Effective treatment of experimental DU-145 prostate cancers with targeted cytotoxic somatostatin analog AN-238.

We evaluated the effectiveness of targeted cytotoxic analog of somatostatin (SST) AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked covalently to SST octapeptide carrier RC-121 in DU-145 human androgen-independent prostate cancers xenografted into nude mice. We also investigated the expression of mRNAs for SST receptor subtypes 2A and 5 (sst2A and sst5) in DU-145 tumors. After 8 weeks of treatment, AN-238 practically arrested the proliferation of DU-145 cancers. The tumor volume in nude mice that received 4 injections of AN-238 at the dose of 150 nmol/kg was 63.4+/-6.7 mm3, nearly 4 times smaller than that in controls which measured 249.1+/-36.3 mm3 (p<0.001). Treatment with AN-238 lowered tumor weight by 68% (p<0.01) compared with the control group and extended the tumor volume doubling time to 184.1+/-69.4 days, versus 32.1+/-6.6 days in controls (p<0.05). No toxicity-related deaths occurred during treatment with AN-238. Cytotoxic radical AN-201 administered alone or in an unconjugated mixture with carrier RC-121 inhibited the growth of DU-145 tumors only after the third and fourth injection and was toxic. The expression of mRNA for sst2A and sst5 was detected in all specimens of control DU-145 tumors and in tumors treated with AN-238. The present study demonstrates the high efficacy of SST-receptor-targeted chemotherapy in a model of human androgen-independent prostatic carcinoma.     

Inhibition of metastatic renal cell carcinomas expressing somatostatin receptors by a targeted cytotoxic analogue of somatostatin AN-238

The effectiveness of chemotherapy targeted to somatostatin (SST) receptors based on cytotoxic SST analogue AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to SST carrier octapeptide, was investigated in human renal cell carcinomas (RCCs). SST receptors, which showed high-affinity binding for AN-238, were found in the SW-839 RCC line with sst2A subtype and in the 786-0 RCC line, which expressed the sst5 subtype. CAKI-1 RCC, which does not express sst2A or sst5, was used as a negative control for testing the specificity of SST receptor targeting. Using microsatellite analysis, AN-238 was shown to selectively inhibit the proliferation of 786-0 line, but not the CAKI-1 RCC line in vitro. The effects of three i.v. injections of 150 nmol/kg of AN-238 or AN-201, given on days 1, 8, and 21, were evaluated in groups of nude mice bearing s.c. xenografts of SW-839 and 786-0 RCC. After 5 weeks, the volumes of SW-839 and 786-0 RCC tumors were decreased by 67.2 (P < 0.05) and 78.3% (P < 0.01), respectively, whereas AN-201 had no significant effect on tumor growth. The inhibition of SST receptor-negative CAKI-1 tumors by AN-238 was only marginal. To investigate the efficacy of SST receptor-targeted chemotherapy in metastatic RCC, three i.v. injections of AN-238 or AN-201 at 150 nmol/kg were given at biweekly intervals to nude mice implanted with 786-0 tumors under the renal capsule. After 6 weeks, the weight of orthotopic tumors treated with AN-238 (55.3 +/- 44.3 mg) was significantly lower (87% reduction; P < 0.001) than that in the control group (414.2 +/- 41.0 mg) or in animals given AN-201 (270.2 +/- 603 mg; P < 0.05). Five of six animals (83%), both in the control and the AN-201 group, developed metastases to lymph nodes, but only one of seven mice (14%) given AN-238 showed lymphatic spread. Lung metastases were found in 83% of controls and 50% of AN-201 treated animals, but none occurred in mice treated with AN-238. This study demonstrates that targeted cytotoxic SST analogue AN-238 provides an effective therapy for chemoresistant neoplasms such as RCC. Because most clinical RCCs express SST receptors, this treatment modality might be beneficial to patients with metastatic disease.     

Glycosphingolipid composition of MDA-MB-231 and MCF-7 human breast cancer cell lines

Much evidence has shown that glycosphingolipids are involved in cellular recognition, regulation of cell growth, and metastasis. In the present study, the major glycosphingolipids of two widely studied human breast cancer cell lines were examined. The MCF-7 cell line has functional estrogen and EGF receptors, is dependent on estrogen and EGF for growth, and is uninvasive, while MDA-MB-231 cells are a model for more aggressive, hormone-independent breast cancer. There was twice as much neutral glycolipid in MCF-7 cells as in MDA-MB-231 cells. The major neutral glycolipids in MDA-MB-231 cells were identified as CTH and globoside. MCF-7 cells also contained as the major neutral glycolipids CTH as well as globoside and two other glycolipids which were tentatively identified as galactosylgloboside and fucosylgalactosylgloboside by exoglycosidase treatments. Conversely, the ganglioside content was four fold higher in MDA-MB-231 cells compared to MCF-7 cells. The abundant gangliosides in both cell lines were GM3, GM2, GM1, and GD1a. A minor monosialoganglioside was detected in MDA-MB-231 cells. The striking 18 fold greater amount of GM3 in MDA-MB-231 cells may have important implications because GM3 has been suggested to be involved in regulation of growth factor functions. In agreement, insertion of ganglioside GM3 into the plasma membrane of MCF-7 cells blocked the growth stimulatory effect of EGF.     

Targeted therapy with a cytotoxic somatostatin analog, AN-238, inhibits growth of human experimental endometrial carcinomas expressing multidrug resistance protein MDR-1

BACKGROUND: Chemoresistance mediated by membrane transporters such as multidrug resistance (MDR-1) glycoprotein remains a challenge in the chemotherapy treatment of advanced or recurrent endometrial carcinoma. Targeted chemotherapy might overcome this resistance. The cytotoxic somatostatin (SST) analog, AN-238, consists of a superactive derivative of doxorubicin (DOX), 2-pyrrolino-DOX (AN-201), linked to the SST analog carrier, RC-121. This conjugate binds strongly to SST receptor subtypes (sst) 2a (sst2(a)) and 5 (sst(5)) and can be targeted to tumors that express these receptors. METHODS: The presence of sst2(a) and sst(5) was determined in 3 human endometrial carcinoma cell lines (HEC-1A, RL-95-2, and AN3CA). Nude mice bearing xenografts of these cancers were treated with AN-238 and its radical, AN-201. The antitumor effects and toxicity were compared. The authors studied the effects of AN-238 and AN-201 on the expression levels of MDR-1, multidrug resistance related protein (MRP-1), and breast carcinoma resistance protein (BCRP) by real-time polymerase chain reaction. RESULTS: The authors demonstrated the presence of mRNA and receptor protein for sst(2a) and sst(5) on HEC-1A, RL-95-2, and AN3CA tumors. AN-238 significantly (P < 0.05) inhibited the growth of these tumors, whereas AN-201 had no effect. Blockade of SST receptors nullified the effects of AN-238. In all 3 endometrial carcinoma lines, AN-238 caused a weaker induction of MDR-1 than AN-201. No major induction of MRP-1 and BCRP occurred after treatment with AN-238 or AN-201. CONCLUSIONS: Targeted chemotherapy with the cytotoxic SST analog, AN-238, inhibited powerfully the growth of endometrial carcinoma, which express SST receptors, regardless of their expression level of MDR-1.     

Administration of a targeted cytotoxic analog of luteinizing hormone-releasing hormone inhibits growth of estrogen-independent MDA-MB-231 human breast cancers in nude mice

Receptor targeted chemotherapy is less toxic and more effective than conventional chemotherapy. Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in about 50% of human breast cancers. Highly potent cytotoxic radical 2-pyrrolinodoxorubicin (AN-201) was linked to the agonistic analog [D-Lys⁶]LH-RH to form cytotoxic LH-RH analog AN-207. We evaluated whether AN-207 could be targeted to the hormone-independent MDA-MB-231 human breast cancers. Nude mice bearing MDA-MB-231 tumors were injected i.v. with 250 nmol/kg doses of cytotoxic radical AN-201, cytotoxic LH-RH analog AN-207, the unconjugated mixture of AN-201 and carrier [D-Lys⁶]LH-RH, [D-Lys⁶]LH-RH alone and vehicle (control). The growth of MDA-MB-231 tumors in animals given a single dose of AN-207 was inhibited significantly (p=0.01) for 3 weeks after injection, whereas tumors in all the other groups grew steadily. All cytotoxic compounds produced leukopenia, but the strongest lymphocyte suppression was caused by cytotoxic radical AN-201. Three weeks after treatment, the presence of mRNA for LH-RH receptors was demonstrated by RT-PCR in all the groups and radioreceptor assays demonstrated high-affinity binding sites for LH-RH on tumor cell membranes of control animals and those treated with AN-201, the carrier peptide alone or in combination with AN-201. At this time point binding assays did not reveal the expression of membrane proteins in tumors treated with AN-207, but 60 days after administration of AN-207, high affinity LH-RH binding sites were found again in MDA-MB-231 tumors. These results indicate that cytotoxic LH-RH analog AN-207 could be utilized for receptor targeted chemotherapy of breast cancers expressing receptors for LH-RH.     

曲格列酮对人乳腺癌MDA-MB-231和MCF-7细胞株增殖影响的研究

目的:探讨过氧化物酶体增殖物激活受体γ(PPARγ)激动剂曲格列酮(TRG)对体外培养雌激素受体(ER)阴性乳腺癌MDA-MB-231细胞及ER+乳腺癌MCF-7细胞增殖的影响。方法:以不同浓度的TRG(0～50μmol/mL)分别处理体外培养的MDA-MB-231细胞和MCF-7细胞48h,MTT法检测细胞存活率;流式细胞术(FCM)检测细胞周期分布;蛋白质印迹法检测PPARγ和ER-α的表达。结果:MTT结果示TRG浓度≥5μmol/mL时MDA-MB-231细胞(P值分别为0.006、0.006、0.000和0.000)和MCF-7细胞(P值分别为0.007、0.006、0.004和0.001)的存活率降低。FCM检测结果表明,经TRG作用后肿瘤细胞主要集中在G1期。以上2种检测结果显示,MDA-MB-231细胞对TRG的敏感程度较MCF-7细胞高;蛋白质印迹法检测结果表明,TRG处理后的MDA-MB-231细胞中PPARγ蛋白的表达水平随药物浓度增加逐渐升高(相对强度分别为2.045、2.600和3.038),MCF-7细胞的ER-α表达水平则随药物浓度增加逐渐降低(相对强度分别为1.164、1.130和0.680)。结论:TRG对ER+和ER-乳腺癌细胞均有抑制作用,且ER-乳腺癌细胞对TRG更敏感。     

Effect of transforming growth factor-beta1 on oestrogen metabolism in MCF-7 and MDA-MB-231 breast cancer cell lines.

Transforming growth factor-beta (TGF-beta) has been shown to inhibit the growth of mammary epithelial cells and may play a protective role in mammary carcinogenesis. In contrast, oestrogens promote the development of breast cancer. Oestrone sulphate (E1S) is a huge reservoir of active oestrogens in the breast being converted to the weak oestrogen, oestrone (E1), by oestrone sulphatase. E1 is reversibly converted by oestradiol-17beta hydroxysteroid dehydrogenase to the potent oestrogen, oestradiol (E2). The aim of this study was to assess the effect of the TGF-beta1 isoform on growth and oestrogen metabolism in the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 human breast cancer cell lines. The results showed that TGF-beta1 significantly inhibited cell growth and stimulated the conversion of E1S to E1 and E1 to E2 in the MCF-7 cell line. In the MDA-MB-231 cell line TGF-beta1 significantly stimulated cell growth and inhibited the interconversions between E1 and E2. In conclusion, the growth inhibitory effect of TGF-beta1 on the MCF-7 cell line would appear to confer a protective effect in breast cancer. However, its ability to increase the amount of E2 would increase the risk of breast cancer. Which of these effects predominates in vivo remains to be explored. The growth stimulatory effect of TGF-beta1 on the MDA-MB-231 cell line probably acts through a mechanism independent of the effect of TGF-beta1 on oestrogen concentrations since this cell line is hormone unresponsive.     

Comparative studies on the polyamine metabolism and DFMO treatment of MCF-7 and MDA-MB-231 breast cancer cell lines and xenografts.

In order to characterize the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and xenografts, their growth kinetic parameters and some biochemical characteristics concerning the receptor status and polyamine metabolism were determined and compared. The doubling times calculated from the growth curves showed higher proliferation rate of MDA-MB-231 cells, both in culture (21 hours) and in xenograft (9.7 days), in comparison to the MCF-7 cells which had values of 32 hours and 11.6 days, respectively. Growth-dependent changes observed in the intracellular putrescine, spermidine and spermine concentrations indicated a higher activity of polyamine metabolism in the MDA-MB-231 cells and xenograft as well. However, biosynthetic key-enzyme ornithine decarboxylase activity (ODC, EC 4.1.1.17) showed neither characteristic differences between the two types of breast cancer, nor consistent relationship with their proliferation rate. Metabolic alterations of the MCF-7 and MDA-MB-231 cell lines grown in vitro were also reflected in the polyamine composition of their culture medium. Independently of their receptor status, both types of breast cancer were responsive to difluoromethylornithine (DFMO) treatment. DFMO inhibited the ODC activity totally and depleted the cellular polyamine levels. MCF-7 cells in culture were more sensitive to the antitumoral effect of DFMO than the MDA-MB-231 line, while the rate of growth inhibition did not differ significantly in the xenografts. The present results provided further evidence on the different polyamine metabolism of ER-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells in vitro and in vivo, suggesting a correlation of hormonal modulation with polyamines as a determinant group of biological response modifiers.     

miRNA-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的增殖与侵袭

目的:研究miRNA-34a(miR-34a)对乳腺癌细胞MCF-7、MDA-MB-231的生物调控作用。方法:采用定量PCR检测人乳腺上皮细胞MCF-10A,乳腺癌细胞株MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a的表达水平。通过miR-34a mimics分别上调MCF-7、MDA-MB-231细胞中miR-34a的表达水平,MTT和Transwell检测肿瘤细胞增殖能力、侵袭力等生物学行为的变化。结果:乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a处于低表达水平。通过miR-34a mimics上调MCF-7、MDA-MB-231细胞中miR-34a的表达后,细胞的增殖能力被miR-34a抑制（P＜0.05）,miR-34a对细胞侵袭有显著抑制作用（P＜0.05）。结论:miR－34a在乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453及Hs578T中低表达,miR-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的细胞增殖和侵袭能力。     

Morphological alterations induced by prostaglandins E1, F2 alpha and A1 in MDA-MB-231 and MCF-7 human breast cancer cell lines

Monolayer cultures of MDA-MB-231 and MCF-7 human breast tumor cell lines were treated with prostaglandins PGE1, PGF2 alpha and PGA1 in a concentration range of 10(-12)-10(-4) M and studied at ultrastructural level. Electron microscopic examinations of both cell lines revealed that PGE1, PGF2 alpha and PGA1 induced morphological changes at concentrations above 10(-8) M. In both the small and large MDA-MB-231 cells, deformation of mitochondrial cristae, increased density of mitochondrial matrix and accumulation of lysosomal-like vesicles were observed. In the nuclei morphological, modifications included, the presence of nuclear bodies, occasional nuclear inclusions, nucleolar budding and the disappearance of the nucleolar granular components. In MCF-7 cells, disorganization of mitochondrial cristae and an increase in their matrix density were also observed. At nuclear level, little or no morphological alterations were observed. The results also indicated that the plasma membranes of both cell lines were the most sensitive organelles to PGs action as in many cells their microvilli were either shortened and spherical in shape or absent.     

Colony-stimulating factor-1 antibody reverses chemoresistance in human MCF-7 breast cancer xenografts

Overexpression of colony-stimulating factor-1 (CSF-1) and its receptor in breast cancer is correlated with poor prognosis. Based on the hypothesis that blockade of CSF-1 would be beneficial in breast cancer treatment, we developed a murinized, polyethylene glycol-linked antigen-binding fragment (Fab) against mouse (host) CSF-1 (anti-CSF-1 Fab). Mice bearing human, chemoresistant MCF-7 breast cancer xenografts were treated with combination chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil; cycled twice i.p.), anti-CSF-1 Fab (i.p., cycled every 3 days for 14 days), combined CMF and anti-CSF-1 Fab, or with Ringer's solution as a control. Anti-CSF-1 Fab alone suppressed tissue CSF-1 and retarded tumor growth by 40%. Importantly, in combination with CMF, anti-CSF-1 Fab reversed chemoresistance of MCF-7 xenografts, suppressing tumor development by 56%, down-regulating expression of the chemoresistance genes breast cancer-related protein, multidrug resistance gene 1, and glucosylceramide synthase, and prolonging survival significantly. Combined treatment also reduced angiogenesis and macrophage recruitment and down-regulated tumor matrix metalloproteinase-2 (MMP-2) and MMP-12 expression. These studies support the paradigm of CSF-1 blockade in the treatment of solid tumors and show that anti-CSF-1 antibodies are potential therapeutic agents for the treatment of mammary cancer.