Induced Breast Cancer Cell Death Research Articles

Abstract C85: ck2 inhibitors in luminal and basal-like breast cancer cells.

Abstract Protein kinase casein kinase II (CK2) is a heterotetrameric serine/threonine kinase involved in proliferation, apoptosis and embryonic development. It is expressed at elevated levels in proliferating tissue, human cancer cell lines and virtually all forms of solid cancers. Recently it has been shown that CK2 inhibition induces apoptosis in cell models of acquired anti-estrogen resistance. These findings suggest that there may be tumor phenotype specific effects of CK2 in breast cancer and consequently phenotype specific sensitivity to CK2 inhibition. As such we sought to determine if luminal and basal cell types display differential sensitivity to selective CK2 inhibitors. Utilizing crystal violet and alamar blue assays, it was found that CK2 inhibition decreased viability of both luminal and basal breast cancer cell lines, with basal-like ER(−) MDA MB 231 cells showing notable sensitivity. Hypertrophic morphologic changes consistent with cellular senescence were noted upon inhibition of CK2. Additionally, flow cytometric analysis revealed an increase in mean vimentin expression in basal but not luminal breast cancer cells following CK2 inhibition. These studies suggest that CK2 utilizes multiple molecular mechanisms to promote tumorigenesis, and that CK2 inhibition may disrupt cell-type specific signaling pathways to induce breast cancer cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C85.

Capsaicin may induce breast cancer cell death through apoptosis-inducing factor involving mitochondrial dysfunction

The majority of breast cancer patients are resistant to chemotherapy or radiotherapy due to the down-regulation or lack of caspase-3 expression. Capsaicin was found to inhibit cancer cell growth in caspase-3-deficient human breast cancer cells. This study aimed to investigate the growth-inhibitive effect of capsaicin and its mechanisms in human breast cancer cell lines, MCF-7 and BT-20. The results showed that cell viability decreased in a dose-dependent manner in both the caspase-3-deficient and non-deficient cells through inducing cell apoptosis and arresting the cell cycle in the S phase. Capsaicin significantly decreased mitochondria membrane potential, induced the cleavage of PARP-1, and decreased procaspase-7 expression in both cells. Apoptosis-inducing factor (AIF) was distinctly released from mitochondria and translocated into the cytoplasm and nucleus in MCF-7 cells (52.9%), but not in BT-20 cells (2%) after treatment with 200 μM of capsaicin for 24 hours. Capsaicin inhibited breast cancer cell growth through inducing cell apoptosis and cell cycle arrest in the S phase. This apoptotic effect could be induced through the mitochondrial pathway, and PARP-1 subsequently cleaved by activation of caspase-7. The application of capsaicin in clinical therapy could be useful for breast cancer patients.

JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells

Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

CG0006, a novel histone deacetylase inhibitor, induces breast cancer cell death via histone-acetylation and chaperone-disrupting pathways independent of ER status

We previously reported that CG0006, a novel hydroxamate-based pan-histone deacetylase inhibitor (HDACI), suppresses the growth of human cancer cells. Here, we tested the ability of CG0006 to inhibit breast cancer cell proliferation in relation to estrogen receptor (ER) status, and examined changes in the expression of cell-cycle regulatory proteins. CG0006 effects on the proliferation of multiple human cancer cell lines were tested using MTT and MTS assays. Changes in estrogen-signaling proteins and cell-cycle regulatory proteins were examined by western blotting and quantitative RT-PCR, and cell-cycle effects were tested using flow cytometry. CG0006 increased histone H3 and H4 acetylation, up-regulated p21 protein, and promoted cell-cycle arrest, inducing G(2)/M-phase accumulation in ER-positive MCF7 cells, and G(1)- and G(2)/M-phase accumulation in ER-negative MDA-MB-231 cells. In both cell types, CG0006 treatment (1 μM) reduced the levels of the estrogen-signaling proteins ERα and cyclin D1, and promoted massive degradation of cell-cycle regulatory proteins. CG0006 down-regulated the histone deacetylase HDAC6 at the protein level in association with a subsequent increase in Hsp90 and α-tubulin acetylation. HDAC6 depletion using small interfering RNA produced a protein-degradation phenotype similar to that of CG0006 treatment. These findings suggest that CG0006 inhibits breast cancer cell growth by two different pathways: a histone acetylation-dependent pathway, and a non-epigenetic pathway that disrupts chaperone function.

Induction of Cancer Cell Death by Self-assembling Nanostructures Incorporating a Cytotoxic Peptide

Nanotechnology offers novel delivery vehicles for cancer therapeutics. Potential advantages of nanoscale platforms include improved pharmacokinetics, encapsulation of cytotoxic agents, enhanced accumulation of therapeutics in the tumor microenvironment, and improved therapeutic structures and bioactivity. Here, we report the design of a novel amphiphilic molecule that self-assembles into nanostructures for intracellular delivery of cytotoxic peptides. Specifically, a cationic alpha-helical (KLAKLAK)(2) peptide that is known to induce cancer cell death by membrane disruption was integrated into a peptide amphiphile (PA) that self-assembles into bioactive, cylindrical nanofibers. PAs are composed of a hydrophobic alkyl tail, a beta-sheet forming peptide, and a bioactive peptide that is displayed on the surface of the nanofiber after self-assembly. PA nanostructures that included (KLAKLAK)(2) were readily internalized by breast cancer cells, in contrast to the (KLAKLAK)(2) peptide that on its own was not cell permeable. (KLAKLAK)(2) nanostructures, but not the peptides alone, also induced breast cancer cell death by caspase-independent and Bax/Bak-independent mechanisms associated with membrane disruption. Significantly, (KLAKLAK)(2) nanostructures induced cell death more robustly in transformed breast epithelial cells than in untransformed cells, suggesting a degree of tumor selectivity. Our results provide proof-of-principle that self-assembling PAs can be rationally designed to generate nanostructures that can efficiently deliver cytotoxic peptides to cancer cells.

Treatment of Breast Cancer Cells with Proteasome Inhibitor Lactacystin Increases the Level of Sensitivity to Cell Death Induced by Human Immunodeficiency Virus Type 1

Upon binding to CD4, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 undergoes conformational changes that facilitate subsequent interactions with the chemokine coreceptor CXCR4 on the T cells. Our previous study showed that HIV-1 induces breast cancer cell death through gp120-CXCR4 interaction without CD4-induced conformational change of gp120. To characterize the structural properties of CXCR4 on breast cancer cells, the structural differences in CXCR4 between breast cancer cell lines and T cells were investigated. Immunoblots of whole cell lysates from breast cancer cell and T cell lines demonstrated that the predominant forms of CXCR4 on the breast cancer cell lines and T cell lines were three species (45, 61, 100 kDa) and one species (45 kDa), respectively. Cell surface biotin labeling revealed that the 100-kDa polyubiquitinated form of CXCR4 is specifically expressed on the surface of breast cancer cell line DU4475 but not T cell line Molt4#8. The treatment of breast cancer cell lines MDA-MB231 and DU4475 with proteasome inhibitor lactacystin leads to increased surface expression of the 100-kDa polyubiquitinated form of CXCR4 and increases the level of sensitivity to cell death induced by HIV-1. These data suggest that the 100-kDa polyubiquitinated form of CXCR4 plays an important role as a trigger for gp120-induced breast cancer cell death.

Methyl-donor nutrients inhibit breast cancer cell growth

Lipotropes (methyl group containing nutrients, including methionine, choline, folate, and vitamin B(12)) are dietary methyl donors and cofactors that are involved in one-carbon metabolism, which is important for genomic DNA methylation reactions and nucleic acid synthesis. One-carbon metabolism provides methyl groups for all biological methylation pathways and is highly dependent on dietary supplementation of methyl nutrients. Nutrition is an important determinant of breast cancer risk and tumor behavior, and dietary intervention may be an effective approach to prevent breast cancer. Apoptosis is important for the regulation of homeostasis and tumorigenesis. The anti-apoptotic protein Bcl-2 may be a regulatory target in cancer therapy; controlling or modulating its expression may be a therapeutic strategy against breast cancer. In this study, the effects of lipotrope supplementation on the growth and death of human breast cancer cell lines T47D and MCF-7 were examined and found to inhibit growth of both T47D and MCF-7 cells. Furthermore, the ratios of apoptotic cells to the total number of cells were approximately 44% and 34% higher in the lipotrope-supplemented treatments of T47D and MCF-7 cancer cells, respectively, compared with the control treatments. More importantly, Bcl-2 protein expression was decreased by approximately 25% from lipotrope supplementation in T47D cells, suggesting that lipotropes can induce breast cancer cell death by direct downregulation of Bcl-2 protein expression. Cancer treatment failure is often correlated with Bcl-2 protein upregulation. These data may be useful in the development of effective nutritional strategies to prevent and reduce breast cancer in humans.

Statin-Induced Breast Cancer Cell Death: Role of Inducible Nitric Oxide and Arginase-Dependent Pathways

Statins are widely used cholesterol-lowering drugs that selectively inhibit the enzyme 3-hydroxy-3-methylglutaryl CoA reductase, leading to decreased cholesterol biosynthesis. Emerging data indicate that statins stimulate apoptotic cell death in several types of proliferating tumor cells, including breast cancer cells, which is independent of its cholesterol-lowering property. The objective here was to elucidate the molecular mechanism(s) by which statins induce breast cancer cell death. Fluvastatin and simvastatin (5-10 mumol/L) treatment enhanced the caspase-3-like activity and DNA fragmentation in MCF-7 cells, and significantly inhibited the proliferation of MCF-7 cells but not MCF-10 cells (noncancerous epithelial cells). Statin-induced cytotoxic effects were reversed by mevalonate, an immediate metabolic product of the acetyl CoA/3-hydroxy-3-methylglutaryl CoA reductase reaction. Both simvastatin and fluvastatin enhanced nitric oxide ((.)NO) levels which were inhibited by mevalonate. Statin-induced (.)NO and tumor cell cytotoxicity were inhibited by 1400W, a more specific inhibitor of inducible nitric oxide synthase (iNOS or NOS II). Both fluvastatin and simvastatin increased iNOS mRNA and protein expression. Stimulation of iNOS by statins via inhibition of geranylgeranylation by GGTI-298, but not via inhibition of farnesylation by FTI-277, enhanced the proapoptotic effects of statins in MCF-7 cells. Statin-mediated antiproliferative and proapoptotic effects were exacerbated by sepiapterin, a precursor of tetrahydrobiopterin, an essential cofactor of (.)NO biosynthesis by NOS. We conclude that iNOS-mediated (.)NO is responsible in part for the proapoptotic, tumoricidal, and antiproliferative effects of statins in MCF-7 cells.

RNAi‐mediated silencing of insulin receptor substrate 1 (IRS‐1) enhances tamoxifen‐induced cell death in MCF‐7 breast cancer cells

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy.

Bisphosphonates induce breast cancer cell death in vitro.

Breast cancer frequently spreads to bone and is almost always associated with osteolysis. This tumor-induced osteolysis is caused by increased osteoclastic bone resorption. Bisphosphonates are used successfully to inhibit bone resorption in tumor bone disease and may prevent development of new osteolytic lesions. The classical view is that bisphosphonates only act on bone cells. We investigated their effects on breast cancer cells using three human cell lines, namely, MCF-7, T47D, and MDA.MB.231, and we tested four structurally different bisphosphonates: clodronate, pamidronate, ibandronate, and zoledronate. We performed time course studies for each bisphosphonate at various concentrations and found that all four compounds induced a nonreversible growth inhibition in both MCF-7 and T47D cell lines in a time- and dose-dependent manner. The MDA.MB.231 cell line was less responsive. Bisphosphonates induced apoptosis in MCF-7 and cell necrosis in T47D cells. The inhibition of MCF-7 cell proliferation could be reverted almost completely by the benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk) inhibitor of caspases, suggesting that the apoptotic process observed in the MCF-7 cell line is mediated, at least partly, by the caspase system. Caspase activity was little changed by bisphosphonates in T47D cells and the inhibitor of caspase did not modify bisphosphonates effects. In summary, we found that bisphosphonates inhibit breast cancer cell growth by inducing cell death in vitro. Such effects could contribute to the beneficial role of bisphosphonates in the treatment and the prevention of tumor-induced osteolysis.