Immune-deficient animals to study "hormone-dependent" breast and endometrial cancer

Athymic (nu/nu) mice are T cell deficient and can accept xenografts of human tumor material. Hormone-dependent tumor growth can be demonstrated in ovariectomized athymic mice by estrogen administration. Estrogen receptor (ER) positive MCF-7 breast cancer cells implanted into the axillary mammary fat do not grow into palpable tumors unless sustained release preparations of estrogen are administered. The non-steroidal antiestrogen tamoxifen, though it exhibits estrogenic properties in the mouse, does not facilitate MCF-7 tumor growth (during short term, i.e. 8 weeks of therapy) and can prevent estradiol-stimulated growth. In contrast, ER negative MDA-MB-231 cells grow with or without estrogen administration and tamoxifen does not control tumor growth. These statements reflect current dogma concerning the value of athymic mice to confirm the hormone dependent growth of cancer cells in vivo. Our aim has been to define the limits of this dogma and to investigate the growth relationship of hormone-dependent and independent cells with their host environment. The potential endocrine or paracine effect of ER negative tumors on the growth of ER positive tumors was evaluated by transplantation on opposite sides of athymic mice or by the inoculation of different ratios of ER positive/negative cells (MCF-7:MDA-MB-231 9:1, 99:1, 999:1). MCF-7 cells could not be encouraged to grow by a rapidly growing MDA-MB-231 tumor on the opposite side of the animal. Similarly ER negative tumors grew out of the mixed tumor inoculates suggesting that ER positive tumors could not be encouraged to grow preferentially by the paracrine influences of ER negative cells. However, estrogen facilitates the growth of an ER positive tumor following inoculation of mixed cell populations. Antiestrogen treatment can blunt estrogen-stimulated growth but cannot control the growth of ER positive/negative containing tumors. ER positive endometrial tumors grow in response to estrogen treatment and some (EnCa101) have been shown to grow in response to tamoxifen or a combination of tamoxifen and estrogen. More unusual though is our recent observation that an ER negative primary endometrial tumor (BR) and its metastasis (BR-MET) grow more rapidly in estrogen-treated athymic mice. This finding seems to have far-ranging consequences for our view of hormone-dependent growth. Either our view of estrogen-stimulated growth needs to be modified or the host is specifically altered during estrogen treatment. We have taken the position that since natural killer cells (present in athymic mice) can be lowered by estrogen this may result in an increased tumor cell survival in the heterotransplant model.(ABSTRACT TRUNCATED AT 400 WORDS)     

11C]Choline as a PET biomarker for assessment of prostate cancer tumor models

[(11)C]Choline has been evaluated as a positron emission tomography (PET) biomarker for assessment of established human prostate cancer tumor models. [(11)C]Choline was prepared by the reaction of [(11)C]methyl triflate with 2-dimethylaminoethanol (DMAE) and isolated and purified by solid-phase extraction (SPE) method in 60-85% yield based on [(11)C]CO(2), 15-20 min overall synthesis time from end of bombardment (EOB), 95-99% radiochemical purity and specific activity >0.8 Ci/micromol at end of synthesis (EOS). The biodistribution of [(11)C]choline was determined at 30 min post iv injection in prostate cancer tumor models C4-2, PC-3, CWR22rv, and LNCaP tumor-bearing athymic mice. The results showed the accumulation of [(11)C]choline in these tumors was 1.0% dose/g in C4-2 mouse, 0.4% dose/g in PC-3 mice, 3.2% dose/g in CWR22rv mice, and 1.4% dose/g in LNCaP mice; the ratios of tumor/muscle (T/M) and tumor/blood (T/B) were 2.3 (T/M, C4-2), 1.4 (T/M, PC-3), 2.5 (T/M, CWR22rv), 1.2 (T/M, LNCaP) and 2.6 (T/B, C4-2), 2.6 (T/B, PC-3), 7.8 (T/B, CWR22rv), 3.2 (T/B, LNCaP), respectively. The micro-PET imaging of [(11)C]choline in prostate cancer tumor models was acquired from a C4-2, PC-3, CWR22rv, or LNCaP implanted mouse at 30 min post iv injection of 1 mCi of the tracer using a dedicated high resolution (<3 mm full-width at half-maximum) small FOV (field-of-view) PET imaging system, IndyPET-II scanner, developed in our laboratory, which showed the accumulation of [(11)C]choline in C4-2, PC-3, CWR22rv, or LNCaP tumor implanted in a nude athymic mouse. The initial dynamic micro-PET imaging data indicated the average T/M ratios were approximately 3.0 (C4-2), 2.1 (PC-3), 3.5 (CWR22rv), and 3.3 (LNCaP), respectively, which showed the tumor accumulation of [(11)C]choline in all four tumor models is high. These results suggest that there are significant differences in [(11)C]choline accumulation between these different tumor types, and these differences might offer some useful measure of tumor biological process.     

Ceramide and glucosylceramide upregulate expression of the multidrug resistance gene MDR1 in cancer cells

In the present study we used human breast cancer cell lines to assess the influence of ceramide and glucosylceramide (GC) on expression of MDR1, the multidrug resistance gene that codes for P-glycoprotein (P-gp), because GC has been shown to be a substrate for P-gp. Acute exposure (72 h) to C8-ceramide (5 microg/ml culture medium), a cell-permeable ceramide, increased MDR1 mRNA levels by 3- and 5-fold in T47D and in MDA-MB-435 cells, respectively. Acute exposure of MCF-7 and MDA-MB-231 cells to C8-GC (10 microg/ml culture medium), a cell-permeable analog of GC, increased MDR1 expression by 2- and 4- fold, respectively. Chronic exposure of MDA-MB-231 cells to C8-ceramide for extended periods enhanced MDR1 mRNA levels 45- and 390-fold at passages 12 and 22, respectively, and also elicited expression of P-gp. High-passage C8-ceramide-grown MDA-MB-231 (MDA-MB-231/C8cer) cells were more resistant to doxorubicin and paclitaxel. Incubation with [1-(14)C]C6-ceramide showed that cells converted short-chain ceramide into GC, lactosylceramide, and sphingomyelin. When challenged with 5 mug/ml [1-(14)C]C6-ceramide, MDA-MB-231, MDA-MB-435, MCF-7, and T47D cells took up 31, 17, 21, and 13%, respectively, and converted 82, 58, 62, and 58% of that to short-chain GC. Exposing cells to the GCS inhibitor, ethylenedioxy-P4, a substituted analog of 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, prevented ceramide's enhancement of MDR1 expression. These experiments show that high levels of ceramide and GC enhance expression of the multidrug resistance phenotype in cancer cells. Therefore, ceramide's role as a messenger of cytotoxic response might be linked to the multidrug resistance pathway.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Synthesis and investigation of a radioiodinated F3 peptide analog as a SPECT tumor imaging radioligand

A radioiodinated derivative of the tumor-homing F3 peptide, (N-(2-{3-[(125)I]Iodobenzoyl}aminoethyl)maleimide-F3Cys peptide, [(125)I]IBMF3 was developed for investigation as a SPECT tumor imaging radioligand. For this purpose, we custom synthesized a modified F3 peptide analog (F3Cys) incorporating a C-terminal cysteine residue for site-specific attachment of a radioiodinated maleimide conjugating group. Initial proof-of-concept Fluorescence studies conducted with AlexaFluor 532 C(5) maleimide-labeled F3Cys showed distinct membrane and nuclear localization of F3Cys in MDA-MB-435 cells. Additionally, F3Cys conjugated with NIR fluorochrome AlexaFluor 647 C(2) maleimide demonstrated high tumor specific uptake in melanoma cancer MDA-MB-435 and lung cancer A549 xenografts in nude mice whereas a similarly labeled control peptide did not show any tumor uptake. These results were also confirmed by ex vivo tissue analysis. No-carrier-added [(125)I]IBMF3 was synthesized by a radioiododestannylation approach in 73% overall radiochemical yield. In vitro cell uptake studies conducted with [(125)I]IBMF3 displayed a 5-fold increase in its cell uptake at 4 h when compared to controls. SPECT imaging studies with [(125)I]IBMF3 in tumor bearing nude mice showed clear visualization of MDA-MB-435 xenografts on systemic administration. These studies demonstrate a potential utility of F3 peptide-based radioligands for tumor imaging with PET or SPECT techniques.     

Syntheses and biological evaluation of topoisomerase I-targeting agents related to 11-[2-(N,N-dimethylamino)ethyl]-2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-one (ARC-31)

Several 11-ethyl-2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-ones with varied functionality on the ethyl substituent have exhibited potent topoisomerase I (TOP1) targeting activity and antitumor activity. The influence of various polar substituents at the 2-position of the 11-ethyl substituent, including N-methylamine, N-isopropylamine, hydroxyl, and hydroxylamino groups, on TOP1-targeting activity and cytotoxicity was assessed. The N-methylamine and N-isopropylamine derivatives were also evaluated as antitumor agents in athymic nude mice with MDA-MB-435 human tumor xenografts. Both compounds were active as antitumor agents upon either parenteral or oral administration.     

Estradiol-stimulated growth of MCF-7 tumors implanted in athymic mice: a model to study the tumoristatic action of tamoxifen

Ovariectomized athymic (nude) mice were inoculated (10(7) cells) with the breast cancer cell line, MCF-7, into the axillary mammary fat pads. Tumors did not grow unless animals were implanted with a 1.7 mg estradiol sustained (8-week)-release cholesterol pellet. Co-implantation with tamoxifen (5 mg, 4-week release) caused an inhibition of estradiol-stimulated growth but did not cause tumor growth when implanted alone. The metabolism of [3H]tamoxifen was determined in the athymic mouse bearing MCF-7 tumors. Metabolites in the liver, uterus and tumor were determined by TLC. The principal metabolite in each of the tissues was 4-hydroxytamoxifen (by comparison of Rfs with authentic standards). Studies with 4-hydroxytamoxifen and N-desmethyltamoxifen (the principal metabolites in patients) showed that each was effective in inhibiting estradiol-stimulated tumor growth. However, tumor growth could be reactivated by treatment with estradiol alone. In a separate experiment, tumor-implanted animals were treated with tamoxifen for 1, 2 and 6 months. Tamoxifen did not cause tumor growth. Nevertheless, tumor growth was reactivated by estradiol on each occasion. These studies confirm the tumoristatic actions of tamoxifen and strongly support the view that therapy must be given indefinitely to patients to control tumor recurrence. The athymic mouse model can be used in the future to determine the efficacy of novel antiestrogens and the development of antiestrogen drug resistance.     

Interleukin-1 directly regulates hormone-dependent human breast cancer cell proliferation in vitro

Direct in vitro effects of IL-1 on hormone-dependent (MCF-7 and ZR-75-B) and independent (HS-578-T and MDA-231) human breast cancer cell proliferation were investigated in short-term and long-term cell cultures. For short-term (48 h) studies [3H]thymidine uptake was used as an index of proliferation, while for long-term (12 day) cultures actual cell numbers were determined. Initial studies, conducted with MCF-7 cells, demonstrated that both forms of recombinant human IL-1 (alpha and beta) at 10(-11) M inhibited [3H]thymidine uptake by MCF-7 by 70%, and by day 7 of the long-term study alpha and beta IL-1 at 10(-11) M inhibited MCF-7 cell growth by 80%. IL-1, while inhibiting the growth of another hormone-dependent breast cancer cell line; ZR-75-B, had no effect on the hormone-independent cell lines MDA-231 and HS-578-T. The differing proliferative responses of the hormone-dependent and independent cells to IL-1 may, in part, be due to the expression of IL-1 receptors on these cells, in that MCF-7 cells express IL-1 receptors [dissociation constant (Kd) = 2.0 x 10(-10) M; receptor density = 2,500 sites per cell and mol wt = 80,000] while the hormone-independent MDA-231 cells do not.     

利用Gaussia萤光素酶建立乳腺癌实时定量检测动物模型

目的：建立-种基于分泌型萤光素酶的实时定量检测实验动物体内肿瘤大小的方法。方法：以分泌型Gaussia萤光素酶（Gluc）为报告基因,以嘌呤霉素为筛选基因,将两者用T2A元件连接后克隆到慢病毒载体,包装慢病毒后感染乳腺癌MCF-7细胞,经嘌呤霉素筛选得到稳定转染细胞MCF-7-Gluc,并检测细胞上清中Gluc活性随时问和细胞数目的变化;将MCF-7-Gluc扩大培养后经皮下注射到雌性BALB／c裸鼠前肢腋下,待肿瘤形成后,检测外周血液中Gluc活性与肿瘤体积的相关性。结果：体外实验显示稳定转染细胞MCF-7-Gluc分泌到细胞上清的Gluc活性与时间和细胞数量在-定范围内均呈现良好的线性关系,体内实验显示裸鼠血液中的Gluc活性与肿瘤体积呈正相关。结论：Gluc技术可作为-种灵活、方便、实时定量检测活体动物体内肿瘤大小的有效工具。     

Identification of a novel aspartic-like protease differentially expressed in human breast cancer cell lines

Four different human breast cancer cell lines were examined to search for genes associated with tumor growth and metastasis. Each of these cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and MDA-MB-435, displays different phenotypic characteristics ranging from poorly to highly tumorigenic and metastatic. The differences in gene expression profiles of these cell lines generated by differential display technique should allow one to identify candidates as putative oncogenes or tumor/metastasis suppressor genes. A novel cDNA expressed in the highly tumorigenic and metastatic cell line, MDA-MB-435, was identified and isolated by this approach. The function for this gene, designated ALP56 (aspartic-like protease 56 kDa), in tumor progression is suggested by the homology of the encoded protein to aspartic proteases, such as cathepsin D. The amino acid residues in two catalytic domains of this family are highly conserved in those domains of ALP56. Northern hybridization indicated that the expression of ALP56 is associated with growth and metastasis of MDA-MB-435 tumors in immunodeficient mice. In situ hybridization of biopsies from breast cancer and colon cancer patients indicated that ALP56 is upregulated in human primary tumors and liver metastasis. These results suggest that this novel gene correlates with human tumor progression.     

Hsa_circ_0092276 promotes doxorubicin resistance in breast cancer cells by regulating autophagy via miR-348/ATG7 axis

Previous study has confirmed that hsa_circ_0092276 is highly expressed in doxorubicin (DOX)-resistant breast cancer cells, indicating that hsa_circ_0092276 may be involved in regulating the chemotherapy resistance of breast cancer. Here we attempted to investigate the biological role of hsa_circ_0092276 in breast cancer. We first constructed DOX-resistant breast cancer cells (MCF-7/DOX and MDA-MB-468/DOX). The 50% inhibiting concentration of MCF-7/DOX and MDA-MB-468/DOX cells was significantly higher than that of their parental breast cancer cells, MCF-7 and MDA-MB-46. MCF-7/DOX and MDA-MB-468/DOX cells also exhibited an up-regulation of drug resistance-related protein MDR1. Compared with MCF-7 and MDA-MB-46 cells, hsa_circ_0092276 was highly expressed in MCF-7/DOX and MDA-MB-468/DOX cells. Hsa_circ_0092276 overexpression enhanced proliferation and the expression of LC3-II/LC3-I and Beclin-1, and repressed apoptosis of breast cancer cells. The effect of hsa_circ_0092276 up-regulation on breast cancer cells was abolished by 3-methyladenine (autophagy inhibitor). Hsa_circ_0092276 modulated autophagy-related gene 7 (ATG7) expression via sponging miR-384. Hsa_circ_0092276 up-regulation promoted autophagy and proliferation, and repressed apoptosis of breast cancer cells, which was abolished by miR-384 overexpression or ATG7 knockdown. In addition, LV-circ_0092276 transfected MCF-7 cell transplantation promoted autophagy and tumor growth of breast cancer in mice. In conclusion, our data demonstrate that hsa_circ_0092276 promotes autophagy and DOX resistance in breast cancer by regulating miR-348/ATG7 axis. Thus, this article highlights a novel competing endogenous RNA circuitry involved in DOX resistance in breast cancer.     

Combination of integrin siRNA and irradiation for breast cancer therapy

Up-regulation of integrin alpha(v)beta(3) has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin alpha(v)beta(3) in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin alpha(v) siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin alpha(v)beta(3)-positive). Integrin alpha(v)beta(3)-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrin alpha(v)beta(3) expression in MDA-MB-435 cells and integrin alpha(v) siRNA can effectively reduce both alpha(v) and alpha(v)beta(3) integrin expression, leading to increased radiosensitivity. Integrin alpha(v) siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin alpha(v)beta(3)-positive cancer types.     

Radiosynthesis,biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel ¹⁸F-labeled 5-resorcinolic triazolone derivative (1, [¹⁸F]PTP-Ganetespib) based on ganetespib. [¹⁸F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [¹⁸F]PEG-N₃ (11) with 37.3?±?5.11% of radiochemical yield and 99.7?±?0.09% of radiochemical purity. [¹⁸F]PTP-Ganetespib showed proper LogP (0.96?±?0.06) and good stability in human serum over 97% for 2?h. [¹⁸F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [¹⁸F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [¹⁸F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [¹⁸F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [¹⁸F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [¹⁸F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

Regulation by estrogen through the 5'-flanking region of the transforming growth factor alpha gene.

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Novel Triterpenoid Isolated from the Root Bark of Ailanthus excelsa Roxb (Tree of Heaven), AECHL-1 as a Potential Anti-Cancer Agent

Background

We report here the isolation and characterization of a new compound Ailanthus excelsa chloroform extract-1 (AECHL-1) (C₂₉H₃₆O₁₀; molecular weight 543.8) from the root bark of Ailanthus excelsa Roxb. The compound possesses anti-cancer activity against a variety of cancer cell lines of different origin.

Principal Findings

AECHL-1 treatment for 12 to 48 hr inhibited cell proliferation and induced death in B16F10, MDA-MB-231, MCF-7, and PC3 cells with minimum growth inhibition in normal HEK 293. The antitumor effect of AECHL-1 was comparable with that of the conventional antitumor drugs paclitaxel and cisplatin. AECHL-1-induced growth inhibition was associated with S/G₂-M arrests in MDA-MB-231, MCF-7, and PC3 cells and a G₁ arrest in B16F10 cells. We observed microtubule disruption in MCF-7 cells treated with AECHL-1 in vitro. Compared with control, subcutaneous injection of AECHL-1 to the sites of tumor of mouse melanoma B16F10 implanted in C57BL/6 mice and human breast cancer MCF-7 cells in athymic nude mice resulted in significant decrease in tumor volume. In B16F10 tumors, AECHL-1 at 50 µg/mouse/day dose for 15 days resulted in increased expression of tumor suppressor proteins P53/p21, reduction in the expression of the oncogene c-Myc, and downregulation of cyclin D1 and cdk4. Additionally, AECHL-1 treatment resulted in the phosphorylation of p53 at serine 15 in B16F10 tumors, which seems to exhibit p53-dependent growth inhibitory responses.

Conclusions

The present data demonstrate the activity of a triterpenoid AECHL-1 which possess a broad spectrum of activity against cancer cells. We propose here that AECHL-1 is a futuristic anti-cancer drug whose therapeutic potential needs to be widely explored for chemotherapy against cancer.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Sensitivity and insensitivity of breast cancer to tamoxifen

Tamoxifen is the endocrine treatment of choice for breast cancer. In several laboratory models in vivo tamoxifen is a tumoristatic agent. When MCF-7 breast cancer cells are inoculated into athymic mice, palpable tumors do not grow unless the animals are treated with estrogen, and tamoxifen inhibits estrogen-stimulated growth. If tamoxifen is stopped, tumors regrow. These results suggest that adjuvant tamoxifen therapy should involve long treatment periods (even lifetime) to prevent tumor recurrence. Unfortunately resistance to therapy and patient relapse inevitably occur, and such disease recurrence involving tamoxifen resistance is difficult to treat successfully. A laboratory model of endocrine therapy failure has been developed. When athymic mice with MCF-7 tumors are treated for 6–8 months with tamoxifen, several tumors grew and continued to grow in tamoxifen-treated mice. These estrogen receptor-positive tumors grow with either tamoxifen or estradiol. Tamoxifen-stimulated tumor growth has been observed in human endometrial tumors implanted into athymic animals. Growth of these tamoxifen-stimulated tumors can be inhibited with the pure antiestrogen ICI 164,384 upon withdrawal of tamoxifen. These data are discussed in terms of treatment strategies for tamoxifen-failed patients.     

Evaluation of a (99m)Tc-labeled cyclic RGD tetramer for noninvasive imaging integrin alpha(v)beta3-positive breast cancer

Integrin alphavbeta3 plays a critical role in tumor angiogenesis and metastasis. Radiolabeled RGD peptides that are integrin alphavbeta3-specific are very useful for noninvasive imaging of integrin expression in rapidly growing and metastatic tumors. In this study, we determined the binding affinity of E{E[c(RGDfK)]2}2 (tetramer) and its 6-hydrazinonicotinamide conjugate (HYNIC-tetramer) against the binding of 125I-echistatin to the integrin alphavbeta3-positive MDA-MB-435 breast cancer cells. The athymic nude mice bearing MDA-MB-435 xenografts were used to evaluate the potential of ternary ligand complex [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate) as a new radiotracer for imaging breast cancer integrin alphavbeta3 expression by single photon emission computed tomography (SPECT). It was found that the binding affinity of tetramer (IC50 = 51 +/- 11 nM) was slightly higher than that of its dimeric analogue (IC50 = 78 +/- 27 nM) and is comparable to that of the HYNIC-tetramer conjugate (IC50 = 55 +/- 11 nM) within the experimental error. Biodistribution data showed that [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had a rapid blood clearance (4.61 +/- 0.81 %ID/g at 5 min postinjection (p.i.) and 0.56 +/- 0.12 %ID/g at 120 min p.i.) and was excreted mainly via the renal route. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had high tumor uptake with a long tumor retention (5.60 +/- 0.87 %ID/g and 7.30 +/- 1.32 %ID/g at 5 and 120 min p.i., respectively). The integrin alphavbeta3-specificity was demonstrated by co-injection of excess E[c(RGDfK)]2, which resulted in a significant reduction in tumor uptake of the radiotracer. The metabolic stability of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was determined by analyzing urine and feces samples from the tumor-bearing mice at 120 min p.i. In the urine, about 20% of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] remained intact while only approximately 15% metabolized species was detected in feces. SPECT images displayed significant radiotracer localization in tumor with good contrast as early as 1 h p.i. The high tumor uptake and fast renal excretion make [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] a promising radiotracer for noninvasive imaging of the integrin alphavbeta3-positive tumors by SPECT.