Apoptosis Of Breast Tumor Cells Research Articles

Abstract C47: 21-OH 6,19-epoxyprogesterone: A new dissociated glucocorticoid with anti-inflammatory properties but without antiapoptotic effects on chemotherapeutically treated mouse mammary tumor cells

Abstract Glucocorticoids (GCs) are widely used as coadyuvants in the treatment of solid tumors with chemotherapics such as paclitaxel (PXL) and doxorubicin (DOXO). However, clinical evidence supports the idea that GCs may induce resistance to these antitumor therapies. To improve cancer treatment quality, intense efforts are being made to design new dissociated steroids (steroid receptor glucocorticoid modulators (SRGMs). These compounds should mimic only the beneficial aspects of glucocorticoid therapy without inducing the undesired side effects in cancer patients. We have previously shown that dexamethasone (Dex), a well-known synthetic GC, inhibits PXL or DOXO induced apoptosis in breast tumor cells through a mechanism involving Bcl-XL and Bcl-2 induction. Here, our goal was to compare Dex effects with those triggered by 21-OH-6,19-epoxyprogesterone (21-OH-6,19-OP), which is a rigid steroid considered a passive GC antagonist. Molecular dynamic simulation studies predicted that 21-OH-6,19OP is unable to induce GR/21-OH-6,19-OP complex homodimerization and/or binding to coactivators, making these complex incapable to directly activate gene transcription (Alvarez et al J Med Chem. 2008. 13:1352). The effects of 21-OH-6,19-OP on apoptosis induced by DOXO or PXL in the epithelial mouse mammary tumor cell line LM3 was determined by analyzing Caspase-3 activity and gene and protein expression of antiapoptotic members of the Bcl family, i.e. Bcl-2 and Bcl-XL. The results show that, contrary to Dex, 21-OH-6,19-OP cannot reverse apoptosis induced by DOXO or PXL in LM3 cells. In fact, 21-OH-6,19-OP and Dex differentially regulate Bcl-2 and Bcl-XL expression. Dex's reversion on chemotherapic induced apoptosis (Dex+DOXO o Dex+PXL) correlates with an increase in Bcl-XL levels, while 21-O-H-6, 19-OP does not affect neither cell death nor Bcl-XL levels. The relevance of Bcl-XL involvement in DOXO resistance of cells treated with Dex was confirmed by knocking-down Bcl-XL with specific siRNA. Silencing Bcl-XL expression directly implicated loss of antiapoptotic GC effects. On the other hand, 21-OH-6,19-OP antiinflammatory action was also evaluated on the lung epithelial tumor cell line A549 by determining Interleukin-8 (IL-8) and Cyclooxygenase-2 (COX-2) expression in the presence of the Tumor Necrosis Factor-alpha (TNF-α). Expression levels of IL-8 and COX-2, measured by qRT-PCR, indicate that 21-OH-6,19-OP may have antiinflammatory effects as it reverses these inflammation markers expression induced by TNF-α in lung epithelial cells. Together, these findings suggest that 21-OH-6,19-OP is a SRGM that keeps glucocorticoid antiinflammatory properties without antiapoptotic effects, thus becoming a novel candidate for treatment of solid tumors as breast cancer. Citation Information: Cancer Res 2009;69(23 Suppl):C47.

Mitotic arrest and JNK-induced proteasomal degradation of FLIP and Mcl-1 are key events in the sensitization of breast tumor cells to TRAIL by antimicrotubule agents

Breast tumor cells are often resistant to tumor necrosis factor-related apoptosis-inducing ligand (tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)/APO-2 L). Here, we describe the sensitization by microtubule-interfering agents (MIAs) to TRAIL-induced apoptosis in breast tumor cells through a mitotic arrest and c-Jun N-terminal kinase (JNK)-dependent mechanism. MIA treatment resulted in BubR1-dependent mitotic arrest leading to the sustained activation of JNK and the proteasome-mediated downregulation of cellular FLICE-inhibitory protein (cFLIP) and myeloid cell leukemia-1 (Mcl-1) expression. The JNK inhibitor SP600125 abrogated MIA-induced mitotic arrest and downregulation of cFLIP and Mcl-1 and reduced the apoptosis caused by the combination of MIAs and TRAIL. Silencing of cFLIP and Mcl-1 expression by RNA interference resulted in a marked sensitization to TRAIL-induced apoptosis. Furthermore, in FLIP-overexpressing cells, MIA-induced sensitization to TRAIL-activated apoptosis was markedly reduced. In summary, our results show that mitotic arrest imposed by MIAs activates JNK and facilitates TRAIL-induced activation of an apoptotic pathway in breast tumor cells by promoting the proteasome-mediated degradation of cFLIP and Mcl-1.

Phloretin Induces Apoptosis in H‐Ras MCF10A Human Breast Tumor Cells through the Activation of p53 via JNK and p38 Mitogen‐Activated Protein Kinase Signaling

Mutations in Ras play a critical role in the development of human cancers, including breast cancer. We investigated the possible antiproliferative effects of the naturally occurring dihydrochalcone phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on H-Ras-transformed MCF10A human breast epithelial (H-Ras MCF10A) cells. Phloretin suppressed H-Ras MCF10A cell proliferation in a dose-dependent manner and induced nuclear condensation in the cells, indicating that phloretin-induced cell death occurs mainly via the induction of apoptosis. Prominent upregulation of p53 and Bax and cleavage of poly (ADP)-ribose polymerase were also detected in the phloretin-treated cells. Finally, phloretin markedly increased caspase-3 activity as well as JNK and p38 mitogen-activated protein kinase signaling. Our findings suggest that the phloretin-induced apoptosis of breast tumor cells contributes to the chemopreventive potential of phloretin against breast cancer.

Down-regulation of RIP expression by 17-dimethylaminoethylamino-17-demethoxygeldanamycin promotes TRAIL-induced apoptosis in breast tumor cells

The Hsp90 inhibitor 17DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) is undergoing clinical trials as an antitumor drug. We show here that treatment of human breast cancer cells with 17DMAG facilitates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Down-regulation of receptor interacting protein (RIP1) is observed upon 17DMAG treatment concomitantly with inhibition of IκBα phosphorylation. Interestingly, RNAi-mediated knockdown of RIP1 expression is sufficient to sensitize human breast tumor cells to TRAIL-induced apoptosis through a NF-κB-independent, mitochondria-operated pathway. Our results indicate that RIP1 is important in maintaining resistance to TRAIL-induced apoptosis in breast tumor cells and highlight the potential therapeutic benefit of the combination of Hsp90 inhibitors and TRAIL against breast tumor cells.

Statins activate a mitochondria-operated pathway of apoptosis in breast tumor cells by a mechanism regulated by ErbB2 and dependent on the prenylation of proteins

Statins are inhibitors of the mevalonate synthesis pathway that induce apoptosis in tumor cells although the apoptotic mechanism activated by statins remains to be elucidated. We have examined the role of the mitochondria-operated pathway of apoptosis in the cell death induced by statins in breast tumor cells and its regulation by protein prenylation and ErbB2 overexpression. Lovastatin treatment down-regulates the expression of Bcl-2 and activates apoptosis through a mitochondria-operated, ErbB2- regulated mechanism. Apoptosis induced by statins is independent of their effects on cholesterol synthesis and involves protein prenylation. Our results indicate that prenylation of apoptosis-regulating proteins is a key event in the survival of breast tumor cells and this requirement could be circumvented in cells overexpressing the oncogene ErbB2.

Flavopiridol Induces Cellular FLICE-Inhibitory Protein Degradation by the Proteasome and Promotes TRAIL–Induced Early Signaling and Apoptosis in Breast Tumor Cells

The cyclin-dependent kinase inhibitor flavopiridol is undergoing clinical trials as an antitumor drug. We show here that pretreatment of different human breast cancer cell lines with flavopiridol facilitates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In breast tumor cells, apoptosis induction by TRAIL is blocked at the level of apical caspase-8 activation. Flavopiridol treatment enhances TRAIL-induced formation of death-inducing signaling complex and early processing of procaspase-8. Subsequently, a TRAIL-induced, mitochondria-operated pathway of apoptosis is activated in cells treated with flavopiridol. Down-regulation of cellular FLICE-inhibitory proteins (c-FLIP; c-FLIP(L) and c-FLIP(S)) is observed on flavopiridol treatment. c-FLIP loss and apoptosis sensitization by flavopiridol are both prevented in cells treated with an inhibitor of the ubiquitin-proteasome system. Furthermore, targeting c-FLIP directly with small interfering RNA oligonucleotides also sensitizes various human breast tumor cell lines to TRAIL-induced apoptosis. Our results indicate that flavopiridol sensitizes breast cancer cells to TRAIL-induced apoptosis by facilitating early events in the apoptotic pathway, and this combination treatment could be regarded as a potential therapeutic tool against breast tumors.

Focal Adhesion Kinase and Protein Kinase B Cooperate to Suppress Doxorubicin-Induced Apoptosis of Breast Tumor Cells

Focal adhesion kinase (FAK) is up-regulated in a variety of cancers, including breast cancer, in association with poor disease prognosis. In the present study, we examined the role of FAK in the control of anticancer drug-induced apoptosis of mammary adenocarcinoma MTLn3 cells. Doxorubicin caused the formation of well defined focal adhesions and stress fibers early after treatment, which was later followed by their loss in association with the onset of apoptosis. Phosphorylation of FAK on tyrosine 397 decreased only during the onset of doxorubicin-induced apoptosis in a Bcl-2 and caspase-independent manner. Doxorubicin also caused an early activation of protein kinase B (PKB). Expression of the dominant-negative acting focal adhesion kinase-related nonkinase (FRNK) sensitized MTLn3 cells to apoptosis caused by doxorubicin. FRNK inhibited the doxorubicin-induced activation of PKB. In addition, inhibition of phosphatidylinositide-3 (PI-3) kinase with wortmannin inhibited the activation of PKB by doxorubicin. Both wortmannin and transient overexpression of the dual lipid/protein phosphatase and tensin homolog deleted on chromosome 10 enhanced doxorubicin-induced cell death. Altogether, these data fit with a model wherein FAK is involved in the doxorubicin-induced activation of the PI-3 kinase/PKB signaling route, thereby suppressing the onset of apoptosis caused by doxorubicin.

The sequential treatment with ionizing radiation followed by TRAIL/Apo-2L reduces tumor growth and induces apoptosis of breast tumor xenografts in nude mice

TRAIL primarily induces apoptosis in cancer cells but not in normal cells. However, some TRAIL-resistant cancer cell lines have recently been discovered. Ionizing radiation may enhance the apoptosis inducing potential of TRAIL in sensitive cells, and sensitize TRAIL-resistant cancer cells. We assessed the influence of sequential treatment of irradiation followed by TRAIL on intracellular mechanisms of apoptosis of breast tumor cells in vitro and on tumor regression in xenografted athymic nude mice. Irradiation augmented TRAIL-induced apoptosis in breast cancer cells through up-regulation of DR5, and subsequent activation of caspases-3, -8 and -9. Inhibition of p53 by siRNA abrogated irradiation-induced DR5 expression, suggesting the requirement of p53 for DR5 induction. The pretreatment of cells with irradiation followed by TRAIL significantly induced more apoptosis than single agent alone or concurrent treatment with irradiation and TRAIL. The sequential treatment of xenografted mice with irradiation followed by TRAIL-induced apoptosis through caspase-3 activation, completely eradicated the established breast tumors, and enhanced survival of mice without detectable toxicity to normal tissues. The sequential treatment with irradiation followed by TRAIL provides an approach to enhance therapeutic potential of TRAIL. Thus, irradiation can be combined with TRAIL in breast cancer therapy.

Human dendritic cells induce tumor-specific apoptosis by soluble factors.

Dendritic cells (DCs) are the most potent antigen producing cells (APCs) for initiation of immune responses including anti-tumor immune responses. In previous reports, it has been shown that DCs efficiently take up and process apoptotic or necrotic bodies of tumor cells. It has also been shown that DCs pulsed with tumor cell apoptotic bodies, lysates or peptides generate potent anti-tumor immune responses. Direct interactions between DCs and viable tumor cells, however, have not been clearly elucidated. We report that monocyte-derived, CD1a+ immature DCs (iDCs) significantly inhibit the growth of breast tumor cells in coculture and transwell experiments in the presence of soluble CD40 ligand (sCD40L), LPS or both. The growth inhibition effects correlated with cell cycle arrest and apoptosis of breast tumor cells. The effects were associated with morphological changes of tumor cells from a round shape to a flat, spindle shape. In contrast, no inhibition of proliferation or morphological changes was observed on normal PBMC, K562 or breast fibroblasts. Interestingly, iDCs undergoing maturation induced by sCD40L+LPS induced a much stronger growth inhibitory effect than iDCs alone or mature DCs treated with sCD40L+LPS. Fractionation of supernatants showed the anti-tumor effects were mediated by a TNF-alpha-dependent and -independent mechanism. Soluble FasL and TRAIL were not involved. Our findings suggest that maturing DCs have the intrinsic ability to induce cell-cycle arrest and apoptosis of breast tumor cells through soluble factors, but not normal cells, in addition to their Ag presentation function.

Placental Transforming Growth Factor-β Is a Downstream Mediator of the Growth Arrest and Apoptotic Response of Tumor Cells to DNA Damage and p53 Overexpression

The p53 tumor suppressor gene and members of the transforming growth factor-beta (TGF-beta) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-beta superfamily, designated placental TGF-beta (PTGF-beta), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-beta is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-beta promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-beta promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-beta overexpression from a recombinant adenoviral vector (AdPTGF-beta) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50-60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-beta overexpression was seen to induce both G(1) cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-beta superfamily and implicate PTGF-beta as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.

P53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells.

Induction of CD95 (Fas/APO-1) and CD95 ligand during chemotherapeutic treatment may contribute to the death by apoptosis of some tumor cells. In this study, we have analyzed the role of the CD95 system in genotoxic drug-induced death of human breast tumor cells. Incubation of the breast tumor cell lines MCF-7 and EVSA-T with doxorubicin or methotrexate caused apoptosis after 48 h of treatment. These drugs induced a marked increase in the level of CD95 mRNA and protein in wild-type p53-expressing MCF-7 cells. On the contrary, the breast cancer cell line EVSA-T that expresses high levels of an inactive form of p53, did not up-regulate CD95 upon drug treatment. Elevation of CD95 expression by DNA-damaging drugs was notably blocked in MCF-7 cells expressing the human papillomavirus type 16 E6 protein (E6 cells) which prevented p53 accumulation upon DNA damage. However, E6 cells were still killed by the drugs. Furthermore, the genotoxic drugs did not induce the expression of CD95 ligand in MCF-7 cells at doses that caused apoptosis in these breast tumor cells. Moreover, drug-induced apoptosis of breast tumor cells was not prevented in the presence of either a CD95 antagonistic antibody or a CD95 ligand blocking antibody. We also observed a strong synergism between lower doses of DNA-damaging drugs and CD95 agonistic antibody in the induction of apoptosis in MCF-7 cells. In summary, our data indicate that drug-induced apoptosis of breast tumor cells occurs by a CD95/CD95L-independent mechanism although by elevating the tumor suppressor proteins p53 and CD95, genotoxic drugs may sensitize breast tumor cells to CD95-mediated apoptosis.