Novel antitumor effect of estradiol in athymic mice injected with a T47D breast cancer cell line overexpressing protein kinase Calpha.

PURPOSE: Resistance to tamoxifen (TAM) represents a significant challenge to the management of breast cancer. We previously reported that the estrogen receptor (ER)-negative hormone-independent T47D:C42 cell line has both elevated protein kinase Calpha (PKCalpha) protein expression and basal activator protein-1 activity compared with the parental ER+ (hormone-dependent) T47D:A18 cell line. Stable transfection of PKCalpha to the T47D:A18 breast cancer cell line results in increased basal activator protein-1 activity, reduced ER function, increased proliferation rate, and hormone-independent growth (Tonetti et al., Br. J. Cancer, 83: 782-791, 2000). In this report, we further characterize the role of PKCalpha overexpression in vivo to elucidate a possible molecular mechanism of tamoxifen resistance. EXPERIMENTAL DESIGN: To determine whether the T47D:A18/PKCalpha cell line would produce hormone-independent tumors in athymic mice, we injected T47D:A18, T47D:A18/neo, or the T47D:A18/PKCalpha20 cell clones bilaterally into the mammary fat pads of athymic mice. Tumor growth was evaluated following treatment with estradiol (E2), TAM, and the pure antiestrogen, ICI 182,780. RESULTS: Mice receiving either T47D:A18 or T47D:A18/neo cells produced tumors that grew in response to E2 treatment, whereas the untreated control and TAM-treated groups showed no tumor growth. Interestingly, mice receiving the T47D:A18/PKCalpha20 clone produced tumors in both the control and TAM groups, whereas tumor growth was inhibited in mice treated with E2. PKCalpha was also overexpressed in an MCF-7 tumor model that also exhibited TAM-stimulated and E2-induced regression. CONCLUSIONS: These results suggest that overexpression of PKCalpha in breast tumors results in hormone-independent tumor growth that cannot be inhibited by TAM treatment. Furthermore, the finding that E2 has an antitumor effect on breast tumors overexpressing PKCalpha is a novel observation that may have important therapeutic implications.     

Overexpression of PKCalpha is required to impart estradiol inhibition and tamoxifen-resistance in a T47D human breast cancer tumor model

We previously reported that stable overexpression of protein kinase C alpha (PKCalpha) in hormone responsive T47D:A18 breast cancer cells produces a hormone-independent/tamoxifen (TAM)-resistant and 17beta-estradiol (E2)-inhibitory phenotype in vivo. Furthermore, overexpression of PKCalpha in T47D:A18 cells also results in cross-upregulation of PKCs beta and delta. In this study, we further characterized the contribution of PKC isozymes alpha, beta and delta to this complex phenotype. To determine whether downregulation of PKCalpha is sufficient to restore the hormone-dependent phenotype in T47D:A18/PKCalpha cells, PKCalpha was selectively knocked down using short hairpin RNA (shRNA). To determine the contribution of PKCbeta or delta to the hormone-independent/TAM-resistant and E2-inhibitory phenotype, stable T47D:A18/PKCbeta and T47D:A18/PKCdelta clones were established. Downregulation of PKCalpha by shRNA in T47D:A18/PKCalpha20 cells also resulted in reduced PKCbeta protein expression in vitro. Tumors established from a T47D:A18/PKCalpha/shRNA stable clone exhibit 50% reduction of PKCalpha protein without concomitant reduction in PKCbeta, and exhibit partial reversal of the TAM-resistant and E2-inhibitory phenotype in vivo. Furthermore, stable overexpression of neither PKCbeta nor PKCdelta in T47D:A18 cells are sufficient to produce hormone-independent growth in vitro or in vivo, nor TAM-resistant and E2-inhibited growth in vivo. Taken together, these results suggest that PKCalpha is required to impart the TAM-resistant and E2-inhibitory phenotype in vivo.     

Methylation of CpG island is not a ubiquitous mechanism for the loss of oestrogen receptor in breast cancer cells.

Methylation has been shown to play an important role in the down-regulation of oestrogen receptors (ER) in breast cancer cells. One critical question that remains unclear is whether methylation can account for the loss of ER expression in cells derived from an ER-positive cell line. This laboratory has established an in vitro cell system using long-term growth of human ER-positive breast cancer cell line T47D in oestrogen-free medium. A clonal cell line, T47D:C4:2 (C4:2), has been characterized. Unlike T47D:A18 (A18), which is a T47D line maintained in oestrogen medium, C4:2 has lost the expression of ER and hormone responsiveness. DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis results confirmed that C4:2 was of the same lineage as A18. These cell lines provide an invaluable system to study the mechanism of ER expression and regulatory pathways leading to hormone-independent growth. The results here clearly demonstrate that the ER CpG island in C4:2 cells remains unmethylated. The loss of ER in the cell line must be due to mechanisms other than methylation. We also evaluated the ER CpG island in the MDA-MB-231:10A (10A) cell line, which is a clone from the MDA-MB-231 line obtained from ATCC and the DNA from the MDA-MB-231 cell line used in the original report. Unlike the cell line from the report, which showed a full methylation pattern in the island, the 10A line only showed a partial methylation pattern in the CpG island. Possible mechanisms pertaining to the heterogeneous methylation pattern of the ER CpG island in the breast cancer cells are discussed.     

Estrone sulfatase activity and effect of antiestrogens on transformation of estrone sulfate in hormone-dependent vs. independent human breast cancer cell lines

Summary The effect of the anti-estrogens ICI 164,384 and tamoxifen on the estradiol (E₂) concentration after incubation of estrone sulfate (E₁-S) with different hormone-dependent (MCF-7 and T-47D) and hormone-independent (MDA-MD-231 and MDA-MB-436) mammary cancer cells, as well as the estrone sulfatase activity in these various cell lines, are presented. The anti-estrogen ICI 164,384 decreased very significantly the concentration of E₂ after incubation of E₁-S with MCF-7 (control, mean ±SE: 100±24 pg/mg DNA; + ICI 164,384 [10^–6M]: 7±2 pg/mg DNA). This effect was much more intense than with tamoxifen. A similar effect was observed with T-47D cells. However, no significant effect was observed in the hormone-independent cells. In the intact cell, estrone sulfatase activity was very intense in the hormone-dependent cells, but very small in the hormone-independent cells. However, this activity became very strong after homogenization in the hormone-independent cells. The data suggest that estrone sulfate can play an important role on the bioavailability of E₂ in hormone-dependent breast cancer, and that understanding the control of estrone sulfatase activity can open new knowledge of the estrogen responses and new possibilities of therapeutic application in breast cancer.     

Differential distribution of protein phosphatase 2A in human breast carcinoma cell lines and its relation to estrogen receptor status.

Protein phosphatase 2A (PP2A) acts as a growth suppressor and is negatively influenced by oncogenic signals. We determined its activity in various human breast carcinoma (HBC) cell types to understand its relationship to estrogen receptor (ER) expression as well as to the distribution of protein kinase C (PKC), an opposing enzyme. PP2A activity was measured using a preferred substrate, histone H1 phosphorylated by PKC. PP2A activity was higher in both the soluble and nuclear fractions of ER-positive cell lines (MCF-7, T47D and ZR-75-1) than in the ER-negative cell lines (MDA-MB-231, Hs578T and BT-20). PP2A multiple forms (2A0, 2A1, 2A2), separated by DEAE-cellulose chromatography and immunoblot analysis of PP2A catalytic subunit, also showed similar differences in these two HBC cell types. In all cases, PP2A distribution was inversely correlated with the PKC activity profile. Moreover, PP2A activity in MCF-7 cells maintained in estrogen-depleted medium was low. Nonetheless, it was induced by a prolonged treatment with 17beta-estradiol, this induction being blocked by the antiestrogens, tamoxifen and ICI-182,780. Studies in both MCF-7 transfectants stably overexpressing ras and MDA-MB-231 transfectants stably expressing ER, suggested that a low PP2A distribution in ER-negative HBC cell types may be related to tumor progression rather than the loss of ER. Conceivably, the presence of high PP2A along with low PKC in ER-positive HBC cell types may be related to the restricted cell growth associated with the retention of a certain degree of differentiation or hormonal control. Conversely, the presence of low PP2A along with high PKC in ER-negative cell types may be related to hormone-independent enhanced cell growth.     

miR-221增强乳腺癌细胞对多西他赛的耐药性

目的:探究miR-221在人乳腺癌细胞T47D和MCF-7多西他赛(docetaxel)耐药性中的作用及机制。方法:qPCR检测多西他赛处理乳腺癌细胞T47D和MCF-7不同时间点,细胞中miR-221含量的变化;qPCR和Western blot检测miR-221 mimics的转染效率。用阴性对照（NC）或miR-221 mimics转染细胞,不同浓度的多西他赛刺激细胞72小时后,MTT法检测细胞对多西他赛的耐药性;PI和Annexin V双染法检测miR-221 过表达对T47D和MCF-7细胞凋亡的影响;qPCR和Western blot检测靶蛋白p27的表达。结果:在T47D和MCD-7中多西他赛刺激明显促进miR-221的表达量升高;miR-221在细胞内可以发挥生物学效应,降低靶蛋白p27的表达;且过表达miR-221明显增强T47D和MCF-7对多西他赛的耐药性,降低其凋亡率。结论:miR-221过表达可明显增强T47D和MCF-7细胞对多西他赛的耐药性。     

Ribozyme-mediated down-regulation of ErbB-4 in estrogen receptor-positive breast cancer cells inhibits proliferation both in vitro and in vivo.

ErbB-4 is a recently discovered member of the class I receptor tyrosine kinase family (ErbB). Little is known about its expression and its importance in human malignancy. To delineate the biological function of ErbB-4 receptors in breast cancer, we used a hammerhead ribozyme strategy to achieve down-regulation of ErbB-4 receptors in various breast cancer cell lines. We observed that down-regulation of ErbB-4 in estrogen receptor-positive (ER+) human breast cancer cell lines (MCF-7 and T47D), which express relatively high levels of ErbB-4, significantly inhibited colony formation. No effects were observed in estrogen receptor-negative (ER-) MDA-MB-453 cells, which express low levels of endogenous ErbB4 and high levels of ErbB-2 and ErbB-3. This occurred despite the fact that fluorescence-activated cell sorter analysis of these latter cells revealed that the expression of the ErbB-4 receptor was completely abrogated by ribozyme treatment. Furthermore, down-regulation of ErbB-4 in T47D and MCF-7 cells significantly inhibited tumor formation in athymic nude mice (P < 0.03 and P < 0.001, respectively). In addition, NRG-stimulated phosphorylation of ErbB-4- and NRG-induced colony formation was significantly reduced in ribozyme-transfected T47D cells. These data provide the first evidence that elevation of ErbB-4 expression plays a role in the proliferation of some ER+ human breast cancer cell lines (T47D and MCF-7) that express high levels of ErbB-4. We have also investigated the expression of ErbB-4 in human primary breast carcinoma specimens, using immunohistochemical staining with an anti-ErbB-4 monoclonal antibody. ErbB-4 expression was found in 60% of the 50 primary breast tumors examined, and high intense immunoreactivity of ErbB-4 was detected in 18% of these primary breast tumors. ErbB-4 receptor expression appeared to correlate with ER+ primary breast tumors. A similar correlation was also observed in the human breast cancer cell lines. These results provide a better understanding of the biological significance of ErbB-4 receptor in breast cancer. Our data suggest that elevation of the ErbB-4 receptor plays a role in ER+ breast cancer cell proliferation. Moreover, ribozyme technology provides a useful tool to delineate the role of a particular gene product.     

Reduction of epidermal growth factor binding in human breast cancer cell lines by an alkyl-lysophospholipid

The effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), an alkyl lysophospholipid derivative, on the binding of epidermal growth factor (EGF) to human breast cancer cell lines (MCF-7, ZR-75-1, and BT-20), the human epidermoid cancer cell line (A431), and the rat fibroblast cell line (NIH3T3) were investigated. The addition of 10 micrograms/ml ET-18-OCH3 to the growth medium reduced the binding of EGF to hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) and A431 but did not change that to the hormone-independent breast cancer cell line (BT-20). ET-18-OCH3 suppressed the EGF-binding prior to the onset of its inhibitory action on cell growth in MCF-7 and ZR-75-1. Scatchard plot analysis demonstrated that ET-18-OCH3 reduced the number of EGF receptor sites without affecting the affinity of EGF receptors in MCF-7 and ZR-75-1. Both EGF-binding and cell growth in NIH3T3 were not changed by treatment with 10 micrograms/ml ET-18-OCH3. These results suggest that ET-18-OCH3 inhibits the growth of hormone-dependent breast cancer cell lines (MCF-7 and ZR-75-1) by reducing the binding capacity of EGF receptors and consequently by disturbing the transfer of a variety of growth-promoting signals.     

Hormonal carcinogenesis in breast cancer: cellular and molecular studies of malignant progression

Summary We have established and characterized a series of variant cell lines in which to identify the critical factors associated with E2-induced malignant progression, and the acquisition to tamoxifen resistance in human breast cancer. Sublines of the hormone-dependent MCF-7 cell line (MCF7/MIII and MCF7/LCC1) form stable, invasive, estrogen independent tumors in the mammary fat pads of ovariectomized athymic nude mice. These cells retain expression of both estrogen (ER) and progesterone receptors (PGR), but retain sensitivity to each of the major structural classes of antiestrogens. The tamoxifen-resistant MCF7/LCC2 cells retain sensitivity to the inhibitory effects of the steroidal antiestrogen ICI 182780. By comparing the parental hormone-dependent and variant hormone-independent cells, we have demonstrated an altered expression of some estrogen regulated genes (PGR, pS2, cathepsin D) in the hormone-independent variants. Other genes remain normally estrogen regulated (ER, laminin receptor, EGF-receptor). These data strongly implicate the altered regulation of a specific subset or network of estrogen regulated genes in the malignant progression of human breast cancer. Some of the primary response genes in this network may exhibit dose-response and induction kinetics similar to pS2, which is constitutively upregulated in the MCF7/MIII, MCF7/LCC1 and MCF7/LCC2 cells.     

Detection of an elevated HER2 expression in MCF-7 breast cancer cells overexpressing estrogen receptor beta1

The estrogen receptor (ER) expression and HER2 amplification are important factors in determining the prognosis and therapy of breast cancer. Interactions between the two signaling pathways for example resulted in ERalpha-dependent regulation of HER2 expression in breast cancer cells. In this study, we investigated to what extent ERbeta is able to affect the HER2 expression. For this purpose, we analyzed HER2 levels in ERbeta1-overexpressing clones of the breast cancer cell lines MCF-7 and SK-BR-3 and of the ovarian cancer cell lines SK-OV-3 and OVCAR-3 by both RT-PCR and Western blot analysis. Treatment with ligand 17-beta estradiol diminished the HER2 expression in MCF-7 wild-type cells, an effect partially inhibited by treatment with 4-OH tamoxifen. MCF-7 breast cancer cells stably overexpressing ERbeta1 exhibited elevated >5-fold HER2 mRNA levels and elevated >3-fold HER2 protein levels even in the absence of estradiol. In contrast, ERbeta1 overexpression did not affect HER2 protein levels in the ERalpha-positive OVCAR-3 ovarian cancer cells and in the HER2 overexpressing, hormone-independent SK-BR-3 and SK-OV-3 cells. By demonstrating the elevated HER2 expression in a hormone-dependent breast cancer cell line overexpressing ERbeta1, our data suggest the presence of cross-talk between the two receptors. This is one of the molecular mechanisms underlying the significant ERbeta/HER2 co-expression observed in recent clinical studies.     

Biological response of the anti-estrogen ICI 164,384 in human hormone-dependent and hormone-independent mammary cancer cell lines

The biological response on proliferation and progesterone receptor (PR) of the anti-estrogen ICI 164,384 [N-n-butyl-N-methyl-11-(3,17 beta-dihydroxyestra-1,3,5(10-trien-7 alpha-yl))-undecanamide] was studied in different mammary cancer cell lines. In the hormone-dependent cancer cell lines (MCF-7 and T-47D) this anti-estrogen significantly decreased cell proliferation, but to reduce 50% of the growth in the MCF-7 cells a very low concentration (10(-9) M) is necessary. Similar effects in the T-47D cell are obtained with a dose of 100-1000 times (10(-6)-10(-7) M). The stimulatory effect in cell proliferation induced by estradiol is also inhibited by ICI 164,384 in both cell lines. This anti-estrogen has no effect on proliferation in the anti-estrogen resistant cell line LY-2, or in the hormone-independent cell line MDA-MB-436. Studies on thymidine incorporation correlate with the effect on cell proliferation. ICI 164,384 also blocks the stimulatory effect on progesterone receptor provoked by estradiol in MCF-7 cells and in T-47D cells which contain high concentration levels of progesterone receptor ICI 164,384 significantly decreases the PR concentrations in both the non-treated and estradiol-treated cells. It is concluded that ICI 164,384 is a full antagonist in the hormone-dependent breast cancer cells, but it has no effect in the anti-estrogen-resistant or in hormone-independent cell lines.     

干扰B7-H4表达通过下调E2F家族相关转录因子抑制乳腺癌细胞的增殖

目的：探讨干扰B7-H4表达对乳腺癌细胞增殖、凋亡、周期以及相关下游分子表达的影响。方法：利用脂质体转染技术分别将特异性靶向B7-H4的siRNA(siB7-H4)及其阴性对照（siNC）转染至对数生长期的乳腺癌T47D和MCF-7细胞,分别命名为T47D-siB7-H4、T47D-siNC、MCF-7-siB7-H4和MCF-7-siNC组。用qPCR法和WB法验证siRNA干扰效果及其对细胞周期分子cyclin D1表达的影响,CCK-8法和FCM分别检测干扰B7-H4表达对T47D和MCF-7细胞增殖、周期和凋亡的影响,qPCR法检测B7-H4干扰对E2F家族相关转录因子表达的影响。结果：成功构建干扰B7-H4表达的乳腺癌T47D和MCF-7细胞。与T47D-siNC和MCF-7-siNC组相比,T47D-siB7-H4和MCF-7-siB7-H4组细胞中B7-H4 mRNA和蛋白表达水平均显著降低、细胞增殖能力显著降低（均P<0.01）,并伴有G1/S期细胞周期阻滞以及cyclin D1表达下调（均P<0.01）,但细胞凋亡率差异无统计学意义（均P>0.05）...     

Loss of pigment epithelium-derived factor: a novel mechanism for the development of endocrine resistance in breast cancer

Introduction

Despite the benefits of endocrine therapies such as tamoxifen and aromatase inhibitors in treating estrogen receptor (ER) alpha-positive breast cancer, many tumors eventually become resistant. The molecular mechanisms governing resistance remain largely unknown. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that displays broad anti-tumor activity based on dual targeting of the tumor microenvironment (anti-angiogenic action) and the tumor cells (direct anti-tumor action). Recent studies indicate that PEDF expression is significantly reduced in several tumor types, including breast cancer, and that its reduction is associated with disease progression and poor patient outcome. In the current study, we investigated the role of PEDF in the development of endocrine resistance in breast cancer.

Methods

PEDF mRNA and protein levels were measured in several endocrine-resistant breast cancer cell lines including MCF-7:5C, MCF-7:2A, and BT474 and in endocrine-sensitive cell lines MCF-7, T47D, and ZR-75-1 using real-time PCR and western blot analyses. Tissue microarray analysis and immunohistochemistry were used to assess the PEDF protein level in tamoxifen-resistant breast tumors versus primary tumors. Lentiviruses were used to stably express PEDF in endocrine-resistant breast cancer cell lines to determine their sensitivity to tamoxifen following PEDF re-expression.

Results

We found that PEDF mRNA and protein levels were dramatically reduced in endocrine-resistant MCF-7:5C, MCF-7:2A, and BT474 breast cancer cells compared with endocrine-sensitive MCF-7, T47D, and ZR-75-1 cells, and that loss of PEDF was associated with enhanced expression of p^Ser167ERα and the receptor tyrosine kinase rearranged during transfection (RET). Importantly, we found that silencing endogenous PEDF in tamoxifen-sensitive MCF-7 and T47D breast cancer cells conferred tamoxifen resistance whereas re-expression of PEDF in endocrine-resistant MCF-7:5C and MCF-7:2A cells restored their sensitivity to tamoxifen in vitro and in vivo through suppression of RET. Lastly, tissue microarray studies revealed that PEDF protein was reduced in ~52.4% of recurrence tumors (31 out of 59 samples) and loss of PEDF was associated with disease progression and poor patient outcome.

Conclusion

Overall, these findings suggest that PEDF silencing might be a novel mechanism for the development of endocrine resistance in breast cancer and that PEDF expression might be a predictive marker of endocrine sensitivity.     

CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.     

Coordinated steroid hormone-dependent and independent expression of multiple kallikreins in breast cancer cell lines

The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well in the pathogenesis of endocrine-related cancers. Previous experiments have shown that many kallikrein genes are under steroid hormone regulation in breast cancer cell lines. We here examine the coordinated expression of multiple kallikrein genes in several breast cancer cell lines after steroid hormone stimulation. Breast cancer cell lines were treated with various steroid hormones and kallikrein (KLK/hK) expression of hK3 (prostate-specific antigen, PSA), hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 was analyzed at the RNA level via RT-PCR and at the protein level by immunofluorometric ELISA assays. We identified several distinct hK hormone-dependent and hormone-independent expression patterns. Hormone-specific modulation of expression was seen for several kallikreins in BT-474, MCF-7, and T-47D cell lines. hK6 was specifically up-regulated upon estradiol treatment in all three cell lines whereas PSA expression was induced by dihydrotestosterone (DHT) and norgestrel stimulation in BT-474 and T-47D. hK10, hK11, hK13, and hK14 were specifically up-regulated by DHT in T-47D and by estradiol in BT-474 cells. Bioinformatic analysis of upstream proximal promoter sequences for these hKs did not identify any recognizable hormone-response elements (HREs), suggesting that the coordinated activation of these four hKs represents a unique expression “cassette”, utilizing a common hormone-dependent mechanism. We conclude that groups of human hKs are coordinately expressed in a steroid hormone-dependent manner. Our data supports clinical observations linking expression of multiple hKs with breast cancer prognosis.     

Effects of low- and high-density lipoproteins on the proliferation of human breast cancer cells in vitro: differences between hormone-dependent and hormone-independent cell lines

The influence of low- and high-density lipoproteins on the proliferation of human breast cancer cells in culture was studied. We compared total cell number after incubation for 48 hr in culture medium containing or lacking plasma lipoproteins. Marked differences were found between hormone-dependent (MCF-7, T-47-D, ZR-75) and hormone-independent (MDA-MB-231, HBL-100) mammary tumor cell lines. The cells also reacted differently on the different lipoproteins offered in the medium. Human low-density lipoproteins (LDL) exhibited a marked stimulation of the growth of hormone-independent cell lines but no or only toxic effects upon the hormone-sensitive lines. Human high-density lipoproteins (HDL) stimulated the proliferation of all cell lines in a dose-dependent manner but hormone-independent cells showed a higher response. These findings point towards different utilizations of nutrients in hormone-dependent and hormone-independent cells.     

Comparative Evaluation of Silibinin Effects on Cell Cycling and Apoptosis in Human Breast Cancer MCF-7 and T47D Cell Lines

Silibinin is a natural polyphenol with high antioxidant and anticancer properties. In this study, its influence on two of the most commonly employed human breast cancer cell lines, MCF-7 and T47D, and one non-malignant MCF-10A cell line, were investigated and compared. Cell viability, the cell cycle distribution and apoptosis induction were analyzed by MTT and flow cytometry, respectively. The effect of silibinin on PTEN, Bcl-2, P21, and P27 mRNAs expression was also investigated by real-time RT-PCR. It was found that silibinin caused G1 cell cycle arrest in MCF-7 and MCF-10A cells but had no effect on the T47D cell cycle. Silibinin induced cytotoxic and apoptotic effects in T47D cells more than the MCF-7 cells and had no cytotoxic effect in MCF-10A cells under the same conditions. Silibinin upregulated PTEN in MCF-7 and caused slightly increased P21 mRNA expression in T47D cells and slightly increased PTEN and P21 expression in MCF-10A cells. Bcl-2 expression decreased in all of the examined cells under silibinin treatment. P27 mRNA expression upregulated in T47D and MCF-10A cells under silibinin treatment. PTEN mRNA in T47D and P21 and P27 mRNAsin MCF-7 were not affected by silibinin. These results suggest that silibinin has mostly different inhibitory effects in breast cancer cells and might be an effective anticancer agent for some cells linked to influence on cell cycle progression.