In this study, adult female Wistar rats were treated with DMBA and the novel organoselenium compounds (1-isopropyl-3-methylbenzimidazole-2-selenone [SeI] and 1,3-di-p-methoxybenzylpyrimidine-2-selenone [SeII]) in the determined doses. The protective effects of novel synthetic organoselenium compounds (SeI and SeII) against DMBA-induced changes in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities and total glutathione (GSH) and malone-dialdehyde (MDA) levels of rat heart and brain were investigated. It was determined that SeI and SeII fully or partially restored enzyme activity. It was also found that lipid peroxidation was also decreased in SeI and SeII treated groups. Consequently, it was determined that novel synthetic organoselenium compounds (SeI and SeII) provided protection of antioxidant activity, and protection against lipid peroxidation measured as MDA in SeI and SeII treated groups was provided by novel synthesized organoselenium compounds. The ability of the organoselenium compounds to prevent oxidative damage induced by DMBA in rats was rationalized.

Estrogen Receptor Expression of DMBA‐Induced Mammary Tumors in Intact and Ovariectomized Lean and Obese Zucker Rats.

Obesity is associated with increased risk for postmenopausal breast cancer. Clinical data suggest that women with Estrogen Receptor negative (ER−) breast cancer have poorer outcome than those with ER+ tumors. The main objective of this study was to investigate whether obesity can be associated with the expression of ER in mammary tumors in the presence or absence of ovarian estrogen. Lean and obese Zucker rats were sham operated or ovariectomized at age 40 days and were gavaged at age 50 days with 65 mg/kg DMBA. Rats were weighed and palpated twice weekly for detection of mammary tumors and killed 135 days post-DMBA treatment. ER immunohistochemistry was performed on paraffin-embedded tumors. The incidence of mammary tumors was 59% in the obese, sham-operated (O/S) rats compared to 30% in the lean sham-operated (L/S) group (P<0.05) and 36% for the obese ovariectomized (O/O) rats while no lean ovariectomized (L/O) rats developed tumors (P<0.001). ER expression was evaluated for all the ductal carcinoma in situ (DCIS) or invasive ductal and lobular carcinoma (IDC) tumors and the results are as follows; L/S group (17% ER – and 83% ER+), O/S group (45% ER− and 55% ER+) and O/O groups had 50% ER− and 50% ER +. These preliminary results suggest that obesity may increase the incidence of ER negative tumors in the obese Zucker rat DMBA mammary tumor model (Supported by Susan G. Komen Breast cancer foundation and ABI to RH)

Ras gene mutations in 7,12-dimethylbenz[ a]anthracene (DMBA)-induced rat sarcomas

Tumor induction in rats by 7,12-dimethylbenz[ a]anthracene (DMBA) will generate malignancies that display reproducible chromosomal abnormalities involving rat chromosome (RNO) 2. Thus, it has been reported that rat DMBA erythroleukemias display RNO2 abnormalities, which in this case were closely correlated to mutations in the Nras oncogene located in RNO2q34. Our cytogenetic analysis in a series of 17 DMBA-induced rat sarcomas showed that 11 (65%) tumors had a significant increase in RNO2 copy number. Furthermore, the incidence of point mutations in codons 12, 13 and 61 of Hras, Kras, and Nras was examined in the same set of sarcomas, and mutations were detected in three (18%) tumors, in codon 61 of Kras (CA A→CA T) (1 of 17) and Nras (C AA→C TA) (2 of 17). We conclude that the high frequency of RNO2 gain was in accordance with previous studies of DMBA-induced rat neoplasms, supporting the idea of a significant role of RNO2 in DMBA carcinogenesis. However, there was no clear-cut relationship between activated Nras and gain of RNO2 material, implying that mutational activation of Nras is not the causative factor underlying the gain of RNO2 copy number in rat DMBA sarcomas, in contrast to what has been suggested for DMBA-induced erythroleukemias.

The Hgfr/Met oncogene is the target for gene amplification in DMBA-induced rat sarcomas

Analysis of chromosome rearrangements in tumors is an important means for revealing genetic pathways underlying tumorigenesis and tumor progression. In a set of 17 rat sarcomas induced by exposure to 7,12-dimethylbenz[a]anthracene (DMBA), we had previously found homogeneously staining regions (which are generally accepted as cytogenetic signs of gene amplification) in 5 tumors, using cytogenetic analysis. By employing comparative genomic hybridization, we detected regional increases in DNA copy number of the proximal part of rat chromosome 4 (RNO4) in the tumors harboring homogeneously staining regions. We detected amplification of the Hgfr/Met oncogene, located at RNO4q21.2, by fluorescence in situ hybridization (FISH) in all five tumors. In four of them, several flanking genes located in the near vicinity of Hgfr/Met, including Cav1 (q21.1), Wnt2 (q21.2?q21.3) and Cftr (q21.3), were also amplified, although amplification was seen in a smaller fraction of the cells than Hgfr/Met amplification. In the fifth tumor (BL150T), Hgfr/Met was amplified in all cells and was the sole amplified gene of those tested. In addition the Hgfr/Met FISH signals in BL150T were tightly clustered and formed compact and intense spots compared with the signals seen in the other four tumors. Application of the free chromatin FISH technique to BL150T showed that the genomic Hgfr/Met probe stained the extended chromatin fibers of up to 1.5 megabases with an almost uninterrupted signal, indicating that the BL150T amplicon was build up solely from Hgfr/Met gene sequences. Our results indicate that the Hgfr/Met oncogene may be the primary target for amplification in a subset of rat DMBA sarcomas.

Peptides and antitumor activity

We developed a group of synthetic analogs of GnRH and Somatostatin to inhibit the tumor growth of different kind. The GnRH analogs decreasing the gonadotroph and steroid hormone levels act on the hormone dependent tumors and influence their growth. One of the most effective antitumor analog was patented under the name FOLLIGEN which inhibited the breast cancer caused by DMBA in rats without any side-effects. Other inhibitory analogs of GnRH with long-lasting effect were effective in the treatment of breast, ovary and prostate tumors. Another analog [alpha-Asp(DEA)]6,Gln8-hGnRH showed a very low endocrine but high antitumor effect in both in vitro and in vivo experiments. Its tritium labeled derivative exhibited specific binding sites on human tumor cell lines. We synthesized the analogs of GnRH-III with effective selective antitumor activity which does not alter the ovarian cycle of rats but inhibits the colony-formation of human breast cancer cell lines and has a significant antiproliferative effect. We also synthesized conjugates of potent GnRH analogs with a branched chain polylysine backbone which induce a 33-35% decrease of cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines and 45-50% inhibition of cell proliferation. Another conjugate decreased the tumor growth of MDA-MB-231 xenografts by 80% in a treatment of 9 weeks and even tumor free animals could be found among the ones treated. Using these radiolabeled peptide hormone analogs we found that human tumor cell lines and xenografts specifically bind the GnRH conjugates. We also synthesized a series of Somatostatin analogs which inhibit tyrosine kinases and the growth of several breast, prostate and colon tumor cell lines. One of our best analogs was a heptapeptide, TT-232, which strongly inhibited the tyrosine kinase activity and the cell-proliferation in different colon tumor cells. However, it did not inhibit the growth hormone release either in vitro or in vivo from rat pituitary cells. The TT-232 was found to be effective on 60 human tumor cell lines, it significantly inhibited the tumor growth on different animal tumor models, and induced apoptosis, as a result of which some animals became tumor free. The TT-232 inhibited the tumor growth of PC3 prostate xenografts with 60% and caused a 100% survival of mice 60 days after the transplantation. It is being preclinically tested at present. We have shown that the new GnRH analogs acting without any hormonal effect and the Somatostatin analogs with strong antitumor and tyrosine kinase inhibitory activity but no hormonal effect may represent a breakthrough in the research of the antitumor peptides, having direct effect on tumor cells.

Amplification and overexpression of the hepatocyte growth factor receptor (HGFR/MET) in rat DMBA sarcomas.

In the present study subcutaneous fibrosarcomas were induced by the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in rats from F1 generation cross breedings of two different inbred strains. Comparative genomic hybridization (CGH) analysis, which allows detection of DNA sequence copy changes, was applied to one of the tumors and it was found that there were increased copy numbers of sequences at chromosome 4q12-q21 in this tumor. We have previously determined that the loci for the hepatocyte growth factor (Hgf) and hepatocyte growth factor receptor (Hgfr/Met), a protooncogene, are situated in this particular chromosome region. Using probes for the two genes in FISH (fluorescence in situ hybridization) and in Southern blots we found that the Hgfr/Met gene was amplified in five of the 19 sarcomas studied, and that the Hgf gene was coamplified in two of them. Northern and Western blots and tyrosine phosphorylation analysis showed that the HGF receptor was overexpressed and functional in all five tumors, as well as in two additional tumors. In summary, both amplification and overexpression of the Hgfr/Met gene was found in about 25% of DMBA-induced experimental rat sarcomas, and HGF receptor overexpression alone was seen in two additional tumors. Possibly this reflects an involvement in paracrine or autocrine stimulation of growth and invasiveness by HGF. Our finding could provide a rodent model system to increased knowledge about causality and therapy, which may be applicable to the sizeable fraction of human musculoskeletal tumors displaying MET overexpression.

Immunohistochemical characterization of pancreatic tumors induced by DMBA in rats

Immunohistochemical characterization of pancreatic tumors induced by DMBA in rats

Steady-state mRNA expression for growth factors in DMBA-induced rat mammary tumors

Steady-state mRNA levels were examined for insulin-like growth factors (IGF) I and II, transforming growth factor α (TGF α) and its receptor and the epidermal growth factor (EGF) receptor in mammary tumors induced by DMBA in rats. An abundant 4.8 kb TGF α transcript was identified in all tumors, along with a 7.5 – 8.0 kb IGF-I transcript. A presumptive 2.9 kb IGF-II transcript was also identified in all tumors. Northern analyses and receptor autophosphorylation studies failed to detect EGF receptors in any mammary tumors. These findings suggest the potential for autocrine or paracrine influences of IGF-I and IGF-II in this tumor model and a possible paracrine influence of TGF α in tumor-induced neovascularization.